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CA 02296765 2000-O1-14 ;
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DEMANDES OU BREVETS VOLUMINEUX
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COMPREND PLUS t7'UN TOME.
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CA 02296765 2000-O1-14
o rcr~us~rns394
MULTIVALENT VACCINE FOR CLOSTRIDIUM BOTULINUM NEUROTOXIN
FIELD OF THE INVENTION
- The present invention relates to the isolation of polypeptides derived from
C'lo.s~ridiurn
s hrriulinum neurotoxins and the use thereof as immunogens Ibr the production
of vaccines.
including multivalent Vaccines. and antitoxins.
BACKGROUND OF THE INVENTION
The genus C'luctridiurn -is comprised of gram-positive, anaerobic. spore-
formine bacilli.
1 U The natural habitat of these organisms is the environment and the
intestine! tracts of humans
and other animals. lndeed, clostridia are ubiquitous: they are commonly found
in soil. dust.
sewage. marine sediments. decayins~ vt:getation. and mud. (.See c.,~~.. P.H.A.
Sheath er crl..
"f 'lu.crriclitrnt." l3c~r;t~c~u'.c .hlcrnuctl tt n~ .St'.stcmulic
l3crrlerirrln,L~v. Vol. ?. pp. 1 I.ll-1?00.
\~l'illiams ~ \'l'ilkins ( 19861. J Despite the identification of
approximately I UU species of
Is <'lnsrr-iclirmu. only a small number have been recognized as etiology=is
agents of medical and
veterinary importance. Nonetheless. these species are associated with very
serious diseases.
includint: botulism. tetanus. anaerobic ccllulitis, gas ganLrene. hacteremia.
pst:udorncmbranous
colitis. and clostridia) gastroenteritis. Table 1 lists some of the species of
medical and
veterinary importance and the diseases with which they arc associated. .as
virtually all of
'?U thaw species have been isolated from fecal samples of apparently healthy
persons. some of
tllCSt."IVOIQICS Illa1' be transient. rather than permanent residents of the
coionic flora.
rAeu: t
. ~t....._:.r:..... c.,....:.~ nr tut~dir~l And Veterinan~ Imnortancc'
'? species Disease
j
C'. cunirrrwcrlcrirmn Bacteriuria (preunant women)
('. ur,~,eruim:nce Infected wounds: Baeteremia: botulism:
Infections of amniotic fluid
Infected war wounds: Peritonitis: Infectious
C'. hco~cuii processes of the eye. ear
and prostate
C'. heiicrinrkikii Infected wounds
i0 c'. hilrrnrcmcrrrv Infected wounds: Abscesses: Gas Gam_rene:
. Bacteremia
C'. t,muiinrun Food poisoning: Botulism (wound. food.
infant)
lJrinan' tract. lower respiratory tract.
C'. hurrriruat pleural cavity. and abdominal
I nfected wounds: Abscesses: Bacteremia
infections:
C'. cuclurerir Abscesses: Infected wounds
CA 02296765 2000-O1-14
PCTlUS97115394
TABLE 1
('Irr.slrirliunr Species Of Medical And Veterinary Importance'
Species Disease
('. ccwni.e Soft tissue infections: Dacteremia
C'. chuulwri BIaCkle~,
C'. c'Irr.crrriliuJilrrnc
r1hdominal, cervical, scrotal, pleural.
and other infections: Septicemia:
Peritonitis; Appendicitis
(' crn~lrleru'mm Isolated from human disease processes,
hut role in disease unknown.
(.. cli//ic'ilc Antimicrobial-associated diarrhea: t'seudomembranous
enterocolitis:
8acteremia: I'voeenic infections
(' /rrllrev Soti tissue infections
C' ,elurnii Sufi tissue infections
(' ~/acwlunun lVound infections: Abscesses: Pcritonis
('. Irrr.rrlru'nrc' Itlfccted war wounds: fiacterernia; Abscesses
I(1 ( lrraullrrenrm Infected war wounds: C.ias !_an~!rrnr:
Gingival placfuc isolate
(' rrnlrrlr.v C~aslmintestinal tract infections
l' irrnrrcurrrrn Gastrointestinal tract Infections: C:nlpycn
la
t' Irrc,s~ulcu'c Penile lesions
<'. I~yuunr Isolated tfnm human disease prUCCSSIS.
but r()Ir in dISeaSI UIIkIIUW'Il.
(' lirltW rrr)r l,atterenlla; I'erltUllltl5: f'111111UI1F11'~
111tCCtIUnS
rrrcrlunrlrnrrrUlflrrlVarIUIIS IIlfeCIIUUS processes
Infected mounds: Gas !~an~~rene: 131ackle_.
(' nrmw I3i~_ head lovinrl: Itedwater
disease Ihovine)
(' rwrrcvrrrr l!rinarv tract infections: Rectal alISCCSSeS
I~cll'tlyllll'I/IC'fllllL3aClCrenlla: PCrIfOnIlll: lIIfCCIOtI
W'(11111d5: AppelldlClllv
(ialS ~~ilnrenC: Anaerobic crII1111115:
('. yc'rJrin,ycuzv tntra-abdominal ahsCCSSC',5: 11f11
tissue infections: food poisunill~~: Necrotizin~
pneumonia; f::mpycma:
~4eninl:itis: t3acteremia: lJterine Infections:
Enteritis necrutans: t.amh
dvsenterv: Struck: ()vine Enlerotoxemia:
('. Inrrrc'hreic'rre f3acteriuria (Pre~nanl women with hacteremia)
('. lrrrri/iorun Abscesses: Infected wounds: fiacterrmia
(' rrrrrrrrvrrrrr
Infections of the abdominal cavity. genital
tract. lun~_. and hiliary tract:
f3acteremia
<' .vcrr'rcrs;u/ru'rrroIsolated from human disease processes.
hut role in disease unknown.
Oas ~.:an~,rene: t3acteremia; Sllpplll'fitll'e
_J ('..v~luicorn IIlteCtlUIIS: ~ICCrotilinS:
r
cnterocolitis: l3raw
('. virr'clc'llir CiaS !~all~~rCllC: wOtllld IIIfCCII<)I1S:
Peltlle Ie111111$: l3aOCrC1111a:
AhSCCSSeS: AhdUllllnill illld Vill,lllill
IIlteCll(1115
. 7 .
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WO 98/08540 PCTNS97/I5394
TABLE !
('Irr.efr'itlirrm Species of Medical And Veterinary lrnponance'
s
Species Disease
Appendicitis: Bacteremia: L3one and soft
tissue infections:
. (' cl,Ircrrrriclc.s lntrapcritoneal infections: infected war
wounds: Visceral ~~as gangrene:
Renal abscesses
Cias Lan==rene: Bacteremia: Endocarditis:
central nervous sastem and
- <'. sl,rrrrmvrc.s plcuropulmonary infections: Penile lesions:
Infected war wounds:
ether pyogenic infections
('. .wrhrerrrnulc' Eiacteremia: Empyema: E3iliary tract.
soft tissue and bane infections
c' ."nhirrvm Liver abscesses: E3acteremia: Infections
resuitin~s due: to hove( flora
Gas ~~anarene: Appendicitis: Brain abscesses:
~ Intestinal tract and soft
. It'I'Irllr)r ' '
( tissue infections: infected war wounds:
i'eriodomitis: Bacteremia
Tetanus: Infected gums and teeth: l'nrneal
ulccrations: Mastoid and
middle ear infections: Intraperitoueal
infections: tetanus neonatorum:
(' r~uurrr Postpartum uterine infections: Soft tissue
infections. especially related
to trauma (includirt~! abrasions and lacerations):
Infections related to
use of contaminated needles
r' rlumrrn.verce'luu'nhwcurrIsolated Irom human disease processes.
hut role in disease unl:nnwn.
' l-unrpilcd front I'.(i. L~.n~wlkirk cu crl "('lcr.s'.sr/ic~cuirrrr".
J'rincyrlc~.s urrcl l'rcunic'c n/ ('Jirric'crl .~lnucnvrhu
1 () Ilmr n~rvrrlry~n. pp. ,?-?.i. star Publishin'_ C'.u., f3elmont. CA I
199?): .I. vephen and R.n. I'etrowski.
.~luvin.v l!'hic'h li'cwrrwc~ .lle~»rhr'c»n~v a»cl Dcre~,t,'rrlutr ('c~Ilc."
ire Ilcrevc'rrerl l'er.ri».,. ?cl ed., pp. 66.07.
lmcrican Sociey for Microhiolo~y 1 I')8(i): R. E3erkow and A..I. I-irtcltcr
tcds.l. "I>rurrrrcrJ I)i.rccrsu~.,."
llcv'c'k ~llr»rrrerl nJ Dicrtvrrr.,r.s a»cl Tlrerup', 16th ed., pp. 1 16-I?G.
Merck Research laboratories. Rahway.
~.J. ( Ic)c)3): and O.II. Si;~mund and C.M. Fraser lcds.). "('Invrriclicrl
IrrJcrc'rin».e." .llc~rc'k l u~m~rvnur'n
1 > tier»rnrl. srh ed.. pp. 39(i.~tt)9_ Merck 8: Cc,.. Rahwav. N.J. (1979).
lit Ilt(11t cases. the pathuecnicitv ut~ these organisms is related to the
release ul~ p(wcrt~ul
rxotuxins etr hi~_hy dcatructim enzvtncs. Indeed, several species (tt~ the
genus ( 'lrr.vriclirr» l
produce toxins and other en-rvmcs uf~ great medical and veterinary
siyiticance. ~C.L..
?() I-lathmav. C'lin. Alicrohiul. Rev. s:66-98 ( 1990).)
1'crhaps hecause ttt~ their significance for human and veterinary medicine,
much
research has keen conducted (tn these toxins, in particular those ut~ ('.
hrrlrrlinrr»r and ('.
cli~jicilc-.
(.: bnltttiur~m
~evcral strains oh ('Irr.slrrclrrrm hmulinrun produce toxins of aiLniticancc
to human and
animal health. ~C.l.. 1-lathew'ay. Clin. llicrohiol. Rev. ;:(il-98 ( 199Q)~
The ct~tccts of these
twins ranLe from diarrheal diseases that can cause destruction of the colon.
to paralytic
etTects that can cause death. Particularly at risk for developine clostridia)
diseases arc
_ ., _
CA 02296765 2000-O1-14
WO 98JOSS40 PCTIUS97115394
neonates and humans and animals in poor health (e~.,s,~., those suffering from
diseases
associated with old age or immunodeficiencv diseases).
('ln.vriclirrm huJUlintrm produces the most poisonous biological toxin known.
'Chc
lethal human dose is a mere 10-'' mglkg bodvwcic_:ht fnr toxin in the
bloodstream. Botulina!
s toxin blocks nerve transmission to the muxles, resulting in tlaccid
paralysis. \~'hen the toxin
rcachrs airway and respiratory muscles. it results in respiratory tailure that
can cause death.
(5. ~lrnon. .I. Infect. Uis. 14:2()1-?OG (1986))
('. huurlirrrrm spores are carried by dust and arc found on vcgetahle> taken
t~rom tire
soil. on ti~esh fruits. and on agricultural products such as honey. t under
conditions favorable
lt) to the organism. the spores germinate to vegetative: cells which produces
toxin. [~. Arnon.
Ann. Rc. Med. ~ I :,41 ( 1 c)8(?11
Botulism disease may he grouped into tour types. based an the method of
introduction
of I()\111 Illl(1 the bloodstream. Food-borne botulism results t~rum ingesting
improperly
preserved and inadequately heated food that contains botulinal toxin. ~Ilterr
mere s~~ cases of
I s I~ooc1-lu~rnc botulism in the t Jnited States between 1 c)76 and 1 c)84. (
K.I.. Mact)onald en crl..
~~m. .I. I~:hiclentiol. 1?~:7~)a (lc)86).J 'The death talc due to hotulinal
toxin i, I?'~:« and can he
higher in particular risk groups. [('.(). Tacket m crl.. nm. .1. Meet. 7(>:7~)-
t ( I ~)H~1), ( V4'ound-
induccel hcaufism results ti-om ('. huJrrlirrunr penetrating traumatized
tissue and proolucing toxin
that is absorbed into the bloodstream. Since Ic)s0. thirm cases of wound
hntulism have been
'?() reported. ('\1.N. ~wart7.. ".~trtrrcrnhic.5prrr~c-l~~nrmirt,~~ l3crculli
T7tu ('lo.wricliu." ph. fp.s-l>~1O. irr
I3.D. 1)avis ce crl..teds.). aliurrrhinlr~,L~n. ~tth edition. .I.13.
I.ippincott ('o. (Ic)c)(I).J Inhalmnon
hotulisrn results when thr toxin is inhaled. Inhalation botulism hay been
reported a, the result
cvf~accidental mpetsure in the laboratory (I:. Ilolzer. hleel. );lin. ~1:17;i
(I~)(~''IJ .md c:ould
arise if the toxin is used as an agent of biological wurtare ( l).lt. (rant rr
crl.. in l3rmrlinrrrra
curd T incrrrrr.c .\'c~uroJnsinv. B.R. UasCiupta. ed.. I'Icnum i'rcss. Nrw
York 1 1 ~)~);1, pp. ~7s-.~76].
Intcetious infant hotufism results from ('. horulinrrnr colonization of the
int~ant intestine with
production of toxin and its absorption into the bloodstream. It is likely that
the bacterium
Lams entry when spores arc ingested and suhsequentU g.erminatc. (~. Arnon. .I.
Infect. 1)is.
f S4:?Ol ( I c)86). ( There ltave been i()0 cases rrportcd since it was tirst
recognized in I ~)7(~.
;() ( M.N. ~wartz. .wrpru. (
CA 02296765 2000-O1-14
WO 98108540 PCTIUS97I115394
Infant botulism strikes infants who are three weeks to eleven months old
(greater than
90% of the cases are infants less than six months). [S. Arnon. .(. Infect.
Uis. 1~:~:201 (198G).]
It is believed that infants are susceptible, due, in large part. to the
absence of the full adult
complement of intestinal microtlura. The benign microtlora present in the
adult intestine
provide an acidic elivironment that is not favorable to colonization by ('.
hrur~linrrnr. Infants
begin fife with a sterile intestine which is gradually colonized by
microtlora. (3ecause of the
limited microtlora present in early infancy. the intestinal environment is not
as acidic.
atllll'IIIF_' lim ('. Irr)nrlirrrrlrr Spore LertttlttatlOn. growth. and toxin
production. tn this regard.
sonic adults who have undergone antibiotic therapy which alters intestinal
microtiora become
1l) nturc susceptible to hotuiism.
:fin additional factor accuuntinL fur infant susceptihiliw to infectious
hutuiism is the
immaturiy of the infant IlttIttUltl'. S1'StCltt. ~I~I1C ntalUfe llttltttllte
S~'Stelll 1S SenSItIZCd to
harterial antigens and produces prutectiw antibodies. Secreturv t~~A produced
in the adult
intestine Ilas tltr ahiliW to ag~~lutinate ve~~etative cells of ('.
hruulirrrrnr. (S. Arnun. J. lntcct.
I s I)is. 1 ~-x:'_'(11 t I r)8h). [ Secretary 1L ~1 ntav also act by
preventing intestinal bacteria and their
products I~l'un1 crosSitlL the cells of the intestine. [~. Arnon. Fpidemiul.
Rev. ;:-is ( 1981 ). [
The inlant 111111t1111C SVSIettl IS l7Ut primed to do this.
('linical Symptoms ol~ infant hotulistn range from ntild paralysis, to
moderate and
smcryparalysis reciuiring huspitalizatiun. to fulminant paralysis. tending to
sudden death. (S.
~U :lrnon. lpidrmiul. Rcv. s:4i t I c)81 ).]
'rhe chicf~ therapy for severe infant botulism is ventilaturv assistance using
a
ntrchcmi~al respirator and concurrent elimination of toxin and bacteria using
cathartics.
enemas. and LaSlCIC la1'QLC. There were G8 hospltaltzattons 111 C'alitornia
f'or infant h~tulisnt
in a sint!le year with a total cost of over $4 million for treatment. ['t~.l,.
l~ranl:ovich and 5.
:lrnun. V4'cst. .I. f~-led. 1 >4: I Os ( I c)9 I ). [
Different strains of C'lo.srniclirrnr hotulinum each produce antigenicatlv
distinct toxin
designated by the letters i1-G. ~crotypc n toxin has been intplicatcd in
?G°~o of th c: cases Of
ii~oti botulism: types (3. E and F have also been implicated in a smaller
percenta~~e of the titod
botulism cases (1!. Sugiyama. ;~~icrobioU. Rcv. 44:419 ( 1980)]. Wound
botulism has been
st) reportedly caused by only types A ur l3 toxins (H. Sugiyama. .~rrpr-cr(.
Nearly all cases of
infant botulism have been caused by bacteria producing either type n or type
f3 toxin.
-5-
CA 02296765 2000-O1-14
WO 98108540 PCTIUS97115394
( Exceptionally. one New Mexico case was caused by ('!us'rrrclium hnurllnum
producing type F
toxin and another by ('lo.sr'riclium hrmrlir7trm producing a type f3-type E=
hybrid.) [S. Arnon.
Epictemiol. Rev. >::15 ( 1981 ). j Tvpe C toxin affects waterfowl, cattle,
horses and mink. ~1'yPe
I) toxin affects cattle. and type E toxin af~feets both humans and birds.
r1 trivalent antitoxin derived from horse plasma is commercially available
from
C'unnaus~ht Industries i,td. as a therapy for toxin types A. B. and h.. 1
iowcver. the antitoxin .
has smeral disadvantages. First. extrenteiy large dusa~~es must h a injected
intravenously
;lndlor intramuscularly. second. the antitctxin has serious side effects such
as acute
anaphvlax)s wh)ch call lead to death. and scrum sickness. Finally. the
efficacy c)f the
I t) antitoxin is uncertain and the treatlnent is costly. jC'.(). ~ ticket n
ul.. Am. .I. Mcd. 711:7c)4
( t ~)8~). (
:1 hcptavalent eyuine botulinal antitoxin which uses only the F(ah')? portion
e)f the
antihculv mc)lecule has been tested by the L)nited States l~1ilitary. (M.
l3aladv. I~~AMROC'
'~imslctmr. 1). O ( Ic)c)1 ).J This was raised against impure toxoicts in
tlu)se lar~~r animals and is
I s nclt a hi~~h titer preparation.
:~ pentavalent human antitoxin has been collected li-om immunized human
subjects fi)r
llSe av a ll'latlttelll t()f llttallt h()tU11S11). ~rlt(; Sllppll' ()f 11115
antlll)\I11 lv 111ltlled and eanll(lI he
eapectec3 to meet the nerds of all individuals stricken with botulism disease.
In addition.
collection of human sera must involve screening out f I1V and c)ther
potentially serious human
?0 pathcyens. ( I'..1. ~chwart, and S.S. Arnon. Western .I. Med. I 56: I c)7 (
I I)~)? ).
infant botulism has been implicated as the cause oi~ mortally 111 solve cases
of Sudden
Infant I)cath Syndrome (SIDS. also known as crib death). ~lO~ is ol~ticictlly
r~co~~ni~rli as
Infant death that is sudden and unexpected and that remained unc;xplained
ctespite cc)mplete
post-nu~rt-rm examination. ~I~hc link of SIDS to inthnt botulism came when
fecal or hloacl
?5 specimens taken at autopsy from SIDS infants were t~und m contain ('.
hv~rrlirrunr organisms
andlor toxin in 3-4% of cases analyzed. (U.R. Peterson eml.. Rev. infect. Uis.
1:6sU
( Ic)7c)).) In contrast. only 1 of 16() healthy infants (().G%) had C'.
hnlulinrrm orLanisms in the
l~ccs and no hotulinal toxin. (~. Arnon m crL, Lancet, pp. 1'_'7;-7(. .tune
17, tc)78.)
In developed ce)untries. SIDS is the number one cause of clcath in children
henvecn
;() one ntunth and'e)ne year old. (~. Arnon cn ul.. Lancet. pp. 1?70-77. .lone
17. 1()78.) R~lore
children die ti~om SIUS in the first year than li-om any other single cause of
death in the first '
_ti_
CA 02296765 2000-O1-14
WO 98108540 PCTNS97/15394
fourteeil years of life. In the United States. there are 8.000-10.000 SIDS
victims annually.
Ici.
Vl.'hat is needed is an effective therapy against 111ta11t botulism that is
li'ee of dangerous
side effects. is available in large supply at a reasonable price. and can be
sate;ly and gently
s delivered so that prophylactic application to infants is feasible.
Immunization of subjects with toxin preparations has been done in an attempt
to
' induct: immunity against howlinal toxins. A C'. hnntlinrcm vaccine
comprisin~_ chemically
inactivated (i.c'.. litrlnaldchvde-treated) type A. B. ('. I) alld F: toxin is
commercially available
lire htllllall tlS:l~C. 1-lowcver. this vaccine preparation has several
disadvantages. First. the
lU efficacy ot'this vaccine is variable tin particular. only 78% ol~recipients
produce protective
levels ef anti-type E3 antibodies followins~ administration of the primary
seriesl. Second.
immunization is pi1111f111 (deep subcutaneous ln(lCUlatlOn IS I'eCllllrl:d IOC
ad1171nISI1'tltllln), with
advcrw reactions hcin;~ common (moderate to severe local rracticlns occur in
approximately
O'~a al~ 1't.'C11711'.IIIS tllllln II1111aI injection: this number rises to
approximately I I'a of individuals
I~ who rect:ivc 11t)OStI'.f 1111C(:ll()nS) [Informational Hrochure for the
I'entavalent (Af3CDE~.)
l3otulinunt l moil. C'enmrs lire Disease (.'ontrol~. ~(~hird. preparation of
the vaccine Is
tlanLertms as active toxin must he handled by laboratory worl:crs.
Vt'hat is naeded are safe and et'ti:ctive vaccine preparations tar
administration to those
:It rill; ol~ mposurc to C'. hululinum toxins.
'? ()
C: clifficilc~
('. cIiJJic~ilr. an or~~anism wllich Lained its name due to dif'licuities
encountered 111 Its
l~tll:llll)11. h:lv 1't,'la:lltll' ht'e11 prtl~'Cn t(1 h c an etiologic
:l~~lllt tlt~ dl:tri'IIC:II tIISI'aSl'.. ( ~Ilf.'alll C'I
ul.. p. I t(,~.). ('. cli/Jicilc' is present in the gastrointestinal tract of
approxilnatelv i'% ol'
healthy adults. anti 10-_,0% of neonates without adverse effect (W warm. at p.
O44): by other
estimates. C'. cliJ)icilc' is a part ot~ the normal gastrointestinal t7ora tlf
?-10'ro ot~ humans. [C.F.
E3rool;s m eel.. (cds. ) "lulcerivn.v' ( 'cr:r.~'ccl ht' : t mterohir
I3crc'reriu." .lamer-. rV lc'Inie'k. d
.lclelher~ '.~ .1-Icclirul alic'rnhiuln,~~t'. I ()th ed., pp. 257-2C1?,
:~ppleton R, Lange. San Matc;o. C'A
( I t791 >. j :1s these organisms are relatively resistant to most commonly'
used antimicrohials.
i() ~~'ht.'ll a patient is treated with antibiotics. the other men thers of
the normal LQSII'(lllltlSL111a)
*rB
CA 02296765 2000-O1-14
vyp ,~glpg~p PCT/US97/15394
flora are suppressed and C'. di/~icile flourishes. producing cvtopathic toxins
and enterotoxins.
It has been found in 25% of cases of moderate diarrhea resultinL lt'om
treatment with
antibiotics. especially the cephalosporins. clindamycin. and ampiciilin. ~M.N.
Swartz at 644.]
Importantly. C'. clif~icile~ is commonly associated with nosocomial
inlections. The
organism is often present in the hospital and nursing home environments and
may be carried
c,n the hands and clothing of hospital personnel who care ti,r debilitated and
Ilttlttllll()(:11111pro1111Sed pallelltS. As many of these patients arc hcin~~
treated with
antimicrohials or other chelnothcrapeutic agents. such transmission of ('.
cli/~icile represents a
significant risk factor ti,r disease. (Engelkirk e~ ul.. pp. l0-(,7.)
('. clif~irilc is associated with a range of diarrhetic illness. ranging ti'om
diarrhea alone
to marked diarrhea and necrosis of the gastrointestinal mucosa with the
accumulation of
inflammatory ells and fibrin. which ti)rms a pseudomembrane in the affected
area. (Brooks
c~ml. > It has been found in over 4>°/. of pseudomenthranous
enterocolitis cases. t~wartz. at
p. l0-4.) his c,eeasionally fatal disease is characterized by (ii,irrhea.
multiple small colonic
is plaques. and toxic ntegacolon. (Swartz. at p, h44.) nlthoueh stoi,l
cultures are sometimes
used ii,r CIIJLIt()SIS. diagnosis is best made by detection oi' the heat
labile toxins present in
kcal filtrates from patients with enterocotitis due to ('. cli/Jirilr. (W
vartz. at p. h4~1-las: and
Brooks rr ul.. at p. ?6U.) ('. cliJ/irile trains are cvtotoxic ii~r
tissucicell cultures and cause
enterocolitis when in_jectcd intracecalU into hamsters. t~u~arto. at p. 644.)
~U The enterotoxicitv of ('. cli~/irilc is primarily due to the action of two
toxins.
llesignated r1 and B. each of approximately x()0.000 In molecular weight. Both
arc potent
cWotoxins. with toxin A pnSSISSIItL direct enteroc~'touovic activity. jl.vcrlv
c-r ol.. Intcrt.
Immun. (,O:~ll3s (199?). Unlike toxin A of (.'. ycrJrinl,~cn.,. an c>r~~anism
rarely associated
with antimicrohial-associated diarrhea. the toxin of ('. cli/)irilr is nen a
spore coat constituent
?s and is nut produced during sporulation. (~wartz, at p. (i~i~4.) ('.
cliJJic'ilc~ toxin A causes
hemorrhage. lluid accumulation and mucosal damage in rabbit ilcal loops and
appears to
increase the uptake of toxin R by thr intestinal mucosa. ~l-u~cin B ll()eS 11
()l CallSe IIlteStlllai
t~llld aCCllllllllatl011. hill Il 1S 1~~~ tlnleS IItOCe IOxIC tltalt l0\In A
l(~ llSSlll' CUlture cells and
eaUSeS Illellthralle damage. ~~ttltOll~,h hl)tlt tClxlIlS llt(illl:e
1111111at' C~Illllar efteC,iS SLICK 111 aCll!)
sU disaggregation. differences in cell specificity occurs.
_g_
CA 02296765 2000-O1-14
WO 9t~540 PCT/US97115394
Both toxins are important in disease. (Borriello cn «L. Rev. Infect. Dis..
I?(suppl.
?):518 (199U): Lverlv cn crl., Infect. Itnmun.. 17:349 (i98S); and Rolte.
Infect. Immun..
S9:1 ??_s ( 1990). j 'hoxin A is thought to act first by hinding to brush
harder receptors.
destroying the outer mucosal layer. then allowing toxin 13 in Lain access to
the underlying
s tissue. These steps in pathogenesis would indicate that the production of
neutralizing
antihudirv a;:ainst toxin A tray he sufficient in the prophylactic therapy of
cn~~n. !-luwevcr.
ctntihodics aLainst toxin B may he a necessary additional component for an
effective
therapeutic a;~ainst lacer stage culunic disease. Indeed, it has been reported
that animals
rccluirc antihodita to both toxin i1 and toxin B to be completely protected
against the disease.
I (f ( Ivim and Rol li;. nbstr. Ann. Meet. ~>m. Soc. Microbial.. f9:(2 (
1987).J
( '. cli/Jioilo leas also been reported to produce other toxins such as an
cnterotoxin
diffi:rcnt from toxins ~1 and B (Ranno c~r «l.. Rev. Infect. Uis.. O(Suppl.
1:51 I-S?0 ( 1984y[. a
Imv molrcufar \1'~SLht l(1\ltl (Rihn rr «/., i3iochcm. liiuphvs. Rcs. ('untm..
l~:l:lo)U-(o)s
t 1984)[. a motiliy altering lactur [.lustus e~ crl.. Ciastrocnterul.. 8.s:8sh-
84; ( 198'_')(. anti
I s perhaps other toxins. Regardless. ( '. rli/~icile gastrointestinal disease
is of pritnarv concern.
It 11 vlLlliflt;altl that due to its resistance to most commonly used
antimicrohials. ('.
clif)ioilr is assoriatc:d with antimicrohial therapy with virtually all
antimicrohial agents
(alth(lll~~h 111(1~t Cl)Iltltt()tll\' ampicillin, clittdamycin and
cephalosporins). It is also associated
with disease in patients under~~oin~~ chemotherapy ltltlt SLICK CUlltpUlllld~
aS Ittl'tlt(1ll'C\atC. ;_
?(I Iluc~n~uracil. cvciuphosphamidc. and duxurubicin. (S.M. Finegolcl c~r
crl.. ('liniwrl (:«icle ~r~
I rr«rrohio ~!7/t'C'll~llT.1'. pp. 88-89. liar I'uhlishing C'o.. Belntont. C:A
( t 9c)?). (
~l r~atment «f ('. cli/ric~ilc~ ciiseasc is problematic. ~.:iven the I11LI7
reSlStattCt'. l)I' the
or~~anism. Oral mctronidazole. hacitracin and vanconwcin have Keen reported to
he ci'fcctivc.
(I~ineguld er crl.. p. 89.) f-lowevrr th crc arc problems associated with
trcattttcttt utilizin~~ these
c:ompuuncis. Vancumvcin is very expensive, some patients arc unable to take
oral medication.
and the relapse rate is high (?0-?5°i~), although it may not occur for
sweral weeks. lcL
( '. cI1//IC'fIC~ disease would br prevented or treated by neutralizing the
effects ul' these
toxins in the ~_astrointestinal tract. Thus, what is needed is an effective
therapy y~ainst ('.
cli~~ic~ilc~ toxin that is free ol~ dank=eruus side effects, is available in
lar~~e supply at a reasunahlc
_g_
CA 02296765 2000-O1-14
PCTIUS97115394
price. and can be safely delivered so that prophylactic application to
patients at risk of
developing pseudomembranous enterocolitis can he el~fectivelv treated.
UES(:RII'TInN OF THE I)RAWINt:S
I~igurc I shows the reactivity of~anti-('. hotrrlimrrn IgY by Vvestern blot.
(~i~;urc ? shows the IgY antibody titer to ('. hrmrlinrrm type n toxoid in
eggs. measured
by E:I.IW ~.
figure .i sltuws the results u1~ ('. clij~irilc~ main :\ neutralization
assays.
I=i~!ure ~ SlleWyS II,e reStlllS (,t~ ('. cliJ%ic~ile~ toxin 13 neutralisation
assays.
l0 i~irurc s shows the resells of ('. cliJJicile~ toxin I3 neutralization
assays.
UiLUre (, is a restriction map of l'. clifjicilc train r1 gene. showin~~
secluences of
l,rimrrs I--t (gyp() ID N()~:l-~4).
I~i~_ure ~ Is 1 N%estern blot ot~ ('. cIiJJicile twin ~~ reactive I,rotcin.
hi'~urc li shows ( '. clij)icile toxin A expression constructs.
I s I~ i~:urc ') chows ( ' cli~Jicilc~ twin A expression constructs.
I~i~~urc lU chows the purification ot~ recombinant (' cIiJJicilr toxin i1.
I~i~_urc 1 1 sht,ws the results oi~ ('. cli/jicilc- toxin i~ nrutraliiation
assays with antibodies
reactive m recombinant toxin A.
I~i~urc 1~ sla,~s the results ti,r a ('. cli/Jicile toxim~ neutralization
l,iate.
?l) I-igurc 1s shins the results ti,r a ('. cliJ)icilr toximl neutralioation
plate.
l~igurc I-1 shows the results ot~ recombinant ('. cIiJJirilr toxin /~
neutralization assays.
I~ i~urc I ~ shows ( '. cIiJJic~ilo toxin A expression constructs.
f~ i;~urc I (, shows a chronutto~=raph plotting ahsorhancr at ''HU not
:ILalt,ll CWentlOn (11111'.
ii,r a h\-1r11~i70-t,BU lgY 1'IV(i preparation.
'_'> I~iLUre 17 shows mo recot3~binant ('. cli/)icilc~ toxin f3 expression
constructs.
IiLUre 18 shows ('. cli/Jicile toxin I3 expression constructs.
I~ inure I c) shwys ( '. cli/jirile toxin l3 expression constructs.
l~igurc '_'() shows ('. cli/)icilr toxin f3 expression constructs.
I~i~~ure ?1 is an SC)~-1'I~(iE '~rl showin~~ the nuritication oC rccomhinant
('. cliJ/iole
;() toxin li lilsiun protein.
CA 02296765 2000-O1-14
WO 98108540 PCTIUS97115394
Figure ?? is an SDS-PAGE gel showing the purification of two histidine-tagged
recombinant C'. cliJ~irilc~ toxin B proteins.
Figure '? > shows ('. clif~icile toxin B expression constructs.
Figure 3~ is a Western blot ol~ ('. cliJ~icilc~ toxin E3 rcactivr protein.
s ri'~ure '_'~ shows ('. hmulirrrun type A toxin expression constructs:
constructs used to
ltrovidr ('. hnrrrlinum or ('. clijJic!!~~ sequences are also shown.
- Fi~~urr ?6 is nn SDS-PAGE Lel stained with C'oomaisse bloc showinL the
purification
ol~ rece~mbinant ('. hn~ulrrurnr type A toxin fusion proteins.
Fi~~ure ?7 Sh(1~4'S ('. hrrlulinrrm mpc /\ toxin expression constructs:
constructs used to
I () provide ( '. hruulinunr sequences are also shown.
I~i~~urc '_'8 is an SDS-I'A(iC gel stained with C'oomaissc hfue showin~~ the
purification
of~ pllisE3ut protein usinL the Ni-NTA resin,
f~igurc '_'t) is an SDS-1'r\(iE gel stained pith C.'()l)IttalvSe hlLle
SftU~t111g Ilte C\preSSl()It (1f~
pllislW o protein in 131.?l(DE3) and BL21(DFi)pl.vsS host Cells.
I~ I~i~~ure ,0 is an SDS-PACiF ~~cl stained with t'c~olnaisse blue showing the
purification
t~l pl lisl3ot protein using a hatch absorption procedure.
l~i~~ure ,l is an SDS-1':\CiC' ~~el stained with Coomaissc blur sltowin~_ the
puriiicaticm
u!~ pl-lisf3c~t and pflisl3ot(nativc) proteins using, a ~(i-N'I~:\ column.
I~i~~ure ;? is an SD5-PACif: ~e:l stained with C'017111a15;iC hlUe
SltO~1'111!~ the purification
?() ol~ pllisl3otA protein expressed in p111sI3otA(syn) kilt lacl~l
T7/pAC'Y('CiroiE3l.?1(E)ES) cells
usin~~ an lI)A coftltn.
f~i~~ur~ ;; is an SDS-PACE: gel stained with C'l)(1t17a11S1'. 111t1e
Slto41'lltg lltl purification
ul~ pl 1is13utr\. pHisI3otl3 anti pEEis(3otE_ proteins ht' IDA
chrctmato~~raphy titllowrd by
chrrmtates~=raphy on S-l00 to rcmow t~c~lding chaperones.
l~ i~~urc :~~ is an SDS-PACi(: gel stained with CoOlttalsse bloc showing the
extracts
derived t~rom plEisE3ot(3 amp T7lacIBL?1(DE31 cells before and after
purification on a Ni-
N'T~A column.
Fi:'ure i5 is an SDS-I'ACiEeel run under native conditions and stained with
('aomaisse blue showing the removal ol'~t'otding chaperones from IDA-puriiicd
fiotE3 protein
i0 urine a S-I()() column.
CA 02296765 2000-O1-14
W~ ,~~pg~p PCTIUS97115394
Figure 3G is an SDS-PAGr gel stained with C'oomaisse blue showing proteins
that
eluted during an imidazole step gradient applied to a IDA column containing a
Ivsate of
pHisButB kan Iaclq T7/pACYCGru/BL31(DF~) cells.
f~ figure ,7 is an SDS-PAGE Lel run under native conditions and stained with
('uomaissc blue showing IDA-purified ButB protein bcti~re and after
ultratiltratiun.
Figure >8 is an SDS-PAGE gel stained with Coomaissc blue showing the
purification
c,t' (3utE protein using a NiNTA column.
I~ieure 3~) is an SDS-PAGE gel stained with ('uurnaissc bloc showinL extracts
derived
f~rum hllisE3utA kan ~I'7 lac/13L?1(DE3) pLysS cells grown in fermentation
culture.
1() figure ~4U is a chromatogram showing proteins present after 1DA-puritiecl
Hot!: protein
was ahplied.to a ~-lUU column.
DEFINI'FION~;
I a t'acilitate understanding of the invention, a number ol~ terms arc defined
hrlc,w.
1 ~ .~~s used herein. the term "ncutralitinL" is used in reference to
antitoxins. par ticulartv
antitoxins cumprisin~ antibodies. which have the abilim to prevent the
patholpical actions of
the toxin against which the antitoxin is directed.
~1s used herein. the term "overproducing" is used in rclercncc to the
production of
clostridia! toxin polvpeptides in a host cell and indicates that the host cell
is hruducing more
?U of the clostridia! twin by virtue of the introduction of nucleic; acid
:it'.Clllt'.tl~W eneOC1111L Sartt
rlustridial toxin pulypeptidc than would be expressed by said bust rcll absent
the introctuction
ot~ said nucleic acid sequences. To allow ease ot~ puriticatiun ot~ toxin
polvpeptides rroduccd
in a bust cell it is preferred that the host cell express or overproduce said
toxin polvpelUidc at
a Icvel greater than I mg/litcr of bust cell culture.
"A host cell capable ut~ expressing a recombinant protein at a level greater
than or
equal to ~% of the total cellular protein" is a bust cell in which the
recombinant protein
represents at !cast ~% ot~ the total cellular protein. ~'o determine what
pcrcenta~~c ui~ total
cellular rrotcin the recombinant protein represents. the tbllowing steps are
tal:cn. ;1 total ut
IU ()I),,",~ units of recombinant host culls (e.,sr.. ?UU ttl of cells at
()f),,,H, :i()lrnlt arr removed
;~() tat a timcpoint known to represent the peal: of expression of the desired
recombinant protein)
to a I .> ml micrutye tube anti peileted for ? min at maximum rpm in a
micrutirge. I'hc
I?
CA 02296765 2000-O1-14
WO ~ro~ PCTIUS97l15394
pellets are resuspended in 1 ml of ~4 mM NaFIPO~, 0.5 M NaCJ. 4U mM imidazole
but'fer
t pH 6.8) containing 1 mg/ml lysozyme. T'he samples are incubated for 20 min
at room
temperature and stored ON at -70°C. Samples are thawed completely at
room temperature
and sonicated ? X 1 U seconds with a I3ranson Sonifier 4~U microtip probe at #
; power
s scttin~~. ~l~hc samples are centrifuged for ~ min. at maximum rpm in a
microfuge. An aliquot
('?U Etl) ot'the protein sample is removed to ?0 Ell 2X sample buffer (this
represents the total
' protein ewract). The samples are heated to 95°C for ~ min. then
cooled and ~ or IU ttl are
loaded onto I'?.~°.~, SDS-P.4G~ eels. Eli~~h molecular weight protein
markers arc also loaded
to allow for estimation of the MW of identified recombinant proteins. After
electrophoresis.
I (1 protein is cletecte:d generally by staining with Coomassie blue and the
stained ~~el is scanned
using a cicnsitomcter to dc1 rmine the percentage of protein present in each
hand. In this
mannrr. the percentage of protein present in the band corresponding to the
recombinant
hrc~tcin of interest may he determined. It is not necessary that ('oomassie
blue be employed
fear the detection of protein. a number of fluorescent dues (e.,~'.. Svpro
orange S-CGS 1
I s ( Molecular I'rohcs. L:u~:enc. URA may hc.~ employed and the stained gel
scann ed using ~t
Iluoruima~.:rr ~~-.<L'.. I~luor Inlager ~I (Molecular Dynamics. Sunnyvalc.
('A)~.
";1 host roll capable of cpressing a recombinant protein as a soluble protein
at a level
~.:reatrr than cn- eclual m U.~s°,;~ of the total soluble cellular
protein" is a host coil in which the
alllt1t1l1I oi~ soluble rcconlhinant protein present represents at least
U.?s°i~ of the total cellular
''() I,rotein. ;1:; used herein "total soluble cellular protein" refers to a
clarified l'EI Ivsate
prepared as described in Example 31(c)(iv). iiriefly. cells arc harvested
tallowin~~ induction
ol~ expna,ion of rccomhinant protein tat a point e>f maximal ~xprcssionf.
~I~i~r cells are
resuspcnded in cell resusprnsion buf'tcr ((~Rf3: ~0 mM Nat'Ua. U.~ M Na(.'l. -
l() mM
imidazole. pl i (i.8) to create a '_U% cell SIISpCnSIUl1 (wet weight of
cellslvolumc of ('Rl'3) and
cell Ivsatcs arc prepared as described in Example 31(c)(iv) (i.e.. sonication
or h(1n10LC111ZatlOtl
lullowud I,y centrifugation). The cell lysate is then flocculated utilizing
polvethvleneimine
( I'E1 ) prior to centrifugation. I'l1 (u ?% solution in di-i,U. pH 7. > with
I 1C11 is added to the
cell Ivsate to a final concentration of U.?%. and stirred for ?t) min at room
temperature prior
m centrifugation lB.sUU rpm in .IAIt) rotor (E3eckmanl liar 3U minute:; at
=i°('~. This treatment
a) removes ItNA. DNA and cell wall components. resulting in a clarified. lom
viscosity Ivsate
("I'l:l C'.I~il'ltllCl l~'SfIIV'). '1'hl l'l'lOnlh111at11 protein present in
the I'll clarified lvsatr is then
_ 13_
CA 02296765 2000-O1-14
WO 98108540 PCTNS97I15394
purified (c:.,~~., by chromatography on an 1DA column for his-ta~~ged
proteins). The amount of
purified recombinant protein (i.c~., the eluted protein) is divided by the
concentration of
protein present in the 1'L:I clarified lysate (typically A mgitnl when using a
?0%, cell
suspension as the startinL material) and multiplied by I()() to determine what
percentage of
total soluhle cellular protein is comprised of the soluble recombinant protein
(see t:xample
~:~n).
.~~s used herein. the term "fusion protein" refers to a chimeric protein
containing the
protein ol' interest (i.r.. ('. hnruiinum toxin A. I3. C. U. E. f~. or (i and
fragments thereof)
.joined to an eao~:enous protein trapment (the fusion partner which consists
of a non-toxin
1() protein). The fusion partner may enhance sotuhilitv of the ('.
l~nrulir7un2 protein as expressed
in a host roll. may provide an of"finite tae to allow purification of the
recomhinant tuslon
protein from the hoa cell or culture supernatant. or hoth. If desired. the
fusion protein may
11~ 1'1111(1Vt'.~I fl'c)111 the: protein of" interest (i.e., toxin protein or
fra~.!ments thereat') prior to
immunization by a uarietv of enzymatic or chemical means known to the art.
I ~ ~~5 ll~(;it heCe111 the term "non-toxin protein" or "nun-toxin protein
accluencc" refers t~
that portion of a i'usion protein which comprises a protein or protein
seclucncr which is not
derived from a bacterial toxin protein.
~I Itc term "protein oi' interest" as used herein refers to the protein ».hosc
expression is
desired within the fusion protein. In a tltsion protein the protein of
interest will h c .joined or
_'() fused with another protein or protein domain, the fusion partner. to
allow tilt enhanced
stability of the protein ol' interest andlor case of purification of the
fusion protein.
:~a used herein. the term "maltose binding protein" refers m lhC 117:11t()Sl
hI11d111L
protein of I:, urrli. I~ portion of the ntaltosc binding protein may he added
to a protein of
interest to ~_cneratc a fusion protein: a portion of the maltose hindin~
protein may merely
enhance the solubility of the rcsultin~ fusion protein when expressed in a
bacterial host. ()n
the other hand. a portion of the maltose binding protein may allow affinity
purification of the
fusion protein on an amylase resin.
:~s used herein. the term "poly-histidine tract" when used in reference to a
fusion
protein refers to the presence oh' two to ten histidine residues at either
lllr amino- or carhoxv-
_~() terminus of a protein of interest. ;~ poly-histidine tract of six to ten
residues is preferred.
The poly-histidinc tract is also defined functionally as bein~~ a number ol'
consecutive histidine '
- 14-
CA 02296765 2000-O1-14
WO 98/Q$540 PCT/US97I1t5394
residues added to the protein of interest which allows the affinity
purification of rite resulting
fusion protein on a nickel-chelatc or IDA column.
:\s used herein. the term "purified" or "to purify" refers to the removal of
contaminants from a sample. I~or example. antitoxins arc purified by removal
of
- 5 rc~ntaminating non-immunoglobulin proteins; they are also purified by the
removal of
immuna~.:lobulin that dues not bind toxin. The removal of nun-immunoglohulin
proteins
andlur the removal of immunoglohulins that do not bind toxin results in an
increase in the
percent ui~ toxin-reactive immunoglohulins in the sample. In another example.
recombinant
twin polvpcptides arc expressed in bacterial host cells artd the toxin
polvpeptides arc purif7ed
I t) by flue removal of host cell proteins: the percent of recombinant twin
polypeptides is thereby
increased in the sample:. Additionally. the recombinant toxin polypeptides arc
purified by the
renewal ol' host cell components such as lipopolysaccharide (c-.,~~..
endotoxin).
f~l~r term "recombinant UNA molecule" as used herein refers to a UNA molecule
which is e:ompriscd o1~ syments of DNA .joined together by means o1~
rnolccular hioloLical
1 > ICChI11c1liCS.
I Ite trim "recombinant protein" or "rccotnhinant polvpeptidc" as used herein
refers to
a proucin nwlecule which is expressed t~rom a recombinant I)N~\ molecule.
~Chc term "native protein" as used herein refers to a protein mhich is
isolated from a
natural ,ourcc us opposed to the production of a protein by recombinant
rncans.
_'() :~, used herein the term "portion" when in reference tc~ a protein (as in
"a portion ol~ a
~~iven hrcUCin") refers to fra~~mcnts csf that protein. The ti~a~~mrnts may
range in sire from
lour amino arid residues m the entire amino acid sequence minus one amino
acid.
:\s used hrrcin "soluble" when in reference to a protein produced by
recombinant
I)NA tcchnolotty in a host tell is a protein which exists is solution in the
cvtoplasnt of the
host cell: if' the protein contains a signal sequence the soluble protein is
exported to the
lcriplasmic space in bacteria busts and is secreted into tits; culture medium
in eucarvotic cells
capable c~l~ secretion or by bacterial host Itossessing the appropriate genes
(i.r.. the kil gene).
In contrast. an insoluble protein is one which exists in denatured form inside
cvtoplasmic
~ranul~s tcalled inclusion bodies) in the host cell. l-Iigh level expression
(i.r.. Lreater than lU-
'() ?0 tt~~~ recombinant proteitt/liter of bacterial culture) of recombinant
proteins often results in
the expressed protein being found in inclusion bodies in the bacterial host
cells. :\ soluble
- IS -
CA 02296765 2000-O1-14
WO 913108540 PCTIUS97/15394
protean is a protein which is not found in an inclusion body inside the host
cell or is found
both in the cytoplasm and in inclusion bodies and in this case the protein may
be present at
high or low levels in the cytoplasm.
A distinction is drawn between a soluble protein (i.r.. a protein which when
expressed
in a host cell is produced in a sulubfe form) and a "soluhilized" protein. i\n
insoluble
recombinant protein found inside an inclusion body may he solubilized (i.o..
rendered into a
soluhlu form) by treating purified inclusion bodies with denaturants such as
guanidine
hydrochloride, urea or sodium dodecvl sulfate (SUS). These denaturants must
then be
removed tram the suluhilioed protein preparation to allow the recovered
protein to renature
IU (refold). Nut all proteins will refold into an active conformation alter
solubilization in a
clenaturant and removal of the denaturant. Many proteins precipitate upon
removal of the
denaturant. BUS may be used to solubilize inclusion bodies and will maintain
the proteins in
solution at low concentration. liowever. dialysis will not always remove all
ul~thc Sl)S ISUS
can form miceilcs which du not dialyze out); tltereforc. Sf)S-suluhilized
inclusiim body
1 ~ protein is soluble hut not refolded.
;\ distinction is drawn between proteins which arc soluble ( i.r., dissolved)
in a
solution devoicf of~ SILItltICanI amounts of ionic detergents (c~.,y.. ~I)~)
ur denaturants (c.,~~..
urea. guanidine hydrochloride) and proteins which exist as a suspension cal'
insoluble protein
molecules cfispcrscd within the solution. A soluble protein will not be
removed from a
?U solution containitt~: the protein by ccntrifueation using conditions
sufficient to remove bacteria
present in a liduid nledtUltl (i.c~., ccntrifutation at I'_'.U()U x ~; for ~-s
minutes). for example.
m test whether uvo proteins, protein ~1 and protein L3. F12'e 1l)111171e II1
1()llltt(111, the Uvo proteins
arc placed into a solution selceied From the group cunstsnn L uI~ I'EW-Na('I
(1'135 cuntainin=
().i M NaC~I). I'BS-NaC'1 containing U.'?'% 'I'ween ?0. I'f3S. 1'RS captaining
U.?°/~ Twcen ?U.
PBS-C' (PISS containing ? mM (.'aC.'I,). PBS-C' containing either U.1 or U.s
°/. Twecn ?U. PBS-
C' containing either U.1 or 0.~% NP-40. PBS-('. cuntainine either U.I or
().~°ro Triton X-IUU.
Pf3S-C' containing U.I°~o sodium deoxvcholate. 'Che mixture containing
proteins :1 and (3 is
then centril~us~ed at ~OOO x L for ~ minutes. ~I~hc supernatant and pellet
li~rmed by
ccntrifugatiun arc then assayed for the presence of protein ~\ and 13. 1I~
protein A is found in
30 the supernatant and not in the pellet (except for minor amounts (r.r.. leas
than lU°/,) as a
result of trapping]. protein is said to he soluble in the; solution tested. 1f
the majority ul'
- IO -
CA 02296765 2000-O1-14
PCT/US97115394
protein I3 is found in the pellet (i.r.. greater than 90%). then protein B is
said to exist as a
suspension in the solution tested.
As used herein. tire term "therapeutic amount" refers to that amount of
antitoxin
rccluired to neutralize the pathologic effects of one ur more clostridia)
toxins in a subject.
~I'hc term "pyrugcn" as used herein refers to a fever-producin~~ substance.
I'yrogena
now he endogenous to the host (c~.y., prostaglandins) clr may he exogenous
compounds (~.~~.,
bacterial endcl- and exutuxins. nonbacterial CO(rtpOt.lndS SlrCh aS
alttl!'ellS and certain steroid
compounds. ctc. ). The presence of pyrueen in a pharmaceutical solution may he
detected
1IS11tL the ('.S. I'harmacopeia (IJSP) rabbit fever test (United States
Pharmacopeia. Vul. XXIt
iU (Ic)c)()) l.!nitccl States Pltarmacupeial C.'onvention. Rucl:villc. MD, p.
151).
fhe term "cndutuxin" as used herein refers to the hi~it nu>lecular wci~ht
complexes
assoeiamt with the: outer memhrartc of ~~ram-negative bacteria. l!npuriticd
endutoxin contains
lipieta. proteins and carhultvdrates. l~liehly purified endutoxin dues nut
contain protein and is
rctcrrcd m as lipupulvsaccharidr (1_I'~). fieeause unpuriti~d endutoxin is of
concern in the
I s production u1~ pharmaceutical compounds (c-.y., proteins produced in E.
cwli using recombinant
I)W tcchncllu~~vl, the tcrrn cndutoxin awsed herein refers to unpurified
endcttuxin. E3acterial
mdutwin is a well knwvn pyrugen.
~1s used hrrein. the term "endutuxin-free" when used in reference to a
cumpusrtlon to
he administered parcntcrallv (with the exception of intrathecal
administration) to a host means
that the cios~ to he delivered contains less than ~ F:l)/l:g body wei~;ltt
(I=DA (iuidclincs filr
I'urcnmral 1)ru;~s ( l)ccemhcr I ~)H7)j. ;~ssutning a weight of 7U kg tier an
adult human. the
d<lsc must cclntain Irss than s~U I:L~ to rncet FDA Guidelines tar parcnter~tl
administration.
f:ndutwin levels are measured herein using the I.imulus Amchocvtc Lvsatc
(L.nl.) test
tl.imulus Antchucytc f..ysate I'vruchrumel". Associates ctf'C.'apc C'c>d. lne.
w-uuds Ilule. ~1~1).
~I~u measure endotoxin levels in preparations of recombinant proteins. 0.~ ml
elf a sulUtlull
cctlnprisin~~ U.s mg of purified recombinant protein in ~0 mM Nal'(),, pIi
7.U. U. iM MaC.'I and
1 ()~/~ glycerol is used in the 1.,~1L assay according to the manufacturer's
instructions for the
l;lldp()Iltt Cltl'111110~~Lnlc ~IIIhUUt diazo-coupling method (lhC SpeCltlC
(:(tmp(711entS llf the huff'er
cclntainin~~ recombinant protein to be anafvzed in the L./1L. test arc not
important: any huftcr
3U havin~~ a neutral pII may he employed (see for example. alternative buffers
cmptuved in
I:;xamples 3~. .lU and ~4s)~. C'umpusitiuns containing less than or cdual trl
than ~s0 endutuxin
_ 17_
CA 02296765 2000-O1-14
PCT/US97/15394
units (EU)Img of purit7ed recombinant protein are herein detined as
"substantially endotoxin-
t~ree." Pret~erably the composition contains less than or equal to 100. and
most preferably Less
than i7r edual to (~0. (IU)/mg of purified recombinant protein. Typically,
administration of
bacterial toxins or toxoids to adult humans for the purpose ut' vaccination
involves doses ot~
about 10-i()0 tlg protein/dose, ~fherefore, administration of 10-i00 ttg of a
purified
rcconthinant prc7tein to a 70 kL human. \vherein said purified recombinant
protein preparation
contains (70 f:U/ntg protein. results in the introduction ol'on)v 0.6 to >()
(:I) (i.e.. 0.? tc7 8.6'%
oi~ the maximum allt,wahle endotoxin burden per parenteral dose).
Administration ot~ 10-50()
try e71~ a purified recombinant protein tc7 a 70 k~ )roman. \vhcrein said
purified rccomhinant
protein preparation contains ?~0 ~U/m~ protein. results in the introduction of
only ?.~ tc, 125
f:U (i.r., ().7 te7 3(,°/, c,h the maximum allowable endot«xin burden
per parenteral dose).
~fhe 1.:11_. test is acceptecl by the ll.S. FI)A as a means c7t detecting
bacterial
mtdotcwins (?I ('.I~.R. s' (7(,0.1U(i -IOS). Studies have shc7\wt that the
LI\I. test is equivalent
c7r superic7r w the 111' rabbit pvru~~en test for the detection c,fendotoxin
and thus the l.nl.
test can horsed as a surrogate for pyro~enicity studies in animals ~(~.C'.
('erason. l')rcys,~c~)a.,':
''l7CI'11f7.1'lll.1'. L. (!. lr.clnr,s,~ cute./ lIC'f74'I'(),tt'liull')11.
Marvel l.)ekkcr. ~etv 1''7rk I I ')8~ ). pp. 1 s0-l i~ (.
I~hc IU):1 l3urcau c,t' Biologics accepts the LAL assay in place '7f the (
!~(' rabbit pyr'7t:en test
sc7 lost,.: as the L_~1I_ assay utilized is shown to be as sensitive as. e7r
ntt7re sensitive as the
rabbit mst ~l~rd. Rr~.. _,~. ?hl.i() ( It)8())~.
'() I~hc term "me7novalent" when used in reference tc, a clostridiul vaccine
r~f~ers t<7 a
vacclltc which is capable ol~ prc7vokin~~ an in tmtlne response in a ht,st
animal directed atainst a
1111'~ll' I\'hl' lll~ t'.ll7Slrlttlal tt7x111. 1 t,l' CxaInDIC_ 1t'
Itl,n,nnilalimo ml a lt.mr v. nh W 1..,...~:._.....
l1-l,l /~ l'7xilt \'aeCllle 111dUeeS Ltltllhl7dleS )n the 1111 n1t1I11Zed
Itt)St \\'hlClt prt7leCl .I;~alnSl It
chailt:n_.:e with type n tt7xin but not against challenge with ype f3. C'. U,
t:. I~ or (i toxins.
then the ype n vaccine is said to he monovalent. In contrast, a "multivalent"
vaccine
provokes an immune response in a host animal directed against several (i.r..
more than c7nc)
clostridiai toxins. Fc7r example, ii' immunization c7t' a host with a vaccine
comprisinL ('.
hnnrti)ltll)r ype ~1 and E3 toxins induces the production of antibodies which
protect the low
against a challenge with both type .~1 and f3 toxin, the vaccine is said to he
multivalent ( i1t
~(> particular. this hypothetical vaccine is bivalent).
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WO 98/08540 PCT/US97I15394
:~s used herein the term "immunogenically-effective amount" refers to that
amount of
an immunogen required to invoke the production of protective levels of
antibodies in a host
upon vaccination.
fhe term "protective level". when used in reference to the level 01~
antibodies induced
' s upon immunization of the frost with an immunogen which comprises a
bacterial toxin. means
a level of circulating antibodies sufficient to protect the frost i~rom
challenge with a lethal dose
c,i~ the mvin.
:1a used herein the terms "protein" and "polypcptide" refer to compounds
cumprisin~~
amino acids .joined via peptide bonds and are used interchangcablv.
The terms "toxin" and "neurotoxin" when used in reference to toxins produced
by
members (i.c~.. species and strains) of the genus ('lu.crniclirrm arc used
interchangeably and
refer t<, the proteins which arc poisonous to nerve tissue.
1'hc mrtn "receptor-binding domain" witch used in reference to a ('.
hrurrlittarm toxin
rcirrs m the carhoxv-terminal portion of the heam~ chant (11, or the ('
fragment) uf' the twin
1 ~ which is prcsumcci to he responsible for the binding of the active toxin
(i.c-.. the derivative
tcwin comprisinz the Ci alld 1. chains joined via disulfide hands) to
receptors un the surface ul~
avnaptosumcs. 'The inceptor-hindin~ domain for ('. hnrrriirtrrm type A toxin
IS tlt'tllted ltt.'Celll
as mmprisin'~ amino acid residues 8(,I through 1?c)G of SC~;Q II) N():'_'8.
~f~he rccept«r_
hindin'; Humain for ('. hruulinnnr ype 13 toxin is defined herein as
comprising amino acid
?0 residues t;-C!i tltrou~~h 1?91 uf~ SE:Q 1I) N0:40 (strain Eklund 17(31.
'I~hc rrccptor-binding
Cll,lttattt t,p ('. I)lJrtlll77rllrl mpc C'I toxin is defined herein as
comprisin_~ amino acid residues
t(>O thruu'.:h l~~)1 ul~ SC:(? lU N():(~0. -l~ltc receptor-binding domain of'
(' hrurrlirurnrmpe C)
toxin is delin cd lterCllt as comprising amino acid residues 8s? through 1?7G
oi' ~L:(~ II)
N():W,. ~I-hc receptor-binding domain of ('. huurlimna type (toxin is defined
herein as
cumprisinu amino acid residues 8;5 through I?5(> of ~C:(,) IU NO:~() (E3eluga
strain). '('hr
receptor-binding domain of ('. hmrrlW arm type F toxin is defined herein as
comprisinC= amino
acid residues 8i3 through 1?7~ of SEQ ID N0:71. 'hhe receptor-binding domain
of t'.
lunrrlirrrrru type (i toxin is defined herein as cuntprisin~ amino acid
residues 8ss tltrou~_h 1?97
u1' Sf:Q IC) N():77. Within a ~~ivrn scrotype, small variations in the primary
amino acid
s0 seduencc ol' the hutulinal toxins isolated from different Slt'altts has
been reported [Whelan c~r
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CA 02296765 2000-O1-14
WO 98/08540 PCTIUS97/15394
crl. ( f ~~)2). .srrhrcr and Minton ( l9c)5) Curr. 'Cop. Microhiol. Immunol.
195:161-194). 7hc
present invention cuntcmplatcs fusion proteins comprisin~_ the receptor-hindin
L domain of C'.
hunrlinum toxins from serutypes A-(i including the variants teund among
different strains
within a ~~iven scrotvpc. ~1'he receptor-binding domains listed above arc used
as the prototype
tar each strain within a scrotype. Fusion proteins containing an analogous
retion trom a
strain mher than the prototype strain are encompassed by the present
invention.
Ivsion proteins comprising the receptor binding domain li.c~.. C' fl~arment)
of botulinal
toxins may include amino acid residues located hevonct the termini ui' the
domains defined
ahom. Ivr example. the pHisBotf~ protein contains amino acid residues 84h-
I?c)1 0l' SE;Q ID
It) .'~l<):~i(): this fusion protein thus comprises the receptor-hindin~
domain !or ('. hruulinrrrrr type
f toxin as detincct above li.r.. Ilc-8~8 through (ilu-1?c)I ). ~imilarlv.
pHisE3otL contains
amino acid residues X27-I?~? 01' SL:Q 1D N():5() and pllisl3ot(i contains
amino acid residues
tSS l - l ~r)7 U~ S(:(~ lI) N():77. l~itus. both rHisliotl= and pf-f isf~oU(i
fusion proteins contain a
Icw amino acids located beyond the N-terminus of the defined receptor-hiu~iin~
domain.
Che terms "native rcnc" or "native gene seyucnces" arc usc;ct tn indicate 1)NA
vellU~lltt.'v ~I1C(1(I111L a particular gene svltich contain the same DNA
seyucnccs as fisund in the
~_cne as isolated Cram nature. In contrast. "synthetic scene scducnccs" arc
I)NA aeyuenccs
which armscd to replace: the naturally occurring DNA seclucnccs vhcn the
naturalf
uceurrin~~ sedurnccs cause expression problems iv a given host cell. Ior
example. naturallv_ -
'(l occurrin~_ DNA seyurnces encoding codons which arc rarclv used in a host
cell may he
replaced to ~.. by site-directed mutagcncsist such that the synthetic I)NA
seeluencc rrpresents
a more i~rec)ucntlv used cotton. ~I~l~c native DNA sequence and the wnthctic
I)N;1 sequence
will pr~t'rrablv encode the salnc amino acid seclttcncc.
~UMMAItY OF THE 1NVCNTIUN
'l~he present invention relates to the production of polypeptides derived Irom
toxins
particularly in recombinant host cells. In one embodiment. the present
invention pro4~ictes a
host cell containing a recombinant expression vector. said vector cncodin~; a
protein
comprising at least a portion of a ('Io.slricliunr hcrrulirurm toxin, said
toxin selected from the
~~roup consisting of mpe f3 toxin and type F toxin. ~I~ht prCSttlt
ttll'ellllt)11 15 11l)I limited by
the nature of W1~11e11Cf:v CnCOdIIIL portions of the ('. hrrrrrlirrrrrn toxin.
These scduenccs may he
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CA 02296765 2000-O1-14
WO 98J~540 PCTNS97115394
derived from the native gene sequences or alternatively they may comprise
synthetic gene
sequences. Synthetic scene sequences are employed when expression of the
native gene
sequences is problematic in a given host cell (c~.,~~., when the native gene
sequences contain
sequences resembling yeast transcription termination signals and the desired
host cell is a
yeast eel I ).
In one embodiment. the lust cell is capable ol' expressin L the recombinant
C'.
' hrnulirrrurl toxin protein at a level greater than or equal to ?% to 40% of
the total cellular
protein and preferably at a level greater than or equal to ~% of the total
cellular protein. In
another embodiment, tire host cell is capable of expressly the recombinant ('.
hourlumrn
I U toxin protein as a soluble protein at a level greater than or equal to
0.25% of the total cellular
protein and prclcrahlv at a lccl greater than or equal to ().~~% t~
10°/. of the total cellular
IroUcW.
~fhc present invention is not limited by the nature of the host cell employed
for the
prootuction ut recombinant ('. lmlrrliruuu toxin proteins. In a preferred
embodiment. the host
I ~ cell is an I: ewli cell. In another preferred embodiment. the host cell is
an insect cell:
particularly preferred insect bust cells arc ,5jrnclulrcmor ~rtryilmrclcr
(Stt)) cells. In another
preferred embodiment. the host cell is a yeast cell: particularly preferred
yeast cells arc I'irhicr
pu.~lnrl.c cells.
In dumber elllb(ldlltletlt. the invention provides a bust cell cuntainini a
1'ecUll7hlllallt
?1> mpressiun vcctcir. said vector encoding a fusion protein comprisinL a turn-
toxin protein
sequence and at Fast a portion of a ('ln.clricliunr hnurlilnrnr toxin. said
toxin selected from the
;~ruup consisting c>I' type 13 twin and type ~ toxin. 'fhe invention is not
limited by the nature
al' the pe,rtiun c~l~ the ('lrr.slriclirrnr hmrrlilurln toxin selected. In a
preferred embodiment, the
portion ol~ the toxin comprises the receptor binding domain (i.e.. the C'
l'ragmcnt of the toxin).
The pl'eSl',Ill invention is not limited by the nature of the non-toxin
protein sequence
employed. In a preferred embodiment. tl7e 11UI1-toxttl prt)ICIII SequCllee
c(1111pr1Se1 a poly
histidinc tract. .1 number of alternative fusion tans or fu siun partners are
known to the art
(c-.,y.. l~llil'. (i~T, protein A. ere.) and may be employed for the
production of fusion proteins
comprisin~_ a portion of a hotulinal toxin.
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CA 02296765 2000-O1-14
gyp yg~p PCT/US97115394
The present invention further provides a vaccine comprising a fusion protein,
said
fusion protein comprising a non-toxin protein sequence and at (cast a portion
of a ('lo.cwiclium
hr~rrrlinerm toxin. said toxin selected from the group consisting of type B
toxin and type h.
toxin. The vaccine tray be a monovalent vaccine (i.e-., containing only a
toxin R fusion
protein or a toxin E fusion protein). a bivalent vaccine li.c~.. containing
bath a toxin B fusion
protein and a toxin E titsion protein) or a trivalent or higher valenev
vaccine. In a preferred
C;Illh()dllllt:llt. the toxin R fusion protein and/or toxin t: fusion prolellT
IS l:Otl1h111ed with a
t~usictn protein comprising a non-toxin protein sequence and at Icast a
portion of ('Ims~niclium
hrurrlinrrm type A toxin. 'hhe present invention is not limited by the nature
of the portion of
1() the ('lo.s~riclirrrn hnrrrlirrann toxin selected. In a preferred
embodiment. the portion of the toxin
comprises the receptor binding domain (i.e.. the C' fragment of the toxin).
The present
invention is not limited by the nature of the non-toxin protein sequence
employed. In a
preferred embodiment. thc.~ non-toxin protein sccluence comprises a poly-
histidine tract. :1
numh~r of~ alternative fusion tags or fusion partners are known to the art (c.
~.. t\-fI3I'. (W'I'.
1 ~ protein ;~. ctc. ) and may he employed fur the generation of fusion
proteins comprising
vaccines. When a fusion partner ti.r.. the non-toxin protein seclucncel is
entploved fc>r the
production of a recombinant ('. hmurlinul toxin protein, the fusion partner
may he removed
from the recombinant ('. hrurrlinul toxin protein ifdcsired (i.~-.. prior to
administration ol'the
protein to a subject) using a variety of methods known to the art (c~.~..
digestion of fusion
:?() proteins containing Ivactor\a or thrombin recognition sites with the
appropriate cnzvmef. ~~
numh~r of the pI;THis vectors employed herein provide an N-terminal his-ta~~
ti~ll<wed by a
fveteuva c:lcava~~c site tree >rxample ?8a): the hotulinal C' I'ragmcnt
secluencrs li~llow the
I~aetorXa site anct thus. IvactorXa can be used to remove the his-tai from the
hotttlinal fusion
protein. In a preferred embodiment. the vaccine is suhstantialU cndotoxin-
f~re~.
he present invention is not limited by the method employed t'~r the generation
of
vaccine comprising fusion proteins comprising a non-toxin protein sequence and
at least a
portion of a ('lo.~nricliurn hn~rrlimnnr te~xin. Tlte fusion proteins may be
produced by
recombinant I)NA means usinL either native or synthetic Lone s~yuenccs
expressed in a mast
cell. ~I'hc present invention is not limited to the production of vaccines
urine recornhinant
.i(1 host cells: cell free in airrn transcriptionitranslation systems may hr
employed for the
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CA 02296765 2000-O1-14
WO 98108540 PCTIIJS971ti5394
expression of the nucleic acid constructs encodin~e the fusion proteins of the
present invention.
An example of such a cell-free system is the commercially available 'I'nTT'"
Coupled
Itcticuluwne Lysate System (I'romega Corporation. Madison. WI). Alternatively,
the fusion
proteins of the present im~ention may he ~~enerated by synthetic means (i.e.,
peptide
J S1'11111~S1S1.
l~he prcaent invention Further provides a method ui' generatirt~~ antibody
directed
' .y~ainst a ('lu.crriclium It()IlllirlrrJlJ toxin comprising: a) providing in
any order: i) an antigen
(;(trltpl'ISlItS'_ a tirSlOtt protein COrnprISlrtg a non-toxin prOtelll
Sl'ClllCnCe and at least a p(trll(trt CtF
a ( 'lrr.vrriclirrrrr hnnrlinrrnr twin, said twin selected from the group
consisting ol' W_ pe 13 toxin
ltt and yp~ L; toxin. and ii) a host: and b) immunizing the host with the
antigen so as to
~.:cneratc an antibody. In a preferred elllb(tdllnent. Lh(; anll!~t'.rt used
t(1 tltttllurtl'!.e the host also
l()Il(11115 ce tirsiun protein comprisin~~ a non-toxin protein sequence and at
frost a portion of
( 'lu.wriciirrrrr hrrlrrlrrrrrm type ;.\ toxin. I~hc present invention is not
lintited by the nature of the
portimt e~l~ the ('lr~.nriclirrm hmurlinrrrrr toxin selected. In a preterred
embodiment. the portion
I ~ al~ the toxin comprises the receptor binding domain (i.c~.. the C'
fra,~ntent of the toxin). 'I~he
prcsrnt invention is not limimd by the nature of the non-toxin protein
sequence employed. In
a preferred embodiment, tlm nets-toxin protein sequence comprises a poly-
histidinc tract. ~1
number ol~ alternative Fusion tags or fusion partners arc known to the art
(e.,L.. MI~P. CJST.
Itrotcin ~\. mc.l :utd may he employed for the generation uF Fusion proteins
comprising
'_(1 vaccines. \\~hen a I~usian partner (i.u.. the non-toxin protein sequence)
is employed tits the
production of a recombinant (' hrtrrrlincr! toxin protein. the fusion partner
may ht removed
I~1'cfltt lltt.' r'et;llrtlhlllaltt ('. hurrrlirnrl toxin protein iF desired
(i.c~.. prior to administration ot~ the
protein m a suhjectt using a variety of methods known to the art Ir.,~~..
digestion uF Fusion
proteins rontainin~~ I=actor\a or thronthin recognition sites with the
appropriate enzyme).
The present invention is nut limited by the nature of the host employed for
the
production of the antibodies of the invention. In a preferred embodiment. the
host is a
ntan tmal. preferably a human. Tlte antibodies of the present invention may he
generated
using nurt-mammalian hosts such as birds, preferably chickens. In a preferred
embodiment
thmtcth<t~t of the present invention Further comprised the step r1 uF
cullectin~_ thr antibodies
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CA 02296765 2000-O1-14
WO 981540 PCTNS9'7l15394
from the host. In vet another embodiment, the method of the present invention
further
comprises the step d1 of purifying the antibodies.
'fhe present invention further provides antibodies raised according to rite
above
methods.
~t~he present invention further contemplates multivalent vaccines comprising
at Ieast
avu recombinant ('. hnrrrlinrura toxin proteins derived from the Lroup
consisting of C'.
hurrrli»rnrr serotypes i\. I3. C. D. E. I:. and (i. 'hhe invention
contemplates bivalent. trivaient,
yuadravalent. pmtavalent. ltcptavalent and septivalent vaccines comprising
recombinant ( '.
I7I)llllrllrll7l t()xlll proteins. I'referuhlv the recombinant ('. hu»rlinrrnr
twin protein comprises
I() the receptor binding domain (i.e.. C' twagment) of the toxin.
DFSCItIPTIUN OF Ttl<F INVENTION
~Chc present invention contemplates vaccinating humans anti other animals with
putypcptidcs derived from ('. by»rlinunr neurotoxins which arc suhstantiallv
cndutuxin-free.
1 ~ T~h~sc hoaulinal peptides are also useful for the production of~
antit<win. :\nti-hcriulinal toxin
antitoxin is awful tier the treatment of patients ei'fected by or at risk of
svntptoms due to the
action ol' (' h»»rlirnrrn toxins. The organisms, toxins and individual steps
of the present
invcnticm arc described scparatrlv h~lmv.
?0 1. ~.7os~rirlimn 5pecics, ClOStridial Diseases And AssOCiatcd Toxins
.~\ preferred embodiment ol' the method of the present invention is directed
twvard
olUainin~~ alihucii~s a~_ainst ('In.vrriclirmr species. their toxins. rnrvmes
car oeh~r metabolic hv_
products. cell wall components. ur svnthctic or recombinant versions of anv
of~ these
compounds. It is contemplated that these antibodies will he produced by
immunization ui'
?s humans ur other animals. It is not intended that the present invention h~
limited to anv
particular twin or any species of organism. In one embodiment. tclxins from
all ('lr~.,vriclirrnr
species arc contemplated as immunugens. L;xamples c~l~ these I(1XII1S
IItCItlde llte Itt.'uCa1111111daSC.'
toxin oi~ ('. htrlt~ricurm. ('. .vorclellii toxins HIT and I.~I~. toxins I\.
13. (.'. I). I. I~. and (i of ('.
horrrlinrrm and the numerous ('. p~r/rin,L~en.v toxins. In one prelerrcd
emheldlment. toxins f\.
-24-
*rB
CA 02296765 2000-O1-14
WO 98/08340 PCT/US97113394
I3. and F ol' C'. haurlirJlJm are contemplated as immunogens. Table ? above
lists various
('in.cn'iclilJnl species. their toxins and some antigens associated 4vith
disease.
TABLE ?
('lostridial Tnrirw
Oreanisnt Toxins and Disease-Associated Antigens
('. hrrlrflMlnll A. B. C,. C,, D. G. F. Ci
C'. hllll'I'IC'fflll Nmraminidase
.4. B. C-.nterotoxin Inot A nor B), M~tilitv
Alterin~~ Factor, Lour
A4olecular Wcir_ht 'toxin. Others
( '. pc'I'jl'llT,~'cI),v(x. (3. E. I. %. 0. v. (~. It. )., rt.
ll
1() t:'. .wrclc~llr
( ' j/crnu'Ifluus tlT, l.T. (x. (i, y
( ~. !lrnav u, lf, y. ~i. (:. .r,. \'. ()
(' W /lllt'lflll C.(. 13. '!, tl
(' hr.crmlvlictnu a. (1. '!. r5. E plus additional cnz\'ntcs
( c hrrurrmn (x. (j. ~. 6
It is tun intended that antibodies produced against (me t(txin \viil only he
used against
that toxin. !t is c(tntemplamd that antihoclics directed against one toxin
(e'.,>,r.. ('. I)rrJl'ills,'C'17.1
wpc /1 enteremxiny my hr used as an effective therapeutic against one (tr more
toxin(sy
?U hr(tduc:ed by (tther members ed' the genus C'hl.sJriclium or other toxin
produeinL organisms
(('. ~ . I3WL~illJl.v LO'rLU.c. .~'IcrJ)h1'InWnc~LUr.c lnn'etl.v.
.~'Jre/)JC)curcul.v 11111IC111.1'. .'iC'lIlC'If)I)C!L'Jc'1'
L'CIIL'tIllC'L'11L'll.l', h.l'L'lJlllJJ11ll111f.1' C1L'1'llt,'llJll.l'll.
(1t11Cf I1.1'L'11Lh1111111J11.1' Sl7eClt.'S. ete.), !t 15 further
c(tntemplatcLl that antibodies dirccteLi a;~ainst the portion eh~ the train
which hinds to
mammalian membranes tc'.,t.. ('. I)CI'~1'll'lf,'L1T.5' enterotoxin A1 ran also
hr used a,.!ainst (uhcr
organisms. It is contemplated that these membrane binding domains are produced
synthrtically and used as immunoLens.
11. ()htainin~ Antibodies In Nnn-Mammals
~1 prclcrmd embodiment oh' the method of the present invention litr ohtainine
antihc,dirs invot'vcs immunization. However, it is also contemplated that
antihudies'could he
obtained from non-mammals without immunization. !n the case where nu
immunisation is
CA 02296765 2000-O1-14
WO 98/08640 PCTlUS97I15394
contemplated. the present invention may use non-mammals with preexisting
antibodies to
toxins as well as non-mammals that have antibodies to whole orLanisms by
virtue of reactions
with the administered antigen. An example of the latter involves immunization
with svllLhcllc
peptides car recombinant proteins sharing epitopes with whole or~_anism
components.
s In a preferred embodiment. the method of the present invention contemplates
immunizing nun-mammals with bacterial toxin(s). It is nut intended that the
present invention
he limited to any particular toxin. In one embodiment, toxin ti~orn all
clustridial bacteria
sources I.ve~r Table ?) arc contemplated as immunoLens. I;xatz~ples eh'these
toxins are C'.
hrr~Iricunu neuraminidase toxin, toxlllS A. f3. ('. O. I. F. and (i from ( '.
hrrrn!i»rr»r .
('. yc~rJrirr,L~cn.s toxins cx. Vii. F, and t. and ('. .svrclellii toxins lIT
and l..T. In a preferred
elllhuC11171t:nt. ('. hwrrlinum toxins A. 13. C'. D. C. and F lur ti~agmcnts
thercutl are
l()nteltlplated as immunogcns.
:1 particularly prelerrcd embodiment involves the use of~ bacterial toxin
protein or
t'ras~mcnts e,l' toxin proteins produced by molecular biological means l i r..
recombinant toxin
l ~ proteins). In a preferred embodiment. the immunuLen comprises the receptor-
hindin~ domain
li.c.. the ~ ~() kt) carhoxv-terminal portion of the heavy chain: al,e~
relerrcd m as the C'
I'ragmcnt> ul~ ('. hnmli»ur» seroypc A neurutoxin produced by recombinant
I)N:1 technology.
In another preferred embodiment. the immunugcn comprises the reccptclr-
hindin~_ domain oi'
('. hurrrli»rr»r scrotvpe li neurotoxin produced by recombinant I)N:1
tcchnulu;_v. In vet
_'() another preferred embodiment. the imlnunogcn comprises the receptor-
binding domain reLion
of l '. hrrrrrli»rrm serotypr F neurotuxin produced by recombinant DNA
technoluw. I n vet
another prclcrrrd embodiment, the immunugen comprises tltr receptor-hinclin~~
doniailv re';iun
ul' l '. hmtrli»rr»r serotvpe C' l ncurotoxin produced by Ceculllhlllalll I)NA
trchnolu~~v. In mt
another prcterred embodiment. the imlnunogen comprises the receptor-binding
domain region
of ('. hnrrrli»r»» serotype C? n curutuxin produced by recombinant UNA
tcChll(llu~=v. In yet
another preferred embodiment. the immunogen comprises the receptor-hindinp
domain region
01' C'. hnrrrlirnr»I serotvpc D neurotuxin produced by recombinant DNA
technology. In vet
another preferred embodiment. the immunuten comprises the receptor-binding
domain region
ul'('. hmrrlinrrrrr serotvpe F neurutoxin produced by recombinant f)NA
tecinu~ley. In vet
:>0 another preferred Clllhodllllellt. the Inlnlull(lLen e(lnlprISeS th v
receptor-binding domain region
of ('. hrrrnli»tr»r serotypc (J neurutoxin produced by 1'eel)111h111a11t I)NA
technology. In a
preferrccl embodiment, the recombinant botulinal toxin proteins arc expressed
as fusion
proteins (o.,~~.. as histidine-rayed proteins). In a still further preferred
embodiment, the
-2b-
CA 02296765 2000-O1-14
~rp gg~p PCT/US97115394
immurio~~en is a tnultivalent vaccine comprising the receptor-binding domain
region of (.'.
hnrcrlinunt toxin ti-om two or more toxins selected from the group consisting
of type A, type
13. type C f includinL C' 1 and C?), ype U. type E, and type F toxin.
When immunization is used. the preferred non-mammal is li~om the class .9w.s.
All
birds are contemplated (r.,~~., duck, ostrich. emu. turkey. etc.). A preferred
bird is a chicken.
Importantly. chicken antibody does not fix mammalian completnent. [.Seer H.N.
Brnson er crl.,
' .I. 111111111111)I. 87:616 ( I ~)( 1 ).] Thus. chicken antibody will
normally not cause a contplemcnt-
dependent reaction. (A.A. I3enedici and K. Yamaea. "Imnrrmns,~lnhulin.~~ crncl
~lruihvcln
I'I'l)ClllC'lll)1T !I) :Jvicrn .Sj~e~cie.c." ht ('lllJtfllrr'lllrl'C'
lnamtrnrrlu,~w (.I..I. Marehaloni. ed.), pp. ;s5-
i() s7s. 131ackwell. (hford (lc)66).] Thus. the preferred antitoxins of the
present invention will
nc» cxhihit complement-related side effects ohserved with antitoxins known
presently.
Vl'hrn hinds arc used. it is contemplated that the antibody will be uhtaincd
from either
the hind scrum ur the e~:~;. :1 preferred embodiment involves c;ollcctiw~ of
the antibody from
the c~~'~. I.ayin~_ hens transport immunoglohulin to the e~~~ walk ( "IcY") in
concentrations
1 ~ cclual m or cxceedin~ that found in scrum. (.S'c~c~ R. f'atterson et crl..
.1. Immunol. 89:27?
t 19h'?): and 1.f3. C'arroll and I3.U. Stollar. J. I3ioi. Chem. ?~8:?~t
(198s).J In addition, the
lar~.:r w~lume of~ e~~~: yolk produced vastly exceeds the volume of scrum thcu
can hr safely
ohtaincd from tire hirer own any given time period. Finally. the antibody
li~orn e~~s~s is purer
and metre iumwpeneous: there is far IeSS n(ln-11T1t11UIlOLIUhUl111 proorin (as
compared to scrum)
?() anti only cme class of immunoglobulin is transported to the yolk.
V~hcn ce,nsicferin~~ immunization with toxins. one may consider modification
ol' the
main, m reduce the toxicity. In this re~~ard, it is not intruded that the
present invention hr
limited by immunization with modified toxin. Unmodified t"native"~ toxin is
;,I~~,
contctnplatcd as an itomuno~_en.
It is also non intended that the present invention be limited by the type of
modification
-- if' nu~ditication is used. The present invention contemplates all types of
toxin modification.
includin~~ chemicut and heat treatment of the toxin. The preferred
modification. however, is
lormaldehvdc; treatment.
It is not intended that the present invention he limited to a particular made
of
;(f immunization: the present invention contemplates all modes uf'
immuniz.~lti~n. including
suhcutan coos. intramuscular. intraperitoneal. and intravenous or
intravasculur in .jcctic»1. tts well
as pr «., administration of immunogen.
7_
CA 02296765 2000-O1-14
~rp gg/~p PCT/US97115394
~1'he present invention further contemplates immunization with or without
adjuvant.
(Adjuvant is defined as a substance known to increase the immune response to
other antigens
when administered with other antigens.) If adjuvant is used. it is not
intended that the present
invention be limited to any particular type of adjuvant -- or that the same
ad.juvant. once used.
he used all the time. While the present invention contemplates all types ot~
adjuvant. whether
used Stparalelv (lr In C()n1b111at1U11S. the preferred use ot~ adjuvant is
IIIe tlsc Ot~ Complete
Freund's .~ld_juvant followed sometime later with Incomplete l~reund's
Adjuvant. Another
preferred use of ad_juvant is the use of Gerbu Adjuvant. -The invention also
contemplates the
use ul' RIBI fowl adjuvant and (:~uil A adjuvani.
I() When immunization is used. the present invention contemplates a wide
variety ot~
immtu~ization schedules. In one embodiment. a chicken is administered toxin(y
on day zero
and suhscducntlv receives toxin(sl in intervals thereafter. It is not intended
that the present
invention he limited by the particular intervals or doses. similarly. it is
not intended that the
presrnt invention he limited to any particular schedule t«r collecting
antihocd. ~i'he preferred
1 s collection time is sometime after day 1 UU.
V4'hcrc hirds arc used and collection of antibody is performed by collcctlng
r~~~~S. the
C:L~!S 117aV' he: stored prior to processing tW antibody. It is preferred that
eggs he stored at 4°C
ter Iess than one near.
It is contemplated that chicken antibody produced in this manner can hr huftcr-
:'_U cstractcd and used analytically. While unpurified. this preparation can
serve as a rcf~erencc
t'or activiy of the antibody prior to t~urther manipulations (c.,~..
immtanoaftinitv purification).
tll. Incrcusin~ The Effectiveness ()f Antibodies
WhCl1 plIrItIC:AtIOII IS LISI'.d. the present invention contemplates
purit'yin~~ to increase the
.'.5 effectiveness ui~ hoth non-mammalian antitoxins and mammalian antitoxins.
Specifically. the
present Invention contemplates increasing the percent of twin-reactive
immunylubulin. ~l~hc
prelcrrcd purification approach ii~r avian antibody is polyethylene glycol
(Pi::(i) wparatiun.
I'he present invention contemplates that avian antibody he initially purified
using
SlI11p1C. 111~~Cpf:IISIVt', procedures. In one embodiment. chicken antihodv
t'rom rigs is purified
;tl by extraction and precipitation with PEG. PECi purification exploits the
differential sutuhilitv
ut' lipids (witiclt are abundant in ep.~ yolks) and yolk proteins in high
concctZtrations of i'l:(i
8U()t). ~1'IIIS(111 NI ul.. lmmunol. Cotnm. 9:495 ( 1 c)8(1). J ~I'hc
technique is rapid. simple. and
relatively inexpensive and yields an immunoplohulin fraction that is
si~:niticantlv purer in
_~g_
CA 02296765 2000-O1-14
WO 98/08540 PCTN897115394
terms of contaminating non-imrnunogfobulin proteins than the comparable
ammonium sulfate
t'ractions c,t' mammalian sera and horse antibodies. The majority of the PEG
is removed tiom
the precipitated chicken immunoglobulin by treatment with ethanol. Indeed, PEG-
purified
antibody is sufficiently pure that the present invention contemplates the use
of PEG-purified
antitoxins in the passive immunization vi' intoxicated humans and animals.
1 V. '1'rc~tment
~I~Itc present invention contemplates antitoxin therapy for humans and other
animals
intoxicated by bacterial toxins. a\ preferred method of treaunent is by
intravenous
l l) administration oh anti-houtlinal antitoxin: oral administration is also
contemplated tier other
cic>stridial antitoxins.
.A. Dosa~c of Antitoxin
It was nc,ted by way of hack~:round that a balance trust he struck N'11111
adn1t11t5tC1'111~~T,
! > mrrcntly avaitahlc antitoxin which is usually produced in large animals
such as horses:
aut'ticicnt antitoxin must he administered to neutralize the toxin. but nut so
much antitoxin as
tc, increase the risk of untcwa,-d side et'Iects. 'l~hese side effects are
caused hv: i) patient
aensitivitv m torcitn (c-.,s,~. horse) proteins: ii) anaphylactic or
immunogenic properties of non-
II11117Lt11c1~~IOhllIln proteins: iii) the complement fixing properties ul'
mammalian antibodies:
and/or iv) the overall burden ol' foreign protein administered. It is
extremely dif'ticult to
strike this balance when. awoted above. the degree ot~ intoxication (and hence
the )eve! of
antimvin therapy needed) can only he approximated.
~I Ite present invcntic,n cnntcmplatcs significantly reducing side; et'fects
su that this
balance is more easily achieved. Treatment according to the present invention
contemplates
reducing side et'tects by using PL:G-purified antitoxin tom birds.
In one embodiment, the treatment of the present invention contemplates tllC
else (,t'
I'l:O-purified antitoxin from birds. ~l'he use of yolk-derived. PECi-purilicd
antibody as
antitoxin allows for the administration ol': I) nontmammalion)-complement-
fixing. avian
antibody: ?) a Icss heterogeneous mixture ol' nUn-InlIl1tI11ULlt7ht11111
proteins: and s) Icss tcltal
stt protein to deliver the equivalent weiLht of active antibody present in
currently available
. antitoxins. The non-man ~nuilian source of the antitoxin makes it uset'ul
for treating patients
who arc sensitive to horse ur other mammalian sera.
_?c~_
CA 02296765 2000-O1-14
WO 98I~S40 PCTIUS97I15394
I3. Delivery Uf Antitoxin
f~IthUll~h It IS not intended to limit the route of delivery. the present
invention
contemplates a method 1br antitoxin treatment of bacterial IntOxICatl011 111
Wl)1C11 delivery of
antitoxin is oral. In one embodiment, antitoxin is delivered in a solid form
(ce.,c,~.. tablets). !n
an alternative embodiment antitoxin is delivered in an aclucous solution. When
an aqueous
suluticln is used. the solution has sufficient ionic strength to soluhilize
antibody protein. yet is
made palatable tile oral administration. The delivery solution may also be
buffered (e.y..
carbonate butter pf i c).S) which can neutralize stomach acids and stabilize
the antibodies when
the antibodies are administered orally. In one embodiment the delivery
solution is an aqueous
solution. !n another embodiment the delivery solution is a nutritional
t<lrmula. I'relerablv.
the delivery SOltltlUll 1S ttltalll tormula. Yct another embodiment
(:~111I1Il1plilt~S llle dclivc:rv of
lyophilized antibody encapsulated or microencapsulated inside acid-resistant
colnpounds.
Methods o1' applying enteric coatings to pharmaceutical compounds are well
known to
the art ~rumFlanies shecializin~= in the coating of pharmaceutical compclunds
arc available: for
Is example. ~l'he C'oatin~~ Place (Verona. W!) and AAI (Wilmington. NC')].
l:ntcric cuatinca
which are resistant to gastric fluid and whose release (i.r.. dlssolutlon of
the ruatin_~ to release
tllc pharmaceutical compound) is pFl dependent arc commercially available
~I~ur eaamhlc. the
polymetllacrylutes lJudragit.k; I. and f-;udragittii~ ~ (R~hm C.imhl-11].
f=udragia t ~ i:s soluble in
intestinal fluid from ptl 7.0: this coating can be used to micruenrapsulatc
lyophilized antitoxin
'_'() antihmii~s and the particles are suspended in a solution having a pll
above or below p!1 7.0
I~or oral administration. The mieropartielcs will remain intact and
undissolved until they
reached the intestines where the intestinal pH would cause tl1e111 to
dlssulvc: thereby releasine
the antitoxin.
l he invention contemplates a method of treatment which can he administered
tile
'_'s treatment ot~ acute intoxication. In one embodiment, antitoxin is
administcrect orally in either
a delivery solution or in tablet form, in therapeutic dosage, to a subject
intclxicatcd by the
hacte:rial main which served as immunogen file the antitoxin.
The invention also contemplates a method of treatment which can he
administered
llrophvlacticallv. In one embodiment. antitoxin is administered orally. in a
delivery solution.
s0 in therapeutic dosage, to a subject. to prevent intoxication of the subject
by the bacterial toxin
which served as immuno~en for the production of antitoxin. In another
embodiment.
antitmin is administered orally in solid form such as tablets or as
microcncapsulated particles.
Microencapsulation of lyophilized antibody using compounds such as
L:udrugit~K~ ( ROI1111
- i (') -
CA 02296765 2000-O1-14
WO 98/08540 PCT/US97115394
Gmbl-il or polyethylene glycol , which dissolve at a wide range of pH units.
allows the oral
administration of solid antitoxin in a liquid form (i.e.. a suspension) to
recipients unable to
tolerate administration of tablets (c~.,s;.. children or patients on feeding
tubes). In one preferred
embodiment the subject is a child. In another embodiment, antibody raised
against whole
bacterial organism is administered orally to a subject. in a delivery
solution. in therapeutic
closa~~e.
Vaccines Against Clostridial Species
'hhe invention contemplates the generation of mono- ;tnd multivalent vaccines
for the
f() protection of an animal (particularly humans) against several clostridiai
species. (7f particular
interest arc vaccines which stimulate the production of a humoral immune
response to ('.
hmrrlirurm. ( '. tetcrui curl (' cliJ~icilc~ in humans. The antigens
comprising the vaccine
~rl:p.l1'~ltl(111 Illa1' he native or recombinantly produced toxin proteins
from the clostridial
apccies listed above. When toxin proteins are used as immunogens they arc:
generally
1 ~ modified to reduce the toxicity. This modification may be by chemical or
genetic (i.c~.,
rrcomhinant DNA technology) means. In general genetic detoxification (i.r..
the expression
«I' nontoxic fragments in a host cell) is preferred as the expression of
nontoxic fragments in a
host cell prcclucles the presence of intact. active toxin in the final
preparation. Ilow~cver.
when chemical modification is desired. the preferred toxin modification is
lornlaldehvde
~() treatment.
The invention contemplates that recombinant C'. hcnulinnm toxin proteins he
used as
anti~cns in mono- and multivalent vaccine preparations. ~e~luble.
substantialfv endotoxin-free
rvcomhinant ('. hmurlimrm toxin proteins derived from serotypes f1. I3 and C:
may be used
individualy (i.e.. as mono-valcnt vaccines) or in combination (i.e., as a
multi-valent vaccine).
In addition. the recombinant t;'. horulirrurrt toxin proteins derived t~on~
scrotpes A. t3 and I:
may be used in conjunction with either recombinant or native toxllls or
toxoids from other
scrotypes of ('. hruulinrrnr. ('. cIiJJicile and C'. trtuni as antigens for
the preparation of these
mono- and multivalent vaccines. It is contemplated that. due to the structural
similarity of ('.
hntulimrnr and ('. te~lGJ7i toxin proteins. a vaccine comprising C'.
diJ~icilc~ and hntrrlinum toxin
s() proteins ( native or recombinant or a mixture thereof be used to stimulate
an immune
respemse a~!ainst ('. hutulintrm. C'. tetcuTi crnc! ('. c!iJJieilc~.
_;I _
CA 02296765 2000-O1-14
PCTNS9'7/15394
The present invention further contemplates mufti-valent vaccines comprising
two or
more botulinal toxin proteins selected from the group comprising recombinant
C'. butulinum
toxin proteins derived from serotypes A. B. C (including C1 and C2). D. E, F
and G.
The adverse consequences of exposure to botulinaE toxin would be avoided by
11n171ttnlZat1011 Uf subjects at risk of exposure to the toxin with nontoxic
preparations which
confer immunity such as chemically or genetically detoxified toxin.
Vaccines which confer immunity abainst one or more: of the toxin types A. E3,
I. 1~
and Ci would be useful as a means of protecting humans ti~otn the deleterious
effects vi' those
('. hr~ltrlimrnt tt)xtn$ known to affect man. Indeed as the possibility exists
that humans could
lU he exposed to any of the seven serotypes of C.'. hv~ulurum toxin (c.~,~..
during biological
warfare ctr the production of toxin in a laboratory setting), multivalent
vaccines capable of
conferring immunity against toxin types A-Ci (including both C'1 and C2
toxins) would be
useful tier the protection of humans. Vaccines which confer immunity against
one or more of
tltc tewill miles C'. D and E would be useful for veterinary applications.
1 > -E-he hotulinal neurotoxin is synthesized as a sinclr polypeptide chain
which is
processed into a heavy (H: -lUU kD) and a Eight (l.: --iU kI)) chain by
clcavacc with protcolytic
enzymes: these m~o chains are held together via disulfide hoods 111 the active
toxin (referred to
as derivative toxin) [B.R. DasGupta and I1. Sugiyama. I310C11C111. lilOp111'S.
Etes. C'ommun.
4R:1 UH ( 1 c)7?): reviewed in B.R. Das(iupta. J. Yhysiol. 84:??U ( 1 ~)9U). I
E. Su~iyama.
?U Microbial. Rev. 44:419 (1980) and C.'.1.. I~athewav. C.'lin. Microhiol.
Rev. _>:(i(~ (1990)[. 'the
heavy chain of the active toxin is cleaved by trypsin to produce two
lia~_ments trrmcd I 1,.
(also referred to as I-1, nr C') and 11~ (also referred to as I1, nr fi). I he
ly. I'ragtnent (-4(~ kD)
cumpriscs the carboxv end of the ll chain. 'rhe III Fragment (-~t~) kl))
comprises the aninm
end and remains attached to the I_ chain (It,~L). Neither I-!~. or H,,1. is
tUxlc. hl~ competes
?> with whole tirrivativc toxin for binding to synaptosomes and therefore 1I~
is said to contain
thr receptor hindinr~ site. 'hhc E I~. and 1-i" fragments of hotulinal toxin
are analogous to the
fragments C' and B of tetanus toxin which are produced by papain cleavage.
~I~hr C' l~ragtnent
of tetanus toxin has been shown to he responsible !or the bindin~_ of~ tetanus
toxin to purified
~an~liusidcs and neuronal cells [i-Ialpern and Loftus. J. t3iol. C'hcm.
?88:11188 (1~)~)_~)).
3() Antisrra raised aLainst purified preparations of isolated hotulinal E-1
and I_ chains have
been shown to protect mtcc against the lethal effects of the toxin: however.
the cFfectivencss
of the w~o antisera differ with the anti-II sera being mare patent (II.
~u~iyama. .srrhrul.
While the different hotulinal toxins show structural similarim to one another,
the different
CA 02296765 2000-O1-14
WO 98JflS540 PCT/(JS97115394
serotypes are reported to be immunologically distinct (i. r., sera raised
against one toxin type
does not cross-react to a significant degree with other types). Thus, the
generation of
multivalent vaccines may require the use of more than one type of toxin.
('. hrurrlinrrnr toxin genes from all seven serotypes have heen cloned and
sequenced
( Minton ( I 995), .crrnr-cr); in addition. partial amino acid sequence is
available for a number of
('. 17(rlrllrrrrrl)1 toxins isolated from different strains within a given
serotype. The C'. hcnulinu»r
lOxIrtS Ct)r7tar11 ahollt 1?50-1300 amino acid residues. On the DNA level. the
overall degree of
homology between C'. hruulinu»r serotypes A, E3. C. D and f; toxins averages
between SO and
(,()% identity with a greater degree of homology being found between I-I chain-
encoding
1 U regions than between those encoding L chains [Whelan cu crl. ( 19c)?)
Appl. Environ.
~~licrohiol. 58:? 345j. The degree of identity between ('. hrrrrrlinr»rr
toxins on the amino acid
Icvcl reflects the level of DNA sequence homology. 'Che most divergent area of
DNA and
amino acid Sc'.qlICIICt: 15 found within the carboxv-terminal area of the
various ('. hunrlirTUm U1
chain '~elllS. ~rht1 portion of the toxin (i.e.. lil.or the C fragment) plays
a major role in cell
1 s hindin~~. ,~ls toxin from different scrotypes is thought to bind to
distinct cell receptor
molecules, it is not surprising that the toxins diverc:e significantly over
this region.
Within a Liven serotvpc, small variations in the primary amino acid sequence
oJ' the
b~tulinal toxins isolated ti-cnn different strains has been reported ~ Whelan
m ul. ( 199?). .wrhrcr
and Minton ( 1995). .wrlrrcrj. The present invention contemplates fusion
proteins comprising
2t) horticms of~ C'. hrrlrrlirttr»r toxins from serotypes A-Ci including the
variants ibund among
different strains within a given serotype. The present invention Provides
oligonuclrotide
primers whici~ may be used to ampliy the C fragment or receptor-binding
re~~ion of the toxin
~~ene i'rum various strains of ('. horulirzunr serotype A, serotvpe 13.
scrotvpe (' (Cl and C2).
srrotvpc I). scrotvpe L, scrotync iv and scrotype G. A large number of
different strains of ('.
?5 horrrlinr»rr serotvpe A. scrotvpc L3. serotype C, serotype D serotype E and
serotypc F arc
available tCUll1 the American Type Culture Collection (ATCC: Rockville. MD).
For example.
the A~I~C'C' provides the followiy: Type A strains: 174 (A'fC:C 3502). 457
(A'l'CC' 17862),
and N("rC 7?7? (ATC.'C 19397): Type f3 strains: 34 (A'TCC 4 3c)). G2A (A~l'CC
794R). NC A
13 13 ( ATC'C 7c)49). I 31 I 4 ( ATCC 8083). 31 37 (ATCC 177811). 1347 (
A'1'C:C' 1784 I ). ?U 17
3() (ATC'C 1784;). ??17 (ATCC' 17844). ?'_54 (ATCC 17845) and V1' 1731 (A'fC C
25765);
Type C strains: ?220 (A'fCC 17782), '?'?39 (ATCC 17783), 2?33 (A~TC.'C' 17784:
a type C'-(~
strain: C'-(3 strains produce C2 toxin). 6G2 (ATCC 17849; a type C'-a strain:
C'-a strains
produce mainly C l toxin and a small amount of C2 toxin). 2021 (A~1'CC 1785U:
a type C'-a
-33-
CA 02296765 2000-O1-14
WO 98/08540 PCTIUS97l15394
strain) and VPl 38()3 (ATCC ?57bb); 'Type D strains: ATCC 9633. 2023 (ATCC
17851 ), and
VPt 599 (ATCC ?7517); Type 1~ strains: A'fCC 43181. 36208 (A'1'CC 9564), ??31
(ATCC
17786). ''??9 (ATCC 17852). ?279 (ATCC 17854) and 2'_'85 (ATCC 17855) and Type
F
strains: '?U2I= (ATCC ?3387). VPI 4404 (ATCC ?5764). VP1 2 383 (ATC'C ?73? 1 )
and
Langeland (A'fCC 3~41~). 'Type Ci strain, 11 3130 (NCFB 301?) may be obtained
from the
National (.'ollection of Food Bacteria (NCFB, AFRO institute of Faod Research.
Reading,
Ilnitcd Kingdoml.
Purification methods have been reported for native toxin types A. 13. C'. O.
E. and F
[reviewed in ti. Sakaguchi. t'harmac. T'her. 19:165 (1983)). As the different
hotulinal toxins
1 () are structurally related. the invention contemplates the expression of
any of the botulinal
IUxlIIS (c.,~~.. types A-(i) as soluble recombinant fusion proteins.
In particular, methods for purification of the type A botulinum neurotoxin
have been
developed [ I...I. Moherg and Iv. Sugiyama. Appl. l:nviron. Microbial. i~:878
( 1 c)78)[.
Immunisation ol' hens with detoxilied purified protein results in the
generation ot~ neutralizing.
Is antihocfics [B.S. 'fhalley rr ul.. in l3vtuli»um a»d 7inunrr.c
11'errrwuuxi».v. fi.R. I)as(iupta. cd..
Plcnunt Press. New York ( 1c)c)3). p. 467).
The currently available ('. hrrlrrli»rrm pentavalent vaccine contprisin~
chemically
inactivated (i.u.. titrmaldehvde treated) type A. R. C. D and L: toxins is
riot adccluate. The
eft3cacv is variable (in particular, only 78% of recipients produce protective
levels of anti-type
?U I3 antibodies fcrllowinL administration of the primary series) and
immunization is paintirl
(deep suhcutan eous inoculation is recluired fur administration), with adverse
reactions being
common (moderate to severe local reactions occur in approximately 6'% of
recipients upon
initial injection: this number rises to approximately I 1°/~ of
individuals who receive booster
injections) [informational Brochure t~tr the Pentavalcnt (ABCDI) L3cttulinum
~l~oxoid, C'c;nters
?s Ior Disease (.'ontrol J. Preparation of this vaccine is dangerous as active
toxin must be handled
by Iaboratorv workers.
In general. chemical detoxification of bacterial toxins using agents such as
f~armalclchvdc. glutaraldchvde or hydrogen peroxide is not optimal tits the
veneration of
vaccines or antitoxins. A delicate balance must he struck between tan much
arid too little
stl chemical modification. If the treatment is insufficient. the vaccine may
retain residual
toxicity. If the treatment is too excessive. the vaccine may lose potency due
to destruction of
native immunoLCnic determinants. .Another major limitation oC using hotulina)
toxoids for the
generation of antitoxins or vaccines is the high production expense. Fur the
above reasons.
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CA 02296765 2000-O1-14
WO 98/540 PCT/US97/15394
the development of methods for the production of nontoxic but immunogenic C'.
butulinum
toxin proteins is desirable.
The (.'. bv~ulinrrrrr and C'. ~ercrnu.s toxin proteins have similar structures
[reviewed in
I=..1. Schantz and E.A. Johnson. Microbiol. Rev. SG:80 ( 1992)]. The carboxv-
terminal 50 kD
t'ragment of the tetanus toxin heavy chain (fragment C) is released by papain
cleavage and has
been shown to be non-toxic and immunogenic. Recombinant tetanus toxin fragment
C has
been developed as a vandidate vaccine antigen [A.J. Makoft~ er «L.
l3iol'i'echnolugy 7:1043
( 1~)8~))I. Mice immunized with recombinant tetanus toxin fragment C were
protected tcom
challenge with lethal doses of tetanus toxin. No studies have demonstrated
that the
recombinant tetanus t~agment C' protein confers immunity against other
hutulinal toxins such
as the (' hrrrrrlin«r» toxins.
Recombinant tetanus ti~agment C has been expressed in L;. calf (A.J. Makoff cr
crl..
E?iiuhhcchnolugy. .wrhrcr and Nucleic Acids Res. 17:10191 ( 1989): J.I.. I
lalpern m crl.. Infect.
(ntrnun. sa:lU(1~ (1990)), yeast [M.A. Romanos e~~ ul.. Nucleic: Acids. Res.
i9:1~101 (1991)]
I ~ and haculuvirus ~ I.G. Charles cr crl.. Infect. Immun. 59:1 h27 ( 1991 )).
Synthetic tetanus toxin
~~cnes had to be constructed to facilitate expression in yeast (M.tl. Rumanus
m crl.. .supra) and
I~'. cwli [f1..1. Makoff cr crl.. Nucleic Acids Rcs., .~~trprcr], due to the
high A-'r content of the
tetanus twin gene sequences. I IiLh A-~f content is a common feature ul~
clostridia) genes
(M.R. I'upohf rr crl.. Infect. lmmun. 59:3673 (i991); H.F. LaPenotiere m crl..
in l3olrrli»rrr» crud
_'() Tcn«nar.s .\ rr»wnnxin.v. B. R. DasGupta. eel.. Plenum Press. New York (
199 ; ), p. 463 ] which
creates expression difficulties in L. coh and yeast due primarily to aitcrcd
colon usage:
fi~eduenw and liu-tuitous pulvadcnvlatiun sites, respectively.
fhc C' fragment of the C'. horarli»u»z type A neurutoxin heavy chain has been
evaluated
as a vaccine candidate. 'fhc ('. I7I)rrlhl7rrr17 type A neurotoxin gene has
been cloned and
sequenced [I).L;. Thompson cu crl.. Eur. J. Biochem. 189:73 (1990)). The C'
fragment of the
type A twin was expressed as either a fusion protein comprising the hotulina)
C t~agment
fused Wlth Ihl'. I11a1tOSe binding protein (MBP) or as a native protein ~H.F.
Lal'enotiere cn crl..
( 199;) .cuhrcr. I I.F. LaPcnotierc m eel.. Tuxicon. 33:1 383 ( 1995) and
Middlcbrouk and i3rown
(1995). C'urr. ~Ii~p. Microbial. Immunol. 195:89-122). Tlte plasmid construct
encoding the
native protein was reported to he unstable. white the fusion protein was
expressed primarily in
- inclusion bodies as insoluble protein. Immunization of mice with crudely
purified MBP
fusion protein resulted in protection against 1P challenge with 3 LDS" doses
of toxin
]l.al'enoticre m «L, ( 1993) and ( 1995), .srrprcr]. However, this recombinant
('. hnrulinrr»r type
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CA 02296765 2000-O1-14
PCTIUS97115394
A toxin C fragment/MBP fusion protein is not a suitable immunogen for the
production of
vaccines as it is expressed as an insoluble protein in F. calf. Furthermore.
this recombinant
(.'. hnnrlinunr type A toxin C fragmentIMBP fusion protein was not shown to be
substantially
t3~ee of endotoxin contamination. f:xperience with recombinant ('. hnhrlinrrm
type A toxin (.'
s fragmentIMBP fusion proteins shows that the presence of the MBP on the
fusion protein
~rcatly complicates the removal of endotoxin from preparations of the
recombinant fusion
protein (.we Ex. 24. inJucr). Expression of a synthetic gene encoding C'.
hn~trlinum type A
toxin C ti-agment as a soluble protein excreted from insect cells has been
reported
[Middlcbraak and Brown ( 1995). .wnhru~; no details regardinL the level of
expression achieved
ur the presence of endotoxin or other pyrogens were provided, Like the
InsoIUbIe protein
expressed in L:. cnli. immunization with the recombinant protein produced in
insect cells was
reported to protect mice from challenge with C'. hnrrrlinum toxin A.
Inclusion body protein must be solubifized prior to purification and/or
administration
to a host. The harsh treatment of inclusion body protein needed to accomplish
this
I s solubili-ratiun may reduce the immunoeenicitv of the purified protein.
Ideally, r~cumbinant
proteins m he used as vaccines are expressed as soluble proteins at high
levels (i.r.. greater
than or equal to about 0.75% of total cellular protein) in E. cwli or other
host cells (e.~~.,
yeast. insect cells. ete.). 'This facilitates the production and isolation of
sufficient quantities of
the imlnunugen in a highly purified form (i.v.. substantially i'rcc ul'
endotoxin or other
~0 pyrogen contamination). The ability to express recombinant toxin proteins
as soluble proteins
in E. orrli is advantageous due to the low cost of growth compared to insect
or mammalian
tissue culture cells.
The ('. hurrrlinrrrn type B neurutuxin gene has been cloned and sequenced from
two
strains of ('. hmulinrrm type B [Whelan c~l crl. (1992) Appl. Environ.
Microbial. sli:2 i4s
2s (Danish strain) and Hutson r~ ul. (1994) Curr. Microbiol.'_B:IUI (Eklund
17B strain)). 'Fhe
nucleotide sequence of the toxin gene derived from the Eklund 1713 strain
(ATCC 25705) is
available from the EMB1,1(ienl3ank sequence data banks under the accession
number X71346:
the nucleotide sequence of the coding region is listed in SEQ ID NU: i9. The
amino acid
sequence al' the C'. hnrulinrrm type Ei neurotoxin derived from the strain
Eklund 178 is listed
30 in S~Q ID NU:40. 'The nucleotide sequence elf the ('. horrrlinum scrotype
13 toxin gene
derived Pram the Danish strain is listed in SEQ ID NC):41. The amino acid
sequence of the
('. lro~trlirzrrna type B neurotoxin derived from the Danish strain is fisted
in SEQ I>7 N0:42.
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CA 02296765 2000-O1-14
PCTIUS97115394
The C'. horulinum type B neurotoxin gene is synthesized as a single
polypeptide chain
which is processed to form a dimer composed of a light and a heavy chain
linked via
disulfide bonds. The liglu chain is responsible fur pharmacological activity
(i.c~.. inhibition of
the release of acetylcholine at the neuromuscular junction). The N-terminal
portion of the
' s heavy chain is thought to mediate channel formation while the C-terminal
portion mediates
toxin binding: the type B neurotoxin has been reported to exist as a mixture
of predominantly
Slllgle C'.halil Wlth S()Ille double chain ( Whelan er crl.. .supra). The ~0
kD carboxv-terminal
portion of the heavy chain is referred to as the C' fragment or the Ilc.
domain. 'The present
invention reports for the first time. the expression of the C fragment of C'.
hnrulinurn type B
toxin in heterolugous hosts (c~.~J., E. culi).
The C'. hurulinlrm type I: neurotoxin gene has been cloned and sequenced twom
a
number of different strains [Poulet or ul. (199?) Biochem. Biophys. Res.
('ommun. 183:1()7:
Whelan cr crl. (1992) tour. J. Biochem. ?04:G~7: and Fujii cu crl. (1993) 1.
Gen. ;vticrohiol.
1 3c):7cy. The nucleotide sequence of the type E toxin gene is available from
the EMF3L
1 > scduence data bank under accession numbers X62089 (strain Beluga) and
X62686 (strain
N(.'~fC' 1 1 ? 1 c)): the nucleotide sequence of the coding region (strain
Beluga) is listed in SI:Q
It NC):~s. The amino acid sequence of the ('. bnrulinurn type E ncurotoxin
derived trom
strain l3cluLa is listed in SEQ ID N():4G. The type I: neurotoxin Lent is
synthesized as a
single lx~lycptide chain which may be converted to a double-chain form (i.r..
a heavy chain
_'() and a light chain) by cleavage with trypsin: unlike the type A
neurotoxin. the type E: .
neurotoxin exists essentially only in the single-chain form. The ~0 kD carboxv-
terminal
portion o!' the: heavy chain is referred to as the C fragment or the Eh.
domain. The present
invention reports for the first time, the expression of the C tragmcnt of ('.
hrrrrrlinurn type F
tu~cin in hetcrolo~:ous busts l~.L~.. E. crrli).
T'he ('. hrrrulinrrnr type C'l. D. F and G neurotoxin genes have been cloned
and
scclucnced. The nuclecftide and amino acid sequences of these genes and toxins
are provided
herein. ~l'he invention provides methods for the expression of the C fragment
from each of
these toxin genes in heterologuus hosts and the purification of the resulting
recombinant
proteins.
The subject invention provides methods which allow the production of soluble
('.
hrnulirrrrm toxin proteins in economical host cells te~.y., L;. c~oli). In
addition the subject
invention provides methods which allow the production of soluble hutulinal
toxin proteins in
yeast and insect cells. Further. methods for the isolation of purified soluble
('. hrnrrlinum
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CA 02296765 2000-O1-14
PCTNS97115394
toxin proteins which are suitable for immunization of humans and other animals
are provided.
'these soluble, purified preparations of C'. bvtulinum toxin proteins provide
the basis for
improved vaccine preparations and facilitate the production of antitoxin.
When recombinant clostridia( toxin proteins produced in gram-negative bacteria
(v.y.,
s E. reli) arc used as vaccines, they are purified to remove endotoxin prior
to administration to
a host animal. In order to vaccinate a host, an immunogenically-effective
amount of purified
Substantially endotoxin-ti~ee recombinant clostridia( toxin protein is
administered in any of a
number ut' physiologically acceptable carriers known to the art. When
administered for the
purpose c1f vaccination. the purified substantially endotoxin-tree recombinant
clostridia( toxin
protein may he used alone or in conjunction with known adjutants. including
potassium alum.
aluminum phosphate, aluminum hydroxide. Gerbu adjuvant (GmDP: C'.C. Biotech
Corp.).
RIBI adjuvant (MfL; lZIBI Immunochemical Research. Inc.). QS2l (Camhrids~e
Bioteclz).
'fh~ alum and aiuminum-based adjutants arc particularly preferred when
vaccines arc to be
administered to humans: however. any adjuvant approved for use in humans may
he
1 ~ employed. 'I~he route of immunization may be nasal. oral. intramuscular.
intraperitoncai or
subcutaneous.
~l~lm invention contemplates the use of soluble, substantially endotoxin-free
preparations of fusion proteins comprising the C' tcagment of the ('.
lmtrrlinunr type A. 13. C,
U. E. F. and (i tux111 as vaccines. In one embodiment. the vaccine comprises
the C fragment
?0 of either the ('. hntulinum type A, B. C, D. E. F, or (~ toxin and a poly-
histidine tract (also
called a histidine tag). In a particularly preferred embodiment. a fusion
protein comprising
the hISLld1111', tagged C fragment is expressed using the pl:~f series of
c~cprcssiol vectors
(Nc~yaLrn). The pET expression system utilizes a vector containing the 'f7
promoter which
encocds the fusion protein and a host cell which can he induced to express the
'1~7 DNA
polymcrase (i.c., a DE3 host strain). The production of C fragment fusion
proteins containing
a histidine tract is not limited to the use of a particular expression vector
and host strain.
Several commercially available expression vectors and bust strains can be used
to eaprrss the
C ti-a~.:ment protein sequences as a fusion protein containing a histidine
tract (Fur example, the
pQE series ( pQE-8. 12, 16, 17. l 8. 30. 3 l , 32, 40. 41, 4-_'. SU, ~ t . ~
'. cu anct /U ) of
,U ~xpressic~n vectors (Qiagcn) which are used with the host strains M t S(pRf-
:P4[ (Qiagen ) and
S(i13~09[pREf4[ (Qia~en) can be used to express fusion prclteins containing
six histidine
residues at the amino-terminus of the fusion protein). Furthermore a number of
commercially
available expression vectors which provide a histidine tract also provide a
protease cleavage
_38_
CA 02296765 2000-O1-14
WO 98108540 PCTIUS97115394
site between the histidine tract and the protein of interest (c.~;~.,
botulinal toxin sequences).
C'leava~c of the resulting tilsion protein with the appropriate protease will
remove the
histidinc ta~~ from the protein of interest (c~.~j., botulinal toxin
sequences) (see Example 28a.
infra). Removal «f the histidine tae may be desirable prior to administration
of the
recombinant hotulinal toxin protein to a subject (c~.~~.. a human).
V1. I)etcction Of Toxin
The invention contemplates detecting bacterial toxin in a sample. The term
"sample"
in the present specification and claims is used in its broadest sense. On the
one hand it is
1(t meant to include a specimen or culture. Un the other hand. it is meant to
include both
biological and environmental samples.
Biological samples may be animal. including human. fluid. soled (e.~~.. stool)
or tissue:
liquid and solid t~m~d products and ingredients such as dairy items.
vegetables. meat and meat
hy-products. and ~astc. Environmental samples include environmental material
such as
i ~ surface ntattcr. soil. water and industrial samples. as well as samples
obtained from trod and
dairy processin~~ instruments, apparatus. equipment, disposable and non-
dispusahl~ items.
l~itrsc ~xamlles are not 1 be construed as IimitinL the sample types
appticablc to the present
111~'t I1LIO11.
Thr invention contemplates detecting bacterial toxin by a campetitiw
immunoassay
~'() method that utilises recombinant toxin A and toxin I3 proteins.
antibodies raised against .
recombinant bacterial toxin proteins. A fixed amount of the recombinant toxin
proteins arc
imlzu~hili~rd to a solid support (c-.,L~.. a nticrotiter plate) tbllowed by
the addition ol' a
biological Salllpll' SLISpCCted of containing a bacterial toxin. The
biological sample is first
mined with affinity-purified or I'E(i iiactionated antibodies directed against
the recombinant
toxin prrnein. A reporter reagent is then added which is capable of detecting
the presence of
antihocl hound to the immobilized toxin protein. The reporter substance may
comprise an
antihocy with binding specificity for the antitoxin attached to a molecule
which is used to
identify the presence of the reporter substance. If toxin is present in the
sample, this toxin
will ce~mpete pith the immobilized recombinant toxin protein !ur binding to
the anti-
recombinant antibody thereby reducing the signal obtained following the
addition ot~ the
reporter reagent. A control is employed where the antibody is not mixed with
the sample.
This gives the highest (or reference) signal.
_ ; c) _
CA 02296765 2000-O1-14
PCTNS97I15394
The invention also contemplates detectinc bacterial toxin by a "sandwich"
immunoassay method that utilizes antibodies directed against recombinant
bacterial toxin
proteins. Affinity-purified antibodies directed against recombinant bacterial
toxin proteins art
immobilized to a solid support (c~.~,J.. microtiter plates). l;ioloLical
samples suspected of
s containing bacterial trains arc then added followed by a washing step to
remove substantially
all unbound antitoxin. The biological sample is next exposed to the reporter
substance. which
hind, tc~ antitoxin and is then washed free of substantially all unbound
rclortcr substance:.
The reporter substance may comprise an antibody with bindin~~ speciticity t«r
the antitoxin
attached to a molecule which is used to identify the presence of the reporter
substance.
l() Idcntitication of the reporter substance in the biological tissue
indicates the presence of the
bacterial toxin.
!t Is alstl contemplated that bacterial toxin be detected by Louring liquids
(~.~~.. soups
and mhcr fluid ti,ods and feeds including nutritional supplements tier humans
anti other
,InimaIW over inuoubili~cd antibody which is directed against thr bacterial
toxin. It is
1 ~ euntemllatcd that the immobilized antibody will be present in ur on such
sullorts as
eartrid~~ua. columns, l,rads. ur any other solid SLILL(lrt Illldltlttt. In om
emhudintcnt. li~lluwing
the ml,asurc «I~ the liduid to the immobiiizcd antibody. unbound toxin is
substantially
rcmomd Iw washinL. l~h~ cvl,osur~ of the liquid is then ~xpascd to a reporter
auhst:utcc
which Jctccts the presence of hound toxin. In a preferred embodiment the
reporter substance
?0 is all 1117\'lttl'. Iluorcscent dvc. ur radioactive compound attached m an
antibody which is
directed against the toxin (i.e., in a "sandwich" immunuassavl. It is also
contemplated that
thr detection system will lm developed as necessary (c.,~~.. the addition ul~
rn7vmc substrate in
m7vmr wstcms: observation u~in~ fluorescent liLht ti>r tluurcsccnt dvc wstemx:
and
cluantitation ul~ radiuactiviy tar radioactive systems).
,;
F.XPER1MENTAL
~l~hc following c:xamlles serve to illustrate certain Lreferred embodiments
and aspects
of thr Lrcscnt invention and arc not to be construed as Iimitin L the scale
thereof.
In the disclosure which titllows. the following abbreviations alllv:
°C' (eicgrees
al i.'enti'~radel: rpm (revolutions Lcr minute): IiBS-l~ween (borate
huf'ti:red saline ronttainin~;
~I'weenl: I3~A (bovine scrum albumin): I:I.ISA (l,'111yllll:-1I17hCd
tlttlttllltlWOrl,t:ltl aSSa\'): (.'HA
Icumlletc Urcunci's ad.juvant): fl~A (incontllete f~reund~s ad.juvant): ILC~
(immulto~~lohulin <i):
1gY' limmunoglobulin Y): IM (intramuscular}; 11' (intrapcritoncal): 1V
(intravenous or
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CA 02296765 2000-O1-14
WO 98108540 Pf"TIUS97/1(5394
intravascular): SC.' (subcutaneous): I~I,O (water): HCI (hydrochloric acid);
LDlrlrt (lethal dose
iur I O()% of experimental animals): as (amino acid): EIPLC (high performance
liquid
chromatography): kD (kilodaltons): grtt (grams): yg (micrograms(: mg
(milligrams): ng
(nano~~ramsJ: yl (microliters): mi (milfilitcrs): mm (millirnctersl: nm
(nanonteters): ).tnt
(micrometer): 1~1 (molar): mM (millimolar): MW (molecular weight): sec
fseconds):min(s)
(minutciminutest; hrls) (hour/hours): MeCI, (magnesium chloride): NaC'I
(sodium chloride);
Na.C'(>: (sodium carhonatc): ()1)_xn (optical densiy at ?8() nm): UD,,~"~
(optical density at 6U()
nm): 1'.~ICiL: (polyacrylamidc gel electrophoresis): PBS (phosphate huffercd
saline ( 1 i0 mM
NaC'I. IU mM sodium phosphate buffer. pIJ 7.?)J: PL~Ci (polyethylene glycol):
PMSF
1() (phenyimethylsultiyl tluoride): SDS (sodium dodecvl sultatc): Tris
(leis(hvclrewmcthyl)aminomethane): l~ItsLlrC~.H> (I-:nsure!t. (toss
L.ahoratorics. ('olumbus ()Il):
I:nlamil K Il:ntamilvi. !~~l~ad Johnson): wiv (weight to volume): viv (volume
to volume):
:lmicon (:\nticon. Inc.. limcrly. ;1:11: :\ntresco (-\ntresco. Inc.. Solos.
()li): I\~I~C'('
Ir\ntcrican I~ypc ('ulturr ('ollc:ction. Rockville. MD): E3BI. (Baltimore
Biologics l.aboratorv.
(a eliuision ol' I3ccton Dickinson). C'ockeysville, A~1D): Becton l7ickinson
(Becton L)ickinson
I,ahwar~. Lincoln Park. ~'.1): BioRad (l3ioltad. Richmond. (';\): l3iotcch (('-
C' (iiotcch ('orp..
I'oway. C':~?: (.'harlcs River (('harlcs River L.ahoratories. VViIlttlngttllt.
M.A): Cocalico
(<'or.rlico I3ioly~irals Inc.. IZcamstown. I'A): (:'yRx (('WRx ('orp.,
Norrross. (it1): I~alron
(c-.~. liawcr Ilcalthcarr ('orp.. N1c(iaw ('ark, IL. and Becton f)ickinson):
l~I)A (I~c;c~c;ral Food
.md 1)ru;~ :~clministration): fisher BioUech (f=isher Biotech. Spriniticld.
N.I): (;IL;('() ((;rand
Islancl (3iulo~_ir (.'ompam°/13RL.. Grand Island. NY): (iihco-BRI,
(Life ~I~ccitnologics. Inc..
(iaithorsi~ur_~. \-1l)): Ilarlctn Sprague I)awlcv (I-iarlan Sprague I)avlcv.
Ine.. Madison. 11t'1):
~-1allinckretdt (~t division ol~ Baxter Ilralthcare ('orp.. Mc(iaw Park. IL):
v1ifliporc (Millipore
('urp.. ~larlhoruugh. MA): New I~Il~laltd BlUlitbS (New L:ns~land Biolahs.
Inc.. Rcverly. MA):
NcwaLCn ( Novagcn. In c.. h-tadison. Vv'I ): I'harmacia ( Pharmacia. lnc..
I'iscatawav. N.1); (~iagct~
((~)iagcn. ('hatsworth. CI1): SasCO (Sasco. Umaha. NI:): Showdex (Shown W nko
America.
Inc.. Nm 1~'urk. N~'): Sigma (5i~~ma Chemical C'o.. St. Louis. ~~9()):
Sterogette (Sterogene.
inc.. :lrcadia. ('~1): ~I~rch l.ah (~I~rch Lab. Inc.. Blacksbur~_. Vn): and
Vaxccll (Vaxcell. In c..
a subsidiary c~f C'yIRX C«rp.. Norcross. (iA).
.sU 1al'hen a recombinant protein is described in the specification it is
referred to in a
short-hand ntanmr by the amino acids in the toxin sequence present in the
recomhinant
protein rounded to tltc; nearest I(). F«r e~camplc. the recombinant protein
pMI318~0-?3O)
et~ntains amino acids 18s? through ?3G? of the ('. cli/~icile~ toxin I3
protein. ~1'hcspecitication
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CA 02296765 2000-O1-14
WO 98/08540 PCTIUS97/15394
gives detailed construction details for all recombinant proteins such that one
skilled in the art
~Vlll IC110H' precisely which amino acids are present Ill a L1Ve11
reCOlllbinatll protein.
I;XAMPLr I
Production ()f high-'Liter Antibodies To ('Iv.S'Ir'flllllll7 c!J/Jirilc~
Organisms In n l(en
lntibodies to certain pathogenic organisms have brell s110~1n l<l be
ef~tcctive in treaties!
diseases caused by those oreanisms. It has not been shown whether antibodies
ran be raised.
as:ainst C'lu.clriclituu clijJicile~. which would be effective in treatin~~
infection by this organism.
I(l ;lccordin=ly, C'. c1J//icilc~ was tested as immunogen tier pruduction of
hen antibodies.
-1-o determin a the best curse for raising high-titer r~~e antibodies against
whole ('.
cli/Jic~ilc- organisms. different immunizing strains and different immunizing
concentrations were
examined. ~I~hc example involved (a) preparation o(' the bacterial immunoLCn.
(h) immunization. (c) purification ot~ anti-bacterial chicken antibodies. and
(ell dmection of
1 ~ anti-bacterial antibodies in the purified IgY preparations.
a) I'rcpars~tion ()f I3actcrial Immuno~cn
('. cli/JirJle strains :l~sic)4 (scrol:roup .A) and ~1:~~)h (seroeroup C')
wcrmriginally
c~htaincd t~rom the A~L('('. ~l~hcse twc, strains were selected hcrausc they
represent two ol~ the
?0 most comntonlv-occurring serogroups isolated from patients with antibioUic-
~tssociatce!
pscudomrmbranou s colitis. ( l)clmec el crl.. J. Clin. Vlicrc~hiul.. ~8( I
l)):'_''_' I () ( I c)c)()). J
;~~lditionallv, both of these strains have been previcmslv charactrrired with
respect to their
virulence in the wrian hamster model tier ('. cliJJicilc~ infection. (I)elmcc
r~ crJ.. .I. Mcd
Microbial.. W:Bi ( 1990). (
~h3~e bacterial strains were separately cultured on brain heart infusion agar
for 48 hours
at 37°(' in a (.ias Pack l0() Jar (f3I31.. C'ockevsville. MDl equipped
mith a Cias Pack Plus
anaerobic envelope (13131.). Uortv-ci,~ht hour cultures were used because they
produce better
growth and the organisms have been found to be more cross-reactive with
respect to their
surface LtIIIlLCl1 prt:Selltiltt(111. ~I~I1C LrCale'.r the degrt:e o1~ cross-
rcac;tiviw ol~ our ILK'
s() preparations, the better the probability of a broad range oi~ activiy
against different
strainsJSCrogroups. (Toms e~ crl.. .I. C'lin. Microbial.. ?Gts):4?O (lc)88).(
'hhe resulting or~.:anisms were removed from the a~~ar suri~ace using a
sterile dacron-tip
swab, and were suspended in a solution containing 0.4% ti~rmaldchvde; in I'1W,
pll 7.'_'. This
_p_
CA 02296765 2000-O1-14
WO 98/08540 PCT/US97115394
concentration of formaldehyde has been reported as producing good results for
the purpose of
preparin~~ whale-organism immunoeen suspensions fur the veneration of
polyclonal anti-(',
cliJ~ic~ile antisera in rabbits. [Delmee e~l crl.. J. C'lin. Mierobiul., '? I
:323 ( 198$); Davies et crl..
Microbial Path" c):141 ( 199()).] In this manner, two separate hacteriaf
suspensions were
prepared. one Fur each strain. The two suspensions were then incubated at
4°C tier I hour.
Irliuwing this period uF tormalin-treatment, the suspensions were centrifuged
at 4.200 x g for
~'() thin.. and llte I'eSUltlltg pellets were washed twice in normal saline.
The washed pellets.
which rcmtaincd ti~rmalin-treated whole organisms. were resuspcnded in fresh
normal saline
such that the visual turbidity oi~ e:1C17 SUSpeltSlun cOCCCSpollded to a #7
NlcFarland standard.
11) (~-1.;1.C'. f~delstein. "J'rnce.s.sins,~ (~linicul ,Sj~ecimen.s./rm
Anuernhio l3crrtoricr: Ivrrlutiun crncl
Iclcrrtilicwtimn l'rric~ecltwc~.s." in S.~'l. Fin could ct crl (cds.)..
Bcrilcy uncl.Srmt'.~~ Dicr,L~nuwic
tliorr~hir~lc~,L~a. pp. 477-s07. C'.V. Mushy C'o.. ( 1990). 'hhe preparation
of McFarland
nrph elometer standards and the corresponding approxintac number of~
c~r~anistm For tacit
tube arc described in detail at pp. 17'_'-17, oFthis volume.( Each of the mu
#7 suspensions
I ~ was then ,pill into two separate volumes. ()ne volume of elicit
SIISpeIISIOIt \1'aS 1'llltlltlf:lrlCall_\'
ud.justed. by the aclditi~n ui~ saline. to correspond to the visual turhidiw
c~f' a # 1 Mcl~arland
standard. ( Icl. ( The it ) suspensions contained approximately , x I f)'
or~:anisms!mf, and the
r7 suspensions cuntainecf approximately ? x l0'' organismsiml. (lcl.( 'f~he
tour resulting
i'(lllel'.Iltl'atllllt-ild,ItICted suspensions uf~ Ibrmalin-treated ('.
cli~jicilr organisms were considered
_'() m he "bacteria) 111111tlIIlULIn SUSpc11S1t)IlS." These suspensions were
used immediately after
prep,lratiun iin the initial immttnizatiun. (.S'ec~ section (h). J
~l~lzr lilrmalin-treatment procedure did nut result in l0()°/a non-
viable bacteria in the
Illlllltlnl>'~lll ~tl5~f.'n51()I1S. In order m increase the level of killin~~.
the tlll'lttallll Cn11eC11tralll)It
and lrn~~th ol' treatment wore both increased tier subsequent immuno~en
prcnarations. as
described below in 'fable 3. (Although viability was decreased with the
stronger formalin
treatment. t ()0'ro inviabiliy of the bacterial immunogcn suspensions was not
reached. ) Alsu.
IIt SLIhSCClllelll II171t1tIItOLel1 preparations, the formaiin solutions were
prepared in normal saline
instead of I'I3S. At day 49, the day of the fifth immunization. the excess
volumes of the tine
prcviems bacterial immunogen suspensions were stored fiwzcn at -7()°('
fir use durin~_ all
s() subsequent innnunizations.
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CA 02296765 2000-O1-14
WO 98/08540 PCTlUS97115394
b) Immunization
t~or the initial immunization. I.0 ml volumes of each of the Four bacterial
immunogen
suspensions described shove were separately emulsified in I.'_' ml volumes of
('I':1 (GIE~CO).
For each of the tour emulsified in tmunogen suspensions. owe liner-month old
White Lc~~horn
hens (pre-laying) were immunized. (lt is not necessary to use pre-laying hens:
actively-laying
locus can also he utifizcd.) Each hen received a total volume of approximately
l .U mi of a
~IItLIt,' ellllllSIIIed immunogen Suspension via lour injections (mo
subcutaneous and nvc)
intramuscuiar) ol' approximately ?50 Ell per site. In this manner. a total 01'
ti,ur different
immunization combinations. using Uvo hens per combination, were initiated for
the purpose of
IO e~'aIUaIInL hotel the eflect of llttn1u111Zlltg e()ltCentratlOn t)lt eL',g
yctlh antibody (lgh')
production, and interstrain cross-reactivity of ICY raised against
Iteterolo~ous strains. 'hhc
lour immunization groups arc summarized in Tahlc s.
~rnttLE 3
Intlntlnization Grtnlns
<iruun t>cait!nationImmunizin!~ Strain :lpproximalr Immunirin~'
Do~c
~.)~t)~t. R I ( ~. CII~~IC'IIt' I .O v I11~ ()r~'i1111511).S.'I)C11
vlrMln 4:>tc)4
.. I.I) ~ fit) 11r;_it(11SI11S.I1C1
C~t7 ~j 1t)(), ~ ( ~. (II~~IC'IIC' I .1 Y I11~ ur'_imism~.IICi,
I ~
4 jS9()
Str1ln
C~t) ~ ist)(). j'7 .. ..
I .1) Y I1) ~ <)fL'i1n1511).S'.'I,Cn
I'hc time point for the first series of immuniratictns was designated .IS
"clay ecru." r111
luhSe(Itlettt IIttItltlnlZallOnS l~tr~ performed as described ahc)ve except
that the bacterial
tntmuna!_cn suspensions were emulsified using IfW ((ilf~i('()) instead of
('f~:l, and for the
later time pc,int Ilnnlunllalloll. llte SIUt'Cd froGelt SUSpe11S1tt11S N'e1'e
115ed instead of frcshU-
prepared suspensions. The immunization schedule used is listed in 'I'ahlc ~
-44-
CA 02296765 2000-O1-14
WO 98108540 PCT/US97/15394
TABLE J
Immunization Schedule
l)ay (7f ImmunizationFormalin-'freatmcnt Immunogen Prcparacion Used
I"~. I hr. Meshly-prepared
I-t I".~. ovcrnisht ,. ,.
I"~. overnight
' ;i In.~~. 4g Itrs.
I o. 7? hrs. .. ,.
stored ti~ozen
1() g; .. .. .. .,
I (>; .. .. .. ..
c) t'urifiraticrn ()f Anti-E3actcrial C.'hicken Antihodics
(iretuhs <tt~ fitur e~~LS wire collected per immunization group hewren days 8U
and R-1
I ~ post-initial immuni~atictn. and chicken immunoglohulin (1gY) was extracted
aecordins; to a
nwdilicatien ul~ the procedure ot~ /\. I'olson m «l.. Immunol. C'ctmm..
c):4c)i ( 19R()). ~1 Lentle
strum cti' distilled water hrctm a scluirt hctttle was used to separate the
yolks 1'rctm the whites.
and the ytlks werr broken by arctErltin~~ them through a i~unnri into a
~~raduatrc! calinder. The
liter individual vetlks were pooled i'nr each grattp. 'hhe Pooled. hrctken
wtlla were blended
''() w ith -t ~ ttluntcs uC eg~L cwraction hut'fcr to imltrovc antibctcH yield
(c;g~_ cxtractictn huffrr i,
I).U1 !\1 sodium phosphate. U.1 M Na(.'1. pli 7.~. containin~~ 0.()()i~yn
thimerosal). and PE:(i
R()Ut) (;\mrescct) mas uddcd tct a concentration uh .s.5'~«. when all the
I'ICi dissolved. the
protein precipitates that turtned were peppered by C~lltflt~tl~atlttll at
!s.()U() x ~~ letr IU minutes.
~I he supernatants were decanted and filtered tltrnu~;h cheesecloth to remove
the lipid layer.
and the PI:C wars added tct the supernatants to a final concentration ut'
I?"i" (the supernatants
wore assumed tct contain s.~'%" PEG). After a second eentrif~u~atinn. the
supernatants were
discarded and the pellets were centr11~11Led a final time to extrude the
remaining f'E:<.~. ~l'hese
crude 1~~~' Pellets were then dissolved in tfte original yolk volume ctf egg
extraction buf~lcr and
stored at :l°C'. .~\s an adctitional control. a preimmune lgY solution
was prepared as described
,(1 above. usin~~ tags collected from unimmunizcd hens.
_c)j_
CA 02296765 2000-O1-14
WO 98/08540 PCTIUS97l15394
d) Detection Uf Anti-Bacterial Antibodies In The I'uriticd ICY
PrcParations
!n order to evaluate the relative levels of specific anti-('. eliJ%ic~ile
activity in the IgY
preparations described above, a modified version oi~ the whale-organism t:LISA
procedure of
s N.V. I'adhvc er crl.. :I. Clin. Microbial. 29:99-iU; (1990) was used. frozen
organisms of hotlt
('. (II~jJC'lJC' strains described above were thawed and diluted to a
concentration of
:lpproximatclv 1 x I ()' organismslml using PI3S, pl i 7.?. In this way. tw'o
separate coating
suspensions were prepared, one ti>r each immunising strain. Into the wells of
c)O-well
microtiter plates (falcon. 1'ro-Bind Assay Plates) were placed 1()U Ell
volumes o!~ the coating
1 () ltlSpe11510115. lit tltlS I11a1111tC, eaelt plate well received a total
clt~ approximately I x I U"
organisms oi' one strain or the other. The plates were then incubated at
~4°C' overnight. The
new nwrnin~. the coating suspensions were decanted. and all wells were washed
three times
tlSlrtL 1'Ii~. In c~rdcr to block nun-spcciiic h11td111L? SItCS. IUU Ell
uf'().s'ri~ (3;~A (~igtna) in !'tW
was then added m each mil, and the plates were incubated lire _' hours at room
temperature.
I > ~f~ltc hlockinL solution w;ts decanted. and lUU hl volumes ot~ the I;~1'
preparations described
above were initially diluted l :s()() with a solution ot~ 0.1 "/" f3~~1 in
PIW. and tltrn serially
dilutes! in 1:; steps. The titllowins~ dilutions were placed in the wells:
I:iU(). t:'_'.~UU.
1:6?.~0()(). I :s l'_'.s()(). and 1:1.s6?.~UU. 'Che plates were a~.!ain
incubated liar '_' buttes at room
temperature. IU11I(1\Vlllg IRIS IlteUhiltl()It, the 1gY-cctntainin~, solutions
were decanted, and the
?U wells wore washed three times using RCiS-'I'ween (U.I M boric acid. ().U?i
~.1 sodium borate.
I.U M Na('I. ().l'~a Twcen-?U). lollowed by two v4~asites using! I'f3~-Twucn
(1).1°... ~I~wccn-?U).
and finally. 1 of vwlshes using I'I3S only. 'ho each well. l0U yl ot~ a 1:71)
dilution oh~ rabbit
:lltll-C:ItIl:kell Jg(i (whole-molecule)-alkaline phosphatase conjugate
(~it:nrt) Idilumd in ll.l'~,~>
(3~n in I'f3S) was added. The plates were again incubated li,r ? lunlrs at
room temperature.
The copulate solutions were decanted and the plates were washed as described
above.
substituting ~U ntM Na,C'U,, pll 9.~ tar tire P13S in the final wash. The
plates were
developed by the addition of I UU tcl ot~ a solution containilt~~ I mL~ml para-
nitrophenvl
pIltlSpltate (~I~=Ills) dissolved in sU ntM Na,C:O;. 10 m~'t 1~'lgC.'I,. pll
~).> to each well. and
incuhatin~= the plates at room telnpcraturc in the dark fire as minutes. I~hc
ahsorhance ul~ each
:,U well w:ls measured at :kIU nln 11S11tL a I)ynatech MR 70(1 plate reader.
In thrs manner. each ol~
the tinlr l~~l' preparations d escrihcd above was tcstccl t'ur reactivity
a~~ainst both of the
ilttmunizin~_ ('. ch~~lC'lIC Slt'alltS: strain-specific. as well as cross-
reactive activity was
determined.
* rE~
CA 02296765 2000-O1-14
PCTIUS97115394
'fable ~ snows the results of the whole-organism ELISA. All four 1gY
preparations
eiem~nstratcd significant levels of activity. to a dilution of 1:~3.SOU or
greater against both of
the immunizing organism strains. Therefore. antibodies raised against one
strain were highly
cross-reactive with the other strain. and vice versa. 7~he immunizing
concentration of
organisms did noU have a siyilicant effect on organism-specific IgY
production. as both
concentrations produced approximately equivalent responses. Therefore. the
lower
immunizin~~ concentration ufapproximately I.~ x I()~ organisms/hen is the
preferred
inununiiin~~ cunccntraticln of the uvo tested. The prcinununc (c~Y preparation
appeared to
p«sscvs rclativrly low levels of ('. cli/Jic~ile-reactive activity to a
dilution of 1:x()0. probably
It) due to prior caposure of the animals to environmental clostridia.
:1n initial vl~Itlll~-()t'~.a111s111 FLISA was perlormed using IgY
preparations made from
ainelc ('i) -1:s~)4. # I and ('f) 4 >~9h. a~ f cg~s collected around day ~U
(data not shown 1.
~peciiic titers were fi~und to he ~ to 10-fold lower than these reported in
Tahle ~. These
results cHnu~nstratc that it is posslhle to hC~111 lnlnlUt11Lt11f, hl'11S
prior to the time that they
I ~ heein to lay r~~~s, and t~ obtain hls:h titer specific IgY I'rotn the
first eggs ti~at are laid. In
cUhcr words. it is n m necessary m wait for the hens t« he=in laving hetore
Ihl 1n1111tII11Zatt011
achcdulc is ,farted.
-47-
CA 02296765 2000-O1-14
WO 98108540 PCTNS97I15394
TABLE 5
Results Of The Anti-C'. diJ~irilc Whole-()r~~anism EL1SA
I!_Y Preparation Dilution Of 43594-Coated Wells43i9G-Coated
IeY Prep Wells
I : 500 I .7-t 6 I .801
I :?.500 I .U~)? I .67(1
('U a isc)), ;:1 1:13.500 U.3U' U.81?
! :62.SUU 0. ! 36 0. ! 7~)
I :3 t 2,i0U U.01'_ (1.080
I : I .s63.s1)U().002 U.U2p
I :500 I .780 1.771
I :?.70U I .U35 I .U78
or) a .,c)4. ~7 I : I ~.soo o. a ss o.~x~
1:63.500 U.US? p, I;?
I :3 I 3.500 O.U33 O.Ua_,
I : I,S63.SU(I(i.U()S ().U24
I :SOU I .36 I .7~)(1
I:?.s()o a s;, I .)77
I : I z.soo a.~a7 n
~ .t;~
( .
I) a;;~)6. '~I
1:62.500 ().U>() U.?a~
1:31?.SUU U.UIU ().U67
i : I .i6?.SUO().UUU t).U:,(,
I:;UO 1.7U' I.sUi
I :?.>UO U.7U(, 0.866
' I:!?.50() 0.210 0
?b?
ll a.ii~)G. r7 .
t
I:6?.SUU U.U3~) (l.U7R
I : i I ?.SUU (LOUD l).U I 7
I : I ,j63.>OU1).UUO ().() I 11
1:500 U.14? 0. ;()~)
I :2.s00 U.O.i? 0.(177
I :13.SU0 O.UU6 U.U?a
I'rcinununc I~;Y
1:62.SOU t>.OU? U.01'_'
1:., I'_.sUO 0.()Ua 1).U I U
I : I . ~(,''.l).i)U? (>.U I 4
~(Il l
t?XAMPLE 2
'Treatment ()I' ('. cli/)icilc~ Infection With Anti-('. c!i/)irile Antihodv
In order to determine whether the immune 1~~Y antihodirs raised aeainst whe)lc
('.
eli/%icilo organisms were ca~tthlr c~l~ inhibiting the infection of~ hamsters
by ( ~. cli~~icilc~.
I ~ hamsters inlcctcd by these hacteria were utiliied. [ l.verlv cu crl..
Infect. lmmun.. ~l):'?'? 1 ~-
'? 1 H ( 1 c)c) I ). ~ ~fhis caam~le im~olved: (a1 determination oh the lethal
dose ut' C'. c!i()icilr
organisms: and (h) treatment of~ infected animals with immune antihudv or
control antihc)dv in
nutritional solution.
-4x-
CA 02296765 2000-O1-14
WO 98108540 PCT/US97/15394
:~) Determination Uf The Lethal Dose Of C. diffrcile Organisms
Determination of the lethal dose of t'. clij%icilc~ organisms was carried out
according to
the model described by D.M. Lyerlv e~ cd.. Infect. Immun.. X9;2215-3218 ( 1991
), (.'. cli~jicile
strain ;1-f(.'C' ~l;i9G (scrogroup C. ATCC) was plated on f3H1 a~~ar and grown
anacrobicallv
( RRI. (ias 1'ak 1 ()U system I at 7°C Ibr ~4? hours. Organisms were
removed trom the agar
aurtacc using a sterile dacron-tip swat and suspended in sterile 0.9°/"
MaCI solution to a
' ~Icnaity of 1 ()~ organisms/ml.
In order to determine the lethal close of ('. cIiJJirile- in tire presence o('
control antibody
and nutritional lormula. non-immutte eggs were obtained ti~ont unimmunized
hens and a 1?%
f'F(i Itrcparation made as described in Example 1(c). This preparation was
redissolved in one
limrth Lhl (rlglllal yolk volume of vanilla flavor I~nsureat,.
~tartinz on day one. groups of tcmale Golden Syrian hamsters (I~larlan Sprague
I)awlyl. ~-~) woks old and wei~~hinL approximately 1()() gm. were orally
administered I ml
ui' tltc pl'llrtllttlltt(:!I'.IlSlll't' K lormula at time zero. ? hours. (
hours. and I() hours. :lt 1 hour.
I ~ animals were orally administered 3.0 mL clindamycin !~iCl (Sigma) in I ml
of water. This
~lru~~ predisposes hamsters to ('. clijJicile infection by altering IIt~
normal tntestrnal flora.. ()n
clay mo. the animals were given 1 ml of the preimntune I~~YIEnsure~!s:
ti~rmula at time zero.
hours. (, hours. and I() hours. /1t 1 hour on day Uvo. different groups of
animals were
inoculatcct orally with saline tcontrol), or 10-'. lUa. 10''. or l0" ('
cli~)icilc~ organisms in 1 ml
'_'(> of~ saline. l~retrn days ;-1'_'. animals were Liven I ml of the
preirnmune Ig~'Il~nsurent~ titrmula
three tinms daily and observed rite the onset of diarrhea and death. L:ach
alllil7ctl was housed
in au individual ca;~r and was oflered limd and water cul lrhuum.
:~elministration cat' 10'' - 10~ organisms resulted in death in ;-.) clays
while the lower
doses ol' 10~ - Il)' or~~anisms caused death in > days. ~'ccal swabs taken
from dead animals
indicated the presence of ('. cliJ~icil~~. Given the effectiveness of the lO'
dose. this number of
ctrr~pnisms was chosen for the following experiment to sec if hvperimmune anti-
('. CJI/~irllC'
antibody could block inlcction.
h) ~I'rcatment Of lnfectcd Animals With Immune Antibody (>r
(.'.ontrol Antibody In Nutritional Formula
~l~hc experiment in (a) was repeated using three groups nl~ seven hamsters
each. Group
A received no.clindamycin or ('. cli/)icile and was the survival control.
<:iroup 13 received
clindamycin. Il)= ('. cli»irilu organisms and preimmunc l~~Y on the santc
schedule as the
)_
CA 02296765 2000-O1-14
WO 98108540 PCT/US97115394
animals in (a) above. Group C.' received clindamycin. 10- ('. cli/Jicilcr
organisms. and
hyperinttttune anti-C'. eli~Jirilc~ I~Y on the same schedule as (iroup E3.
'I~he anti-(' cli//icile IgY
was prepared as descrihed in Example l except that the I?'%~ I'hCi preparation
was dissolved
in one lirurth the original vollc voluttte of )rnsureltt;.
All animals were observed for the onset of diarrhea or other disease symptoms
and
death. C:ach animal was housed in an individual cage and was ohli;red trod and
water crcl
lihinrrrr. The results are shown in 't'ahlc 6.
TABLE 6
The E:I~tect O1~ Oral Feedin!~ ()f' Hvperimmune I~sY Antibody un ( '.
~li/Jic~ilr Infection
I Animat Group Time To Diarrhea''time 'fo
() ()early'
A tire-immune I,.Y only no diarrhea no deaths
f3 C'lindamycin. ('. ~li/%ic~ilc. ,(1 hrs .7~) hrs.
prrirnmune I~_Y
(' t'lindamycin. (' c1i/)icilc. immune~.~ I1C5. sG hrs.
I!~Y
i ? Nte:m of seven animals.
I lamsters in the cc>!ntrul ~~roup f1 slid not develop diarrhea and rwtained
healthy tlurin_=
the eaperimentai period. hamsters in groups 13 and C' clcvele~peci diarrhcal
disease. ;lnti-(.'.
cli/Jicil~~ l;~Y' did not protect the animals from diarrhea or death. all
animals succumbed in the
same time interval as thc~ animals treated with preimmune ls_Y. Thus. while
immunisation
?() with whole ur~~anistns apparently can improve sub-lethal wnnptoms with
particular hactcria
(sre IJ.S. Patent No. 5.()80.89 to li. Tukc>ro). such an approach clous ncn
prays to he
productive to protect against the Itahal el'tects of ('. cli//iculr.
CXAMPL(: 3
Production ol' ('. hml~linunr Type l~ AtttItV\IIt In urns
i(1
Itt order to determine whether antihodies could be raised against the toxin
produced by
clostridial pathogens. which would he effective in treatine clostridial
diseases, antitoxin to ('.
hrr~ulinrrm type n toxin was produced. This example involrs: la) toxltt
nloclllication: fh)
immunization: (c) antitoxin collection: td) antigenicim assessment: and (e)
assay oi~ antitoxin
titer.
->0-
CA 02296765 2000-O1-14
PCTIUS97/15394
Toxin Modification
('. hmulintrrrr type A toxoid was obtained from B. R. DasGupta. From this, the
active
ype r1 neurotoxin (M.Vf. approximately ISO kD) was purified to greater than
99% purity.
according to published mclhods. [E3.R. DasGupta & V. Sathvamoorthv. Toxicon.
?'':41
( 1984). J The neurotoxin was detoxified with formaldehyde according to
published methods.
~ 13.R. ~ingtt &: fi.R. Das(upta. ~l~oxicon. 27:40; ( 1989). ~
ti) Immunization
( ~ hwtrlhrtrm toxoid for immunization was dissolved in PBS ( 1 ms'iml) and
was
IU rmulsitietl mith an approximately equal volume of C'FI1 (CiIE3C()) for
initial itttlnunization or
I1 ~'1 liar hcmster immunization. ()n day iero. two white Ieghorn hens.
«htaincd from local
breeders. were each injected at multiple sites (intramuscular and
subcutaneous) with l Inl
inactivated toxoicl emulsilied in 1 ntl C'F:1. ~ubsequrnt booster
IIttItllllll7iltt(tltS ~lCr~ IttaCle
:1(:ehl'Cillt;' Ict the 1C11111N'InL :iCltedule tC)r dal' Ctt~ InIeCtl(tn alld
t(1x(IICI aItlOUlll: CIa1'5 1-~ :111d ~ l -
().; my: clay ! 71 - U.7~ m~~: days .i94. 401. 409 - U.?j mg. t)ne hen
received an additional
h(x~strr c~i~ U. I sU ntg on day i4-4.
c) .Antitoxin Collection
~I~mal yolk immunogiohulin (l~~y) was extracted as described in Fxantplc 1(c)
and the
~() I~'1'' hcllrt was dissol~~cd in the oriLinal yolk volume of PF3S whit
thinterctsctl.
cl) :lnti~cnicity Assevsmcnt
1:~';_s were collected t~roln day 4U9 throus!h day 4? i to assess whether the
toxoid was
sui~licicnllv immunogenir m raise antibocd. L:ggs from the two hens v<crc
pcu~lcd and
antibody was collected as described in the standard PL:G protocol.
~I~xat11p1C~ l(c).)
:~IttILI'.111CI1~' ol' the botulinai Ioxllt was assessed on Western blots. -
fhe I ~0 kI) detoxif led
type :1 ncuroto xin and unmodified, toxic. ;UU kD hotulinal wpe r1 complex
11(1x111 llscCt tbr
Intragastric route administration liar animal gut neutralization experiments:
see E-:xample (i)
were separated c>n a SDS-pe~tyacrvlamide reducing gel. The Western hlen
technique was
pertormecl according to tht: method ctf 'rowhin. [l 1. '1-owhin m ul.. I'roc.
Natl. :lead. Sci.
- l'~.~1. 70:~43iU ( 1979).f 'I~cn Et~~ samples W ('. hnlulirttun cntnplex and
toxoicl were dissolved
in SDS reducing sample buffer ( I % ~I)S. 0.~% 3-mercaptocthanol. ~0 mMt 'I-
ris. pL 1 O.R. I U'%
;:lycrvl. 0.()?5% w/v hromphem'l blue. IU% [3-ntercaptoethanol). heated at
~)5°C tier lU ntin
_ jl _
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WO 98/08540 PCT/US97115394
and separated on a I mm thick ~% SDS-polyacrylamide s:el. [ K. Weber and M.
Ushorn."Proteins uncl ,Suclium Uode~cvl .SulJute: Aloleculcw l~l~'c.~i~~Mr
DeeermincrtirnT rrn
Polvcrcr.vlumiclc (ieLwnul Relcrrccl >'rr~ccclurc~.s." in The I'rnrein.v. 3d
Edition (1-I. Neurath ~
R.L. Ilill. eds). pp. 17c)-??a, (Academic Press, NY. 1~)7j).~ fart of the yf
was cut oFf and
the proteins Were stained with Coumassic l3lu e. ~l~ite proteins in the
rcrnainder of the gel were
transferred to nitroceilulosc using the Milliblot-SDE electru-blotting system
(Millipore)
according to manuf'acturer's directions. The nitrocclluluse was temporarily
stained with I()'%
I'cmceau ~ [5.13. ('arroll and A. l.aughon. "I'I'rJCIIlL'rl(lr7 Crr9cl
I'rrl'I~rC'Urlrll? r)I I'()h'C'hJl7ClI
.~I mihuclic~.s rrr Ihc~ fvnrri,snr .S'c~~~nrcnrr ry ,Q-,ycrluclu.siclcr.se
Irtr.viurr I'rrm~in.v. " in DA;-i ( 'lur7ira,sw .~t
lU !'rcrrticorl .Ilyruuch. Vol.lll. (D. Glover. ed.), pp. 89-1 1 1. IRL.
Press. Oxford. ( lc)H7)] to
visualize th c lanes. then destained by running a gentle stream ctt~ distilled
water over the blot
for wveral minutes. ~l~hc nitrocellulose was immersed in t'13~ containing
i°/~ f3SA overnight
at -1°(' m block any rcmainin g protein binding sites.
The blot was cut into strips and each strip was incubated with the appropriate
primary
is antibody. 'l~hr avian anti-('. hrrtulinunr antibodies [described in Icl)
and prr-immune chicken
antibody (as control) were diluted (:l'_'~ in 1'135 containin!~ t mgiml 13W1
for ? hours at room
temperature. I llc hleris were washed with two chin ges each of large volumes
ol~ 1'I3S. I3tiS-
~fween and I'R5. successively ( 10 minlwash). Ooat anti-chicken IgCi alkaline
phosphatasc
cun,jugatcd secondary antibody (fisher L~iutech) was diluted l:sl)() in I'I3~
r(111talttltti 1 mLiml
'_'U 131A and incubated with the blot for ? hours at room temperature. -l~he
hlcns mere washed
with wu changes rash h~ large volumea of PISS and L313S-~I'ween. litllowed by
ane chanLe of
I'I3S anti U.1 lit Tris-! tt:'I, pli ~).~. Riots were developed in f~rcshlv
prepared all:alinc
phusphatasc substrate hut'fer t l()U It~_/ml nitrobiue tetrazolium (5irma).
51) Etgiml ~-hronu~-~l-
chloro-;-indolvl phosphate (sigma). s mM Mg(:'l, in i() mM Na,('t).. pl-1
c).s).
?~ -I'he Western blots are shown in E~igure I. 'l~he anti-(' honrlinrrm I~~Y
reacted to the
toxuid to ~~ive a broad intlnunoreactivc hand at about I~i~-1 ~U kD un the
reducing ~~cl. ~I-his
tuxoid is rd~ractive to disulfide cleavage by reducing a~ucnts due to
litrmalin crosslinking. The
immune l;~Y reacted 45'ILIt lltl,' aCtll'e t()x111 (:Untplex. a 97 kU ('
hn~trlirarrnr type A heavy chain
and a ~s kD light chain. The preimmune: Iglr' was unreactive m th a ('.
hurrrlinrrnr compirx or
;(> toxuicl in the VI%estern blot.
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c) Antitoxin Antibody Titer
The tgY antibody titer to C'. hmtrlinlrm type A to~;oid of~ e~~s harvested
between day
-I()') anti ~4? ; mas evaluated by I:LISA, prepared as tiillows. Ninety-siv-
well Falcoli pro-bind
plates were coated overnight at ~4°C with lU0 Etl/well toxoid [I3.R.
Singh & R.R. Das Ciupta.
~fmicutt _'7:-tU_; ( 198~))~ at 2.~ ttg/ml in I'IiS, pH 7.~ containing ().005%
thilnerosal. 'the
li~llowin;~ day the molls were hlockcd with PI3S containing ( % BSA for 1 hour
at 37°C'. The
I~~Y from immune or prcimmunc eggs was diluted in PBS containing I"a BSA and
0.05°/.
~1 »mn ?0 and the plates were incubated for 1 hour at i7°C'. 'I he
plates were washed three
times Svith PISS cctntalnlng 0.0>% Tween 2U and three times with PBS alone.
~111caline
lt) phosphatase-conjugated :oat-anti-chicken Ig(i (Fisher t3iotech) was
diluted 1:760 in I'I3S
containin~~ I ~~,~~ fiSA and O.Oa°/> T~wen '_'0. added to the platys.
and incubated l hour at 37°C'.
l~hc plates were washed as before, and p-nitrophenvl phosphate (Sigma) at l
mg/ml in 0.()5 M
\a,('();. pf-i c).s. If) m~-1 MgC'I, was added.
l~ltr results arc shown in higurc ~. C'Itleketl5 11111t1111t1Zed with the
te~xoid generated
I ~ hi~~lt tit rs o(~ antihW y to the itnmunogcn. Intportantlv. c~_~s from
lltltll 1111111111t1Z.ed IteltS had
ai;_nificant anti-imlnuno~mn antibody titers as compared to preimmune umtr«I
eggs. t~hc antl-
(' hrmrlirrrrnr I~~l' possessed si~~nificant activity. to a dilution ctl~
1:03.750 c>r greater.
EXAMPLE .t
'() I'rcparation ()h Avian L:g~~ Yolk Immunoglobulin In An Urally
Administrahlc form
In orcler to a(IIttIItIStl:l' avian IgY' antibodies orally !()
eXllel'1I11CIllal mice. an et~fcctivr
drlivery lormula lur the I~~1~' had to hr determined. The concern was that it'
the crude l~_1'
was dissolved in I'I3S. the saline in I'BS would dehydrate the mice. which
might prove '
Iwrmf~ul over the duration oh the study. Therefore. alternative methods ol'
oral administration
of I~~Y wore tested. The example involved: (a) isola-lion ot~ immune IgY: (h)
soluhilization
of 1~~1~' in water or I'BS. includin~~ subsequent dialysis of the IgY-E'BS
solution with water to
eliminate or reduce the salts (salt and phosphate) in the hut'f~r: and (c)
comparison ot~ the
c)uantity and activity of~ recovered IgY' by ahsorhance at ''8() nm and
1'Atif:, and enwmc-
;l) linked immtlnoassav (ELISA). .
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PCTNS97115394
a) Isolation Of Immune IgY
In order to investigate the must effective delivery formula for lgY, we used
IgY which
was raised against ('rnrcrlrr.s clrrri.vcrrc mrr-i/icrr.v venom. Three eggs
were collected from hens
immunized with the ('. chn-i.c.~ur.s rc~rnijicu.s venom and 1gY was extracted
from the yolks urine
the moctitied ('olson procedure described by Tltallev and ('arroll
~fiio/'I'echnologv_ . 8:r)3~1-9;g
( I~)9())~ as described in Example t(c).
Chc egg yolks were separated from the whites. pooled. and blended whit titer
volumes
of 1'I3~. I'owolered PECi 8()O() was added to a concentration of 3.>'%a,. The
mixture ~~~as
centrifuged at l0.()UO rpm tits 10 minutes to pellet the precipitated protein.
and the
I() wpernatant was filtered through cheesecloth to rename the lipid layer.
!'owdered PI:G 8f)()U
was added to thr supernatant to bring the: tinai I'ECi concentration to I?'%
(ussunnng a I'FG
concentration oh 3.5'ra in the supernatant). 'hhe 1?% f'fai/l~~l' mixture was
divided into two
~cpal w~lunms and centrifuged to pellet the IgY.
h) ~uluhitizatiun ()f The I~;Y In Water ()r 1'B~
()ne pellet was resuspendcd in 1!? the original yolk volumr oi~ I'I3~, anti
the utlmr
pellW was rrsuspended in I /'' the original yolk vole me of water. -I'he
pellrts were then
centrifuged to remove any particles ctr insoluble material. ('hc I~~1' in 1'1W
wlutic~n dissctl~ed
readily hut the traction rcsuspended in water remained cloudy.
-'() In order to satisfy anticipated sterility requirements fits orally
administered antihctdies.
the antibody solution needs to he filter-sterili7cd (as an altcrnativr t« brat
sterilization which
would cirstrctv the antibodies). I'hc preparation oi' IgY rcsuspended in water
mar tcto rlctudv_
m pays throu~_h either a ().? ur ()..~; ym membrane filter. set 1(1 ml e,l~
thr 1'i3~ rrsuspcnded
fraction was dialyzed overnight at room temperature against ''st) ml ctl'
water. 'I'lte tullcwvin~_
morning: the dialysis chamber was emptied and rclilled with '_s() ml of fresh
11.( ) titr a second
dialysis. 'thereafter. the virlcis oi~ soluble antibody were cletcrmined at
()I~_~~~ and arc
compared in fable 7.
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WO 9810$540 PC'T/US97115394
TABLE 7
nNns.n~om.n !1f L.V v:_i_u nr
__ __ ..~... .... ..w.cms
Fraction Absorbance OC 1:10 Dilution
At 380
nm Percent Recoven~
f'I3S cJiasolvecJ
1.149 100io
II_(> dissolved
4.746 61ro
J'E3~ dissolved:'!-I
O dialyzed
: 4.885 77/,.
R esuspcnding _thc pellets in PBS Ibllowed by dialysis against water recovered
more
antibody than directly resuspendin'~ the pellets in 'eater (77°/"
versus tit%). Lquivalent
It) volumes ol~ the l~.:lr' preparation in 1'BS or water 'were compared by
PAGE. and these results
"err in accordance with the ahsorhanec values (data not shown).
cl :~ctivity ()f I~1' Prepared With Different solvents
:1n 1:1.1:1 way perlormed tc~ compare the binding activity ut~ the 1gY
catracted by
etch prctccdure described aho'°c. ('. (hrl'r.''.S'lr.S' rerni/ictr.s
(('.cl ~. ) venom at ?.i Itgiml in I'!iS
was used t« ecru each well ui' :t ~)(-'yell microtiter plate. ~l~ltc
rcntaining protein binding sites
were hlocl:ed with I'BS containing ~ ntg/mi BSA. Primary antibody dilutions t
in 1'(3~
containing.: 1 mgitnl I3'~~~1 'mre added in duplicate. Alter ? hours of
incubation at room
temperature. the unbound primary antibodies were removed by 'vashiny the wells
with I'BS.
13I3S-T'vcen. and I'I3S. The species specific secondary antibody (goat anti-
chicken
immunoglohulin alkalim-phosphatasc conjugate (Sigma) was diluted 1:760 in ('BS
containinL
m;;!ml F3S.~ and added to each 'veil of the microtiter plate. After ? hours
ul' incubation at'
room temperature. the unbound secondary antibody 'vas removed by 'washing the
plate as
hciitrr. and t~rcshly prepared alkaline phosphatase substrate (Sigma) at 1
cg/tnl in ;0 mIV1
Na,t'(J;. IU mlvl MgC'1,. pIl c).s was added to each well. ~I~Itc color
development was
measured on a Vynatcch MR 7UU Inicroplate reader using a X12 nm filter. ~I~hc
results are
S11()1''tl 111 ~I~ablc 8.
'I'hc binding assay re.°sults parallel the recovery values in Table 7.
with 1'135-dissolved
t;~Y shmving slightly more activiy than the PBS-dissolvedll I:O dialyed
antibody. The
water-dissolvcd~ antibody had considerably less binding activity than the
other preparations.
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EXAMPLE 5
Survival Of Antibody Activity Atter Passage Through The Gastrointestinal Tract
In order to determine the feasibility of oral administration of antibody. it
was of~
interest to determine whether orally administered If:Y survived passage
through the
~_astrointestinal tract. 'rhe example involved: (a) oral administration of
specific itntnune
antibody mixed with a nutritional formula: and (h) assay ohantihodv activity
extracted from
i~cces.
TAI3L,E 8
) () Anti~scn-I3indine Activity Of' I_Y Prepared With f)ilierent lolvrmc
Dilution t'reimmune I'EiS DissolvedILO Dissolvedt~HS~I~.()
I :5(1(1 (LUUS I .748 I .577 1.7.t?
1 :_'.sU(1 (1.U(14 0.644 !)..i4~J U.G(16
I : I'_.5(l(1(I.UU I U. 14.i U.U54 tLU~)U
I :(,?.5()U U.U01 (LU25 t).UU7 U.UIh
I : : I ?,5()(1U.U I 0 U.(IUU I).IIUU U.()U?
:v) Oral Administration Of Antibody
hhe I~Y preparations used in this example arc the some 1'IW-dissolved/1-i.(>
dialv~cd
?() antivenom materials obtained in Exampic ~t above. mixed with an equal
volume et' 1:11tat1111~sr'.
l~wc, mice were used in this experiment. each roct:ivin~ a dilli:rcnt diet as
t~ellwvs:
) > water and ii~o~l as usual:
f immune IzY Preparation dialyzed against water and mixed I : I with lntamil~N
.
(.fhe nlll:e ~~t'.r~ given the corresponding mixture as their only source of
li)od and water).
~J
b) Antibody Activity After Ingestion
~ll~tcr both mice had in~csted their respective lluids. each tube vyas
refilled with
approximately IU ml of the appropriate tluid lirst think in the murnin~. 13v
mid-morning
there was about ~l to s ml of liquid Icft in each tube. At this point stool
samples were
sU collected troth each n )ouse. weighed: ~llld dlSSUIVCCI in approximately
s0() Etl I'BS per lUU mg
stool sample. Une hundred and sixty m~~ of control stools (1u~ antibody) and
c)c) m~ of .
experimental stools (specitic antibody) in 1.5 ml micrefut~c tubes were
dissolved in 8U() and
sUU Etl pEi~. respectively. ~I~hc samples were heated at 37°C for lU
minutes and vortcxed
vi~~urouslv. The experimental stools were also broken up with a narroH
spatula. Mach sample
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WO 98108540 PCT/US97115394
was centrifueed for ~ minutes in a microfuge and the supernatants. presumably
containing the
antibody extracts. were collected. The pellets were saved at 3-8°C in
case future e~ctracts
were needed. Because the supernatants were tinted. they were diluted five-leld
in I'BS
(a111talnltl~_ I mg/ml 13SA for the lllltla) d11U11011 It1 the eIIZYITIe
11111T1U170dSSa1' (FLISA). The
primary ewracts were then diluted five-tbld serially from this initial
dilution. 'l~he volume of
primary extract added to each well was 190 ~tl. The ELISA was performed
exactiv as
- ~Icscribtd in Eaatnple -I.
TABLE 9
SpecIIlC Allllt)O(tv ACtivitt Allr~r t~wcc~.rN 't'hr.,...,~. TI,., i-
.,.....~:.....-. .__~ .~.
1 Dilution 1'reimmune IgY . ...__ ...
t) Control Fecal L:XP. Fecal Extract
Extract
I : ; t) c l.o()o
U.U3..
I:_'s O.UI(> ~ 0 U.UIG
1: I'_; U 0 U.00~)
I :6_'; U
f ).OOS U.()1) I
I a .~ i ~: r) t) u.oou
~
-hllere was some active antihc~dy in the fecal extract I'!'Olll the llll)llSe
given the specific
amihmiy in (~:nfatnil )i: ti~rmula. but it was present at a very low level.
~incc the samples were
assayed at an initial I:s dilution, the binding observed could have been
higher with Icss dilute
?0 samples. ('onseducntlv. the mice were allowed to continue ingesting either
reLUlar li>od and
water ur tlm specific 1~'Y' in Enfamil k: li)rmula. as appropriate. so the
assay could be repeated.
~\noth~r l~:l.l~n plate was coated overnight with i tlg/ml of ('.cl.r. ICIlOn1
111 I'I3S.
~I~I~r I~uilowin~~ nu~rnine the I::I_lW 1 plate was hlocl:ed with ~ m~~inil
(3W \. and tl)e fecal
samplrs wrrc extracted as hetbre. except that instead of heating the extracts
at ,7°C, the
samples were kept on ice to limit proteolysis. The samples were assayed
undiluted initially.
and in sX serial dilutions thereafter. Otherwise the assay was carried out as
before.
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TABLE 10
SpCCItIC Antibody SLIrVIVCS Passa«e 7~hrnmnh Tlra r:~crrn:nro~r:....1 T...._.
.. ... ..__.
Dilution t'reimmune IgY Control Extract
i:xp. Extract
undiluted U tl
U03
. 0.379
I : i - () tl U.07I
I :? i O.OOU 0 U.U?7
I : I ?; U.UU3
U.U 17
I :6? i U.U()() tl U.U08
1:31 ? S U.OU3
U.OU_
The experiment confirmed the previous results. with the antibody activity
markedly
higher. the control fecal extract showed no anti-('.cl r. activity. even
undiluted. while the
fecal extract t~rom the: anti-('.cl. J. I~;YlFntamilcH:-fed mouse shoNmd
considcrahlc anti-('.cl r.
activity. ~fhis experiment (and the previous experiment) clearly demonstrate
that active l~sY
I~ antihocly survives passa~~c throu~~h the tnousc digestive tract. a finding
mith l;tvorahlc
1111plICal1(lllS tier IhC StICCeSS Ot~ t~~Y antibodies administered orally as
a tlzeraprutic or
prophylactic.
CXAMPLI? 6
:'-() Ire t~'ian ~ieutrali~ation ()f ~I~vpc ('. hurrrlirrrnn
I'vpc A Neurutoxin 13y nvian Antitoxin nntihodv
~t'his example demonstrated the ability oi~ E'E:(.i-purifi~cl antitoxin.
collected as
described in Fxampic i. to n eutralizc the lethal el~icct oi~ ('.
hrurrlirrrrnr neurotoxin type ~1 in
:'.> mice. ~E~o determine the oral lethal dose (I.DICItl1 al~ toxin n. groups
ol' E3.'~1..E3/e mice were
~~iven different doses ot~ toxin per unit body weight (average body weight ot~
'_'-3 ~~rams). l~or
oral administration. toxin A complex. which contains the ncurotoxin associated
with other
non-toxin proteins was used. This complex is markedly more toxic than purified
ncurotmin
when given by the oral rattle. ~ 1. ()hishi c~J ul.. lntcct. Immun., l 6:1 UO
( I c)77 j. J ( '. hrrJulirrunr
:;U toxin type f1 complex. obtained t~n~m Eric .tohnson (llniversitv ()f
Vv'isconsin. Mtadisonl was
?~U Etg/ml in :iU mM sodium citrate. pl t >.~. specific toxicity ; x I t)'
mouse L.D=,Ilmg with
parentcral administration. ilpproximately 4U-~0 ng/gm body Wight vas usually
fatal within
48 hours in mice maintained on conventional fiuul and water. When mice vvcre
'_iven a diet
ucl lihirum of only Enfamil(k) the concentration needed to produce lethality
way approximately
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WO 98/08540 PCT/US97/15394
?.~ times higher ( 125 ng/gm body weight). Botuiinal toxin concentrations of
approximatefv_
?00 n~:lgm body weight were fatal in mice fed EnfamiiC~ containing preimmune
IgY
(resuspended in L;ttfamilu~~ at the original yolk volume).
~T'he oral LDIt", of ('. hvrrrlinnm toxin was also determined in mice that
received
known a117UllIltS Ol' a mixture of preimmune IgY-Ensure~~t delivered orally
through feeding
needlrs. t )sing a ?? ~_auge feeding needle, mice were given ?SO )tl each c~f'
a preimmunc
' 1~_~'-f:nsure~n mixture (preimmune 1gY dissolved in ll4 original yolk
volutnc) I hour before
anct I.''_' hour and ~ hours after administering botulinal toxin. ~l'oxin
concentrations given
orally ranged ti-om approximately 12 to 312 ng/gm body weight (0. ~ to 7.5 ~tg
per mouse).
1() liotulinal toxin conttplex concentration of approximately 40 nglgm body
weight ( 1 Etg per
ntousc ) was lethal in all mice in less than 3( hours.
f wo ,rrULIpS (t I' BALI3/c mice. 1 () per group, were each given orallyl
stt7Lie (lose l1(~
y~_ oacl) ul' hmulina! toxin complex in I()0 Etl of ~0 tnM sodium citrate pl-1
i.;. 'fhe mice
r~cciveci ?sU )tl treaunents of a mixture of either preimmune or immune l~~Y
in f:nsurc k. ( I!4
I ~ c~ri4=foal wlk molume) 1 hour before and 1!? hour. 4 hours. and 8 hours
alter botuiinal toxin
administration. The mice received three treatments per day t<tr m'o more days.
The mice
wcrr cthscrucd tier ~)(~ hours. The survival and mortality are shown in 'fable
1 1.
TABLf. 1 I
Neutral vat inn ()f IW rulin;rl ~f'~,.~n, n ~~> n;,.,.
I uxin 1)usr nntilx~dy~ Type Number Number ()f' Mice
n~ ~~m W f' Mice Alive Dead
.1 t .h nun-immune tt ! 0
-ll.t, ;uui-hutuliml Itl
toxin
:111 mice treated with the preimmune IgY-Fnsureu mixture died within 4O huurs
post-
toxin administration. The avera~~(: time of death in the nucc w'as J? hours
post toxin
administration. '(~r(:atments of preitnmune I~;Y-Ensur(:ii3:~ mixture did heft
continue: beyond ?4
(tours due to extensive paralysis of the mouth in mice of this group. In
contrast, all ten micr
treated with the immune anti-botuiinal toxin IgY-Ensureu mixture survived past
~)(~ huurs.
Only 4 mice in this group exhibited symptoms of botulism te)xicity (two ntice
about ? days
3() after and two niice ~4 (lays after toxin administration). Tltese mice
eventually died ~ and (~
days later. Six at' the mice in this inttnunc group displayed no adverse
effects to the toxin
and remained alive; and healthy long' term. Thus. the avian anti-botulinal
toxin antibody
dctnonstrated very good Itrotcciictn froth the lethal cl'tects of the toxin in
thr experintcntal
mtcc.
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EXAMPLE 7
Production Uf An Avian Antitoxin Against ('ln.wriclirrm cliJ~icile Toxin A
Toxin A is a potent cyotoxin secreted by pathoctcnic strains of l '.
cfiJ~icile~. that plays a
direct role in damming gastrointestinal tissues. In more severe cases of ('.
clij)icile
intoaicati~n. pseudomembranous colitis can develop which may he fatal. 'This
would be
prcuented by neutralizing the effects of this toxin in the ~!astrointestinal
tract. As a first step,
antihculies were produced against a portion of the te)xltl. The e~camplc
involved: (a)
conjugation ol' a synthetic peptide of toxin A to bovine serum albumin: (h)
immunization of
Itl hs_~ns with the peptide-f3SA conjugate; and (c) detection of antitoxin
peptide antibodies by
L:I.ISn.
a) Conjugation Of A Synthetic Peptide ()f Toxin A 'fo I3ovinc
scrum Albumin
is The synth ctic peptide (.'Q'FIDGhItYYFN-Nli, (~I:(~ II) N():8?) was
prrpared
tunumrciatlv (Multiply Peptide ~ystcms. San DieLC~. ('A) and validated tc~ he -
Rtf% pure by
hieh-pressure Liquid chromatoLraphy. The eleven amino acids ti~llowivg
thewstcinc residue
rcpresmt a consensus sequence of a repeated amino acid sequence t'ound in 'I
«xin A. ( Wren
cv ul.. Infect. Immun.. s9a I S 1-3165 ( 1991 ). ~ -("he cvsteine was added to
facilitate
?() u~n_jyatiem to carrier protein.
In order to prepare the: carrier fir conjugation. 135n (sigma) was dissolved
in U.()I M
~iaP(),,. pll 7.U to a final concentration of?U I17L/ll7l and n-
maleimidohcniovl-~-
hydroxvsuccinimide ester (ME3~: 1'iercc) was dissolved in N.N-dimcthvl
ti,rn,~unidc to a
c:OIICW tfalti111 t)f i mgiml. Ml3S solution. U.SI ml. was added to s.?5 ml of
the E3SA solution
and incubated tar i() minutes at room temperature with stirring cwrv >
minutes. The MIiS-
activatcd E3~A was then purified by chromatography on a E3io-(iel P-IU column
(I3io-Rad: 4U
ml bed w,lume) equilibrated with sU mM NaPU,. pIl 7.U buffer. I'cak fractions
were pooled
(h.0 mll.
I.y~philizc:d toxin n peptide (?U mL) was added to the activated 1~5A mixture.
stirred
;t) until the peptide dissolved and incubated , hours at room temperature.
Within ?t) tninutcs.
the rcacticm mixture became cloudy and precipitates ti~rmc~l. Al'tcr ; ht~urs.
the reaction
mixture was ccntritueed at 1().U()U x g for 10 min and the supernatant
unalyicd for promin
C(111tCllt. No significant protein could he detected at 28U nm. The copu~.:atc
precipitate was
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washed three times with PBS and stored at 4°C. A second conjugation was
performed with
15 m~_ of activated BSA and 5 mg of peptide and the conjugates pooled and
suspended at a
peptide cc~nccntration ul' 10 mg/ml in 10 mM Nal'O;. pH 7.?.
b) immunization Of Hcns With Peptide Conjugate
7~wo hens were rich initially immunized on day zero by injection into tmu
- suhcutaneuus and w~u intramuscular sites with l mg of peptide; conjugate
that was emulsified
in C'F:1 (G1BC()), 'hhe hens were boosted on day 1~ and day 21 with 1 mg of
peptide
conju~,Ite cmulsificcE in IFA ((_iIBC'U).
Detection Of Antitoxin Peptide Antibodies By CLISA
E~~~' wars purified tram two e~.:gs obtained before immunization (pre-immune)
and two
y_~~s obtained 31 and _,'? days after the initial immunization using PECi
E~ractiunatiun as
drscrihrcl in E:xamplc I.
Wells of a c)h-well microtiter plate (Falcon 1'ro-Bind Assav Plate) were
coated
'wcrlti~~ht at ~i°(' with 1l)() pglml solution ul' the toxin A
synthetic peptide in I'E3S, pH 7.'_
prrparcd I,v dissulvinL I m~! of the peptide in I.U ntl ui' H,U and dilution
of 1'IW. 'fhe prc-
immunc anti immune IgY preparations were diluted in a live-fold series in a
buffer containing
I';~r I'IC~ ROOU and 0. I % ~~wecn-?0 (viv) in PE3S, pl-I 7.'_'. The wells
were blocked for '_'
?() hours at roam temperature with 1 SO Etl u1' a solution containing :i"/o
(vlv) ('arnatiomk: notttat
dry milk and 1'% PECi 8000 in PBS. pll 7.'_'. After incubation for ? hours of
rnr,r"
temperature. the wells ware washed. secondary rabbit anti-chicken l~C-alkaline
phusphatasc
I 1:7;(1) ctddcd. the wells washed tlLillll and the color development obtained
as described in
l~:xampl~ i . ~l~hc results arc shown in '~ahle 1?.
TABLE 12
Re:fcliviW ~ llf' lov lUnh T'.,..:., n,._.:.i..
_ _...... . _r......
Dilution Uf I'EG Prcp Absorbance At alU
nm
Preimmune Immune Anti-I'epcidc
t : I o0 0.o I .~ n's
I : aoo o.o(Ia cl.u ;~
y() I :~~oo 0.004 u.oo>
Clearly. the immune antibodies contain titers against this repeated epitopc of
toxin A.
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EXAMPLE 8
t'roduction Of Avian Antitoxins Aeainst
('lu.sn'iclitcm cliJJicile Native Toxins n And R
l~o determine whether avian antibodies are ef'f'ective for the neutralization
of ('.
eIiJJicilc toxins. hens were immunized using native ('. cliJJicile~ toxins A
and E3. The resulting
egg yoil: antibodies were then extracted and assessed for their ability to
neutralize toxins A
and 13 in virrn. The Example involved (a) preparation of the toxin immunogens,
(h)
immuniTation. tc) purification of the antitoxins. and (d) assay of toxin
neutralization activity.
a) I'reps>tration Of The Toxin Immuno~;enr
l3oth C'. cliJJiril~ native toxins r1 and 13. and ('. cIiJJicilr toxoids.
prepared by the:
treatment of~ the native toxins with formaldehyde. were employed as
immunogens. ( ~. diJJirilc~
toxoicls i1 and 13 were prepared by a procedure which was modified ti'om
puhtished methods
Il;hrich r~ crl.. Infect. lmmun. ''B:lU~1 (1980). separate solutions lin Pfi~)
oi'native c'
cliJJioile~ toxin ~\ and toxin !3 (Tech Lab) were each adjusted to a
concentration of U.?U mgiml.
and formaldehyde was added to a final concentration of 0.4'%. I~he
toainitornlalcdhvdc
solutions mere then incubated at ,7°C' 1i)r 40 hrs. Uree formalclchvde
was then rcmovml From
the resulting toxoid solutions by dialysis against I'1W at 4°C'. In
previously published reports.
:?() this dialysis step was not performed. Therefore, I~rce formaldehyde must
have been present in
their toxoid preparations. 'I~he toxoid solutions were conccntraW d. clslllg a
C'mtriprcp
c:onccntrator unit (Amiconl. to a final toxoid cc)ncentration ul'~.() mginll.
I~h~ uvo r~sultin~_
preparations were designated as toxoid A and toxoid 13.
l '. cliJJirilc~ native toxins were prepared by concentrating stock
stlllltl()!ls ol' twin :1 and
.''_s toxin 13 l~I~rch L.ab. lne). using ('cntriprcp concentrator units
llmicon?. to a final
CUlleelttl'atlUll ()f 4.U ntglml.
h) Immunisation
.l..llc f first Uvo immunizations were perti)rmed using the toxoid n and
toxoid 13
sU immuno;;cns described ahoy e. r1 total of i different immunisation
combinations were
clnploved. I~or the first immunization group. 0.? m1 of toxoid n was
emulsified in an cclual
volume of Titer- Max adjuvant (t'vtRW. 'biter ~9at was used in order to
conserve the amount
Ut' 1111111unUgell llSed. allCi t() ~Illlpllf~' the Inlmllnl7at1U11
prUCedtll'e. TI1I1 1It11t1t11117atIUl1 gt'Ullp
_ ()y _
*rB
CA 02296765 2000-O1-14
WO 98/08540 PCT/US97/15394
was designated "CTA." F'or the second immunization group. 0.1 ml of toxoid B
was
emulsified in an equal volume of Titer Max ad.juvant. This group was
designated "CTB."
For the third immunization group. 0.? ml of toxoid A was first mixed with 0.2
ml of toxoid
f3, and the resulting mixture was emulsified in 0.4 ml of Titer Max ad_juvant.
This croup was
clcsignatcd "C'TAI3." In this way. three separate immunogcn emulsions were
prepared, with
eaClt trtllUISIUII COnIaltlln~l, a final concentration of 2.0 mg/ml of toxoid
A (CTA) or toxoid B
' (C'TI3) or a mixture Ut~?.0 mg/ml tuxuid A and ?.0 mg/m) toxoid B (C~I~AB).
( )n da>' (). 1~'hite I.eghorn hens, obtained from a local breeder. were
immunized as
tullowv: Group CTA. l~aur hens were immunized. with each hen receiving ~OOl.tg
of toxoid
:1. via t«o intramuscular (l.M.) injections of 50y1 of C7'A emulsion in the
breast area.
Group CTI3. ()nc hen was immunized with 200ftg of toxoid B, via two l.M.
injections of
sOEtl ut~ C~~~I3 emulsion in the breast area. Group CTAB. Four hens were
immunized. with
each llcn receiving a miwurc containinu 200yg of tuxoid A and ?UUty of toxuid
I3. via nsw
!.'\1. In.lccaons «t' lOOftl of C'1~AR emulsion in the breast area. The second
inununizatiun was
t~ prturmccl i weeks later, on clay .i5. esactlv as described for the first
immunization above.
!n order to determine vyhcthcr hens previously immunized with ('. clijjirile
toxoids
could tc,lrrate whseclucnt !master immunizations using native toxins. a singly
hen i~rom croup
C'~1~;11~ vy;r.s immunized lur a third time. this time using a mixture of the
native twin A and
Ilatl\'t twin L3 described in section (a) above (these toxins were not
formaldehyde-treated. and
'-O were us~cl in their active form). This was dam in order to increase the
amount (titer) and
al~tinitv c,l' snecitic antitoxin antibody produced by the hen over that
achieved by It111171.It1IZltlg
with tmuids only. ()n day (~?. 0. I ml of a toxin mixture was hrcpared which
contained
?O()u~~ of native toxin ,~ and ?OOEt;~ of native toxin Ci. 'l~his toxin
mixture was then
umulsilicci in ().1 Inl of ~l~itcr A~av adjuyant. /v sin~te C'~('ALi hen was
Ihell InlIllUnl7ed will
'_5 the rl'Sll11111L Illlnllt110i!ell enll.Il510n. via two I.M. injections of
100y1 each. into the breast area.
~fhis hen was marked with a wine band. and observed fey adverse effects fur a
period oi~
alproxitnately I week. after which time the hen appeared to be in ~~oud
health.
I3ccause the C~I~AIi hen described above tolerated the booster immunization
with native
toxins A and (3 with no adverse; effects. it was decided to boost the
rcmainine hens with
?() native toxin as well. ()n day 70. booster immunizations were performed as
follows: (Group
CTA. :\ 0.? ml volume ol~ tire ~4 m~.:iml native toxin A Sl>Illtl()n wilS
1I11lIlSltlcd in an equal
volume of Titer Mas adjuyant. f:ach of the ~ hens was then immunized with-
'?0()Etg oi' native
toxin A. as described for the tuxoid A immunioations above. Group C_'T13. r1
s0~r1 volume
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of the ~t mg/ml native toxin B solution was emulsified in an equal volume of
Titer Max
adjuvant. ~I'lte hen was then immunized with ?OUEtg of native trnin E3. as
descrihcct for the
toxoid R immunisations above. C;soup CTAB. r1 0.1 i mI vulutne of the 4 mg/tnl
native
toxin A solution was first mixed with a U.15 ml volume the ~1 mg/ml native
toxin E3 solution.
The resulting toxin mixture was then emulsified in 0.3 ml of Titer Max
adjuvant. T'he .s
retnaining hens (tJtc hen with the wing band was nut immunized this time) were
then
intmuniied with ?UUEtg of native toxin A and 2UOEtg of native toxin ii as
described liar tile
tuxoid ~1-T tuxoid E3 immunizations (( TAf3) above. ()n day 8~, all hens
received a second
booster immunization using native toxins, done exactly as described ier the
first boost with
1 t) native toxins above.
111 hens tolerated both booster immunizations with native toxins with no
adverse
ul~fccts. :1s previous literature references describe the use of f<trmalcichvd
e-treated tuxoids.
thin is apparently the lirst time that any immunizations have been performed
using native ('.
cIiJJic~ilc~ toxins.
I
c) Purification ()f Antitoxins
E:~.:;_s were collected from the hen in group ('TR 1 ()- I ? days ti~lluwing
the second
immuniiatiun with toxoid (day .;~ immunization described in section (h)
ahuvel. and i~rom the
hms in ~~ruups C'T~1 and C'TAE3 2U-21 days following the second immunization
with toxoid.
?U 1-v horsed us a pre-immune (negative) control. eggs Were aIS() C(tIICCted
1ro11t ttlltrttItlLIItIGC(~
hens from the same Clock. E:gg yolk immunoglohulin ( IgY) was extracted t~rctm
the ~4 groups
ul~ cgzs as described in Example ) (c). and the final Ig~' pellets were
suluhilircd in the
original yolk volume u1' 1'(3~ without thimcrosal. lmnurtantlv. thimerusal was
ewlmi~~~i
because it would have been toxic to the ('f-IO cells used in the toxin
ncutraliration assays
described in section (d) below.
d) Assuy Of Toxin Neutralization Activity
l he toxin neutralization activity al' tire IgY solutions prepared in section
(c ) above was
determined usinL an assay system that was modified from published methods.
~F:hrich m crl..
;() Infect. lmmun. ?8:1U41-lUd; (lc)c)2): and Mc(iec rr «!. Microh. Path.
I_':3;i-;41 (It)t)2).l
As additional controls, affinity-purified goat anti-('. cIiJJicilr toxin n
(~l~cch I.ah) and affinity- -
puritied ~uat anti-('. cIiJJicilo toxin t3 ('Inch Lab) were also assayed tier
toxin neutralization
activity.
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The IgY solutions and goat antibodies were serially diluted using F 12 medium
(G113C0) which was supplemented with 2% FCS (GIBCO)(this solution will be
referred to as
"medium" fir the rct~ainder of this W ample). The resuitiy antibody solutions
were then
mixecf with a standardized concentration ot~ either native ('. cIiJJirile
toxin /\ (Tech L.ab), or
native ('. cliJJirile toxin B (-Ccch Labl. at the concentrations indicated
below. (=allowing
incahation at 37°(.' for 6U min.. l UUErI volumes of the toxin ~
antibody mi~aures were added
ro the wells o1' ~)f~-wall microtitcr plates (Falcon Microtest III) which
contained ?.S x IUD
('hincsu I'IanlSIl',1' Uvary (C'110) cells per wall (the C'I1(~ cells Nrre
plated on the previous da_v
m allow them to adhere to tltc plate wells). 'the final concentration of
toxin, or dilation of
I(1 antihuciy indicated below refers to the final test concentration oi'each
reagent present in the
respective microtiter plate wells. -hoxin reference wells werr prepared which
contained Cl-IO
cells and toxin A ur toxin l3 at the same concentration used for the toxin
plus antibody
mixtures (these vsclls contained no antibody). Separate control wrlls were
also prepared
which uuntaincd C'f 1() ells and mcdiutn cmlv. The tISSLI~' plates \Vt:Ce
lllerl 111Cllhaled t~(7I' I 8-
1 ~ _'-1 hrs. iv a ;7°C'. humidified. ~°~" ('()= incubator. On
the toilowinp day. the rc:mainin~
adherent lviclhlcl cells in the plate wells were stained LISI11L U.?"/.
crystal violet (Mallinckrodt)
Hissc~lwel in "~;~ ethanol. I«r 1() thin. 1~\eeSS 5111111 11'aS then renulvud
by rinsin~~ with water.
and tlm stained culls vvcre sulubilized by adding lUUyl of I",a ~p~
(dissc>Ivcd in water) tc
each wall. I~hu absorhance of~ each well was then measured at s7(? nm. and the
p~rcrnt
'U cytomvicitv ut~ each test sample or mixture va~as calculated tlslllg flee
following formula:
% CHO Cell Cytotoxieity = [ 1 - ( _Abs. Sample )~ X 100
Abs. Control
(!nliku previous reports which qaantitatc results visually by counting cell
rounding b_v
microscopy. this Lxamplu utilized spectrophotometric methods m cluantitate the
('. cliJJicile
'_'a toxin bioassay. In order to determine the toxin ~1 neutralirin~~ activity
of the ('Tn. C'TAE3.
and pre-immune I~Y preparations. as va~ell as the at~finiy-purified l:oat
antitoxin f1 control.
dilutions c~f' these antibodies were reacted against a U. l ly~,~ml
concentration of native main f1
(this is the approx. s0"/" cytatoxic ciasu ul~ toxin A in this assay svstcml.
~I~hu results arc
shown in Ui~~urc .i.
3O C'ompletc neutralization of to\Irl A occurred with the C~1~A !gY (antitoxin
A, above> at
cfilutions ol~ 1:8() and lower. while significant neutralization occurred out
to the 1:320 dilution.
-(i~-
CA 02296765 2000-O1-14
WO 988340 PCTlUS97115394
The CTAB IgY (antitoxin A + toxin B, above) demonstrated complete neutrali-
ration at the
1:320-I:IOU and lower dilutions. and significant neutralization occurred out
to the 1:128U
dilution. The commercially available affinity-purified goat antitoxin A did
nut completely
neutralize toxin A at any of the dilutions tested. but demonstrated
significant neutralization
out to a dilution of 1:1.28U. '1'hc preimmune 1gY did not show any toxin A
neutralizing
activiy at arty of the concentrations tested. These results demonstrate that
Is~~' purified'from
c~=~~s laid by hens immunized with toxin A alone. or simultaneously with toxin
A and toxin B.
is an cftectiye toxin A antitoxin.
'hhe lOxllt I3 neutralizing activity of the CTAI3 altd hre-IInmLlIte IgY
preparations, and
1() also the affinity-purified goat antitoxin B control was determined by
reacting dilutiuns of
these antibodies against a concentration of native toxin I~ of U.1 ng/ml
(approximately the
St)'%, cyutoxic dose o1~ toxin B in the aSSay SySlent). 'I'lte reS11115 art'.
SIl(tl~.'11 IIt I~ieure a.
('omhlcte neutralization of toxin 13 occurred wth Iltc C' I AI3 Ig~'
(antitoxin n ~ toxin
Ii. above) at the 1:40 and lower dilutions. and significant neutralization
occurred out to the
I s I :s'_'U dilution. 'I'hc affinity-purified yat antitoxin f3 demonstrated
complete neutralization at
Hilutiuns ul' 1:64U and lower, and significant neutralization uc;curred trot
to a dilution of
I:?.shU. The preimmune IgY did pert show arty toxin I3 neutralizing activity
at any of the
concentrations tested. These results demonstrate that I~~Y purified from c~~gs
laid by hens
itttntuni~cd simultaneously with toxin A and CUXIn f3 is an eftectiyc toxin B
antitoxin.
'() )n a separate study. the toxin B ncutraliziy actiyiy of C"I'IB. C"1':113.
and pre-itnmunc
I~_Y Itreparatictns was determined by reacting diiutiuns uf~ these antibodies
against a native
main fi concentration of ().1 ttgiml I approxirnatey I UU'% cytotuxic dose ol'
toxin l3 in this
assay system). The results are shown in 1=i~ure ~.
~i~~niticant neutralization of toxin B occurred with the C'~fI3 Ig~' (anW uxln
I3. above)
?5 at dilutiuns of 1:8U and lower. while the CTAB IgY (antitoxin A I toxin fi.
above) was bound
tct hays siyiticant neutralizing activity at dilutiuns of 1:40 and lower.
'I~hc prcimmunc IgY
did not show any toxin B neutralizing activity at any u1' the concentrations
tested. 'These
results dentunstrate that I~~Y purified front eggs laid by hens immunized with
toxin I3 alone.
or simultan c:uuslv with toxin A and toxin B. is an el~fectivc toxin f3
antitoxin.
;()
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CA 02296765 2000-O1-14
PCT/US97/15394
EXAMPLE 9
In oian Protection Uf Golden Syrian Hamsters From
C'. cliJficile Disease Rv Aviatt flntitoxins Against ('. cllJ~icilc Toxins A
And D
The must extensively used animal model to study ('. cllJficilr disease is the
hamster.
~Lyrly cn «!.. Infect. Immun. =17:;49-35? (19')2).-) Several other animal
models for
antihionic-inefuced diarrhea exist. hut nc3ne mimic the human lbrm of the
disease as closely as
the hilrllSter model. ~R. I-clcetv. ".~lnlnru! rtluclc~!.v nJ,~lnrihlunr-
lncluc'cc! ('nli~i.v." iu C). Zalc and
M. Sande (eds.). L.:rlrcrimertl«! aloclc~Ls ire .9nlintior'nh!«!
('hento~lremcrJy. Vol. ?. pp.(il-72.
1 () ( I c)XW, ~ In this model. the animals are first predisposed to the
disease by the oral
administration of an antibiotic, such as clindamvcin. which alters the
population of normallv-
c>ccurrin__ ~~astrointcstinal flora (Fekey. at 61-7?). Following the oral
challenge of these -
aninutls with viahlc ('. cllJJlc!!r organisms. the hamsters develop cecitis.
and hemorrhage,
trlccratiun. and inllammati«n are evident in the intestinal ntucosa. (I.verlv
m crl.. Infect.
Itlln1l1I1. -47:_,4')_;i'? ( Ic)gi),~ ~~hc animals become lethargic. develop
severe ciiarrhca. and a
higlz pcrccnta~c ot~ them die ti'om the disease. ~Lyerly c~r ml.. infect.
ttnmun. =t7:s4')-;j'?
I t c)Si 1. ~ This model is therefore ideally suited fir the evaluation ol'
therapeutic agents
~icsi_~ncd li>r the treatment or prophvlaxis of ('. cli~jicile disease.
The ahilii c,f' the avian ('. cliJJlrlIc~ antitoxins. described in L:xample I
shove, to
-'(~ protect hamsters ti'um ( ', cliJJic!!e disease was evaluated using the
~iofden 5vrian hamster
nuofel cat' ('. c!i/Jicllr infection. The i:xample involved (a) preparation of
the avian ('. c!lJJicile
antitoxins. (h) in river protection ol'hamsters fiom ('. cllJJic'ilr disease
hv_ treatment with avian
antitoxins, anti fc) Iun~__tcrm survival of treated hamsters.
a) I'reparution Of The Avian C. rlifjrcile Antitoxins
l:~~gs were collected from hens in groups CTA and C'TAB described in Example 1
(b)
shove. ~1 o he used as a pre-immune (negative) control. eggs u~erc: also
purchasedfrom a local
aupcrmarl:m. I:~~g yoll: immunu~~lohulin (f~:Y) was extracted ti'om the i
~~roups of r~_«s as
described in f;xample 1 (e). and the final 1gY pellets were soluhiiizcd in cme
fi~urth the
?() original woll: volume ol' Irnsure ~i nutritional formula.
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CA 02296765 2000-O1-14
WO 98108540 PCT/US97/15394
b) Irr vivo Protection Of Hamsters Against C.: difficile Disease By
Treatment With Avian Antitoxins
-hhe avian C'. c.li~ficile antitoxins prepared in section (a) above were
evaluated te,r their
ahilit t(, protect hamsters f~roln ('. cliJ~irile disease using an animal
model system which was
modified t~rom published procedures. [Fekety, at l)1-72: BOrrielio en ul.. J.
Med. Microbiol..
~-I:ss-(o ( i()R71: i~im r~ «L. Int'ect. Inu7lun.. »:29H4-?99'' ( ! ()87):
I3orriello er crl.. J. Med.
1'1icrobiOl.. ?x:1()1-1()() (lc)RR): Dclmee and Avesani. .I. Med. MicrohiOl..
3 >:RS-9U (199U);
and l..yrrly c'r crl.. lnti:ct. Immun.. i():'?? I ~-2218 ( I c)91 ). [ lvr the
Study. three soparate
experimental ~~roups were used, with each group consisting OI' 7 female
("rOlden wrian
f () hamsters ( C.'harles River), approximately 10 weeks old and wci~hine
apprOximatelv l UO ams.
oath. ~fhr ihrcc ~~roups were designated "CTA." "C'hAf3" and "I're-immuno."
'I~hese
dosignatiOns corresponded to the anutOxin preparations with which the animals
in each group
were trcatod. ~ctcal animal was housed in an individual caLC. and was
e,i~fered ti,od and water
cul lihinrm throu~:h the entire length eh~ the study. ()n day I. each animal
was orally
l ~ administered 1.0 ml e)C one Oh the three antitoxin preparations (prepared
in section (a) above)
at tho ii,llmvin~; timepoints: (t hrs., =t hrs.. and R hrs. ()n etav ?. the
day i trcatrnent was
ropeated. ( )n city 3, at the () hr, timepoint. each animal was al:ain
administorod antitoxin, as
descrihod ahow. :1t I Ilr.. each animal was orally administered .s.(> 111g
(11~ ciind;lmvcin-1ICI
(~iunlal in 1 ml Of water. ~l'his treaullent predisposed the animals t<,
infection with ('.
Ch~~l(.'lI~'. ;1S a cUllLrc)! 1(,r pUSSlble CIldUgeIlUllS ('. cliffrCll('
e()IU1117aI1U11. :lll addlllUllal a111111a1
1'1'(1111 the value Slllptllellt (UIILrCated) W'aS aISU adnllnlStered >.()
I11L (,t~ Clllldally'CI11-I'l~~l 111 tile
same manner. This clindamycin control animal was loft untreated (an(t
uninfoctod) for the
1'elllalll(ler <,1~ the study. :>t the ~I hr. anct 8 hr. tinlepuints. the
animals were administered
antitoxin as described above. ()n day ~t. at the U hr. timepOint. each animal
was a~!ain
administered antitoxin as described above. At 1 hr.. each animal was orally
challnged with 1
ml (lt~ C'. cliJ/iculc inoculum. which contained approx. 100 ('. cliJ/icile
strain ~t3j9(, organisms
in sterile saline. ('. cJi~)icilc strain ~s59(i. which is a serOgroup ('
strain. was chosen because
it is representative ot~ one ot~ the most t~requetltlv-Occurring scrOtroups
isolated from patients
with antibiotic-associated pseudomemhranous colitis. [I)cltnee c~t crl.. .I.
Clin. MicrohiOl..
,U '_'8:22 I U-?'' I ~4 ( I c)Ri). ~ In addition. this strain has been
provicuslv elemonstrated t(l he virulent
in the I1a11111e1' model oi~ infection. ~I)eimee and Auesani. .I. Mo(l.
~~licrobiol.. .;;:85-c)() '
( 1 ()c)U). ~ ;lt the 4 hr. and 8 hr. tinlepoints. the animals were
administered antitoxin as
descrihccl above. ()n day's ; throus~h t s. tile animals were administered
antitoxin 3x per day
-(~R-
CA 02296765 2000-O1-14
WO 98/08540 PCT/US97115394
as described for day 1 above. and observed for the onset of diarrhea and
death. ()n the
mornin~~ of day 14. the final results of the study were tabulated. These
results are shown in
'fable 1 s.
Representative animals from those that died in the Pre-Immune and ('TA groups
were
~ nccropsicd. Viable ('. clij~ic~ilc~ organisms were cultured tom the ceca of
these animals. and
the gr(WS patholoLy of the gastrointestinal tracts of tlleSe a171111aIS waS
CanSIStl:nt 11'Ith that
' cxpcctcct for ('. clij~icile~ disease (inflamed. distended, Ivemorrhagie
cccum, filled with watery
diarrhea-li(;e material). In addition. the clindamycin control animal remained
healthy
throu~~lwut the entire study period. thcrrtore indicating that the hamsters
used in the.studv had
It) ncn previously been colonized with endogenous ('. cliJjicilr organisms
prior to the start of the
study. I ollowin~~ the final antitoxin treatment nn day 13. a singly surviving
animal ti~om the
("C:1 _~Jwup. and also from the C'~I~.Af3 group. was sacrificed and
necropsicd. >Vcr pathology
wars nmrd in either animal.
TABLE 13
~('.~ ~ n......i...
Treatment CiruuP Nn. Animals lVo. Animals
Surviving Dcad
I're-Immune t
<'TA (Antitoxin A only ; .,
C'1'Af3 (Antitoxin n - Antitoxin7
~0 13)
l~r~atmrnt of hamsters with orally-administered toxin f~ and toxin t3
antitoxin Igroup
('~1'~1I3) aocccssfully protected 7 tort crf7 (I()0°/~) ol'thc animals
from ('. cIiJJioilc~ disease.
~I~rcatn~cnt ol~ hamsters with orally-administered toxin A antitoxin (group
U'T~11 protected i
out ol' 7 (71'a) of these animals tram ('. clijJiciie disease. Treatment usinL
pre-immune IrY
was nut protective against ('. cliJjicilr disease. as only I out ol' 7 (
14'ro) of Ihesc animals
survived. These results demonstrate that the avian toxin A antitoxin and the
avian toxin A -~
toxin E3 antitoxin effectively protected the hamsters t~otn ('. cli/jticile
cliscasc. 'fhcsc results
~tIW1 1lt'~'~~11 that allhallgh the neutralization of toxin A alune confers
some dct~ree of
protection a~~ainst ('. cli~~icilc~ disease, in order to achieve maximal
protectionsimultaneous
?() ~lntJll1~J11 ~1 and antitoxin I3 activity is lleCeSSar1'.
cl Long-Term ~un~ival ()f Treated H:~msters
It haS been previously reported in the literature that hamsters treated with
orally-
adtninistcrcd bovine antitoxin l~~(i concentrate arc protected from ('.
cliJ%icilc disease as long
-(~9-
CA 02296765 2000-O1-14
WO 9810834(1 PCTIUS97115394
as the treatment is continued. but when the treatment is stopped, the animals
develop diarrhea
and subsequently die within 72 hrs. [Lyeriy e~ ell.. Infect. Inttnun..
X9(6):?? t S_2318 ( 1991 ). J
In order to determine w~hethrr treatment of ('. cliJ)ici!e disease using avian
antitoxins
prOllll)te1 lOlll~-terln Sllt'VIVaI 1011(114'lllg the discontinuation ol~
treatment. the 4 survivittg
animals in group C.'T'A. and the (i surviving animals in group C"hAI3
w°ere observed for a
pericul of I 1 davs (?64 hrs.) foflowin~! the discontinuation of antitoxin
treatment described in
section ( h? abewc. All hamsters remained hcalthv through the entire post-
treatment period.
This result demonstrates that not onlv does treatment with avian antitoxin
protect against the
onset of ('. cli/~ic~ile disease (i.e~., it is ei~fective as a prophylactic).
it also promotes Icing-term
1 (? survival hevctnd the treatment period. and thus provides a lasting cure.
EXAMPLE 1 (1
In vinn ~l~rcatmcnt ()I' L;stahlished ('. c!i/~iri!o Inti:ctiun In (~oldcn
wrian IlanlSlefS ~1~1117 /~\'I~tll Atltlt()~tl7S AgaltlSl ('. cli//irilc~
l~uxins :\ ~\nd 13
l
hhe ahilitv ot~ the avian ('. cli/Jici!~ antitoxins, described in Example !i
above, to treat
an established ('. cli~jicilc~ infection vas evaluated using the (~c>Iden
'ivrian !talllSter 111UdeI.
The Ixamplc involved (a) preparation of the avian C'. cli~)irilc antitoxins.
(h) 111 win treatment
c~f~ hamsters with established ('. clij)icilo infection. and (cl histulotic
cvaluaticln of cecal IISSUe.
?U
I'rcpar:>Ition ()f The Avian C: rliffrcile Antitoxins
C:r~_s were collected from hens in group C'~I~AL3 described in l~:vamplc H th)
above.
which were inttnttnized with ( '. cli»irilo toxoids and native toxins ;~ and
E3. f~=gs purchased
from a local supermarket were used as a pre-immune (negative) mmtroi. 1~.~.:g
yolk
~5 inununeylohulin (IgY) was extracted from the ? groups of eggs as described
in Example 1
fc). and the final 1gY pellets were soiuhiliccd in one-fourth the original
volk volume of
I~IlSll1'~'.k' nutritional formula.
b) Ire nivo Treatment ()T Hamsters With fstablisite~ C: rliffici!c~
1 nfection
The avian ('. cliJ~icile antitmins prepared in section (al above were
evaluated for the
ahilitv to treat established ('. cli;!/ic~ile infection in hamsters uvng an
':1t11lnal nu~del system
v
CA 02296765 2000-O1-14
WO 98108540 PCTIUS97115394
which was modified from the procedure which was described for the hamster
protection study
in Example 8(b) above. ~ '
For the study, tour separate experimental groups were used, with each group
consisting
ul~ 7 female Golden Syrian hamsters (C'harles River). approx. 10 weeks old.
weighing
approximately ! 00 gms. each. Each animal was housed separately. and was
offered food and
water crcl lihinrn~ through the entire length of the study.
C)n day 1 of the study. the animals in all four groups were each predisposed
to ('.
cli/~ioilc- infection by the oral administration n1' i.0 mg of clindamvcin-I
IC"I (Sigma) in 1 ml of
water.
I() O)n day ?. each animal in all (our groups was orally challenged with 1 ml
of
('. cli//ioilo inoculum. which contained approximately Il)() ('. cIiJJicilc~
strain ~ i59(~ organisms
in sterile saline. (', cli/)ic~iie strain ~4;a96 was clZOSen because it is
representative of one of the
mast l~rc:e~uent)v-occurrin~~ scrogroups isolated from patients with
antibiotic-associated
pscudomctnbranous colitis. [Deltnee en crl.. .1. Clin. Microhiol.. '_'8:2210-
221~t (Ic)9()),J In
addition. as this was the same ('. clillic'iJo strain used in ~tll of the
Previous E~.~camplrs above. it
was a~uin uacd in order to provide experimental continuity.
()t~ R.r)~ ~ of the study (?.t hrs. post-infection), treatment was started
i~or men of the
lour groups ui~ aninrtls. Each animal of one ~!roup was orally administered I
.0 ml of the
C'TnI3 l~~l~' preparation (prepared in section (a) above) at the following
timepoints: 0 hrs.. 4
-'() hrs.. and H hrs. The animals in this group were designated "CTAB-24." The
animals in the
second group were each orally administered I.() ml of the pre-immune I~:Y
preparation (also
hrcpared in section tal above) :1t the Satlle timepoints as for the C'~InlI3
group. flicse animals
were elesignated "1'rc-?-1." 1i()I11111L \1'v1S done to the remaining tvo
~=roups ol~ animals un clay
'-? ( )n day ~I. ~l8 hrs. post-infection. the treatment described for day 3
above was repeated
for the C'T~1I3-2~t and I're-2~ groups. and was initiated for the remaining:
Uvo groups at the
same timepoints. 'f~he final two Lroups of animals were designated "("I~f~I3-
48" and "I're-~18"
respectively.
()n days ; through ~). the animals in all four groupswcrc adminiacrcd
antitoxin ur
;() pre:-immune I~Y. W per day. as described for day 4 above. l~he fimr
experimental groups are
summarized in Cable l ~4.
_71 _
CA 02296765 2000-O1-14
WO 9810$540 PGTIUS97115394
TABLE 14
Exoerimentat '1'reatmrnr csrnnnc
C_iroup DesignatiunExperimental Treatment
CTAf3-24 Infected. treatment wlantitoxin I;~Y started
rin 24 hrs. post-infect ion.
I're-24 Infected. treatment wlpre-immune l~_Y started
!ii? ~d hrs. post-infection.
C'TA13-4ti Infected, treatment wiantitoxin I~~Y stm~ted
rir~ 48 hrs. post-infection.
I're-48 infected. treatment wlpre-immune !Y started
~rr~ 4A hrs. post-infection.
.411 animals were ohscrved for the onset of diarrhea and death through the
conclusion
1 U of the study on the morniy of day 1 (). 'The results ol~ this study arc
displayed in Table 1 ~.
TABLE IS
f:xnerimental Uutcom~--t)av tn
f-rcatment Group No. Animals Survivin Nu. AnIntals Dead
C' fAf;-24 6 t
I're-?.1 p
C"TA (3-4 H 4 ;
Pre-4 H
Ei~~hty-six percent of the animals which began rcceivin~ treatment with
antitoxin 1~~'
_'() at 24 hrs. post-infection (CTAfi-24 above) survived, while s7°%
oh~ the animals treated with
antitoxin I~~Y nartin~ ~8 hrs. post-infection (CTAB-48 abovcl survival. In
contrast. none oi'
the animals receiving pre-immune 1~Y starting 24 1)rs. post-infection (l're-?-
l ahovel survived.
and only '?9°/, of the animals which bean receiving treatment with pre-
immum I~:Y at 4R
hrs. post-infection (I're-48 above) survived through the conclusion of the
study. These results
demonstrate that avian antitorins raised ct~ainst ('. efiJJicilr toxins A and
l3 arc capable of
successfully treating established (.'. c!iJ)icilo infections in vitro.
c) Histologic Lvaluation Of Cecal Tissue
In order to t~urthcr evaluate the: ability of tl)e yY preparations tested in
this study to
;() treat cstahlisftec~ ('. clijJicile infection. histologic evaluations ~erc
performed c,n cecal tissue
specimens obtained from ri~presentative animals from the study described in
section (h) ahove.
Immediately i~ollowiy death. cecal tissue specimens mrc rt:momd i~rc)m animals
which died in the 1're-?~ and I'rc-~8 groups. Following the completion ol~ the
study. a
rrprcscntativr surviving animal was sacrificed and cccal tissue specimens were
removed team
CA 02296765 2000-O1-14
WO 98108540 PCTNS97/15394
the CT'AB-24 and CTAB-48 groups. A single untreated animal from the same
shipment as
those used in the study was also sacrificed and a cecal tissue specimen was
removed as a
normal control. A11 tissue specimens were fixed overnight at 4°C in 14%
buttered formalin.
The fixed tissues were paraf'tin-embedded. sectioned. and mounted on glass
microscope slides.
The tissue sections were then stained using hematoxytin and eosin (Il and 1;
stain), and were
examined by light microscopy.
' l upon examination. the tissues obtained f'rorn the: CTAB-?4 and CTAB-48
animals
ahoweci no pathology. and were indistinguishable tr~om the normal control.
This observation
provides further evidence tbr the ability of avian antitoxins raised against
('. cliJjicile~ toxins A
1(Wand Ii to ct'fcctiyely treat established ('. clijJicilc~ infection. and to
prevent the pathologic
consequences which normally occur as a result of C', clij)icile disease.
In contrast. characteristic substantial mucosai dama~c and destruction was
observed in
the tissues c~l'the animals tram the I're-?4 and 1're-~i8 groups which died
ti~om C'. cliJJicilc~
disease. Formal tissue architecture was obliterated in these two preparations.
as most ot~ the
mucosal !aver was observed to have sloughed away. and there were numerous
larec:
hcmnrrhaLic areas containing massive numbers ot~ erythrocytes.
EXAMPLE: I 1
Cloning And Expression Uf C'. cliJJicile 'hoxin A f~ra~ments
?0
I-I~c toxin A Lene has been cloned and sequenced, and shown to encode a
protein oi'
predicted A~IUb' of ;08 kd. ~l7ove ce ul.. Infect. Immun.. ~8:48tf-4H8
(Ic)c)0).~ (liven the
expense and difficulty <H~ isolating native toxin A protein. it would he
advantageous to use
srmplc and inexpensive pre~caryotic expression systems to produce and purify
high levels of
recombinant toxin A protein fbr immunization purposes. Ideally, the isolated
recombinant
protein would he soluble in order to preserve native antipenicity. since
sofuhilizcd inclusion
body proteins often do not told into native conformations. 'I'o allow ease of
purificaticm, the
recombinant protein should he expressed to levels greater than 1 mL/liter ot~
E. cwli culture.
~l~u determine whether high levels of recombinant toxin A protein can be
produced in
l:. cull. f'raLnu~nts of the toxin A scene were cloned into various
prokaryotic expression
vectors. and assessed t«r the ability to express recombinant toxin A protein
in E. cwli. 7'hrcc
prokaryotic expression systems were utilized. 'these systems were chosen
because they drive
expression of either titsion (pMAI,c and pGI:X2.'I') or native (pL:T? ia-c)
protein to hi~!h levels
. 7; _
CA 02296765 2000-O1-14
WO 9810854U PCT/US97115394
in >'. cwli. and allow affinity purification of the expressed protein on a
iigand containing
column. Fusion proteins expressed from pGEX vectors bind glutathione agarose
beads. and
are eluted with reduced glutathione. pMAL fusion proteins bind amylase resin,
and are eluted
with maltose. A poly-histidine tae is present at either the N-terminal
(pF'!'16b) or C-terminal
(pET? 3a-c1 end of pET fusion proteins. 'This sequence specifically binds Ni,~
chelate columns,
and is eluted with imidazole salts. L:xtensive descriptions o1' these vectors
arc available
[Vl'illiams el ell. ( 19c)S) U~V9 C'lunin,~~ ?: Exprc~.ss~im7 ,S'vsremu.s.
Glover and I lames. eds. IRL
Press. (>xford, pp. I S-~8J, and will not be discussed in detail here. The
Example involved (a)
cloning of tire toxin A uene. (b) expression of large fragments of toxin A in
various
I U prokaryotic expression systems. (c) identification of smaller toxin A gene
fragmems that
express efficiently in l:. rnli. (d) purification of recombinant toxin A
protein by affinity
chromatography, and (e) demonstration of functional activity of a recombinant
fragment. of tire
toxin A ~enc.
15 a) C.'lunin~ Uf The Toxin A Gene
~\ restriction map of the toxin A gene is shown in Figurr O. 'I-he encoded
protein
contains a carhoxy terminal ligand binding region. containing multiply repeats
of a
carbohydrate hindinn domain. (yon Eicltel-Streihcr and Saucrborn. Gene ~)h:tU7-
f 13 (lc)90).]
The toxin ,A gene was cloned in three pieces, by using either the polvmerasc
chain reaction
?0 (PCR) to amplify specific regions. (regions 1 and ?. (inure 6) or by
screenins~ a constructed
~.:rnomic library for a specific toxin A nene ttanment (region s. Figure U).
~I he sequences of
the utilized l'C.'R primers are fI: i' (iCiAAA'1°f
TA(i("I'(iC'n(iC'~1~1~C"I~(inC.' ;' (~I;(~ 1I)
N().:l ): P'_': s. TCTAGCAAA'fT('CiC'TTrT Ci'I°TCiAA >' (SFQ IO
N().:?): I'3; 'i.
(.'T('(iC'A~I'A'f/1<iC'ATTAGA(.'C .;' (SL:Q ID N0.:3): and P4: ~.
2~ C'I'A'1'C'TACiCiCC'TAAACiTAT 3' (SEQ ID N0.:4). These regions were cloned
into
prokaryotic expression vectors that express either titsion (pMAl.c and
pCiEX?'1') or native
(pI:T?sa-c) protein to high levels in L'. rnli. and allow affinity
purification of the expressed
protein on a ligand containing column.
C'Il).SII'IChi1l17 CIIf,IC'll~' VPI strain 10463 was obtained Pram the
A'I'C'C' (nTC'C' It4i25i)
30 and grown under anaerobic conditions in brain-heart infusion medium ((3BL).
Hinh
molecular-weight C'. cli~ficilc DNA was isolated essentially as described by
Wren and
'I'ahaqchali ( 1987) .I. Clin. Microbial.. ?~:?40'?, except protcinase K and
sodium dodecvl
sulfate (5DS) was used to disrupt the bacteria. and cctvltrimethylammoniunt
bromide
- 74 .
*rB
CA 02296765 2000-O1-14
WO 98108540 PCT/US97/15394
precipitation [as described in Ausubel et al., Current Protncols~ in
Mvleculcrr Bivloy ( 1989)]
was used to remove carbohydrates from the cleared lysate. The integrity and
yield of genomic
DNA was assessed by comparison with a serial dilution of uncut lambda DNA
after
electrophoresis on an agarose gel.
Fragments I and 2 were cloned by PCR. utilizing a proofreading thermostable
DNA
polymerise (native /yr polymerise; Stratagene). The high fidelity of this
po)vmerase reduces
the mutation problems associated with amplifcation by error prone polymerises
(e.~~., 7ag
polymerise). PCR amplification was performed using the indicated PCR primers
(Figure O)
in 50 pl reactions containinL 10 ntM Tris-EiCI(8.3), 5U mM KCI. 1.5 mM MgCI.,,
?OU ttM
Il) each dNZ'P. U.2 tW1 each primer. and 50 ng C'. cJif~icile~ genomic DNA.
Reactions were
cwcrlaid with 100 yl mineral oil. heated to 94°C for 4 min. 0.5 yl
native p/rr paiymerase
(~tratageneJ added. and the reaction cycled 30x at 94°C for I min.
50°C.' for 1 min. 72°(_' for
-t min. followed by 1 () rnin at 7?°C'. Duplicate reactions were
pooled. chloroform extracted.
and ethanol precipitated. After washing in 70% ethanol. the pellets were
resuspended in 50 yl
15 TI: butler [ 1 U ntM 'hris-HCL. 1 mM EDTA pH B.OJ. Aliquots of l Opl each
were restriction
cli~~estcd with either ~cuRllllitrcli (fragment 1) or EcvRIll'.stl (fragment
?). and the
appropriate restriction fragments were gel purified using the Prep-A-Gene kit
(BioR adl, and
ligated to either l:ruRlLSnrcrl-restricted p(iEX2T (Pharmacia) vector
(fragment 1 >. or the
L:cwRlll'.~~tl PMAIc (New England I3iolabs) vector (fragment ?). E3oth clones
are predicted to
?() produce in-Irime fusions with either the glutathione-S-transterase protein
(pGFX vector) or
the maltose binding protein (pMAL, vector). Recombinant clones were isolated.
and
ce~nfirmed by restriction digestion. using standard recombinant molecular
biuloy techniques.
~5ambrool: ur crl.. :llulccurltrr ('lorrin,~~. ,~1 l.ubrrrmur.n ;hlcrnucrl (
1989). and designated pGA3U-
Oh0 and pMAWoO-11()0. respectively (.vee Uigure O for description o!'thc clone
designations).[
Fragment 3 was cloned Irom a genomic library of size selected P.stl digested
('. clifficilc- Lcnomic DNA, using standard molecular biology techniques
(Sambrook et ul.).
(liven that the fragment 3 internal I'.st( site is protected from cleavage in
C'. cliJjicile genomic
DNA [Price rr crl.. C.'urr. Microbiol.. 16:5-C~0 (1987)], a 4.7 kb t~agrnent
from P.stl restricted
('. cllJftC'ilC' gcnomic DNA was Lel purified. and ligated to l'.stl
restricted, phosphatase treated
3(J p()Cr) UNA. The resulting genomic library was screened with a
uligonuclcotidc primer
- specific to fragment 3, and multiple independent clones w ere isolated. The
presence Of
ti-agment 3 in several of these clones was confirmed by restriction digestion.
and a clone ol'
- the indicated orientation (Figure 6) was restricted with
I3crrnl~llNirrclIII. the released fraement
-75-
CA 02296765 2000-O1-14
PCTIUS97/15394
purified by gel electrophoresis. and ligated into similarly restricted pE:T?3c
expression vector
DNA (Novagen). Recombinant clones were isolated, and confirmed by restriction
digestion.
This construct is predicted to create both a predicted in frame fusion with
the pE'T protein
leader sequence. as well as a predicted C.'-terminal poly-histidinc affinity
tag. and is designated
pI'A1 i00-?b8U (see E~igure 6 fbr the clone designation).
h) Expression t)f Large Fragments ()f Toxin A In C call
Protein expression i~rom the three expression constructs made in (a) was
induced. and
analyzed by Western blot analysis with an affinity purified. goat polyclona)
antiserum directed
against the toxin A toxoid ('Tech L,ah). The procedures utilized for protein
induction. SDS-
I'AGL;. and V1~'cstern blot analysis arc described in detail in 1~'illiatns
em! (Ic)c)5). .sr,l~,-cr. In
brief. i ml ?X Y'1' (16 L tryptonc. )0 g yeast extract, s ~. NaCI per liter,
pII 7.; +- 1()0 ttgjml
ampicillin were added to cultures of bacteria (E3L?1 fbr pMAI and p(iE:X
plasmids, and
f3L?1(DE~:s)I.vs~ tier pL:'T plasmids) containing the appropriate recombinant
clone which were
I ~ induced t« express recombinant protein by addition of IPT(i to 1 mM.
C'ultures were grown
at s7°C'. and induced when the cell density reached 0.5 017,,,~~~.
Induced protein ~vas allowed
to accumulate fbr two hrs alter induction. Protein samples were prepared by
pclleting 1 ml
aliduots of bacteria by crntritttgation ( I min in a microfuge). and
resuspcnsion oh' the pcllcted
bacteria in 150 Etl ol' '?x SDS-PAGE sample buffi:r [ Williams cn crl. (
lc)c)5)_ .crr~mu[. The
?1) samples were heated to 95°C fur ~ mitt. the cooled and ~ or 10 ftl
aliquots loaded on 7.5%
SDS-I'A(~1:; ~:cls. I3ioRad high molecular weight protein markers were also
loaded. to allow
l'.Slllllatt()Il ()f the MW of identified fusion proteins. After
electrophoresis. protein was
dciected either ~~enerally by staining gels with C'oomassic bloc. or
specifically. by hlottine to
nitrocellulose liar Western blot detection of specific immunoreactive protein.
N'cstern blots.
(performed as described in E:xamplc s) which detect toxin A reactive protein
in cell lvsates of
induced protein I~om the three expression constructs are shown in Higure 7. fn
this figure.
lanes 1- ; contain ccl l lysates prepared from !:, cwli strains containing
pl'A 1 100-28(U in
f31?I(I)Ea)lwf~; cells: lanes ~l-6 contain cell lysatcs prepared from !:. cwli
strains containins~
pI'A 1 I ()()-?8(U in I31? 1 (DL:3)IvsS cells: lanes 7-~) contain cell Ivsates
prepareel from I:~. cv~li
.;0 strains cuntainin~~ pMA30-66U: lanes 10-12 contain cell lysates prcparecl
ii-om L:. cwli strains
containin~~ pMA660-1 1()0. The lanes were probed with an af'finitv puriiieei
~~oat antitoxin A
polycional antih~~dy ('Tech I,ab). C'untrol iysates from uninduced cells
(lanes I. 7. and 10)
contain very little immunorcactive material compared to the induced samples in
the remaining
-76-
CA 02296765 2000-O1-14
WO ~/~~ PGT/US97115394
lanes. The highest molecular weight band observed for each clone is consistent
with the
predicted size of the full length fusion protein.
Each construct directs expression of high molecular weight (HMW} protein that
is
reactive with the toxin A antibody. The size of the largest immunoreactive
bands from each
sample is consistent with predictions of the estimated MW of the intact fusion
proteins. This
demonstrates that the three fusions arc in-tTame. and that none of the ClotleS-
c017ta1t1 CIUI11I1g
' artifacts that disrupt the integrity of the encoded fusion protein. I
lowever, the Western blot
demonstrates that fusion protein ti~om the two larger constructs (pC;A3U-b64
and pPAI 100-
?CiRO) arc highly dc~traded. Also, expression levels of toxin A proteins from
these two
1 () constructs are low, since induced protein bands are not visible by
C'oomassic staining (not
shown ). ~;evcral other expression constructs that fuse large sub-regions of
the toxin A gene to
either pI~IAI_c or pE'f?3a-c expression vcetors, were constructed and tested
for protein
induction. These constructs were made by mixing gel purified restriction
fragments. derived
i~rom the expression constructs shown in Figure b. witf~ appropriately cleaved
expression
vectors, li~.:ating. and selecting recombinant clones in which the toxin A
restriction t'ragments
lord li~~ated tcr~e;ther and into the expression vector as predicted for in-
t'ramr fllslonS. The
expressed toxin A interval lvltl1111 Il7t'St CnnStrUCIS arC Show'll In Figure
8. as well as the
internal restriction sites utilized to make these constructs.
As used herein. the term "interval" refers to any portion (i.c'.. any se~:ment
of the toxin
'-() which is less than the whole toxin molecule) of a clostridiai toxin. In a
preferred
tlllhl)ctIr7lCIlI. "interval" Peters to portic)ns of C'. cli/j'icilo toxins
such as toxin ~1 ur toxin E~. It
is alsc) contemplated that these intervals will correspond to epitopes oi'
II111111111<)IU_L1C
llllp()t'(aIICC. SLIClI aS alltILCIIS (1r t1n111U110~e11S a~alt7Si wltlCl7 a
ncutrallilng allllhOdV CLSpOtISe lS
H'fcctcd. It is not intended that the present invention be limited to the
particular intervals or
seducnces described in these Irxamples. It is also contemplated that sub-
portions of intervals
(c'.,L~.. an epitope contained within one interval or which bridges multiple
intervals) be used as
compositions and in the methods of the present invention.
In all cases. Vl~'cstern blot analysis of each of these constructs with goat
antitoxin A
antihodv (Tech Lab) detected FIMW tilsion protein of the predicted size (not
shown). 'hhis
confirms that the reading frame of each of these clones is not prematurely
terminated. and is
. tilscd in the correct frame wlth the titsion partner. I-lowever. the Western
blot analysis
resealed that in all cases, the induced protein is highly deguded, and. as
assessed by the
absence of identitiahle induced protein hands by Coomassic Blue staining, are
expressed only
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at low levels. These results suggest that expression of high levels of intact
toxin A
recombinant protein is not possible when lame regions of the toxin A gene are
expressed in
F.. cvli using these expression vectors.
c) High Level Expression Uf Small Toxin A Protein Fusions In
E. call
Experience indicates that expression difficulties are olten encountered when
large
(greater than 100 kcl) fragments are expressed in l:. cwli. ,~1 number of
expression constructs
contninin~~ smaller hagments of the toxin A gene were constructed, to
determine if small
regions of~ the gene can be expressed to high levels without extensive protein
degradation. A
summary of~ these expression constructs arc shown in figure 9. All were
constructed by in-
framc flrS1l7r1S Of' convenient toxin A restriction fragments to either the
pMAI,c or plrT?3a-c
vectors. Protein preparations frorn induced cultures of each oi' these
constructs were analyzed
by hoah C'oomassie Blue staining and Western analysis as in (hl above, In all
cases. higher
1 ~ levels of intact. full length fusion proteins were observed than with the
larger recombinants
from section (h).
dl Purification Uf Recombinant Toxin A Protein
Lame scale (SUO ml) cultures of each recombinant from (c) were grown. induced.
and
?U soluble and insoluble protein fractions were isolated. The soluble protein
extracts were
affinity chromatographed to isolate recombinant fusion protein. us described (
Williams c-r crl.
( 19c)4). .~rrpru]. In brief. extracts containing tagged pf:'h fusicms were
ci~rumatcy_raphed un a
nickel chclate column, and eluted using imidazolc salts as described by the
distributor
(NovaLen). L:xtracts containing soluble pMAI_ fusion protein were prepared and
chromatographed I11 CUlllllln buffer ( lU mM NaPO" U.SM NaCI. lU 111M (i-
mercaptoethanol.
pt-1 7.?) over an amylase resin column (New England Biolahs). and eluted with
column
buffer containing IU mM maltose as described [Witliams r~ crl. ( 1995).
.vupr~cr]. Vlrhen the
expressed protein was found to he predominantly insoluble. insoluble protein
c:xtracta were
prepared by the method described in U.xamplc 17, in~i~cr. The results arc
summarised in 'hahle
3U IO. I~iLUre 10 shows the sample purifications of recombinant toxin A
protein. )n this figure.
lanes I and ? contain MRI' l~usiun protein purified by affinity purification
o1~ soluble protein.
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CA 02296765 2000-O1-14
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TABLE 16
Puriticatinn of t~nrnrnhinn.W'.,..:., w n-_._:-_
,"-
Clone '~" Protein Yield Affinity/ Intact Soluble
. purified Soluble Yield Intact
Solubility ~,, Fusion Protein''Insoluble
Protein Fusion
Protein
pMA30-270 Soluble 4 mg1500 mls t0% NA
1'MA30-300 Soluble 4 mg/500 mIs S-10% NA
pMA300-660 Insoluble NA 10 me/500
ml
pMAO60-I 100 Soluble 4.i ntg/500 SO.~ NA
mls
pMA 1 100- Soluble I 8 mg/S00 10% NA
I G 10 mls
pMA 1610-I fioth 22 mg1500 90% 2U mgUO ml
R70 mls
I() pMAl4>0-IR70 insoluble ..-._ NA 0.2 m~;~50U
ml
pl' Soluble A 90% NA
I I(10-14>0 (l.l mg1500
mis
pPA I I (l0- Soluble 0.03 meI500 90~ h A
I 870 mls
pMA I R7(l-2(iR(L
(loth 12 mg!500 80% NA
mls
rt'aIR70-368()Insoluble __-.. NA 10 mgi500
I ml
''" pP = pf-_T?3 vector. pM-pMAl.c vector, A=toxin A.
"" R,rsed on I.i OD,", - I m~~ho! (extinction coefficient of MIiP).
''' Iatimatcd tit' Cuumassic stainin_; of SDS-PAGE xcls.
Lanes s and 4 contain M$N fusion protein purified by solubilization of
insoluble inclusion
'-() bodies. The puriticd fusion protein samples are pMA I 870-2h80 (lane 1 ).
pMA6G0- I I ()() (lane
'). pMA 30U-G00 (lane s) and pMAI450-1870 (lane 4).
Poor yields of affinity purified protein were obtained when poly-histidinc
ta~~gcd plT
vectors ~~et'e used to drive expression (pPAI 100-140, pI t 10()-1870).
However, signiticant
protein yields wore obtained from pMAI, expression constructs spanning the
entire toxin A
gene. and yields of full-Icn gth soluble fusion protein ranged from an
estimated 200-400
Etg/500 ml culture (pMA30-300) to greater than 20 mg/500 ml culture (pMA16i0-
1870).
only one interval was expressed to high levels as strictly insoluble protein
(pMA300-GGO).
-I~hus, although high level expression was not observed when using large
expression constructs
ti-om the toxin A gene. usable levels of recombinant protein spanning the
entire toxin A gene
were obtainable by isolating induced protein from a series of smaller PMAI.
expression
constructs that span the entire toxin A tene. This is the first dr:monstration
of the t~asLbLI(t_V
- of exprNSSin~~ recombinant toxin A protein to high levels in !~. cwli.
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c) Ncmagglutination Assay Using The Toxin A Recombinant
Proteins
'('he carboxy terminal end C(111S15t1t1~ of the repeatin~~ units contains the
hema~;~lutinatiol, activity or binding domain of C'. cliJ~icilc- toxin n. 'fo
determine whether
the expressed toxin n recombinants retain functional activity.
hema~glutination assays were
Icrtitrmeei. Two toxin A recombinant proteins. one contuinin~ the binding
domain as either
,uluhle at~tinitv puritied protein (pMA 1870-268()) or SDS solubilized
inclusion body protein
(pI'A187f1-2680) and soluble protein frc,m one region outside tltat domain
(pMAI 100-1010)
\Yere tt.'Sled USI11'~ a described procedure. (FLC.'. hrivan el. crl.. Inlcct.
lmmun.. x;:573
( 1986). C'itrated rabbit red blood cells (RIZI3C.')(Cocalicol were washed
several times with
~fris-hol'i'er ( 0.1 M Tris attd s0 mM NaCI ) by centrifugation at 4~0 x g
)i~r 10 minutes at 4°
C'. n 1 '!a RRI3(' suspension was made ti~ottt the paclccd cells and
resuspended in ~Cris-hut'fer.
l)ilutiow oh~ the recombinant proteins and native toxin ~1 ('l~ech l.ahs) were
ntadc in the ~l~ris-
huf~tcr and added in duplicate to a round-bottomed ~O-well microtiter plate in
a tinal volume
1 s h~ 1 ()() X11. ~l~o each well. >0 pl of the 1 % RR13C' suspcnsie~n was
added. mixed by gentle
tappin~~. and incubated at 4°C' tits i-~ Jt(,IICS. SILnttleanl
h~ma~~lutinatirm occurred curly in
the recctmhinant proteins containinL the binding domain IpMA 1870-?(,80) and
native toxin
:1. The recombinant protein outside the binding domain (pMA I 1(>0-161())
displayed no
hrma~'~lutination activity. ( )sing eeluivalent protein concentrations. the
hemae~iutination titer
2(l t'or main .~1 was 1:''iG. while titers tier tits soluble and insoluble
r~ceunhinant proteins ut~ the
bindinL domain were 1:?~(i and about 1:1000. C.'Icarly. the reromhinant
proteins tested
retain ed functional activity and were able to bind RRI3C's.
EXAMPLE 12
Ivnctional Activity C)T IgY Reactive Against 'Toxin n Recombinants
~i~hc expression eH~ recombinant toxin A protein as multiple i~ra~:ments in
l:.cwli has
c(cn tonstratcd the feasibility oC generating toxin A antigen throu!_h use of~
recombinant
methodologies ( Example I 1 ). The isolation oi~ these recombinant proteins
allows the
s0 immunoreactiyity ol~ each individual subregion of the toxin A prcaeilt to
he determined ( r. r..
in a antibody pool directed aLainst the native toxin r1 protein). This
identifies the re~ictns (iT
any) tier which little' or no antibody response is elicited when the vyhole
protein is used as a
inunmuycn. ~lntibodi~s directed against specific lra~ments cri' the train A
protein can be
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CA 02296765 2000-O1-14
WO 9t~/08540 PCT/US97/15394
purified by affinity chromatography against recombinant toxin A protein. and
tested for
neutralization ability. This identifies any toxin A subregions that are
essential tier producinc
neutralizing antibodies. Comparison with the levels of immune response
directed against
these intervals when native toxin is used as an itnmunogen predicts whether
potentially higher
titers of ncutraliziy antibodies can be produced by using recombinant protein
directed against
a individual region. rather than the entire protein. hinally, since it is
unknown whether
untihodies reactive to the recombinant toxin A proteins produced in Example l
1 neutralize
toxin ~1 as effectively as antibodies raised against native toxin A ()examples
~) and l(J). the
protcctivc ability ol~ a pool of antibodies affinity purified against
recombinant toxin f\
1 tJ t~ragments was assessed for its ability to neutralize toxin A.
this (:xamplr involved (a) epitopc mapping of the toxin i1 protein to
determine the
titre uf~ specific antibodies directed against individual subrcgions of the
toxin A protein when
native tewin :\ prcncin is used as an immunogen. (h) at'tiniy purification of
IgY reactive
.y~ainst recombinant proncins spanning the toxin A gene. (c) toxin A
neutralization assays with
1> ul'tinity purified I~~Y reactive m recombinant toxin A protein to idcntif:v
suhrcgions of the
main .t protein that induce the production of neutralizing antibodies. and
determination of
mhcthcn rcnnplete neutralization of toxin A can be elicited with a mixture oi~
antibodies
rcactrvc to rcconthinant toxin a1 protein.
~~) E:pitopc M~ppinl; Uf The Toxin A Gene
i'hc affinity purification ol~ recombinant toxin A protein specific m defined
intervals ol'
tlm W vin :1 protein allows cpitope mapping o1' antibody pool, directed
against native toxin A.
This has nc,t previously been possible. since previous expression ol' twin A
recombinants i~as
h een assessed only by UVestern blot analysis, without knowlccigc of the
expression levels c,f'
~> the rotcin c.~ _
p [ ~,t,.. won l:ichcl-~treihcr m crl. J. Cicn. Microbial.. 135:jj_~,4 (
Ic)g~))). 'Thus. hi«h
c,r low reactivity of recombinant toxin A protein on Western blots may reflect
protein
mpression level dit7ercnccs. not immunorcactivim differences, (liven that the
purified
rccomhinunt protein generated in L:xamplc 1 1 have bccll duantrtatcd, the
issue oi' relative
immunorractivitv of individual regions of the toxin n protein was precisely
addressed.
.,lJ hoe the purposes of this f:xamplc. the toxin A protein was subdivided
into h intervals
( 1-6), numbered liom the amino (interval 1) to the carboxyl (interval 6)
termini.
The recombinant proteins corresponding to tlzese intervals were from
expression clones
- (see I:xatnple I 1(d) for clone designations) pMA30-300 (irtterval 1 ).
pMAS(JO-GCO (interval
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WO 98/08540 PCTNS97115394
~). pMA6G0-1100 (interval 3), pPA1100-1450 (interval 4), pMA1450-1870
(interval S) and
pMA 1870-2680 (interval (~). These C clones were selected because they span
the entire
protein fi~om amino acids numbered 30 through 2680, and subdivide the protein
into 6 small
intervals. Also, the carbohydrate binding repeat interval is contained
spcciiicallv in one
interval (interval G). alluwin~ evaluation of'the immune response specifically
directed against
this rc:~_ion. Vdestcrn blots o1' 7.5"i° SDS-PAGE gels. loaded and
electrophorcsed lNlth defined
cluantities ut' each recombinant protein. were probed with either goat
antitoxin A poiyclonal
antibody ('I'rch t.ah) or chicken antitoxin A polyclonai antibody [pC."I~~1
ICY. l~:xamplr 8(c)].
The hl«ts were )trcparcd and developed with alkaline phosphatase as previously
described
[ Wiliiams r~ «l. ( I c)95). .strpr«[. r1t Least c)0% of all reactivity. in
either goat or chicken
antibody pools, was teund to he ciirecicd against the ligand hindin~ domain (
interval 6), The
rrmainin~_ imntunoreactivitv was directed against all five remaining
intervals. and was similar
in both antihoctv cools. except that the chicken antibody showed a much lower
reactivity
ct!sainst interval ? than the goat antibody.
1 ? This clearly demonstrates that when native toxin A is used aS an
IlttntllnUil'tl IIt LUiIIS
c>r chickens. the bull: of~ the irnmunc response is directed a~ainU the
li~ancf hinetin~ clnmain of
the promin. with the re:nlainin~ response distrihtlted throughout the
romainirn~ '!:> elf' the
hrotcln.
'-(> h) Affinity Purification ()f IgY Reactive Against Recombinant
'Toxin A Protein
:1t'tinitv columns. containing recombinant toxin A protein from the O defined
intervals
in (a) chow. mere made and used to (if at'finity puritw antihcuiies reactive
to each individual
interval t'rom the C'~l~A I'~Y preparation [l:xamplc 8(c)[. and (ii) deplete
interval specific
untibadics from the C"hA t~~Y preparation. Aftiniy columns were made by
coupling I ml 01'
I'f3S-washed Actigel resin (Sterogcne) with region specilic protein and 1/1()
final volume of
Ald-cuuplin~ solution (1M SlldIUll1 C\'altOhOCOhydrICfl',1. ~1'he total
t'citolt spccllie protein
added to each reaction mixture was 2.7 nt~ (interval I). ? IttL (intervals ?
and ). 0.1 m~
(interval 4). 0.? mg (interval i) and 4 mg (interval G). I'rotcin tier
intervals 1. .i. and b was
30 ut~tiniw purified pMAI litsion lrotrin in column but~fcr (see L.xatnplc I I
). lntcrval 4 was
at'iinitv purified poly-histidine containinL pCT titsion in I'R~: intervals ?
and ; were loom
inclusion body preparations of insoluble pMAI, fusion protein. ciiatv~cd
extcwivcly in P13S.
Alicluots eh' the supernatants from the coupling reactions. bclurc and alter
coupling. were
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CA 02296765 2000-O1-14
PCT/US97115394
assessed by Coomassie staining of 7.5% SDS-PAGE gels. based on protein band
intensities.
in all cases greater than SU% coupling efficiencies were estimated. The resins
were poured
into ~ ml E3ioRad columns. washed extensively with PF3S, and stored at
4°C.
.~IIqLIOIS Uf the C'T~1 IgY polvclonai antibody preparation were depleted for
each
individual region as described below. A ?U ml sample uf~ the CTA IgY
preparation [Example
Hlc)~ was dialyzed extensively against 3 changes of !'BS ( ! liter for each
dialysis). quantitated
' by .Ihsc~rbance at OD_~", and stored at =t°C. Six i ml aliquots of
the dialyzed'IgY preparation
~~crc removed. and depleted individually for each of the six intervals. h;ach
1 ml aliquot was
passed aver the appropriate aft3niy column, and the eiuatc twice reapplied to
the column.
I U ~I~hc eluatc was collected. and pooled with a 1 ml PBS vyash. Bound
antibody was eluted
i'rnm thr column by washin~~ with ~ column volumes of ~ M Ciuanidinc-EICI (in
10 mA~! ~I~ris-
I IC'l. pl l R.()). The column was reequilibrated in PRS, and the depleted
antibody stock
reapplied as dcscrihrd above. The eluate was collected. pooled with a 1 ml
PfiS wash.
cluantitated by ahsorhance at ()1)"",. and stored at 4° <'. !n this
manner. 6 aliquots of the CTA
) ~ I~~Y preparation vycrc individually depleted for each of the ( to~:in A
intervals. by two rounds
ol' al'iiniy depletion. The spcciticiw of each depleted stock was tested by
1~'estern blot
analysis. ~.1ultiplc 7.5"% SDS-PAGE gels were loaded vyith protein samples
corresponding to
all h train ~1 suhre~;ions. .~ftcr clcctrophorcsis, the gels were plotted. and
protein transfer
confirmed by I'onceau ~ staining [protocols described in llvilliams er «l.
(Ic)c)5). ,~~i,hr«[. After
?l) E~locking the blots 1 hr at ?U°C in I'E3S+ ().l% 7~ween 20
(1'f3S'h) containinL ~'% milk (as a
h)ewkin~_ hut'tcr). ~4 mi ol' either a I!SUO dilution of tl3e dialyzed CTA !gY
preparation in
hlcxkin,~ Uut'tcr. or an equivalent amount of the six depleted antibody stocks
(uslnL OI)=~,~ to
standardlzc antibody concentration) vyere added and the blots incubated a
further 1 hr at room
temperature. The plots vsure vyashed and developed with alkaline phosphatasc
(using a rabbit
'' anti-chicken alkaline phosphate conjugate as a secondary antibody) as
previously described
[ Williams c~W 1. ( 19c)5), .v«pr«J. In all cases, only tire target interval
was depleted for antibody
reactivity. and at Icast 9()% of the reactivity to the tarLet intervals was
specifically depleted.
Region specific antibody pools wrre isolated by affinity chromatography as
described
h clovy. l~cn mls ui' the dialyzed ('TA tgY preparation were applied
sequentially to each
3U aftiniy column. such that a single lU ml aliquot was used to isolate recion
specific antibodies
specific to each of the six suhrcgions. 'I-he columns Here sequentially washed
with 1()
volumes ol' PL3S. 6 volumes of I3IiS-Tween, 10 vollllncs of T(3S. and elated
vyith ~ ml
nctiscp elution media (Sterogenc). The eluate was dialpcd extensively against
several
_g;_
*rB
CA 02296765 2000-O1-14
PCT/US9~/15394
changes of PBS. and the affinity purified antibody collected and stored at
4°C. The volumes
of the eluate increased to greater than 10 mls during dialysis in each case.
due to the high
viscosity of the ~lctisep elution media. Aliquots of each sample were 20x
concentrated usinlr
(.'cntricon 30 microconcentrators (Amieon> and stored at 4°('. 'fhe
specificity of each region'
specific antibody pool was tested. relative to the dialyzed C'~I~A IgY
preparation. by Western
blot analvsls. exactly as described above. except that ~ ml samples of
blocking buf~ter
ce~ntainin~~ It)U pl region specific antibody (unconcentrated) were; used
instead of the depleted
C'~l~A I~_~' preparations. Each affinity purified antibody preparation was
specific to the defined
interval. crept that samples purified against intervals 1-i also reacted with
interval G. This
may he ctue to nun-specific binding to the interval 6 protein, since this
protein contains the
repetitive lirand binding domain which has been sho~s~n to bind antibodies
nonspecifically.
) Lvcrlv c~~ ul.. C.'urr. Microbiol.. 19:303-306 ( 1989). )
I~h~ reactivity c~f each affinity purified antibody preparation tn the
correspondin_;
proteins was appro~cimatrly the same as the reactivity of the l/i()t) cliluted
dialylcd C'T'A IgY
I ~ preparation standard. (liven that the specilic antibody stocks were
diluted 1!=t(), this would
indicate that the unconcentrated at'finity purified antibody stocks contain
I/IU-1/2U the
concentration of specilic antibodies relative to the startin~~ ("TA tcY
preparation.
c) 'Toxin A Neutralization Assay lJsing Antibodies Reactive
'-t) Toward Recombinant Toxin A Protein
1'he ('l IU toxin neutralization assay (Example 8(ct)) was used to assess the
ahiliw_ of
the ctepleted ur enriched samples generated in (h) ahoae to ncutraliie the
cwmuxicitv ul toxin
:1. 'I he general ability o1' aftiniW purified antibodies to neutralize toxin
A was assessed by
mixin~~ tey~cther aliquots of al! 6 concentrated stocks o1' the 6 affinity
purified samples
~~enerated in (h) above. and testing the ability of this mixture to neutralize
a toxin A
concentration of 0.1 ftg/ml. 'The results. shown in Figure 1 1. demonstrate
almost complete
neutralization of toxin n using: the affinity purified (Af) mix. Sumc epttopes
4wthtn the
recombinant proteins utilized fir affinity purification were probably lost wh
en the proteins
were denatured before af~finiy purification )hy Guanidine-!f('I treatment in
th) above). 'Thus.
3U the neutralization ability of antibodies directed against recombinant
protein is probably
underestimated using these af~tinit~~ purified antihoclv pools. 'This
cvperintent demonstrates
that antibodies reactive to recombinant toxin A can neutralize cvtotoxicity,
suggesting that
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WO 98/08540 PCTIUS97/l5394
ncutralizine antibodies may be generated by using recombinant toxin A protein
as
immunoeen.
In view of the observation that the recombinant repression clones of the twin
A gen c
divide the protein into O suhregions. the neutralizing abilim of antibodies
directed against each
individual reLion was assessed. The neutralizing ahilitv of antihadies
directed al:ainst the
ligand hindinr domain of toxin A was determined first.
In the toxin neutralization experiment shown in Flgtlre 11. interval (~
specific
antihoctius ( interval 6 contains the ligand hinding domain 1 were depleted
ti~om the dialyzed
I'hCi preparation. and the et~I~ct on toxin neutralization assayed. Interval 6
antibodies. were
lO depleW d either by utilizing the interval 6 depleted C:TI\ IgY preparation
t~ont (h) ahovc ("-6
aCl~. cdpleted" in I~iLUrc: 1 1 ), or by addition of interval O protein to the
C'T~1 ILK' preparation
tcstimated m he a I () ii~ld It101i1C exCeSS ()1'er alttl-Itttcf~'aI C)
In1117U17UglOlllllllt present in this
prrparatinnf W competitively cnmpete for interval 6 protein ("-h prot
depleted" in Figure I 1).
lit l1<1111 iltSlattee5, rrnuwal cof interval O SpeeltlC altLlb(1(ilc'.S
rcdUCCS tlt~ llelltl'alllat1011
W'ticicncy relative to the starting CT.'\ lgY preparation. 'This dcnulnstratcs
that antihodies
directed a~_ctinst interval (i cuntrihute to toxin neutralization. since
interval (~ corresponds tc~
the Ii;_~tnci hindin~~ domain ot~ the protein, these results demonstrate that
antibodies directed
a~~ainst thin re~~ion in the Pf.:(i preparation contrihute to the
neutralization ol~ toxin A in this
assay, l Imcvcr. it is significant flan alter removal of these antibodies. the
I'1~(i preparation
'_(1 retains aiLniticant ahiliy to neutralise toxin A (Figure I 1 ). This
Iteutralization is probahly
due to the ~tlll(lit ()t~ anllb(ICIIIS SpceItlC to other regions of the toxin
A protein. since at Icast
~)()'.'~~ ol~ the IILaIICI hllldllt;~ rr<_ion reactive antibodies mere rcmcwed
in tlm depleted son tple
prepared in th) above. This conclusicm was supported by comparison of~thc
toxin
ncutraiization oi~ the at'tiniW purified f1l'1 mix comparcct to af~iinity
purified intewal l>
antihncly alone. ~\Ithou~,~h some neutralization ability was ohservcd with A('
interval 6
antihoclies alone, the neutralization was significantly less than that
ohserved with the mixture
at~ all (~ :\I' antibody stctcla (not shown).
Crl1'en tltal Ilte J711x Ol~all six affinity purified samples almost
contplctelv neutrali~cd
the cyutoxiciy' ol~ toxin r1 (lvigure 1 ! ). the relative importance of
antibodies dirceted as!ainst
train f\ intervals 1-s within the mixture was determined. This was assessed in
Uvo ways.
First, samples cuntainine affinity purified antihodies rcprcsentin4~ s of the
O internals were
prepared, such. that each individual region was depleted from one sample.
Figure 1?
dernonstrates a sample neutralization curve. comparing the n eutralization
ability uC at'tinitv
_8j_
CA 02296765 2000-O1-14
WO 98/(ISS40 PCTIUS9'f/ti5394
purified antibody mixes without interval 4 (-4) or ~ (-5) specific antibodies.
relative to the
mix oi~ all to affinity purified antibody stocks (positive control). While the
removal of interval
spcci(ic antibodies had no ctfcct on toxin neutralization (or intervals I-;.
not shown). the
loss 0l' interval d specific antibodies significantly reduced toxin
neutralization tEigure 1?).
Similar results were seen in a second experiment. in which affinity purified
antibodies.
directed a~_ainst a single region. mere added to interval (, specific
antibodies. and the effects
on toxin neutralization assessed. Only interval a specific antibodies
si~~nificantlv enhanced
neutralization when added Eo interval C specific antibodies (figure Is). These
results
demonstrate that antibodies directed against interval ~l (corresponding to
clone pl'A1 lUU-1450
in I~i~~ure ~)) are important for neutralization of cvtotoxicitv in this
assay. I:pitope mapping
has shown that only iovv levels oi~ antibodies reactive to this region
arelgeneratcct wh-rn native
toxin ~\ is used as an immunogcn [E:xamplc 1?(a)~. It is hypothesized that
intmunization with
rccc,mhinant protein specific to this interval will elicit higher titers ol'
nt;utralizin~~ antibodies,
In summ.u. . this a11a1\'SIS has identified two critical regions ol~ the toxin
,~ prc,tein a!~ainst
v1'111CI1 Itl,'Ult'allLltt~.'. itlltlbpdleS are produced. as assayed by the
C.'t10 neutralization assay.
FXAMPLI~: 13
Production i\nd Evaluation ()I~ Avian Antitoxin
Against ('. CII~~IC'IIL Recombinant Toxin A I'olvpcptidc
?()
In Ivampl~ 1''. we demonstrated neutralization of toxin i\ mediated
cvtotoxicitv by
al~tinim purified antibodies reactive to recombinant toxin A protein. ~I-o
determine whether
antibodies raised as~ainst a recombinant polypeptidc fragment c,l~ ('.
cIiJJicile~ toxin A may hr
cl'tcctiw in trcatin= clostridial diseases. antibodies to recombinant toxin A
protein representing
the bindin_; domain were generated. Two toxin A hindinL domain recombinant
poypeptides,
e~pressin~~ the binding domain in either the pMAl..c (pMA187U-2O8U) or pL:T
'_'~(pl'A187U-
_'h8U> vrctor. were used :1S IntlllllttU~T.CI1S. The pMAI. protein was
af'tinitv puriticef as a soluble
product )I~xample 1'_'(d)) and the pE'i~ protein was isolated a5 1115<,Itlhle
IItC1lI51l,tt bodies
)Example I'_'(d)) and solubilized to an immunologicallv active protein using a
proprietary
;() metltos_i described in a pending patent application ((I.S. Patent
Application Serial No. _
()8/I?c).U?7). This L:xampie involves (a) immunization. (h) antitoxin
collection. (c) .
determination of antitoxin antibody titer, (d) anti-recombinant toxin ~\
neutralization of toxin
i1 henta~~~~lutination activity in ri~rn. and (e) assay of in ni~ro toxin i\
ncutralirin~ activiy.
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Immunization
The soluble and the inclusion body preparations each were used separately to
ilnmunizc hens. Both purified toxin A polvpeptidcs were diluted in YBS and
emulsified with
approximately equal volumes of CFA for the initial immunization or !FA for
subsequent
hoostrr immunizations. On day zero. filr each ot~ tile recombinant
preparations. W ~o egg
layng white Leghorn hens (obtained from local brcedcrl were each itl.jected at
multiplC sites
( intramuscular and Sltbct1ta11eUl1S) with 1 m! ot~ rt',COtllblnallt
ad_tU~'allt nllxtule c'.Ulltaltllnl_'
apllfU\Illlittt'.11' ().> f0 I .J nl~!~ (1t' CCCUlllhtllant IOxlll /~.
f30011Cr 1111n1U111T,allUClS (11' I.() 111
were '~ivrn nn days l~l a,ld dVV ?8.
h) Antitoxin C.'ollcciion
I~oUal yolk immune 1g7' was extracted as described in tile standard Pf:(i
protocol (as in
I~:xamplr 1 > and the final f~~~' pellet w,ls dlssoIVCd in sterile 1'fW at the
ori~!inal yolk volume.
i~his material is cicsi~~natcd "ilnmunC recombinant I~~1'~~ or "immune l~~Y."
1;
C) ,lntitoxin ~1ntil>,odv 'Titer
~(~a c(mcrmine if the recombinant toxin A protein was sut'ticicntlv
immunogellic to raise
antibodies in hens. tilt antihocd titer of a recombinant toxin A polrpcptidc
was determined by
f~(.I~A. 1:~_~~s ti-otll both hens were coIIcCtcd nn day 3'_'. the yolks
pooled and the antibody _
-'() was iwlatcd using 1'F(i as described. The immune recombinant 1gY antibody
titer mas _
dctermimd I~c~r the soluble recombinant protein containin g the maltose
bindin~~ protein t~USIUtl
~mllcratcd in p-Mal (pMA1870-?(18()l. 'Vinetv-six well Falcon I'ro-hind plates
were coated
omrni~_ht at ~4°(' with lt)U Etl iwcll cli~ toxin A rccomhlnant at ?.~
Elg /l.ll in !'13S containing
t).Uj'~,' tllimcrosal. ,~lnclther plate was also Coated with maltose binding
protein (MI31'I at the
same concentration. to permit Comparison ot~ antibody reactivity to the Cusion
partner. The
next day. the wells were blocked with I'fW Containing I"a bovine serum albumin
(13~A) ter 1
hour at ;7°('. IgY isolated from immune or preimmunc c~_g's was diluted
in antibody diluent
( l'f3~ ec111talllillg I ".% BSA and U.(l;~ ~ Tween-2U), and added to the
blocked wells and
incubated tier I'hour at >7°C' . The plates were washed three times
mith PI-W with U.Ui°~~
a) ~fvveml-_'U. thCn three III11LS l4IIh PB~. Alkaline phosphatase Conjugated
rabbit anti-Chicken
lgCi (~i,'nlct) diluted I:IUUU in antibody diluent was added to the'plate.
anti incubated for 1
hour at ,7°C. '1"he plates were washed as before and substrate was
added. [p-nitrophcnyl
hhosphatc (~igma)~ at I tllL/1111 in ().l)iM N;I_[();, hl.) 9.> and 1() mM
MgC'l,. 'fhe plates
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were evaluated quantitatively on a Dynatech MR 300 Micro I:PA plate reader at
410 nm
about 10 minutes after the addition of substrate.
Based on these ELISA results. high antibody titers were raised in chickens
immunized
with the toxin A recombinant poiypeptide. 'The recombinant appeared to be
highly
immunogenic. as it was able to generate high antibody titers relatively
cluickly with few
immuni:cations. lmtnune IgY titer directed speciticallv to the toxin A portion
of the
recombinant was higher than the immune IgY titer to its fusion partner. the
maltose hindins;
protein. and significantly higher than the prcimmune lgY. LI.,ISA titers
(reciprocal of the '
highest dilution of IgY generating a signal) in the prcimmune IgY to the MI3P
or the
lU recombinant was <1:3U while the immune IgY titers to ML3I' and the toxin A
recombinant
were i :18710 and :~ 1:93750 respectively. Importantly. the anti-recombinant
antibody titers
~.:enerated in the hens against the recombinant polypeptide is much higher,
compared to
antibodies to that region raised using native toxin A. l'he recombinant
antibody titer to
rc~.ion 1870-268() in the C~l'A antibody preparation is at least tier-told
Icw~er compared to the
1 ~ recombinant generated antibodies ( 1:18750 versus -1:9 370). Thus, it
appcan a better
immune response can he generated against a specific recombinant usin;~ that
recombinant as
the immunogen compared to the native toxin A.
This observation is significant. as it shows that because recombinant portions
stimulate
the production of antibodies. it is not necessary to use native toxin
molecules to produce
'_U antitoxin preparations. 'Thus. the problems associated with the toxicity
of the native toxin arc
avoided and large-scale antitoxin production is lhcilitated.
d) Anti-Recombinant Toxin A Neutralization ()f Toxin A
Hemagglutination Activity Irr Vilro
?? ~1'oxin A has hemagglutinatim; activity besides cytotoxic and cnterotoxin
properties.
Specifically. toxin A ag6lutinates rabbit erythrocytes by binding to a
trisaccharide (gal 1-3I31-
4(ileNAc) on the cell surface. (H. Krivan c~ crl.. Infect. Imrnun.. >s:s7~-S81
(l~)gb).~ We
examined whether the anti-recombinant toxin A (immune I~zY. antibodies raised
against the
insoluble product expressed in pE'T) can neutralize the hema~_~_lutination
activity oh' toxin A in
3(1 vitro. 'fhc hemaLglutination assay procedure used was cicscrihcd by Il.t'.
Krivan er crl.
Polyethylene glycol-fractionated immune or preimmune IgY ware pre-absorbed
with citrated
rabbit erythrocytes prior to performin~~ the hemagglutination assay because we
have found that
lgY alone can agglutinate red blood cells. Citratcd rabbit red blood cells
(RRI3C's)(Cocaiico)
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were washed twice by centrifugation at 450 x g with isotonic buffer (0.1 M
Tris-I-iC.'I. 0.05 M
NaCI. pl-I 7.''). RRI3C-reactive antibodies in the IgY were removed by
preparing a 10%
RRI3C' suspension (made by addin~~ packed cells to immune or prcimmune ELY)
and
incubating the mixture te,r I hour at 37°C. The RRBC's were then
removed by centrifugation.
'veutraiization c~t~ the hemagglutination activity ot~ toxin A by antibody was
tested in round-
hottomed c)(,-moll microtiter plates. Twenty-fire yl of toxin ;\ ( il Etg /ml)
(Tech L.ab) in
isotonic huhttr was mixed N ith an equal volume of dit~ferent dilutiona of
immune or
preimmunc I;~l' in isotonic buffer. and incubated for I~ minutes at room
tc:mperaturc. 'I~hen,
i!) Ell ul~ a I'.i> RRf3C' lUSpertSll)It Irt IS<ltOltre butler was added and
the mixture was incubated
1() li,r ~ hours at =1°('. l~llSllll~ Cc)Itlrcll wells containing the
final concenlratlUn (1t' ~) Fy/ml of
toxin :1 ,Il~trr dilution without l~~l' were also included. liemagglutination
activity was
asscswl visually. with a ciii'fusc matrix of RRB("s coating the bottom ot~ the
well
rcl,rrsmtin~~ a positive hcnta,.:~~lutination reaction and a tiLht button of
RRE3("s at the hc,ttont
«t~ the wall rrl,rrsentin~~ a nca!ativc reaction. ~I~hr anti-recombinant
immune IgY' ncutraliLCd
toxin :1 Itrnta'~~;lutination activity. ;~ivinL a ncutraliration titer ot~
1:li. l towcvcr. prcinttnunc
1~~~' was unahlv to ncutralirc the hcnta~.;~~lutination ahiliW ol~ toxin f1.
r) lvxay ()f' lu 6 itr« Toxin A Neutrali~in~ Activity
l hu ability of the anti-reromhinant toxin n Is~Y (immune l~~Y alltlh(1d1e5
1'alSed aeainst
~'t) I,~~1:1 I t;7t)-?(,fit), the soluble recombinant bindin~~ domain protein
expressed in pMi\L.
Wsi!~namcl ;IS rlnti-toy. :\-? in hiLUrc I-t . and referred to as recombinant
re~~ion p attd prr-
ilmttutto i~_1~. prepared as dearrihed in F~xantple 8(c) ahoye. m ncutrali~e
the'cwotoxic activity
~,I~ tclxin :\ was assessed in ri~rn using the C'f iC) cell cytcttoxicitv
assay. and toxin ~\ l~I rclt
l.ah) at a concentration ol~(l.lEy~ml. a, clscrihed in L:xamplr !3(d) al,ovc.
:\s additional
controls. the anti-native toxin A 1~~~' ((.'T~\) and pre-immune I~.:Y
preparations described in
Lxample S(y above were also tested. The results are shown in Tieurc I~I.
l~lte anti-recombinant toxin ;\ IgY demonstrated only partial nrutrali~ation
of the
ryotoxic activiy ot~ toxin n. while the pre-immune 1gY did not demonstrate any
significant
neutralizilt;~ actiyitv.
,1)
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EXAMPLE l~t
In vian Neutralization Of (', cIiJJiciIr ~I~o~cin A
The ability e7i~ avian antibodies (!~Y) raised aLainst recombinant toxin ;1
binding
CI()171a111 Ill IICUIt'illlZC the enterotoxin activity ol' (', cliJ~icilc~
toxin A was evaluated irr vinn
using Cic7ldcn Syrian hamsters. 'fhe Example involved: (a) rrcparation of the
avian anti-
1'<'Cl)117h117a11t t0\In A 1~Y tar oral administration: (h) in oinu
prc)tectiun oi' hamsters ti-om c'.
cli//icilc~ toxin A cnterotc)xicity ht' treatment ot~ toxin A with avian anti-
recombinant toxin A
1~;Y: and (c) histulcyic evaluation of hamster ceca.
:~) !'reparation Of The Avinn Anti-Itecombin:mt 'Toxin A 1);1
('or <)ral Administration
L;_~~s were cc)Ilertcd t'rom he n5 Nllll:l7 Il~id t7~e11 1111171t11117.1',d
with tile rcec7mhinant <'
cli//ic~ilc~ train r1 I~ra~ment pMA I 87()-2680 (described in L;xan7ple I s.
above I. .1 second group
ul~c~~_s purchased at a local supermarket was used as a pre-immune (ne~ativet
control. L:L
y)Ik imnlune)~_lc)hulin (I~Y1 was ewractcd ht' 1'FCi From the hv(1 groups (>f
e~~~~s as described
in L:xampic Htcl. and the final I~~Y pcIIWs were soittbili7ed in unc-ti)urth
the ori~~inal v_ olk
w)lunle using (). I ~9 carhc)nate buffer (miwure c7t~ NaI IC'(), and
1\a,('();). pll () ;. f~he basic
carbonate hut~icr was used in order to protect the toxin ~1 from the acidic pl
I ul~ the stomach
~() ~Ill'If()11117CI1t.
h) In vine 1'r0tcction ()f Hamsters A);ainst C: ~lifjicil~~ Toxin A
1~:nterotoxicity By Treatment Of Toxin A With Avian Anti-
rccomhinant Toxin A 1gY
In order to :ISSI:SS the ability ol' the avian allll-re(:(7177h111a11I l()\1l7
n I~Y. prvparcd in
s~ctiun (a) ahc)ve to neutralize the in ni)~~u enterotoxin activity c)f toxin
A. an in oi)v~ toxin
ncutralizatie)n model Has developed usiny~e)ldcn Syrian hamsters. This nu)del
was based un
published values tile the minimum amount of toxin A required to elicit
diarrhea (Q.()8 mr
toxin A'hz body wt. ) and death ((). I C) mg tc)xin f~il~~ hc)dv wt. l in
l7alllsters when
() administered orally (I.verlv en crl. Infect. Immun.. ~17:;4c)-:s? ( lc)8s).
hoe the study. lour separate experimental groups wrrmscd. with each Lroup
consistit7_~
ot~ 7 tcmal~ (~()Iden Syrian hamsters (C'harics River). al7prox. three and
c)ne-hatf~ weeks ofd.
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WO 98!08540 PCT/US97/15394
weighing approx. ~0 gms each. The animals were housed as groups of 3 and 4,
and were
offered trod and water ucl lihimm through the entire length of the study.
Ivr each animal. a mixture containing either lOEtg of toxin A (0.2 mg/Kg) or
;O~tg of
main i\ ((>.b meiKg) (('. cli//icilc~ toxin ~\ was obtained from 'i~ech Lah
and 1 ml of either the
anti-recombinant toxin A IgY or pre-inttnune IgY (from section (a) above) was
prepared.
These miwures were incubated at ,7°C for 60 min, anei wore thin
administered to the animals
I» (I)~ oral room. 7~hc animals wrrc then observed for the onset of diarrhea
anct death for a
periml of ?.t hrs. lollowin!~ the administration of the toxin ~1+-1'~Y
mixtures. at the end ot~
which time. the following results mere tabulated and shown In -(~ahle ( 7:
rnB~E t7
Study Outcome At ?4 f lours
Study Outcome at ?4 Hours
tixpcrimental Group ,.--
,_ t-lealtft>r Diarrhea= Dead'
y Twin A - :\ntitoxrn A«ainst interval G 7 1) 0
,r) ry~ fuxiu .1 - Antitoxin Against Interval G ; () tt
l() y Toxin ~\ ~ I're-lrnmunc Scrum t)
?() u~; -toxin ~ ~ I're-Immune () i
' :\nimals rrmained healthy throu!_h the entire ?4 hour study peri<xi.
nmmalv developed diarrhea, but did nat die.
Animals developed diarrhea. and subsequently died.
1'retrcatment u!~ toxin A at hoth doses tested, using.: the anti-rccomhinant
toxin ~\ Igl'.
prevented all overt SvnlptOtttS ()h dtSeaSe ttt hamsters. Therefore.
pretreatntcnt of C'. cli//irilc'
tetxin r\. using the anti-rccomhinant toxin A 1~~~'. rtcutralized the in
t~irci enterotoxin activity
e~f' the toxin ;1. In contrast. all animals from the two groups which received
toxin f\ which
had hccn pretreated usin~~ pre-in tmune 1gY developed disease symptoms which
ranged tram
diarrhea to death. The diarrhea which developed in the ~ animals which did not
die in each
of thr mo pre-immune ~_roups, spontaneously resolved by the end of the 24 hr.
study period.
c) liistoio~ic !:valuation Oi Hamster Ceca
'(1 In order to titrtl)er assess the ability of anti-rccornbinant toxin A i~_Y
to protect
hamsters ti'om the entcrotoxin activity of toxin A. histologic evaluations
were performed oft
_ the coca of hamsters from the study described in section (h) above.
Three groups of animals were sacrificed in order to prepare histolugical
specimens.
T7re first group consisted of a single representative animal taken from each
of the ~I ~~roups of
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WO 98/08540 PCT/US97/15394
surviving hamsters at the conclusion of the study described in section (b)
above. These
animals represented the ?4 hr. timepoint of the study.
The second group consisted of two animals which were not part of the study
described
above. hut \vere separately treated with the same toxin A f pre-immune I~~Y
mixtures as
clrscrihed for the animals in section (b) above, Both of these hamsters
developed diarrhea.
and \vcrc sacrificed 8 hrs. at'tcr the hnti; Ut' ad1171n1StraLlUl1 l7 t' the
main A + Pre-immune 1gY
mixtures. ~1t the time ut~ sacrifice. both animals were presenting symptoms of
diarrhea.
TItCSI', a1111tta1S rl'prCSl.'111t'.d the acute phase ul' the study.
I~hr final s~ruup consisted of a single untreated hamster ti'(lltt fltC
SFllttc'. 1111ptttelll C)t
1() animals as those used fir the t\vo previous groups. This animal served as
the normal control.
samples eh' cecal tissue were removed from the 7 animals eirscrihed ahc>ve:.
and were
lixed overnight at =I°C' using 10°/, hul'fcred tormalin. ~Citc
fixed tissues wore parat'tin-
cnthccicled. sectioned. and mounted on glass micrclscope slides. 1'lte tlSStll
Sl(;tI1111S 1t'l'rC then
,taincd using hcmatoylin and eosin (II and F stain 1. and \vcre cxantincd by
li~~ht microscopy,
1 ' ~f'hc tissues obtained i~rom the two ?=t hr. animals which rcciivrcl
mixtures rontainina
uithrr I(>EI,; yr sUtcg c>i~ toxin A and anti-recombinant toxin ~1 IgY'
\1'c'1't Illlll~IlllLIIISItFtf)ll' from
the normal control. both in terms ol~ ~~ross pathulous. as well as at the
microscopic level.
'I'hcsc observations provide t'urthcr evidence ictr the ahiliy ul~ anti-
rc:comhinant toxin A IeY to
et'I'ectivcly neutralize the in niw~ enterotoxin aetiviy of ('. cliJ/ic'ile
toxin ;~. and thus its abilitv_
?t) to prevent acute ur lasting toxin j~-induced patholuLV.
In contrast. the tissues from the t\vo ?4 hr. animals \vhich received the
toxin .~ + pre-
immunc f~~l~' mixtures cHmclnstraW d sib=niticant pathology. In both ol~ thm
~~ruups. the
ntucosal layer \vas observed to he Il'.11 organized than in thmurmal control
tissm. I h~
cytoplasm ul' the epithelial cells had a vacuolated appearance. anct Laps
\verc prevent between
the epithelium and the underlyin~~ cell laycn. The lamina prupria was largely
absent.
Intestinal villi and crypts \vrrc signiiicantlv diminished. and appeared to
have been overgrown
It\' ~l 111a11a1' la\'trr (1t~ lpltllC1t~11 CCIIS and tibrohlasts.
l~flt'1'Ct01'l. .11I111)LI~!h tlttSl' ~111t111a15 tt1'CI'tl\'
appeared to recover i~ront the acute symptoms of toxin ~~ intoxication.
lasting pathologic
alterations to tltc cecal mucosa had occurred.
;() ~l'he tissues ohtaincct from the t\vo acute animals which received
mixtures of train A
and pre-immune IgY dentonstratcd the most significant pathology. :1t the gross
pathological
level. both animals were observed m have severely distended coca which w ere
tilled with
watery. diarrhea-like material. ~~t the microscopic Irvel, the animal that was
~:iven the
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WO 981t~8540 PCT/US97/1i5394
nuxture containing lOEtg of toxin A and pre-immune )gY was found to stave a
mucosal !aver
which had a ragged. dammed appearance. and a disorganized. compacted duality.
The crypts
were lar~~elv absent. and numerous breaks in the epithelium had occurred.
There was also an
intlux ol~ erythrocytes into spaces helvecn the epithelial layer and the
underl)'ing tissue:. The
animal mhich had received the mixture containing 3()Elg of toxin f1 and pre-
immune IgY
ctcntonstratcd the most severe patholctLy. 'The cecal tissue tlt~ this anlrnal
had an appearance
vrry similar m that observed in animals which had died ti~om C'. cliJJicile
disease. Widespread
~lcstructiun ul' the rnucosa was noted. and the epithelial layer had sloughed.
I Iemorrhagic
areas containing Large numbers ot~ erythrocytes Here very prevalent. All
semblance' of normal
I() tissur architecture was absent t~rom this specimen. In terms of the
presentation of~ pathologic
mcnts. this in lim hamster model u1~ toxin A-intoxication cctrretates very
closely with the
patholey~ic mnscduences ut~ ('. cli/Jicile disease in hamsters. 'hhc results
presented in this
l~:vampu dmtonstrate that while anti-recombinant toxin .A (interval () IgY iv
capable oh~~nlv
lrlrtiallv Il~llt!'LIIIGIIIL the cvtotoxic activity ott~ ('. eliJ%ic~ilo toxin
A. the same antihctd_v .
~t~tcctiwly neutralizes I()()% of tire in rime enterotetxin activity of the
toxin. Vl~'Itile it is not
intended that this invention he limited to this mechanism. this may he dur to
the cvtotoxicitv
anti muromvicitv c,l' ('. cli/Jicilu ~I~uxin ~1 as tvo sc:paratc and distinct
hicloeical t'unctiuns. '
FaAMPLt? 1;
In t 'inn Neutralization OI' ('. UiJJicilo ~I~uxin .~ E3v
rlntihoolics ;l~~ainst IW c:omhinant Toxin A I'olvpc~ptidcs
~fhc ahiliy ot~ avian antibodies directed against the recombinant ('.
cli~Jioilc- toxin i1
I~ra~=ntent I 87f)-?(>80 (as cxpressect by hMA 1870-?68(): sec ~xampic I s >
tc, neutralize the
cntcrc,tc,vic activity ot~ toxin n was demonstrated in Example 14. The abiliy
ol~ avian
antibodies ( I);Ys) directed aiainst other recombinant toxin A epitopes to
neutralize: native
toxin .1 in uvu was next evaluated. Tltis example invnlvcd: (al the
preparation ut' tg~'s
a~~ainst rcc;omhinant toxin /1 pulypepiidcs: (hl ire non protection ot~
Italttslers against toxin n
by treatment with alai-recombinant t<txin ~1 IgYs and (cj ctuantiticatiun
ot'spccific antibody
re,ncentratiult in CT:1 and Interval (, I~~Y t'EG preparations.
_ 'bite nucleotide seducncc ot~ the coding region oh the entire train .~1
protein is listed in
~L:Q lI) N():i. The amino acid sectuence of the entire toxin A protein is
listed in SL:Q II)
N<):h. ~i~he amino acid SC; CIl1e11Cf; CIItISISIIn~! ot~ amino acid residues I
87() through ?O8() of
_ c), _
CA 02296765 2000-O1-14
WO 98108540 PCT/US97115394
toxin :\ is listed in SFQ fD N0:7. The amino acid sequence consisting of amino
acid
I'eS1d1.1eS I R70 thrott~h 19(,0 of toxin A is listed in SEQ II) N():R.
:1) Preparation Of 1gY's Against Recombinant Toxin A
Polvpeptidcs
f:~~~s were collected t'rom I.eghorn hens which have been immunized with
recombinant
( '. cli~~ic~ilc~ toxin ~1 polypcptide fragments encompassing the entire toxin
I1 protein. 'fhe
pnlypcptide ira~menis used as imrnunogens were: i ) pMA IR70-?G80 (Interval
l>). ?) p1'A
1 1 ()()- I ~4s1) (Interval ~ ). and i) a mixture of fragments consistine of
pMA 30-;00 ( Interval I ),
It) pMn s0U-6h0 (interval ?), pM~1 h(~()-1100 (Interval ;) and pMn 14>0-1R70
(Interval 5).
This mixture ot~ immunogens is referred to as Interval I?_ss. ~I~hc location
of each interval
within thr toxin ~~ ntOleCllle 1S 511014'11 ttt higurc 1 ~;~. In I~i~;urc l
gin. the fi~llcvin~.~
abbreviations are used: pi' reters to the p1;-r?3 vcct~r lNew l:n~,:land
L3ioLahs): pM refers m
the p~~1~11.'s-~ vector (New l:n~~lattd fiiol.ahs): .~ refers to toxin .~: thr
numbers refer tc> the
1 ~ amino aciei interval exprcsscd in the clone. (For example. the designation
pMA;()-;00
indicates that the rmomhinant clone encodes amino acids :U-;()() uf~ toxin ,~1
,end the vct:tor
used was pMAl.m'_e).
~I~Im recombinant prow ins were ~eneratect as ctcscrihcd in E'.xaltlple 1 I.
i~hr I~Ys were
cxtrac;md and soluhiliicd in 0. I M carbonate buffer pl l c).5 li,r oral
administration :1s described
'_'() in Example l.~(a). -hhe IgY reactivities against each indiviclual
recombinant interval was
maluamd by L~LISA us described in I:xamplc 1 ;(c).
h) !rt ~ivn Protection Vf Hamsters Against Toxin
A lay Treatment With Anti-Recombinant Toxin A Antibodies
'i~he ahiliy of antibodies raised against recombinant toxin i\ polypeptides to
provide in
ai~~n prmcction against the cnterotoxic activity of toxin i1 was examined in
the hamster model
system. l~his assay was performed as described in hxamplc I~(h). t3ricllv. for
carp ~l0-s0
<~ram tcmalc Golden wrian hamster (('harles River). 1 ml ol' I~~Y ~lX (i.o..
rcsuspended in II4
of the l)t'1~_tllil yolk volume) I'f:(i prep aLa111St Interval 6. interval ~
c?r Itltcrval I'_'3> was
,U mixed with ,0 Et~ (I.I)"", oral dose) ol' ('. cli~ficilv toxin A S heck
I.ah). I'reimmune l~Y
mixed with toxin A served as a negative control. nntihodies raised against (
'. clij)ioilo toxoid
:1 (Example R) mined with toxin /~ (CTA) served as a positive control. 'l~he
miwurc was
incubated for I hour at 37°C' then orally administered to li~htlv
etherizcd hamsters usinL all
CA 02296765 2000-O1-14
WO 98108540 PCT/US9711i5394
I 8G ieedin~ needic. The animals were then observed for the onset of diarrhea
and death for
a period of approximately '_'4 hours. The results are shown in Table 18.
TABLE 18
Stndv ()ulcnntc~ AftNr ~d W nme
'hrcatmcnt group f-Icalthy' Diarrhea- Dead'
1'rcimmunc () (
)
. ~..t.~ ~ () ()
Interval (, ~, ,
mtr~.,l .t o
I r,
I () Interval i?3s () t) 7
' -1nimal shoos nn ai~_n of illness.
Animal developed diarrhea. trot did not die.
nninrn developed diarrhea and died.
I're-treatment (,I' t(txin A with Iph's against Interval 6 prevented diarrhea
in (, of 7
ltamxmrs anal cc,mplctelv prevented death in al! 7. In contrast. as with
preimmune I'; Y'. I~~'s
a;~ainst Interval -1 unci Interval I?:_', had no effect cot the (Inset (tf
diarrhea and death in ihc
hamsters.
'() c) (~>uantitication ()f Specific Antibody Concentration In (:"I':1
:lnd Interval 6 18Y Pf:G Preparations
lu, determine the purity (,f I~~Y PECi preparations. an aliduot ofa pMAI1170-
?68()
(Interval (t) I~~1' I'I~.(i ltrcparatiun mas chrctmatographcd usiy llpl.t' and
a I~\~'-~i(>; sirinL
column (~It(,dcW. f hr rcsultin g profile of absorbance at ?80 nm is shown in
I~ipurc Ih. '-1'he
,in~_ic lar~m peak cc,rrespands to the pmciicted MW' ol' Izl'. Intcrratien
c,f' the .Irca under the
ainile Ittr~sc peak showed that greater than ~)5% of tltc protein eluted from
the ce,lumn was
present in this single peak. This result demonstrated that the majority
(=w)5'%) of the material
absarbin~ at ?80 not in the PECi preparation corresponds to Irl'. '1-heretore.
ahs(trbance at
?80 not cart be used to determine the total antibody concentration in l'L(i
preparations.
;() I o dcaermine the concentrati(tn of Interval 6-spcctUc anttbodtes
(expressed as percent
c,h total antibody within the CTA and pMr11870-?(,80 (Inten~al G) f'I-'.Ci
preparations. defined
duantities ctl~ these antibody preparations wer(.~ aftiniy purified un a pI'A
187()-?(t80(l i) (shc,wn
schenttttically in f~i~ure lsl3) af~iiniw column and the specilic antihctdics
were yuantificd. In
t~irurc ist3 the titllowin~ abbreviations arc used: pI' refers to tire
p(~'I'?3 vctaor (New Ett~land
i3iol.ahs): pM refers to the pMAI.'"-c vector (New l:ngiand (3ioL.abs): pV
refers to the pCiEX
_c~5-
CA 02296765 2000-O1-14
WO 98108540 PCTIUS97115394
vector (Pharmacia): pB refers to the I'inPoint'"'' Xa vector (1'romega): A
refers tct toxin A: the
numbers refer to the amino acid interval expressed in the clone. 'fhe solid
black ovals
represent the ME3P: tltc hatched ovals represent glutathionc ~-transfcrase:
the hatched circles
represent the hicnin tag: and Fif II-I represents the poly-histidinc tai.;.
nn affinity column containing recombinant toxin A repeat protein was made as
follows. l~e~ur ntl of PE3~-washed .~cti~:el resin (Sterogenel was coupled
with s-IU mg of
pl'A 1 R7t)-?Ct8U inclusion body protein prepared as described in L:xample (
17) and dialyzed
inlet 1'I3~~ in a l; ml tube (Faiconl containing 1/10 final yetlurtte ,~Id-
eouplinL solution ( 1 M
sodium cyancthctrcthydride). Alidu<ns ctf the supernatant ti~otn the
coupling:, rcacticms. helore
I11 and after couplins~, were assessed by C'oomassie ,twining ctf'
7.S°.~~ ~t)~-I'n(;f: gels. Based
upon pre»ein hand intensities. greater than b mg ol' recombinant prcttcin vyas
coupled to the
resin. The resin was poured into a IU ml column t(3ictRad). washed extensively
with I'f3S.
pre-eluted with ~ M guanidine-F~C1 (in 1() ntM ~I~ris-I~C'I, pll fl.U:
U.UUi~,. thimerosal) and rr-
ccluilihratcd with I'IW. ~I~hc cctlurnn was stored at 4~C'.
1 ~ ,Aliquots of a pMA187U-?G81) (Interval h) or a (.'TA IgY pctlycional
antibody
preparation (1'I;(i prep) were ai~tittiy purilicd on the ahctyc al~liniW
column as titlluws. ~I~hc
column v,ls attached to an ( ~V monitor I1SC'()) and washeJ with 1'Li~. Iutr
pMn I87U-2680
!gl' puriticatic»t. a ?X I'I(i prep (liltcr sicrilitcd using a U.~li tl
liltcr: appntximatcl_y >UU me
total IiY) was applied. The column was washed with PI3~ until the baseline was
re-
~0 cstahlishccf I the column flow-tlu~ou~~h vyas saved), washed with
l3fi~~l~wuen m elute
nunsprciticallv binding antibodies and re-equilibrated vyith 1'E3'i. E3uund
antihctdy was eluted
from tltc column in ~I M Luanic(inc-f ICl ( in I U mM 'Kris-f IC'l. pl (
f;.(): 1).U()ia," thimerosal ).
~I~hc entire elution peak mas cctllcctcd in a Is ml tube Il~alcanl. Il~c
column w,rs rc-
equilihrated anJ the column eluatc was rc-chromato«raphrd as dcsrrihed above.
The antihuc(y_
prcparatietn was quantified by ('V ahsorhance (the elution huffi:r was used m
iero the
spectrctphcttctmetcr). 'fcual purified antibody was approximately ') m~~ and 1
mss t~rctm the first
and second chromatcyraphy passes. respectively. 'l~hc low yield f~rum the
second pass
indicatcc( that most specific antibodies were removed by the first round ct1'
chromatography.
The estimated percentage ~tt~ Interval (~ specitic antibodies in floe
pfVIA187U-?(tRU PI:Ci prep is
:;U approxintatcly '"%u.
~I~hc percentage ctl~ Interval 6 specific antibodies in the ("I':1 f')~(i prep
was determined
(LII111'l.IItL the same column and methodctlugv described above) let he
approximately U.~'a of
total l~l''.
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CA 02296765 2000-O1-14
PtrT/US97I15394
A 4X PCG prep contains approximately ?0 tngiml I~~Y. Thus in b) above.
approximately 4t)0 Et~ specific antibody in the Interval G I'CG prep
neutralioed 30 Etg toxin A
117 Vll'fl.
EXAMPLE IG
In 1'iru ~freattnent Ot' ('. cli/Jirilr Disease In
Hamsters By Recombinant internal h Antibodies
fhe ahiliy of antihocties directed against rccumhinant interval G ot~ toxin A
to protect
1() hamsters in oiml from l'. cli»icile disease was examined. T~fis example
involved: (a)
I,ro(,hylactic treatment of ('. cliJJic~ile~ disease alld (h) therapeutic
treatment ol~ ('. cli/Jicilu
ciiaeas~.
~r) Prophylactic Treatment ()f G difficilc~ Disease
I~his mperimcnt was Itertormcd as described in Exantplr c)(h). 7~hree Lroups
each
cansistin~~ uf~ 7 female I ()() ;_ram Syrian hamsters (C'harles River) were
prophylacticallv treated
with either prcimmutze I;~Y's. lgYs against native toxin n and f3 (C'1'nI3:
sec Example H (a)
and (h)~ ~,r l~~l's atainst Interval h. I~~Ys mere prepared as -iX PI:Ci
preparations as ~iescrihed
in I-:xantl,lr ~)(a).
-'() i~hu ~tninutls were orally dosed ; times daily. rouLhl_v at ~l hour
intervals. li,r I'_' days
with 1 ml antibcxl preparations diluted in Ensure~ai;. 1.!sing lSltIltaIlS
()t~ SI,<:C:ItIC ailtlt70d~, .
cvncmtration t~rum Iv:vample I ~(c; I. each dune of the Interval ( antihodv
prep contained
al,Proxitnatcly -i()U ~t~ ut specific antibocy. ()n day ? each hamster was
predisposed to (..'.
cliJ)ic~ilc~ infection by the ora) administration of~ ;.() mg ul~ C'lindamyrin-
11C'I (Si~tma> in I ml
ol~ water. (>n clay i the 11a111SI1'fS ~ll'I'C Orally challenged with I ml ol'
('. cli//ic~ilc~ inuculum
strain ATC'(' -ISj9G in sterile saline containing approvimateiv 1()0
organisms. ~1'Ite aninrtls
were then ohscrved fi,r the onset of diarrhea and suhsequcnt.dcath during the
treatment
period. ~fhe results arc shown in Table I ~).
_c)7_
CA 02296765 2000-O1-14
~rp gglpg~p PCTIUS97115394
TABLE 19
I.cthalitv After 12 Davs Of l'rcatmem
Treatment Group Number Animals Alive Number Animals Dead
f
Prcimmunc 0 7
C'1'A I3 6 I
Imerval 6 7 t1
Treatment ol' hamsters with orally-administered IgYs against Interval h
auccessf'ullv
protected 7 trot ol' 7 ( 10()'%) ol' the animals from ( '. cliJJicilr disease.
()ne of the hamsters in
I () this group presented witlt diarrhea which subseduentlv resolved during
the course of
treatment. ;~s shown previously in L:xample ~). antibodies to native toxin f\
and toxin I3 were
highly protective. In this I~.wmple. 6 trot ot~ 7 animals survived in the
C'~I'A13 treatment Lroup.
:111 ot~ the hamsters treated with preimmune sera came clown with diarrh ea
and died. -I hr
,urviw~r, io both the C'T.~13 and Interval 6 groups remained healthy
thruu!~Ite,ut a I ~ clay pust-
I s trcatmmt period. In particular. (i out of 7 Interval 6-treated hamsters
survived at Icast
weeks all~r termination ol' treatment which suggests that these antihndies
provide a lun~:-
lastin~ cure. I'itcsc results represent the first demonstration that
antibodies generated aialllsL a
1'e(:()Itth111a111 ry~ion ol' toxin n can prevent C'l)t1I) when aclminiUercd
passively to animals.
l~hcsc results also indicate that antibodies raised against interval U alon a
may hr suhlicicnt to
''() protect alltlttalS from ('. cli/Jirilc disease when adminisUrrd
prophvlacticallv.
I'rrviuuslv others had raised antibodies against toxin :1 h~ actively
11111ttt11111t1t~~
h~lnlS(~1'v a~~ainst a recombinant polvpeptidc located within the Interval l>
rcLion ~I.verlv.
I).M.. r~ crl. ( l Nc)U) C'urr. Microhiol. ? I :?c)J. higure 17 shows
achmtaticallv the iocatitm of
the l.vcrlv. er crl. infra-Interval t recombinant protein (cloned into the
pl!C' vector) in
comparison with the complete Interval 6 construct (pMAl87()-?O8()) used herein
to generate
ncutrali~in~~ antibodies directed against toxin A. In higure I7. the solid
black oval represents
the 1~1I3P which is bused to the toxin A interval G in pMA1870-2680.
l h~ Lvcrlv. ur crl. antibodies (infra-Interval h) wore only able to partially
protect
hamsters a~_ainst ('. clij)icilr infection in terms ctt' survival (=4 out c~('
8 animals survived) and
,s(I furthermore. these antibodies did not prevent diarrhea in any u(~ the
animals. ~ldditicmalU.
an111taIS treated with the infra-Interval h antibodies [I.vcrlv. n ul. (
Ic)c)()). .,y~rcr) died when
treatment w as removed.
In contrast. the cxp criment shown above demonstrates that passive
administration oi~
anti-Interval (~ alttihodies prevented diarrhea in (, out of 7 animals and
completely prevented
-98-
CA 02296765 2000-O1-14
WO 98108540 PCTIUS97/115394
death due to CDAI~, furthermore. as discussed above. passive administration
ctf tire anti-
Interval (, antibodies provides a lone lasting cure (i.e~.. treatment could be
withdrawn without
incident 1.
h) Therapeutic Treatment Uf C. ~(iJfrcile Disease: Ire Vivo
Treatment Uf An Cstablished C. ~lifftcile infection In
' Hamsters With Recombinant Inten~al G Antibodies
Tltc ability ot- antibodies against recombinant interval G of toxin .~ to
thcrapeuticaliy
treat ('. ~liJ)ic~ilc~ disease was examined. -fhe experiment was performed
essentially as
I U clcscrihed in E:xamplc 1 U(h). -Three groups, each containing seven to
eight female Golden
Syrian hamsters ( 1 ()U E~ each: C.'harfes River) were treated with either
prcimmunc 1gY', IgYs
a;~aittst native toxin ;1 and toxin t3 (C.'-frlE3) and lgYs against Interval
1. -The antibodies were
hrep.lrcd as dcacrihcd above as ciX PIOi preparations.
fhc hamsters were first predisposed to ('. cli/~icilc~ infection with a 3 mg
dose oi'
I ~ (.'linciantwin-fiC'I (Si~~ma) aClIttIItISter-ed orally in I ml W' water.
;lpproximatclv 24 hrs later,
the animals were orally challenged mith 1 ml of ('. cliJ%icilc~ strain A'fC'C'
.)s5<)h in sterile
Sallllt ec)IttaltllltL approximately ?Ul) ur__anisms. One day after infection,
the presence of toxin
n anti li wus determined in the ti:ces of the hamsters using a commercial
intmunoassa_v kit
((~yuclunr ~~-l3 hPA. C.'ambridLC l3iotcch) to verify estabiishntrnt of
infection. I~aur
'_U ntc:mhcrs W~ each group were randomly selected and tested. feces from an
uninfected hamster
was tested as cl negative control. .~11I infected animals tested positive tar
the presence of- toxin
aecnrelin~_ tc> the manulacturer's procedure. -The initiation of- treatment
then started
approximately ?.~ hr P,~st-inti:etion.
I~hr animals wore dosed daily at roughly 4 hr intervals with 1 ml antibody
preparation
diluted in i.:nsure li ( Ross (_ahs). -1'hc amount of specific antibodies
given per dose
tdctermined by aftinim purification) was estimated to he about ~LUU pg of anti-
Interval G igY
(for animals in the Interval 6 group) and 1UO Erg and 7U Elg of anti-toxin A
(Interval O-
speciticl and anti-toxin 13 (Interval s-specific: see fixample 19),
respectively. 1'or the C'T-Afi
preparation. fltc animals were treated fir 9 dais and that observed file an
additional :l d,.lvs
sU tier the presence ot- diarrhea and death. Z-he results indicating the
number ol~ survivors and the
- nunthcr of dead 4 days post-infection arc shown in 'fable '_'().
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CA 02296765 2000-O1-14
WO 98108540 PCTIUS97115394
TABLE 20
In a)m ~fherapeutic Treatment With Interval 6 Antihodiet
Treatment Group Number Animals AliveNumber Animals Dead
f'rcimmune ~4 ;
CTAf3 R (1
Interval 6 8 a
Antiho(iies directed against both Interval () and C'TnI3 successiullv
prevented death
from ( '. cli~/i('ile when therapeutically administered 24 hr after infection.
'This result is
IU significant since matte investigators hcgin therapeutic treatment ot~
11a111Ster5 w'ltlt exlSlltlg
CII'lli~ (c'.~.. \'allC()111yC1n, phcneltamyclns. IiaCUnll(:1115. llC.) H !tr
1711 t-Ilttet'11011 ~~\4'a1111)Il. c'/
ccl. ( I c)c) I ) Ilntimicrahial Agents and Chemotherapy 35:1 I U8 and 1 f
9X()) .I. Antibiotics 42:94 ~.
forty-t\vc) prrcrnt of hamsters treated with prcimmune l~.Y' died rr()n1
(.'I)AI). While
the anti-Interval 6 antibodies prevcnte(i death in the treated hamsters. they
did nut eliminate
1 ~ all svmptc)ms ut~ C'DAD as 3 vnimals presented with slight diarrhea. In
addition. unc ('~1-AH-
treated and e)nc prcimmunc-treate(t animal also had diarrlt~tt 1 ~ (lavs post-
inlcction. These
I'l,'SIIIIS 111dICat~ that anti-Interval (> antihodies provide an et~dctivc
means c)t~ therapy' I'c)r
C'I)t11).
?() EXAMPLE 17
Induction ()1' Toxin A Neutralizin~~ :Yntibodies Re(luirrs ~e)fuhlc Interval 6
Protein
:1s shown in I~.xamplcs 1 I (d1 and 1 ~. CxpreSSll)It (1t~ rC'.C()Ittblllalll
proteins in L:. c'c~li
now result in the production oh either soluble c)r insc)lublr protein. It'
insoluble prc)tein is
pre)duced. the recombinant protein is sc)luhilized prior tc) immuniratiun ut~
anltnals. ~I~e
determine \\helher. one or both of the soluble or insoluble recombinant
pre)teins could horsed
to gcnrratc neutraiiring antibodies to toxin A, the foll()\vin~_ experiment
was perfbrmcd. This
example invulve(1 a) expression of the luxtn A repeats and subl~ras~tttcnta
()t~ th ese repeats in F..
c~c~li wing a variety ot~ expression vectors: b) identification e)f
rece)mbinant toxin A repeats and
3U sub-res_ions tc) which neutralizing antibodies hind: and c) determination
ut~ the n eutralizatic)n
ahilitv ot~ antihc)dics raised aLainst soluble and ins~luhle tc)xin i~
I'epl.'ttl 1111m11I1()LCI7.
- 1 UU -
CA 02296765 2000-O1-14
PCT/US97113394
:~) Expression Of The Toxin A Repeats And Subfra~ments Of
These Repeats In E. cnli Using A Variety Of Cxpression
Vectors
The In terval G immuno~en utilized in tramples i ~ and i G was the pMA 1870-
2680
prcttcin, in which the toxin A repeats arc expressed as a soluble fusion
protein with the MBP
(clcscrihed in Example 1 1 ). (nterestin~ly. expression of this region (from
the ,SpI site to the
rnd of~ the repeats. see FiLUre i ~B) in three other expression ccmstructs, as
either native
(1~1'.~I1i70-2680). poly-llis ta~~ed (pPA1870-2680 (1-I)) or hiotin-ta~~8ed
(pf3A1870-2680)
proteins resulted in completely insoluble protein upon induction of the
hacterial host (see
hi~,:urc I ~l3). ~f ite host strain 13i.?I (Novagcn) was used for expression
ctf pI3A1870-2Ggp and
lutst strain fit.? 1 ( UI: i ) ( Nova~~ett) was used for expression ctf' pl'A
1870-2680 and pPA I 870-
?(>80t I t ). These insoluble proteins accumulated to high If'~'~1S 111
IItCIUSicllt hodies. Expression
ul~ rec<Imhinant (tIaSIttIdS 111 F.. orrli host cells grown in ?X 1'T
Iltedlullt lvaS performed as
dcscrihed [1~'illiants. m crl. (199s1..wrpncr~.
I' ;\, summarioeci in figure I~t3. e~cpression of f~agntents of the toxin A
repeats (as
either ~.tcrminal .~p~1-I:c~uRl ti~aLments. or C-terminal l:crrRl-end
fragments) also yielded
high lccls e~f~ insoluhlr protein usilt~ p(ihX (p(iA 1870-? 1 c)0). Pinpoint'"-
Xa ( pBA I 870-21 c)()
attd pI3:\??s0-?(,g0) and plT expression systems (pl'A1870-'?(c)()). I~hc
pC~IX and pE'T
expression ystcms are described in Example 11. 'I~hc Pinl'oint'"-?~a
expression system drives
_'0 the cpressiun ul~ fusion proUCins in L'. cwli. Ivsion proteins fi~c~m
1'inI'ointl"-Xa vectors
contain a biotin tai.: at the amino-terminal end and can he affinity purified
Sttftl.inl<'~' Sctfi
IZeIcaW .llldllt t'('~Ilt tf'romc~a) 1111deC Ittlld denalUl'InL cOltdlllt)1tS
(; ml\~1 hiotinJ.
~I~Itc sultlhility uf~~xprcsscd prcncins from the pl'Ci187()-21c)0 and
pl'A187()-2190
cprcssion cclnstructs was determined af~tcr induction of~ recombinant protein
expression under
conditions reported to enhance protein solubility (These conditions comprise
<~roWh o1' th r
host at reduced temperature (30°C) and the utilization of high ( 1 mM
IP'f(i) or low (0. I mM
11'~f(.i) rctncentrations oh inducer [VVilliams c°~ u/. ( lc)95).
.vtrprcrJ. ;111 expressed recombinant
toxin ~\ )trotein was insoluble under these conditions. Thus. expression of
these t~ra~~ments oC
the Iclxin ~\ repeats in pt~~T and pCiI:X capression vectors results in the
production of insoluble
1'ccomhinant protein even when the host cells are grown at reduced temperature
and urine
I«~~cr concentrations of the induccr_ Althouih expression of these fragments
in pMal vectors
yi~ldeet affinity puritiable soluble thsion protein. the protein was either
predominantly
insoluble (pMA1870-21c)0) or unstable (pMA2250-2650), Attempts to solubilize
expressed
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CA 02296765 2000-O1-14
WO 98108540 PCT/US97115394
protein ti~om the pMA I 870-2190 expression construct using reduced
temperature or lower
induces concentration (as described above) did not improve fusion protein
solubility.
C'ollcctivelv. these results demonstrate that expression of the toxin A repeat
region in
E. C'(~Il 1'eSUItS Ill the production of insoluble recombinant protein. when
expressed as either
large (aa 1870-2080) or small (aa 1870-?190 or as ??~0-2(80) fragments. in a
variety of
expression vectors (native or poly-his tagged pE'T. pGL:X or f'in1'oint'"'-
Xa,vectors), utilizing
~.row-th conditions shown to enhance protein solubility. The exception to this
rule were
1'usions with the Mfil'. which enhanced protein solubility. either partially
(pMA1870-2190) or
tllllv t I,!~-1.11870-2080).
h) Identification Of Recombinant TOxin A Itepexts And ~ub-
Rc~~ions '1'o Which Ncutralizinl; Antibodies l3ind
~Imin A rrprat regions to which neutralizing antibodies hind were identified
by
utilizin~~ recombinant toxin !1 repeat region proteins cvpresscd as soluble ur
insoluble I,rcnrins
1 s to deplete proUcctivc antihociics from a polvclonat pool of antibodies
against native C'. cli/Jicilc~
toxin :1. :\n in W m ItSSitv \1'aS developed to evaluate proteins ter the
ability t« hind
ncutralirin~~ antibodies.
The rational tier this assay is as ti~l)ows. ltccomblnant proteins were first
pre-mixed
with antii,odics n_~ainst native toxin A (C'TA antibody: generated in I:xamplc
8) and allowed
?0 to tract. W hsccluentlv. ('. cli/Jicile toxin A was added at a
concentration lethal te, hamsters
and the miwurc was administered to hamsters via IP injection. li'thc
recombinant protein
contains mutraii~inL epitopes. the C"i~A antibodies would lose their ability m
hind toxin A
resultin<.: in cliarrhea andior death of the hamsters.
1'he ttssav was pertermed as follows. The lethal dose of toxin A when
cleliverect orally
to nine ~tI) tee s0 g Golden Syrian hamsters (Sasco) was determined to he 10
to .i0 )tg. ~I~hc
I'f:(i-purified C"1~A antibody preparation was diluted to O.sX concentration
ti.e.. the antibodies
were diluted at twice the original yolk volume) in 0.1 M carbonate hutti:r. pH
~).~. l'hc
antibodies were diluted in carbonate butter to protect them from acid
degradation in the
stomach. I~hc concentration of O.~X was used becausr it was Iiluncl to he the
lowest effective
st) concentration against toxin A. 'hhc concentration of Interval 6-specific
antibodies in the O.~X
C"1'A prep was estimated to he l0-IS Et~ItW (estimated using the mcti~od
described in -
hxample 1 S).
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PCTIUS97/15394
The inclusion body preparation (insoluble Interval 6 protein: pPA1870-?680(H))
and
the soluble Interval G protein [pMA1870-2680; see Figure 1~~ were both
compared for their
ability to hind to neutralizing antibodies against C'. diJjicile toxin A
(CTA). Specifically. t to
tn,.: ct(~ recombinant protein was mixed with ~ ml of a O,SX CTA antibody prep
(estintated
tel contain h(T-7() Ey of~ Interval (-specilic antibody'). rafter incubation
for 1 hr at 37°C', CTA
(-l~rch I.abl at a final concentration of ,U Ltglml was added and incubated
for another 1 hr at
s7°C'. ()ne ml ctf this mixture containing: i0 Etg of toxin A (and 10-
IS ttg of Interval C~-
specitic antihc)cty was administered c)rally tet 40-j0 g (ioldcn Syrian
hamsters (Sasco).
IW cclmhinant proteins that result in the loss of neutralizinf: capacity of
the CTA antibody
I () would indicate that those proteins contain neutralizing epitopcs.
Preimmune and CTA -
antihcteties thctth at O.sX) without the addition of any recombinant protein
served as negative
and positive cuntrclls. respectively.
t ~~~1 other inclusion body preparations, hclth expressed as insoluble
products in the
p1:~1~ vector. ,verc tasted: one containin~~ a different insert (toxin t~
fragment) other titan
I ~ Interval (1 called pPB 18>0-''U7() (see !=inure 1 ti) which serves as a
contrcll tile insoluble
Interval (. the cltlter was a truncated version of the Interval to region
called pl'A I 87()-? I c)0
( W a (~ i~~tlrr I _i13 ). ~t~he results of this experiment arc shown in
~l~ahlc ? I .
TABLE 21
t31ttd111!_ ()1~ IVrlItralILllll AtltlbOdlcc Rv Snlmhl.. Inrnrv.,l /
O.~..r":., e....~.. r~...__.__
__ .. ..._... .....,.~rw _-,
Treatment Group' Nealthv' .".""",. rumrs
Diarrhea' Deud'
E'reinlnnmc Ah () ; ,
<.' T'.1 r1 h :T I U
<'~I~A Ah - Int b (.soluble!I , ,
rwrA Ah !rat h (insoluble)s r) U
C'~T'A Ah + pP81850-3070 5 ()
C'TA Ah - pPA I R7U-2190 > 1) U
(' rlrJ/lrilc toxin A (CTA) eras added to each group.
Animals showed no si~~ns ell' illness.
'() Animals <tevetoprd diarrhea but did not die.
' Animals developed diarrhea and died.
1'rcimmune antibody was ineffective against toxin A. while anti-C'~I~.1
antlbodtes al a
clilute O.~X concentration almost completely protected the hamster against the
enterotoxic
effects of C'TA. ~~he addition oi' recombinant proteins pPF3l 8iU-2070 or p1'A
1870-? I ~)0 to
the anti-('TA antibod\' had no effect upon its protective ability, indicating
that these
recomhtnant proteins do nett hind tc) neutraltzlng antibodies. (>n the ctthcr
hand. recombinant
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Interval 6 protein was able to bind to neutralizing anti-CTA antibodies and
neutralized the in
riw~ protective effect of the anti-CTA antibodies. Four out of five animals in
the group
treated with anti-CTA antibodies mined with soluble lnterva! 6 protein
exhibited toxin
associated toxicity (diarrhea and death). Moreover. the results showed that
Interval O protein
1111ISI he eaprcsscd as a soluble product in order I<Ir it to bind to
neutralizing alai-C'hA
.11111h()dleS SInCC', the addition of insoluble Interval O protein had n~
effect on the nculrallzltlL
capacity o1' the C"rA antibody prep.
c) Determination Of Neutralization Ability Uf Antibodies
Raised Against soluble And Insoluble 'Toxin A Repeat
Immuno~cn
l'u determine it' neutrafizin~~ antibodies are induced against soluhilizcd
inclusion
l,cuiies. insoluble toxin :1 repeat protein vas solubili~ed and specific
antibodies were raised in
cltickcns. Insoluble pl'A1870-?G8U protein was soluhilired usiy the method
describccl in
1 ~ Vl,'illiam:; rml. ( 199s). .ctcprcr. I3rictlv. induced cultures (>()(> ml)
were pcllctcd by
ucntril~uzation at .i.()UO X g tim IU min at 4°('. 'The cell pellets
were resusp cncicd thorouLltlv
in IU ml elf inclusion hctdv sonication buffer ('_~ mM HI:PES pli 7.7. lt)U mM
KC'l. 1?., mM
MgC~I.. _'()'~,;~ Llvcerol. 0.1°/~ (viv) Nonidct I'-40. I mM I)T'I~).
T~It~ StlSpe1t51()II llvtS transferred
tct a iU tttl non-glass centrifuge tube. Dive hundred EIl of ! () mgiml
lvsowme was added and
?() the tubes were incubated on ice for 30 min. The suspension was then froien
at -70°(' I'or at
Fast I hr. ~l'hc suspension was thawed rapidly in a water bath at room W
ntpcrature and then
placed on ice. The suspension was thrn sonicatcd using at least eight 1 s sec
bursts of the
microprobe (l3ranson Sonicator \lodel i~iu. ~4st.)) «ith intermittent cuoliry~
on ice.
The sunicatecl suspension was transferred to a 3> ml ()akrict~m tube and
ccntril'ugcd at
'_'j 0.000 X ~~ tier 10 min at ~t°(' to pellet the inclusion bodies.
The pellet was \vashed ? times by
pipcttin~~ or vortexing in fresh. ice-cold RIPA buffer ~U.1% SUB. 1°.a>
Triton X-10(). 1°~~
,odium dcoxvcholatc in TE3S ('~ mM 'Iris-C1 pI-1 7.~. 1s0 mM Na('1)~. 'fhe
inclusion bodies
were reccntriftlged after cash wash. 'I'hc inclusion bodies were dried and
transicrrcd using a
small metal spatula to a l > ml tube ( Falconl. (>nc ml of 10'%~ BUS was added
and the pellet
;(1 was soluhilircd by gently pipcttin~_ the solution up and down using a l ml
micropipcttctr. 'I~hc
SItIIIhIII'/.iltlUll w'LlS facilitated by beatify the sample to c)s°('
when necessary.
()nee the inclusion bodies were in solution. the samples were diluted with ~)
volun tes
of 1'(3S. ~I~hc protein solutions were dialyzed ovcrniLht against a 1 t)U-tiUd
volume of I'13S
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CA 02296765 2000-O1-14
PCTNS97115394
contaitiin~.: U.OS"/o SDS at room temperature. The dialysis buffer was then
changed to PBS
containing U.O1'%" SDS and the samples were dialyzed for scvera) hours to
overnight at room
temperature. The samples w'cre stored at 4°C until used. Prior to
further use. the sarnplcs
were \varnted to room temperature to allow any precipitated S()~ to go back
into solution.
~l~l)t' IItC'.IllSlOIt hodv preparation ~e'as Used to immunize hens. The
protein was diai_vzed
into I'135 and emulsified with approximately equal volumes of C(:A fitr the
initial
!tt)11tt1111La11C)It l)1' II~A for suhscCluent hooster immunizations. ()n day
zero. for each of the
recombinant recombinant preparations. l 'o egg laving white l.cehorn hens were
each injected
at multiple sites (IM and SC) with I mt of recombinant protein-acf.juvant
mixture containing
I() approximately U:a-1.~ mg oh recombinant protein. Booster immunizations of
I.0 mg were
~~iv'rn oi~ days I-1 and day ?R. (ggs were collected on day i'? and the
antibody isolated urine
('l:O as duacrihvCt in (:xample I~(a). Ilith titers of toxin'A specific
antibodies were present'
las assayed by I:l.l~,~. tISIItL lltl' Ittellt()d deSCrlheCl in F:vample 131.
'(~iters were detcrtttined
file h<ah anti bodies against recombinant polypcptidcs pPn187()-?(t8U and
pM/1187(1-?(t8U and
1 s were litund tc> h(: comparable at .~ ! :(?.a()0.
:lntihodi~s a;~ainU soluble Interval 6 (pMA 1870-?G8U) and insoluble interval
()
~(inclttsion body). pl'i11870-?hBU~ were tested for ncutralizin~ abifitv
against toxin A using
the in nim assay described in (:xample 1>(h). frcimmune antibodies and
atltlh(tdlel aLatltSl
lU\llt :~ (('~1:'~) SeI'1'l:Cf ilv lleLalll\'e attd p(1SILIVe COnlr(tlS.
reSpCCt1\'el\'. ~lte 1'eStIltS a!'e SIt()41'It
-~() Ilt hal,ll
TABLE 22
~lllltl'~lill:lilllll ()t t~(1\I11 n (j\ /~Itllb(1(IIr'V Aminet Cnlmhl..
I~r"r.~..l ~ n..~...:.. m..,.i_ ,
_ _ ._.__... ...m rmcr
Antibody it'eaunent Group h(ealth\' ..... .....v :-r IIVIIrS
t)iarrhcu~ Dead'
I'reinununc I () :(
CTn
() 0
tnterv~ll 6 (Soluble)' ; () ()
Interval 6 (Insoluble) () ,
_ ;
AItInIiIIS Sh()wed no sign of illness.
?() ,animal developed diarrhea but did nltt die.
' /lnintal devcl«ped diarrhea and. die(l.
-l()(1 urn ml.
Antibodies raised against native toxin A were protective while prcimmune
antibodies
had little rffcct. r1s found usiy the in oi~ro C'HO assa\' [described in
Example 8(d)j where
antibodies raised against the soluble Interval O could partially itcutraliic
the effects of~ toxin A.
here thry \verc able to completrly neutralize toxin A in virll. In contrast.
the antibodies
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CA 02296765 2000-O1-14
WO 98108540 PCT/US97/15394
raised against the insoluble Interval G was unable to neutralize the effects
of toxin A in viva
as shown above (Table ??) and In ri~rn as shown in the CHO assay described in
Example
8(d) J.
These results demonstrate that soltthle toxin A repeat in7tnunogcn is
necessary to
induce the production of neutralizing antibodies in chickens. and that the
generation of such
soluble immunogen is obtained only with a specific expression vector (pMal)
containing the
tc)xin ~1 ry_ion spanning as 187U-3()8U. ~I~itat is to say. insoluble
prUll'II7 tllal IS 51t171('C~UI:ItII~'
W )IIIhIIILICI CtUCS 17l)I rcSUII ill a IUxln A an tigcn that -Ill elicll a
IlellIralIZlltg allllt7UdV.
EXAMPLE 18
Cloning And Expression C>f The ('. cliJ)ioilr Toxin E3 (i'ene
~fhr toxin 13 gene has been cloned and sedue.~nced: the amino acid seduencc
deduced
t~rl)m the cle)ncd nuclcuticle seclucncc predicts a MVI~' ol' ?6c).7 I:I) li)r
tcwin )3 ~ I3arroso m crl..
L; Vucl. ;lcicts I~es. 18:4U()4 (Ic)9U)). The nucleotide sectuence of the
ec)dinl! rr'~ic)n e)f the entire
twin f3 ~_rm is listed in SEQ 1D N():9. The amino acid sequence u1~ the entire
toxin f3
protein Is listed in ~Ia? ID NO: l0. The amino acid seclucnce consisting of
amino acid
residues 1 tt~U through ?,()U of toxin f3 is (istcd in ~if:() lU N():l 1. ~I
he amino acid secluencc
ce)nsistin~_ c)I~ amino acid residues 1750 through ?sGU of toxin f3 is listed
in ~f:(,> IU N():l?.
'U (iiwn the expense and difficulty e)l~isolating native toxin B protein. it
would he
advanta~~ce>us to use simple and inexpensive prucarvotic expression systems to
prc)ducc and
ruril'v hi~~lt levels ut~ r~cc)mhinant toxin ii protein I~c)r immunization
pureosca. I~leallv. the
isolated 1't,'L'()111hltlalll prUlellt 14UU1(t hl S(tl11171C Ill UI'der to
preserve native antigenicity. since
solubili~rd inclusion body prc)teins otters du not ti)Id into native
cclnt'ormations. indeed as
shown in L.xam171c 17. neutralizing antihoctics against rrcctmhinant main A
were only obtained
when sc)tuhie rccomhinant toxin A polypeptides were used as tile immunogcn.
~l~u allow case
oh' purification. the rccomhinant protein should be expressed te) Imuls
~~reater than I mglliter
of I:'. w~li culture.
fo clctermine whether bleb levels c)f recombinant toxin I3 protein ce)uld he
l7roduced in
() l.. onli. 1'ra<.:ments of the toxin I3 gene were clone=d into various
prokaryotic c~cpressiott
vectors. and aW(:SSCd tell' the ability to express recombinant tct~in 13
protein in I:. cc~li. 'l~his
I'xample involved (a) cloning of the toxin E3 gene and (h) expression c)f the
toxin I3 gene in
I:. c~uli.
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CA 02296765 2000-O1-14
WO 98108540 PCT/US97115394
Cloning Of The Toxin B Gene
The toxin B gene N~as cloned using PCR amplifecation from C'. cli~~icilc
~~enomic DNA.
Initially. the gene was cloned in twc~ overlapping tcagments. using primer
pairs I'S/P6 and
f'7/I'8. ~I~hr location of these primers along the toxin I3 gene is shown
schematically in Figure
i H. -t~ltc sequence of~ each of these primers is: I'>: ~'
TAGA.AAAAATGGCAr'\tITG'I~ s' (SF;Q
II) N():1 I ): 1'(; s' TT'1'CATCTTCiTA GAGTCAAAG s' (SEQ ID NO:1?):
I'7: s' (~~11~(~CCAC'AAGA'fOA'I'TTAGTG ;' (SEQ 1D NO:I ,): and
1'8: ~' ('~fAA~fTGAGC'TGTA'1'C'AGGATC 3' (SEQ ID NC>:14).
Fipurc I R aISO SJt()1~'S the location of the followin~~ primers aloll~ the
toxin f3 hem: I'9
Il) vyhich consists of the sequencr i' ('(iGAATTCCTA(iAAAAAA'f(:i(iCAA A~fCi
s' (SIrQ ID
N():I~): I'lU which consists ot~the sequence ~' CiCI~C'TAGAATOA
C'C:ATAA<iC:T:\C.iCCr1
~' (~I:Q 1U N(>:I6): I'1 1 l4ltlCll C(lllSIStS (tf the sequence
s' ('(i(ir\~1~1"I'(.'(ii\C'rT'f(iCi~fACiAAA(iCiTGGA i' I~I~Q lU N():17): PI i
vy111Ch C(111SIStS Ot
tire srcluenc~ s' ('(:r(iAA'I"fC'CiC~TI~ATT'ATCT'I'AA(iGATCi 3' (51:Q 1D
N():18): and I'14
I s which consists of the sequence s' C'(iCiAATTCTTGATAf\CTGGA~f TTGT'GAC' >'
(SF:Q ID
V(): I ~)). ~l~hc amino acid scclucncc consisting of amino acid residues I R52
throuLh ? iO? ol'
toxin L3 is listed in SfQ IU NO:?U. l~he amino acid sequence consisting ctf
amino acid
residues 17ss through ? i6'? of toxin 13 is listed in SEQ fU N():?l .
('lo.clriclirrnr cliJ~ioile~ VPI strain IU4G; was obtained t'rctnt ihc
r\merican Type Culture
?(1 ('ollcction li\'fC'C' -l;?;i) and ~~rovyn under anaerobic; conditiow in
hrain-heart int~usiun
medium (Becton Uicl:iltsonl. Hi~_h molecular-weight ('. cliJ~icile DNA vyas
isolated esSe11t1aIIV
as drscrihecl [ ~4'ren and Tahaclchali ( 1 c)R7) .f. C lip. ~~licrohiol..
'?s:?4()'' J. except I ) 1 ()U EtI:/ml
protrinasr h in t).s",~s SDS Ovals used to disrupt the bacteria and 2)
cetytrilnethylammonlum
hrotnidc (('~I~AIi) precipitation Jas described by Ausuhcl e~ crl.. (=ds..
('rrrrern I'rvnncols irr
.1-lulerulcw I3imln,~t~, Vol. ? ( 1980) ('urrent I'rotoculs) was used to
remove carbohydrates from
the cleared lysatc. l3rietly. atier disruption of the hacteria with protcinasc
K and SDS. the
solution is adjusted to approximately 0.7 M NaCI by the addition of a II7
volume of SM
NaC:I. :\ 1110 yoiun tc o1' CTAI3/NaCI ( 10% C:'I'A13 in U.7 M NaC.'I)
solution was added and
the sulttti~n was mixed thorou~_hlv and incubated 10 min at 65°C'. An
tclual yolultt~ of
c:hlon~lorlmisoamyl alcohol (?q:l l vyas added and the phases were thoroughly
mixed. The
or~~anic and aqueous phases were syarated by centrifugation in a nticrof'uec
for i min. '1-hc
aqueous supernatant was renewed and ewracted with phenol/chlorotorm/ isoamyl
alcohol
l'~:?~: I ). The phases were separated by centrifugation in a microt'us~c for
> min. The
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CA 02296765 2000-O1-14
WO 981x8540 PCT/US97115394
supernatant was transferred to a fresh tube and the DNA was precipitated with
isopropanol.
'Che DNA precipitate was pelleted by brief centrifugation in a microtilgc. The
DNA pellet
was washed with 70~/> ethanol to re111oVe residual C~I'llL3. The DNA pellet
was then dried
and redissulved in TI: buffer ( 1(I mM Tris-FOCI pFIB.U. I mM IDTA). The
integrity anti
yield c>1~ renotnic DNA was assessed by comparison whit a serial dilution c)f
uncut lambda
UNA alter electruphuresis on an agarose gel.
'fovin I3 I'ragmcnts were cloned by 1'CR utilizing a prouf~rcadittg
thermustahle UNA
lulymcrasc ~nativc l'/ir pulvmerasc (~tratagene)). 'I'hc hi~~h lidcliw c)f
this polvmcrase
reduces the mutation prl>h1tI17S aSSOClated with amplilicatiun by e:rrc)r
prune pulvmerascs (c~.,c,~.,
I () 7irc) pc)lymerascl. 1'CR amplification was performed using the 1'('R
primer pairs I'> (SI:(,) IU
N(~:I I 1 with I'G (St:Q In N():1?) and I'7 (SI:Q ID i~():13) with I'8 (~l:Q
ID N():14) in ~0
)Il reactions cuntainin~~ I() mM Tris-I1('1 pH8.3. >U mM KC'I. 1.; ntM1
1~I~~C'I,. ?UO EtM c)f'
rash d\'1~P. ().? EIM caclt primer. and .iU n~: ('. cli//ioilr ~~enumic I)NA,
IW actic)ns were
overlaid with IU() Itl mineral oil. heated to 94°C li)r d min. l).>Itl
native I'lir pulvmerase
1 ~ (Strata«cnrl was added. and the reactions were cycled 30 11111cs at
~)4°(' li)r t min. S()°(' fi)r I
rain. 7''°C' (? min fi)r each kh c)1'scctuencc to he arttpliticd).
fi)llumed by I(1 min at 7?°C'.
1)upficate reactions were pooled. chlurutitrm c~ctractcd. and ethanol
precipitated. otter
washin~_ in 7(>"/« Uhanul. the prllets wrr~ rcsuspcndcd in s(1 Itl 'fl~.
huf'ler ( 1 () m~1 1'ris-f lC'I
pl~R.(l. 1 n1M rU'1'A).
'_() 'I'hc I';IPI) ampiificatietn product was cloned into p(!('Ic) as outlined
below. lUyl
aliyuots <)I' UNA were gel purified using the Prep-a-(ienc kit (f3iuRaH). and
Il;~ate:d to .~'mcrl
restricted plJ(' 1 ~) vector. Itecc)mhinant clones were isolated and coot
firmed by restriction
~fi~_estiun usin!~ standard rccumhinant molecular hiuluev techniques
(~arnhruok cn crl.. Ic)89).
Inserts flotm twct independent isolates were identified I11 WIIICh II)e
111\111 13 insrrt was oriented
'_'i arch that the vector l3crrrrlll and .5'crrl sites were ~~ anct s'
c)ricntrd. respcctivelv (pl~(.'Rl()-
1 i iU). The insert-containing l3crrol-IILScre~l fragment way cloned into
similarly cut ph'f?3a-
vector DNA, and protein expressi<)n was induced in small scale cultures I s ml
) of identif ied
l:lt)lleS.
-IU)tal protein extracts were isolated. resolved on ~U~-I'A(iL ~_els. and
toxin 13 protein
;() identilied by V~'cstern analysis utilizing a goat anti-toxin l~ a1'tinitv
purified antihctdv (Tech
l.ab). Procedures ibr protein induction. SI)~-PA(ib;. and WcsW rn blot
analysis were
perti)rmed as described in Vv'illiams ur crl. ( 1990. .srrl)rcr. In brief. s
ml cultures of bacteria
grown in ?XYT containing I UU Etgiml ampicillin containin~~ the appropriate
recumhinant clone
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CA 02296765 2000-O1-14
WO 9$/08540 PCT/US97/15394
were induced to express recombinant protein by addition of IPTG to f mM. The
~. cvli hosts
used were: BL21(DES) or BL21(DE3)LysS (Nova~!en) for pET plasmids.
(.'allures were induced by the addition of IPTCT to a tinal concentration of I
.0 mM
when the cell density reached 0.~ OD,,,H,. and induced protein was allowed to
accumulate for
two hrs alter induction. Protein samples were prepared by pelletin g 1 1111
aliquots of bacteria
by centritit~~ation ( 1 min in microfuge). and resuspension of the petleted
bacteria in I50 f.tl of
' '_'~ SOS-I'ACiC sample buffer (0.12 mM Tris-HCI pH G.8. '_' mM ED'rA. ~'%
SI)S. 20%
gfyccrcll. ().0?S'% hromophenol blue; (3-mercaptoethanol is added to i% hetore
use). The
samples were boated to ~)>°C for ~ min, then cooled and s or IO ~t(s
loaded on 7.S% SDS-
lU PA(iL gels. high molecular weiLht protein markers (BioRad) were also
loaded. to allow
estimation ut' the MVV «t~ identified tilsion proteins. After electrophoresis.
protein was
detected either Lcnerallv by stainine the gels with C.'oc»nassie Blue. or
specifically. by blotting
t« nitroccllulos~ tier Western blot detection of specific inttounoreactive
protein. The MW et'
induc:cd twin li rcaclivr protein allowed the integrity ut' the toxin I3
reading ti-ame to he
1~ determined.
The pl:~I~?:h recombinant (pI'B1U-I~30) expressed hiLh MVv' recombinant toxin
Q
reactive hrmein. ccmsistent with predicted values. 'hhis confirmed that ti~ame
tcnninatin~
errors had not occurred durin~_ I'C'R amplification. :~ ph;T'? ~h expression
clone containing the
I()-17s()aa interval uf~ the toxin F3 gene was constructed. by fLlsloll of the
F.cmItV-,Syel
t'ra~~mrnt of the I'7!1'8 amplification product to the l'>-L~w~RV interval c~f
the ('~!I'G
amplification product Isee I~i~~ure 18) in pl'BI()-ISSU. The inteLritv oflhls
clone lpPRl()-
17;0) m,ls confirmed by restriction mapping. and Western hint detection of~
expressed
recc>mhinant toxin E3 nr<nein. !_cvels of induced protein li~nm both pPI310-
li;() and pPBlO-
17>() were teu~ low to facilitate purification of usable amounts ol~
recombinant toxin I3 protein.
'I~hc remainin~~ 1760-2 iO0 as interval was directly cloned lllto pMAI_ or
pl;T expression
vectors ti'(1111 the I'7/('H amplification product as described below.
b) I:xprcssiun Of The Toxin t3 C:ene
i) ()vervicw ()f Expression Metlmdulo~ies
fragments of the torin E3 gene were expressed as eitlZer native or fuSll)Il
pr(7te111S Ill E.
cwli. Native prcUCins were expressed in either the pT_'r?sa-c or plTlOh
expression vectors
(Nova~~en). T'Ite pl:T?i vectors contain an extensive polylinker sequence in
all three reading
ii~amcs (a-r vectors) followed by a C.'-terminal poly-histidinc repeat. The
pl:TlGh vector
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CA 02296765 2000-O1-14
WO 98/08540 PCTIUS97/t5394
contains a N-terminal poly-histidine sequence immediately ~' to a small
potylinker. The
poly-histidine sequence binds to Ni-Chelate columns and allows affinity
purification of tagged
tarLet proteins (Williams m ul. ( 1995). .s:rprcr(. '1'hcse affinity tags arc
small ( 10 as for
pETICh. ( as tbr pET2s) allowing the expression and affinity purification oi~
native proteins
s with only limited amounts of foreign sequences.
An N-terminal histidine-tagged derivative of pL'l~l(b containing an extensive
cloning
cassette was constructed to facilitate cloning of N-terminal poly-histidine
tagged toxin 13
expressing constructs. This was aceompiished by replacement of the promoter
region oi' the
pl.'f?3a and h vectors with that of the pETIGb expression vector. I~,ach
vector was restricted
I() with 13,i,~111 and r1'd~~l. and cite reactions resolved on a 1.2 % agarosc
gel. The pE:Tl6b
promoter region (contained in a 200 by I3,s,~1I1-l~'cle~l fragment) and the
promoter-Icss pET?3 a
or h vectors were cut from the gel. mixed and Prep-A-Cicn c (RioRad) purified.
The eluted
DNA gas ligated. and transtbnnants screened for promoter replacement by ;\mr!
digestion «1~
purified plasmid DNA (the pETIGh promoter contains this sltc. the pE'r':
pr~mater does
l s not). -these clones (denoted pETI-Iisa or b) contain the pL~f 1Gh promoter
(consisting ol~ a
p~I~7-lac promoter. rihosome binding site and poly-histidinc ( I()aa)
sequence) fused at the ~Vdc~l
site to tlm cWensive pE'f2 i polylinker.
All Mi3P fusion proteins were constructed and expressed in the pMAI.'"-c or
pMAI.'"-h? vectors (New England E3iolahs) in which the protein of interest is
expressed as a
?f) C.'-terminal fusion with MC3f. All pET plasmids u-crc expressed in either
the R1.?1(I)ES) or
RL? l ( I)lr s )I,yS repression hosts, while pMal plasmids were expressed in
the 131.? I host.
f.ar~_o scale (s()t) mls to i liter) cultures of each recombinant were grown
in ?~ ~"('
heath. induced. and soluble protein fractions were isolated as described (
Wiiliams. et al.
( 1995). .wrhrcr). 'I"hc soluble protein extracts were aftiniy chromatographed
to isolate
?5 recombinant tilsion protein,. as described (Wiltiams et ul.. ( 1995) .srrpr-
u(. In hrief~. extracts
containin~~ tagged pE'1' fusions were chromatographed on a nickel chclatc
coiunm, and eluted
using imidazole salts or low pEl (pll 4.()) as described by the distributor
(NovaLCn or (~iagen).
E~:xtracts containing soluble pMAI, fusion protein were prepared and
chrornatc>s~raphcd in 1'R~
huffier over an amvlose resin (New f=ngland Riolahs) column. and eluted with
Pf3S containin~_
;() 10 mM maltose as described ( Williams er ul. ( 199i). .srq~ru~.
CA 02296765 2000-O1-14
WO 98108540 PCT/US97I15394
ii) Overview Of Toxin B Expression
in both large expression constructs described in (a) above. only low level
(i.e., less
than 1 mg/liter oC intact or nondegraded recombinant protein) expression oC
recombinant
protein was detected. A number of expression constructs containing smaller
fragments of the
toxin F3 gene were then constructed. to determine if small regions of the gene
can be
expressed tn high levels (i.e.. ~~reater than 1 mg/liter intact protein)
without extensive protein
' clcgradation. ;111 were constructed by in frame fusions of convenient toxin
Q restriction
!'ragmenis to either the pMAL-r. pL:T?3a-c, pL:Tl6h or pCTIlisa-b expression
vectors. or b_v
engineering restriction sites at specific locations using PCR amplification
[using the same
l() conditions described in (a) ahove[. In all cases. clones were verified by
restriction mapping.
and. where indicated. DNA scquencin~;.
Protein preparations from induced cultures of each ol' these constructs were
analyzed.
by ~I)~-1'i1(il~:. W estimate protein stahilitv (C:oomassic lilac staining)
and immunc~reactiviw_
against anti-toxin 13 spccilic antiserum t Western analysist. higher levels of
intact (i.r.,
ru>ndcgradcd). t'ull Icn~~th !'union proteins were observed with the smaller
constructs as
compared with the larger recombinants, and a series of exprcssiun constructs
spanning the
entire toxin l3 gene were constructed ( Figures I $. 19 and ?() and 1'ablr ?
3).
(.'onstructs that expressed significant levels of recombinant toxin !3 protein
(greater
than I ntgilitcr intact recomhinant protein) in I'. cwli were identified and a
series of these
clones that spans the toxin B gene arc shown in Figure t c) and summarised in
'!'ably ? 3.
-I'hesr c:luncs were utilized W isolate pure toxin B recombinant preUcin
f'rorn the entire totin L3
gem. ~i'~niticant protein yields were obtained from pM.4L. mpression
constructs spanning the
mtirc toxin l3 gene. and yields of f'trll length soluble fusion protein ranted
ti-om an estimated
I m!,:ilitcr culture (pMfil 1()()-1 ~_;0) to greater than ''() mgilitcr
culture (pMB17~0-?36U).
Representative purifications of MBP and poly-histidine-tagged toxin E3
recombinants
are shown in figures '?1 and '_'~. l~igurc ?1 shows a C'oomassie Blue stained
7.s".% SDS-
PlUlL: ~.:ul on which various protein scunples extracted fi~om hactcria
harboring ph-111$i()-
36(t were rlectrophoresed. ~ampics were loaded as follows: lane i : protein
extracted from
uninctuced culture: l_anc ?: induced culture protein: lane ;: total protein
t'rom induced cultttrc
3() lll~ter S()111CalrUn: Lane ~4: soluble protein: and Lane ~: eluted
affinity purified protein. Fi~~ure
?? depicts the purification ol' recombinant proteins expressed in bacteria
harboring either
pPB I 8~()-3301 ( Lanes 1-3 ) or pPB 1760-2 3b0 ( Lanes 4-O). Samples were
loaded as fol lows:
uninduced total protein ( Lanes 1 and 4): induced total protein ( Lanes '_'
and ~): and eluted
CA 02296765 2000-O1-14
WO 981118540 PCT/US97/15394
aftiniy purified protein (Lanes 3 and G). The broad ranLe molecular weiLht
protein markers
(Bioltad) are shown in Lane 7.
thus. although high level expression was not attained using large expression
constructs
from the toxin B gene. usable levels of recombinant protein w~cre obtained by
isolating
induced protein from a series of smaller pMAL expression constructs that span
the entire
toxin I3 gene.
Thcsc results represent the first demonstration of the feasibility of
expressing
recombinant toxin B protein to high levels in E. culr. :\s well_ mpression ol'
Slllall t'eglUilS Uf
the putative ligand binding domain (repeat region) of toxin B as native
protein yielded
1() insoluble protein, while large constructs. or fusions to MBI' were soluble
(ligurc 19),
demonstrating that specific methodologies arc necessary to produce soluble
fusion protein
t~rom this interval.
iii) Clone Construction And Cxpression Details
1 ~ .~\ portion of the toxin Ii acne containing the toxin B repeat region
~aminu :ICid
residues 1X~2_?362 of toxin f3 (SI:Q IU N():2U)~ was isolated by I'C:R
ampliticaticm of this
interval of the toxin B gene from ('. cliJ~irile renomic UNfI. The scyuencr,
and location
within the toxin Ii gene, of the m~o I'C'R primers ~P7 (~t(~ fU N():l ;) and
I'ti (S1-:Q It)
N():1~)~ used to amplify this red=ion arc shown in figure l~.
?() UNA From the I'C.'R amplification was purified by chloroform extraction
and ethanol
precipitation as described above. The UNA was restricted with .\prl. and the
cleaved U'Vn
was resolved by agarose gel electrophoresis. 'I~he restriction di~.:esti~m
with .~prl cleaved the
s.b kb amplitication pmduet into a 1.8 kb doublet band. This dcrublm band was
cut from the
gtl and mixed with appropriately cut. gel puritied pMALc or pFT?.pb vector.
These vectors
'_'~ were prepared by digestion vyith flincIIlI. tilling in the overhanging
ends using the Klenow
enzyme. and cleaving with .t7xrl (pMllLc) or NhcU Ipp.T?~b). 'fhe gel purified
UNA
I~ragmcnta were purified using the Prep-A-Ciene kit (E3ioRad) and the i)NA was
ligated.
transformed and putative recombinant clones analyzccl by restriction mappin~~.
pf:~l~ and pMal clop cs containing the toxin B repeat insert (aa interval 17sU-
2 36t) of
s() tr~xin t3) were verified by restriction mapping. using enzymes that
cleaved specific sites within
the toxin (3 region. In both cases fusion of the toxin t3 .~pe~l site with
either the compatible
l7orl site (pMa.l) or compatible .\'hc~l site (pET) is predicted to create an
in frame fusion. This
was cuntirmed in the case of the pMB175U-2360 clone by 1)NA scyuencing of the
clone
_ I I? _
CA 02296765 2000-O1-14
a PCT/US97115394
,junction and 5' end of the toxin Li insert using a MBP specific primer (New
England
Biolabs). In the case of the pET construct. the fusion of the blunt ended
toxin I3 3' end to
the tilled NincIIII site should create an in-frame fusion with the C-terminal
poly-histidine
sequence in this vector. The pPB 1750-2360 clone selected had lust. as
predicted. the Hirrcllll
site at this clone ,junction: this eliminated the possibility that an
additional adenosine residue
was added to the s' end of the PCR product by a terminal transferase activity
of the PJir
' polymerise. since fusion of this adenosine residue to the tilled Hiracl(II
site would regenerate
the restriction site (and was ohserved in several clones).
()nc liter cultures of each expression construct were grown. and fusion
protein puriticd
1() by af'tiniy rhromatoeraphy cm either an amylase resin column (pMAI.
constructs: resin
supplied by New L:ngland L3iolabs) or Ni-chelate column (pE~f constructs:
resin supplied hs~
(>iagcn or Ncwagen) as descrihcd (VJilliams c~ ul. (199~)..srrlr-crj. The
integrity and purity of
the fusion proteins were determined by C'uomassie stainin g of SUS-1'A(if~
protein gels as well
as Vl'estern hlm analysis with either an affinity puriticd goat polvclonal
antiserum ('fcch L.ab).
1 ~ ur .c chicken polvclunal I'E(i prep. raised against the toxin Ii protein
(CTI3) as described
ahoy in I:aamplc 8. In both cases. affinity purification resulted in yields in
excess of 2() m~~
protein per liter culture. of which greater than c)U% was estimated to hc;
lull-lell~_th
recomhinant protein. It should he noted that the poly-histidine afliniW tagged
prmein was
released ti~c~m the (~iaem Ni-NTA resin at low imidaTOle concentration ((O)
mM).
neccssitatinL the uw ol~ a ~l0 mM imidazule rather than a 60 mM imidazole wash
step during
purification.
:1 periplasmicallv secreted version of pMI31750-? 360 was constructed by
replacement
ot~ the promoter and MI31' cc~ctin~~ rc~=ion ot~ this construct with that from
a related vector
(pMill.~v-p'': Nmv l~yland l3iolabs) in which a signal seduenec is present at
the N-terminus
of the Mt31'. such that fusion protein is exported. This was accomplished by
substitutine a
I3,L~lI1-l:cwRV promoter fragment from pMAL.-p~ into pM131750-2 36(). The
yields of secreted.
affiniy purified protein (recovered from osmotic shock extracts as described
by ftiggs in
( 'rrurc-nr I'rrnucvrlv in A~Ir)IeL'irllrr' I3inlrr,~~u. Vol. 2. Ausubel. cn
crl.. Lids. ( 1989). C.'urrcnt
Protocols. pp. I 0.6.1 - 16.6.14] tt'om this vector (pML3p1750-? 36()) were
6.5 mg/liter culture.
;() of which ;()°/. was estimated to be lull-length fusion protein,
_ ~I~hc interval was also expressed in two non-overlapping t~ra~;rnents.
pMI31750-lc)7()
was cc~nstructcd by introduction of a t~ameshift into pMfi 1750-2 360. by
rcstricaion with
llincllll. tilling in the overhanging ends and rcligation of the plasmid.
Recombinant clones
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WO 98/08540 PCT/US97/15394
were selected by IUSS Ut' the HinclIlI site, and further restriction map
analysis. Recombinant
protein expression t'rom this vector was more than 20 mglliter ol' greater
than 90% pure
protein.
The complementary region was expressed in pMB 1970-2360. 'This construct was
created by removal of the 170-1970 interval at' pMR 17>U-23OU. This was
accomplished by
restriction of this plasmid with EewRI (in the pMalc pulylinker ~' to the
insert) and III, tilling
in the overhanging ends. and religation of the plasmid. The resultant plasmid.
pMB 1 c)7U-2360,
was made using both intracellularly and secreted versions of the p~ti31750-2
3(iU vector.
No fusion protein was secreted in the pMBp197U-2360 version. perhaps due to a
1l) ~onti~rmational constraint that prevents export of the t'USIOIl protein. l
lowever, the
intracellularly expressed vector produced greater than ~4Umg/(iter ot~ greater
than 90°/, full-
length t'usic~n protein.
('unstructs to precisely express the toxin t3 repeats in either pMalc
(pMBl8S()-2 3(iU) or
pl:~I~ l Oh ( pl'I318s0-? 3GU) were constructed as follows. The I)N~ interval
includinL the toxin
I ~ B repeats was I'CR amplified as described above utilizing t'C'f2 primers f
14 (~i-:~) II) N():19)
and I'R (~I-:(~ IU Nt>:I~t). I'rirner f'14 adds a F,'cwRl site inunediatelv
tlanking the surrt uf'the
toxin fi rcprats.
The amplified t~ragment was cloned into the FiI'7 Blue I-vector (Novagen) and
l'CC()Illblllflllt clones in which single copies ot~ the PCR tragment were
inserted in either
't) orientation were selected (p~l'71850-2 36U) and confirmed by restriction
mapping. ~1'he insert
was mined t'rom nvo appropriately oriented independently isolated p'I'71830-
2300 plasmlds.
with I~W~ItI (s' end of repeats) and I'.vtl lin the t7anking polvlinker ot~
the vector). and cloned
into i:cwRl/I'.wl cleaved pMalc vector. The resulting construct (pMB1850-23OU)
was
confirmed by restriction analysis. and yielded ?U mg/l of soluble tilsion
protein [greater titan
9U% intact (i.e.. nondes!raded)J after af'tinity chromatography. Restriction
uf' this plasmid with
l-lif~cllll and religation of the vector resulted in the removal of the 1 c)70-
33OU interval. The
resultant construct (pMB185U-lc)70) expressed greater than 7U mg/liter ot'c)Uc
~~ lilll length
('union hrutein.
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CA 02296765 2000-O1-14
PCTYUS97/15394
The pPB1850-2360 construct was made by cloning a EroR1 (filled with Klenow)-
l3crmHI fragment from a pT71850-2360 vector (opposite orientation to that used
in the
pMF31850-2360 construction) into Vilc~I (filled)/l3umH1 cleaved pETl6b vector.
Yields of
affinity purif7cd soluble fusion protein were 15 mg/[iter. 01' greater than
90% full length
f~USIUII protein.
Several smaller expression constructs from tl~e 1750-2070 interval were also
' constructed in Ills-tagged pET vectors. but expression of these plasmids in
the BL21 (DE3)
host resulted in the production of high levels of mostly insoluble protein
(see Table 23 and
Figure l ')). These constructs were made as follows.
pPB1850=1970 was constructed by cloning a B,~lI1-HincIlIl fragment of pPB1850-
2360
into I3,~~III,'IfirxJIlI cleaved pl:T236 vector. pPB185U-2070 was constructed
by cloning a
R,t,~/Il-I'mUl fragment of pPB 1850-?3G0 into B~~IIIIHincll cleaved pI:T2 3h
vector. pPB [ 750-
1')70(c1 was constructed by removal of the internal Hincl<I1 fragment ofa
pPR1750-3360
vector in which the vector HincIlll site was regenerated during cloning
(presumably by the
1 ? addition of~ an A residue to the amplified I'(.'R product by terminal
transterase activity of I'Jir
l~olvmerase). The pPB1750-1970(n) construct was made by insertion ol'the
insert containing
the ;~~clc I-fliucJ111 fragment of pPB! 750-2 3C0 into identically cleaved
pETHish vector. All
constructs were confirmed by restriction digestion.
An expression construct that directs expression of the 10-470 as interval of
toxin Ii
-20 was constructed in the pM ale vector as follows. A Nlre~1 (a site 5' to
the insert in the pf:T2 3
vectors-,-1/IIl (filled) fragment of the toxin B gene fram pPBIU-[SS0 was
cloned into ,.L?~crl
tc:cuopatihlc with a1'hc~1)IHincllll (filled) pMalc vector. The integrity of
the construct tpMRlO-
-l7(l) was verified by restriction mapping and DNA sequencing of the 5' clone
.junction using
a MBP specific DNA primer (New England Biolabs). Ilowever. all expressed
protein was
25 degraded to the MBP monomer MW.
A second construct spartnins~ this interval (aa 10-470) was constructed by
cloning the
I'C:R amplification product from a reaction containing the Pc) (SEQ ID NUNS}
and P10 (SI:(~
Il) N():l6) primers (Figure IR) into the pETI-Iisa vector. This was
accomplished by cloning
the P('R product as an F.'caRl-blunt fragment into F.coRl-Hir~rll restricted
vector DNA;
30 recombinant clones were verified by restriction mapping. Although this
construct (pPBlO-
5?0) allowed expression and purification (utilizing the N-terminal
polyhistidine affinity tag) of
intact fusion protein, yields were estimated at less than 500 ftg per liter
culture.
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CA 02296765 2000-O1-14
WO 98108540 PCT/US97115394
Higher yield of recombinant protein from this interval (aa 1 ()-~?0) were
obtained by
expression of the interval in two overlapping, clones. The 10-330aa interval
was cloned in
both pE~.THisa and pMalc vectors. using the l3umHl-.~1~I(I1 (filled) DNA
fragment ti'om pI'R10-
~?0. l'his fragment was cloned into Buml-11-Nincllll (filled) restricted pMalc
or l3unrHl-llincll
restricted pL:THisa vector. Recombinant clones were verified by restriction
mapping. f Iigh
level exprcwion of either insoluble (pL;7~) or soluble (pMal) lilsion protein
was ohtained. 'total
yields ef fusion protein li'om the pMBlO-33U construct (Figure lIi) were
20'mg/litcr culture.
eh which 1 ()'% was estimated to be full-length fusion protein. :~Ithoug.h
yields of this interval
were hie:her and -()0% full-length recombinant protein produced when expressed
from the
11) pFT construct. the pMal fusion was utilized since the expressed protein
\vas sotublc and thus
n~orc likely to have the native conformation.
The pMB260-520 clone was constructed by cloning l:ruRl-.l~hcrt cleaved
amplified
I)NA t'rom a I'CR reaction containing the !'11 (SE:Q li) N():17) and 1'IU
(~I=Q ID N():1h)
f)NA primers (Figure 18) into similarly restricted pMalc vector. Yields
ol~afiinitv purified
I s protein were I0 mg/liter. of which approximately 50% was estimated to hr
full-length
recombinant protein.
'hhc aai 10-1 1 1 U interval was expressed as described below. This entire
interval was
expressed as a pMal fusion by cloning the rVhrl-IlincIlll fragment ef pllClilO-
153() into ,t7or1-
Ilinclfll cleaved pMalc vector. The integrity of the construct (pME3510-111())
was verified by
20 restricti(In mapping and DNA scquencin g of the ~' clone junction using a
MBI' specific 1)N.~
primer. The yield o1' affinity purified protein was ?~ mglliter culture. oi~
which ~':~, was
estimated u~ he tilll-length lilsion protein ( i 111g/lltel'I.
l~t) altelllpt t(7 l7hlilln 111L11er \'IrldS. this m~ion \.vas expressed II1
I\\() ti'agmrnts (aaJ 10-
820. and !~?0-1 110) in the pMalc vector. ~fhe pME3510-820 clone was
cnnstructed by
insertion of a .S'crc'1 (in the pMalc polylinker s' to the insert)-Illul L)NA
fragment from
pMB5l0-1 1 10 into .S'crclhSlrrl restricted pMalc vector. The pME382(1-t 1 IO
vector was
constructed by insertion of the llpui-llincllll fragment el~ pll(.'R10-I i;0
into I3crrnHl (filled)
Ilinclfll cleaved pMalc vector. ~l'he integrity of these constructs were
verified by restriction
mapping and DNA sequencing of the ~' clone ,lLIlleIl()Il LISIIIL a MIiP
specific DNA pritncr.
s0 Recombinant protein expressed from the pME3510-820 vector \~~as highly
unstable.
Ilowcvcr. high levels (20 mg/liter) of =~()0'% Dull-tengtls fusion preucin
w~rrc e~btaincd i'rom the
pMBf320-1105 construct. The combination of partially degraded pMB51(1-111()
protein
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CA 02296765 2000-O1-14
WO 9t310t3540 PCTIUS97115394
(enriched for the S 10-820 interval) with the pMB820-1 1 ! 0 protein provides
usable amounts oC
recombinant antigen from this interval.
The aa1100-1750 interval was expressed as described below. The entire interval
was
expressed in the pMalc vector tcom a construct in which the ,4ccl(filled)-
.Sjwl fragment of
pPBlO-1750 was inserted into .S~arl/.lhcrl (Xhal is compatible with .Sj~el:
.Surf and filled Acct
sites are both blunt ended) restricted pMalc. The integrity of this construct
(pMBI 100-1750)
was verified by restriction mapping and DNA sequencing of the clone .junction
using a MBP
specific DNA primer. Although IS mg/liter of affinity purified protein was
isolated from cells
harborin~~ this construct, the protein was greater titan 99"/, degraded to
MI3P monomer size.
I() A smaller derivative ofpMB110()-1750 was constructed by restriction
ofpMBI100-
1750 with ,4j11I and .Serif (in the pMalc pofylinker ;' to the insert).
filling in the overhanging
ends. and rclieating the plasmid. The resultant clone (verified by restriction
digestion and
1)NA arc.luencing) has deleted the aal 5,0-1750 interval. wls designated pM131
100-1 S >0.
pnlti 1 1 UO- I 530 expressed recombinant protein at a yield of greater than
~0 tnglliter. of which
I ' ;°i~> was ~stimate~i to b~ full-length fusion protein.
-hhrec constructs were made to express the remaining interval. Initially. a
L3.~pHI
(tilled)-.~p-I twagmcnt from pl'B10-1750 was cloned into EcnRl(lilled)L~hcr1
cleaved pMalc
vector. 1'he integrity of this construct (pMB157U-1750) ~,~as verified by
restriction mapping
and DNA scqucncine of the 5' clone.junction usinL a MBI' specific DNA primer.
Erpression
'O ot~ recomhinant protein from this plasmid was very low. approximately i mg
affinity purified
protein per liter. and most was degraded to MBP monomer site. This region was
subscducntly expressed from a I'('R amplified DNA fragment. :1 I'CR reaction
utilizing
primers 1'IS ~SEQ ID N():18: Pls was engineered to introduce an EcoR1 site s'
to amplified
t«xin 13 scducnceaj and 1'8 (S1:(,~ ID N():14) was performed on ('. clijjicilc-
penomic I)NA as
-'S described ahoy. The amplified tcagment was cleaved with EcwRI and .SJ~cp.
and cloned into
f:cwRi/.17m1 cleaved pMalc vector. The resultant clone (pMB1530-1710) was
verified by
restriction map analysts, and recombinant protein was expressed and purified.
~l~hc yield was
~~rcatcr than ?0 mg protein per liter culture and it was estimated that 25"/"
was full-Iens_th
fusion protein: this was a significantly higher yield than the ori~!in.~l
pMi3157()-1750 Clottc.
e(1 The insert of'pMf31S30-1750 (in a EcnRl-Scrll fragment) was transferred to
fife pLTfiisa
vector (Ec~oRll.i'lrul cleaved. .l?ml and .ScrlI ends are compatible). No
detectable fusion
protein was purified on Ni-Chelate columns from soluble lysates ol' cells
induced to express
fusion protein from this construct.
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CA 02296765 2000-O1-14
WO 98108540 PCTIUS97115394
TABLE Z3
Summary Of Toxin B Expression Constructs''
Clone Affinity 'fagYield (mglliter) "
,~ hull Length
pPBlO-17j0 none low (estimated ,
from Western
plot hyh.)
pPBlO-I5;0 none low (as above) ~
pMB 10-t70 MBP I smyl ' 0,0
pl'B I ll-52U poly-his O.sm'el ''0/"
I,('t110-330 poly-his ~20m'ml (insoluble)90i~
Ir:lll3 / r)-j3rlA4L3P ? rl nr,tvl I rl'.'6,
I 0 lnt~l3?fil!-~3U A1BP l unr,x~l
lrAll35I0- I A4f3P '.inr,~vl j ,;;
l 111
pMl3s 10-830 MBP degraded (bv Western
blot
h~~bl
h t 113,~~'rl-! ,1llJl' 'rlnr,sel 'NJ.~,.
t I r)
pMBI 100-17;0 MBP I~mg~l 0"r
I J lr.lll3lll)I)-Ij3l)A4BP .lumt~l j;;,
IMBIi70-)7i0 MBP 3nterl ' ;"i~
pl'BIS.iO-1760 poly-his no purified protein
detected
y t1131 s;rl-l ~L1I3P ?r)nrs,'il
-ill
h l lt~ l;a- A~l~h ~ ~n~rr,,.,;1 ~ no~i,
~.~r a
?() i,Mf3p17;0-?3G0 MBP 6.smail (secreted)
pl'B 1710-336() poly-his .'_Om;~il ~90%
pM B I 7s0- I M BP .,(hn", I
c)70
[,Mfi I X70-23fi0MBI' 4(hngi I v)0iu
pMBplc)70-33GU MLIf' (no secretion) NA
pMf1 t Ai0-?360 MBP ''Om~ll ~90/.
pPB I 850-?360 polyh is I ~nr'~ I ,~)0%
rMB I HSO- I MBP 70ma~1 ~90/~
X70
pPt318>0-1970 poly-his ~IOmt:'I (insoluhlt:)~c)0.;,
pPBi8~0-2070 poly-his ~IOtn,;il (insoluble)~)0'~~
?() p('B17s0-Ic)70(c)poly-his Illm,.rl (insoluble)c)0/.
pl'Bl7s()-1970(n)poly-his ~IOmg.l tinsoluhlelv)U%
C.'lones in italics are clonea currently utilized to purify reconthinant
protein from each selected
interval.
ij
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EXAMPLE I9
Identification. I'uritication And Induction ()f Neutralizin g
Antibodies Against Recombinant C'. clif~icilc~ Toxin B Protein
~t~o determine whether recombinant toxin B polvpeptide tcagments can ~~enerate
neutralizing antibodies, ypically animals would first be immunized with
recombinant proteins
and anti-recombinant antibodies are ~rnerated. These anti-recombinant protein
antibodies arc
then tested for neutralizing ability in aiuu or irt airr«. f)cpendin~ on the
intmunogenic nature
of the mcomhinant polypcptide. the Leneration of high-titer antibodies against
that protein
IU may take several months. To accelerate this process and identify which
recombinant
Itolypeptide(s) may he the best candidate to generate neutralizing
alltlbodles. dr_~pleticm studies
wcr~ Imrformecf. ~peciticallv. recomhinant toxin B polypeptide were pre-
screened by testinc
vyhcthcr they have the ability to bind to protective antibodies from a C.'TB
antibody
preparation attd Ilellee deplete those antibodies of their neutralizing
capacity. ~f'hose
I ~ recombinant polvpcptidcs ti~und to hind C TI3, were then utilized to
~.;eneratc neutralizing
antihodies. This Example involved: a) identification of recombinant suh-
regions within toxin
ti to which ncutraiizin~: antibodies bind; b) identification oi' toxin 13 sub-
recion specific
antibodies that neutralize toxin f3 in riw: and c) generation and eyaluaticm
of antibodies
reactive to recombinant toxin t3 polypeptides.
~U
a) Identiiicatiun Uf Recombinant Sub-Regions Within Toxin B
'To Which Neutralirin~ Antibodies Bind
~uh-I'ILIOI1S ~~'tthln t0\111 B to vyhich neutralizing antihodies hind vyere
ictentitied by
utilizins~ recombinant toxin R proteins to deplete protective antibodies froth
a polyclonal pool
of antibodies against native ('. cli~Jicile toxin B. An in viro assay was
developed to evaluate
protein preparations for the ability to hind neutralizing antibodies.
Itecomhinant proteins were
first pre-mixed with antihodics directed against native toxin 13 (C.'~1 I3
antibody: sec f:xample 8)
atld allowed to react fete one hour at ;7°C'. Subsequently. C'.
cli//iril~~ toxin t3 (C'ff3; Tech
I.ab) vyas added at a concentration lethal to hamsters and incubated i~or
another hour at 37°C'..
s(1 Alter incubation tltis mixture vyas injected intraperitoneally (Il') into
hatosters. ll' the
recombinant polypeptide contains n eutrafizing epitopes, the ('Tf3 antibodies
will lose its
ahility to protect the hamsters against death from CTB. if partial or complete
protection
_ 1 1 c) _
CA 02296765 2000-O1-14
WO 98108540 PCT/US97/I5394
occurs with the CTB antibody-recombinant mixture, that recombinant contains
only weak or
non-neutralizing epitopes of toxin B. This assay was performed as follows.
Antibodies against CTB were generated in egg laying Leghorn hens as described
in
Example 8. 'fhc lethal dosage (LU "",) of ('. cli~%icilo toxin B when
delivered I.P. into 4Ue
female Golden Syrian hamsters (Charles River) was determined to be ?.~ to ~
Ftg. Antibodies
generated against C TB and purified by 1'L:G precipitation could completely
protect the
hamsters at the 1.1'. dosage determined above. The minimal amount of CTB
antibody needed
to al'fitrd good protection against ~ l.tg of C:TB when injected I.P. into
hamsters was also
cletermined ( 1 X PE(i prep). These experiments defined the parameters needed
to test whether
IU a given recombinant protein could deplete protective C'fB antibodies.
The cloned regions tested for neutralizing ability cower the entire to~cin B
gene and
were designated as Intervals (INT ) I through 5 (see Figure f ~)).
Approximately equivalent
final concentrations ot~ each recombinant polypeptide were tested. The
tbllowing recombinant
pulypeptldcs were used: 1 ) a mixture of intervals 1 and 2 (INT-I. ?); '') a
mixture of
1 ~ Intervals ~4 and ~ (INT-4. ~) and 3) Interval 3 (IN'I'-;). Recombinant
proteins (each at about
1 ntg total protein) were first prcincuhated with a final C~I~Ei antibody
concentration of 1X
~i.r.. pellet dissolved in original yolk volume as described in Example 1(r))
in a tinal volume
of ~ mls lire I hour at 37°(.'. Twenty-five Ftg of CTB (at a
concentration of ~ Ftg/ml). enough
C"1'B to kill 5 hamsters. was then added and the mixture was then incubated
for 1 hour at
-?0 ;7°C. Five. ~4Ug female hamsters (Charles River) in each treatment
group were then each
LIYCII 1 ml of the mixture I.1'. using a tuberculin syringe with a ?7 gauge
needle. The results
of this experiment are shown in Table 24.
TABLE 1d
t'31T1d1f1f3 ()t~ NPl~rrnls~~n" ent~iw~l;.... O., rwm -
_._.._.__._.. .,.. ....",,m
' _
f reaunent (;roup Number Of' Animals Number Of' Animals
Alive Dead
CT13 antibodies
C.'TB antibodies
INT1.3
C.'TI3 antibodies
INT4
,
C"TI3 antibodies + p
INT 3
.0 .
('. rli/Jicilr toxin B fCTE3) was added to each group.
As shown in Table 24, the addition of recombinant proteins from IN'f-1. 2 or
INT-4, 5
had no effect on the In 1'lv!) protective ability of the C.TF3 antibody
preparation compared to
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WO 98/08540 PCTIUS97/15394
the C'TB antibody preparation alone. In contrast. INT-3 recombinant
polypcptidc was able to
remove all of the toxin B neutralizing ability of the CTB antibodies as
demonstrated by the
death of all the hamsters in that group.
The above experiment was repeated. using two smaller expressed fragntents
(pMI3
170-1970 and pMB 1970-2360, set Figure l9) comprising the IN'f-3 domain to
determine if
that domain could he further subdivided into smaller neutralizing epitopes. In
addition. titli-
lcngth IN~f-; l7c,iypeptidc expressed as a nickel tagged protein (p1'B1750-
2360) was tested for
neutralizing abiliy and compared to the original IN'T'-3 expressed MBP fusion
(pMB 1750-
~360). ~('hc results arc shown in -fable ?5.
TABLE 25
Renwval Of Neutrali~inu Antihn~~im ti., uo.,~~. r~._...:._:._.- .,
Treatment Group' Number Uf Animals Number ()f Animals
Alive Dead
t't3 iltltlb(ldleS ; If
~'r(3 FIt7t117t,dIeS ~
p('t3l~i~-X3(7()
cwrt~ anuhodies pM~ I7so-?36Ut)
C"t't3 amibodics pM(i1970-3360; .,
C'Tt3 antibudirs - pMt317iU-1970
t' ~li/licvl~~ ttwin Ii (C'Tf31 was added to each ,.:roup.
~()
The results summarized in Table ?5 indicate that the smaller polvpcptidc
fragments
within the 1NT-.s domain. pMB17~0-1970 and pMB1970-2360. partially lose the
ability to
hind to anct remove neutralizing antibodies from the CTI3 antibody Pool. '(-
hcse results
demonstrate that the full length 1NT-; poiypeptide is required to completely
deplete the CTI3
'_'3 antibody pool uh neutralizing antibodies. This experiment also shows that
the neutralization
epitope ol' (NT-3 can be expressed in alternative vector systems and the
results are
independent of the vector utilized or the accompanying fusion partner.
Other Interval 3 specific proteins were subsequently tested for the ability to
renwve
neutralizing antibodies within the CTR antibody pool as described above. T'he
Interval 3
3t specific protein s used in these studies are summarized in Figure ?3. In
Figure ?3 the
Ibllow-in~~ abbreviations are used: pl' refers to the pET?3 vector: pM re(crs
to the pM~lL.c
vector: t3 refers to toxin B: the numbers refer to the amino acid interval
expressed in the
clone. 1-he solid black ovals represent the MBP: and I-I1H represents the poly-
histidinc tag.
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W'~ ~ PCT/US97/15394
Unly recombinant proteins comprising the entire toxin I3 repeat domain
(pMB17S0-
23G0. pPB 1750-2300 and pPB 180-2~(~0) can bind and completely remove
neutralizing
antibodies from the C'1'B antibody pool. Recombinant proteins comprising only
a portion of
the toxin R repeat domain were not capable of completely removing neutralizing
antibodies
li-om the C"rI3 antibody pool (pMB17S0-1970 and pMI3lc)70-2;60 could partially
remove
neutralizine antibodies while pMB18~0-170 and pI'fi18a0-2070 tailed to remove
any
neutralizing antibodies from the C'I-B antibody pool).
~Chc above results demonstrate that only the complete ligand binding domain
(repeat
region> of the toxin 13 gene can hind and completely remove neutralizing
antibodies from the
C"TI3 antibody pool. These results demonstrate that antibodies directed
against the entire toxin
13 repeat re~_ion arc necessary tUC II? 1'!t'l) toxin neutralization (see
Figure ?;: only tire
recombinant proteins expressed by the pMB1750-23f~t1. pl'B 17i()-? iGU and
pl'I31850-? ;h()
vectors arc capable of completely removing the neutralizing antibodies from
the C'I'B
,mtiboy hc~l f.
I s Thcsc results represent the first indication that the entire repeat rceion
of toxin B
wc~uld he necessary for the Leneration of antibodies capable of neutralizing
toxin F3. and that
sub-regions may not be sutiicient to generate maximal titers of neutralizing
antibodies.
Identification Of Toxin Q Sub-Region ~ihecific Antibodies
'-() That Neutralize Toxin I3 In Vivo
'i o determine if antibodies directed against the toxin B rehear region are
sufficient for
ncutra)iration. region specific antibodies within tire ("fI3 antibody
preparation were affinity
purified. and tested tier in uiw~ nctttralization. Affinity columns containine
r~combmant toxin
13 repeat proteins were trade as described below. A separate affinity colunln
was prepared
?5 using each of the Iollowin g recombinant toxin B repeat proteins: pi'1317s0-
2 i(~0. pl'i318i0-
?360. pMI317>0-1970 and pMB 1970-2360.
Ivr each affinity column to be made, four m) ol' PIi~-washed Actigel resin
(Steros!ene)
was coupled overnight at room temperature with S-!() mg of affinity purified
recombinant
protein ( f first rxtensivelv dialyzed into PBS) in I ~ ml tubes t halcon>
containing a 1 /10 final
0 volume Ald-couplin;_ solution ( I M sodium cyanohorohydride). nliduots of
the supernatants
f~l'Otll the C:(ltlpllll~ reactions. before and after coupling. were assessed
by ('oamassie staining
of 7.~'%~ ~t)~-f'A(iE Lcls. t3ased on protein hand intensities, in all cases
greater than s0'%
coupling cfticiencies were estimated. The resins were poured into l() ml
columns (BioRad).
_ 122
CA 02296765 2000-O1-14
WO 98108540 PCT/US97/15394
washed extensively with PBS. pre-eluted with 4M guanidine-HCl (in 10 mM 'I'ris-
HCI, pH
8.0) and reequilibrated in PBS. The columns were stored at 4°C.
Aliquots of a C'TB IgY polyclonal antibody preparation (PEG prep) were
affinity
huritied on each of the four columns as described below. The columns were
hooked to a (1V
monitor (ISCO). washed with PBS and 40 ml aliquots of a ?X PEG prep (filter
sterilized
using a ().45 ft filter) ~~ere applied. The columns were washed with PBS until
the baseline
value was re-established. 'the columns were then washed with BI3Stvveen to
elute
nonspecifically hinding antibodies, and rccquilibrated with PBS. Bound
antibody was eluted
from the column in 4M ~uanidinc°-HCI (in IUmM Tris-I-1C1. pHB.U). The
eluted antibody was
lU immediately dialyzed against a 100-fold excess of PBS at 4°C' tier 2
hrs. The samples were
thcll HIaI1'Ztd extensively against at least 2 changes of PBS. and affinity
purified antibody was
collected and stored at 4°C. The antibody preparations were quantified
by l1V absorhancc.
'1h c clarion volumes were in the range of 4-8 ml. All affinity purified
stocks contained
similar total antibody concentrations. ranging from 0.?5-0.35% of the total
protein applied to
I ~ the columns.
The abiliy of the affinity purified antibody preparations to neutralize toxin
B i~ viva
was cletermined using the assay outlined in a) above. Affinity purified
antibody was diluted
1:1 in PB!~ hetore testing. The results arc shown in 'fable ?l.
In all cases similar levels of toxin neutralization was observed, such that
lethality was
--'0 clelaycd in all groups relative to preimmunc controls. This result
demonstrates that antihodies
reactive to the repeat reLiun of the toxin E3 gene are sufficient to
neutralize toxin E3 in rims.
'1hc hamsters will cyentuallv die in all groups. but this death is maximally
delayed with the
('TE3 1'E:(i prep antibodies. Thus neutralization with the affinity purified
(~1P) antibodies is
nol as complete as that ohscryed with the CTB prep before affinity
chromatography. This
_'S result may be due to lass of~ activity during guanidine denaturation
(during the elution of the
antibodies ti~om the affinity column) or the presence of antibodies specific
to other regions of
the toxin E3 ~e,zc that can contribute to toxin neutralization (present in the
C.'TB E'I:G prep).
- l23 -
CA 02296765 2000-O1-14
WO 98108540 PCT/US97115394
TABLE 26
Neutralization Of Train R Rv Affinirv P~~rl~:o,1 n...;l.,.,~:....
Treatment group' Number Animals Number Animals
Alive'' Dead"
Preimmunc' ()
CTB':400 Etg 5 0
C~fB (AI' on pE'B1750-23GU):- ; ()
875 Et~;
('TB (AP on pMB1750-1970):- ~ ()
R75 Etg
CTB (AI' on pMB197U-23601:= s
500 tte
1 () ' (' eliJjic~iJ~~ toxin B {CTB) (Tech Lab: at 5 tynml, ?5 tt'; total) at
lethal ccmcentration t«
hamsters is added to antibody and incubated for one hour at ,7~(.'. After
incubation this
mixture is injected intraperitoncallv (IP) into hamsters. t:ach treatment
~aroup received toxin
premixed with antibody raised a~~ainst the indicated protein. as either: '-lX
antibody i'ECi prep
or 'affinity purified (AP) antibody (trotn CTB PEG prep. on indicated
columns). The amount
1J ol'sprciiic antibody in each prep is indicated: the amount is directly
determined f«r affinim_
purified preps and v estimated tin the 4X CfB as described in t:xamplc Is.
l he numbers in each t!roup represent number of hamsters dead or alive. ? hr
past II'
administration of toxinrantibodv mixture.
?U
-I~hv observation that antibodies at~finiy purified against the nun-
uverlappin~~
pML~l7i()-Ic)70 and pMf3lc)70-2360 proteins neutralized toxin R raised the
pussthtltty that
either I ) antibodies specific to repeat sub-regions arc sufficient to
ncutraliie toxin l3 or ?)
sub-region apecitic proteins can bind most ur all repeat specific antibodies
present in the CTB
25 polvclunal pool. This would Iikelv be due to conformational similarities
between repeats.
since he~nuolupy in the primary amino acid sequences between different repeats
is in the range
of only '_'s-7>°/. (I:ichcl-Strciber. cn crl. I lc)9?) Mulcc. (ien.
(imctic~ ?ss:?60~. l hcsc
possibilities were tested by allinity chromatography.
The ("T'B PEG prep was sequentially depleted 2X un the pMB 17;()- I c)70
column: only
s0 a small elution peak was observed after the second chromatography.
indicating that most
reactive antibodies were removed. 'This interval depleted C"I-Ti preparation
was then
chromatographed on the pPB l R50-? ,GO column: no antibody bc~unci to the
column. 'hhc
reactivity of the C.'Tf3 and C'1'B (pMB1750-1970 depleted) preps to pl'fil7~U-
2;60. pl'B1850-
i60. pME3l7aU-1970 and pML3197O-2_,GU proteins was Ihen determined by I:L.IS;1
using the
3~ protocol ~icscribed in L~xample l s{c). Hrietly. c)U-well microtitcr plates
( haleun, I'ro-Hind
Assay Plates) were coated with recombinant protein by adding 10() )tl volumes
of~ protein at
I-? ).tglml in 1'BS containing U.OOS% thimerosal to each well and incubating
overnight at 4°C.
The nest morning. the coating suspensions were decanted and the wells were
washed three
- 124 -
*rB
CA 02296765 2000-O1-14
WO 98108540 PCT/US97/15394
times using PBS. In order to block non-specific binding sites. 100 ftl of 1.0%
BSS (Sigma)
in PISS (blocking solution) was then added to each well, and the plates were
incubated for 1
hr. at 37°C'. '1-he blocking solution was decanted and duplicate
samples of 1 SO ~tl of diluted
antibody was added to the first well of a dilution series. The initial
tCSIttIL SCrlIIl1 dilution
"~~s ( 1/?OU for CTB prep, (the concentration of depleted C'fB was
standardized by ()I~,%«) in
blocking solution containing U.S% Tween 20. followed by° S-Fold serial
diiutions into this
solution. This was accomplished by serially transferring 30 ~tl aliquots to
120 ~tl buffer.
roving. and repeating the dilution into a fresh well. Alter the final
dilution. >U yl was
renewed From the well such that all wells contained 12U Ell final volume. A
total of S such
dilutions were performed (4 wells total). The plates were incubated for i hr
at 37°C.
Following this incubation, the serially diluted samples were decanted and the
wells were
washed three IIlI7CS USlllg PBS containing O.S% Tween ?0 (PRST'), followed by
two 5 min
washes wing i3BS-~E~ween and a final three washes using I'~iS'T. To each well.
IUU yl uF
I'IUU() Hiluted secondary antibody (rabbit anti-chicken !g(~ alkaline
phosphatase (Sigma)
1 ~ dilutrct in blocking solution containiy ().S% Tween ?U~ was added, and the
plate was
incubated 1 hr at 37°C'. The con.jugatc solutions were decanted and the
plates were washed 6
tithes in E'RS'1~. then once in s0 mM Na=C'O;. 10 mM MgCI,. pH 9.5. The plates
were
deyelupcd by tlm addition ol' I UU y) of a SUIUtIOtt Ccllltainlllf I m~~/ml
para-vitro phem_ I
ni~osphate (Sigma) dissolved in SU mM Na,CO;. IU mM MgCI,. pH9.S to each well.
'l~he
-'() plates were then incubated at room temperature in the dark for s-~4s min.
The absorhcnc_v oh
each well was measured at ~lU nm using a Dynatech MR 7U0 plate reader.
\s predicted by the aFtiniy chromatography results. d rpletion of the ('TB
prep «n the
pMB t 7sU-197() column renewed all detectable reactiyiy t~ the pMB 1970-23GU
protein. The
reciprocal purification of a C'~I'B prep that was depleted on tl2e pMB197U-
?;6U column
'-? vi~ldcd no hound antibody when chromatographed on the pMBl7Sp-1970 column.
These
results demonstrate that all repeat reactive antibodies in the CTB polyclonal
pool recognize a
conserved structure that is present in non-overlapping repeats. Although it is
possible that this
conserved structure represents rare conserved linear epitopes. it appears mare
likely that the
neutralizin~~ antibodies recognise a specilic protein confi~rmati~n. TI7ls
conclusion w'as also
su~~gested by the results of Western blot hybridization analysis ol' CTB
reactiviW_ to these
recombinant proteins.
Vl~estern blots of 7.S°,'° SUS-PAGE gels. loaded and
clcctrophorescd with defined
quantities of each recombinant protein, were probed with the C'7'ti polyclonal
antibody
CA 02296765 2000-O1-14
WO 98/OSS40 PCTJUS97I15394
preparation. The blots were prepared and developed with alkaline phosphatase
as described in
Example 3. The results are shown in Figure 24.
Figure 24 depicts a comparison of immunoreactivitv of IgY antibody raised
against
either native or recombinant toxin B antigen. Equal amounts of pM131750-Ic)7()
(lane 1 ),
pMI31970-2360 (lane 2). pPBi8~0-2360 (lane 3) as well as a serial dilution of
pPB1750-2360
(lanes 4-li comprising iX. 1/lOX and 1/100X amounts, respectively) proteins
were loaded in
duplicate and resolved on a 7.~% SDS-PnCil: gel. The gel was blotted and each
halt' was
hybridized with PECi prep IgY' antibodies from chickens immunized with either
native CTR or
pPR1750-? 3G0 protein. Note that the full-length pMB1750-1070 protein was
identified U111V
I(1 by antibodies reactive to the recombinant protein (arrows).
although the ('T13 prep reacts with the pP131750-2 3G0. pl'B I 85U-2300. and
pMB 1970-
''360 proteins, no reactivity to the pMI317~0-1970 protein ~uas observed
(Figure ?4). (liven
that all repeat rractive antibodies can be bound by this protein durin~~
al~finim
chromatography. this result indicates that the protein cannot told properly on
Western blots.
I ~ Since this eliminates all antibody reactivity. it is unlikely that tllc
repeat reactive antihoelies in
the C'TI3 prep recognize linear epitopes. 'This may indicate tlmt in order to
induce protective
antihe~dics. recombinant toxin 13 protein will need tn hr properly folded.
r) Generation And Evaluation Of Antibodies Reactive To
'-t) Recombinant 'toxin I3 Poly~pelrtides
i) Generation Of Antibodies Reactive To
Itecnmbinant 'Toxin I3 Proteins
Antibodies against recombinant proteins were generated in c~=g laving i.eghorn
hens as
described in Example 1 3. Antibodies were raised f using l~rmnds ad.juvant
(Clihco) unless
?5 otherwise indicated) against the following recombinant proteins: I ) a
mixture of Interval 1+2
proteins t see Figure t 8): ? ) a mixture of interval 4 and ~ proteins I see
Figure 18): 3 )
pMBlc)70-'? 360 protein: 4) pP13175()-2360 protein: ~) pMR17i0-?360: O)
pM1317s0-?360
('I'itcrmax adjuvant (Vaxcell)]: 7) pME317~0-2360 (Clcrbu adjuvant
(E3iotechlj: 8) pMI3p17:i0-
?360 protein: ~)) pI'I31850-2360: and 1t)) pMB1850-?360.
30 Chickens were boosted at least ~ tInICS wllh reCOtllblnallt protein until
Fl.ISA
reactivity (usin~~ the protocol described in b) ahllye 41'Ith till;
a\i:eptl(111 lhal 1110 plates were
coated with pl'I317~0-2360 protein] of poivclonal PF.CI preps was at )cast
ectual to that of the
CTB polvclonal antibody PEG prep. ELISA titers were determined for the I'I~:(i
preps from
_ 12(, -
CA 02296765 2000-O1-14
WO 98/08540 PCTIUS97/15394
all of the above immunogens and were found to be comparable ranging from
1:12500 to
:G2~00. 1-ligh titers were achieved in all cases except in G) pMI31750-2360 in
which stronu
titers were not observed using the Titermax adjuvant, and this preparation was
not tested
tirrther.
i
ii) Evaluation Of Antibodies Reactive To
Recombinant Proteins 13y Western Blot
Ht~bridization
Western blots of 7.5% SDS-PAGE: gels, Joaded and electrophorcscd with defined
lU quantities ot'recombinant protein (pMB1750-1970, pl'BIRSU-23GU. and pMB1970-
2360
proteins and a serial dilution of the pl'B1750-2360 tc~ allow quantification
of reactivity). were
probed with the C'~hE3. pl'B 170-2360. pMB 1750-236(1 and pMB l 970-2360
polyclonaJ
antibody preparations ( t'rom chickens immunized using Freunds adjuvant). The
blots were
hreparcd and developed with alkaline phosphatase as described shove in h).
I? :~s shoNr in figure ?4. the C"I~B and pMB197U-?3G() preps reacted strongly
with the
pJ'131 7s()-?;(,(), pl'l31 Rs0-?3G(), and pMB 1970-2360 proteins while the
pI'B 1750-2360 and
pMBlc)70-?.iGO ((ierhu) preparations reacted strongly with a1I ti~ur proteins.
The Wcstcrn
blot rcactiviy ch~ the pPBl7i()-2360 and pMB1970-2360 (Crerbu) preparations
were equivalent
to that ef~ the C'Tli preparation. while reactivity of the: pMB 1970-? 3G()
preparation was °10%
_'(1 that of the C"fl; prep. Despite equivalent L,LISA reactivitics only weak
reactivity
(approximately I°,'~) to the recombinant proteins were observed in 1'EG
preps icom two
independent ~,~roups immunized with the pMBl7>0-236(1 proncin and one ~~roup
immunized
with the pNlli I 7i()-? 3GU preparation using Freunds ad.juvant.
.lftiniw purification N~as unfired to determine if' this dil'ferencc in
immunoreactivitv by
Western blot analysis reflects differing antibody titers. Fifty m) ?X PE.(i
preparations tTOm
chickens immunized with either pM1317s0-2360 or pMB1970-2 3GU protein were
chromatographed on th c p1'B1750-2360 affinity column from h) shove. as
described. The
yield ~~f affinity purified antibody (°/~ total protein in preparation)
was equivalent to the yield
obtained from a C"fB PF:C~ preparation in b) shove. ~rhtts. dif'ferenccs in
Vl%estern reactivity
3(1 reflect a dualitative dihlerencc in the antibody pools. rather than
quantitative differences..
These results demonstrate that certain recombinant prcneins are more
ct'lcctive at generating
high affinity antibodies (as assayed by Western blot hybridization).
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CA 02296765 2000-O1-14
WO 98108540 PCT/US97I15394
iii) In Vivo Neutralization Of Toxin Q Using
Antibodies Reactive To Recombinant Protein
~fhe in vivo hamster model [described in Examples ~) and 14(b)J was utilized
to assess
the neutralizing ability of~ antibodies raised against recombinant toxin 13
proteins. The results
from three experiments are shown below in Tables 27-?9.
The ability of each immunogen to neutralize toxin B in rir« has been compiled
and is
ahown in fable 30. As predicted from the recombinant protein-CTI3 premix
studies ('fable
?~t) only antibodies to lntervai i (1750-23GG) and not the other rcgiow ot~
toxin Ii (i.e..
intervals 1-s) arc protective. Unexpectedly. antibodies generated to 1NT-3
region expressed in
I 0 pMAI._ vector ( pMB 1750-23GU and pMpB 1750-2360) using Freunds adjuvam
were non-
neutralizine. 'this observation is reproducible, since no neutralization
wasmbscrved in mc,
independent immunizations with pMI31750-23GU and one immunization with
pMpR175U-
~Gt). The tact that iX quantities oi~ af~tinity purified toxin f3 repeat
spccilic antibodies lrom
pMfil7s()-?sG0 P1:(i preps cannot neutralize toxin 13 while 1X quantities
ofat'linitv purified
is anti-C'TI3 antibodies can (Table 2H) demonstrates that the differential
ability uf' C~1'13
antibodies m neutralize toxin 13 is due to qualitative rather than
cluantitativc differences in
these antibody preparations, Only when this region was expressed in an
alternative vector
(pl'L317s()-~sGU) or using an alternative adjuvant with the pMf317s0-?;G()
protein were
neutralizinL antibodies generated. Importantly, antibodies raised using
i~rcunds ad.juvant to
-_'U pl'13185U-?.sGO. which contains a ti~agment that is only 10() amino
:tl:ICtS SIllLfllt'.l' Ihal1
recombinant pl'1317iU-?3GU. arc unable to neutralize toxin 13 in viw, ('Fable
?7): note also that
the name ucctor is trscd ti>r both pl'B I $~0-23GU and pl'F317ip-? ;G(J.
leg _
CA 02296765 2000-O1-14
WO 98I08S40 PCT/US97/15394
TABLE 27
In L7vn Neutralization Of Toxin f3
Treatment Group" Number Animals Alive'' Number Animals Dead"
I'reimmurte 0
CTf3 ~ .
r)
INTI+2
0
_ 5
INT4~5
i
pMB 17:10-2360 p
pMB 1970-2360 0
IO S
pPtlI750-2360 -
0
( ~ ~~~!!~~'i~~ toxin 13 (CTL3) (at 5 )reiml: 25 Icg total: Tech Lab) at
lethal concentration to
hamsters is added to antibody and incubated for one hour at 37°C. After
incubation this
mixture is injected intrapcritoneally (IP) into hamsters. f:ach treatment
~~roup received toxin
I ~ premixed with antibody raised against the indicated protein. as a aX
antibody PE:(i prep.
" I he numbers in each croup represent numbers of hamsters dead or alive. _'
hours post II'
administrsttron of to.vin/antibody mixture.
TABLE 28
'r() /17 t'inrr IVPtetr~lim~~.,.. rW 'r....:_ r, "
........ ~ ~.,. mmmomes
""'"", ~-um~eu
Treatment <iroupv Number Animals Number Animals Dcad''
Alive"
I'reimmunel I ) 0
C'T'E3( I ) ~
0
pP81750-2;G0( I ) s
r)
I.5 m;_ anti-pM131750_2360(2)I
4
I.i m;~ anti-pM81970-2360(2)
(>
:()() )y~ anti-CTRr2) 5
()
c cli/lic~ile~ toxin R (CTE3) (at 5 Fr;_iml: 25 )rg total:Tech I,ab) at lethal
concentration to
'() hamsters Is added to antibody and incubated for one hour at .i7°C.
After incubation. I ml of
this mixture is injected intraperitoneally (IP) into hamsters. Each treatment
group received
toxin premixed with antibody raised a~~ainst the indicated protein, as either
( I ) 4X antibody
PEG prep or (2) affinity purified antibody (on a pP81750-2360 resin), either
I.5 nrza~roup
(iuni-pMB1750-2;60 and anti-pMDt970-2;G0: used undiluted affinity purified
antibody) or 350
F~_ ~m'rrup (anti-(.'TB, repeat specific: used I/5 diluted anti-('-fB
antibody).
'fhe numbers in each group represent numbers of hamsters dead or alive. 2 hr
post-IP
administration of toxiniantibod>~ mixture.
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TALiLE 29
Generation ()f Neutralizing Antibodies lltilizin~_ The (ierbu Adjuvant
Treatment Groups Number Animals Alive''Number Animals Dead''
Preimmunc (I ;
C-TB i
pMB 1970-33G(1 0 ;
p M B I R 5(1-3; Elf l I ;
PPfi I 85()-3.ib(i (1 s
pMB17i0-3360 (Gerbu ~ p
1 adll
(f
c'. r.li~/ic~ilu toxin B (CTB) ('tech Lab1 at lethal concentration to
11~'lntSlt'r'S IS 1dded m antibody
and incubated for one hour at 37°C. Atter incubation this mixture is
injected intraperitoncally
(11') into hamsters. Each treauncnt ~~roup received toxin premmrd with
antibody raised a<,ainsr
thr indicated protein. as a 4X antibody f EG prep.
I hr numbers in each rzroup represent numbers of h;unstcrs dead ur aliyr. ?hrs
post II'
adminiatr.uion of toxin antibody mixture.
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TABLE 3U
In Y'ivn Neutrali~arinn nr r"..:., n
Antigen In viva
lmmuno~_en A d,juvantTested Preparation
tJtilized Neutrali
For AP i
''
- Preimmurte NA' PE zat
on
G NA
rto
C'TB (native) TitermaxPEG
-.
NA
yes
C'ffi 'I'itermaxAP
(natives
pPBl7;o_?;GU w
s
('T'13 7'itermaxAP
(nativW
pPB 185(1-?36Uvc
s
C'Tf3 'fitermaxAP pPBl7io-1970 yes
(native)
C-TB 'fitermaxAP
(natives
pPB197U-3360 vcs
1() lreunds PE(i
pME317iU-3_,60
NA
no
pML317iU-2sG() Frcunds AP
pPfi t 7iU-2_;6Urto
pMBl7sU-?;(,t) Gcrbu Pf:G -
NA
vcs
pMB Freund t PEG NA -
I
c)7U-?36U
nu
pMf31)7(1-3.i6() hrcuads Af pl'E317s0-?
i61)
. no
pl'fi Freunds PE(i
1760-?-iliU
NA
pl'B I~reundsl'ECi NA .
lBSU-?-~6U
no
ph-tli Frcunds PFG A-
lBSU-?.iht)
N no
IV Freunds PEG NA
f
I
-?
nu
I\'1' l-reundsPEG NA
.t
~
'
()
_
t:ither I'f-'G preparation (PEG) or affinity purified antibodies IAP).
1'm' ~Irnotes complete neutralization (U.~; dead) while 'na' denotes nu
neutralization !~ ~ dead)
al toxin l3. ? hours post-administration of mixture.
'' S
'\:~~ tlellUtlS 1101 applicable.
f~he pf'1317ip_?3(,p antibody pool confers significant in nivn protection.
equivalent to
that obtained with the affinity purified ('1-(3 antibodies. This correlates
with the uhserycd high
aflinity of this antibody pool (relative to the pMB175()-? 3G(1 er pMf31970-
?;p() pops) a,;
~() assayed by Western blot analysis (I~i~ure ?4). These results provide the
first demonstration
that in Ivtw neutralizing antibodies cnn be induced using recombinant toxin l3
protein as
jmmunu~mn.
.l.ltr failure ul- high concentrations of antibodies raised against the
pw11317s0-? 3(tl)
protein lusin~ hrcunds ad.juyant) to neutralize. ve~hile the use of Cierhu
adjuvant and
pMRl7s()-'36O protein generates a neutralizing response. demonstrates that
conformation or
presentation ut' this protein is essential for the induction ol~ neutralizin~~
antibodies. '1'hcsc
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results are: consistent with the observation that the neutralizing antibodies
produced whets
native C"~B is used as an immunogen appear to recognize conformational
cpitopes [see section
h) above, -THIS 15 the first dCIttUllSlratlon Iltat llte COItfOrJttatl()It Or
presentation of
recomhinant toxin 8 protein is essential to generate high titers ol'
neutralizing antibodies.
EXAMPLE 20
Determination Of' Quantitative And Qualitative
f)ifterences E3ctween pM1317~U-?3hU. piv91317aU-?,OU (Crbu)
<)r pl'f3175U-?3GU 1gY Polyclonal Antibody t'rcparations
In L;xample 19. it was demonstrated that toxin L3 neutralioin~.: antibodies
could he
=enerated LI111tg SpeCltlC reC()n1h111al11 t0xln (3 proteins (pl'1317aU-? 3GU)
or specific ad.juvants.
~lntihodies raised a~_ainst pM13175U-2 3(t0 were capable u1~ neutralizing the
entcrotoxin effect
<h~ twin li when the recombinant protein was used m immuniie hens in
ce~njunction with the
I ~ ( ierhu adjuvant. hut not when hrcunds ad.juvant was used. T-o determine
th c basis tier these
antigen and ad,juvant restrictions. toxin f3-specific antibodies present in
the nem~alirinh and
nun-neutralising I'f:G preparations were isolated by aftiniw chromatography
and tested for
c)ualitativr ur quantitative ditTercnccs. The example involvrd al purification
of anti-toxin li
specific antihoctics i~rum pMI317sU-2360 and pI'I317iU-?3(U I't~.(.i
preparations and h) iu uilvl
'() ncutrali~ation of toxin t3 using the affinity purified antibody.
a) 1'urificution Ofvpecific Antibodies From pMR1750-23611 :end
pl'Ii1750-2360 PE(: Preparations
Tu purify and determine the concentration o1~ specilic antibodies (expressed
as the
percent of total antibody) within the pPB1750-23(~U (Ivreunds and t~erbul and
pPHl7~U-?36U
f'ECi preparations. detincd quantities of these antibody preparations were
chromatographed on
an aftinity column containing the entire toxin Ei repeat region (pf'Lil7~U-
23OU). The amount
of af~tinity purified antibody u~as then quantified.
:\n affinity column containing the recombinant toxin 13 repeat protein.
p!'f317aU-? 3hU.
() was made as tullows. four ml of PI3S-washed Aetigel resin (~terogene) was
coupled with ~
m~~ of pt'817~()-3 3OU aftinitv purilied protein (dialysed fuse, 1'135:
estimated to he greater than
95% titll length tilsion protein) in a is ml tube (t~alcun) containing= I/I(7
final volume Ald-
coupling solution ( 1 IVI sodium cyanohorohydride). Aliquots of the
supernatant from the
_ 1;~ _
CA 02296765 2000-O1-14
WO 98J08540 PCT/US9?/15394
couplinL reactions, before and after couplin g, were assessed by Coomassie
staining of 7.5%
SDS-PAGE gels. Based on protein hand intensities. greater than 95%
(approximately ~ mg)
of recombinant protein was coupled to the resin. The coupled resin was poured
into a 1 U ml
column t BioRad). washed extensively with PBS, pre-eluted with 4M guanidine-
LIC I ( in 1 U
mM Tris-I-1C1. pli 8.U: 0.005°/~ thimerosal) and re-equilibrated in
I'BS and stored at 4°C.
Alicluots ot~ pM817SU-23GU. pML31750-2360 (Gerbu) or pl'B 1750-2 360 1gY'
polyclonal
antih«dy preparations (PEG preps) were at3inity purified on the shove column
as follows.
The column was attached to an t IV monitor (1SC0). and washed with E'BS. forty
ml aliquots
ot~?X 1'fCi preps (liltcr sterilized using a U.4~ It filter and quantified by
t7I),~" heli~re
1t) chromatcyraphy) was applied. The column was washed with P(iS until the
baseline vvas re-
e,tahliahcd lthe column flow-through was saved). washed with BBSTween to elute
nonspcciticallv hindinL antibodies and re-equilibrated with PBS. Bound
antihodv was cfuted
Drum the column in 4M guanidine-l t(.'I ( in I () mM Tris-1 I~ L, pH 8.U.
U.O()5°~~ thimerosal J and
1110 ~11111'~ elUtl()Il peak collected in a I~ ml lobe (Falcon). The
Cl)llllnll 11'i3S re-eCIUlllbfalt'.d.
1 ~ and the column cluatc re-chrc~matographed as described shove. l~he
antibociv preparations
wcr~ quantified ht' lIV ahsorhance /the elution buffer was used m ~cro the
apcctrophotometcrl. .lpproximatcly I U fold higher concentrations uf~ total
purif icd antihodv
was ahtaincd up«n elution uf' the first chromatography pass relative m the
second pass. ~I hr
low yield t~rum the second chromatography pass indicated that most ot~ the
specific antibodies
?() w cry renuwcd by the lust round o1~ chromatography.
I'mls eh al'finitv purified specific antibodies were prepared by dialysis of
the column
elutes at'tcr the first column chromatography pass for the pMB17s0-2sGU.
pMI31750-23101
((terhm en- pl'L317>0-23G0 I~~Y' polyclonal antibody preparations. l~ltc
elutes were collected
un ice and immediately ciialyred aLamst a 1UO-fold volume of 1'135 at
4°C' for ? hrs. The
,ampl~s were then dialyzed a~~ainst s changes of a OS-fold volume ul' PI3S at
4°C'. I)ialvsis
was performed for a minimum ol' 8 hrs per change of PBS. 'fhe dialyzed samples
were
collected. crntrif~uged to remove insoluble debris. quantified by ()L7,~". and
stored at 4°C'.
The percentage ot~ toxin B repeat-specific antibodies present in each
preparation was
determined using the quantitications ol~ antibody yields t'rotn the first
column pass (amount of
~(1 specific; antibody recovered after first passltotal protein loaded). The
yield of repeat-specific
at~tinitv purified antibody (expressed as the percent of~ total protein in the
preparation) in: I )
the pMB i 7;0-? 3G0 PING prep was approximately 0.~°/>. ?) the pMB i
7s0-2s(,0 (Oerhu) prep
was approximately ?.;'%. and ;) the pPB1750-2iG0 prep was approximately
t).4°/..
., .
- 1 ~ _~ -
CA 02296765 2000-O1-14
WO 98108540 PCT/US97115394
1'uritication of a CTB IgY polvclonal antibody preparation on the same column
demonstrated
that the concentration of toxin B repeat specific antibodies in the CTB
preparation was 0.35°/~.
These results demonstrate that 1 ) the use of (ierbu adjuvant enhanced the
titer of
specific antibody produced against the pMBl7S()-2 36U protein S-fold relative
to immunization
s using Freunds ad.juvant. and ?) the differences seen in the in riw~
neutralization ability of the
pMf317iU-? 3GU (non neutralizing) and pl'B17SU-2360 (ncutralirin~~l anct CTB
(neutralizing)
I'L:(i preps seen in Example 19 was not due to dltteCenCCS 111 the titers ctf
repeat-specific
antibodies in the three preparations because the titer ol~ repeat-specific
antibody was similar
for all three preps: therefore the differing ability of the three antibody
preparations to
1() neutralize toxin I3 must reflect qualitative differences in the induced
toxin I3 repeat-specific
antibodies. I'u confirm that qualitative differences exist bcm~een antibodies
raised in hens
immunized witii different recombinant proteins andlor different ad.juvants.
the same amount of
at~tiniw purilicd anti-toxin 13 repeat taa IH70-? 36U 01~ toxin f3)
atttihocties 1'r<tm the different
preparations was administered to hamsters usinL the in viau hamster nutdel as
dcacrihcd
1 s below.
b) lu vivre heutralitati0n Uf Toxin !3 UsinK Affinity Purified
Antibody
The in viv~> hamster model was utilized to asSCSS the neutralizing ability c~C
the affinity
?U purified antibodies raised against recombinant toxin fi proteins purified
in (al above. :\s well.
n ~~7v IgY I'ECi preparation from a second independent immunization utilizing
the pl'I317SU-
~_i6U anti~.!en with Freunds adjuvant was tested titr in ui~~rmeutraslization.
The results arc
shown in -1'ahle .i 1.
Tltc results shown in '('able _~ 1 demonstrate that:
?S I ) as shown in Example 19 and reproduced here. I.S mg ui' affinity
purified
antibody from pMB17SU-? 360 immunized hens using Iweunds adju4~ant does
nut neutralize toxin I3 fJ1 S'fl'!). l-fowever. 3U() tcg of a!'finiU purified
antibetdv
from similarly immunized hens utilizing CJerbu ad.juvant demonstrated complete
neutralization of toxin !3 ur rilv~. This demonstrates that (ierhu ad,juvant.
in
~(1 addition to enhancin,_ the titer of antibodies reactive to the pMi317sU-
?360
antitcn relative to l~reunds ad,juvant (demonstrated in (a) above). also
cllhances
the yield of neutralizing antibodies to this antigen. greater than S fold.
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?> C'omplete in aivo neutralization of toxin B was observed with I.~ m~ of
affinity purified antibody from hens immunized wlth pPB17~0-3360 antigen.
hut not mith pMB1750-2660 antigen. when Freunds adjuvant was used. This
demonstrates. usinL standardized toxin B repeat-specific antibody
concentrations. that neutralizing antibodies were induced when pP1317~0-2 36U
hut nut pMI317~0-2360 was used as the antigen with Frcunds adjuvant.
' s) C'ompfcte in rirn neutralization was observed with 300 Et~ of'pMB17~0-
2360
(Cierhu) antibody. but not with 300 ~t~ of pPB17;0_2360 (hreunds) antibody.
l~llus the pR9B1750-2 36U ((ierbu) antibody has a Iti~her titer of
neutralising
antibodies than the pPBl7~U-2360 (Freunds) antibody.
-t) ('ompfetc neutralization of foxier 13 was observed using 300 yg o1' C"fg
clntihody ~aftinity purified tA1')) but not 100 Erg CT)3 antibody (A!' or
I'I:G
hrcp). This demonstrates that Lreater than IOU ~t~~ of toxin R repeat-specific
.lntibocfy tanti-C"I~B1 is necessay to neutralize ?~ ~tc~ toxin I3 in aion in
this
I ' clssay. and tltat af'linity purified antibodies specific to the toxin B
repeat interval
neutrali-rc toxin (3 as effectively as the PFP prep of I~Y raised against the
entire C'TB protein (shown in tltis assay).
~a was observed with the initial pl'B17S0-?3G0 (IgY) 1'L:(i preparation
I E:xalnple 1 ~)), contplete neutralisation was observed with a I~~' f'f-:(i
t~reparation isolated from a second independent group of pPBl7~U-23GU
( Frcunds) immunized hens, 'this demonstrates that ncutralizinL antibodies are
reproducibly produced when hens art immunized with p!'E317iU-? ;lO) protein
IIIIIIlInL Freunds ad,juvant.
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TABLE 31
In viva Neutralization OC Toxin B Using AClinity Puritied Antibodies
Treatment Group'' Number Animals Number Animals Dead''
Alive''
Preimmunc' ()
CTB ( 3U0 Ey~)' i 11
C'TR ( I UU Ey,1- I '1
pMI317;U-33GU fG) (~ m~_)-; ()
ItME317sU-2.i GU ((i) ( ; (t
I.i m~=Y
rMBl7sU-2360 (Ci) (3UU i 0
N~;1'
I pMI317iU-23GU (F) ( I.i ()
() m~~)'
pPB 17it1-2360 (t~) ( I ; tt
.i m;~l
pl'I317iU-2.i6U (f) (;()()I
ly;)-
('TI3 ( lU() Ec_)' '- .
pl't317iU-?3 GU (F11~t1() ; It
y=)
I>
(' cli//icih~ toxin f~ (C'~TIi) (~I'cch Lah) at lethal concentration to
hamsters ('_> )t;;) w;ts added to
tlmantibody (amount oC specific antibody is indicated) and incubated lbr one
hour at .7°C.
.1t'ter incubation. this mimure was injected If into hamsters I I's total mix
ipected per
hamstrrl. Fach treatment ~_roup received toxin premixed with antihoch_ raised
a_ainst the
indicatrd protein ((i w~erbu ad.juvant. f~--1-reunds adjuvantt. ' indicates
tltr antthody v, as a -IX
I_Y I'f.(i prcl: ~ indicates the antibody was ~l~tinity purified on a
pl'I318s()-?.il>U resin: and
indicates that thr :tntihody was a I X.I~~Y Pf:G prep.
' lhmumbers in each =group represent numbers of hamsters dead or alive. _' hrs
post II'
:utministration at toain:antibodv mixture.
t?XAMYLI; 21
Diagnostic t:n71'111e Immunoassays for ('. cliJJicilc~ 'l oxins 11 Anti l3
() The ability of the recombinant toxin proteins and antibodies raiset.l
against these
recombinant proteins (described iv the above examples) to form the basis of
111aLlle)Sfle aSSaYS
hor the detection W~ clostridial twin in a sample was examined. Two
immunoassay fitrmats
were tested to ctuantitativelv detect ('. cli/Jicile to~(in r1 and toxin 13
t'rom a hioloLical
specimen. ~fhe first tbrmat involved a competitive assay in which a fixed
amount ot~
3i recombinant toxin A or fi vav immobilized on a solid support lc'. ~.,
microtiter plate v.~ellsl
f'olloveci by the addition ol' a toxin-containing hiolo~~ical spcimen mixed
with aCtinity-
petrified or E'E:C~ fractionated antibodies as:ainst recombinant toxin ~\ or
ti. t('toxin is present
in a specimen. this toxin will compete with the immobilized recombinant toxin
protein t~or
l3G -
CA 02296765 2000-O1-14
WO 98108540 PCT/US97/15394
hindine to the anti-recombinant antibody thereby reducing the signal obtained
following the
addition of a reporter reagent. The reporter reagent detects the presence of
antibody bound to
the immobilized toxin protein.
In the second format. a sandwich immunoassay was developed using affinity-
purified
antibodies to recombinant toxin A and B. 'T'he affinity-purified antibodies to
recombinant
toxin :1 and E3 were used to coat microniter wells instead of the recombinant
polypeptidcs (as
mas done in the competitive assay format). Biological samples containing toxin
A or R were
then added to the wells followed by the addition of a reporter reagent to
detect the presence
ul' hound t()XI11 111 the well.
a) Competitive Immunoassay For The Uctecti0n Uf C: ~liJficile
Toxin
Recombinant toxin r\ or t3 was attached to a solid support by coating ~)(,
well
microtiter plates v~ith the toxin protein at a concentration of lEtgiml in
I'BS. The plates were
I s incubated overnight at ?-8°C'. ~fhe IollowinL morning. the r«atinf!
solutions were removed
and the remaining protein bindinu sites on the wells were blocked by tilling
each wu>II with a
I'f3~ st~lutic~n containing O.s'%' (i~,A and 0.05% Tween-?0. Native ('.
cIiJJic~iJc~ toxin ~1 or B
( fcch I.ahl was diluted to 4 ~ylml in stool ertracts from healthy Syrian
hamsters (Sasco).
~I~hc; stool mracts were made by placing fecal pellets in a l s ml centrifuge
tube: PBS was
'_'0 added ut ? ml/pcllet and the tube was yorte~ced to create a uniform
suspension. The tube was
then ccntrif~ugrd at ?OOU rpm for ~ min at room temperature. ~I~hc supernatant
was removed:
this pmprises the stool extract. fifty frl of the hamster stool extract
w°as pipetted into each
wc;ll ol~ the microtiter plates to serve as the dilucnt for serial dilutions
of the ~l pp/ml toxin
samples. ()ne hundred Etl of the toxin samples at ~ ~tg/mi was pipctted into
the first row of
vyclls in the microtiter plate. and ~U yl aliquots were removed and diluted
serially down the
plate in duplicate. An equal yc~lume of affinity purified anti-recombinant
train antibodies [ !
n~;/wel( ol' anti-pV1A1870-2h80 antibody was used for the detection of toxin
~1: 0.5 ngwvell of
anti-pMI317~0-23Ot)(<icrhul was used for the detection of toxin B) were added
to appropriate
wells. and the plates were incubated at room temperature for ? hnurs with
gentle agitation.
s0 Wells serving us ny~atiue control contained antibody but no native: toxin
to compete for
hindins~.
Unbound toxin and antibody were removed by washing the plates 3 to ~ times
with
PBS cotUaining ().(l~% Tween-20. f()110Wrt1L the lyaSl7 SICp. 100 ltl of
rabbit anti-chicken IgCi
IJ7 -
CA 02296765 2000-O1-14
WO 98108540
PCTIUS97I15394
antibody conjugated to alkaline phosphatase (Sigma) was added to each well and
the plates
were incubated for 2 hours at room temperature. The plates were then washed as
before to
remove unbound secondary antibody. Freshly prepared alkaline phosphatasc
substrate ( 1
mgiml p-nitrophenvl phosphate (Sigma) in iU mM Na,C'(>:. pl-1 ~).s: 1() mM
MLC1=) was
a added to each well. Once sufficient color developed. the Plates were read on
a 1)ynatech
VIR7UU microtiter plate reader using a 410 nm filter.
~~hr results are summarized in Tables 32 and 3 i. 1. r the I'e5U1C5 S1117w11
111 'liable o?.
the wells were coated with recombinant toxin i1 protein (pMAIH7()-?O8()). I~h~
amount e~t~
native toxin n added (present as an addition to soluhilized hamster stool) to
a Liven well is
1() indicated (U to ?UU ng). Antibody raised against the recombinant toxin A
protein. pMn1870-
?680. was affinity purified on the an affinity column containin~~ pt'A1870-
?68U Ldcscrihed in
f~xumple 2U). As shown in Table i'_'. the recombinant toxin r\ protein and
affinity-purified
antitoxin can be used for the basis of a competitive immunoassay tier the
detection of toxin A
in biological samples.
) s vimilar results were obtained using the recombinant toxin fi. pI'B I7sU-?
~(,U. and
antihudirs raised a~~ainst pMB17s0-2i(0((icrhu). For the results sh«wn in -
f.lble ss. the
wells were coated with recombinant toxin li protein (pI'1317~()-?3(iU). ~1~11c
allulttnt of native
toxin li added (present as an addition to solubili~ed hamster stool) to a
Liven well is indicated
(U to ?U(1 ng). nntihodv raised against the recombinant toxin R protein.
pME3t7~0-
'U '3(~0(( ~crbu). was affinity purified on the an af~tinity column containing
pl'Ei f 8s()-? >(~U
(described in L:xampfe ?U). ~1s shown in ~t'able 3. the rcconthinant toxin li
protein and
affinity-purified antitoxin can he used for the basis ol~ al Wlllpellll\'e
In111111no,1v1.1~ fi,r the
detection oi~ toxin 13 in biological samples.
In this competition assay, the reduction is considered slLnltlcant over the
background
?s levels at aII pollltS: therefore the assay can be used to detect samples
containinL less than 1?.s
ng toxin A/well and as little as sU-lUU ng toxin tilwell.
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CA 02296765 2000-O1-14
PCTlUS97I15394
TABLE 32
Competitive Inhibition Of Anti-C. dij/icile Toxin A Iiy Native Toxin A
n~~ Toxin AlWell OD"" Readout
?00 U.17G
100 0.253
() 0.240
i 0.259
1'_'.5 0.309
G.25 0.367
ll) .i.l3i 0.417
(1 O.i90
TABLE 33
( umpetitivc Inltihitiun ()f nnti-C. cliJ/iolr Tuxin B By N;nim i~oxin l3
n~ Twin t3; Well OD"" Readout
200 t1.39?
100 O.sG6
>t) t).G(>7
0.778
I'_'.s 0 970
6.2 s I).003
i.l?S 1.040
0 I.Oii
-hhese competitive inhibition assays demonstrate that native C'. cli/~icilo
toxins and
recombinant ('. ciifJicilr toxin proteins can compete fir binding to
antibodies raised against
recombinant ('. clijIicile toxins demonstrating that these anti-recombinant
toxin arrtihodics
provide ett~cetive diagnostic reagents.
:() h) sandwich immunoassay For The Detection ()t' C rlifficilc~
Toxin
- ~1t'finity-purified antihcuiics a~_ainst recombinant toxin i1 or toxin I3
were immohilircd
to c)O »~ell microtiter plates as ti~llows. The wells were passively coated
overnight at 4°C'
_ with affinity purified antibodies raised against either pMA1870-?b8() (toxin
A) or pM1317~Q-
_ 13() _
CA 02296765 2000-O1-14
WO 98!08540 PCTIUS97I15394
?360(Cicrbul (toxin B). The antibodies were affinity purified as described in
Example ?0.
The antibodies were used at a concentration of I ttglml and 100 ~tl was added
to each
microtiter well. The wells were then blocked with 200 Eel ut~ 0.5% BSA in PBS
for 2 hours
at room temperature and the blocking solution was then decanted. Stool samples
from healthy
s wrian l3amsters were resuspended in PBS, pll 7.4 (? ml l'l3S/stool pellet
was used to
rcsuspend the pellets and the sample was centrifuged as described above).
'l~he stool
suspension was then spiked with native ('. c!i/)icile~ toxin A ctr B (~fceh
Labl at 4 Etg/ml. 'fhc
stool suspensions containing toxin (either toxin 11 or toxin B) were then
serially diluted two-
fuld in stool suspension without toxin and 50 Eel was added in duplicate: to
the coated
f 0 microtiter wells. Wells containing stool suspension without toxin served
as the nes=alive
control.
('h r elates were incubated for ? hours at room temperature and then were
washed
three tunes with I'B~. ()ne hundred Eel uf~ either goat anti-native W xin A or
~ctat ant!-llatlve
toxin 13 l lmh l.ah) dilute! 1:IUUU in PBS containing I°'° f3~n
and O.Ui°/, ~IVvecn ?U was
is added tct cae:h well . The plates were incubated for another ? hours at
roam temperature.
I he plates were then washed as before and 100 Eel of alkaline phnsphatasc-
cctn.jugated rabbit
anti-goat Ig(i I('appcl. Durham. N.C'.) was added at a dilution H~ I:IUUU,
l~hc platen were
incubated fur anoth er ? hours at room temperature. The plates were washed as
hetitrc then
developed by the addition of 100 fel/well of a substrate solution containing I
mg,%ml p-
?U nitrophenvl phosphate (sigma) in ~0 mM Na,C()., pl-i 0.~: lU mM MgC'I,. 1-
!te ~thsctrhance of
such wrll was measured using a plate reader (I)vnatrchl at -11() nm. The assay
results arc
shown in I~ahles ;.1 and i5.
'T'ABL~ 3d
('. cli//icilr lo~cin A Detection In Stool tJsins: At'tinitv-Purified
Antibodies Acainst Twin A
n 'Toxin AlWell --- OU,~" Readout
2oU U.9
I t)(> (1.H
;(1 t1.7,
'-~ (1.71
.i0 ~ I ~ ~ - _ (LSt)
r,.'; (Lay t -
o t1
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WO 98/08540 PCT/US9'1115394
TA8LE 35
e' cli/jicilc~ Toxin B Detection In Stool Using Affinity-Purified Antibodies
A«ainst Toxin R
m_ Toxin B/Well OU"" Readout
3U0 I .3
I UO 0.97.s
50 0.887
' '_ > U.84 (i
I'.5 U.GS I
G.? ~ 0.431
I I1 U 0.004
I
l-hu results shown in Tahles s:~ and 35 show that anUbodles raked against
recombinant
twin :1 and tcwin 13 li'agments can be used to detect the presence ol' ( '.
cJiJJic~ilr toxin in stool
samples. ~I~hesc antihodics form the hasis for a sensitive sandwich
immunoassay which is
capahle «t' detecting as little as l.?i n~~ of either toxin A ar (3 in a ~0
ttl Stool sample. As
shown ahcwc in Tables s~1 and 3s. the background for this sandwich immunoassay
is
cwremelv Imv: therefore. the sensitiviW ot~ this assay is much tower than O.?s
ng toxin/well.
It is likely that toxin levels oh~ 0.; to i.() pg/wcll could be ctctected by
this assay.
The results shown above in Tables 33-35 demonstrate clear utility ot~ the
rccomhinan t
''() rca~=ents in t '. cliJ)icilo toxin dctcction systems.
EXAMPLI22
(.'onstruction And f~xpressic~n ()C (', hrmrlintrnr C' Fragment lesion
1'rotcins
?5 The ('. hnmlinrrnr type n llelll'OlOxlll gene has been cloned and sequenced
[Thompson.
rt crl.. l;ur. .I. I3iochem. 189:73 t Ir)r)())~. 'the nucleotide sequence of
the tUxltl ~enc is
available t'rom the FMf3L/Gent3ank s~qucnce data banks under the accession
number X2066:
the nucleotide sequence ot~ the cudin~~ region is fisted in SCQ lI) NO:?7.
~l~ttr amino acrd
seclucnee ui~ the ('. hvtarlimrm wpc n neurotoxin is listed in SL:Q 117
N():?8. '7~he ypc n
s() neurotoxin ~~ene is synthesized as a single polypeptidc chain wltich is
processed to term a
diner composed of a light and a heavy chain linked via disulfide bonds. The 50
kD carb~xv-
terminal portion of the heavy chain is referred to as the C fragment or the I
I,. domain.
Previous attempts by others tc> express polypeptides comprising the C
i~ragment of ('.
hrmrlintrnt type n toxin as a native polypeptide (c~.,s,~.. not as o fusion
protein) in E. coil have
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WO 98108540 PCTNS97I15394
been unsuccesstitl [H.F. LaPenotiere. v al. in Bolulinum and Tercrnu,v
rVem~otvxin.r. UasGupta.
Ed.. Plenum Press. New York ( 199 3). pp. 463-46C]. C:xpression of the C
fragment as a
fusion ~~itlt the T. coli MBP was reported to result in the production of
insoluble protein
(H.F. LaPenotiere. er crl,. .suhru).
In order to produce soluble recombinant C'. fragment proteins in E. cwli.
fusion proteins
comprising a synthetic C fragment eenc derived from the ('. hurrrlinum type-A
toxin and
wither a portion of the ('. cliJJicile~ toxin protein or the MBi' were;
constructed. this example
involvc:ci a) the construction of plasmids encoding (.' Iragment fusion
proteins and b)
expression of ('. hnlcrlinrrm C' fragment fusion proteins in F.. cwli.
a) ('.onstruction Of Ptasmids Encoding C Fragment Fusion
Proteins
In f:xampfe 11. it was demonstrated that the ('. cli~Jicilr toxin A repeat
domain can be
el'ficientlv expressed and purified in F.. cwli as either native (expressed in
the pE::'1' ?3a vector
1; in clone pI'A1870-2G8()) or fusion (cspressed in the pMAl,c vector as a
fusion with the l:.
cwli Mf31' in clone pMAlR7()-268()) proteins. lesion proteins comprising a
fusion between
the MIi1'. portions af' the ('. di/Jirile toxin A repeat domain (shown m he
expressed as a
soluhlc fusion Protein) and the C fragment of the C'. horrrlirrunr ype A toxin
were constructed.
r1 Iltsion protein comprising the (.' fragment of the ('. horulinrrrn type n
toxin and the MBI'
?0 was also cc»tstructcd.
I~iLUrc: ?s provides a schematic representation of the hotulinal fusion
proteins along
with the donor constructs containinL the ('. clif~icile toxin A sequences or
('. hutulmrrnr ('
fragment scclucnces which were used to _~eneratc the hotulinaf fusion
proteins. In higurc ?~.
the solid hexes represent ('. cli/~ic~ite toxin A gene seduences. the open
boxes represent ('.
2s hnnrlirrum C' fragment sequences and the solid black ovals represent tltc
F. ruli M13P. When
the name for a restriction enzyme appears inside parenthesis. this indicates
that the restriction
site was destroyed during construction. ~1n asterisk appcarin~ with the name
for a restriction
enzyme indicates that this restriction site was recreated at the cloning
.junction.
In lviLUre 25. a restriction map of the pMA1870-2(80 and p1'nl 1()0-2l8()
constructs
,t) (described in Example 1 1 ) which contain sequences clcrived from the ( '
cli~/irilr toxin n
repeat clomatn arc shown: these constructs were used aS the source (1I~ ('.
cliJ)irilr toxin A gene
sequences tier the construction of plasmicis encoding fusictns between the (
'. Imrtrlirurrn C'
fragment ~~rtte and the ('. cli~jioilc~ toxin A gene. The pMA1870-2080
expression construct
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CA 02296765 2000-O1-14
WO 98108540 PCT/US97115394
expresses high levels of soluble. intact fusion protein (?0 mg/liter culture)
which can be
affinity purified on an amylose column (purification described in Example 1
ld).
The pAlterBot construct (Figure 2~) was used as the source of (.'. hmuliraum C
fragment ~_ene sequences f'or the botulinal fusion proteins. pAIterBot was
obtained from J.
s Middlebroul: and R. Lemley at the U.S. Department of Defense. pAlterliot
contains a
synthetic ( '. hrnrrlir~inr~ C' twagment inserted in to the PALTER-1 (N;
vector ( Protncga). This
synthetic C' fragment gene encodes the same amino acids as does the naturally
occurring C'
fragment gene. The naturally occurrin~~ (.' tiagment sequences. Like most
clostridial genes. are
extremely A'T rich (~l'ltompson m crl.. .srrprcr). This high AIT content
creates expression
I() difficulties in !:. ccrli and yeast due to altered codon usage frequency
and fortuitous
pulvadenvlatiun sites. respectively. In order to improve the expression of ('
ti-agment proteins
in I:~. cmli. a wnthetic version of the gene was created in which the non-
preferred cuctuns were
replaced with preferred cudons.
I'lte nucleotide sequence of the ('. hurulir;'rrnr C' fragment gene sequences
contained
within p:\Iterf3ut is listed in SI~Q IU NO:'??. The first six nucleotides
IAT(iGCT) encode a
117e1111()Iltlte and alantne restduc. respectively. These two amino acids
result from the insertion
of thr (' h~mrlirrrrm C' fragment sequences into the pALTI:R'J vector and
l,ruvide the initiator
mcthiunirn residue. The amino acid sequence of the ('. hrmrliraruu C' fragment
encoded by the
seducnces contained within pAltcrlW t is listed in SCQ ID NO:?3. The first w~u
amino acids
?U lMet i\lal are encoded by vector-derived sequences. From the third anonu
acid residue
c,nward 1:\r~~l. the amino acid seduencc is identical to that found in the l'.
hunrlirrum type A
tU\I11 ~_C11~.
I he pMA l 870-'_'68U. 11'A 1 1 (1()-2080 and pAlterBut constructs were used
as progenitor
plasmicis m make expression constructs in which t~agments of th~~ ('.
cliJjicilr toxin A repeat
_'~ domain were expressed as genetic fusions with the ('. houulirrrrnr C'
fragment gene using the
hMAI..-~ wpression vector (New E:nLland Biol.abs). The pMAI_-c expression
vector
generates fusion proteins which contain the MBP at the amino-terminal end of
the protein. A
l:UllSh'llel. pME3ut. in which the ('. hruulinrrm C fragment iene was
expressed as a fusion with
unit' the ~~13P was constructed (FiLUre'~). Fusion protein expression was
induced Irom E.
_~(1 cwli strains harhoring the above plastnids. and induced protein vvas
at'tinitv purified un an
amyluse resin column.
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WO 98108540 PCTIUS97115394
i) Construction Uf pBlueBot
In order to facilitate the cloning of the ('. hmrrlinru>r C fragment gene
sequences into a
number of desired constructs. the botulinal gene sequences were removed tiom
pAIterBot and
were inscrtc;d into the pt3lucscript plasmid (Stratagenel to s~cncrate
pE3iue13ut (I~i~~ure ?5).
s pl3lucl3ot was constructed as t«Ilows. bacteria containing the pAlterl3ot
plasmid were gown
in medium containing tetracycline and plastnid DNA was isolated using the
(?IAprep-spin
I'lasmid fit ((~iagen). ()nc microgram of pAIterI3uo DNA was digested with
;\~cwl and the
t'CSUIlIItl: s' recessed sticky end was made blunt using the Klenuw fragment
of t)NA
pulvmerase 1 (here after the Klenow tiagmcnt). ~I~hc pA lterl3ut DNA was then
di~~ested with
It) I~incllll m release the botulinal gene sequences (the But insert) as a
blunt (filled ,~'rul sitc)-
I-lincIlII fragment. pl3lucscript vector DNA was prepared by digesting '_'0()
n~= of pt3lucscript
DNA with ,5'rrrcrl and flinclIII. ~l~hc digestion products Iroltt both
plasmids were resolved on an
~tLaros~ ~_cl. ~hhc appropriate fragments were renewed t~rom the gel. mixed
aitd huritied
utilitin~~ the Prep-a-W ne kit (l3ioRadl. The eluted DNA was then li~_atcd
usin~_ ~T-l I)NA
is li~asc and used to transtorm competent I)IiSa cells ((iihcu-13111.). Must
cells were made
c;umpetent fur transt«rmatiem using the calcium chloride protocol ul'
Samhruul: ur crl.. .orrp-cr at
l.f(?-1.8s. Recombinant clones were isolated and confirmed by reatrictiun
di!_cstian using
standard recombinant molecular bioluey techniques t~ambrouk cr crl. .strlmcr).
'hhe resultant
clone, p131ucIW t. contains several useful unique restriction sites Ila11k11tL
the Rut insert (i.e..
'_U the ( '. hrurrlinrrnr C' f~raLment sequences derived from pAIterHot 1 as
shown in I~ i~~urc ? ~.
ii) (.'.onstruction ()f C: difjcile l G hnlulirrrrnr I
NBP Fuxion Proteins
t'unstructs encoding fusions between the C'. cliJ~irilr toxin r1 gene anti the
t'. hnrulinunr
'_'s C' lragntcnt gene and the MI3t' were made utilizing the same recombinant
D NA methodology
outlined above: these fusiolt proteins contained varying amounts of the ('.
c!i/%icilc toxin A
repeat domain.
I~hc pMAf3ot clone contains a '_'.4 kb insert derived ti-um the: ('.
clif)ic~ilc toxin A gene
Mused to the (iut insert (i.e. the C'. hrnrrlirarrrn C' lratmcnt sequences
derived from pAlterf3ot).
;(> pMABW (Figure '_'s) was constructed by mixing gel-purified 1)NA from
:\~rnllllimllll di~~ested
pl31uel3ut (the l.'_' kh But fragment). ,~pcUINurI digested pl'Al lUt.)-2O8()
(the ?.~ kb ('. cliJ~ic~ilc~
toxin A repeat fragment) and .1'hcrlltliracllll digested pMAI.-a vector.
Recombinant clones
were isolated. confirmed by restriction digestion and purified using the
(~lAprcp-spin t'lasmid
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CA 02296765 2000-O1-14
gyp ggJpg~p PCTNS97115394
Kit (Qiagen). This clone expresses the toxin A repeats and the botulinal C
fragment protein
sequences as an in-frame fusion with the MBP.
The pMCABot construct contains a l.0 kb insert derived tiom the C'. diJficile
toxin A
gen a Fused to the Bot insert (i.c~. the ('. hultelinum C ti~a~ment sequences
derived tiom
s pAIterBot). pMCABot was constructed by digesting the pMABot clone with
F'coRt to
remove the i' end of the ('. cli/~irilc~ toxin A repeat (see Figure '_'~, the
pMAL.-c vector
contains a I~ewRl site s' to the C'. cli~~icilc~ insert in the pMAf~ot clone).
The restriction sites
were tilled and rcligated together after gel purification. The resultant clone
(pMCABot.
Fi~~ure ?s) ~_enerated an in-ttame titsion between the MI3P and the remaining
s' portion of the
1 U ( '. cli/Jicilr toxin A repeat domain fused to the Rot gene.
t ire pMNAL3ot clone contains the I kb SIeIIEcnRI (tilled) fragment from the
('.
cliJjicilc~ toxin A repeat domain (derived from clone pPAI 1UU-2680) and the
!.~' kh ('.
l~rmrlimrrrr C' Fragment gene as a .\~onl (filled)/tfinc1l11 fragment (derived
from pAIterBot).
I~hest two li-agtncnts were inserted into the pMAL-c vector digested with
.l7nrlltlincllll. The
1 s twe, inserI fragments were ~;encrated by digestion of the appropriate
plasmid with F.coR1
(pl'nI lUU-?C8U) or :\'onI (pAIterBot) followed by treatment with the Klenow
FraLmcnt. i\tter
treatment with the Klenmv fragment. the plasmids were digested with the second
enzyme
(either .Sj~c~! or tlincllll ). All three Fragments were gel purified, nixed
and frep-a-CTcne
purified prior to ligation. (allowing ligation and transformation. putative
recombinants were
'_'U analyzed by restriction analysis: the EcwRl site was found to he
regenerated at the fusion
junction. as was predicted fi>r a fusion between the tilled EcrrRl and r\~onl
siW s.
;\ construct encodin~~ a Fusion proUein between the bmulinal C' Fra~~ment
~:cnc and the
MBI' ~~cn~ wus constructed (i.c~.. this Fusion lacks any ('. cli()irilr toxin
~\ ~enc sequences) and
termed pMI3ot. ~1'he pMBot construct was made by removal ol' the ('.
clijJicile toxin A
sequences from the pMAf3ot construct and fusing the C Iragmen t ttene
sequences to the MBP.
This was accomplished by digestion of pMABot DNA with .Sltrl (located in tltc
pMALc
I,otylinker s' to the ,(heft site) and ,1'hcrl (located i' to the Nutl site at
the toxA-Rot fusion
,junction). tilling in the .(heft site using the Klenow tcagment, gel
purifying the desired
restriction t~r:lLlll~tlt, and ligating the blunt ends to circularize the
plasmict. following ligation
sU and transti>rmation. putative recombinants were analyzed by restriction
mappini of the Bot
insert (i.r. the ('. hrntrlintr»t C t~agment sequences).
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WO 98108540 PC'TIC1597115394
b) Expression Of C botuliunm C Fragment Fusion Proteins In
E. coli
Large scale ( 1 liter) cultures of the pMAL-c vector, and each recombinant
construct
described above in taj were ~~mw~n. induceci, and soluble protein fractions
were isolated as
s described in Example t8. 'hhe soluble protein extracts were chromatographed
on amylose
at~tinim columns to isolate recombinant fusion protein. 'I~he puriticd
recombinant fusion
proteins were analysed by running samples on SDS-PAGE gels lulluwcd by
Coomassie
staining anct by Western blot analysis as described (Williams err crl. (
lc)94) .,tyncr~. In brief.
extracts were prepared and chromatugraphcd in column buffer ( !U mM Nal'U,.
U.s M NaCI.
IU lU mM ~3-mercaptocthanol. pH 7.'_') over an amylase resin (New (~nLtand
Biolahs) column.
and eluted with column buffer containing lU mM maltose as described [Williams.
co ul.
( Ic)c)4). .,'lrlJl'(l[. An ADS-PA(iF pcl containins~ the purified protein
samples stained with
C'oomassie blue is shown in figure '_'G.
In li;~urc ?G. the tollowiy samples were loaded. I.ancs I-h contain protein
puritied
1 s from 1~~. ur~lr CUlll~1tt11t1~ the pMAI.-c, pl'A1870-2G8U, pMAl3at.
pMNAI3ut. pMCAI3oU and
pMl3ut plasmids. respectively. Lane 7 contains broad range mcllccular weight
protein markers
( L3ioRad ).
l~he protein samples were prepared ii~r electrophoresis by mixing s yl u1~
uluvd protein
with j yl of ?X SI)S-PACE sample buffer (U.13> mM 'Kris-Fi('I. pli 6.8. '_' mM
ED'FA. 6%
~U SDS. ?()'% ~Ivcerol. U.U'_'s'% hromophenol blue: (3-mercaptocthanul is
added m 5°~~ heti~rc
use). The samples were heated to c)s°t' for ~ min, then cooled and
loaded on a 7.>% a~_arose
ADS-!'A(if~; gel. Broad sauce molecular weight prrncin markers wrre also
loaded to alUw
intimation of the MW of~ identified t'usicm proteins. At'tcr clcctrolhoresis.
protein was
detected ~enrrallv by staining the gel with Coomassie blue.
?s In all casca the 1'IClds w'el'e 111 l:XCeSS Uf 2U mg tilsion protein per
liter culture (sic
'Fable sb) and. with the: e~cception of the pMCABot protein. a hi'_h
pcrccntape (i.c~.. greater
than ?U-~U% of total eluted protein) of the eluted fwiem protein was of a MW
predicted for
the full length fusion protein (figure ?O). It was estimated (hv visual
inspection) that Less
than IU'i~~ of the pM(.'AL3ot i~usion protein was expressed as the full icn~th
fusicln protein.
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CA 02296765 2000-O1-14
PCT/US9'7l15394
TABLE 36
Yield Of Affinity Purified C'. lturulinum C Fra~_ment ! MBP Fusion Proteins
Construct Yield (mgJlitcr of Percentaee ()f Total
Culture) Soluble Protein
pMAEiot 24 s.O
pMCABot 34 S.0
pMNABot 40 ~ s
pMBot ?3 5.0
hMA 1870-2680 40 4.8
I t) These results demonstrate that bleb Level expression ef intact ('.
hrntrlinum C
I~ra'~ment-t '. cli~/icilr toxin A fusion proteins in ~~. unli is feasible
using the pMAL-c
mprcssiun system. These results are in contrast to those reported by H. Iv.
L.aPenotiere. rml.
I ! c)c)s ). .~rrpor. I n addition, these results show that it is not
necessary to fuse the hooutinal ('
f~ra,~mmt Lone to the (.'. cli/~icilo toxin A gene in order to produce a
soluble fusion protein
! s using the pMAI.-c system in I:. cull.
In order to determine whether the above-described hotulinal titsion proteins
were
rrcn~~nizcd by anti-('. hurrrlinrun toxin /~ antibodies. 1~'estern blots were
performed. Samples
e:~ntainin~~ affinity-purified proteins from I:. cwli containinL the pMAfiot.
pM(.'ABot.
pMNARut. pMBoU. pMA I 870-3680 car pMALc plasmids Overt: analyzed. SDS-PAGE:
gels
?0 ~7.9a acrviatnide) were loaded with protein samples purified from each
expression construct.
.~ltcr rl~ctruphoresis. the gets were blotted and protein transi~r was
contirtned by 1'onccau
atainin~~ (as described in L:xamptc 1?h1.
lollcwing protein transfer. the blots were htocked by incubation for 1 hr at
?0°(' in
hlockin:: buffer [PBS~t~ (Pf3S containing t).I% Tween ~() and s% dry milk?[.
The blots were
then incubated in 10 ml of a solution containing the primary antibody: this
solution comprised
a 1/5()() dilution of an anti-('. hrmnlinunr toxin A 1gY PEG prep (described
in Example s) in
hluckin~= buffer. The blots were incubated ter 1 hr at room temperature in the
presence of the
primary antibody. T'he blots were washed and developed using a rabbit anti-
chicken alkaline
phosphatasc conjugate ll3oehringer Mannheim) as the secondary antibody as
fullow~s. ~I'ltc
~() rabbit anti-chicken antibody was diluted to 1 ug/ml in blocking buffer
(It) ml final v~lum~
per hlon) and the Mots were incubated at room temperature fi)r I hour in the
presence of the
secondary antibody. 'T'he blots were then washed successively with I'f3S'f.
l3EiS-'1'ween and
- ~t) mM Na.,('();. pH 9,5. The blots were then developed in freshly-prepared
alkaline
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CA 02296765 2000-O1-14
WO 98108540 PCTIUS97115394
phosphatase substrate buffer ( 100 Etglml nitro blue tetrazolium. ~0 Etg/mf 5-
bromo-chloru-
indolvlphosphate. ~ mM MgCI, in ~0 mM Na,CO,, pl-E 9.~). Development was
stopped by
floodin~~ the blots with distilled water and the blots were air dried.
This Western blot analysis detected anti-t'. hmrrlinunr toxin reactive
proteins in the
pMAl3ut. pM(:'AI3ui. pMNABot and pMBot protein samples (corresponding to the
predicted
full Ic;n~~tit proteins identified above by Coomassie staining in i~ipure ?().
hut not in the
pMA 1 1 t)(1-?Ofi() or pMALc protein samples.
These results demonstrate that the relevant fusion proteins purified un an
amylase resin
as described above in section a) contained immunoreactivc t' h~nrrliuum ('
fragment protein as
I t) prcdictrd.
I~.XAMPLE 23
Generation Uf Neutralizin~~ Antibodies
f3v Nasal Administration Uf pMl3ot Protein
l;
l~hc ability ut~ the recombinant hutulinal toxin proteins produced in W ample
?'' to
Slllnlllalt'. :1 1l SIt;mIC 1111117u11t'. reSp(lnSe against hotulinal toxin
epitopcs was assessed. ~I-his
example: invoivccf: al the evaluation of the induction of scrum I~.:(i titers
produced by nasal or
oral administration of hotulinal toxin-containing t'. cli()icile toxin A
fusion proteins and b)
~.() the lr7 S'll'() neutralization of ('. I)l)Ir(lirtrrm tye A ncurotoxin by
anti- recombinant t'.
I)lrrrlIII711J11 (' t~ra~~ment antibodies.
Evaluntiun ()f The Induction Uf Scrum !~(: Titers Produced
liv Nasal ()r ()r~l Administration Uf I3otulinal Toxin-
=!5 Containing C rliJftcilc~ 'Toxin A H usiun Nroteins
fix groups containing five G week old CF icmale rats f ('harics ItivcrJ per
group were
immuniicd nasally or orally with one of the following three combinations using
protein
prepared in i~xarnplc '_'?: ( 1 ) ?SU Itg pME3ot protein per rat (nasal and
oral); ?) ''s0 pg
pMAIicit protein per rat (nasal and oral): s) 1?5 Et~; pMl3ut admixed with 1?s
Eye pMA1870-
() ?ti8f) per rat (nasal and oral). A second set of > s~roups cuntainin~~ _i
('F I~rntale rats/gruup
were immuni-red nasally or orally with one of the followitt~~ combinations (4)
?SU Etg '
pMNAf3ot protein per rat (nasal and oral) or ~) ?SU ~y pMAI.-c protein per rat
(nasal and
oral ).
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CA 02296765 2000-O1-14
W O 98108540 PCT/US97115394
The fusion proteins were prepared for immunization as follows. The proteins
(in
column buffer containing 10 mM maltose) were diluted in 0.1 M carbonate
buffer, pH 9.~ and
administered orally or nasally in a 200 yl volume. ~l~hc rats were lightly
sedated with ether
prior to administration. The oral dosing was accomplished usiy a 20 gauge
feeding needle.
s The nasal dosing was performed using a 1'-200 micro-pipettor (Giisun). ~hhe
rats were
huosted 1 ~l days after the primary immunization using the techniques
described above and
were hled 7 days later. Rats ti'om each group were liehtlv etherizcd and hled
from the tail.
fhc hlcu~d was allowed to clot at 37°(.' for 1 hr and the serum was
collected.
The scrum froth individual rats was analyzed using an ELISA to determine the
anti-C'.
hmulinurtt type A toxin IgG serum titer. The ELISA protocol used is a
modification of that
dcscrihcd in hxample lsc. Briefly. ~)(~-well microtiter plates (Falcon. Pro-
Bind Assay Plates)
were coated with ('. hnurlirttrnt type n toxoid (prepared as described in
Example sa) by
placin~~ Il)() yl volumes of ('. huttrlinrrnt type n toxoid at '.~ ~tu/ml in
PBS containing
(>.()Oi",« thimerosal in each well and incubating overnight at 4°C. The
next morning, the
I s cc,atin~= suspensions were decanted and all wells were washed three times
urine PI3S.
In order to hlock non-specific binding sites. 100 ~tl of blocking solution
(0.5% BSA in
I'l3Sj was then added to each veil and the plates were incubated for 1 hr at
s7°(.'. The
hluckin~_ se~lution was decanted and duplicate samples of 150 EII of diluted
rat serum added to
the first wetl ~>i' a dilution series. The initial testing serum dilution was
1 a0 in hlockim;
?0 ,olutie~n containing U.i% Tween ?0 followed by S-f()ld dlIULIUnS IlltO
tIllS St)lut1011. ~rlllS waS
accomplished by serially transferring 30 Ell aliquots to l~O ~.ll hIOCkIIIL
SOllltlOn COtlta1111ng
U.s°/~ I~ween ?0. mixin~~. and repeating the dilution into a Iresh
well. After the final dilution.
s() E11 was removed from the well such that all wells contained 130 Ell final
volume. A total
of 3 such dilutions were performed (~i wells total). 'hhe plates were
incubated t hr at i7°C'.
?J 1'()llt)w'InL LhIS IIICUhat10t1, the serially diluted samples were decanted
and the wells were
washed six times using ('BS containing U.5% '1-wecn 20 (i'BST). ~f~u each
well. l0U Ell of a
rabbit anti-Rat lgCi alkaline phosphatase (Sigma) diluted (1/1000) in blocking
buffer
containin~~ U.~% Tween ?0 was added and the plate was incubated for 1 hr at
s7°C. The
conjugate solutions were decanted and the plates were washed as described
above, substituting
30 s() 171M Na,CO.. pl-( 9.~ for the PBST in the final wash. The plates were
developed by the
addition W' 10() tll of a solution containing I mg/ml para-vitro phenyl
phosphate (Sigma)
dissolved in ~0 mM Na,CO;. 10 mM MgCI,, pH 9.5 to each well, and incubating
the plates at
roam temperature in the dark for ~-its min. The absorbency ol' each well was
measured at
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CA 02296765 2000-O1-14
tt
Rnulc Nasal t)ral
ttt
luununi>:uti,ttt
pMliot pMlit,t&
lutruunnsmtIMMl.INfpMliot K pMAI)tN pMl)ot pMAlx71)-pMAliu1
pMAlx711- (rNn
2hRp
I )ilutitrn
1 . :() t111xU I Ilall I 11311II U(U) 11 I~NI 11 (1x11IL 1
>11
1 I ,a a a 17 u.:xu a 54n o Itz~ n u7n n.a:u a uz7
I ';It I).IItINIt.?xl) 11.2(>It11 Irlf1It fl?II11 (rl()(11114
I :7st) I1 (N17 1> Itx4 (1119(11111(1()tl f1114U (1111 fl 1)(17
rr Ituta ~ = I
I muJ
~untW rs rcprcscul tltc a,cra_m values oMtuutcJ Inmt Int I:1.11A pl:uca.
O;tndartlimJ ulilvinc Ilm prcinuuuur cmurml
TABLE 38
()etermination ()I' Anti-('. hrnrrlrnrrrn i ype A toxin Serum I!~(i 'titers
tW Ilrnvin<_r Immunization With l'. h(rlulrnam L )~ra!.!tneW -C'ontainin~~
Fusion i'roleins
::0 Route ol' Nasal (>ral
Inunurtiz.~tion
fnununo!_cnf'RE-IMMUNEpMBot I)MABot pMNABoI pMNAi3ot
17i luti<,n
l:-;U 0.040 O.Si7 U.01() U.UtS O.UItI
I : 1 i(1 (1.0()c) 0.~8.i (1.001 (1.UU_i U.OU?
:'-s l:7sU U.U(11 0.14(1 ().(IUO II.UUt) (LUUU
I::t7iU U.uUU U.U4U ().()UU ILUUU U.UUU
i: IZaIS I I
~t-tSlla~
The above 1:I,ISA results demonstrate that reactivity against the botulinal
fusion
;U proteins was strongest when the route of administration was nasal: only
weak responses were
stimulated when the botulinal fusion proteins were given orally. Nasally
(telivcred pM()t and
pMI3m atimixed with pMA187()-268() inv()ked the greatest serum 1gO response.
~l~h(ac results
show that only the pMBot prenein is necessary to induce tIllS Cl'Sp(111SC.
since the addition ot~
the pMA I 870-2080 protein did not enhance antibody response (~l'able i7).
Placement at- the -
;s C'. cli~~iciler toxin A fra~~ment hetween the MBP and the C'. h()mrrir7nn)
(' ('raiment protein
WO 98108540 PCT/US97/15394
410 nm usin g a Dynatech MR 700 plate reader. The results are summarized in
Tables 37 and
,8 and represent mean serum reactivities of individual mice.
~r:~w;t.e a7
I)rlcrminatimt 1)t Anti-(' hunrlrrrunr 'I ypc A Iwin Scrum Ig(i Titers I
ollmwin~, Innnunti;ttit,n 14'ilh ('. lurrrrfnrrrnr (' I~ragmcnt-(n nnaiuing
I~ is on I'rutcins
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CA 02296765 2000-O1-14
WO 98/08340 PCTIUS97I15394
dramatically reduced anti-bot IgG titer tsce results using pMABot, pMCABot and
pMNABot
proteins).
This study demonstrates that the pMBot protein induces a strong serum IgG
response
directed against C'. horulinunr type A toxin when nasally administered.
h) In Vivo Neutralization Of C. botalinuy Type A Neurotoxin
' 13y Anti- Kecombinant C botrrlinunr C Fragment Antibodies
Thr ability ut' the anti-C'. hnlulrrnrm type A toxin antibodies generated by
nasal
administration ui' recombinant hotulinal fusion proteins in rats (Fxamplc 2?)
to neutralize ('.
I () hnnrlirturn type A toxin was tested in a mouse neutralization model. The
mouse model is the
art accepted method ii~r detection of hotulinal toxins in body tiuids and fbr
the evaluation of
anti-hutulinal antibodies (E:.J. Schantz and U.A. Kautter. .l. Assoc. ()f~f.
Anal. C'.heln. 61:96
( Ic)c)()) and Investis!atiunal New Drug (BB-INU-3703) application by the
Surgeon General ot~
the I)cpartmcnt of the Armv to the Federal hood and I)ru~; AdministrationJ.
The anti-('.
hr~rtrlirtunr ypc A toxin antibodies were prepared as follows.
Rats t~rom the group riven pMBot protein by nasal administration were boosted
a
second time with ?s0 Etg pMBut protein per rat and serum was collected 7 days
later. Serum
i~ram one rat t'rum this group and ti-om a preimmune rat was tested for anti-
C' hnrttlrrtrtm type
:~ toxin mutralizin~: activity in the mouse neutralization model described
below.
~'U The I.D;" ui~ a solution ot~ purified C' hWarliratrm type A toxin complex.
obtained from
Dr. h:ric ,luhnson 1 university uF VVISCOIISIn Madison), was determined using
the intrapcrituneal
( 1 f' ) n~etlu~cl oi~ Schantz and Kautter J.I. Assoc. Oi~f. Anal. C.'hem. O I
:96 ( I c)7R ) [ using I 8-3?
gram fcn~ale fC'R mice and was t«und to be 3500 l.D~"Ilnl. The determination
ut~ the LU~" was
hcrti~rmed as ti~llows. A 'fvpc: A toxin standard was prepared by dissolving
purified type A
?s toxin complex in 2~ mM sodium phosphate buffer, pl~I 6.8 to yield a stock
toxin solution of
s. l s x 1 U' LU;"/mg. 'f he OI),,~ ut' the solution was determined and the
concentration was
adjusted to lU-30 Elg/ml. 't'hc toxin solution was then diluted l:lU() in gel-
phosphate ( 3U mM
phosphate. pIl G.4: U.?% ~~elatin). Further dilutions of the toxin solution
were lade as shown
hclo» in -l'ahle ,9. Two mice were injected If' with ().5 m1 of each dilution
shown and the
s() mice were observed fer symptoms uf~ botulism for a period of 72 hours.
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E
CA 02296765 2000-O1-14
PCTlUS97115394
TABLE 39
Determination Ot' The Ll)." Of Purified C' hrrralinum Type A Toxin Complex
Dilution Number Dead At 72 hr
I :320
i I :640
1:1280
1:3560 0!3 (sick after 73 hr)
I :5120 03 l no symptoms 1
1 () From the results shown in Table 39. the toxin titer was assumed to he
between 2560
L, I);"lml and il?0 L.Di"Iml (or about 384U I.Ua"Itnl). ~fhis value was
rounded to 3~OU
I_D;"/ml fir the sake of calculation.
~fhc amount of neutralizing antibodies present in the serum of rats immunized
nasally
with pLll3ot protein was then determined. Serum From mo rats boosted with
pMHot protein
1, as described above and preimmune scrum from one rat was tested as follows.
~l~hc toxin
standard was diluted 1:IUU in gel-phosphate to a final concentration Wit' ssU
Lly"/ml. ()ne
milliliter W' the diluted toxin standard was mixed with ?~ Ell oi' serum from
each of the three
rats and ().'' ml of gel-phosphate. 'fhe mixtures were incubaterd at roam
mmperaturc fir 30
111111 Vvlth (1(;CilSlUllal mixing.:. Each of two mice were injected with !I'
with l)., ml of the
?U mixturos. The mice w~cre observed for signs of holulism fur 7? hr. Mice
receiving serum
from rats immunized with pME3ot protein neutralized this challenge dour. l~-
1icr receiving
preimmunc rat scrum died in less than ?4 hr.
~I'hr amount of neutralizing anti-toxin antibodies present in the scrum of~
rats
immunized with pMl3ot protein was then ~uantitated. Serum antibody titrations
were
part~ormed by mixing U.1 ml of each of the alllIbUdV dilutions (sec ~l~ablc
~U) with U.1 ml of a
1:10 dilution of stock toxin solution (3.i x lUy 1.I)s"Iml) with 1.U ml of gel-
phosphate and
injectinL U.~ ml 1P into ? mice per dilution. l~he mice were then observed for
signs of
hotuiism for 3 days (7? hr). 'fhe: results are tabuiatcd in Tahlr s~).
r1s shown in 'i'ahle al) pME)ot serum neutralized ('. hmrrlirurm type A toxin
complex
:~U when used at a dilution of 1:3~'U or less. A mean neutralizing value of
1GA IlJlml was
obtained ii~r the pMBot serum (an ItJ is defined as l(l.OUU nu~us~ L.I);").
This vaiuc translates
to a circulating serum titer of about _>.7 IlJlmg. of serum protein. This
neutralizing titer is
c:omparahle to the commcrcialiy available bottled concentrated (('onnaught
l..uboratorics. L.td.)
horse anti-('. hmulimrm antiserum. A 1U ml vial of Connaught antiserum
contains about 20U
CA 02296765 2000-O1-14
WO 98108540 PCTIUS97115394
mg/ml ol' protein:each nil can neutralize 750 IU of (:'. hvmlu~um type A
toxin. After
administration of one vial to a human. the circulating serum titer of the
(:onnaught
preparation would be approximately ?5 lU/ml assuming an average serum volume
of 3 liters).
Thus, the circulating anti-('. hrurrlirrunt titer seen in rats nasally
immunized with pMBot
protein ( 16t; lUlml) is (i.7 time higher than the necessary circulation titer
of anti-C'. hurtrlinum
antibody needed to he protective in humans.
TABLE 4U
c~uantittttion Of Neutralizing Antibodies In pMBot Sera
pMBot'
""-
Dilution Rat 1 Rat.2
I t :?U ?!2 3;1
()
I :4U ?;~, y~1
I :xl) 1l1 1'1
I : I GU ? ~~ 1'1
1 7 /1~ 1111,
I.3_U
I a54() U!'_' U,~?
f : I ?RU O'3 t)"
I :'_ 56Q 0!? (~ r,
Ntunbers represent the ntunber of mice surviving at 7? hours which rercived
scrum taken from
~l) rats immunized with the pMl3ot protein.
These mice survived but mere sick atier 73 hr.
fhesu results demonstrate that antibodies capable of neutralizing C'.
hrrrulirrunr ype A
toxin are induced when recomhinant ('. hmulinum C fragment fusion protein
produced in IJ:
''S c~r~li is used as an immunogcn.
EXAMPLE 2a
Production Of' Solubic C'. hmulinum C Uragment
Protein ~ubstantiallv Free Of l:ndotoxin Contanunation
()
f:xample ?3 demonstrated that neutralizing antibodies are generated bs
immunization
with the pMBot protein expressed in F.. cull. These results showed that the
pMl3ot fusion
protein is a good vaccine candidate. However. immunogens suitable for use as
vaccines
should he pyrogen-free in addition to having the capabilim of inducing
neutralizing
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PCTlUS97/15394
antibodies. Expression clones and conditions that facilitate the production of
C'. horuli»um C
fragment protein for utililization as a vaccine were developed.
The example involved: (a) determination of pyrogen content of the pMt3ot
protein:
(h) generation of C.'. hvtulira:mn C' fragment protein free of the MBl': (c)
expression of C'.
hrurrlirmm C fragment protein using various expression vectors: and (d)
purification of soluble
(' hrnulimrm C fragment protein substantially free of signilicant cndotoxin
contamination.
a) Determination Uf The I'yrogen Content Of The pl1'IBnt
Protein
I () In order to use a recombinant antigen as a vaccine in humans or other
animals. the
antigen preparation must be shown to he free of pyrogens. ~I'hc most
significant pyrogen
present in preparations of recombinant proteins produced in gram-negative
bacteria. such as E.
onii. is endotoxin (F.C. I'earson. I'ary~en.s: e~ndmarin,v. L,1L ~e~.c~ius,~
cure! eleyt~ry,~w~fuinn.
( lc)8>) Marcel Dekkcr. New York, pp. '' >-56J. 'I'ct evaluate the utility of
the pMF3ot protein
I ~ as a vaccine candidate, the endotoxin content in MI3i' fusion proteins was
determined.
I~he endotoxin content of recombinant protein samples was assavccl lltlllztng
the
I.imulus assay (L..AL kit: Associates of Cape Cod ) according to the
manuf'acturer's
instructions. Samples of affinity-purified pMal-c protein and pMA1870-?GR()
were found to
contain high levels of endotoxin (=e0.000 F:U/mg protein: I:11 (endotoxin
unit)). This
'0 suggested that MRP- or toxin A repeat-containing fusions with the hotulinal
(' fragment
should also contain high levels of endotoxin. Accorclinf:lv. removal o(~
rndutoxin from
aflinim-purified pMal-c and pMBot protein preparations was attcrnpted as
t'olluw~s.
samples of pMal-c and pMfiot protein were dcpyroeenated with pUvmyxin to
determine if the endotoxin could he easily removed. The ti~llowine amount of
protein wets
?i treated: '_'~) ml at 4.8 OD,ri"/ml for pMal-c and 19 mls at 1.44 ()D,H"/ml
ter pMBot. The
protein samples were dialv~ed extensively against Pl3S and mixed in a ~0 ml
tube (Falcon)
with 0.~ ml PIiS-equilibrated polymvxin I3 (Affi-Prep I'oiymyxin. I3ioRad).
The samples
were allowed to mix by rotating the tubes overnight at 4°C'. The
polvmvxin was pclleted by
centrifugation tbr 30 min in a bench top centrifuge at maximum speed (
approximately ?OOU x
s0 ~) and the supernatant was removed. The recovered protein (in the
supernatant) was
quantified by ()U,r", and the endotoxin activity was assayed by 1..rll.. In
both cases only
approximately l/i of the input protein was recovered and the polymyxin-treated
protein
retain cd significant endotoxin contamination (approximately 70()0 E~.tJhng of
pMl3ot).
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CA 02296765 2000-O1-14
WO 98108540 PCTIUS9'7/15394
The depyrogenation experiment was repeated using ati independently purified
pMal-c
protein preparation and similar results were obtained. From these studies it
was concluded
that si~_niticant levels of endotoxin copurities with these MBP tusion
proteins using the
amvlosc resin. Furthermore. this endotoxin cannot be easily removed by
polymyxin
treatment.
These results suggest that the presence of the MBP sequences on the fusion
protein
complicated the removal of endotoxin i~ram preparations o!' the pMBot protein.
b) Generation Uf C. hotuliaum C Fragment Protein Free Of
1 () The MBP
It way demonstrated that the pMBot fusion protein could not be easily purified
from
contaminatin~~ enctotoxin in section a) above. The ability to produce a
pyrogen-flee (e.,~~.,
cndotoxin-free) preparation of soluble hotulinal C fragment protein free of
the MBP tag was
nrxt invc:stiLatcd. ~1'hc pMBot expression construct was designed to
facilitate puritication ol'
I ~ the hmulinal (.' fragment from the MI3P tag by cleavage of the fusion
protein by utilizing an
rntineercd factor Xa cleava~~e site present between the MBP and the hotulinal
t' fragment.
The Farmr !(a cleavaLC: was pcrti~rmed as follows.
Factor Xa (Mew hngland Biolahs) was added to the pMBot protein (using a U.I-
!.U%
Factor Xa/pMBot protein ratio) in a variety of buffer conditions (c.,y.. PBS-
NaCI (PI3S
?U cuntainin~ U.s M NaC'1). I'BS-NaCI containing 0.2% Tween ?0, PHS. 1'BS
containing 0.?%
'IVveen ?U. I'I3S-(' (1'13S containing ? mM CaCI,). PF3S-C containing either
0.1 car O.S
~l~ween ?U. I'BS-t' containing either U. I or U.5% NP-40. PBS-C' containing
either ().1 or U.S%
-triton \-10(). f'BS-C' containing U.1'% sodium dcoxvcholatc. PBS-C containing
t).1'% SDS].
The Ivctor Xa di~cstions were incubated for 13-72 hrs at room temperature.
?S The extent of cleavage was assessed by Western blot or Coomassie blue
staining of
proteins following electrophoresis on denaturing SDS-PAGE gels. as described
in L:xample
'_'_'. C'lcavage reactions (and control samples of uncleaved pMBot protein)
were centrifuged
t'or ? min in a microfuee to remove insoluble protein prior to loading the
samples on the gel.
The Factor ,~a treated samples were compared with unclcaved. unccntrifuged
pMl3ot samples
.st) on the same gel. ~fhe results of this analysis is summarized below-.
I 1 Most (about ~)U%) pMBot protein could be removed by centritiyation, even
when unclcavcd control samples were utilized. This indicated that the pMBot
fusion protein
was non titlly soluble (i.c~., it exists as a suspension rather than as a
solution). (This result was
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CA 02296765 2000-O1-14
PCTNS97I15394
consistent with the observation that most affinity-purifted pMBot protein
precipitates after
lon g term storabe ('? weeks) at 4°C. Additionally. the rna.jority ( i.
c~.. 75%) of induced
pMBot protein remains in the pellet after sonication and clarification of the
induced L. cull.
ltcsuspension of these insoluble pellets in fBS followed by sonication results
in partial
solubilization of the insoluble pMnot protein in the pellets. J
?) The portion of pMBot protein that is fully in solution (about 1 U% of pMBot
protein) is completely cleaved by Factor Xa. but the cleaved (released)
botulinal C fragment is
relatively insoluble such that only the cleaved MBf remains Dully I11
SUlttt1011.
None of the above reaction conditions enhanced solubility without also
1 U reducing effective cleavage. Conditions that effectively solubilized the
cleaved botulinal C
fragment were not identified.
:k) The use of U.I% SI)S in the buffer used for Factor Xa cleavage enhanced
the
soluhilitv of the pMBot protein (all oi' pMRot protein was soiuhle?. ltowever.
the presence of
the Si)~ preventod any cleavage of the fusion protein with Factor Xa.
Is ~) nnaivsis of pelleted protein from the cleava~~e reactions indicated that
both i'ull
length pMBot (i.e-.. uncleaved) and cleaved hotulinal C fragment protein
precipitated during
incuhation.
l~hcse results demonstrate that purification of soluble heauiinal C' i~ragment
protein utter
clc;ava~e of the pMBot fusion protein is complicated by the insolubility of
both the pMl3ot
?U protein and the cleaved hotulinal C fragment protein.
c) hxprcssion Uf C. botulurum C Fragment l)sing Various
Expression Vectors
In order to determine if the solubility of the botulinal C' liagment was
enhanced by
expressing the C fragment protein as a native protein. an N-terminal dis-
tagged protein or as
a fusion with glutathione-S-transferase (GST), alternative expression plasmids
were
constructed. 'l~itese expression constructs were generated utilizing the
methodologies described
in ixartyfe ??. Figure 37 provides a schematic representation o1~ the vectors
described helow.
in i~iyre ?7, the tollowin~! abbreviations are used. pl' refers to the
pI~.T?:~ vector.
30 pHIS refers to the pE'I'Hisa vector. pBlue refers to the pBluescript
vector. pM refers to the
pMAI.-c vector and pG refers to the pGEX3'h vector (described in Example I 1
). ~l'he solid
black lines represent C'. hmulinunr C' fragment gene sequences: the solid
black ovals represent
the Ml3P: the hatched ovals represent CiST: "IiHHHH" rrprcscnts the posy-
histidine tar. In
- 156 -
CA 02296765 2000-O1-14
PCT/US97115394
Figure ?7. when the name for a restriction enzyme appears inside parenthesis.
this indicates
that the restriction site was destroyed during construction. .fin asterisk
appearing with the
name for a restriction enzyme indicates that this restriction site was
recreated at a cloning
.junction.
i) Construction Of pYBot
In order m express the ('. hnrulimrm (' it~agment as a native li.c~., non-
t~us~d) protein.
tlm pI'fiat plasmid (shown schematically in Figure ?71 was constructed as
follows. The C'
(~1'L1L111Cn1 SelItteIlCeS present in pAltcrtiot (Example ?'_') were renewed
by di~~estion of
I U pAIterI3eU with V'~mi and Ilincllll. The V'cvUlflincllll C fragment insert
was Iigated to
l~E~fllisa vector Icfcscrihed in E.:xamplc l8h) wloch was di~~estcd with
r~'rcrl and NinclIII. This
li~~ation creates ao espressicln construct in which the n~c~nl-rncocled
toethionine ~f the botulinal
C' f~ra~~mrnt is the initiate~r colon and directs expression of the native
hotulinal (' fragment.
The li'~ation products were used to transform competent 131.? 1 ( f.)f: ;
)p(.ys~ cells 1 Novaeen ).
1 ~ R~comhin~tnt clones were identified by restriction mapping.
ii) (.'onstruction t)f pHisBot
I n order to cypress the ('. hmulinrrm C fragment containing a poly-histidinc
tag at the
amino-terminus of the recombinant protein. the pl-IisRrn plasmid (shown
schematically in
'_'() f iturc ?71 was c:unstructed as follows. The V'crUIHincllll botulinal C'
(~ragmcnt insert from
pnlterhoU was li~~ated into the pl=Tl lira vector which was di~~ested with
,1'Irrl and IIincIlll.
-l~hc .~'wrl lon the C' fragment insert) and rVlac~l (on the pl:THisa vector)
sites mere filled in
usin~_ the hlrnow fragment prior W li!=ahem: these sites were then blunt end
ligated (the ~~'clel
site was re~~encrated at the clone juneticm as predicted). ~l~hc li~;ation
products were used to
'?5 transform competent t3L?1(DEi)pl.vs~ cells and recombinant clones were
idcntitied by
restriction mapping.
~l~hc resulting pllisBot clone expresses the botulinal C fragment protein with
a
histidim-ta~~~wd N-terminal extension having the following sequence:
Mct(i1v11isllis
l-IisHisl~iisttisf-iisHisHisliisScrSer(~IyHislle(ituGIyArgl'iisMetAla. (5EQ
ll) N():?~): the amino
:(t acids enee~clrd by the hotulinal C' fragment gene arc underlined and the
vector encoded amine
acids arc presented in plain mpg. The nucleotide sequence present in the
pf:Tllisa vector
which encodc,~s the pliisBot fusion protein is Listed in SEQ ID NU:''S. The
amino acid
sequence of the pl iisfiot protein is listed in SEQ II) NO:?O.
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CA 02296765 2000-O1-14
WO 98/48540 PCTIUS97115394
iii} Construction Uf pGBot
The botulinal C fragment protein was expressed as a fusion with the
glutathione-S-
transferase protein by constructing the pGBot piasmid (shown schematically in
Figure 27).
This expression construct was created by cloning the ,r1-nrlhSull (.' fragment
insert present in
pL3lueHot (Example 22) into the pGEX3T vector which was digested with ,Snarl
and .l7anl.
The :~'oU site (present on the botulinal fragment) was madr blunt prior to
ligation using the
hlencw ti~aLment. The ligation products were used to transtitrm cornpetent
f31_? l cells.
Each of the above expression constructs were tested by restriction di'ucstion
to confirm
the intcLriw of the constructs.
1() large scale ( 1 liter) cultures of pPBot [BL21(OL:3)pl_ys~ host], pEIisHot
[BL?1(UF-i)pLysS host] and pCil3ot (BL31 host) were grown in ?X 1'T medium and
induced
(115Irti If'TCi to ().8-1.() mM) for 3 hrs as described in Lxamplc ?'_'.
'l~otal. soluble and
insoluble protein preparations were prepared from 1 ml aliquots ctf each
lar~~c scale culture
[ W'illiams c~t crl- ( 1994). .wrp-crJ and analyzed by SUS-1'A(iE. No obvious
induced hand was
1 s detectable in the pl'Bot car pHisE3ot samples by C'oomassie staininL.
while a prominent
insoluble band of the anticipated MW' was detected in the p(iL3ut samplr-
~oluhle ivsatrs o1~
the p(il3ot IarLt,' scale ( resuspendcd in l'L3S) or pi lisL3ot largr scale [
resuspendcd in Novagcn
1X binciin~~ buffer (i mM imidaxolr. 0.~ M NaC'I. '_'0 mM ~I'ris-Il('l. pll
7.c))[ cultures were
prepared and used to affinity purify soluble affinity-tagged protein as
follows.
,~~ .L.Itc r(il3ot lysatc was aftiniy purified on a ;~lutathione-agarose resin
II'harmacial
exactly its described in smith and C'orcoran (Current E'rotocols in Molecular
l3iolo~y.
~upplc~ttcnt ?$ (Ic)c)4). pp. 1(i.7.1-10.7.7[. The pl-Iisl3ot protein was
purified on the f-Ifs-l3ind
resin ( Ncwacen 1 utilizing the 1-iis-hind buffer kit (Nen~ayn I rxactlv as
ctcscrihed by
manufacturer.
samples from the purification of both the pCiE3ot and pEiisl3ot proteins
(including
uninduccd. induced. total. soluble. and affinity-purified eluted prcttr:in)
were resolved stn SDS-
PnCiLgels. Following electrophoresis, proteins were analyzed by C'oomassie
staining or by
Western blot detection utilizing a chicken anti-('. hrtrrrlirrrrm i~vpe n
toxoid antihoclv (as
described in Example ?3}.
These studies showed that the p(it3ot protein was almost entirely insoluble
under the
utilized conditions. while the pi-IisBot protein was salable. rlflinity
purification of the
pilisE3ot protein on this lirst attempt was inefficient. both in terms oi'
yield (most of the;
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WO 98108540 PCT/US97/15394
immunoreactive botulinal protein did not bind to the His-bind resin) and
purity (the botulinat
protein was estimated to comprise approximately 20% of the total eluted
protein).
d) Purification Of Soluble C, botulint~nr C Fragment Protein
Substantially Free Uf Endotuxin Contamins~tion
the above studies showed that the pEIisBot protein was expressed in F.. cwli
as a
soluhlc protein. 1-lowever. the affinity puritication of this protein on the
Ills-bind resin was
very inctticient. In order to improve the affinity purification of the soluble
pl-EisBot protein
(in tams of hoth yield and purity). an alternative poly-histidinc binding
affinity resin (Ni-
!U N'fr1 resin: (?iagen) Vv'aS Lltlllzed. The Ni-NTA resin was reported to
have a superior hinding
attinim (h,,= I x lU~'' at pH B.U: (,)iagcn user manual) relative to the His-
bind resin.
1 soluble lysate (in Novagen IX binding buffer) troth an induced I liter ?X
Y'I'
culture was prepared as descrihed above. Briefly. the culture oi' pl-iisBot
(E31?1(DCa)pLvsS
host J w av grown at 37°C' to an OD~,,N, of ().7 in 1 liter of ?X YT
medium containing I UU
l ~ yLiml ampicillin. s~l~ Etgiml chloramphenicol and 0.2% glucose. Protein
expression was
induced by the addition of IPT(i to 1 mM. Three hours atter the addition of
the IPTCi. the
cells were cooled for 1 ~ min in a ice water bath and then centrifuged I U min
at ~()UU rpm in
a .IA I () rotor ( E3ccl:man) at ~I°C. 'fhe pellets were rcsuspended in
a total wlume of 4U mls
W vagm 1 \ binding butter ( > mM imidazole. U.5 M NaCI. ?U mM Tris-E-1C'1. pUl
7.c)).
'_'U transli:rred m.ww ss ml Uakridge tubes and fiozen at -70°C' for at
least 1 hr. The tubes
mere thawc~i and the cells were lysed by sonication (4 X 2U second bursts
using a E3ranson
~oniiicr -ts() ~~ith a power setting ot'6-7) on ice. The suspensi<m was
clarified by
ccntritiy~aticn for 20 min at c).000 rpm ( l0,UU0 x ,t,~) in a .IA-l7 rotor
((3eckman).
The soluble lysate was hroueht to U.1% NP4() and then was batch absorbed to 7
mI of
a 1:1 slurry of Ni-NTA resin:binding buffer by stirring for I hr at
4°('. The slurry was
poured into a column having an internal diameter of I or 2.~ cm (I3ioKad). The
column was
lhCt1 \~'aSh~d sequentially with I.i 1171s of Novagen 1X binding buffer
containing U.1% NI'4U.
1 s ml of Novagen 1 X binding bttffcr. 15 m) wash buffer (GU mM imidazole.
(l._i M NaC'l. ?0
mM 'hris-EIC'I. pli 7.~)) and l~ ml NaHPO, wash buffer (s() mM NaHP(),, pli
7.U. U.; 111
?() NaC'1. lU °r~ ~Ivccrol). 'The bound protein was eluted by
protonation of the resin using elution
butter (5U mM NaHPO,. pEi 4.U. 0.3 M NaCI, 10 % glycerol). -fhe eluted protein
was stored
at 4°C'.
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CA 02296765 2000-O1-14
WO 98108540 PCT/US97115394
Samples of total, soluble and eluted protein were resolved by SDS-PAtiE.
Protein
samples were prepared for electrophoresis as described in l;xample ?'?b.
Duplicate gels were
stained with C.'oomassie blue to visualize the resolved proteins and C'.
hnrulirnrnr type A toxin-
reactive protein was detected by Western blot analysis as described in
f:xample '_'?b. A
i representative Coomassie stained gel is shown in Figure ?R. In Figure ?8.
the Ibliowing
SaIllpleS ~~'Crl loaded on the I?.5% acrvlamide gel. Lanes 1--J C011t~1tt1
reSI7CCIIVCII' total
protein. soluble protein. soluble protein present Ill tile Ilo~~~-lhrotl~il of
the Ni-N~I~A column
and affinity-purified pl~isl3cri protein (i.r.. protein released from the Ni-
N'I~n resin by
profanation). Lane s contains hi~.h molecular weight protein markers
(Liiultad).
I U ~I~hr purification of pHisf3oi protein resulted in a yield of 7 mg of
affinity purified
protein from a I liter starting culture of BL?1(DE3)pLysS cells harhorin~~ the
pliisBot
plasmid. ~fhe yield of purified pHisl3ot protein represented approximately
U.4"/° of the total
soluble protein in the; induced culture. :lnalysis of the purified pf-lisL3ot
protoin by SDS-
I'n(if- rmeatcd that at least c)0-c)i°ra, ot'the protein was present as
a siylc band (I~is~urr ~R) of
l s the predicted MW (SO kD). 'hhis s0 kD protein band was immunoreactivc with
anti-('.
hrurrlinmn mpc ~1 toxin antibodies. 'hhe extinction cocfticient of the protein
preparation was
dctcnnincd to hr l.~ (using the fierce f3C'.n assay) or 1.4s (usin~~ the
l.owrv assay) ()t),;'~~ per
1 111t'./n11 solution.
~amplcs of pI-1 neutralized eluted pHisl3ot protein were resolvc,~d on a I~B
RUS HI'LC
_''.t) column t~hudvxl. .~ithough His-tagged proteins are retained by this
sizing column (perhaps
due to the inherent metal binding ability of the proteins), the relative
mohilim of the pl-lisliot
protein was consistent with that expectccf for a non-aggrcgatc;d protein in
solution. Most of
the induced pH isl3ot protein was dctcrtnincd to be soluble under the growth
anti solubilicatioe~
conditions utilized above (i.c~., greater than 90'%" of the pf-lisRot protein
was found to hr
's soluble as judged by comparison of the levels of pl-fisliot protein seen in
total and soluble
protein samples prepared from (3L21(DL:3)pLysS cells containing the pflisRot
plasmid).
~iDS-I',~Cif~ analysis of samples obtained after centrifugation. extended
stc~raRc at -30°('. and
at least '_' cycles of freezing and thawing detected no protein loss tdue to
precipitation).
indicatin'_ that the ptlisBot protein is soluble in the elution huftvr (i r..
>(> m~-1 Na111'()'. pll
,0 .1.(). U.s A~ NaCI. 40 °/~ glycerol).
Determination oi' endotoxin contamination in the affinity purified pliisE3ot
preparation
loiter pH ntutrali~.ation) using the I.nL assay (Associates ol~ C'apr (''od)
detected no
significant endotoxin contamination. 'l~he assay was performed usinL the
endpoint
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CA 02296765 2000-O1-14
WO 98/08540 PCT/US97115394
chromogenic method twithout diazo-coupling) according to the manufacturer's
instructions.
This method can detect concentrations of endotoxin greater than or equal to
U.03 ELJ/ml (El!
refers to endotoxin units). The LAL assay was run using U.~ ml of a solution
comprising 0.5
mg pHisBot protein in ~U mM NaHPO.,. pE~f 7.U. 0.3 M NaCI. 10 % glycerol: 3U-
6U Ell were
s cletected in the 0.5 ml sample. Therefore. the affinity purified pllisBot
preparation contains
(,()-12U ELJ/m~ of protein. f DA (uidelines for the administration of
parenteral druLS rec)uire
that n c:amposition to he administered to a human contain less than ~ ELJIkg
body weight (~Thc
average httillall body vycight is 70 kL: therefore up to ~~l9 Ell units can he
delivered in a
parental dose. ). Because very small amount of protein arc administered in a
vaccine
1 t) preparation (generally in the range of 1 U-SUU ug of protein).
administration of affinity
purified pl-EisBot containing 6U-120 FlJlmg protein would result in delivery
ot~ only a small
perccnta;~c oi~ the permissible endotoxin load. For example. administration of
1 U-S00 Etg of
purified pl lisi3ot to a 7U 1cL human. where the protein preparation contains
60 El.llmg protein.
results in the introduction ot~ only U.(~ to i0 ELJ (i.e.. U.? to 8.6% of the
maximum allowable
I ~ cndotoxin burden per parcnteral dc~sc (less than s I:UII:g body vyeight)J.
hhr above results demonstrate that endotoxin (l.P~) does nca copuril'v with
the
pE-lisI3ut protein usinL the above purification scheme. !'reparations of
recombinantly produced
pl-lisl3ua protein containing lower levels of endotoxin (less than ur equal to
? Fll/ mg
recombinant proUein-1 ma>~ be produced by washing the Ni-NTA column with wash
buffer until
'_'() the ()I~,~" returns m baseline levels (i.c~., until nn more 1.1V-
absorbing material comes off u(
the column).
~I'Ir above results illustrate a method for the production and purification
of~ soluble.
huutlinal C' ti~aLment protein substantially free of endotoxin.
EXAMPLE 2S
Optimization Of The Expression And Purification ()f pi-lisBot Protein
The results shown in Example 24d demonstrated that the pl-lisl3cri protein is
an
excellent candidate for use as a vaccine as it could be producul as a soluble
protein in L:. cwh
i0 and could b~ purified free of pyrogett activity. In order to optimize: the
expression and
purification of the pl-IisEiot protein. a variety of growth and purification
conditions were
tested.
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CA 02296765 2000-O1-14
qrp 9g/pg~p PCTNS97l13394
a) Growth Parameters
i) Host Strains
The influence of the host strain utilized upon the production of soluble p1
(isBot
protein was investigated. A large scale purit3cation of pllisBot was performed
(as described
in Example ?4d above using the BL31(UL:3) host (Nova~en) rather than the
I3L?1(I)L~3)pLysS bust. The deletion of the pL,ysS plasmid in the BI_21(DC3)
host yielded
higher Icyels of expression due to de-repression of the plasmid's T7-Iac
promoter. Hlowever.
the yield of affinity-purified soluble recombinant protein was very Ic~w
(approximately O00
ty/ liter culture) when purified under conditions identical to those described
in Example ?4d
1 () ahwc. This result was due to the tact that expression in the BI.? l
(UI:3) host yielded very
hi~~h level expression of the pl(isBot protein as insoluble inclusion hodles
as shown by ~DS-
1'ACilr analysis olprotein prepared from induced BL?I(UI:3) cultures IFigure
?~), lanes l-7.
described below). '1-hcae results demonstrate that the pllisBot protein is nm
inherently tonic
to 1:. cwli cells and can he expressed to high levels using the appropriate
promoterihost
1 ~ rumhination.
Figure ?~) shows a Cuomassie blue stained SUS-I'.ACiL: ~.el ( 1?.s'%a
arrvlamide) onto
which extracts prepared from I3L,?1(UE3) cells containin~~ thc> pl-lis(iot
plasmid were loaded.
Mach lane was loaded with ?.i Ltl protein sample mixed with ~.~ tll uI~?X ~I)S
sample buffer.
The samples were handled as described in Example 2?h. The following samples
were applied
?U m the ~~cl. L_anes 1-7 contain protein isolated from the BL?1(()I'3) host.
I.ancs 8-14 contain
proteins isolated from the F3L21(I)F~a)p(.ys~ host. Total protein wits loaded
in lanes 1. ?. 4.
(,. H. I U and 1'_'. Soluble protein was loaded in Lanes ;. ~. 7. 9, 1 I and I
. Lane I contains
protein I'l'Oltl uI1111ducCd 17t7S1 cells. Lanes 2-1 3 contain protein from
host cells induced for s
hours. 1('TCi was added to a final concentration olU.l mM (Lanes (-7). ().; mM
(Lanes 4-i)
c,r 1.U InM (Lanes 2. 3. R-1 3). 'rhe cultures were grown in 1,13 broth (Lanes
8-c)). ?X Y1
broth (Lanes lU-11) or terrific broth (Lanes I-7. 12-13). The pIIisI3ot
protein seen in Lanes
s. ~ and 7 is insoluble protein which spilled over from lanes ?, a and (~.
respectively. I (igh
molecular weight protein markers (L3ioRad) were loaded in Lane 14.
;~ variety of expression conditions were tested to determine il the (3I.? 1(
DF 3) bust
,() could be utilized to express soluble pFiisRot protein at suitably high
levels ( i.c~.. about I ()
111rltlll). ~(~I1C CUIIdIIlOIIS aIECrf:d were temperature (growh at 37 ur
30°C'), culture medium
(?X Y~f. I.Li or ~I~errif~c broth) and inducer levels (U.1. U.3 or 1.() mM
lI''('G). All
CUt11h117atIOnS t)1 these variables were tested and the induction levels and
solubility vyas then
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CA 02296765 2000-O1-14
WO 98/08540 PCT/US97115394
assessed by SDS-PAGC analysis of total and soluble extracts [prepared fiom 1
ml samples as
described in Williams e~ al., (t994), seryraJ.
nll cultures were grown in 15 ml tubes (Falcon #2057). All culture medium was
prewarmed overnight at the appropriate temperature and were supplemented with
f 00 Etglml
ampicillin and 0.2% glucose. Terrific broth contains t2 g/1 hacto-tryptone. ?4
glt bacto-yeast
extract and ! ()U roll) of a solution comprising U.17 M KH,PU,. 0.7? M K,1
IPt>,. C'uftures
' were grown in a incubator on a rotating wheel (to rnsurc aeration) to an
UI),,,H, of
approximately 0.4. and induced by the addition of IPTG. In all cases. high
level expression
ul~ insoluble pHisf3ot protein was observed. regardless of temperature. medium
or induccr
1 () c;ollc:ettlraltoll.
~i'hc ~l~fect oh varying the concentration of~ IP~1'G upon ?X YT cultures
grown at 2p°C
was then investigated. 11''fG was added to a final concentration ot~ either 1
mM. (). l mM.
t>.0~ mM or ().()I mM. :\t this temperature, similar levels oC pHis f3cn
pre>tcin was induced in
the presence od' either I or U.l mM IPTCi; these levels of expression was
lc».vcr than that
! > uhservccl at higher temperatures. Induced protein levels were reduced at
0.()5 mM IPT'(i and
.Ihsent at t).()I mM lPT(~ (relative to I.0 and 0.1 mM IPTG inductions at '_'
3°t'). However.
n« conditicms were observed in which the induced pHisBot protein was soluble
in this host.
Thus. ~tltlunyh expression levels are: superior in the BL?I(DE31 host (as
compared to the
I3L? 1 ( t)L: i )I)LVS~ hnSl). conditions that facilitate the production of
soluble protein in this host
?() ce~ulci nm he identified.
~I hesc results demonstrate that production of soluble pl-fisBut protein was
achieved
usin;~ the 13L? I ( DF3 )pl.ysS host 111 COI11t111CI10r1 ~1'Ith till' T7-lac
promoter.
ii) Effect ()f Varying Temperature, l~lcdium And
IPTG Concentration And Length Uf Induction
The eti'ect crowing the host cells in various mediums upon the expression of
recombinant hotulinal protein Crom the pHisRot caprcasion construct [in the
I3L21(I)Ia)pl,ysS
host) wets investigated. I3L?1(DE 3)pLysS cells containing the pI-Iisl3ot
plasmid were grown
in either I.IB. ?X Y~I~ or 'terrific broth at 37°C'. 'Che cells were
induced usiy I mM IPT'ti for
30 a 3 hr induction period. (expression of pl-iisBot protein was found to he
the highest when the
cells were grown in ?X YT broth (see Figure ?9. lanes 8-13).
The cells were then grown at i()°C.' in 2X YT broth and the
concentration of~ IPTti was
varied from I Ø U. ; or (>.1 mM alld the length of induction was either i or
s hours.
- I (i3 -
CA 02296765 2000-O1-14
PCT/US97115394
Expression of pHisBot protein was similar at all 3 induces concentrations
utilized and the
levels oi' induced protein were higher after a ~ hr induction as compared to a
3 hr induction.
Using the conditions found to be optimal for the expression of pElisBot
protein, a large
scale culture was grown in order to provide sufficient material tier a large
scale purification of
s the pllisBot protein. ~I'hrce 1 liter cultures were grown in 2X YT medium
containing l0U
Itg/ml ampicillin. ;-1 Etgiml chloramphenicol and U.''°/. glucose.
'T'he cultures were grown at
,U°C and were induced with 1.U mM lt'TG tbr a ~ hr period. The cultures
were harvested
and ~t soluble lysatc were prepared as described in Example I?;. n large scale
purification
was performed as described in Example 24d with the exception that except the
soluble lysate
was batch absorbed tbr , hours rather than for 1 hour. The final yield was 13
tng pl-lisBot
proteiniliter culture. 'I'hc pEiisBot protein represented 0.75°/~ of
the total soluble: protein.
The shove results demonstrate growth conditions under which soluble pllisBot
protein
is produced (i.r.. use cal' the E3E.21(UES)pl.vsS host. ?X 1'~1' medium.
sU°C. l.U mM ll'T(i for
hnurs>.
~~i
h) Optimization Of Purification Parameters
t~or optimization of purification conditions, lame scale cultures 13 X 1
liter) were
s!rown at i()°C' and induced with I mM I('TG for 5 hours as described
above. The cultures
were pooled. distributed to centrifuge bottles, cooled and pelleted as
described in Example
:'_U ?-Id. ~l~l~e cell pellets were froien at -7U°(.' until used. Each
crll prllet represented I!s ol' a
liter startine culture and individual bottles were utilized for each
cytimizati~n experiment
descrihect below. This standardised the input bacteria used lin- each
experiment. such that the
yields W' affinity purified pllisRot protein could he compared hcuveen
dii'fercnt optimization
caperimcnts.
?>
i) Binding ~pccificity (pll Protonation)
,~1 lysate of pHisIiot culture was prepared in PBS IpII 8.t)) and applied to a
, ml Ni-
NTA column equilibrated in I'BS (pEl 8.U) using a iluw rate of U.'_' ml/min (s-
4 eolumn
vulumes/hr1 using an Econo chromatcyraphy svstelll (BioRadl. ~I~hr column was
washed with
,(l I'BS (pI-I 8.U) until the absorbance (()U,;~") of the elute was at
baseline levels. 'I~hc llow rate
was then increased m ? ml/min and the column was equilibrated in 1'BS (plI
7.U). r1 pf~l
gradient (hf 1 7.U to 4.() in I'E3S) was applied in order to elute the hot111d
pI)tsEiot protein from
the column. Fractions were collected and aliquots were resolved on ~I)~-
I'ACiI: gels. The
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CA 02296765 2000-O1-14
WO 98108540 PCTIUS97/15394
PAGf: Lets were subjected to Western blotting and the pHisBot protein was
detected using a
chicken anti-('. hntulinum Type A toxoid antibody as described in Example 2?.
H'rom tho Western blot analysis it was determined that the pllisBot protein
begins to
elute i~rotn the Ni-NTA column at pH 6.0, 'This is consistent with the
predicted elution uf~ a
s Ills-rayed protein monomer at pH 5.9.
'I~hese results demonstrate that the pH at which the pl lisI3ot protein is
protonated
(released) from Ni-N~I~A resin in Pi3S buffer is pli 6.U.
ii) I3indin~ Specificity (Imidazolc Competition)
I() In order to detine puritication conditions under which the native G. c«!i
proteins could
be rcmovc:d from the Ni-NTr1 column while leaving the pHisTiut protein bound
to the
column. the following experiment was performed. A lvsate of pFlisBot culture
was prepared
in iU nW9 Nalll'U,. t).~ M Na('1. 8 mM imidazole (pl-1 7.U). T~ttis lysate was
applied to a 3
m! Ni-M7~~1 column equilibrated in ~() mM NaHPU,. 0.5 M NaCI (pF1 7.U) using
an Ecuno
1 ~ chromatography system (I3ioRad). A tlow rate of U.2 ml/min (3-4 column
volumes/hr) was
utilizrcl. l~hc column was washed with s0 mM NaI~iPU,. U.~ M NaC'1 (pH 7.U)
until the
absorbancc of the elute rrturncd to baseline. The flow rate was then increased
to ? elll/Illln.
hhr column was eluted using! an imidazole step gradient [in ~t) mM NaHI'U,.
U.S M
NaC'1 (pl l 7.U)[. f:lution steps were 2U mM. 40 mM. 60 mM. 8U mM. I00 mM. ?UU
mM. 1.U
'_'t) M imidazolr. iullowed by a wash using U.l mM EDTA (to strip the nickel
from the column
and remove any rcmaininL protein). In each step. the wash was continued until
the ()I),~"
rrturncet to baseline. Fractions were resolved on SOS-I'rlCif: gels. Western
blotted. and
pHisl3ut protein detected using a chicken anti-('. hrrrrrlinunr Type A toxuid
antibody as
described in Example ?~'. Duplicate gels were stained with C'oumassie bloc to
detect eluted
protein in each traction.
The results of the t'AGE analysis showed that most of' the non-specifically
binding
bacterial protein was removed by the 30 mM imidiazoie wash. with the
rcmainins~ bacterial
proteins being removed in the 4U and 60 mM imidazole washes. The pIIisIW t
protein bes~an
to elute at IU() mM imidazole and was quantitatively eluted in ?UO mM
imida~ole.
~1'hesc results precisely defined the window of imidazole wash stringency that
optimally 1'CI11UYCS G. cvrli proteins f~rum the column while specifically
retainin~t the pl~isBnt
protein in this.huffer. These results provided conditions under which the
pHisl3ot protein can
he purified free of contaminating host proteins.
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CA 02296765 2000-O1-14
PCTIUS97/15394
iii) Purification Buffos And Optimized
Purification Protocols
A variety of purification parameters were tested during: the development of an
optimized protocol I-or batch purification of soluble pliisC3ot protein.
~I~lte results of these
analyses arc summarized below.
Batch puritications were performed (as described in l:xamplc ''4d) usin~~
several
buffers to determine ii' alternative buf'f'ers could be utililcd for binding
of the pt-fisE3ot protein
to the Ni-N~I"A column. tt was determined that quantitative binding of
pHisliot protein to the
Ni-NTA resin was achieved in either Tris-HCI (plI 7.c)) or NaEiPC), (pII 8.U)
buffers.
1 t) Binding of the pf-iisliot protein in NalifU, bui~fer was not inhibited
using ~ mM. 8 mM or 60
mM imidazule. (quantitative elution of hound pllisl3ut protein was obtain cd
in buffers
containing ~U mM NalifU,. U.3 M NaC'.1 (pH 3.5-4.U). with or without lU'r~
s~lyccrol.
Ulowmcr. ~luantitation of soluble affinity purified plvisBut protrin before
and attcr a l~rceze
thaw (following several weeks storage of the affinity puriticd elute at -
?U°(.') revealed that
I > c)4°/~ of the protein was recovered using the glycerol-containing
hui~tcr. hut only 6R°/a of the
protein was recovered when the buffer lacking glycerol was employed. This
demonstrates
that glycerol enhanced the solubility of the pllisBot protein in this low pl~l
hufi'er when the
eluted protein was stored at freezing temperatures tc~.,~~.. -2U°C').
Neutralization of~ pl-l by
addition uf~ NaEi,PU, buffer did not result 1lt ObV1Ul15 protein
precipitation.
~0 It was determined that quantitative binding ol' pl Iisl3ot protein using
the hutch fi~nnat
occurred alter i hrs (Figure 3U), but not after 1 hr u1' bindin~_ at
~l°(' (the resin wets stirred
Haring binding=>. Fiturc 3U depicts a Coomaisse blue stained ~I)~-fA(iI: Lcl
t7.s".~~,
acrvlamictcl containing samples of proteins isolated durin~.~, thr
puritication ul~ pflisi3ut protein
from lysatc prepared from the BL?1(OF3)pl.vsS boat. i:ach lane was loaded with
> ~tl of
'_'s protein sample mined with ~ )tl of 2X sample buffer and processes! as
described in f:~camplc
'_?b. Lane 1 contains hiLh ntulecular weight protein markers ( l3ioRad). Lanes
? and s
contain protein eluted from the Ni-NTA resin. Lane ~ colllalns soluble protein
after a _~ hr
hatch incubation with the Ni-NTA resist. Lanes ~ and 6 contain soluble and
total protein.
rcspcctiwlv. figure _iU demonstrates that the pHisHot protein is completely
soluble ~comparc
3U lanes 5 and 6 which show that a similar amount of the sU I:C) pf (isE3ot
protein is seen in
both: if a substantial amount (greater than 2U%) of the pHisBot protein were
partially
insoluble in the host cell. more pllisBot protein would be seen in lane 6
(total protein) as
compared to lane ~ (soluble protein). Figure 3U also demonstrates that the
pHisi3ut protein is
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completely removed from the lysate after batch absorption with the Ni-NTA
resin for 3 hours
(compare Lanes 4 and 5).
The reported high affinity interaction of the Ni-NTA resin with liis-tagged
proteins
(K~= 1 x !0-'' at pl-I 8.01 suggested that it should be possible to manipulate
the resin-protein
complexes without siLnificant release of the bound protein. Indeed, it was
determined that
after the recombinant protein was bound to the Ni-N1'A resin. the resin-
pHisBot protein
complex was highly stable and remained bound following repeated rounds of
centrifugation of
the resin for ? min at I h00 x ,~~. When this centrifugation step was
performed in a ~0 ml tube
(Falcon). a tight resin pellet formed. 'this allowed the removal oCspent
soluhle Ivsate by
1 () pouring off the supernatant followed by resuspension of the pellet in
wash buffer. I=urther
washes can be performed by centrifugation. The ability to perform additional
washes permits
the dwelopmcnt of protocols for batch absorption of large volumes of lysatc
with removal of
the lysatc h~in_~ performed sitnply by centrifugation following binding of the
rccomhinant
hrotcin m the resin.
Is .1 simplified. integrated purification protocol was developed as follows. A
soluble
lysate was made by resuspcnding the induced cell pellet in binding buffer [i0
m11-4 NaIIPO,,
t).S ~1 NaC'l. (0 mM imidazolc (pH 8.0)[, sonicating ~4 x ?0 sec and
centrifuging for ?0 min
ut 10.00() x ,L~. NI'-40 v<as added to 0.1% and Ni-N7'A resin (equilibrated in
binding buffer)
was added. L~.ight milliliters of a I ; I slurry (resin:binding buffer) was
used per liter of
?0 startiy culture. The miwure was stirred for 3 hrs at 4°C. The slurry
was poured into a
column havin~~ a I cm internal diameter It3ioRad), wotshed with binding butler
containin~~
().1 "~" '.11'40. then binding buf~ier until haselinc was established (these
steps may alternatively
he performed by centritii=anon of the resin. resuspcnsion in hinding buf~fcr
containing NI'd0
followed by centrifugation and rcsuspension in bindinL buffer). Imidazole was
removed by
~~ashinL the resin with ~0 mM NaHPO_,, ().3M NaCI {pH 7.()). Protein bound to
the resin was
eluted using the same butter (~0 mM NaHPO" 0.3M NaC:I) having a reduced pl-1
(ptl( ;.s-
4.0 ).
n pilaf purification was performed following this protocol and yielded 18
mgllitcr
affinity-purified pllisRot. 'the pHisE3ot protein was greater than ~)U'% pure
as cstitnated by
:() C~oomassie staining oi~ an SDS-PA(if~ gel. This represents the highest
observed yield of
soluble affinity-purified pl~(isBot protein and this protocol eliminates the
need for separate
imidazolc-containing binding and wash buffers. In addition to providing a
simplified and
. ctticicn t protocol for the affinity purification of recombinant pl-IisBot
protein. the above
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WO 98108540 PCT/US97115394
results provide a variety of purification conditions under which pHisBot
protein can be
isolated.
EXAMPLE 26
p 'hhe pHisBot Protein is An Effective Immunugen
In E:xamplc '?3 it was demonstrated that neutralizing antibodies arc
s~enerated in mouse
serum alter nasal immunization with the pMBot protein. llowcver, the pMBot
prcUein was
fbund to cupurify with significant amounts of endotoxin which could not be
easily removed.
I~f) The pHisl3ut protein. in contrast. could be isolated free of si~_niticant
endotoxin contamination
making pf~fist3ot a superior candidate tilr vaccine production. ~I~o further
assess the auitahilitv
of pl lisliot as a vaccine. the immunogenicity of the pliisBot protein was
determined and a
comparison of the relative immunoLCnicity of pMt3ot and pt~iWrn proteins in
mice wax
pcrtormcct as ti~llows.
1 ~ ~fwo groups of~ eight RAI.Bc mice were immunized with either pMtiot
protein or
~flisl3ot hrutein using (ierhu (iMDt' adjuvant (CC' Biotech). pMfioo protein
(in I'IiS
containing 1 () mM maltose? or pHisBot protein ( in >0 mMNaHI'(),. (). ; M
Na('I. 1 O'~o
glycerol. pl-t 4.()) was mixed with (icrbu adjuvant and used l0 1111111un1'le
illiCe. E~:ach mouse
received an II' injection of 100 til antigen/adjuvant mix (:i0 Eig antigen
plus 1 EtL adjuvant) on
"'~7 clay (). Mice were boosted as described above with the exception that the
route of
administration was I M on day I ~1 and 28. The mice were bled an day 77 and
anti-( ~.
I)IIIIIIIItIIll1 I~vpe A tuxuid titers were determined using serum collected
from individual mice
in each ~~roup (as described in E;xampte 2s). The results are show-n in Tahle
4I.
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r:',st.E: ar
~Illi-(' hrlrrelrnmn type .A I'ow,id scram Ig(i 'l~itcrs In (ndivitlual Micc
(mmuniwtl H ith pMlit,l or pl lisl3ot Protein
I'reimmunc' pMDon pHisfiot=
Sample Sample. Sanlplc
Dilution t)ilution Dilution
\ltnlse
n I';11:2?III 1:62?l1I 1:2601:12111:62sUI 1:2511I-t20I
12~I1 sll io h2o
s I o u.lt)oa u.1x171 u.7tn)a mt)~
f,7x u;: :7a .i2o
.
I.Ir,I0.9310.2;4I)n7;1:1)11.x29).41)t)ILI.i4
(
I.thJf14xIl,lt)U.11:lII 1112:1Il.lsi11.1?2
~9h
7 I I.Ixt)1l.i.iJILllr,7I l).xaf1n (Lrlt)11
f22 52 ~.rx
1 I.())II().2xt)11.1167I I.ixfl().xtl;t123s
612 r,2t)
1 n Il.t)130.242!) 11 I fl.t)s2ILJ770
l) O(,t>07 .1x5 146
;
- (1411111.2311.11xf1111JI.?2a11.72611.264ILIIHt)
s a.m7 cl.2aanll:xu.nlaI<m 1)x27ILIO,nn2t1
~I~:111
I itrrno.lxuo21 unllf)Ixl2I 1)u,.m nu;7 I Irxt)1,n..clln
Iw ln.l ~Ix I
l:r
l;
I
Ilt:
pnnnnnnlr
sumplc
represents
the
awr:r,c
lioln
?
scls
of
Juplicute
wrlls
txuuainine
serum
tinm
a
individuld
nu,use
111111111111Letl
,villl
fett,111111t1:1111
.l/rlrtrn'rr,t'11t't'trl'
elllert,ln\Itl
I~
(Sl:lj)
i1I111!_l'11.
IillS
atltl!_CIl
Lv
It111111It1t11l,LIC:1111'
Iltlrelalt'.tl
Im
!'
hmlerllrllrll!
Itt\111
:11111
prnVItIW
a
lltlllrn)
W
rt1111
~I) \waec of tluplic:m well,
hhc results shown above in 'fable ~ 1 demonstrate that both the pMBot and pI-
IisRot
protrins arc immunoe:enic in mice as 100% oi~ the mice 18/8) in each group
seroconverted
from non-immune to immune status. The results also show that the average titer
of anti-C'.
'_'; hr)rrrirnrr))r ~I~vpe A toxoid lgG is ?-_, told higher after immunization
with the pllisFiot protein
relative to immunization with the pMBot protein. This suggests that the pl-
IisBot protein may
be a supt:rior immunogen to the pME3ot protein.
CXAMPLI: 27
3U Immunization With The Recombinant
pl~isBot Protein Generates Neutralizing Antibodies
~I~hl I'CSUIIS SIII~Wn Irl Example ?6 demonstrated that both the pllisl3ot and
pMBot
proteins were capable of inducing hi~~i~ titers of anti-C'. l7nrulinrrnr type
A to~coid-reactive
antibodies in immunioed hosts. The ability of the immune sera from mice
immunioed with
either the pHisf3ot or pMBot proteins to neutralize ('. hmulinurrr type A
toxoid in aim) was
determined using the mouse neutralization assay described in Example ?3b.
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'the two groups of eight BALBc mice immunized with either pMBot protein or
pl-iisBot protein in (;xample 2G were boosted again one week after the
bleeding on day 77.
1'he boost was performed by mixing pMBot protein (in PBS containing IU mM
maltose) or
pHisBot Protein (in ~0 mM NaHPO,. U.s M NaCI. lU% s~lvcerol. pII 4.U) with
Cierbu
s adjuvant as described in Example ?6. Each mouse received an I1' injection of
lUU Itl
antigenladjuvant mix (~0 yg antigen plus 1 pg adjuvant). 'fhc mice were bled O
days titter
this boost and the serum ti-om mice within a group was pooled. SerUnt from
preimmune mice
was also collected (this serum is the same serum described in the teotnote to
'fable ~I ).
'The presence of neutralizing antibodies in the pooled or prcimmune scrtun was
1 U detected by challenging mice with ~ L.DS" units of type A toxin mixed with
I UU Ell of pooled
serum. Tht; CltallellS!e waS performed by mixing (per nl(lusC to he injected)
lUU yl of'scrum
tiwm each pool with lUU Ill of purified type A toxin standard (SU I.Oj" iml
prepared as
dcscrihcd in f:xamplc ? ih) and SUO )tl of gel-phosphate. 'l~he mixtures were
incubated tbr 30
min at room temperature with occasional mixing. Each of tour mice were ipected
Il' with
1 s the mixtures (t).7 ml/mousc). ~l'he mice were ohservcd for signs c,1'
hotulism ii~r 7'_' hours.
Mice receiving toxin mixed with serum from mice immunized with either the pl
iisl3ot or
hMf3ot proteins showed no signs of hotulism intoxication. to contrast, mice
receiving
prcimmune scrum died in IeSS than 24 hours.
These results demonstrate that antibodies capable of neutralizing l '.
lmnclinum type A
?U toxin are induced wizen either of the recombinant (.'. hcmrlinum ('
traement proteins pH isHot
or pMt3ot are used as immunogens.
EXAMPLE 28
Cloning And U:xpression t)f The C' I~ragmcnt o1~ ('. ht)Itlllrlifl7l
~s Serotype A Toxin In E. cwli Utilizing A Native Gene hragntcnt
1 n Example 2? above. a synthetic gene was used to express the C' fragment of
( '.
h«rnli»i»n scrotypc A toxin in G'. cwli. The synthetic gene replaced non-
preferred (i.r.. rare)
colons present in the (' fragment gene with colons which are preferred by 1:~.
cull. The
,U 51'Iltltetle Lclle V1'aS generated because it was been reported that genes
which have a his~h A/Z'
content (such as most clostridial genes) creates expression dil'ticulties in
f. cwli and yeast.
rurthermore. LaPenoticrc m crl. suggested that problems encountered with the
stability (non-
fusion constructs) and solubility (MRI' fusion constructs) of the (.' fragment
of ('. hutulinum
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CA 02296765 2000-O1-14
WO 98108540 PCTNS97/15394
serotypc A toxin when expressed in E. rnli was most likely due to the extreme
A/T richness
of the native C'. hvtulir~um serotvpe A toxin gene sequences ( La!'enotiere.
cu crl.. .supra).
In this example. it was demonstrated that successful expression ol' the C
Iwagment of
('. hrmrlinum type A toxin gene in E. cwli does not require the elimination of
rare colons
(i.r.. there is no need to use a synthetic gene). This example involved a) the
cloning of the
native C' fi~agmem ~f the ('. hn~erlirmm serotype A toxin gene and
construction of an
cxpresston moor and b) a comparison of the expression and purification yields
of C'.
hrutrlirrtrrtr serotypc A C fragments derived from native and synthetic
expression vectors.
IU a) Cloning Uf The Native (: Fragment Of The (: hotulirrum
Scrotype A '('oxin Gene And Construction Uf An Expression
Vector
The seroype A toxin gene was cloned fiom ('. hnlrrlirrum ~enomic DN,A using
I'CR
ctmplitication. The tollowing primer pair was employed: s'-C(~CCA'I'G(~CTAC
Is A~I~T~I~rTA'fCI~ACATTTAC.'-s' (>' primer. Ncwl site underlined: SFQ IU
N():?<)) and
~~-W'AA(~('TT'CTT(~AC'AVA(.'TCA'1'(~TA(~-3' (s' primer. Nindlll site
underlined: SEQ IU
'~( ):3()). ( '. hnnrliraum ype A strain was obtained tcom the American '(~ype
('allure (.'ollection
(.1-I~C'('# ( c):97) and grown under anaerobic conditions in Terrific hroth
medium. Iliuh
molecular-weight ('. hrrltrlintrm DNA was isolated as described in Example 1
1. The integrity
'_() and yield of genomic DNA was assessed by comparison with a serial
dilution ol' uncut lambda
t)NA alter electrophoresis on an agarose gel.
Th c gene tragmcnt wax cloned by I'CR utilizing a proofreading th crmostable
UNA
pc~lymcrase (native l'Jir polvmcrasct. 1'CR amplitication was pertitrmed using
tire above
primer pair in a ~OUI reaction containing IOmM Tris-HCI (pH 8.s>. sUmM KCI.
l.~mM
'-S MeC'I,. ?U()IeM each dNTP. O.?~.M each primer. and SOng ('. hutulirtum
genomic DNA.
Reactions ~~ere overlaid with 1 ()Olcl mineral oil, heated to 94"(' 4 min.
U.5~1 native I'yt
polymerasc (Stratagenc) was added. and thirty cycles comprising c)4"C for I
min. s0°(' for 2
min. 7'?°C' for 2 min were carried out followed by 10 min at
72°C. An aliquot ( I ()Ltl ) of the
reaction mixture was resolved on an agarosc gel and the amplified native C'
fragment gene
was gel purified using the Prep-A-Gene kit (BioRad) and ligated to p(:'RScript
vector DNA
(Stratagcne). Recombinant clones were isolated and contirmed by restriction
digestion, using
standard recombinant molecular biology techniques /Sambrook et crl. ( 198c)),
.,urprcr]. In
addition, the sequence of approximately ,00 bases located at the ~' end of the
C fragment
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CA 02296765 2000-O1-14
WO 98108540 PCT/US9'7115394
coding region were obtained using standard DNA sequencing methods. The
sequence
obtained was identical to that of the published sequence.
An expression vector containing the native C'. hotulinunr serotype A C
fragment gene
was created by ligation of the A~cml-!find))) fragment containing the C:
fragment gene from the
pC'RScript clone to ~Vhel-IlindlIl restricted pETHisa vector (Example 18b).
~l'he Ncwl and
:1'hc~l sites were tilled in using the Klenow enzyme prior to ligation: these
sites were thus
!lent-end Grated together. The resulting, construct was termed pl-Iisl3otA
(native). pHisBotA
(native) expresses the ('. hmulinum scrotype A C' fragment with a his-tagged N
terminal
~'xt211S1u11 wllleh has the ti,llowing sequence:
McOTIvIIisHisllisllisllisllisH
isHisHisHisSerScrGIyF(isllcWlrrC~Ivilrt~HisMetAla (SEQ ID
N():?~1. where the underlining represents amino acids encoded by the ('.
hourlirunir C'
fragment gene (this N terminal extension contains the recognition site tile
hactorXa protease.
shown in italics. which call be employed to renewed the polyhistdinc tract
from the N-
tcrminus af~ the fusion protein). The pHisRot (native) construct expresses the
identical protein
I ~ as the pl-tisIiot construct (Ex. ?4c: herein after the pliisBotA1 which
contains the synthetic
>;clle.
The predicted DNA sequence encoding the native ('. hamlimrn~ serotypc A C'
fragment
~~ene contained within pilisL)utA (native) is listed in ~LQ II) N():sl (the
start of translation
(A'l~O) is located at nucleotides 1()$-110 and the stop of translation (TAA)
is located at
'0 nucleotidesi4~)~l-l~l~)(, in SC:Q ID N():3l) and the corresponding amino
acid sequence is listed
in SE~:Q ID N():?O (i.r.. the same amino acid sequence as that produced by
I,I~istiotA
mntainin~~ synthetic <_cne sequences).
ly Comparison Uf The Expression And Purification Yields ()f
C: butulintrni< Serotypc A C Fragments Derived Frorn Native
And Synthetic Expression Vectors
Recombinant plasmids containing either the native ur the synthetic ('.
hultrlinunr
scrotype A C' fragment ~~enes were transtbrmed into l:. cull strain
I31?1(DI:3) pLysS and
protein expression way induced in I liter shaker flask cultures. ~l utal
protein extracts a ~erc
30 isolated. resolved tin ADS-I'ACiE. gels and ('. hunrlirarmz (' fragment
protein was identified by
WeSterll allall'S1S LltIIILlIIL a eIllChcil allll-('. hmullmnn aeroypc A
tuxuid antiserum as
descrihed in Example 3'?.
. 17
CA 02296765 2000-O1-14
PCT/US97/15394
Briefly. I liter (2XYT + 100 Etg/ml ampicillin and 34 ug/ml chloramphenicol)
cultures
of bacteria harboring either the p1-IisBotA (synthetic) or pHisBotA (native)
plasmids in the
Rl?1(DE3) pL.vs~ strain were induced to express recombinant protein by
addition of IPTG to
I mM. C'ulturcs were grown at 3U-32°C'. IPTG was added when the cell
density reached an
s ()D~,,N, (l.S-I.0 and the induced protein was allowed to accumulate for 3-4
hrs otter induction.
'rhc cells were cooled for 1 s min in a ice water bath and then centrifuged
for 10 min
at s0()0 rpm in a JA t 0 rotor ( Beckman I at 4°C. The eel l pellets
were resuspended in a total
volume of 4() mls iX binding buffer (~i0 mM imidazole. 0.5 M NaCI. 50 mM
NaPO,, pli
t1.0). transferred to two ~U ml Oakridge tubes and frozen at -70°C for
at least 1 hr. The tubes
were then thawed and the cells were Ivsed by sonication (using tour successive
?() second
bursts) on ice. 'hhe suspension was clarified by centrifugation ?0-30 min at
9,000 rpm
( 1 O.U()O,f,~) in a .lA-i 7 rotor. The soluble lysate was batch absorbed to 7
ml of a t :1 slurry of
NiNTA resin:hinding buffer by stirring ?-~l hr at 4°C'. The slurry was
centrifuged for 1 min
at ~0(),t~ in 5() ml tube ((=alconl, resuspended in ~ mls binding buffer and
poured into a ?.5 cm
I ~ diameter column (BioRad). The column was attached to a UV monitor (ISC())
and the
column was washed with bindinf; buffer until a baseline was established.
Imidazoie was
renewed by washing with ~OmM Nal'O,. 0.3 M NaCI. 10% glycerol, pH 7.0 and
bound
protein was eluted using sOmM NaP(),, 0.3 M NaCI. 10% glycerol. pI-1 3.~-4.U.
fhe eluted proteins were stored at 4°C. Samples of total, soluble, and
eluted proteins
?() were resolved by ADS-f'ACiI. l'rotcin samples were prepared for
electrophoresis by Itllxfl7~
I)fl t<Ual (~I~) or soluble (S) protein with 4 Efl PBS and ~ y! ?X SDS-PAGE:
sample buffer, or
tel eluted (C) protein and s Efl ?X SDS-PAGE sample buffer. ~l'he samples were
heated to
~)~' C for s min. then cooled and ~ or 10 Ids were loaded on 12.5% SUS-PAC'IE
gels. Broad
range nwlecular weight protein markers lE3ioRad) were also loaded to allow the
MW of the
?s identified fusion proteins to be estimated. After electrophoresis. protein
was detected either
generally by staining fiefs with C'oomassie blue, or specifically, by blotting
to nitrocellulose
for Western blot detection of specific immunoreactive protein.
l~or Vl4'estern blot analysis. the gels were blotted. and protein transfer was
confirmed by
I'onceau S staining as described in Example ??. Alter blocking the blots for l
hr at room
:0 temperature in blocking buffer (PRST and 5% milk), 10 ml of a 11500
dilution of an anti-C.'.
hnte~lirrrrm toxin A 1~;Y PEG prep (Lx. 3) in blocking buffer was added and
the blots were
incubated for an additional hour at room temperature. The blots were washed
and developed
using a rabbit anti-chicken alkaline phosphatase conjugate (Boehringer
Mannheim) as the
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CA 02296765 2000-O1-14
WO 98108540 PCT/US97115394
secondary antibody as described in Ex. 22. This analysis detected t:'.
borrrlinum toxin A-
reactive proteins in the plIisBotA (native and synthetic) protein samples
(corresponding to the
predicted full lEngth proteins identified by Cuomassie staining).
A gel containing proteins expressed from the pHisBot and pHisBot (native)
constructs
s during various sta~!es of purification and stained with Coomassie blue is
shown in Figure 31.
In Figure ,l. lanes I-4 and ~) contain proteins expressed by the pHisI3otA
construct (i.e., the
synthetic gene) and lanes ~-8 contain proteins expressed by the pHisBotA
(native) construct.
Lanes I and 5 contain total protein extracts; lanes 2 and b contain soluble
protein extracts;
lanes .i and 7 contain proteins which flowed through the NiN'1~A columns:
lanes 4, 8 and ~)
l(J contain protein eluted from the NiN'hA columns and lane l0 contains
molecular weight
markers.
Tlre above purification resulted in a yield of 3 mg (native gene) or 1 1 me
(synthetic
Ll'11e) ol'affinity purified protein from a t liter starting culture, of which
at bast 90-9>°/, of
the protein was a single band of the predicted MW (~Okd) and immunoreactiyitv
tier
I > recombinant ( '. hmulinum serotvpe A C' fragment protein. Other than the
level of expression.
no difl'crence N~as observed hctween the native and the synthetic gene
expression systems.
These rrsults demonstrate that soluble ('. hanrlinum scrotype A C' l'raLmcnt
protein can
be expressed in f:. cwli and purified utilizing either native or synthetic
Bern se~lucnces.
'(I EXAMPLE 29
Generation Uf Neutralizing Antibodies lJsing A Recombinant
('. hmurlinrrrn Serotype A C' Fragment Protein Containing A Six Itesiduc tlis-
~I~aL
In Lxample 27. neutralizing antibodies were generated utilizing the p1(isf3otA
protein.
which contains a histidine-tagged N-terminal extension comprising I (1
histidine residues. 'fu
determine ii' the generation of neutralizing antibodies is dependent on the
presence of this
particular his-tag, a protein containing a shorter N-terminal extension
(comprising 6 histidine
residues) was produced and tested for the ability to generate neutralizin f:
antibodies. 'This
example involved a) the claning and expression of the pOHisBotA(syn) protein
and h) the
-3() generation and characterization of hvpcrimmune scrum.
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WO 98108540 PCTlUS97/15394
a) Cloning And Expression Of The p6HisBotA(syn) Protein
The p6HisBotA(syn) construct was generated as described below: the term "syn"
designates the presence of synthetic gene sequences. This construct expresses
the C frgament
of the ('. hcuulir?run serotype A toxin with a histidine-tagged N terminal
extension having the
following sequence: MetHisHisfiisHisHisHisMetAla (SI:Q ID N0:33); the amino
acids
encoded by the hotuiinal C fragment gene are underlined and the vector encoded
amino acids
are presented in plain type.
(~XHis oligonucleotides ji'-'fATGCATC:ACCATCAC:CA'tCA-3' (SEQ ID NO:33) and
~'-C'ATG'1'CiA~rGGTGATGG'T'GATGCA-3' (SEQ fD N0:34) were annealed as fUIIUU's.
One
() microgram of each oligonucleotide was mixed in total of ?0 l.tl f X
reaction buffer 2 (NEB)
and the miwure was heated at 70°C tier ~ min and then incubated al
42°C.' for ~ min. l'he
annealed oligonucleotides were then ligated with gel purified l4'cleIINirrdlIl
cleaved pET23h
( ~I~7 hromotcr 1 or pFT21 b ('r7lac promoter) DNA and the eel purified
NewIItIirrdlll C'.
hrmrlinrrnr serotvpe A (.' fragment synthetic gene fragment derived from
pAltcrBot (I:x. ??).
) ~ Recombinant clones were isolated and confirmed by restriction digestion.
The DNA sequence
encoding the 6X his-tagged BotA protein contained within pGHisBotA(syn) is
listed in SEQ
II) N0: 3s. The amino acid sequence of the p6X1-lisliotA protein is listed in
Sf:Q fD N0:36.
The resulting recombinant pGXIUisBotA plasmid was transfbrmcd into the
f3I.21(DE3)
pLvsS strain. and l liter cultures were grown, induced and harvested as
described in hxampEe
?0 ?8. f Its-tagged protein was purified as described in Example 28, with the
ibllowing
modifications. ~f'h~ binding buffer (BB1 contained ~ mM imidazole rather than
40 mM
imidarole and NI'40 was added to the soluble lysate to a linal concentration
of 0.1%. 'I~hc:
hound material was washed on the column with BB until the baseline was
established, then
the column uas washed successively with BB+?0 mM imidazoie and BB+40 mM
imidazole.
?> 'fhe column was eluted as described in Example 28.
In the case of the pE:T?3-derived expression system, nigh level expression of
insoluble
61-lisBotA protein was induced. The pL:T?I-derived vector expressed lower
levels of soluble
protein that bound the NiNTA resin and eluted in the 40 mM imidazole wash
rather LhaI1
during the iou p1-I elution. These results (i.v., low level expression of a
soluble protein) are
30 consistent u-ith the results obtained with pfiisBotA protein (Ex. ?5): the
pl~isBotA construct,
like the pET? t-derived vector. contains the T7lac rather than T7 promoter.
The 6HisBotA protein thus elutes under less stringent conditions than the lOX
histidin c-containing pI-IisBot protein 1 100-200 mM imidazolc: Ex. ?5)
presumably due to the
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reduction in the length of the his-tag. The eluted protein was of the
predicted size [i.c~..
slightly reduced in comparison to pHisBotA protein].
b) Generation And Characterization Of Hypcrimmune serum
s Eight BALBc mice were immunized with puritied 61)isRotA protein using Cierbu
GMDP ad_juvant (C'C' Biotech). The 40 tnM imidazole elution was mixed with
Cierhu
ad,juvant and used to immunize mice. Each mouse rrceived a subcutaneous
ipcction of~ IOU
yl antigen/adjuvant mix (!2 pg antigen + 1 ~tg adjuvant) on day 0. Mice were
wbcutancouslv
boosted as above on day 14 and bled on day 28. C'ontro) mice received pl
iisRotB protein
(prepared as described in Ex. 35 below) in Gerbu adjuvant.
Anti-('. hntulirrrrm serotype A toxoid titers were determined in serum from
individual
mice t~rom each group using the I:LISA described in Example 2 is with the
exception that the
initial testing scrum dilution was 1:100 in blocking hut'fi:r containing 0.>%
~I~ween ?U.
lullowed by serial 5-told dilutions into this butter. The results ot~ the
~I.ISi1 demonstrated
I ~ that seroconversion (relative to control mice) occurred in all 8 mice.
The ability eel' the anti-('. Imrrlinum serotype A C t~agment antibodies
present in scrum
t~rom the immunized mice to neutralize native (.'. hmrrlinrrnr type A toxin
was tested using the
mouse neutralization assay described in Example 23b. The amount of
neutralizing antibodies
present in the serum ot~ the immunized mice was determined usinL scrum
antibody titrations.
?0 The various serum dilutions (0.01 ml) were mixed with 5 L,Di" units of ('.
hotulinum type A
toxin and the mixtures were injected IP into mice. The neutrali-raticms wrre
perforrned in
duplicate. ~l~hc mice were then observed for signs of botulism It~r ~4 days. i
Jndiluted serum
was ti~und to protect 100% ot~ the injected mice while the l :10 diluted scrum
slid not. This
corresponds to a neutralization titer oi~ O.US-0.~ IU/ml.
'?5 These results demonstrate that neutralizing antibodies were induced when
the
6liisBotA protein was utilized as the imntunogen. Furthermore, these results
demonstrate that
arroconversion and the generation of neutralizing antibodies dues not depend
on the spcciiic
N terminal extension pmsent on the recombinant ('. huurlinrrm type A C'
fragment proteins.
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EXAMPLE 30
Construction Of Vectors For The Expression Of His-Tagged
('. hmulinum Type A Toxin C Fragment Protein Using the Synthetic Gene
S A number of expression vectors were constructed which contained the
synthetic C'.
hmulinernr type A toxin C fragment gene. These constructs vary as to the
promoter (T7 or
'f7lac) and repressor elements (laclq) presem an the plasmid. The T7 promoter
is a stronger
nronu~ter than is the '1'7lac promoter. The various constructs provide varying
expression
levels and varying levels of plasmid stability. This example involved a) the
construction of
expression vectors containing the synthetic C.'. hmulinum type A C fragment
gene and b) the
determination of the expression level achieved using plasmids containing
either the kanamycin
resistance ar the ampicillin resistance genes in small scale; cultures.
a) Construction Of Expression Vectors Containing The
synthetic C. botulirrunr Type A C Fragment C:ene
Expression vectors containing the synthetic ('. hnmlu7una type A C fragment
gene were
engineered to utilize the kanamycin resistance rather than thc,~ ampicillin
resistance gene. This
was clone t~>r several reasons including concerns regarding the presence of
residual ampicillin
in recottthinant protein derived icom plasmids containing the ampicillin
resistance gene. In
?() addition. ampicillin resistant plasmids are more difficult to maintain in
culture: the ~3-
lactamase secreted by cells containin~~ ampicillin resistant plasmids rapidly
degrades
extraccllular ampicillin. allowinL the Lrowth of plasmid-negative cells.
.A second altered leature of the expression vectors is the inclusion of laclq
gene in the
hlasmid. This repressor lowers expression from lac regulated promoters (the
chromosomally
?5 located. lactose regulated T7 polymerasc gene and the plasmid located T7lac
promoter). This
down regulates uninduced protein expression and can enhance the stability of
recombinant cell
lines. The final alteration to the vectors is the inclusion of either the 'f7
or 1'7lac promoters
that drive high or moderate level expression of recombinant protein.
respectively.
The expression plasmids were constructed as follows. In all cases. the protein
s~ expressed is the pHisBotA(syn) protein previously described. and the only
differences between
constructs is the alteration of the various regulatory elements described
above.
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i) Construction Of pHisBotA(syn) kan T7iac
The pHisBotA(syn) kan T7lac construct was made by inserting the ScrIUl.Yhul
fra~:ment
containin t the C'. homlinarm type A C fragment from plwlisBotA(syn) into
pET?4 digested with
.Scrplhl'hnl (Novagcn: fragment contains kan gene; and origin of replication).
~fhe desired
construct was selected for kanamycin resistance and confirmed by restriction
digestion.
ii) Construction Of pl-IisBotA(syn) kan laclq T7lac
l~hc pllisl3otA(syn) kan laclq T7lac construct was made by inserting the
,17~u1IHindII1
fragment containing the ('. horrlinerm type A C fragment from
pliisBotn(syn)kan~1'7lac into
It) the pE'1'?4a vector digested with XhutlHindIII. 'fhe resulting construct
was confirmed by
restriction dieestion.
iii) C:unstruction t)f pHisBotA(syn) kan lacly T7
1-he pl iisBotA( syn) kan laclc~ T7 construct was made by inserting the
,l'hulllfindl tl
1 ~ fragment containing the ('. hvlrrlinunt type I1 C fragment from
pIIisBotA(syn) kan lacld T7lac
into ,l7nrl/IlindllI-digested pliisl3otB(syn) kan laclq T7 (described in L:~c
;7c h claw). ~flze
resulting wmstruct was confirmed by restriction digestion.
h} Determination Uf The Expression Level Achieved lJsinfi
_'U I'lasmids Containing Either The kanamycin Resistance Or
The Ampicillin Resistance Genes In Smail Scale Cultures
()ne liter cultures of pHisBotA(syn) kan ~I~7)acllil2l(OE3)nl,ysS and pl-
IisE3otAtsvn)
amp ~I~7lac/I3l?1(DE3)pL,vsS [this is the previously designated pllisBotAfsvn)
construct) were
~~rown. induced and his-tagged proteins were purified as described in Esamplr
?8. No
'_'s differences in yield or protein integrity/purity were observed.
These: r~~sults demonstrate that the antigen induction levels from expression
constructs
were not affected by the choice of ampicillin versus kanamvcin antibiotic
resistance genes.
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EXAMPLE 31
Fermentation Of Cells Expressing Recombinant Botulinal Proteins
a) Fermentation Culture Of Cells Expressing Recombinant
Botulinal Proteins
Fermentation cultures were grown under the followin 8 conditions which were
optimized fur growth of the BL.21(DE3) strains containing pFT derived
expression vectors.
An overnight 1 liter feeder culture was prepared by inoculating of 1 liter
media (in a 2L
shaker flask) with a fresh colony grown on an LB kan plate. The feeder culture
contained:
6U0 nUs nitrogen source [2U ~m yeast extract (BBL) and 40 ~m tryptone
(BBL)1600 mlsJ. 200
mls ~X fermentation salts (per filer: 48.5 gm K,HPO,, 12 gm NaH,PU,~H,O, i pm
NH,C1.
2.~ pm NaC'1). 180 mls dH,U. 20 mls 20"/0 ~,lucose. ? mls I M MgSU,, S mls
O.OSM CaCI,
and :) mls of a 10 mglml kanantycin stock. All solutions were sterilized by
autoclaving.
except the kanamycin stuck which was filter sterilized.
1 ~ :1n aliquot (~ ml) of the feeder culture broth was removed prior to
inoculation, and
~~rown fur ? davs at 37°C' as a culture broth sterility control. Growth
was not observed in this
control culture in any of the ferntentations performed.
I~hc inoculated feeder culture was gown for 1?-I~ hrs (UN) at 3U-,7°C'.
Care was
takrn t« prevent ovcrsaturation of this culture. The saturated feeder culture
was added to 10L.
?U ul' fermentation media in termenter (BioflolV. New Brunswick Scientific.
I:ellsOn. N.1) as
follows. The termentcr was sterilized 120 min at 121°C with dH,U. The
sterile water was
rcmm~c~3. and Icrmentation media added as follows: 6 liters nitro~ett sourer.
'_' liters ~X
fermentation salts. ? liters ?"/o tlucose. 20 m1s 1 M MgSU,. i0 mls O.U~ M
CaCI=. 2.~-3.~
mls ~~laco) P 400 antifoam (PPG Industries Inc.. Gurnee. I1.), 40 mls lOm~/ml
kanamycin and
I() mls tracr elements (8 gm FeSO,~7H,U. 2 gm MnSO,~H,U. 2 ~m AIC1;~6Ei,0. 0.8
gm
C'oC'1~6H_O. (1.4 gm ZnSO.,~7I-1,0. 0.4 gm Na,MoO,~2H,0, U.2 gm CuC'.1,~?Ii,O.
0.2 gm
NiCI,. 0.1 gm H,B0,/200m1s 5 M HCl). All solutions were sterilised by
autoclaving. except
the kanamvcin stock which was filter sterilized. Fermentation media was
prcwarmed to 37°C.'
before the addition of the feeder culture.
;0 After the addition of the feeder culture. the culture was fermented at
:~7°C, 40U rpm
agitation. and 10 t/min air sparging. 'The DO, control was set to 20"/" PID
and dissolved
oxv~en levels were controlled by increasing the rate of agitation f~e-om 4()0-
SS0 rpm under
I)U, control. DO, levels were maintained at greater than or equal to 20"/"
throughout the
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entire fermentation. When agitation levels reached 500-60U rpm the temperature
was lowered
to 30°C to reduce the oxygen consumption rate. Culture growth was
continued until
endogenous carbon sources were depleted. In these fermentations. glucose was
depleted first
[monitored with a glucose monitoring kit (Sigma)], followed by assimilation of
acetate and
other acidic carbons [monitored using an acetate test kit (Boehringer
Mannheim)]. Uuring the
assimilation phase. the pH rose from 6.b-6.8 (starting pH) to 7.4-7.5, at
which time the bulk
of the remaining carbon source was depleted. 'This was signaled by a drop in
agitation rate
(from a maximum of 700-800 rpm) and a rise in DO, levels =~30'ro. This
corresponds to a
ODh,", reading of l8-20/ml. At this point a fed batch mode was initiated, in
which a teed
solution of 50% glucose was added at a rate of approximately 4 ~~m
glucose/liter/hr. 'hhe pH
was adjusted to 7.0 by the addition of 25% H;PO, (approximatcty 60 mls).
Culture growth
was continued and reached peak oxygen consumption within the next 3 hrs of
growth (while
the remaining residual non-glucose carbon sources were: assimilated). This
phase is
characterized by a slow increase in pl i. and air sparging was increased to 1
~Llrnin. to keep
1 > the maximum rpm below 85U. Once the residual acidic carbon sources are
depleted the
agitation rate decreases to G~0-750 rpm and the pH begins to drop. I1H control
was
maintained at 7.0 PID by reLUlatcd pump addition of a sterile 4M NaOII
solution which was
consumed at a steady rate for the remainder of the fermentation. Growth was
continued at
;0°(', and the cultures were grown linearly at a growth rate of 4-7
OD~"" units/hr. to at least
?i) 81.5 ()U,,"" units/ml (>sOg/1 dry cell weight) without induction. Antifoam
(a 1:1 dilution with
filter sterilized 100% ethanol) was added as necessary throughout the
fermentation to prevent
foaming.
During the fed batch mode. glucose was assimilated immediately (concentration
in
media consistently less than 0.1 gm/liter) and acetate was not produced In
SI~LTnItICallt levels by
2.~ the pC:~r plasmidIBL? 1 (DE i ) cell lines tested (approximately 1
gmlliter at end of
fermentation: this is lower than that observed in harvests from shaker flask
cultures utilizing
the same strains). This was fortuitous, since high levels of acetate has been
shown to inhibit
induction levels in a variety of expression systems. The above described
conditions were
found to be highly reproducible between ferlncntations and utilizing different
expression
plasmids. As a result. Llucosc and acetate level monitoring were no IonLer
preforlned during
fermentation.
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b) Induction Of Fermentation Cultures
Induction with IPTG (250 mg-10 gms, depending on the expression vector and
experiment) was initiated 1-3 hrs after initiation of the glucose feed (30-50
ODD,",/ml). The
growth rate atier induction was monitored on a hourly basis. Aliquots (~-10
ml) of cells were
harvested at the time of induction. and at hourly intervals post-induction.
Optical density
readings were determined by measuring the absorbance at 600 nm of 10 yl
culture in 990 Ltl
PBS versus a PBS control. The growth rate after induction was found to varv
depending on
the expression system utilized.
() c) Monitoring Uf Fermentation Cultures
f~ermcntation cultures were monitored using the following control assays.
i) Colony lHorming Ability
.'fin alicluots of cells were removed icom the cultures at each timepoint
sampled
I ~ (uninduced and at various times after induction) were serially diluted in
PBS (dilution 1=1 S
yl cellsl,, ml PBS. dilution ? = t ~ yl of dilution l/3 ml I'BS. dilution i =
s or ( yl of
c.lilution ?/3mls PBS) and l0U yl of dilution 3 was plated on an LB or TSA
(trypticase soy
agar) plate. T'hc plates were incubated ON at 37°C and then the
colonies are counted and
scored tier macro or micro growth.
2(f
ii) Phenotypic Characterization
C'alonies growing on LI3 or ~fSA plates (above) from uninduced and induced
timepoints were replica plated onto LB+kan, L.H+chloramphenicol (fbr
fermentations utilizing
L.vsS or p~ICYC'Gru plasmids). L.13+kaw+-ImM IPTG and LB plates, in this
order. 'I'lze plates
''s were gown 6-8 hrs at 37°C and growth was scored on each plate for a
minimum of 40-50
veil isolated colonies. The percentage of cells retaining the plasmid at time
of induction (i.e..
uninctuced cultures immediately prior to the addition of 1PTG) was determined
to be the #
colonies LB+Kan (or chloramphenicol) platel# colonies I_B plate X 100%. 'rhe
percentage of
cells with mutated pET plasmids was determined to be the Il colonies
Lti+Kan+lI'T(i plate/#
s() colonies I_I3 plate X 100%. Colonies on all LB plates were scored
morphologically fbr E.
cwli phenotype as a contamination control. Morphologically detectable
contaminant colonies
were not detected in anv fermentation.
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iii) Recombinant BotA Protein induction
A total of 10 ODD", units of cells (c~.~~., 300 ~tl of cells at UD,,""---
~O/ml) were removed
from each timepoint sample to a 1.~ ml microfuge tube and pelietcd for 3 min
at maximum
rpm in a microfuge. The pellets were rcsuspended in 1 ml of 50 mM Nafll'(),,
U.S M NaCI,
~IOmM imidazole buffer (pH 6.8) containing 1 mg/ml lysozyme. ~l'he samples
were incubated
for ?U min at room temperature and stored UN at -70°(.'. Samples were
thawed completely at
room temperature and sonicated ? X IU seconds with a Iiranson 5onitier 4sU
microtip probe
at # s power setting. The samples were centriFuged for i min at maximum rpm in
a
microfugc.
IU nn aliquot (2U Etl) of the protein samples were removed to 2U ~tl ?X sample
buffer,
hetbre or after centrifugation. for total and soluble protein extracts,
respectively. ~fhe samples
were heated to ~)~°C fee :i min. then cooled and ~ or 10 yl were
ie>aded clnto 13.x'%
sI)s-1'n(ir gels. Nigh molecular weight protein markers (BioRad> were also
loaded to allow
for estimation of the MW of identified fusion proteins. Aficr elcctr~phoresis.
protein was
I ~ detected either generally by staining gels with C'oomassie blue. ur
speciticallv, by blotting
onto nitrocellulose Ias described in L:x. 38) for Wcstcrn hlot detection of
spccilic his-tagged
proteins utilizing a NiN'1'~1-alkaline phosphatase conjugate exactly as
descrihcd by the
manufacturer 1(~iagcn).
'() iv) itecombinant Antigen Purification
nt the end of each tertnentation run. I-lU liters of culture were harvested
from the
icrmentrr and the bacterial cells were pelleted by centrifugation at (U()0 rpm
tier 10 1n1r1 !11 a
.InIU rotor (Beckman). 'The cell pellets were stored frozen at -70°(:'
or utiliicd immediateU
without freezing. Cell pellets were resuspended to I ~-3(l°/~ weight to
yoUume in resuspcnsion
:'_S buffer (~~enerally s0 mM NaPO~. U.s M NaCI. 4UmM imidazole, pH (i.8) and
lysed utilizing
either sonication or high pressure homogenization.
l~or sonication. the resuspcnsion buffer was supplemented with lysozvme to I
mglml.
and the suspension was incubated ier 30 min. at room temp. The sample was then
frozen ()N
at -70°('. thawed and sonicated =t X ?U seconds at microtip maximum to
reduce viscosity.
a0 For honuy~enization. the cells were lvzed by 2 passes through a honwgenizer
(Bonnie
Mini-lah type 8.3U li) at 60U Bar. C'r11 lysates were clttriticd by
ccntritirgation ii~r 3U I11111 al
I U.UOU rpm in a .IA I U rotor.
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For 1DA chromatography, samples were flocculated utilizin g polyethyieneimine
(PEI)
prior to centrifugation. Cell pellets were resuspended in cell resuspension
buffer (CRI3: 50
mM Nal'U,, U.5 M NaCI. 40 mM imidazole, pH G.8) to create a 20% cell
suspension (wet
weight of cellslvolume of CRB) and cell lysates were prepared as described
above (sonication
s ~ or homogenization). PEI (a ?% solution in dH,O, pl-I 7.~ with HCl) was
added to the cell
Ivsate a final concentration of 0.?%, and stirred for 20 min at room
temperature prior to
~ centrifueation (8.500 rpm in JAIO rotor for 30 minutes at 4°C'). This
treatment removed
RNA. DNA and cell wall components. rcaulting in a clarified. low viscosity
lysate ("PFI
clarified lysate").
l0 His-tagged proteins were purified from soluble Eysates by metal-chelatc
al'tinity
chromatography using either a NiNTA resin (as described in Ex. 28) or an IDA
(iminodiacetic
acid) 1'tS111 aS described below.
lL)A resin affinity puritications were performed utilizing a low pressure
chromatography wstem (ISCn). ~'1 7 mi (small scale) or 70 ml (large scaly)
C'helating
f ~ ~cphar~sc Ivast Flow ( I'harmacia) affinity column was poured: in
addition, a second guard
column was poured and attached in line with the first column (to capture Ni
ions that leached
off' the aftiniw column). The columns were washed with 3 column volumes of
dII,O. The
guard column was then removed and the affinity column was washed with 0.; M
NiSU, until
rcsistivim was established. then with dII,U until the resistivitv returned to
baseline. The
?0 columns were reconnected and eyuilihrated with cell resuspension buffer
((.'RI3; s0 ntM
NaI'(),. ().5 M NaC'I. 40 mM imidazole, p1~ 6.8). The clarified sample (in
CRI3) was loaded.
blow rates were ~ mllmin tbr small scale columns and 20 ml!min f«r lar~_c
scale columns.
~lftcr sample loading. the column was washed with CRI3 until a baseline
established and
hound protein was eluted with elution buffer (~0 mM NaPU,, 0.5 M NaCI. 800 mM
?5 imidazolc. '_'()% glycerol, pEI 6.8 or 8.0), Protein samples were stored at
4°C or -?0°('. ~1'he
yield of eluted protein was established by measuring the OD"~" of the
elutions. with a I mglntl
solution o1' protein assumed to yield an absorbance reading of 2Ø
I~he IDA columns may be reLenerated and reused multiple times (= 10). 'To
regenerate
the column. the column was washed with 2-3 column volumes of II,O, then 0.05 M
FDTA
.,0 until all ol~ the bluelareen color was removed followed by a wash with
dH,O. The IDA
columns were sterilized with 0.1 M NaUH (using at least 3 column volumes hut
not more
than i0 minutes contact time with column packing material). then washed with 3
column
volumes 0.0~ M NaP(~,,. pl-1 ~Ø then dII,O and stored at room temperature in
?U % ethanol.
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EXAMPLI? 32
Construction Of' A Folding Chaperone Ovcrexpression System
Co-overexpression of the E. cnli GroI:L/GroE:S folding chaperons in a cell
expressing
a recombinant foreign protein has been reported to enhance the solubility o1'
sotnc foreign
proteins that arc otherwise insoluble when expressed in F.. rnli [Ciragcrouu
cu crl. ( 1992) Proc.
Natl. Acad. Sci. USA 80:10344]. The improvement in solubility is thought to be
due to
chaperone-mediated binding and unfolding of insoluble denatured proteins. thus
allowing
multiple attempts for productive refolding of recombinant proteins. Bv
overexpressing the
IO chaperones, the unfolding/refolding reaction is driven by excess chaperone,
resulting, in some
cases. in hither yields of soluble protein.
In this rxamplc, a chaperone overexpression system. compatible with pE~C
vector
expression systems. was constructed to facilitate testing chaperone-mediated
solubilir-anon of
('. hcmrlinrrnr type A proteins. This example involved the c:luninL of the
Cirul:L/I:S opcron
I s and construction of a pLysS-based chaperone hyperexprcssion system.
The GroEL/GroES operon was fCR amplified and cluncd into the pC'ItScript
vector as
described in Example 28. The following primer pair was used: 5'-('GCA'1
ATGAA~CA'I~TCGTCCATTC~CA'rti-3' (SEQ 1D NO: 37) [~' primer. start colon oi'
grol:S
gene converted to lVilel site (underlined)] and 5'-GGAAC~CTTGCAGGGC'AA7'
T~1CATCATG
2U (SEQ ID NC):38) ( 3' primer. stop colon of groEL gene italicized, en
gineered I/indlll site
underlined). f=ollowing amplification, the chaperone opcron was rxciscd as an
:1'clollflindlll
fragment and cloned into pET23h digested with N'cle~l and Ilindlll. )'his
construction places
the Gro operon under the control of the T7 promoter of the pI'T'~' 3 vector.
The desired
construct was confirmed by restriction digestion.
?5 The 'f7 promoter-Gro operon-T7 terminator expression cassette was then
excised as a
I3LIIIll3.,lWl (tilled) fragment and cloned into I3umH1 (compatible with
I3~~lII)INindIlI (filled)
cleaved pl_ysS piasmid (this removed the T7 lysozvme gene). 'fhe resulting
construct was
designated pAC YCGro. since the plasmid utilizing the pAC YC l 84 origin from
the piysS
plasmid. Proper construction was confirmed by restriction digestion.
~0 pACYCGro was transformed into BL21(DL;3). cuiturea were gown and induced
with
1 mM IPTG as described in preceding examples. Total and soluble protein
extracts were
generated from cells removed before and after IPTG induction and were resolved
on a 12.5 °,'o
SDS-PAGE gel and stained with Coomassie blue. 'This analysis revealed that
high levels of
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WO ~ro~ PCT/US97l15394
soluble GroEl and GroES proteins were made in the induced cells. These results
demonstrated that the chaperone hyper-expression system was functional.
EXAMPLE 33
s (i<rowth Uf ButA/pACYCGro Cell Lines 1n Fermentation Cultures
lt~duction of BL? 1 (DE i) cells lacking the LysS plasmid which contained BotA
expression constructs grown in shaker flask or fermentation culture resulted
in the expression
n1~ primarily insoluble BotA protein. E~crmentatiun cultures were performed to
determine if
I t) the simultaneous overexpression of the Ciro operon and recombinant C'.
hnrlrlimrm type A
proteins t f3otA proteins) resulted in enhanced soluhility of the recombinant
I3otA protein.
~l'his example involved the fermentation of pHisBotA(syn)kan laclq
T7lac/pACYCGro
t3t.~1(Dlrsl and pl-Iisl3otA(svn)kan Iaclq T7IpACYC(iru BL21(DE3) cell lines.
1'he
lermentatiuns were repeated exactly as descrihed in Example ,I.
Chloramphenicol (s~
I ~ Ey~ml ) was included in the feeder and fermentation cultures.
a) Fermentation ()f pHisBotA(syn)kan laclq T7lac/pACYC(~ro
13L21(DE3) Cells
lvr lerlnentation of cells containing plasmids comprising the 'r7lac promoter.
?0 induction was with ? gms IP'TG at 1 hr post initiation of glucose teed.
'fhe UD,,"" was .ii at
time ul' induction. then 48.x, GI.~. 67 at I-s hrs post induction. Viable
colony counts
decreased from U- i hr induction [? 1 ( i s ). 0. 0. U: dilution , utilized 3
~I of dilution ? cells]
1l'IIh 11t1111ht1'v 111 parenthesis tOC the lndlCallllL I111Cr(7cOlOmeS. Uf ?8
colonies scored at the
time ul' induction. ?3 retained the pllisE3otA(syn)kan lacltl ~r7lac plasmid
tkan resistant), ??
contained the chaperone plasmid (chloramphenicol resistant) and nu colonies at
induction
grca on IPTG+lvan plates (no mutations detected). These results were
indicative of very
S1r011~~ pronuacr induction. since colony viability dropped immediately after
induction.
I~otal and soluble extracts were resolved on a 1?.s"/" SI)S-I'ACiE gel and
stained with
C'oomassie. High level induction of Giro chaperones was observed. hut very low
level
s() expressian of soluble EiotA protein was observed, increasing from I to
4.() hrs post inducticm
(no expression detected in uninduced cells). The dramatically lower expression
of the RotA
antigen in the presence of chaperone may be due to promoter occlusion (i.e..
the stronger 'f7
promoter on the chaperone plasmid is preferentially utilized).
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b) Fermentation Of pHisBotA(syn)kan lacly T7/ pACYCGro
BL21(DE3) Cells
A fermentation utilizing the T7-driven IiotA expression plasmid was pcribrmed.
Induction was with 1 gm IPTG at ? hrs post initiation of~ glucose feed. The
UU,,"" was 41 at
s time ot~ induction. then ~1.~. hl.~. W.5 and CMG at 1-4 hrs post induction.
Viable colony
counts decreased froth (l-4 hrs induction [71. 1 (34). I ( 1 ). 1, U: dilution
3 utilized 6 yl
dilution ? calls) with numbers in parenthesis for the uninduced timepoint
indicating
microcolonies. Uf G~ culonies scored at the time of induction. al) 6~ retained
both the
pHisBotA(syn)kan lactcl T-77 plasmid (kan resistant) and the chaperone plasmid
1() (chloramphenicol resistant) and no colonies at induction grew on lE'TG+Kan
plates (no
mutations detected).
~I'utal and soluble extracts were resulved on a I?.5% ADS-PAGE: gel and
stained with
C'oomassie. I-lieh level induction of Gro chaperones and moderate level
expression of~ soluhle
I3otA protein w°as observed. increasing from 1 to 4.0 hrs post
induction (no expression
1 > detected in uninduced cells).
;1 PL1-clarified lysate (0.?'% final cocnentration f'F:l) [8sp ml from is(I gm
cell pellet
(? liters t'ermentation harvesi)~ was purified on a large scale IUA column. A
total of 78 mL
ut~ protein was eluted. f~:W racts t~rom the purification mere resolved cm a
1'_'.s!% SUS-I'AGf:
gel and stained with C'oomassie. The elution was found to contain an
approximately 1:1 mix
'0 ut' IiotAichaperonc protein (Figure i?). I'EI Eysates prepared in this
manner were typically 1G
()U,~"iml. This was estimated to be 8 mg proteitvmi of lysate (hv I3t'A
assay). Thus. the
eluted recombinant I3otA protein represented U.S~% of~ the total suluhlc
cellular protein
applied to the column.
In Figure s?, lane 1 contains molecular weight markers. Panes ?-~) contain
extracts
~s from pllisL3otA(svn)kan laciq T-7lpAC'YCGro/BL?1(DI:i) cells hefi~re or
during purification
on the IDA column. lane ? contains total protein extract: lane .s contains
salable protein
extract: lanes 4 and ~ contain PFI-clarified lysates (duplicates): lanes 6 and
7 contain tlow-
through from the (DA column (dupiicatcsl and lanes X and ~) contain IUA
colcrr1711 clule (lane
~) contains 1110 the amount applied to lane 8).
p0 'i~hesc results demonstrate. that although the majority oh~ the E3otA
protein produced
was insoluhle. 3U mg/liter of soluble recombinant E3otA protein can he
purified utilising the
pHisliotA(syn)kan lacld T7/pAC'YC(iro/BL21(I)F i) expression wstcm.
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EXAMPLE 34
Purification Uf Recombinant BotA Protein From Folding Chaperones
In this example of size e~cclusion chromatography was used to purify the
recombinant
I3utA protein away from the fiUdin~~ chaperones and imidazolc present in the
IDA-purified
material ( Ev. 3.i ).
Tu enhance the solubility of the recombinant BotA protein during scale-up. the
protein
was cu-expressed with folding chaperones (Ex. 33). As observed with the
recombinant I3ot13
protein ( IW ample ~0 below). the fi~ldin~~ chaperones cu-eluted with the
recombinant BotA
1 () protein during th c Ni-IDA purification step. Because the recombinant
I3otA and BotB
proteins have similar molecular weights (about 1/IU the size of the non-
reduced folding
chaperone) and the imidazole step gradient strategy was unsuccessful in
purifying BotB away
from thr folding= chaperone (see I:x. ~U). size exclusion chromatography was
examined for the
abiliy tc~ purilj the recombinam BotA protein away from the folding
chaperones.
1 ~ .~1 column (?.~ x ?~ cm) containing Sephacryl S-l0U HR (Pharmacia) was
poured (bed
w~lumc 1 IU ml). Proteins having molecular weights greater than 100 K are
expected to elute
in the void volume under these conditions and smaller proteins should he
retained by the
heads and elute at different tithes. depending on their molecular weights. -
I'u maintain
soluhiliy of the purified BotA protein. the Sephacryl column was cduiiibratcd
in a buffer
?U lutvin;~ the same salt concentration as the buffer used to elute the BotA
protein from the IDA
column li.c~.. sU mM sodium phosphate. U.5 M NaCI. IU% glycerol: all reagents
from
~~tallinkrodt. ('hesterfield. MU).
Dive mil)iliters ot'the IDA-purified recombinant BotA protein (Ex. 33) was
filtered
throu~~h a U.4~ a syringe filter. applied to the column and the equilibration
buffer was
pumped through the column at a flow rate of 1 ml/minute. Eluted proteins were
monitored
by absorbance at 28U nm and collected either manually or with a fraction
collector (BioRad).
Appropriate fractions were pooled, if necessary, and the protein was
quantitated by absorbance
at 280 nm and/or BC'A protein assay (Pierce). The isolated peaks were then
analyzed by
native and/or SDS-PAGE to identify the proteins present and to evaluate
purity. The (uldinL
s0 chaperone eluted first. followed by the recombinant BotA protein and then
the imidazole
beak.
SDS-PAGE analysis ( 13.~°io polyacrylamide, reduced samples) was used
to evaluate
the purity of the IDA-purified recombinant BotA protein before and after S-IUO
purification.
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Figure 33 shows the difference in purity before and after the S-100
purification step. In
Figure 33. lane 1 contains molecular weight markers (f3ioRad broad range).
Lane ? shows
the IDA-purified recombinant BotA protein preparation. which is contaminated
with
significant amounts of the foldin L chaperone. Following ~-lU0 purification.
the amount of
s ti~lding chaperone present in the BotA sample is reduced dramatically (lane
~). Lane 4
contains no protein (i.e.. it is a blank lane): lanes s-8 contain samples of
IDA-purified
rcconWinant E3otH and E3otE proteins and are discussed in/i~cr.
I-:ndotoxin levels in the S-100 purpled I3otA preparation were determined
using the
I_;11. assay (Associates oi' Cape C.'od) as describe in Example 2-1. The
purified BotA
preparation was found to contain ??.7 to 45.~ EIl/mg recombinant protein.
Tltl'Se reSUIIS de1t10115tratc that S(Le eXCIUSIUIt Cltr()malUfraphy N~aS
SIICCesSfUI in
puridin~~ the recombinant f3otA protein from folding chaperones and imidazolc
following an
initial IDA purification step. Furthermore. these results demonstrate that the
~-lUU purified
f3otn protein was substantially tree ol' endotoxin.
li
CXAMPLC 35
(.'loving And Expression Uf The (' Fragment
Ut"fhe C'. hurulirterm Scrotypc E3 Toxin (~enc
?U I~hc ( '. hcuulir7rrm type 13 ncurotoxin gene has been cloned and seducnccd
~ VJhelan c~
girl. ( I ~)~)?) flppl. E:nviron. Microbial. ~8:? i4~ and l lutson rml. ( 1
c)94) C'urr. ~-1icrobiol.
_'8: l01 ~. l'hr nucleotide sequencr of the toxin scene derived I'rt~m the
E~:kluncE I 7fi strain
(.~'fC'C' ~'~76s) is available from the Ei~~IBLICienfiank sequence data banks
under the
acccssiun number X71343: the nucleotide sequence of the coding region is
listed in SI:Q ID
'_'S NU:3c). 'I~he amino acid sequence of the (', horulinum type B ncurotoxin
derived from the
strain h.klund 17I3 is listed in SL:Q ID NU:40. The nucleotide sequence of the
('. horrrlinum
serotvpc E3 toxin gene derived from the Uanish strain is listed in SI;Q ID
N():41 and the
corresponding amino acid sequence is listed in SEQ ID N():4?.
'fhc 1)NA sequence encoding the native ('. hvnrlrnurn serotvpe f3 (' i~ragment
gene
s0 clcrivcd from the Eklund 17B strain can he expressed using the pF.TtEish
vector: the resulting
coding: region is listed in SE:Q 1f) NU:43 and the corresponding amino aci~E
sequence is listed
ill SF:Q 1D NU:44. The UNA sequence encoding the native C'. hnnrlimrm scrotypc
B ('
fragment gene derived from the Danish strain can be expressed using tile pI~TI-
Iish vector; the
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PCTIUS97I13394
resulting coding region is listed in SEQ ED N0:45 and the corresponding amino
acid sequence
is fisted in SEQ ID N0:4G. The C frgament region from any strain of ('.
bntr.rlinum serotype
B can he amplified and expressed using the approach illustrated below using
the C: fragment
derived tiwm C'. horarlinum type B 2017 strain.
The ('. hmulintun type B neurotoxin gene is synthesized as a single
polypeptidc chain
which is processed to form a dimer composed of a light and a heavy chain
(inked via
disulfide bonds: the type B neurotoxin has been reported to exist as a mixture
of predominatly
aingle chain with some double chain (V~'helan eo crl.. supra). The ~0 kD
carboxv-terminal
portion of the heavy chain is referred to as the C fragment or the H~. domain.
L:xpression of
I l) the C' !'ragmcnt of C'. hvrrrlirrrrm type B toxin in hcterologous hosts
(c~.lr., F.. rrrli) has not been
previously reported.
The native C' fragment of the (.'. hmulinrrm serotype E3 toxin gene was cloned
and
mpressie~n constructs were made to facilitate protein expression in L'. cvli.
This example
invoUvcd I'C'R amplification of the ~!ene, cloning, and construction of
expression vectors.
1 ~ ~I-he C' f~ragmcnt ch' the ('. hrnrrlinrrnr scrotype B ( BotB) toxin gene
was cloned using
the promcols and ~11t1d1tI1111S described in >rxamplc ?8 for the isolation of
the native BotA
gene. ~fl~c ('. hnrrrlinum tvpr E3 ?U17 strain was obtained from the American
'fvpe Culture
('ullcction (:~ l~C'C' ~ 1784x). Thr ti~llowing primer pair was used to
amplil'v the l3otl3 gem:
'-C'(iC.'C';1~TC~(~C'I'CiA~I'AC'An'I~AC'~I~AATAGAA AT(i-~' (s' primer.
engineered :'~'onI site
?0 undcrlimd (~I~:Q lI) N():47)j and ~'-GC'AAG
C'TTTT.=IT'fC.'ACiTCC'ACCC't"I~CATC-s' [s'
primer. engineered hlindllI site underlined, native gene termination colon
italicized (SLQ ID
!v():~X)[. ~I~tcr cloning into the pC'Rscript vector, the
,~'In~I(filled)atfindlll ti~aLment was
clunccl into pIrTFIish vector as described for BotA C fragment gene in Example
?8. The
resulting construct was termed pl-IisRot().
?> pt-lisBeriB expresses the BotB gent sequences under the transcriptional
control ui~ the
~I~7 lac promoter and the resulting protein contains an N-terminal IUXf-lis-
tae affinity tag. 'hhe
pllisE3ouB expression construct was transformed into I3L?1(DE3) pLys~
competent cells and I
liter cultures were grown. induced and his-tagged proteins were purified
utilizing a NiNTA
resin (eluted in low pf l elution huffed as described in Example ?R. Total.
se~luble and
3() ptlriticd proteins were resolved by SDS-PAGE and detected by Coomassie
staining and
Western blot hybridization utilizing a chicken anti-(.'. hrmrlinum serotype B
tc~xoid primary
antibmiv (generated by immunization of hens using ('. hunrlinrrm serotype 13
toxoid as
described in Example i). Samples c~f l3otA and BotE: C fragment proteins were
included on
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the gels for MW and immunogenicity comparisons. Strong immunoreactivity to
only the
BotB protein was detected with the anti-('. hmulintrm serotvpe B toxoid
antibodies. The
recombinant BotB protein was expressed at low levels (3 mg/liter) as a soluble
protein. The
luritied BotI3 protein migrated as a single band of the predicted MW (i.c., --
sOkUl.
These results demonstrate the cloning of the native ( '. hurtrlinum serotype B
C
I~raLment gene. the expression and purification of the recombinant BotB
protein as a soluble
his-ta~~f:ed protein in E. cw/i.
EXAMPLE 3G
Ccneration Uf Neutralizing Antibodies Using The Itecomhinant pH isBotl3
Protein
Thr ability of the purified pflisBot protein to generate neutralising
antibodies was
rxamined. Nine EiAI_Be mice were immunized with BotB protein (purified as
described in
f:x. >~) using (irrhu CiMI)I' adjuvant (C'C.' Biotech(. ~l~i~e low Pli elution
was mixed with
1 ~ (ierhu ad.juvant and used to immunize mice. Each mouse received a
suhcutanectus injection
of 1(H) yl anti~~rnlad.juvant mix ( 1~ frg antigen + l leg ad.juvant) on day
(1. Mice were
subcutaneouslv boosted as above on day 1=1 and bled un day 28. Mtice were
suhseduentlv
hmst~d I-'_' wec:la after bleeding, and were then bled on day 7U.
Anti-('. hu~ulinnm serotvpe B toxoid titers were determined in day ?8 scrum
from
~l) individual mice from each group using the ELISA protocol outlined in
Example '_'c) with the
reception that the plates were coated with ('. hululin:rm serowne l3 toxoid,
and the primary
altllhl)ll~ 1S'aS a chicken anti-('. l~rrntrlinrrm serotvpe R toxoid.
Scroconvcrsion relative tet
control mice immunized with pI-IisBotl: antigen (described bclowl~ was
observed with all 9
mice itttmuni-r_ed with the purified pHisBotl3 protein.
-hhc ability of the anti-BotB antibodies to neutralize native C'. hnnrlinum
type I3 toxin
was tested in a mouse-('. hrnrrlirnrrn neutralization model using pooled mouse
serum (see I:x.
~h). The LI7;" of purified ('. horrrlirarrnr type B toxin complex (f)r. Laic
.lohnson. University
of WISCU11S111. Madison) was determined by a intrapcritoncal (II') Method
(Schantz and Kautler
( lc)78). .vrrprcrJ win(: 18-?? ~~ female ICIt mice. The amount of
neutralizing antibodies present
~() in the serum of the immunimd mice was determined using scrum antibody
titrations. ~Chc
various serum dilutions (0.01 ml) were mined with i LDt" units eri' ('.
hwulirurm type I3 toxin
and the miWures were injected IP into mice. ~I~hc neutralizations ~~crc
performed in duplicate.
'fhe mice were then observed for signs of botulism tits ~1 days, Undiluted
serum (day 28 or
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WO 98108540 PGTIUS97115394
day 70) was found to protect 100% of the injected mice while the I : I O
diluted serum did not.
This corresponds to a neutralization titer of 0.0~-0.5 iU/ml.
These results demonstrate that seroconversion occurred and neutralizing
antibodies
were induced when the pllisBotB protein was utilized as the irnmunogen.
EXAMPLE 37
Construction ()f Vectors To facilitate l~xprcssion
()f Ills-Tagged Botli Protein In fermentation Cultures
1 t1 ~1 number of expression vectors were constructed to facilitate the
expressiun of
recombinant Rotes protein in larLe scale fermentation culture. These
constructs varied as to
the strength of the pronuUCr utilized (T7 or T7lac) and the presence of
repressor elements
larlq ) on the plasmid. ~f'he resultinL constructs varied in the level of
expression achieved and
in plasmid stability which tacilitated the selection of a optimal expression
system for
1 ~ li:rmcntatiun scalcup.
The I3ot13 expression vectors created for fermentation culture were engineered
to
utilize tlm l:anamycin rather than the ampicillin resistance gene. and
contained either the T7 or
~f7lac premuUCr. with or without the laciq gene tbr the reasons outlined in
Example i0.
In all cases, the protein expressed by the various expression vectors is the
pHisl3ot 13
hrotcin described in Example is. with the only differences between clones
being the alteration
of various rc~~ulatorv elements. using the designations outlined below. the
pllisE3otI3 clone
( l:x. ; ~ ) is mluivalent to pl 1is13otB amp -I'7lac.
(:onstruction Of pllisl3atB kan '1'7lac
ptlisBotl3 kan T7lac was constructed by insertion of the f3t~Illltfindlll
ti~aement of
pl iisBouli which contains the BotB ~~ene sequences into the pYA 1870-?680 kan
1'7lac vector
which had been digested with B~~lll and Hindlll (the pl'A I 870-2680 kan
~I'7lac vector contains
the pi:T?~4 kan Lenc in the pET'_'s vector. such that no laclq gene is
present). Proper
I:UnSII'ttCIII?rl ()f pHisBotB ban T7lac was confirmed by restriction
ciigcstion.
;0
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PCTIUS97I15394
b) Construction Uf pHisBotB kan iacIq T7lac
pHisI3otB kan IacIy 'f7lac was constructed by insertion of the
13,s,~lll/Hindlll fragment
01' pHisBotl3 which contains the BotL3 gene seyuenccs into similarly cut
pf:T'?4a vector.
Proper construction of pHisBotB kan laciq ~f7lac was c~ntirmed by restriction
di~~estion.
c) Construction Of pHisl3otB kan laclq T7
pHisI3otB kan laclq T7 was constructed by inserting the NdelIXhoI fragment
ti~om
pl-lisl3otlkan lady T7lac which contains the BotR gene sequences into
similarly cleaved
pl'A 187()-2080 kan lacld T7 vector (this vector contains the T7 promoter, the
same N-
1() terminal his-taL as the l3ot constructs, the ('. cJi/7icile tcwin A
insert. and the kan laclq genes:
this cloning replaces the ('. cJi~)icile main A insert with the I3cltR
insert). I'ropcr construction
was confirmed by restriction digestion.
Ivxprcssion of recombinant BotB protein from these expression vectors and
purification
ol~ the l3otf3 protein is described in Example 38 below.
1
EXAMPLE 38
f~erlncntation And I'uritication ()f Rcculnhinant f3otI3 1'rotcin t!tilizinc
'I~hc
pl-IisI3otR kan laciq T7lac, pf~isl3otB kan ~f7lac And pl-iisf3eltE3 kan
laclcl I~7 Vectors
=!0 ~flzc pHisl3otf3 kan laclq T7lac. pf-IistiotB kan T7lac and fiotli kan
Iaelcl l~7 constructs
~illl ll'allSlOrtlled IlltO tl7t', I3121(Uf: i) Stl'aln~ ~Ylre Lr()wll 111
termelltatlnll ellltllreS I() dlterlnln(:
the utility ot~ the various constructs for large scale expression and
purification W~ soluble l3otf3
protein. ;111 lcrmcntations were pcrtorlncd as described in f ample s 1.
a) Fermentation Of pHisl3otB kan laclq T7lac/13121(DE3) Cells
'fhc icrmentation culture was induced 45 min post start of glucose feed with l
gm
II''r(i (final concentration = 0.4 mM). pfl was maintained at (,.s rather than
7Ø ~fhc ()U,,""
was 27 at time of induction, then i~. _i8. and 40 at I-; hrs post induction.
f)uplicatc platings
of diluted 1 hr induction samples (dilutions were prepared as described lx. s
1. dilution 3
p0 utiliLCd 3 Ell W''dilution ? cells) on 'fSA and Lf3 ~-kan hlatcs yielded 89
'hSA colonies and 81
kan colonies (90% kan resistant).
Total and soluble protein extracts were resolved on a i 2.5°/> SDV-
I'A(:iF ~,cl and total
protein was detected by staining with C'uomassic blur. l.ow Icvel induction of
insoluble I3ot
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WO 98108540 PCT/US97/15394
E3 protein was observed. increasing tiom 1 to 3 hrs post induction (no
expression was detected
in uninduced cells).
h) Fermentation Of pHisI3otB kan T7lac/B121(DF3) Cells
s The fermentation culture was induced I hr post start of glucose teed with 2
gm IPTG
t tinal concentration = 0.8 mM). pI i was maintained at 6.5 rather than 7Ø
The OD,,,H, was
'_'-1.i at time of induction. then 31.x. ~?. and 33 at 1-3 hrs past induction,
respectively.
Duplicate platings of diluted 0 hr and 2 hr induction samples (dilutions were
prepared as
described C:x. s 1: dilution .i utilized 3 (tl of dilution ? cells) on -I'SA
and LE3+kan plates
1 () yielded s? TSA colonies and 54 kan colonies (all kan resistant) for
uninduced cells. and 1
'1'~A colony and 0 kan colonies ? hr post induction. 'These results were
indicative of strong
induction. since viable counts decreased dramatically ? hrs post induction.
'( mal and soluble extracts were resolved on a 10% SDS-PAGE gel and total
protein
was detected by staining with ('<aomassie blue. Moderate induction of
insoluble I3utB protein
I ~ was observed. increasing from I to ; hrs post induction (no expression was
detected in
uninduced cells).
c) Fermentation Of pEiisBotB kan laclq T7/B121(DF:3) (:ells
'fhe tcrmentation was induced ? hr post start of glucose loud with ~ ~~m iPT(i
(final
?U conecntratic?n = l.O mM). pII was maintained at 6.5 rather than 7Ø The
UDh"" was ~ts at
time uf~ induction. then ~17. 50. and 5t) and » at I-4 hrs post induction.
respectively. Viable
cnlunv counts decreased after induction (9G. i. 1. ?. 3: dilution 3 utilized 3
yl of dilution
~c:flsl. t)1' O3 colonies scored at the lime of induction. all bs retaining
the Buts; plasmid (kan
resistant) and n(1 COIUnICS al It7dllCtIUt1 LCCW OIl (PT(i + Kate plates Ino
mutations detected).
~fe~tai and soluble extracts were resolved on a 12.5% SDS-('ACiE gel and total
protein
was detected by staininL with Cootnassie blue. Moderate level induction of
insoluble l3otl3
protein was erhserved. increasing from 1 to 4 hrs post induction (lower level
expression was
detected in uninduced cells. since the 'f 7 rather than T7lac promoter was
utilized).
d) Purification Of pHisBotB Protein From pHisBotB amp
T7lae/B121(DE3) Cells
Soluble recombinant BotB protein was purified utilizing NiNTn resin from 80 ml
of
- cell tsate ~~enerated ti~om cells harvested from a pl-IisBotli fermentation
(using the p((isBotB
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WO 98108540 PCTIUS97115394
amp T7lac/Bl2t(DE3) strainj. As predicted from the small scale results above.
the majority
of the induced protein was insoluble. As well. the eluted material was
contalninatcd with
multiple E. cwli contaminant proteins. A Coomassie bloc-stained SDS-PAGI: gel
containing
extracts derived t'rum pllisBotB amp T7lac/Bl2l(DEV,3) cells brl~ore and
during purification is
s shown in figure 3:L In figure 34. lane 1 contains broad range protein MW
markers
(BioRad). Lanes ?-s contain extracts prepared from pliisl3otB amp
'r7lac/B121(DI:3) cells
grown in fermentation culture: lane ? contains total protein: lane 3 ccmtains
soluble protein:
lane 4 contains protein which did not bind to the NiN'hA column (i.c~.. the
flow-through) and
lane ~ contains protein eluted from the NiNI'A column.
IU Similar results were obtained using a small scale IDA column utilizing a
cell ivsate
from the pl 1is13utB kan lacIq 'r7 fermentation described shove. ?~0 mls h~ a
?U% wlv I'F;l
clarified Ivsate (sU gms cell pellet) of botR kan laclq ~I~7/8121(I)E:3) cells
were purified on a
small scale Il)A column. The total yield ui~ eluted Protein was 21 mg protein
(assuming I
mg/nti S(llLlllt111 ~ ? OD,~"Iml). When analyzed by SUB-I'AW~. alld
C'(1t1111aSSIC StJ111111~!. the
1 s l3otR protein was found t~ comprise approximately s0% of the eluted
protein with the
remainder hein~~ a ladder of 1. onli proteins similar to that observed with
the NiN'I~.<1
purilicatiun.
l~hc NiN~fn alkaline phosphatase conjugate was utilized to detect his-tagged
proteins
on a W'cstcrn blot containing total, soluble, soluble (f'f:l clarified).
soluble (at~ter IUA column)
_'U and elution samples t~rom the IDA column purification. 'hhe results
denumstratcd that a small
percentage e~f~ fiutt3 protein was soluble, that the soluble prmein was ncn
prccinitated by I'CI
treatment and was quantitatively hound by the II)A column. since a 1 liter
Irrtnentahon
harvest yielded a (~7.~ ~=m cell pellet. this indicated that the yield ol'
soluble at'tinitv purified
RotE3 protein from the lUA column was 14 mg/litcr.
~j
EXAMPLE 39
(.'u-Expression Of Recombinant l3cnl 1'roaeins
,4nd Holding C.'haperones In Fermentation Cultures
sU lermentations were performed to determine if the simultaneous
uverexpression ot~
t(lldlllg (:llaperOlleS (i.c~.. the (iro operon) and the f3cnB protein
resulted in enhanced solubility
of~ the I3ot 13 protein. ~fi~is example involved fermentation of the p)
IisI3utBkan laciq
'r7lac/pAC'YCGro E3L? 1 ( DE3). pHisI3otI3 kan T7lac/pA(.'YCCiro 131? 1 (I)E3)
and pl Iisl3otBkan
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Nrp 9g~pgs4p PC'TlUS97/15394
laclq T7! pACYCGro BL21(DE3) cell lines. Fermentation was carried out as
described in
Example 31; 34 yglml chloramphenieol was included in the feeder and
fertncntation cultures.
a) Fermentation t)f pHisl3otlitcan laclq T7lac/pACYCGro
BL21 (DE3) Cells
Induction was with ~ gms IPTG at I hr 1 ~ min post initiation of the glucose
feed.
I~he ()D,,"" was 38 at time of induction. then 50, 58.5. 6? and G8 at I-4 hrs
post induction.
Viable colony counts decreased during induction (?4. 0. 0. ?. 0 at 0-4 hr
induction: dilution 3
utilized 3 tcl of dilution ? cells). Uf ?:l colonies scored at the time of
induction. ?=1 retained
It) the ButE3 plasmid (kan resistant). ?-1 contained the ci~aperone plasmid
(chloramphcnicol
resistant) and no colonies at induction grew on IPTGi-Kan plates (no mutations
detected).
t~utal and soluble extracts were resolved on I?.5% SDS-PAt~F; gels and were
either
stained with (.'oomassie blue or subjected to Western blotting (his-tagged
proteins were
ctctectcd utilizing the NiNTA-alkaline phosphatase conjugate). 'This analysis
revealed that the
1 s tyro chaperones were induced to hiLh levels. hut very low level expression
of soluble Butf3
hrutcin was observed, increasing trum I to 4.0 hrs post induction (nu
expression detected in
uninducect cells. induced protein detected (»1IV Ufl Western blot). The
dramatically lower
expression ul' Botf3 protein in the presence of chaperone may he due to
promoter occlusion
(i.e.. the stronger 'h7 promoter on the chaperone piasmid was pretercntially
utilised).
?0
h) Fermentation Of pHisI3otB kan T7lac/pACYCGro/I3121(DF3)
Cells
IIIdlIClll)Il W'aS lVIll1 ~ t:ms If TOi at 1 hr punt initiation of the glucose
teed. The ()D,,""
was 3:.s at time of induction, then ~~~1. 51, 58.5 and 69 at I-4 hrs post
induction. Viable
colony counts decreased after 2 hrs induction (43. 65. 74. U (70), 0 (70) at
tf-4 hr induction;
bracketed numbers represent microcolonies; dilution 3 utilized 3 Etl of
dilution ? cells). Most
colonies at induction retained the Botl3 plasmid (kan resistant)and the
chaperone piasmid
(chlorantph enicol resistant) and no colonies at induction grew on IPTCi+Kun
plates (nu
mutations detected).
30 ~l~utal and soluble extracts were resolved on a 12.5'% SDS-fACiE~: eel and
subjected to
Western blotting: his-tagged proteins were detected utilizing the NiNTA-
alkaline phosphatase
conjugate. This analysis revealed that the. Ciro chaperones were induced to
high levels and
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PCTNS97/15394
low level expression of soluble Bat B protein was observed. increasing from t
to 4.0 hrs post
induction ( no expression detected in uninduced cells).
A small scale IDA purification of BotB protein from a 2~0 ml Pl:l clarified I
S°~o w/v
extract ( :7.5 gm cell pellet) yielded approximately 1?.5 Itlg protein, of
which approximately
s s0°/> was Bntf3 protein and 50% was CiroEl, chaperone (assessed by
C'oomassie staining of a
10'%~ 5DS-PAGE gel). The NiNTA alkaline phosphatasc c<mjugate was utilized to
detect his-
taggcd proteins on a Western blot containing total, soluble. soluble (I'FI
clarified), soluble
fatter lDA column) and elution samples from the IDA column purification. The
results
demonstrated that all of the BotB protein produced by the pl-lisBotB kan
1() T7lac/pAC'Y('(iru/Bl2l(DE3) cells was soluble: the BotR protein was not
precipitated by I'EI
treatment c:ltd was quantitatively hound by the 1DA column. ~incc a l liter
fermentation
harvest yielded a 7~ gm cell pellet. this indicated that the yield o1~ soIUbIc
aftinttv purified hot
f3 protein fiom this tcrntentation was 13.~ mg/liter. These results also
demonstrated that
additional purification steps are necessary to separate the chaperone proteins
t~rom the BotR
1 s protein.
c) F'crmcntatiun Of pHisI3otBkan Ittclq
T7lpA('YC(:ro/I3L21(UF3) Cells
Induction was with 4 gms IPTCi at 2 ltr post initiation of the glucose feed.
~I~ite (>D,,""
?0 was ~tO at time ot~ induction. then ~h. G,. G9 and 7t.~ at 1-~l ltrs pout
induction. Viably colony
cc>unts cfccreascd after induction tab. 3(~), s. (). 0 at 0-4 hr induction:
bracketed numbers
represent microcolonies: dilution ; utilized 3 Etl of'dilution 2 cells). All
ts;/s;) colonies
scored at the time of induction retained the liotl3 plasmid (kan resistant)
and the chaperone
plasmid (chloramphenicol resistant) and no colonies at induction grew on
tP'I~G+f~an plates
''s (no mutations dNtected).
Total and soluble extracts were resolved on a 10% S1)~-I'AGI.: gels anct
Western
blotted and his-tagged proteins were detected utilizing the NiN'1'A-alkaline
phosphatasc
conjugate. 'this analysis revealed that the Ciro chaperones were induced to
hlLit levels
(observed by ponceau S stainin~~l, anct a notch higher expression of soluble
L3ot R protein
;0 tcompared to expression in the pHisl3ott3 kan T7lac/pAC.'YC.'Oro
fer~ttc:ntatioy was observed
at all timepoints. including uninduccd cells (some increase in l3otl3 protein
levels were
observed at~ter induction).
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A small scale IDA purification of BotB protein from a IOU ml PEI clarified IS%
wiv
extract ( l ~ gm cell pellet) yielded approximately 40 mg protein. of which
approximately 50%
was BotB protein and 50% was CiroEL chaperone. as assessed by C'oomassie
staining of a
10°/> SDS-PAGE gel. The NiNTA alkaline phosphatasc conjugate was
utilized to detect his-
- s tagged proteins on a Western blot containing total, soluble, soluble (PEI
clarified), soluble
tatter IDA column) and elution samples from the 1DA column purification. The
results
. demonstrated that a significant percentage (i.c~., -1()-20 °ro) of
I3otB protein was voluble, that
the soluhiiized protein was not precipitated by PCI treatment and was
quantitatively bound by
the IDi1 CUlltnlt7. Since a 10 liter fermentation yielded a 108 gm cell
pellet. this indicated that
I (1 thr vielct ol' soluble affinity purified BotB protein from this
fermentation was 144 mglliter.
In a scale up experiment. ? liters of a 20% wlv I'EI clarified lysate,~ of
pl~IisI3otB kan
laclcl l~7/pAC'YWirolI3L?1(DES) cells were purified on a large scale IDA
column. The
purilication was performed in duplicate. 'l~he total yield of RotB Protein was
?'_0 and .~'_'~
m~~s prouein in the two experiments (assuming i mg/ml solution = ?.0
UD,%"/ml). This
I ~ represents 0.7°/r. ur I .0%. respectively. of the total soluble
cellular protein (assuming a I'I:I
Ivstatc havinL a concentration of 8 mg protein/ml and that the eluted material
c«mprises a 1:1
miwurr et' BoUB and folding chaperone). The NiNTA alkaline phosphatasc
conjugate was
utilizrd m detect 1115-taLged prole111S 011 a Western blot contamtng total,
soluble. soluble (YEI
clarified). soUuhic (after (DA column) and elution samples from the llO1
column purification.
?t) 1 hese results clcmonstrated that a significant percentage (i.e.. -~-1 (>-
20 '%) of the I3otL~ protein
was se~luhlc, that the soluhilized protein was not precipitated by PEI
treatment and was
yuantitativclv hound by the II)A column. Since a t liter icrmentation harvest
yielded a 108
,~m crll pellet. this indicated that the yield of soluble at~tinity purified
BoUB protein from the
IarLe scale purification v,.~s OO mg or 89 mglliter. l~hese results also
demonstrated that
2s further purification would be necessary to remove the contaminating
chaperone protein.
The above results provide methodologies for the purification ot~ soluble BrnB
protein
from fermentation cultures. in a forth contaminated predominantly with a
single E. cwli
protein (the folding chaperone utilized to enhance solubility). In the next
example. tllethOds
arr provided for the removal of the contaminating chaperone protein.
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WO 98108540 PCT/US97/15394
EXAMPLE ~0
Removal Of Contaminating Folding Chaperone ('rotein
From Purified Recombinant C'. J)nlIllil?feel) 'I'ypc Ii Protein
s In ibis example size exclusion chromatography and ultratiltration was used
to purify
recombinant BotB protein from the folding chaperones and imidazole in IDA-
puritied
material.
~l~u enhance the solubility of the recombinant I3otB protein during scale-up.
the protein
was co-expressed with folding chaperones (see E;x. i9). During the Ni-fI)A
purification step.
1 () the ti~ldinp chaperones co-eluted with the BotB protein in 8U0 n~M
imidazole: therefore, a
second purification step was required to isolate the BotB free of folding
chaperones. Lane s
of Fiyrc ss contains proteins eluted from an IDA column to which a lysate of
pllisl3ott3 kan
lac(q T7/pAC'YC'Ciro/BL21(DC3) cells had been applied: the proteins were
resolved on a
-1-1 >°% polvacrvlamide pre-cast gradient eel f I3io-Rad. I lercules.
C'A ) run under native
1 ~ c;onditions and then stained with Coomassie blue. !n Figure 3~. lanes I
and 4 contain proteins
present in peak I and peak ? from a 5ephacryl S-10() column run as described
below: lane
1S hlallk.
As seen in lane .> of Figure 3~. the IDA-purified sample consists primarily of
the
folding chaperones and the l3otB protein. The tact that the chaperones and the
Eiot B antigen
?U appear as ww distinct hands under native conditions suggested they were not
cnmplcxed
tcyethcr ancf therefore. it should be possible to separate them. using either
a gradient of
imicfaze~lc concentrations or size exclusion methods.
In order t~ determine whether a Lradient of imidazole concentrations could be
used to
separate the chaperone from the I3ot13 protein. a step gradient using imida-
rolc at ?t)0. 4(lt).
?s 000, and 800 mM in ~U tnM sodium phosphate. ().5 M NaCI and 10 % glycerol.
pt-I 6.8 was
applied to an IDA column (containing proteins bound from a iysatc of pllisBotB
kan lacJq
~f7lpAC.'YCGroIBL21(DE:3) cells). By narrowing the range of imidazoie
concentrations. it
was hoped that the BotB and chaperone proteins would diffcrcntiallv elute at
different
concentrations al' imidaie.~le. 1-eluted proteins were numitored by ahsorbance
at ?80 nn~ and
30 collectecE either manually or with a traction collector (BioRadl. 1'rmein
was found to elute at
30U and 400 mM imidazole only.
Figure 3O shows a Coomassie stained SUB-PAVE gel containing protein eluted
durin~~
the imidazolc step gradient. lane I contains broad range MW markers
(l3ioltad). Lane
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qrp 9g/pgsqp PCT/US97115394
contains BotB protein purified by IDA chromatography of an extract of
pHisBotB/BL21 (DE3)
pC.ysS cells grown in shaker t7ask culture (i.e., no co-expression of
chaperones: I:x. 35).
lane i contains a 20% wiv PEI clarified lysate of pHisButB kan Iaclq
~f7lpACYCGru1BL21(DC3) cells (i.c~.. the lysate prior to purification by IDA
chromato~~raphyl. Lanes 4 and ~ contain protein which eluted at ?00 or 400 mM
imidazole,
respectively. Lane 6 is blank. I_atZes 7 and 8 contain 1IS the load present in
lanes 4 and 5.
.-1s shown in Figure 3G. both the chaperone and the ButB protein eluted in 200
mM
imidazule. and more chaperone elutes in 400 mM imidazole. however no
concentration of
IlllldazOle tested permitted the elution of I?;otB protein alone.
Consequently, no significant
I() purification was achieved using imidazole at these c:uncentrations.
Because of ihc considerable difference in molecular w~cif:hts between the
folding
rhapcrune. which is a multimer with a total molecular weight around 400 kD (as
determined
cm a ~hooi~x KE3 H04 sizing column by HPLC), and the recombinant BotB protein
(molecular
weight around i0 kD). size exclusion chromatography was new examined for the
abilitv_ tn
I s separate thesr proteins.
u) Size Exclusion Chromatography
~1 column containing Sephacrvl ~-1()0 I1R (S-100) (Pharmacia) was poured (Z.5
em x
_'~ cm : - 1 10 ml bed volume). The column was equilibrated in a buffer
consisting u1~
?U phusplurte buffered saline ( IOmM potassium phosphate, I50 mM NaCI. pl l
7.?) and 10
~,Ivcrrul (Mallinkrodt). Typically. s ml of the IDA-purified BotB protein was
filtered through
n 0.~~ ~i svrinLe filter and applied to the column. and the eduilibration
butler was pumped
thruuLh the column at a flow rate of I ml/minute. fluted proteins were
monitored by
ahsurbancc at 280 nm and cullrcted either manually or with a fraction
collector. Appropriate
?s tubes were pooled, if necessary, and the protein was quantitated by
absorbance at 280 nm
anctlur by BCA protein assay. 'rhe isolated peaks were then analyzed by native
and/or
ADS-PAGE to idrrntify the protein and evaluate the purity.
F3ecause of its larger size. the folding chaperone eluted first, followed by
the
recombinant ButB protein. A smaller third peak was observed which tailed to
stain when
s() analyzed by SDS-PAGE: and therefore was presumed to be irnidazolc.
- ADS-I'AGL: analysis ( l?.i°ru pulyacrylamide. reduced samples) was
used to evaluate
the purity of the IDA-purified recombinant BotB protein before and after S-
1()0 purification.
. 'The results are shown in Figure s3.
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CA 02296765 2000-O1-14
WO 98108540 Pl'rTIUS97/15394
In Figure 33. lane 1 contains broad range MW markers (BioRad). Lane ~ contains
IDA-purified BotB protein. Lane 6 contains IDA-purified BotE3 protein followin
p. S-100
purification. Lane 7 is blank (lanes 2-4 were discussed in Fx. 3~4 above).
The results shown in Figure 33 show that the IDA-purified BotB is
si~~niticantly
contaminated with the folding chaperone (molecular weight about 60 kl) under
reducing,
conditions: lane 6). Ivllowing S-l0U purification. the amount of folding
chaperone present in
the l3olB sample was reduced dramatically (lane 7). Visual inspection of the
C'oomassie
stained SI)S-PAGIr gel revealed that after S-100 purification. a c)0"~~ of the
total protein
present was BotB.
l0 The lDA-purified BotR and the S-lUU-purified BotB samples were analyzed by
E~IPLC
cm a size exclusion column tShodex KB 804); this analysis revealed that the
RotE3 protein
represented (i4'% of th a total protein in the IDA-purified sample alld that
following S-IUU
purification. the liotl3 protein represented ='95"/" of~ the tonal protein in
the sample.
The IDA-purified RotB material was also applied to a ACA 44 (Spectral'ur.
Houston,
I, ~l'X) c«lumn. l~he ACA ~~4 resin is equivalent W the S-100 resin and
chromatography using
the A('A -t-t resin was carried out eaactlv as described above fer the S-100
resin. -l~he AC.'A
-1~4 resin was found to separate the recombinant l3ott3 protein t~rom the
Ibldin~ clutperone.
The .~C'~1 -L4-purified tie~tf3 sample was anaiyred fur endotoxin using the
f..Al, assay
(Associates of Cape t'od) as describe in txample ?~i. Two aliqctuts of thr
AC'.~1 ~4-purified
20 l3utt3 preparation were analyzed and were found to contain either a8 to I
Ih l~:li/m~,
rccomhinant protein or ~)4 to 18c) ELJ/mg recombinant protein.
These results demonstrate that size exclusion chronlatoLraphy can hr used to
purify the
1'l'CUIllhlllatlt l3cUB protein fiwm the folding chaperone anct imidazoie in
It):1-purified material.
b) llltralfiltration For The Separation Uf Recombinant i;~tl3
Protein And Chaperones
I:Itratiltration was examined as an alternative method for the separation
recombinant
I3o113 proUein and folding chaperones in IDA-purified material. Vv'hile in
this example only
mixtures of l3otB and chaperones were separated by ultratiltration. this
technique is suitable
'~0 for use with recombinant (3otA and l3otfproteins as well provided that the
wash huftcrs used
are altered as necessary to take into account different requirements tier
solubiliy.
The recombinant E3otFi protein and folding chaperones were separated using a
ovo-step
sequential ultratiltration method. The first membrane used hart a Ill)i11111a1
IIlUlet:lllaC Welght
-2UU-
CA 02296765 2000-O1-14
PGTIUS97l15394
cutotf (MWCO) of approximately 100 kD; this membrane retains the larger
folding chaperone
while allowing the smaller recombinant protein to pass through. The addition
of several
volumes of wash huffer may be required to efficiently wash the recombinant
protein through
the membrane. The second step utilized a membrane with a nominal MWC'O of
s approvimatelv l0 kD. During this step. the recombinant antiLen was retained
by the
membrane and could be concentrated to the degree desired and the imidazole and
excess wash
huffcr passed through the membrane.
Twenty-seven milliliters of an 1DA-purified BotI3 preparation was
ultratiltercd through
a ~7 mm Y'M 100 ( 100 kD MWC:O) membrane (Amicon) in a ~0 ml stirred cell
(Arnicon).
(() 'The membrane was washed in dd I1,0 prior to use as recommended by the
manufacturer. Six
uolumcs of lU~% glycerol in PBS were washed through to remove most of the
recombinant
(W tLi protein and this wash was collected in a separate vessel. The resulting
BotB
protein-rich filtrate was then concentrated I?-fold using a YM 10 ( l0 kD
MW('O) membrane
(.~lmice~n). to a Iinal w~lume of 1~ ml. The YM IUO and YM 10 concentrates
were analvzcd
1 ~ alone wvth the lysate starting material by native PAGE using a ~1 - I S%
pre-cast gradient gel
( (BioRad ). (~hc results arc shown in Figure 37.
tn Figure .;7. lane I contains IUA-puritied I3ott3 derived Irorn a shaker
flask culture
(i.r.. no cu-expression «f chaperones: L;x. 3~); lane 2 contains a 20% wlv
('FI clarified Ivsate
ul~ p(~ist3ott3 kan laclcl T7/pAC YCGro/f3L21(DE3) cells; lane 3 shows the
lvsate of lane i
?0 after II);'~ purification: lane 4 contains the YM 10 concentrate and lane ~
cemtains the YM
1 ()() concentrate.
.(~11C I'eStlllS SllUwrl In Figure 37 demonstrate that the recombinant I3otf3
prcucin can be
purified away tiwm the ti~IdinL chaperone by ultratiltratiun through a t()0 kU
MWC'O
membrane ( lane ~). leaving the chaperone protein in the I ()0 kD concentrate
( lane > ).
Analysis ~f the sample in lane ~ also showed that very little of tlZe I3otB
protein was retained
by the 1 OU kI) M WC:O menibranc after G volumes of wash buffer had been
applied.
~(~he I3utB samples fbllowing !DA chromatography and following ultratiltration
through
the YM 100 memhranc were anlyzed by 1-1PLC on a size exclusion column (Shodcx
KB 804):
this analysis revealed that the BotB protein represented O4% of the total
protein in the lUA-
s0 purified sample and that following ultratiltration through the YM 10()
mcmhranc. the F3otl3
protein represented >9C~% of the total protein in the sample.
The Both protein purified by ultratiltration through the YM 100 membrane was
examined Ibr cndotoxin using the LnL assay (Associates of Cape C'ud) as
describe in
l_
CA 02296765 2000-O1-14
PCTlUS97I15394
Example 24. Two aliqouts of the YM 100-purified BotE3 preparation were
analyzed and were
found to contain either 18 to 36 EU/mg recombinant protein or 125 to 250 EUlmg
recombinant protein.
The above results demonstrate that size exclusion chromatography and
ultratiltration
s can he used to purity recombinant botulinal toxin proteins away from tblding
chaperones.
EXAME'LE 41
C.'loning And Expression Of T'he C Fragment
Uf The C'. hcnrrlinum Serotype F: 'l~oxin Cime
fh a ('. hmarlinrnrr type E: neurotoxin gene has hem cloned and sequenced from
several
different strains [Poulet co ul. ( 1992) E3iochem. f3iophys. Res. C'otnmun.
183:107 (strain
l3clugal: ~'helan ce crl. ( lc)c)?) L;ur. J. Ciiochem. 304:657 (strain NC'TC'
1 121c)): fvjii ri crl.
( Ir)9()) 1~-1icrobiol. Immunol. s4:1U41 (partial sequence of strains Mashike.
twani and C)taru)
1 s and Fujii m ul. ( 19c)3) .I. Cien. Microbial. 139:70 (strain Mashike)~.
The nucleotide sequence
oh the wpc Itoxin gene is available from the EMBI_ sequence data bank under
accession
numbers \62(l8c) (strain Beluga) and X626$3 (strain N('TC' 11210). The
nucleotide sequence
of the codin__ region (strain E3eluga) is listed in SE:Q ID NU:4c). The amino
acid sedum ce of
the ('. hrmrlimrm type I: ncurotoxin derived from strain Belgua is listed in
SE:Q ID NU:50.
.!U ~I~hc nucleotide sequence of the coding region (strain NCTC 11?19) is
listed in ~E(? ID
NU:51. The amino acid sequence of the (', hntulinum type E ncurotoxin derived
from strain
NCTC' I !?Ic) is listed in SL:Q ID N():52.
~I'hr ()NA sequence encoding the native ('. hnnrlimrnr serotvpc E-: ('
li;tgmcnt gene
derived front the Beluga strain can he expressed as a histidinc-tagged protein
using the
='_5 plTllisb vector: the resulting coding region is listed in SEQ ID N():5 3
and the corresponding
amino acid sequence is listed in SE:Q ID N0:54. ~l'he DNA seeluencc encoding
the C'
fragment of the native ('. hrnrrlrnunr serotype C gene derived from the NCTC 1
1219 strain
can be expressed as a 111S21dlne-lil~~,Cd ftISIOI1 protein using the pr'1'1-
lish vector: the resulting
coding rcLion is listed in SL~,Q IU NO:55 and the corresponding amino acid
sequence is listed
:i(f in SE(l ll) NU:56. 'fhe C' fragment region from any strain of ('.
hnnrlinrrnr scrotype L: can he
amplified and expressed using the approach illustrated below wine the ('
fragment derived
from ( '. hu~uliraurn type E 223 l strain l A~I'CC ~ 177$6).
_ ? p? _
CA 02296765 2000-O1-14
WO 98108540 PCT1US97115394
The type E neurotoxin gene is synthesized as a single polypeptide chain which
may be
converted to a double-chain form (i.e., a heavy chain and a light chain) by
cleavage with
trvpsin: unlike the type A neurotoxin. the type E neurotoxin exists
essentially only in the
single-chain forth. The 50 kD carboxv-terminal portion of the heavy chain is
referred to as
the C' t~ra~~ment or the H,. domain. Expression of th c C fragment of C'.
hmtrlintrnt tvpc f~. toxin
in hcterologous hosts (r.,s,~.. E. cvli) has not been previously reported,
flte native C' fragment of the ('. bttlulinum scrotype E toxin (BotE) gene was
cloned
and inserted into expression vectors to facilitate expression of tire
recombinant BotE protein in
L-. c~uli. 'This example involved PC R amplification of the gene. cloning, and
construction of
1 () expression vectors.
The BotE: scrotvpe gene was isolated using PCR as described for the BotA
serotype
=ene in Example 28. 'fhc C'. bcmtlintun type E strain was obtained from the
American Type
('allure ('ullcction (ATCC # 17780: strain 2231 ). 'fhc following primer pair
was used in the
I'C'R amplification: ;'-CGCCA'fGGCTC'TTTCTTCTTAT ACACiATGA~I'-3' (S' primer,
Is engineered ,\'onl site underlined) (SEQ 1D N0:57) and
~~-W'A:'ICiCTI'TT;1TTTT~1'C'T'I'GCC'~1-1'C.'CA7'G-3' (3' primer. engineered
llindlIl site
underlined. native gene termination colon italicized) (SEQ II) N():~8). The
I'CR product was
insertcc( into pC'Rscript as described in Example 28. The resulting p('Rscript
BotE clone was
contirtncd by restriction digestion. as well as, by obtaining tire sequence of
approximately 300
?() bases located at the s' end of the C fragment coding reLion using standard
DNA sequencing
methods. The resulting Both sequence was identical to that of the published
C'. horulinemt
type 1=, tewin sequence [ Whelan er of t 1992). .strprcr].
The ;\'helttilled)/NindIII fragment from a pCRscript Both recombinant was
cloned into
pETHish vector as described for I3otA C fragment in Example 28. The resulting
construct
was termed pH isl3otE. pHisBotE expresses the BotE gene under the control of
the T7 lac
promoter and tltc rcsuiting protein contains an N-terminal IOXHis-tag affinity
tag.
The pl~isBotE expression construct was transformed into BL21(DE3) pLysS
competent
cells and l liter cultures were grown. induced and his-tagged proteins were
purified utilizing a
NiN7'A resin (eluted in low pli elution buffer) as described in Exantplc 28.
Total, soluble
s() and purified proteins were resolved by SDS-PAGE and detected by Coomassie
staining. The
results are shown in Figure 38.
In I~i~;ure 38, lane 1 contains broad range MW markers (BioRad): lane 2
contains a
total protein extract: lane 3 contains a soluble protein extract: lane ~4
contains proteins present
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CA 02296765 2000-O1-14
WO 98108540 PCTIUS97/15394
in the llow through from the NiNTA column (this sample was not diluted prior
to loading and
therefore represents a load ~X that of the load applied for the total and
soluble extracts in
lanes ? and i): lane ~ contains proteins eluted from the NiN'I'A column; lane
6 contains
protein eluted lion a NiNTA column which had been stored at -20°C for 1
year.
s The pllisBotF protein was expressed at moderate levels (7 mg/litcr) as a
totally s«luble
protein. 'E~he purified protein migrated as a single band of the predicted
Mlll~.
Vv'estern blot hybridization utilizing a chicken anti-('. horulinunr serotvpe
h. toxoid
primary antibody (generated by immunization of hens as described in Example 3
using C'.
I»nulirrunr serotypc E toxoid ) was also performed on the total, soluble and
purified E3otE
proteins. Samples of BotA and BotE3 C fragments were also included on the gels
to facilitate
MW and immunogenicity comparisons. Strong immunoreactivity was detected using
the
anti-('. hrrnrlinrrm type E: toxoid antibody only with the l3otL: protein.
These results demonstrate that the native f3otI~ gene scduences can he
expressed as a
soluble his-tagged protein in F. emli and purified by metal-chelation affinity
chromatography.
I
EXAMPLL ~2
Generation ()f Neutralising Antibodies LJsing ~i'he Itccombinant pllisBotE
Protein
The ahility of the purified pHisBotE protein to generate neutralizing
antibodies was
?0 examined. Nike BAI.Bc mice were immunized with BotE~ protein (purified as
described in
Lx. ~41 ) using <ierbu GMDE' adjuvant ((:C Biotech>. The law ply elution was
mixed with
(~crbu acl_juvant and used to immunize mice. Each mouse received a
subcutaneous in.jceticm
of 10() til antigen/adjuvant mix (s~ Etg antigen + l Eig ad.juyant) on day ().
Mice were
subcutancously boosted as above on day 14 and bled on day ?8. Mice were
subseduently
hoosted and bled on day 70.
Anti-('. hmulinrrm serotype F toxoid titers were determined in day'8 scrum
iiom
individual IlllCe from each group using the EL1SA protocol outlined in Example
?9 with the
exception that the plates were coated with C'. horrrlinum serotype L: toxoid.
and the primary
antibody was a chicken anti-('. hmulinum serotypc E toxoid. Seroconversicm
relative to
;() control mice immunized with the p6xllisBotA antigen (E:x. ?9)) was
observed with all 9 mice
immunized with the purified pFIisE3otl: protein.
The ability of the anti-I3atL antibodies to neutralize native ( '.
hrnrrlirrrrm type I: toxin
was tested in a mouse-C'. hmrrlinum neutralization model using pooled mouse
serum (sec Ex.
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?3b). The LDsc, of purified C'. hotulinum type E toxin complex (Dr. Eric
Johnson. University
of Wisconsin. Madison) was determined by a intraperitoneal (IP) method
[Schantz and Kautler
( 1978). supra using I 8-22 ~ female ICR mice. The amount of neutralizing
antibodies present
in the serum of the immunized mice was determined using serum antibody
titrations. The
various serum dilutions (0.01 ml) were mixed with S LD;" units of C'.
hotrrlinum type F toxin
and the mixtures were injected IP into mice. The neutralizations were
perfi~rmed in duplicate.
-hhe mice were then observed fior signs of botulism for ~4 days. l.Jndiluted
serum from day 28
did nut protect. while undiluted. 1/10 diluted and 1/100 diluted day 70 serum
protected (1005
of animals) while !/1000 diluted day 7() serum did not. This corresponds to a
neutralization
titer ch' S0-500 1l.Jlml.
These results demonstrate that seruconversion occurred and neutralizing
antibodies
were induced when the recombinant BotI: protein was utilized as the immunogcn.
CaAMPLE 43
1' Construction ()f Vectors 'I'o Facilitate E=xpression
()f Ills-Tagged fiotl: Protein In Fermentation Cultures
.~\ number ut~ expression vectors were constructed to facilitate the
expression of
recombinant f3otl: protein in large scale fermentation culture. These
constructs varied as to
?0 the str-cnph of the promoter utilized (~1~7 or T7lac) and the presence of~
repressor elements
f lac(cl ) un the plasmid. 'the resulting constructs varied in the level of
expression achieved and
in ~IF1SI111C1 stability which facilitated the selection of a optimal
expression system tier
lurmentation scaleup. 'this example involved a) construction of liotE
expression vectors and
h) determination of expression levels in small scale cultures.
,5
Construction Of QotE Expression Vectors
~fhe RutE expression vectors created tbr fermentation culture were engineered
m
utilize the kanamycin rather than the ampicillin resistance gene. and
contained either the T7 or
T7lac hromuter_ witlj or without the laclq gene for the reasons outlined in
Example iU.
~() In all cases. the protein expressed by the various expression vectors is
the pHisf3otE
protein described in Ixamplc ~4 f . with the only differences between clones
being the alteration
of various regulatory elements. using the designations outlined below. the pl
fisHotl; clone
(I:x. ~41 ) is equivalent to pf-IisButl; amp T7lac.
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i) Construction Of pHisBotE kan lacty T7lac
pI-lisBotE kan laclq T7lac was constructed by inserting the .~i7~uI/HindlIt
fiagment of
pHisI3otE which contains the I3otE gene sequences into ,~hcr1/tfindllI-cleaved
pCT24a vector.
Proper construction was confirmed by restriction digestion.
ii) Construction Of pllisBotE kan T7
pI 1is13utE: kan '1~7 was constructed by ligating the Botl:-containing
.17~u1/,Supi fragment
of pHisf3utf: kan laclqT7lac to the 'f7 promoter-containins~ .17or1/.S'crpl
fragment of pf~T23a.
Proper construction was confirmed by restriction digestion.
iii) Construction Of pHisBotl: kan laclqT7
IaFlisBotfkan IacIqT7 was constructed by inserting the 13,L~lIIIt-IindIIl
i~ragment from
pHisfiutE kan 'f7 which contains the ButE gene sequences into L3,y11(/ttindlIl-
cleaved pL;T?4
vector. I'rc~per construction was confirmed by restriction digestion.
I
b) Determination Uf BotE Expression Levels In Small Scate
Cultures
I~hr three ButE kan expression vectors described above were transtormcd into
B121(UE ~) competent cells and 50 ml (2XY'1~ + 40 Etglml kan) cultures were
crown and
() induced with I1'PG as described in Example 28. Total and soluble protein
extracts from
beti~re and after induction made as described in Example 28. 1'hc total and
soluble extracts
were resolved on a I?.5'% SDS-I'nGE gel. and his-tagged proteins were detected
on a
Vv~estern hlut utilizing the NiNTIt-alkaline phosphatasc conjugate as
described in Fxamplc
31(c)(iiil. 'The results showed that all three BotF cell lines expressed his-
tagged proteins of
's the predicted MW ter the BotE protein upon induction. The results also
demonstrated that
the two constructs that contained the T7 promoter expressed the I3otf: protein
before
induction. while the T7lac promoter construct did not. Upon induction, the T7
prumotcr-
containing constructs induced to higher levels than the T7lac-containing
construct, with the
pl-1is13utl~ kan laclq'r7lBl?I(DI:3) cells accumulating the maximal levels of
Rotl: protein.
,U
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EXAMPLE 44
Expression And Purification Of pHisBotE Frotn Fermentation Cultures
Based on the small scale inductions performed in Example 43. the pHisBotE kan
laclq
T7/B1? 1 ( UF3 ) strain was selected for fermentation scaleup. This example
involved the
Icrmentation and purification of recombinant BotE C fragment protein.
A fermentation with the pHisBotE kan laclq T7IB121(DF3) strain was performed
as
described in Example 31. The fermentation culture was induced 2 hrs post start
of the
glucose teed with 4 gm IPTG (final concentration = I.G mM). 'fhe OD,,"" was 42
at time of
induction. then 4G.5. 48. ~s and ~4 at I-4 hrs post induction. Viable colony
counts decreased
Irom ()-4 hr induction [ 131. 4 (28). 7 (3), 7. 8: dilution 3 utilized 6 Ltl
of dilution ? cells;
bracketed colonies arc microcolonies). All (32/32) colonies scored at the time
of induction
retained the I3otE plasmid (kan resistant) and no colonies at induction grew
on IPTCi+Kan
plates c no mutations detected). These results were indicative of strong
promoter induction,
I S since colony viability reduced after induction. and the culture stopped
growinL during
icrmcntation (stopped at ~~l OU,,""imp).
Total and soluble extracts were resolved on a 12.5% SDS-PAGE gel and total
protein
vvas detected by staining with Coomassie blue. 'flze results are shown in
Figure 39.
In Figure i9, lane 1 contains total protein from a pNisBotA kan T7
Iac/Bl2l(UE3)
?() LysS fermentation (Ex. 24). Lanes ?-9 contain extracts prepared from the
above pliisBotE
kan lacld -h7/Ii121(UE3) fermentation; lanes 2- 4 contain total protein
extracts prepared at U. 1
and ? Iwurs post-induction. respectively. Lane ~ contains a soluble protein
extract prepared at
hours p«st-induction. Lanes O and 7 contain total and soluble extracts
prepared at 3 hours
pUSt-IndU~tlon. respectively. Lanes 8 and 9 contain total and soluble extracts
prepared at 4
hours post-induction. respectively. Lane 10 contains broad range MW markers
(BioRad).
~I~hc results shown in Figure 39 demonstrate that moderate level induction of
totally
wluble Bot L: protein was observed. increasing from 1 to 4 hrs post induction
(no expression
was detected in uninduced cells). From a 2 liter fermentation harvest a ISO gm
(wet wt) cell
pellet tvas obtained and used to make a PEI-clarified lysate ( 1 liter in CRR.
pl t 6.8). The
~0 lysate was applied to a large scale II)A column and 200 mg of BotE protein.
which was
fbund to be greater than 95% pure ( as judged by visual inspection of a
C.'oontassie stained
ADS-PAGE gel). was recovered. This represents 2.5% of the total soluble
cellular protein
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(assuming a PEI lysate having a concentration of 8 mg proteiniml) and
corresponds to a yield
of 1 UU mg t3otE protein/liter of fermentation culture.
The above results demonstrate that high levels of the recombinant l3otE
protein can he
expressed and purified from fermentation cultures.
CXAMPLC 4~
Removal Of Imidazole From Purified Recombinant BotF Protein !'reparations
The expression oh recombinant BotE protein. unlike the l3otA and E3otB
proteins, did
1 (.) not reduire the presence of foldins~ chaperones to maintain solubility
durint scale-up. A size
exclusion chromatography step was included however to remove the imidazole
from the
sample and exchan~!e the IUA elution buffer tim one consistent with the RotA
antigen.
;~ Sephacrvl ~-1()U l-IR tS-IUU: Pharmacia) column was poured t'_.> cm x ?~
crtl: bed
volume ~ 1 tU ml). Under these conditions. the E3otE protein should he
retained by the beads
to a Iesser degree tlZan the smaller imidazolc. therefbre the !)otE protein
should elute from the
column hcfbrc the imidazolc. ~fhe column was equilibrated in a butter
consisting of iU mM
sodium phosphate. U.~ M NaC'I, and lU% glycerol tall reagents from
Mallinkrodt). Five
milliliters ol~ the lUA-purified f3otL: protein (L:x. 4:1) was filtered
through a (>.-1> ~~. syringe
filter and applied to the S-lU0 column. and equilibration buffer was pumped
through the
~0 column at a flow rate of l mi/minute. Fluted proteins were monitored by
ahsorhance at 280
nm, and collected either manually or with a fraction collector. .~pprnpriate
tubes were pooled
il' necessary. anti the protein was quantitated by ahsorhance at ?8t) nm anJmr
l3(':1 protein
assay. The isolated peaks were then analysed by native and/or SE)~-I'ACiI: m
identify the
proteinls) and evaluate the purity.
:?> Figure 40 provides a representative chromatogram showing the purification
of~
II)A-purified BotE on the S-lUU column. Even though folding chaperones were
not
over-expressed with this antiLCn. a small amount of protein eluted at a time
consistent with
the folding chaperones expressed with E3otA and Botl3 proteins (Giro) tree the
first peak). The
second peak in the chromatogram contained the E3otl: protein. and the third
peak was
aU presumably imidazole. This presumed imidazole peak was isolated in
comparable levels in
lUA-purified I3otA and IiotB protein preparations as well.
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These results demonstrate that size exclusion chromatography can be used to
remove
imidazole and traces of contaminating high molecular weight proteins from IDA-
purified
BotE protein preparations.
The S-lUU-purified BotE protein was tested tbr endotoxin contamination using
the
I_.41, assay as described in Example 24. 'This preparation was found to
contain 64 to 128
I-:tllmg recombinant protein and is therefore substantially tree of cndotoxin.
The 5-IOU purified BotE was mixed with purified preparations of BotA and E3otB
proteins and used to immunize mice: ~ Elg of each Bot protein was used per
immunization
and alum was included as an adjuvant. After two itnmunizations with this
trivalent vaccine.
l0 the immunized mice were challanged with ('. hruerli»um toxin. The immunized
mice
contained neutralizing! antibodies sufticicnt to neutralize between IU().UUU
to 1.UUU.UUU (.D<<,
of either toxin A car t0\itl B and between I .UUU to l U.UUO I.O", ai' toxin
L:. The titer of~
nrutraliring antibodies directed against toxin E would be expected to increase
following
subsequent boosts with the vaccine. ~I~hesc results demonstrate that a
trivalent vaccine
I s containiy recombinant f3otA. BotB and BotE proteins provokes neutralizinc:
antibodies.
L:XAMPLE a6
Expression U1~ 1'he C' ):ragmcnt C)f The ('. hrmnlrnrrr»
~c:rotvPe C' 'toxin Gene And Generation Uf Neutralizing Antihodi~s
?U
~f hr ( ~. hrnnli»rn» type C' I neurotoxin gene has been cloned and scclucnced
[himura cn
crl. ( I c)c)U) I3iochcm. Biophys. Res. C'nmtn. l 71: I 3U4]. The nucleotide
sequence c~t~ the toxin
=enc derived from the C'. hmulirrrr»r wpc C strain C-Stockholm is available
from the
1:MI31./(irnl3ank sequence data banks under the accession number l)c)U210: the
nucleotide
scquenrc of the coding region is listed in SEQ ID NU:59. -fhe amino acid
sequence of the C'.
hnrlrlirrrrrrr tyre C'1 ncurotoxin derived ti'om this strain is listed in SEQ
II) NU:60.
l'hc DNA sequence encoding the native C'. bmuliraru» seronvpe ('1 C.' fragment
gene
derived from the (.'-Stockholm strain can he expressed using the pE~1'Elisb
vector: the resulting
coding region is listed in SEQ ID NU:(1 and the corresponding amino acid
sequence is listed
;U in Sf:Q JO NU:62. The C' feagment region tiom any strain of ('.
hrurrli»urrr scrotypc C' can be
alllplllled and expressed USlng the approach illustrated below using the C
ti~a~~ment derived
from ( '. hrrrrrlirrum tvpe C' C-Stockholm strain. Expression of the C'
ti~agment of'('. hurulimrnr
type ('1 toxin in heterologous hosts lc~.,l,~.. l.: calf) has not been
previously reported.
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The C fragment of the (.'. hcuuhnum serotype C:1 (BotC 1 ) toxin gene is
cloned using
the protocols and conditions described in Example 28 for the isolation of the
native BotA
gene. A number of C'. hotulinum serotype C' strains (expressing either or both
C1 and C2
toxin) arc available from the ATCC' [c~.),~., 2220 (ATCC 17782). 2'_';9 (ATCC
1778x), 2223
(A~l'C'C 17784: a type C-[3 strain: C-(3 strains produce C'? toxin). 662
(ATC(..' 17840: a type
C'-cx strain: C'-w strains produce mainly C I toxin and a small amount of C'2
toxin). 2021
(ATC'C' 17850: a type C'-cx strain) and VI'I 3803 (ATCC ?~7G(fJ.
Alternatively, other type C'
strains may he employed for the isolation of sequences encoding the C fragment
of ('.
honrlimrm scrutypc C' toxin.
The following primer pair is used to amplify the BotC acne: ~'-CGCC:ATGGC
TTTA'fTAAAAGATA'I'AA'CTAATG-i' [5' primer, engineered .Ncwl site underlined
(SFQ ID
N():l;)) and ~'-GC:AA(CTTTTA'hTCACTTACACiGTAC' AAAA('C'-s~ [p' primer.
engineerccl hlindlll site underlined. native gene termination cudon italicized
(SEQ II)
'v():lW)~. lvlluwing I'C'R amplification. the I'CR product is inserted into
the pC'Rscript vector
I'. ~ and then th r l.i kb l~ras~ment is cloned into pl:Tllish vector as
described for tW tA C fragment
~_ene in Example 28. The rcsultin t construct is termed pllisButC'. Proper
construction is
confirmed by I)NA srduencin~ of the I3otC sequences contained within pi
Iist3utC'.
pHisl3utC' expresses the ButC Lene sequences under the lean scriptiunal
control of the
T7 lac pronuocr and the resulting protein contains an N-terminal IOXI-lis-tae
affinity ta~~. ~I~hc
'0 pHisE3utC' expression construct is transformed into 131.21(DE3) pl..vs~
competent cells and I
liter cultures are ~~rown. induced and his-tagged proteins arc purified
utilizing a NiNTA resin
(eluted in ?~0 mM imidazole, ?0% glycerol) as described in f=.xample '_'8.
I~mal, soluble and
purified proaeins arc resolved by SDS-I'ACiI: and detected by C'uumassie
staining and Western
hlut hvhridizatiun utilizing a Ni-N~r~1-alkaline phosphatase crnyu~atc
(Qiagcn) which
1'CCU~',171ZeS hIS-La~'L:ed protC111S aS dCSCrihed In Example 31(c)(Ill).
~fhis analysis permits the
determination of expression levels of the p11is13otC protein (i.c~., number of
mgiliter expressed
as a suluhle protein). The purified E3utt.' protein will migrate as a ain~lc;
band of the predicted
MW (i.c~.. ~--~OkD).
The level of expression of the pHisBotC protein may he modified (increased) by
,0 substitution ui' the ~l'7 promoter fur the ~I'7lac promoter. or by
inclusion of the iaclq gene un
the expression plasmid. and plasmid expressed in B1.21 (DE : ) cell lines in
fermentation
cultures as descrihcd in Example 30. If' only very low levels (i.~-.. Ims than
0.5%) uf' soluble
pHIISB(11C~ protein arc expressed LISI11~ the abUVf: expreSSIUlI 5~'Ste111S,
the pF-lisl3otC construct
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may be co-expressed with pACYCGro construct as described in Example 32. In
this case, the
recombinant BotC protein may co-purify with the folding chaperones. The
contaminating
chaperones may be removed as described in Example i4. Preparations of purified
pHisBotC
protein are tested for endotoxin contamination using the I_r1L assay as
described in F;xample
s ~~1.
The purified pl iisBotC.' protein is used to generate neutralizing antibodies.
BAI,Bc
mice arc; immunized with the BotC' protein using Gerbu (iMDP adjuvant (C: C:
Biotech) as
described in Example 36. The ability of the anti-BotC.' antibodies to
neutralize native ('.
hnrulir~rrm type C' toxin is demonstrated using the mouse-('. hnrulirzrrrrr
neutralization model
1() described in Example .iG.
GXAMPL)C 47
Expression Of The C Fragment Of The ('. hrrrulinrurr
Serotypc D Toxin Gene And Generation Uf Neutralizing Antibodies
1;
The ('. hrrrrrlirrrrnr type D neurotoxin gene has been cloned and sequenced
(Sunagawa
et al. f ! 992) .f. Vet. Med. Sci. ~4:90s and Binz cu al. ( 1990) Nucleic
Acids Res. 18:5556).
The nucleotide sequence of~ the toxin gene derived from the CBIb strain is
available from the
FME3LiGcnBank sequence data banks under the accession number 549407; the
nucleotide
'_'(1 sequence of the coding region is listed in SEQ 1D N0:6~. The amino acid
sequence of the C'.
hurrrlrnrrm type U ncurotoxin derived from the CB16 strain is listed in SI:Q
ID NU:6G.
I'he UNA sequence encoding the. native ('. horulinrurr serotype U C' fragment
gene
derived from a BotU expressinL strain can be expressed using the pETIIisb
vector: the
rrsultin~= coding region is fisted in SEQ IU N0:67 and the corresponding amino
acid sequence
is listed in SEQ ID NU:68. 'fhe C' fragment region from any strain of (.'.
hnruliraum serotype
U can be amplified and expressed using the approach illustrated below using
the C tcagment
derived from ('. hrrrulirrum type D CB I G strain. Expression of the C'
fragment of ('.
hnrrrlirrrrnr type U toxin in heterologous hosts (e~.~~.. E. culi) has not
been previously reported.
~l~hc C' fragment of the ('. horrrlirrrurr serotype D (Boll)) toxin gene is
cloned using the
_s(1 protocOs and conditions described in Lxample ~8 1'or the isolation of the
native ButA gene.
A number of C'. hcrrulirrrrnr type D strains are available t~rom the A'fC'C'
(c~.s,~., ATCC 9GS i.
?0?3 (A~l'CC 17$51). and VPl 5995 IATCC 2717)].
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CA 02296765 2000-O1-14
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The following primer pair is used to amplify the BotD gene: ~'-CGCCATGGC
TTTATTAAAAGATATAATTAATG-3' [5' primer. engineered Nco1 site underlined (SEQ tD
N0:63)~ and 5'-GCAAGCTTTT..~CTC7'ACCCATCC.'fCIGATCCCT-;' [3' primer,
engineered
Ilindlll site underlined. native gene termination codon italicized (SEQ fI)
N():69)~.
s Following I'Ctt amplification, the PCR product is inserted into the
pC:Rscript vector and then
the I.~ kh tiagment is cloned itno pETHisb vector as described fir Bot~1
C.1'ragment gene in
Example 28. The resulting construct is termed pHisl3otU.
pHisBotU expresses the BotD gene sequences under the transcriptional control
of the
T7 luc promoter and the resulting protein contains an N-terminal l UXIVis-tag
affinity tag. 'hhe
f0 pHisHotD expression construct is transformed into HL21(UE3) pl.ysS
competent cells and I
liter cultures arc grown, induced and his-tagged proteins arc purilicd
utiliiinc~ a NiN'rA resin
as described in Example 28. Total, soluble and purified proteins are resolved
by SUS-PAGE
and detected by C'oomassie staining and Western blot hvhridiTation utilizing!
a Ni-NTA-
alkaline phosphatase conjugate (Qiagen) which recognizes his-tagged proteins
as described in
1 ~ Example s I (c)( iii). This analysis permits the determination of
expression levels of the
pl-IisBotU protein (i.c.. number of ms~/liter expressed as a soluble protein).
l~he purified HotU
protein will migrate as a single band of the predicted MW (i.c-.. -~OkI)).
The level of expression of the pHisBotU protein may he modified (increased) by
substitution of the T7 promoter for the T7lac promoter. or by inclusion of the
laclq gene on
?U the expression plasmid. and plasmid expressed in B1.21(DE3) cell lines in
fermentation
cultures us described in Example 30. If only very low levels (i.e.. less than
about U.5~/~) of
soluhle pHisHotl) protein are expressed using the above expression svstcros.
the pflisl3otU
construct may he co-expressed with pACYCGro construct as described in Example
3?. In
this case, the recombinant BotU protein tnay ca-purify with the folding
chaperones. l~he
?5 contaminating chaperones may be removed as described in I:xamplc ;4.
(reparations of
purified pHisBotD protein are tested for endotoxin contamination using the LAL
assay as
described in Example 24.
1'he purified pHisBotU protein is used to generate neutralizing antibodies.
BALBc
mice are immunized with the E3otD protein using (icrbu CiMUI' ad_juvant (C'C'
Biotech) as
sU described in Example 36. l~he ability of the anti-BotD antibodies to
neutralize native ('.
hnltrli~anm type U toxin is demonstrated using the mouse-C'. lmur~linarna
neutralization model
described in f:xample 36.
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EXAMPLE 48
Expression Of The C Fragment Of The C'. hrrrulinum
Serotype F Toxin Gene And Generation Of Neutralizing Antibodies
The C'. homlinrrm type F neurotoxin gene has been cloned and sequenced [East
er crl.
( Ic)9?) !-'EMS Microbial. Lett. 96:2?5]. The nucleotide sequence of the toxin
gene derived
_ Iron the ?031 strain (AT'CC 23387) is available from the EMBLI(icnBank
sequence data
banks under the accession number M929()6: the nucleotide sequence of the
coding: region is
listed in SFQ 1D N():70. The amino acid sequence of the (.'. hrnrrliru»rr type
F ncurotoxin
It) derived fi'orn the 202F strain is listed in SEQ ID N0:71.
The I)NA seduence encoding the native C'. hnlulirrrr»r scrotype F C fragment
gene
derived from the 2()?F strain can be expressed using the pE'I'Hisb vector: the
resulting coding
rezion is listed in SCQ ID N0:73 and the corresponding amino acid sequence is
listed in SE:Q
)U Nt):7:. ~l~hc C' tcagment region tiom any strain of C', hmrrlir7u»r
serotype F can be
1 ~ amplified and expressed using the approach illustrated below using the C
iraLment derived
from ( ' hruulirrrrm type F 202F strain. Expression of the C' fragment of ('.
hrnrrlirurnr type I-'
toxin in heteCC)lllgOLIS hosts (c.~,~., L: cwli) has not been previously
reported.
~I~hr C' 1'ra~~ment of the ('. hurrrlinum serotype I~ (Botf~) toxin genr is
cloned wine the
protocols and conditions described in Example 28 for the isolation of the
native BotA Gene.
'-U hhr ('. hnrrrlinr»rr tvpe fr ?OAF strain is obtained from the American
~l~vPe (.'allure Collection
(f~'1~C'(~ '? ~-;87). Alternatively. sequences encoding the BotF toxin may be
isolated from anv_
I3ot1 rxprcssing strain (c~.,s,~.. VPI 4404 (ATCC '_'S7(~4). VPl ?3H'' (A'i'CC
?73'_' 1 1 and
Langrtanct (A'1'CC ;5415)].
The following primer pair is used to amplify the BotF gene:: 5'-
C(iC.'C'A~fGCi(..'
?5 ~fA~rT("I~AA'I~~fATA'1~ATTTTAA~I'ACi-3' (5' primer, engineered ;1~'cwl site
underlined (SFQ
1D N():74)( and 5'-GCAAGCTTTC;4'fTCTTTCCATC.CATTCTC-3' [3' primer. engineered
l~indlll site underlined. native gene termination codon italicized (SFQ ID
N0:75)(.
FollowinL fCR amplification, the PCR product is inserted into the pC.'Rscript
vector and then
the 1.5 kh Iragmeni is cloned into pETHisb vector as described I«r f3otA ('
fragment gene in
3(1 Example ?8. The resulting construct is termed pHisI3otF:
pI-IisI3otF expresses the BotF gene sequences under the transcriptional
control of the
'f7 lac Promoter and the resulting protein contains an N-terminal lOXliis-ta~~
aftinitv ta~~. The
pHisEiotl~ expression construct is transformed into BL21(DF:3) pLysS competent
cells and 1
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WO 98108540 PCTIUS97/15394
liter cultures arc grown. induced and his-ta6ged proteins arc purified
utilizing a NiNTA resin
as described in Example 28. Total. soluble and purified proteins arc resolved
by SDS-PAGE
and detected by Coomassic staining and Western blot hybridization utilizing a
Ni-NTA-
alkalinc phosphatase conjugate ((?iagen) which recognizes his-tagged proteins
as described in
Example 31(c)(iii). This analysis permits the determination of expression
levels of the
pl~isBotF protein (i.e., number of mgllitcr expressed as a soiuble protein).
The purified BotF
protein will migrate as a single band of the predicted MW (i.e.. ~-iOkD).
'the level of expression of the plvisBotF protein may be modified (increased)
by
SIIbSIIILttI()t1 Of the T7 promoter for the T7lac promoter, or by inclusion of
the Iaclq gene cm
1 U the expression plasmid. and plasmid expressed in I3L? 1 (DE3) cell lines
in fermentation
cultures as described in Example 30. If only very low levels (i.c., less than
about U.5%) of
soluble pE lisf3otF protein are expressed using the above expression systems.
the pI IisBotF
construct may he co-expressed with pACYCCiro construct as described in Example
32. In
this case. the recombinant BotF protein may co-purify with the folding chapcro
n cs. The
1 s contaminating chaperones may be removed as described in f:xample 4.
f'rcparations of
purified pt-lisBotF protein arc tested for endotoxin contamination using the
Li\L assay as
dcscrihed in Example 24.
~I'hc purifcd pf lisBotEv protein is used to generate neutralizing antibodies.
I3ALRc
mice arc immunized with the Both protein using (icrbu (iMDP adjuvant (CC
Biotech) as
?() described in E:aample 3G. The ability of the anti-Botl antihodics to
neutralize native ('.
hrmrlinrrrn type h toxin is demonstrated using the mouse-('. hr~rulinunr
neutralization model
descrihccE in E:xamplc il.
EXAMPLh. 4c)
Expression Of The C Fragment Uf The ('. hrurrlinum
Serotvpe G Toxin Gene And Cicncration Of Nwtralizing Antibodies
~fhe ('. Jmurlinum type (i neurotoxin gene has been cloned and sequenced
(Campbell eu
crl. ( 199;) Biochimica et Biophysics Acta 1216:487 and Binz e~ crl. ( 199())
Nucleic Acids lies.
s(1 18:SSibj. The nucleotide sequence of the toxin gene derived from the 1
13/30 strain (NCFF3
3012) is available from the E:MBLI(,enBank sequence data banks under the
accession number
X741 G?: the nucleotide sequence of the coding region is listed in SEQ ll)
NU:76. 'fhe amino
_?I4_
CA 02296765 2000-O1-14
WO 98108540 PCT/US97/15394
acid sequence of the C'. bmulinunr type G neurotoxin derived from this strain
is listed in SEQ
ID NO:77.
The DNA sequence encoding the native C'. butulinum serotype G C.' fragment
gene
derived from the 113/30 strain can be expressed using the pETHisb vector; the
resulting
coding region is listed in SFQ II) N0:78 and the corresponding amino acid
sequence is listed
in SEQ ID N0;79. The C.' fragment region from any strain of C'. hvtulinuna
serotype G can
be amplified and expressed wine the approach illustrated below using the C
fragment derived
I~t'U111 ( '. hurulinum type Ci 1 13/ 30 strain. Expression of the C.'
iraLntent of C'. IJarrrllnrrnl type
Ci toxin in heterologous hosts (c~.,t,~.. E. call) has not been previously
reported.
1 t1 The C fragment of the C'. hunrlinum serotypc G (BotG) toxin gene is
cloned using the
protocols and conditions described in Example 28 for the isolation of the
native BotA gene.
T~hr ('. hrnrrlinrrrrr type C~ 113/30 strain is obtained from the NCFl3. 'fhe
following primer
pair is used to ampltfv the BotCi~ gene: i'-CGCCAT'GGCTGAC' ACAA'fTTTAA'rACA
AWI~-s' [5' primer, engineered r1~'rui site underlined (SEQ IO N0:80)j and
1 ~ 3'-CiC'CTC'VAGT7:~t'rTCTGTCCA~I'CCTTCATCCAC-3' [3' primer. engineered
.l?ruI site
underlined. native gene termination codon italiciacd (SEQ fD N0:81 )].
Ivllowine E'CR
amplification. the 1'CR product is inserted into the pCRscript vector and then
the 1.~ kb
fragment is cloned into pETI-lisb vector as described for BotA C' fragment
gene in )~xample
~8 with the exception that the sequences encoding Bot(i are excised from the
pCRscript
'?0 vector by digestion with 74'crrl and ,l7ml and the rb'roI site is blunted
(the BotG sequences
contain an internal lliudlll site). This ,'~'c~oE(til(ed)lllrrrl fragment is
then ligated to the
pE:Ti Iisb vector which has been diLCSted with lv'lrel and ,Sell and the ~1-
lrc I site is blunted.
The rcsultin~_ construct is termed plfisl3otG.
pllisl3ot(i cypresses the Rote Lone sequences under the transcriptional
control of the
'f7 lac promoter and the resulting protein contains an N-terminal IOXHis-tag
affinity tag. The
pl-lisBotG expression construct is transformed into BL21(DE3) pLysS competent
cells and 1
liter cultures arc grown, induced and his-tagged proteins are purified
utilizing a NiNTA resin
as described in Example ?8. 'Total. soltthlc and purified proteins are
resolved by SI)S-('ACrE
and detected by C'oomassie staining and Western blot hybridization utilizing a
Ni-N'fA-
:~0 alkaline phosphatase conjugate (Qiagen) which recognizes his-tagged
proteins as. dcscrihed in
Example 31(c)(iii). This analysis permits the determination of expression
levels of the
hHisBotG protein (i.c~., number of mg/litcr expressed as a soluble protein).
The purified BotG
protein will migrate as a single band of the predicted MW (i.c.. -SOkD).
- 2I5 -
CA 02296765 2000-O1-14
PC'TIUS9'7/15394
The level of expression of the pHisBotCi protein may be modified (increased)
by
substitution of the T7 promoter for the T7lac promoter, or by inclusion of the
laclq gene on
the expression plasmid. and plasmid expressed in Bi.?1(DE3) cell lines in
fermentation
cultures as described in Example 30. If only very low levels (i.o., less than
about 0.5%) of
soluble pH isBotCi~ protein arc expressed using the above expression systems,
the pHisl3otCi
construct may be co-expressed with pACYCCiro construct as described in Example
3?. In
this case. the recombinant Bot(i protein may co-purify with the folding
chaperones. The
contaminating chaperones may be removed as described in Irxample 3~1.
Preparations of
purified pllisBotCi protein are tested for endotoxin contamination using the
LAI. assay as
described in E:xampie 24.
The purifed pH isBotCr protein is used to generate neutralizing antibodies.
F3ALBc
mice are immunized with the BotG protein using Cierbu CiMl7P adjuvant (('C
f3iotechl as
descrihect in Example 3(~. The ability of the anti-BotCi antibodies to
ncutrali~c native ('.
hmrrlirurnr tvpr (i toxin is demonstrated using the mouse-(.'. hmrrlinrrnr
neutralization model
I s described in Lxample 36.
EXAMPLE 5U
t-:xpression Of Recombinant Botulinai Toxin Proteins In Fucarvotic (lust
C.'ells
:?0 Recombinant hotulina) C lra~!ment proteins may he expressed in eucarvotic
host cells.
such as yeast and insect cells.
a) Expression In Yeast
Botuiinal C.' fragments derived from serotypes A, B, C'. D. l~. F and G may be
expressed in yeast cells using a variety of commercially available vectors.
lclr example, the
pPIC3K and p1'IC9K expression vectors (Invitrogen) may be employed ii~r
expression in the
methyiotrophic yeast. Piclricr pu.swri.v. When the pI'IC3K vector is employed.
expression of
the hotulinal C fragment protein will be intracellular. When the pl'ICSK
vector is employed.
the hotulinal C fragment protein will be secreted (the alpha factor secretion
signal is provided
sO on the pPIC9K'vector).
17NA SeqllellceS cllcOd1111 the desired C fragment is inserud into these
vectors using
techniques known to the art. Briefly. the desired botuiinal expression
cassette (including
sequences encoding the his-tag: described in the preceding examples) is
amplified using the
_~Ih-
CA 02296765 2000-O1-14
WO 98/08540 PCTIUS97115394
PCR in conjunction with primers that incorporate unique restriction sites at
the termini of the
amplified fragment. Suitable restriction enzyme sites include ,fncrBl. EcvRI.
RvrII and NuU.
When the botutinal C fragment is to be expressed using the pPIC3K vector, the
initiator
methionine (ATG) is provided by the desired Bot gene sequence and a Kozak
consensus
s sequence is engineered upstream of the ATG (c~.~r., ACCATGG).
~Che amplified restriction fragment containing the hotulinal C fragment gene
is then
CIUtICd into the desired expression vector. Recombinant clones are integrated
into the Pichiu
hcr,wvri.,~ gcnome and recombinant protein expression is induced using
methanol following the
manufacturer's instructions (Invitrogen Pichia expression kit manual).
1 (1 C'. hmulinum genes are A/T rich and contain multiple sequences that arc
similar to
yeast transcriptional termination signals (e.g., TTTTTATA). If' premature
transcription
termination is observed when the botulinal C fragment genes are expressed in
yeast, the
transcription termination signals present in the C fragment genes can be
removed by either
site directed mutagenesis (utilizing the pALTER system; Promega) or by
construction of
1 ~ synthetic genes utilizing overlapping synthetic primers.
The botulinal C tcagment genes may be expressed in other yeast cells using
other
commercially available vectors [e~.l,~., using the pYES? vector (Invitrogen)
and .S. ve~rewi,siue~
cells ( Invitrogen)).
?0 h) Expression in Insect Cells
I3otulinal C' fragments derived from serotypes A, B. C. D. E. F and G may be
expressed in insect cells using a variety of commercially available vectors.
Uor example, the
pE3iueBac4 transter vector (Invitrogen) may he employed for expression in
,Sj~ncl~yuc~ru
jmr~~iperclu (,~~)) insect cells (baculovirus expression system) (equivalent
baculovirus vectors
?5 and host cells are avaialble from other vendors, e.l,~., Pharmingen, San
Diego, C'A). Botulinal
C fragments contained on Nrollflindlll fragments contained within the pHisBotA-
G
expression constructs (described in the preceding examples) are cloned into
the pBIueBac4
vector (digested with N'crrl and Hlndltl); the Nrot site present on the C
tragmcnt constructs
overlaps with the start codon of the fusion proteins. In the case of botulinal
C:' tcagment
st) clones that contain internal Hirzdlll sites (c~.~~., using the BotG
sequences described in Ex. 49).
the C tragment gene is contained within a NcoIIXhnI fragment on the pHIisI3ot
construct.
'this ~\-cwl/,l7~ul fragment is excised from pHisBot and inserted into
pBlueBac~4 digested with
~\~'c.wl and S'crll. Recombinant baculoviruses are made and the desired
recombinant C fragment
CA 02296765 2000-O1-14
WO 98/08640 PCT/US97I15394
is expressed in S~9 cells using the protocols provided by the manufacturer
(lnvitrogen MaxBac
manual). 'fhe resulting constructs will express the pl-iisBot protein
intracellutarly (including
the N-terminal his-tag) under the control of the polyhedrin promoter. For
extraccllular
secretion of botulinal C fragment proteins. the C fragment seduenccs from the
pHisBot
constructs arc cloned into the pMelBacB vector (lnvitrogenl as described above
for the
pBlueBacd vector. When the pMeIBacB vector is employed. the his-tagged
botulinal C
t'ragment proteins are secreted (utilizing a vector-encoded honeybee mclittin
secretion signal)
and contain a nine amino acid extension at the N-terminus.
Ills-tagged botulinal C fragments expressed in yeast or insect cells are
purified using
1() metal chclation columns as described in the preceding! examples.
T~rom the above it is char that the present invention provides compositions
and
methods for the preparation of effective multivalent vaccines against C'.
hnrulint.rm neurotoxin.
It is also contemplated that the recombinant botulinal proteins be used fir
the production of
1 ~ LlnIItOXInS. nl1 publications and patents mentioned in the above
specification are herein
incorporated by reference. Various modifications and variations of the
described method and
system of the invention will be apparent to those skilled in the art without
departing from the
scope and spirit «f the invention.
_ ?lg _
I
CA 02296765 2000-O1-14
PC'T/US97I15394
SEQUENCE LISTING
(1) GENERAL
INFORMATION:
J (i) APPLICANT: Williams, James A.
Thalley, Bruce S.
(ii) TITLE OF INVENTION: Multivalent Vaccine For Clostridium
Botulinum Neurotoxin
' lU
(iii) NUMBER OF SEQUENCES: 82
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Medlen & Carroll
(B) STREET: 220 Montgomery Street, Suite 2200
(Cl CITY: San Francisco
iD) STATE: California
(E) COUNTRY: United States of America
(F) ZIP: 94104
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
Ivi> CURRENT APPLICATION DATA:
(A1 APPLICATION NUMBER: US
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Carroll, Peter G.
LB) REGISTRATION NUMBER: 32,837
(C) REFERENCE/DOCKET NUMBER: OPHD-02959
(i::) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (415) 705-8410
(B) TELEFAX: (415) 397-8338
~t0
(2) INFORMATION
FOR SEQ
ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
!ii) MOLECULE TYPE: DNA (genomic)
iU
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GGAAATTTAG
CTGCAGCATC
TGAC
24
~J !2) INFORMATION
FOR SEQ
ID N0:2:
(i> SEQUENCE CHARACTERISTICS:
tA) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
' W (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
TCTAGCAAAT
TCGCTTGTGT
TGAA
24
' (~) INFORMATION
FOR SEQ
ID N0:3:
70
.?~g_
CA 02296765 2000-O1-14
WO 98ItJ8540 PCTNS97115394
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
j (D) TOPOLOGY: linear
(ii)MOLECULE TYPE: DNA (genomic)
(xi)SEQUENCE DESCRIPTION: :3:
SEQ ID N0
lU
CTCGCATATA 20
GCATTAGACC
(2) INFORMATION
FOR
SEQ
ID
N0:4:
]j (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE; nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
'_'
U
(ii)MOLECULE TYPE: DNA (genomic)
(xi)SEQUENCE DESCRIPTION: :4:
SEQ ID N0
CTATCTAGGC 19
CTAAAGTAT
(2) INFORMATION
FOR
SEQ
ID
N0:5:
(i) SEQUENCE CHARACTERISTICS:
i() (A) LENGTH: 8133 base
pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii)MOLECULE TYPE: DNA (genomic)
lix)FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..8130
~U
(xi)SEQUENCE DESCRIPTION:
SEQ ID N0:5:
ATG TCT TTA ATA TCT AAA GAA GAG AAA CTC GCA TAT AGC 48
TTA ATA ATT.
Met Ser Leu Ile Ser Lys Glu Glu Lys Leu Ala Tyr Ser
Leu Ile Ile
~J i 5 10 15
AGA CCA AGA GAA AAT GAG TAT AAA CTA ACT AAT TTA GAC 96
ACT ATA GAA
Arg Pro Arg Glu Asn Glu Tyr Lys Leu Thr Asn Leu Asp
Thr Ile Glu
20 25 30
jU
TAT AAT AAG TTA ACT ACA AAC AAT AAT AAA TAT TTG CJ1A 144
AAT GAA TTA
Tyr Asn Lys Leu Thr Thr Asn Asn Asn Lys Tyr Leu Gln
Asn Glu Leu
35 40 45
~j AAA AAA CTA AAT GAA TCA ATT GAT ATG AAT AAA TAT AAA 192
GTT TTT ACT
Lys Lys Leu Asn Glu Ser Ile Asp Met Asn Lys Tyr Lys
Val Phe Thr
50 55 60
TCA AGC AGA AAT AGA GCA CTC TCT AAA AAA GAT ATA TTA 240
AAT CTA AAA
(>()Ser Ser Arg Asn Arg Ala Leu Ser Lys Lys Asp Ile Leu
Asn Leu Lys
65 70 75 BO
GAA GTA ATT CTT ATT AAA AAT TCC AGC CCT GTA GAA AAA 288
AAT ACA AAT
Glu Val Ile Leu Ile Lys Asn Ser Ser Pro Val Glu Lys
Asn Thr Asn
()~ 85 90 95
TTA CAT TTT GTA TGG ATA GGT GGA GAA GTC AGT GAT ATT GCT CTT GAA 336
Leu His Phe Val Trp Ile Gly Gly Glu Val Ser Asp Ile Ala Leu Glu
100 105 110
- 220 -
CA 02296765 2000-O1-14
PCTIUS97/15394
TAC ATA CAATGG GAT AAT TAT CTG 384
AAA GCT ATT GCA AAT
GAA ATT
AAA
Tyr IleLysGlnTrp AlaAspIleAsn AlaGluTyr Ile LysLeu
Asn
115 120 125
TGG TATGATAGTGAA GCATTCTTAGTA AATACACTA AAG GCTATA 932
AAA
Trp TyrAspSerGlu AlaPheLeuVal AsnThrLeu Lys AlaIle
Lys
130 135 140
GTT GAATCTTCTACC ACTGAAGCATTA CAGCTACTA GAA GAGATT 480
GAG
' 10 Val GluSerSerThr ThrGluAlaLeu GlnLeuLeu Glu GluIle
Glu
195 150 155 160
CAA AATCCTCAATTT GATAATATGAAA TTTTACAAA AGG ATGGAA 528
AAA
Gln AsnProGlnPhe AspAsnMetLys PheTyrLys Arg MetGlu
Lys
165 170 175
TTT ATATT,TGATAGA CAAAAAAGGTTT ATAAATTAT AAA TCTCAA 576
TAT
Phe IleTyrAspArg GlnLysArgPhe IleAsnTyr Lys SerGln
Tyr
180 185 190
ATC AATAAACCTACA GTACCTACAATA GATGATATT AAG TCTCAT 624
ATA
Ile AsnLysProThr ValProThrIle AspAspIle Lys SerHis
Ile
195 200 205
CTA GTATCTGAATAT AATAGAGATGAA ACTGTATTA TCA TATAGA 672
GAA
Leu ValSerGluTyr AsnArgAspGlu ThrValLeu Ser 'ryrArg
Glu
~10 215 220
ACA AAT TCT TTG AGA AAA ATA AAT AGT AAT CAT GGG ATA GAT ATC AGG 720
?() Thr Asn Ser Leu Arg Lys Ile Asn Ser Asn His Gly Ile Asp Ile Arg
225 230 235 240
GCT AAT AGT TTG TTT ACA GAA CAA GAG TTA TTA AAT ATT TAT AGT CAG 768
Ala Asn Ser Leu Phe Thr Glu Gln Glu Leu Leu Asn Ile Tyr Ser Gln
245 250 255
GAG TTG TTA AAT CGT GGA AAT TTA GCT GCA GCA TCT GAC ATA GTA AGA 816
Glu Leu Leu Asn Arg Gly Asn Leu Ala Ala Ala Ser Asp Ile Val Arg
260 265 270
~lI)
'."TA TTA GCC CTA AAA AAT TTT GGC GGA GTA TAT TTA GAT GTT GAT ATG 864
Leu Leu Ala Leu Lys Asn Phe Gly Gly Val Tyr Leu Asp Val Asp Met
275 280 285
CTT CCA GGT ATT CAC TCT GAT TTA TTT AAA ACA ATA TCT AGA CCT AGC 912
Leu Pro Gly Ile His Ser Asp Leu Phe Lys Thr Ile Ser Arg Pro Ser
290 295 300
TCT ATT GGA CTA GAC CGT TGG GAA ATG ATA AAA TTA GAG GCT ATT ATG 960
Ser Ile Gly Leu Asp Arg Trp Glu Met Ile Lys Leu Glu Ala Ile Met
305 310 315 320
AAG TAT AAA AAA TAT ATA AAT AAT TAT ACA TCA GAA AAC TTT GAT AAA 1008
Lys Tyr Lys Lys Tyr Ile Asn Asn Tyr Thr Ser Glu Asn Phe Asp Lys
J? 325 330 335
CTT GAT CAA CAA TTA AAA GAT AAT TTT AAA CTC ATT ATA GAA AGT AAA 1056
Leu Asp Gln Gln Leu Lys Asp Asri Phe Lys Leu Ile Ile Glu Ser Lys
340 345 350
O)
AGT GAA AAA TCT GAG ATA TTT TCT AAA TTA GAA AAT TTA AAT GTA TCT 1104
Ser Glu Lys Ser Glu Ile Phe Ser Lys Leu Glu Asn Leu Asn Val Ser
355 360 365
OS GAT CTT GAA ATT AAA ATA GCT TTC GCT TTA GGC AGT GTT ATA AAT CAA 1152
Asp Leu Glu Ile Lys Ile Ala Phe Ala Leu Gly Ser Val Ile Asn Gln
370 375 380
_ 771 _
CA 02296765 2000-O1-14
PCTIUS97115394
GCC TTG ATA TCA AAA CAA GGT TCA TAT CTT ACT AAC CTA GTA ATA GAA 1200
Ala Leu Ile Ser Lys Gln Gly Ser Tyr Leu Thr Asn Leu Val Ile Glu
385 390 395 400
J CAA GTA AAA AAT AGA TAT CAA TTT TTA AAC CAA CAC CTT AAC CCA GCC 1298
Gln Val Lys Asn Arg Tyr Gln Phe Leu Asn Gln His Leu Asn Pro Ala
405 410 415
ATA GAG TCT GAT AAT AAC TTC ACA GAT ACT ACT AAA ATT TTT CAT GAT 1296
1() Ile Glu Ser Asp Asn Asn Phe Thr Asp Thr Thr Lys Ile Phe His Asp
420 425 430
TCA TTA TTT AAT TCA GCT ACC GCA GAA AAC TCT ATG TTT TTA ACA AAA 1344
Ser Leu Phe Asn Ser Ala Thr Ala Glu Asn Ser Met Phe Leu Thr Lys
435 440 445
ATA GCA CCA TAC TTA CAA GTA GGT TTT ATG CCA GAA GCT CGC TCC ACA 1392
Ile Ala Pro Tyr Leu Gln Val Gly Phe Met Pro Glu Ala Arg Ser Thr
450 955 460
ATA AGT TTA AGT GGT CCA GGA GCT TAT GCG TCA GCT TAC TAT GAT TTC 1440
Ile Ser Leu Ser Gly Pro Gly Ala Tyr Ala Ser Ala Tyr Tyr Asp Phe
465 470 475 480
ATA T,AT TTA CAA GAA AAT ACT ATA GAA AAA ACT TTA AAA GCA TCA GAT 1488
Ile Asn Leu Gln Glu Asn Thr Ile Glu Lys Thr Leu Lys Ala Ser Asp
485 490 495
TTA ATA GAA TTT AAA TTC CCA GAA AAT AAT CTA TCT CAA TTG ACA GAA 1536
Leu Ile Glu Phe Lys Phe Pro Glu Asn Asn Leu Ser Gln Leu Thr Glu
S00 505 510
CAA GAA ATA AAT AGT CTA TGG AGC TTT GAT CAA GCA AGT GCA RAA TAT 1589
Gln Glu Ile Asn Ser Leu Trp Ser Phe Asp Gln Ala Ser Ala Lys Tyr
iJ 515 520 525
CAA TTT GAG AAA TAT GTA AGA GAT TAT ACT GGT GGA TCT CTT TCT GAA 1632
Gln Phe Glu Lys Tyr Val Arg Asp Tyr Thr Gly Gly Ser Leu Ser Glu
530 535 540
fit)
GAC AAT GGG GTA GAC TTT AAT AAA AAT ACT GCC CTC GAC AAA AAC TAT 1680
Asp Asn Gly Val Asp Phe Asn Lys Asn Thr Ala Leu Asp Lys Asn Tyr
545 550 555 560
TTA TTA AAT AAT AAA ATT CCA TCA AAC AAT GTA GAA GAA GCT GGA AGT 1728
Leu Leu Asn Asn Lys Ile Pro Ser Asn Asn Val Glu Glu Ala Gly Ser
565 570 575
AAA AAT TAT GTT CAT TAT ATC ATA CAG TTA CAA GGA GAT GAT ATA AGT 1776
~() Lys Asn Tyr Val His Tyr Ile Ile Gln Leu Gln Gly Asp Asp Ile Ser
580 585 59D
TAT GAA GCA ACA TGC AAT TTA TTT TCT AAA AAT CCT AAA AAT AGT ATT 1829
Tyr Glu Ala Thr Cys Asn Leu Phe Ser Lys Asn Pro Lys Ann Ser Ile
~J 595 600 605
ATT ATA CAA CGA AAT ATG AAT GAA AGT GCA AAA AGC TAC TTT TTA AGT 1872
Ile Ile Gln Arg Asn Met Asn Glu Ser Ala Lys Ser Tyr Phe Leu Ser
610 615 620
GO
GAT GAT GGA TCT TTA GAA TTA AAT AGG ATA
GAA ATT AAA TAT CCT
GAA
1920
Asp Asp Gly SerIleLeu Glu Leu Asn Arg Ile Glu
Glu Lys Tyr Pro
625 630635 640
VS AGA TTA AAA AAGGAAAAA GTA AAA GTA ATT GGA GGT 1968
AAT ACC TTT CAT
Arg Leu Lys LysGluLys Val Lys Val Ile Gly Gly
Asn Thr Phe His
645 650 655
777
* rE4
CA 02296765 2000-O1-14
PCTNS9711S394
AAA GAT GAA TTC AAC ACA AGC GAA TTT GCT AGA TTA AGT GTA GAT TCA 2016
Lys Asp Glu Phe Asn Thr Ser Glu Phe Ala Arg Leu Ser Val Asp Ser
660 665 670
J CTT TCC AAT GAG ATA AGT TCA TTT TTA GAT ACC ATA AAA TTA GAT ATA 2064
Leu Ser Asn Glu Ile Ser Ser Phe Leu Asp Thr Ile Lys Leu Asp Ile
675 680 685
TCA CCT AAA AAT GTA GAA GTA AAC TTA CTT GGA TGT AAT ATG TTT AGT 2112
Ser Pro Lys Asn Val Glu Val Asn Leu Leu Gly Cys Asn Met Phe Ser
690 695 700
TAT GAT TTT AAT GTT GAA GAA ACT TAT CCT GGG AAG TTG CTA TTA AGT 2160
Tyr Asp Phe Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Ser
1J 705 710 715 720
ATT ATG GAC AAA ATT ACT TCC ACT TTA CCT GAT GTA AAT AAA AAT TCT 2208
Ile Met Asp Lys Ile Thr Ser Thr Leu Pro Asp Val Asn Lys Asn Ser
725 730 735
~ ()
ATT ACT ATA GGA GCA AAT CAA TAT GAA GTA AGA ATT AAT AGT GAG GGA 2256
Ile Thr Ile Gly Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly
740 745 750
~J AGA AAAGAACTT CTGGCTCACTCA ATAAATAAA GAA 2304
GGT GAA
AAA
TGG
Arg LysGluLeu LeuAlaHisSerGly TrpI1'eAsnLyeGlu Glu
Lys
755 760 765
GCT ATTATGAGC GATTTATCTAGTAAA TACATTTTTTTTGAT TCT 2352
GAA
i0 Ala IleMetSer AspLeuSerSerLys TyrIlePhePheAsp Ser
Glu
770 775 780
ATA GATAATAAG CTAAAAGCAAAGTCC AATATTCCAGGATTA GCA 2400
AAG
Ile AspAsnLys LeuLysAlaLysSer AsnIleProGlyLeu Ai.a
Lys
iJ 785 790 795 8U0
TCA ATATCAGAA GATATAAAAACATTA CTTGATGCAAGTGTT AGT 2448
TTA
Ser IleSerGlu AspIleLysThrLeu LeuAspAlaSerVal Ser
Leu
805 810 815
CCT GATACAAAA TTTATTTTAAATAAT AAGCTTAATATTGAA TCT 2996
CTT
Pro AspThrLys PheIleLeuAsnAsn LysLeuAsnIleGlu Ser
Leu
820 825 830
TCT nTTGGGGAT TACATTTATTATGAA TTAGAGCCTGTTAAA AAT 2544
AAA
5er IleGlyAsp TyrIleTyrTyrGlu LeuGluProValLys Asn
Lye
835 840 845
nTA ATTCACAAT TCTATAGATGATTTA GATGAGTTCAATCTA CTT 2592
ATA
?(1 Ile IleHisAsn SerIleAspAspLeu AspGluPheAsnLeu Leu
Ile
850 855 860
GAA AATGTATCT GATGAATTATATGAA AAAAAATTAAATAAT CTA 2640
TTA
Glu AsnValSer AspGluLeuTyrGlu LysLysLeuAsnAsn Leu
Leu
865 870 875 880
GAT GAGAAGTAT TTAATATCTTTTGAA ATCTCAAAAAATAAT TCA 2688
GAT
Asp GluLysTyr LeuIleSerPheGlu IleSerLysAsnAsn Ser
Asp
885 890 895
G(1
ACT TACTCTGTA AGATTTATTAACAAA AATGGTGAGTCAGTT TAT 2736
AGT
Thr TyrSerVal ArgPheIleAsnLys AsnGlyGluSerVal Tyr
Ser
900 905 910
H? GTA GAAACAGAA AAAGAAATTTTTTCA TATAGCGAACATATT ACA 2784
AAA
Val GluThrGlu LysGluIlePheSer TyrSerGluHisIle Thr
Lys
- 915 920 925
_ '77 j _
CA 02296765 2000-O1-14
PCT/US97115394
AAA AGT ATA AAG AAT ATA GAT GGT 2832
GAA ACT AGT ATT GTT
ATA ACA AAT
Lys Glu IleSer Ile Lys Asn IleIle Asp Gly
Thr Ser Thr Val
Asn
930 935 940
J AAT TTA TTGGATAATATA CAG TTA CATACT CAA AATACA 2880
GAT TCT GTT
Asn Leu LeuAspAsnIle Gln Leu HisThr Gln AsnThr
Asp Ser Val
945 950 955 960
TTA AAC GCAGCATTCTTT ATT CAA TTAATA TAT AGCAAT 2928
TCA GAT AGT
lU Leu Asn AlaAlaPhePhe Ile Gln LeuIle Tyr SerAsn
Ser Asp Ser
965 970 975
AAA GAT GTACTGAATGAT TTA AGT TCAGTT GTT CTTTAT 2976
ACC AAG CAA
Lys Asp ValLeuAsnAsp Leu Ser SerVal Val LeuTyr
Thr Lys Gln
jj 980 985 990
GCT CAA CTATTTAGTACA GGT TTA ACTATA GAC ATCCAA 3024
AAT TAT TCT
Ala Gln LeuPheSerThr Gly Leu ThrIle Asp IleGln
Asn Tyr Ser
995 1000 1005
TTA GTA AATTTAATATCA AAT GCA AATGAT ATA GTACTA 3072
GTA ACT AAT
Leu Val AsnLeuZleSer Asn Ala AsnAsp Ile ValLeu
Val Thr Asn
1010 1015 1020
CCT ACA ATAACAGAGGGG ATA CCT GTATCT ATA GACGGA 3120
ATT ACT TTA
Pro Thr IleThrGluGly Ile Pro ValSer Ile AspGly
Ile Thr Leu
1025 1030 1035 1040
ATA AAC TTAGGTGCAGCA ATT AAG TTACTA GAA GACCCA 3168
GAA GAC CAT
iU Ile Asn LeuGlyAlaAla Ile Lys LeuLeu Glu AspPro
Glu Asp His
1045 1050 1055
TTA CTA AAAAAAGAATTA GAA GCT GTGGGT TTA ATAAAT 3216
AAG GTT GCA
Leu Leu LysLysGluLeu Glu Ala ValGly Leu IleAsn
Lys Val Ala
j1 1060 1065 1070
ATG TCA TTATCTATAGCT GCA ACT GCTTCA GTT ATAGGT 3269
GTA ATT GGA
Met Ser LeuSerIleAla Ala Thr AlaSer Val IleGly
Val Ile Gly
1075 1080 1085
GCT GAA GTTACTATTTTC TTA TTA ATAGCT ATA GCAGGA 3312
CCT GGT TCT
Ala Glu ValThrIlePhe Leu Leu IleAla Ile AlaGly
Pro Gly Ser
1090 1095 110 0
ATA CCT TCATTAGTTAAT AAT GAA ATATTG GAT GCAACT 3360
TTA CAT AAG
lle Pro SerLeuValAsn Asn Glu IleLeu App AlaThr
Leu His Lys
1105 1110 1115 1120
TCA GTG GTAAACTATTTT AAT CAT TCTGAA AAA TATGGC 3408
TTG TCT AAA
J() Ser Val ValAsnTyrPhe Asn His SerGlu Lys TyrGly
Leu Ser Lys
1125 1130 1135
CCT CTT AAA GAA TTAGTT ATT GATTTA 3456
ACA GAT CCT GAT
GAT
AAA
ATT
Pro Leu LysThrGluAsp Asp Lys LeuVal Ile AspLeu
Ile Pro Asp
JJ 1140 114 5 115 0
GTA ATA TCA ATA AATTCG GGAACA 3504
GAA GAT ATA
TTT AAA
AAT CTA
AAT
Val Ile Ser IleAsp Phe Asn AsnSer Lys GlyThr
Glu Asn Ile Leu
1155 1160 1165
O()
TGT AAT ATA GCA ACT 3552
TTA ATG GGT
GAG
GGG
GGA
TCA
GGA
CAC
ACA
GTG
Cys Ann Ile Ala Gly Thr
Leu Met His Gly
Glu Thr
Gly Val
Gly
Ser
117 0 1175 1180
r
C)> AAT ATA TTT ATT 3600
GAT TTC CCT
CAC TCA
TCT
CCA
TCT
ATA
AGT
TCT
CAT
Rsn Asp Phe Ile a
Ile His Phe Pro
Ser
Ser
Pro
Ser
Ile
Ser
Ser
His
1185 1190 1195 1200
CA 02296765 2000-O1-14
WO 98108540 PCT/US97115394
TCA TTA TCA ATT TAT TCT GCA ATA GGT ATA GAA ACA GAA AAT CTA GAT 3648
Ser Leu 5er Lle Tyr Ser Ala Ile Gly Ile Glu Thr Glu Asn Leu Asp
1205 1210 1215
J TTT TCA AAA AAA ATA ATG ATG TTA CCT AAT GCT CCT TCA AGA GTG TTT 3696
Phe Ser Lys Lys Ile Met Met Leu Pro Asn Ala Pro Ser Arg Val Phe
1220 1225 1230
TGG TGG GAA ACT GGA GCA GTT CCA GGT TTA AGA TCA TTG GAA AAT GAC 3744
lU Trp Trp Glu Thr Gly Ala Val Pro Gly Leu Arg Ser Leu Glu Asn Asp
1235 1240 1245
GGA ACT AGA TTA CTT GAT TCA ATA AGA GAT TTA TAC CCA GGT AAA TTT 3792
Gly Thr Arg Leu Leu Asp Ser Ile Arg Asp Leu Tyr Pro Gly Lys Phe
~J 1250 1255 1260
?()
TAC TGG AGA TTC TAT GCT TTT TTC GAT TAT GCA ATA ACT ACA TTA AAA 3840
Tyr Trp Arg Phe Tyr Ala Phe Phe Asp Tyr Ala Ile Thr Thr Leu Lys
1265 1270 1275 1280
CCA GTT TAT GAA GAC ACT AAT ATT AAA ATT AAA CTA GAT AAA GAT ACT 3888
Pro Val Tyr Glu Asp Thr Asn Ile Lys Ile Lys Leu Asp Lys Asp Thr
1285 1290 1295
~J AGA AAC TTC ATA ATG CCA ACT ATA ACT ACT AAC GAA ATT AGA AAC AAA 3936
Arg Asn Phe Ile Met Pro Thr Ile Thr Thr Asn Glu Ile Arg Asn Lys
1300 1305 1310
TTA TCT TAT TCA TTT GAT GGA GCA GGA GGA ACT TAC TCT TTA TTA TTA 3984
i0 Leu Ser Tyr Ser Phe Asp Gly Ala Gly Gly Thr Tyr Ser Leu Leu Leu
1315 1320 1325
TCT TCA TAT CCA ATA TCA ACG AAT ATA AAT TTA TCT AAA GAT GAT TTA 4032
Ser Ser '1'yr Pro Ile Ser Thr Asn Ile Asn Leu Ser Lye Asp Asp Leu
~J 1330 1335 1340
TGG ATA TTT AAT ATT GAT AAT GAA GTA AGA GAA ATA TCT ATA GAA AAT 9080
Trp Ile Phe Asn Ile Asp Asn Glu Val Arg Glu Ile Ser Ile Glu Asn
1345 1350 1355 1360
4t)
GGT ACT ATT AAA AAA GGA AAG TTA ATA AAA GAT GTT TTA AGT AAA ATT 4128
Gly Thr Ile Lys Lys Gly Lys Leu Ile Lys Asp Val Leu Ser Lys Ile
1365 1370 1375
GAT ATA AAT AAA AAT AAA CTT ATT ATA GGC AAT CAA ACA ATA GAT TTT 9176
nsp Ile Asn Lys Asn Lys Leu IIe Ile Gly Asn Gln Thr Ile Asp Phe
1380 1385 1390
TCA GGC GAT ATA GAT AAT AAA GAT AGA TAT ATA TTC TTG ACT TGT GAG 4224
J(1 Ser Gly Asp Ile Asp Asn Lys Asp Arg Tyr Ile Phe Leu Thr Cys Glu
1395 1400 1405
TTA GAT GAT AAA ATT AGT TTA ATA ATA GAA ATA AAT CTT GTT GCA AAA 4272
Leu Asp Asp Lys Ile Ser Leu Ile Ile Glu Ile Asn Leu Val Ala Lys
1910 1415 1420
~)()
TCT TAT AGT TTG TTA TTG TCT GGG GAT AAA AAT TAT TTG ATA TCC AAT 4320
Ser Tyr Ser Leu Leu Leu Ser Gly Asp Lys Asn Tyr Leu Ile Ser Asn
2925 1430 1435 1440
TTA TCT AAT ACT ATT GAG AAA ATC AAT ACT TTA GGC CTA CAT AGT AF,A 4368
Leu Ser Asn Thr Ile Glu Lys Ile Asn Thr Leu Gly Leu App Ser Lys
1445 1450 1455
CA 02296765 2000-O1-14
WO 98108540 PCTlUS97115394
AAT ATA GCG TAC AAT TAC ACT GAT GAA TCT AAT AAT AAA TAT TTT GGA 4416
Asn Ile Ala Tyr Asn Tyr Thr ASp Glu Ser Asn Asn Lys Tyr Phe Gly
1460 1465 1470
J GCT ATA TCT AAA ACA AGT CAA RAA AGC ATA ATA CAT TAT AAA AAA GAC 4464
Ala Ile Ser Lys Thr Ser Gln Lys Ser Ile Ile His Tyr Lys Lys Asp
1475 1480 1485
AGT AAA AAT ATA TTA GAA TTT TAT AAT GAC AGT ACA TTA GAA TTT AAC 4512
lI) Ser Lys Asn Ile Leu Glu Phe Tyr Asn Asp Ser Thr Leu Glu Phe Asn
1490 1495 1500
AGT AAA GlIT TTT ATT GCT GAA GAT 11TA AAT GTA TTT ATG AAA GAT GAT 4560
Ser Lys F~sp Phe Ile Ala Glu Asp Ile Asn Val Phe Met Lys Asp Asp
1505 1510 1515 1520
?t)
F~TT AAT ACT ATA ACA GGA AAA TAC TAT GTT GAT AAT AAT ACT GAT AAA 4608
lle Asn 'I'hr Ile Thr Gly Lys T'yr Tyr Val Asp Asn Asn Thr Asp Lys
1525 1530 1535
AGT ATA GAT TTC TCT ATT TCT TTA GTT AGT AAA AAT CAA GTA AAA GTA 4656
Ser Ile Asp Phe Ser Ile Ser Leu Val Ser Lys Asn Gln Val Lys Val
1540 1545 1550
AAT GGATTATATTTA GAATCCGTATACTCA TCTTACCTTGATTTT 4704
AAT
Asn GlyL.euTyrLeuAsn GluSerValTyrSer SerTyrLeuAspPhe
1555 1560 1565
GTG AAAAATTCAGATGGA CACCATAATACTTCT AATTTTATGAATTTA 4752
i(! '~JalLysAsnSerAspGly HisHisAsnThrSer AsriPheMatAsnLeu
1570 1575 1580
TTT TTGGACAATATAAGT TTCTGGAAATTGTTT GGGTTTGAAAATATA 4800
Phe LeuAspAsnIlc:Ser PheTrpLysLeuPhe GlyPheGluAsnIle
i~ 1585 1590 1595 1600
-~( I
AAT TTT CTA ATC GAT AAA TAC TTT ACC CTT GTT GGT AAA ACT AAT CTT 4848
Asn Phe Val Ile Asp Lys Tyr Phe Thr Leu Val Gly Lys Thr Ann Leu
1605 1610 1615
GGA TAT GTA GAA TTT ATT TGT GAC AAT AAT AAA AAT ATA GAT ATA TAT 4896
Ciy Tyr Val Glu Phe Ile Cys Asp Asn Asn Lys Asri Ile Asp Ile Tyr
1620 1625 1630
TTT CGTGAA TCGTCATCT ACTATATTTAGC GGA 4944
TGG AAA
AAA AGC
ACA
Phe GlyGlu Lys ThrSerSerSer SerThrIlePheSer Gly
Trp Lys
1635 1640 1645
AAT GGTAGA GTT GTAGTAGAGCCT TATAATCCTGATACG GGT 4992
AAT ATA
~() Asn GlyArg Val ValValGluPro TyrAsnProAspThr Gly
Asn Ile
1650 1655 1660
GAA GATATA ACT TCACTAGATTTT TATGAACCTCTCTAT GGA 5040
TCT TCC
Glu AspIle Thr SerLeuAspPhe TyrGluProLeuTyr Gly
Ser Ser
1J 1665 1670 1675 1680
ATA GATAGA ATA AATAAAGTATTG GCACCTGATTTATAT ACA 5088
TAT ATA
IIe AspArg Ile AsnLysValLeu AlaProhipLeuTyr Thr
Tyr Ile
1685 1690 1695
T~GT TTAATA ATT AATACCAATThT TCAAATGAGThCTAC CCT 5136
AAT TAT
Ser LeuIle Ile AsnThrAsnTyr SerAsnGluTyrTyr Pro
Asn Tyr
1700 1705 1710 r
GAG ATTATA CTT AACCCAAATACA CACI1AAAAAGTAAAT ATA 5184
GTT TTC
Glu IleIle Leu AsnProAsnThr HisLysLysValAsn Ile
Val Phe
1715 1720 1725
- 22G -
CA 02296765 2000-O1-14
WO 98108540 PCT/US97/15394
AAT TTA GAT AGT TCT TCT TTT GAG TAT AAA TGG TCT ACA GAA GGA AGT 5232
Asn Leu Asp Ser Ser Ser Phe Glu Tyr Lys Trp Ser Thr GIu Gly Ser
1730 1735 1740
GAC TTT ATT TTA GTT AGA TAC TTA GAA GAA AGT AAT AAA AAA ATA TTA 5280
Asp Phe Ile Leu Val Arg Tyr Leu Glu Glu Ser Asn Lys Lys Ile Leu
1795 1750 1755 1760
CAA AAA ATA AGA ATC AAA GGT ATC TTA TCT AAT ACT CAA TCA TTT AAT 5328
Gln Lys Ile Arg Ile Lys Gly Ile Leu Ser Asn Thr Gln Ser Phe Asn
1765 1770 1775
AAA ATG AGT ATA GAT TTT AAA GAT ATT AAA AAA CTA TCA TTA GGA TAT 5376
Lys Met Ser Ile Asp Phe Lys Asp Ile Lys Lys Leu Ser Leu Gly Tyr
1780 1785 1790
'?0
ATA ATG AGT AAT TTT AAA TCA TTT AAT TCT GAA AAT GAA TTA GAT AGA 5424
Ile Met Ser Asn Phe Lys Ser Phe Asn Ser Glu Asn Glu Leu Asp Arg
1795 1800 1805
CAT CAT TTA GGA TTT AAA ATA ATA GAT AAT AAA ACT TAT TAC TAT GAT 5472
Asp His Leu Gly Phe Lys Ile Ile Asp Asn Lys Thr Tyr Tyr T;~r Asp
1810 1815 1820
~J GAA GAT AGT AAA TTA GTT AAA GGA TTA ATC AAT ATA AAT AAT TCA TTA 5520
Flu Asp Ser Lys Leu Val Lys Gly Leu Ile Asn Ile Asn Asn Ser Leu
1825 1830 1835 1840
TTC TAT TTT GAT CCT ATA GAA TTT AAC TTA GTA ACT GGA TGG CAA ACT 5568
iU Phe Tyr Phe Asp Pro Ile Glu Phe Asn Leu Val Thr Gly Trp Gln Thr
1845 1850 1855
ATC AAT GGT AAA AAA TAT TAT TTT GAT ATA AAT ACT GGA GCA GCT TTA 5616
Ile Asn Gly Lys Lys Tyr Tyr Phe Asp Ile Asn Thr Gly Ala Ala Leu
1860 1865 1870
ACT AGT TAT AAA ATT ATT AAT GGT AAA CAC TTT TAT TTT AAT AAT GAT 5664
Thr Ser Tyr Lys Ile Ile Asn Gly Lys His Phe Tyr Phe Asn Asn Asp
1875 1880 1885
.1(1
GGT GTG ATG CAG TTG GGA GTA TTT AAA GGA CCT GAT GGA TTT GAA TI1T 571
Gly Val Met Gln Leu Gly Val Phe Lys Gly Pro Asp Gly Phe C~lu Tyr
1890 1895 1900
TTT ~;CA CCT CCC AAT ACT CAA AF,T AAT AAC ATA GAA GGT CT.G GCT ATA 5760
Phe Ala Pro Ala Asn Thr Gln Asn Asn Asn Ile Glu Gly Gln AIa ile
1905 1910 1915 1920
GTT TAT CAA AGT AAA TTC TTA ACT TTG AAT GGC AAA AAA TAT TAT TTT 5808
O) Val Tyr Gln Ser Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr TJr Phe
1925 1930 1935
GAT AAT AAC TCA AAA GCA GTC ACT GGA TGG AGA ATT ATT AAC AAT GAG 5856
Asp Asn Asn Ser Lys Ala Val Thr Gly Trp Arg Ile Ile Asn Asn Glu
1940 1945 1950
AAA TAT TAC TTT AAT CCT AAT AAT GCT ATT GCT GCA GTC GGA TTG CAA 5904
Lys Tyr Tyr Phe Asn Pro Asn Asn Ala Ile Ala Ala Val Gly Leu Gln
1955 1960 1965
G(!
GTA ATT GAC AAT AAT AAG TAT TAT TTC AAT CCT GAC ACT GCT T1TC ATC 5952
Val Ile Asp Asn Asn Lys Tyr Tyr Phe Asn Pro Asp Thr Ala Ile Ile
1970 1975 1980
TCA AAA GGT TGG CAG ACT GTT AAT GGT AGT AGA TAC TAC TTT GAT ACT 6000
Ser Lys Gly Trp Gln Thr Val Asn Gly Ser Arg Tyr Tyr Phe Asp Thr
1985 1990 1995 2000
-2?7_
CA 02296765 2000-O1-14
PCTNS97115394
GAT AAT GGT GAT GGTAAA 6048
ACC TAT CAC
GCT AAA
ATT ACT
GCC ATT
TTT
Asp Ile Asn Gly IleAsp GlyLys
Thr Ala Tyr His
Ala Phe Lys
Thr
2005 2010 2015
TTT TATTTT GATAGT TGT GTAGTG AAA GGTGTG TTTAGTACC 6096
GAT ATA
Phe TyrPhe AspSer Cys ValVal Lys GlyVal PheSerThr
Asp Ile
2020 2025 2030
TCT AATGGA TTTGAA TTT GCACCT GCT ACTTAT AATAATAAC 6144
TAT AAT
I()Ser AsnGly PheGlu Phe AlaPro Ala ThrTyr AsnAsnAsn
Tyr Asn
2035 2040 2045
ATA GAAGC~TCAGGCT GTT TATCAA AGT TTCTTA ACTTTGAAT' 6192
ATA AAA
ile GluGly GlnAla Val TyrGln Ser PheLeu ThrLeuAsn
Ile Lys
2050 2055 2060
C~GT AAAAAA TATTAC GAT AATAAC TCA GCAGTT ACCGGATTG 6240
TTT AAA
Gly LysLys TyrT'yr Asp AsnAsn Ser AlaVal ThrGlyLeu
Phe Lys
2065 2070 2075 2080
CAA ACTATT GATAGT AAA TATTAC TTT ACTAAC ACTGCTGAA 6288
AAA AAT
Gln ThrIle AspSer Lys 'I'yrTyr Phe ThrAnn ThrAlaGlu
Lys Asn
2085 2090 2095
GCA GCTACT GGATGG ACT ATTGAT GGT AAATAT TACTTTAAT 6336
CAA AAA
rla AlaThr GlyTrp Thr IleAsp Gly Lys'ryrTyrPheAsn
Gln Lye
2100 2105 2110
~,CT AACACT GCTGAA GCT ACTGGA TGG ACTATT GATGGTAAA 6384
GCA CAA
il)Thr AsnThr AlaGlu Ala ThrGl.y Trp ThrIle AspGlyLys
Ala Gln
2115 2120 2125
~..~?.AT11TTAC TTTAAT AAC ACTGCT ATA TCAACT GGTTATACA 6 4 3 2
ACT GCT
Lws TvrTvr PheAsn Asn ThrAla Ile SerThr GlyTyrThr
Thr Ala
::130 2135 2140
.
ATT AT'rAAT GGTAAA TTT TATTTT AAT GATGGT ATTATGCAG 6480
CAT ACT
Ile lleAsn GlyLys Phe TyrPhe Asn AspGly IleMetGln
His Thr
2145 2150 2155 2160
F,TA GGAGTG TTTAAA CCT AATGGA TTT TATTTT GCACCTGCT 6528
GGA GAA
lle GlyVal PheLys Pro AsnGly Phe TyrPhe AlaProAla
Gly Glu
2165 2170 2175
-I~:'1ATACGGAT GCTAAC ATA GAAGGT CAA ATACTT TACCAAAAT 6575
AAC GCT
Asn 'I'hrAsp AlaAsn Ile GluGly Gln IleLeu TyrGlnAsn
Asn Ala
2180 2185 2150
GAA TTCTTA ACTTTG GGT AAAAAA TAT TTTGGT AGTGACTCA 6624
AAT TAC
~f)Glu PheLeu ThrLeu Gly L~ysLye Tyr PheGly SerAppSer
Asn Tyr
219 5 2200 220 5
AAA GCAGTT ACTGGA AGA ATTATT AAC AAGAAA TATTACTTT 6672
TGG AAT
Lye AlaVal ThrGly Arg IleIle Asn Lyr>Lys TyrTyrPhe
Trp Asn
ij 2210 221 5 2220
AAT CCTAAT AATGCT GCT GCAATT CAT TGCACT ATAAATAAT 6720
ATT CTA
Asn ProAsn AsnAla Ala AlaIle His Cy~Thr IleAsnAsn
Ile Leu
222 5 223 0 223 5 2240
(~l
)
GAC AAGTAT TACTTT GATGGA ATT CAA TATATT 6768
AGT CTT AAT
TAT GGA
Asp LysTyr TyrPhe Gly Ile Gln GlyTyrIle
Ser Leu Asn
Tyr
Asp
2245 2250 2255
( ACT ATTGAA AAA 6816
AGA
AAT
AAT
TTC
TAT
TTT
GAT
GCT
AAT
AAT
GAA
TCT
Thr IleGlu Ann Lys
Arg Asn
Phe
Tyr
Phe
Asp
Ala
Asn
Asn
Glu
Ser
2260 2265 2270 _
* rE,
CA 02296765 2000-O1-14
PCT/US97115394
ATG GTA ACA GGA GTA TTT AAA GGA CCT RAT GGA TTT GAG TAT TTT GCA 6864
Met Val Thr Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala
2275 2280 2285
J CCT GCT AAT ACT CAC AAT AAT AAC ATA GAA GGT CAG GCT ATA GTT TAC 6912
Pro Ala Asn Thr His Asn Asn Asn Ile Glu Gly Gln Ala Ile Val Tyr
2290 2295 2300
CAG AAC AAA TTC TTA ACT TTG AAT GGC AAA AAA TAT TAT TTT GAT AAT 6960
I() Gln Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp Asn
2305 2310 2315 2320
GAC TCA AAA GCA GTT ACT GGA TGG CAA ACC ATT GAT GGT AAA AAA TAT 7008
Asp Ser Lys Ala Val Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr
2325 2330 2335
TAC TTT AAT CTT AAC ACT GCT GAA GCA GCT ACT GGA TGG CAA ACT ATT 7056
Tyr Phe Ann Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile
2340 2345 2350
?0
GAT GGT AAA AAA TAT TAC TTT AAT CTT AAC ACT GCT GAA GCA GCT ACT 7104
Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Ala Glu Ala Ala Thr
2355 2360 2365
GGA TGGCAA ACTATTGATGGT TATTACTTTAAT ACTAACACT 7152
AAA
AAA
Gly TrpGln ThrlieAspGly LysLysTyr'fyrPheAsn ThrAsnThr
2370 2375 2380
TTC ATAGCC TCAACTGGTTAT ACAAGTATTAATGGTAAA CATTTTTAT 7200
>()Phe IleAla SerThrGlyTyr ThrSerIleAsnGlyLys HisPheTyr
2385 2390 2395 2400
TTT AATACT GATGGTATTATG CAGATAGGAGTGTTTAAA GGACCTAAT 7248
Phe AsnThr AspGlyIleMet GlnIleGlyValPheLys GlyProAsn
2405 2410 2915
GGA TTTCAA TACTTTGCACCT GCTAATACGGATGCTAAC AACATAGAA 7296
Gly PheGlu TyrPheAlaPro AlaAsnThrAspAlaAsn AsnIleGlu
2420 2425 2430
-1(1
GGT CTv,GCT ATACTTTACCAA AATAAATTCTTAACTTTG AATGGTAAA 7344
Gly GlnAla IleLeuTyrGln AsnLysPheLeuThrLeu AsnGlyLys
2435 2440 2445
AAA TT~':TAC TTTGGTAGTGAC TCAAAAGCAGTTACCGGA CTGCGAACT 7392
L,;rsTurT: PheGlySerAsp SerLy AlaValThrGly LeuArgThr
r s
X450 2455 2460
ATT CATGGT AAAAAATATTAC TTTAATACTAACACTGCT GTTGCAGTT 7440
J Ile AspGly LysLysTyr'fyrPheAsnThrAsnThrAla ValAlaVal
2465 2470 2475 2480
ACT GGATGG CAAACTATTAAT GGTAAAAAATACTACTTT AATACTAAC 7488
'fhr GlyTrp GlnThrIleAsn GlyLysLysTyrTyrPhe AsnThrAsn
~J 2485 2490 2495
WI
ACT TCT ATA GCT TCA ACT GGT TAT ACA ATT ATT AGT GGT AAA CAT TTT 7536
Thr Ser Ile Ala Ser Thr Gly Tyr Thr Ile Ile Ser Gly Lys His Phe
2500 2505 2510
TAT TTT. AAT ACT GAT GGT ATT ATG CAG ATA GGA GTG TTT AAA GGA CCT 7584
Tyr Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lye Gly Pro
2515 2520 2525
OJ GAT GGA TTT GAA TAC TTT GCA CCT GCT AAT ACA GAT GCT AAC AAT ATA 7632
Asp Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile
2530 2535 2540
- 229 -
CA 02296765 2000-O1-14
PCTIUS9'7115394
GAA GGT CAA GCT ATA CGT TAT CAA AAT AGA TTC CTA TAT TTA CAT GAC 76$0
Glu Gly Gln Ala Ile Arg Tyr Gln Asn Arg Phe Leu Tyr Leu His Asp
2545 2550 2555 2560
AAT ATA TAT TAT TTT GGT AAT AAT TCA AAA GCG GCT ACT GGT TGG GTA 7728
Asn Ile T'yr Tyr Phe Gly Asn Asn Ser Lys Ala Ala Thr Gly Trp Val
2565 2570 2575
ACT ATT GAT GGT AAT AGA TAT TAC TTC GAG CCT AAT ACA GCT ATG GCT 7776
Thr Ile Asp Gly Asn Arg Tyr Tyr Phe Glu Pro Asn Thr Ala Met Gly
2580 2585 2590
GCG AAT GGT TAT AAA ACT ATT GAT AAT AAA AAT TTT TAC TTT AGA AAT 7829
Ala Asn Gly Tyr Lys Thr Ile Asp Asn Lys Asn Phe 'ryr Phe Arg Asn
)j 2595 2600 2605
?()
GGT TTA CCT CAG ATA GGA GTG TTT AAA GGG TCT AAT GGA TTT GAA TAC 7$72
Gly Leu Pro Gln Ile Gly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr
2610 2615 2620
TTT GCA CCT GCT AAT ACG GAT GCT AAC AAT ATA GAA GGT CAA GCT ATA 7920
Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile
2625 2630 2635 2640
CcT TAT CAA AAT AGA TTC CTA CAT TTA CTT GGA AAA ATA TAT TAC TTT '7968
Arg '?''; r C~ln Asn Arg Phe Leu fiis heu Leu Gly Lys Ile Tyr Tyr Phe
2645 2650 2655
cGT AA': AAT TCA AAA GCA GTT ACT GGA TGG CAA ACT ATT AAT GGT AAA 8016
;(1 Gly Asn Asn Ser Lys Ala Val Thr Gly Trp Gln Thr Ile Asn Gly Lye
2660 2665 2670
GTA TF~T TAC TTT ATG CCT GAT ACT GCT ATG GCT GCA GCT GGT GGA CTT 8064
Val Tyr Tyr Phe Met Pro Asp Thr Ala Met Ala Ala Ala Gly Gly Leu
iO 2675 2680 2685
-I~)
TTC GAG ATT GAT GGT GTT ATA TAT TTC TTT GGT GTT G7\T GGA GTA AAA 8112
Phe Glu Ile Asp Gly Val Ile Tyr Phe Phe Gly Val Asp Gly Val Lys
2690 2695 2700
GCC CCT GGG ATA TAT GGC TAA 8133
Ala Pro Gly Ile Tyr Gly
2705 210
('.: ) INFORMATION FOR SEQ ID N0:6
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2710 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
i _s
- Met Ser Leu Ile Ser Lys Glu Glu Leu Ile Lys Leu Ala Tyr Ser Ile
1 5 10 15
Arg Pro Arg Glu Asn Glu Tyr Lye Thr Ile Leu Thr Asn Leu Anp Glu
25 30
Tyr Asn Lys Leu Thr Thr Asn Asn Asn Glu Asn Ly.s Tyr Leu Gln Leu
35 40 Y5
Lys Lys Leu Asn Glu Ser Ile Asp Val Phe Met Asn Lys 'ryr Lye Thr
50 55 60
Ser Ser Arg Asn Arg Ala Leu Ser Asn Leu Lys Lys Asp Ile Leu Lys
65 70 75 BO
_ 7 j~ _
CA 02296765 2000-O1-14
WO 98108540 PCTIUS9~I15394
Glu Val Ile Leu Ile Lys Asn Ser Asn Thr Ser Pro Val Glu Lys Asn
85 90 95
Leu His Phe Val Trp Ile Gly Gly Glu Val Ser Asp Ile Ala Leu Glu
J 100 105 110
'I'yr Ile Lys Gln Trp Ala Asp Ile Asn Ala Glu Tyr Asn Ile Lys Leu
115 120 125
Trp Tyr Asp Ser Glu Ala Phe Leu Val Asn Thr Leu Lys Lys Ala Ile
130 135 140
Val Glu Ser Ser Thr Thr Glu Ala Leu Gln Leu Leu Glu Glu Glu Ile
145 150 155 160
li
Gln AsnPro GlnPheAspAsnMetLys PheTyrLysLysArg MetGlu
165 170 175
Phe IleTyr AspArgGlnLysArgPhe IleAsnTyrTyrLys SerGln
?() 180 185 190
Ile AsnLys ProThrValProThrIle AspAspIleIleLys SexHip
195 200 205
Leu ValSer GluTyrAsnArgAspGlu ThrValLeuGluSer TyrArg
21U 215 220
Thr AnnSer LeuArgLysIleAsnSer AsnHisGlyIleAsp IleArQ
~25 230 23 5 ~40
i()
Ala AsnSer LeuPheThrGluGlnGlu LeuLeuAsnIleTyr SerGln
295 250 255
Glu LeuLeu AsnArgGlyAsnLeuAla AlaAlaSerAspIie ValArg
iO 260 265 270
Leu LeuAla LeuLysAsnPheGlyGly ValTyrLeuAspVal AgoMet
275 280 28 5
Leu ProGly IleHisSerAspLeuPhe LysThrIleSerArq ProSer
~90 295 300
Ser IleGly LeuAspArgTrpGluMet ileLysLeuGluAla IleMet
305 310 315 32U
.~ i
~.'JS't';Lys LysTyrIleAsnAsnTyr ThrSerGluAsnPhe IlspLys
r
325 330 335
Leu AspGln GlnLeuLysAspAsnPhe LysLeuIleIleGlu SerLys
340 345 350
Ser GluLys SerGluIlePheSerLys LeuGluAsnLeuAsn ValSer
355 360 365
App LeuGlu IleLysIleAlaPheAla LeuGlySerValIle AsnGln
370 375 380
Ala LeuIle SerLysGlnGlySerTyr LeuThrAsnLeuVal IleGlu
385 390 395 400
()()
Gln ValL~rsAsnArgTyrGlnPheLeu AsnGlnHisLeuAsn ProAla
405 410 415
_ lle GluSer AspAsnAsnPheThrAsp ThrThrLysIlePhe IsisAsp
~)J 420 425 43U
Ser LeuPhe AsnSerAlaThrAlaGlu AsnSerMetPheLeu ThrLys
435 440 445
CA 02296765 2000-O1-14
PC1'IUS97/15394
Ile Ala Pro Tyr Leu Gln Val Gly Phe Met Pro Glu Ala Arg Ser Thr
450 455 460
Ile Ser Leu Ser Gly Pro Gly Ala Tyr Ala Ser Ala Tyr Tyr Asp Phe
965 470 475 480
Ile Asn Leu Gln Glu Asn Thr Ile Glu Lys Thr Leu Lys Ala Ser Asp
485 490 495
Il) Leu Ile Glu Phe Lys Phe Pro Glu Asn Asn Leu Ser Gln Leu Thr Glu
500 505 510
Gln (~lu Ile Rsn Ser Leu Trp Ser Phe Asp Gln Ala Ser Ala Lys Tyr
515 520 525
Is
Gln PheGlu LysTyrValArg Asp GlyGly SerLeuSerGlu
'I'yr
'I'hr
530 535 540
Asp AsnGly ValAspPheAsn LysAsnThrAlaLeu AspLysAsnTyr
?f)545 550 555 560
Leu heuAsn AsnLysIlePro SerAsnAsnValGlu GluAlaGlySer
565 570 575
:~ys AsnTyr ValHis'I'yrIle IleGlnLeuGlnGly AspAspIleSer
580 585 590
'I';rrGl.uAla ThrCy~AsnLeu PheSerLysRsnPro LysAsnSerIle
!i95 600 605
;f>
Ile IleGln ArgAsnMetAsn GluSerAlaLysSer '1'yrPheLeuSer
G10 615 620
:,sp AspGly GluSerIleLeu GluLeuAsnLysTyr ArgIleProGlu
5~5 630 635 640
t,rg LeuLys AsnLysGluLys ValLysValThrPhe lleGlyHipGly
645 650 655
~;:~sAspGlu PheAsn'1'hrSer GluPheAlaArgLeu SerValAspSer
660 565 670
~eu >erAnn GluIleSerSer PheLeuAspThrIle LysLeuAspIle
675 680 685
~;
..-,rYroLv_sAsnVaLGluVal AnnLeuLeuGlyC's Asnhtor.PhPSer
690 695 'l00
Tvr AspPhe AsnVal:~luGlu ThrTyrProGlyLye LeuLeuLeuSer
J()706 710
715 720
.le MetAsp LysIleT'hrSer ThrLeuProAspVal AsnLysAsnSer
725 730 735
W Ile ThrIle GlyAlaAsnGln TyrGluValArgIle AsnSerGluGly
740 745 750
Arg LysGlu LeuLeuAlafiisSerGlyLyeTrpIle AsnLysGluGlu
755 760 765
Of
1
Ala IleMet SerAspLeu5er SerLysGluTyr!le PhePheAspSer
770 775 780
Ile AspAsn LysLeuLysAla LysSerLysAsnIle ProGlyLeuAla
~)J7A5 790 795 800
Ser IleSer GluAspIleLys ThrLeuLeuLeuAsp AlaSerValSer
805 810 815
CA 02296765 2000-O1-14
PG"TNS97115394
.Pro Asp Thr Lys Phe Ile Leu Asn Asn Leu Lys Leu Asn Ile Glu Ser
820 825 830
Ser Ile Gly Asp Tyr Ile Tyr Tyr Glu Lys Leu Glu Pro Val Lys Asn
S 835 840 845
Ile Ile His Asn Ser Ile Asp Asp Leu Ile Asp Glu Phe Asn Leu Leu
850 855 860
IU Glu Asn Val Ser Asp Glu Leu Tyr Glu Leu Lys Lys Leu Asn Asn Leu
865 870 875 880
Asp GluLys TyrLeuIleSerPhe GluAspIleSerLys AsnAsnSer
885 890 895
Thr TyrSer ValArgPheIleAsn LysSerAsnGlyGlu SerValTyr
900 905 910
Val GluThr GluLysGluIlePhe SerLysTyrSerGlu HisIleThr
?() 915 920 925
Lv GluIle SerThrIleLysAsn SerIleIleThrAsp ValAsnGly
s
_ 930 935 940
Asn L~uLeu AspAsnIleGlnLeu AspHisThrSerGln ValAsnThr
945 950 955 960
Leu AsnAla AlaPhePheIleGln SerLeuIleAspTyr SerSerAsn
965 970 975
i()
Lys Asp Val Leu Asn Asp Leu Ser Thr Ser Val Lys Val Gln Leu Tyr
980 985 99D
Ala GlnLeu PheSerThrGlyLeu AsnThrIleTyrAsp SerIleGln
ij 995 1000 1005
Leu ValAsn LeuIleSerAsnAla ValAsnAspThrIle AsnValLeu
1010 1015 1020
Pro TrrIle ThrGluGlyIlePro IleValSerThrIle LeuAspGly
1025 1030 1035 1040
ile AsnLeu GlyAlaAlaIleLys GluLeuLeuAspGlu HisAspPro
1045 1050 1055
:~
i
Leu LeuLys LysGluLeuGluAla LysValGlyValLeu AlaIleAsn
1060 1065 1070
Met SerLeu SerIleAlaAlaThr ValAlaSerIleVal GlyIleG1y
J() 1075 1080 1085
Ala Glu Val Thr Ile Phe Leu Leu Pro Ile Ala Gly Ile Ser Ala Gly
1090 1095 11D0
Ile Pro Ser Leu Val Asn Asn Glu Leu Ile Leu His Asp Lys Ala Thr
1105 1110 1115 1120
Ser Val Val Asn Tyr Phe Asn His Leu Ser Glu Ser Lys Lys Tyr Gly
1125 1130 1135
(~()
Pro Leu Lys Thr Glu Asp Asp Lys Ile Leu Val Pro Ile Asp Asp Leu
1140 1145 1150
Val Ile Ser Glu Ile Asp Phe Asn Asn Asn Ser Ile Lys Leu Gly Thr
()J 1155 1160 1165
Cys Asn Ile Leu Ala Met Glu Gly Gly Ser Gly His Thr Val Thr Gly
1170 1175 1180
- 233 -
CA 02296765 2000-O1-14
WO 98108540 PCT/US97115394
Asn Ile Asp His Phe Phe Ser Ser Pro Ser Ile Ser 5er His Ile Pro
1185 1190 1195 1200
Ser Leu Ser Ile Tyr Ser Ala Ile Gly Ile Glu Thr Glu Asn Leu Asp
1205 1210 1215
Phe Ser Lys Lys Ile Met Met Leu Pro Asn Ala Pro Ser Arg Val Phe
1220 1225 1230 _
1() Trp Trp Glu Thr Gly Ala Val Pro Gly Leu Arg Ser Leu Glu Asn Asp
1235 1240 145
Gly Thr Arg Leu Leu Asp Ser Ile Arg Asp Leu Ty~r Pro Gly i~y~ Phe
1250 1255 1260
~i
Tyr Trp Arg Phe Tyr Ala Phe Phe Asp Tyr Ala Ile Thr Thr Leu Lys
1265 1270 1275 1280
Pro Val Tyr Glu Asp Thr Asn Ile Lys Ile Lys Leu Asp Lys Asp Thr
1285 1290 1295
Arg Asn Phe Ile Met Pro Thr Ile Thr Thr Asn Glu Ile Arg Ann Lys
1300 1305 1310
Leu Ser Tyr Ser Phe Asp Gly Ala Gly Gly Thr Tyr Ser Leu Leu Leu
1.315 1320 1325
:~er Ser Tyr Pro Ile Ser Thr Asn Ile Asn Leu Ser Lys Asp Asp Leu
1330 1335 1340
_i()
Trp Ilu F~he Asn Zle Asp Asn Glu Val Arg Glu Ile Ser Ile Glu Asn
1345 1350 1355 1360
Gly 'I'hr Ile Lys Lys Gly Ly:~ Leu Ile Lye Asp Val Leu Ser Lys Ile
1365 1370 1375
Asp Ile Asn Lys Asn Lys Leu Ile Ile Gly Asn Gln '1'hr Ile Asp Phe
1380 1385 1390
Sez- Vly Asp Ile Asp Asn Lys Asp Arg Tyr Ile Phe Leu 1'hr Cys Glu
1395 1400 1405
Leu Asp Asp Lys Ile Ser Leu Ile Ile Glu Ile Asn Leu Val Ala Lys
1410 1~115 1420
.~ 1
S~r T~,~r Ser Leu Leu Leu Ser Gly Asp Lys Asn Tyr LPU Ile Ser Asn
1425 1430 1435 1440
Leu Ser Asn Thr Ile Glu Lys Ile Asn Thr Leu Gly Leu Asp Ser Lys
~() 1445 1450 1455
Asn Ile Ala Tyr Asn Ty~r Thr Asp Glu Ser Asn Asn Lys Tyr Phe Gly
1460 1465 1470
J~ Ala Ile Ser Lys Thr Ser Gln Lys Ser Ile Ile His Tyr Lys Lys Asp
1475 1480 1485
Ser Lys Asn Ile Leu Glu Phe Tyr Asn Asp Ser Thr Leu Glu Phe Asn
1990 1495 1500
(~0
Ser Lys A?,~p Phe Ile Ala Glu Asp Ile Asn Val Phe Met Lys Asp Asp
1505 1510 1515 1520
Ile Asn Thr Ile Thr Gly Lys Tyr Tyr Val Asp A~n Ann Thr Asp Lys
()J 1525 1530 1535
Ser Ile Asp Phe Ser Ile Ser Leu Val Ser Lys Asn C1n Val Lys Val
1540 1545 1550
-?34-
* rF3
CA 02296765 2000-O1-14
PCT/US97/15394
Asn Gly Leu Tyr Leu Asn Glu Ser Val Tyr Ser Ser Tyr Leu Asp Phe
1555 1560 1565
Val Lys Asn Ser Asp Gly His His Asn Thr Ser Asn Phe Met Asn Leu
1570 1575 1580
Phe Leu Asp Asn Ile Ser Phe Trp Lys Leu Phe Gly Phe Glu Asn Ile
1585 1590 1595 1600
Asn Phe Val Ile Asp Lys Tyr Phe Thr Leu Val Gly Lys Thr Asn Leu
1605 1610 1615
Gly Tyr Val Glu Phe Ile Cys Asp Asn Asn Lys Asn Ile Asp Ile Tyr
1620 1625 1630
l;
Phe Gly Glu Trp Lys Thr Ser Ser Ser Lys Ser Thr Ile Phe Ser Gly
1635 1640 1645
Asn Gly Arg Asn Val Val Val Glu Pro Ile Tyr Asn Pro Asp Thr Gly
1650 1655 1660
Glu Asp Ile Ser 'rhr Ser Leu Asp Phe Ser Tyr Glu Pro Leu Tyr Gly
1665 1670 1675 1680
Ile Asp Arg Tyr Ile Asn Lys Val Leu Ile Ala Pro Asp Leu Tyr Thr
1685 1690 1695
Ser Leu Ile Asn Ile Asn Thr Asn Tyr Tyr Ser Asn Glu Tyr Tyr Pro
1700 1705 1710
i()
Glu Ile Ile Val Leu Ann Pro Asn Thr Phe His Lys Lys Vai Asn Ile
1715 1720 1725
Asn Leu Asp Ser Ser Ser Phe Glu Tyr Lys Trp Ser Thr Glu G.Ly Ser
jJ 1730 1735 1740
Asp Phe Ile Leu Val Arg Tyr Leu Glu Glu Ser Asn Ly~ Lys Ile Leu
1745 1750 1755 1760
Gln Lys Ile Arg Ile Lys Gly Ile Leu Ser Asn Thr Gln Ser Phe Asn
1765 1770 1775
.~ i
Lys Met Ser Ile Asp Phe Lys Asp Ile Lys Lys Leu Ser Leu Gly Tyr
1780 1785 1790
lle Met Ser Asn Phe Lys Ser Phe Asn Ser Glu Asn Glu Leu Asp Arq
1795 1800 1805
Asp tiffsLeuGly PheLysIleIleAspAsn LysThrTyrTyrTyr Asp
1810 1815 1820
Glu AspSerLys LeuValLysGlyLeuIle AsnIleAsnAsnSer Leu
1825 1830 1835 1840
~J Phe TyrPheAsp ProIleGluPheAsnLeu ValThrGiyTrpGln Thr
1895 1850 1855
Ile AsnGlyLys LysTyrTyrPheAspIle AsnThrGlyAlahla Leu
1860 1865 1870
()()
Thr SerTjrrLys IleIleAsnGlyhysHis PheTyrPheAsnAsn Asp
1875 1880 1885
Gly ValMetGln LeuGlyValPheLysGly ProAspGlyPheGlu 'I'yr
6J 1890 1895 1900
Phe AlaProAla AsnThrGlnAsnAsnAsn IleGluGlyGlnAla Ile
1905 1910 ' 1915 1920
CA 02296765 2000-O1-14
PCT1US97115394
Val Tyr Gln Ser Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe
1925 1930 1935
Asp Asn Asn Ser Lys Ala Val Thr Gly Trp Arg Ile Ile Asn Asn Glu
1940 1945 1950
Lye Tyr T'yr Phe Asn Pro Asn Asn Ala Ile Ala Ala Val Gly Leu Gln
1955 1960 1965
I() Val Ile Asp Asn Asn Lys Tyr Tyr Phe Asn Pro Asp Thr Ala Ile Ile
1970 1975 1980
Ser LysGly TrpGlnThrVaI AsnGl.ySerArgTyr TyrPheAspThr
1985 1990 1995 2000
!i
Asp ThrAla IleAlaPheAsn GlyTyrLysThrIle AspGlyLysHis
2005 2010 2015
Phe 'TyrPhe AspSerAspCys ValValLysIleGly ValPheSerThr
?() 2020 2025 2030
Ser AsnGly PheGluTyrPhe AlaProAlaAsnThr TyrAsnAsnAsn
2035 2040 2045
Ile GluGly GlnAlaIleVal TyrGlnSerLysPhe LeuThrLeuAsn
2050 2055 2060
Gly L,y~Lys TyrTyrPheAsp AsnAsnSerLysAla ValThrGlyLeu
2065 2070 2075 %OBO
;()
Gln ThrIle AspSerLysLys TyrTyrPheAsnThr AsnThrAlaGlu
2085 2090 2095
Ala AlaThr GlyTrpGlnThr IleAspGlyLysLys Tyr'I'yrPheAnn
i~ 2100 2105 :'.110
'I'hrA=,nThr AlaGluAlaAla ThrGlyTrpGlnThr IIeAspGl.rLys
2115 2120 2125
-l!)I,y~ TyrTyr PheAsnThrAsn ThrAlaIleAlaSer ThrGlyTyrThr
'?130 2135 2140
Ile IleAsn GlyLysHisPhe T'yrPheAsnThrAsp GlyIleMe~Gln
2195 2150 2155 160
.~
i
lie GlyVal PheLysGlyFro AsnGlyPheGluTyr PheAlaProAJ.a
2165 2170 2175
hen Tl:rAsp AlaAsnAsnIle GluGlyGlnAlaIle Leu'I'yrClnAnn
2180 2185 2190
Glu PheLeu ThrLeuAsnGly LysLysTyrTyrPhe GlySerAspSer
219 5 2200 2205
L,y~ AlaVal ThrGlyTrpArg IleIleAsnAsnLys L,ysTyrTyrPhe
2210 221 5 222 0
O)
Asn Pro Asn Asn Ala Ile Ala Ala Ile His Leu Cys Thr Ile Asn Asn
2225 2230 2235 2240
Asp Lys T'yr Tyr Phe Ser T;~r Asp Gly Ile Leu ~ln Asn Gly Tyr Ile
2245 2250 2255
Thr Ile Glu Arg Asn Asn Phe Tyr PhP Asp Ala Asn Asn Glu Ser Lys
()J 2260 2265 4270
Met Val Thr Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala
2275 2280 2285
- 23G -
CA 02296765 2000-O1-14
WO 98/08540 PCT/US97I15394
Pro AlaAsnThr HisAsnAsnAsnIle GluGlyGlnAlaIle ValTyr
2290 2295 2300
Gln AsnLysPhe LeuThrLeuAsnGly LysLysTyrTyrPhe AspAsn
2305 2310 2315 2320
Asp SerLysAla ValThrGlyTrpGln ThrIleAspGlyLys LysTyr
2325 2330 2335
1() Tvr PheAsnLeu AsnThrAlaGluAla AlaThrGlyTrpGln ThrIle
2340 2345 2350
Asp Glv~ysLys TyrTyrPheAsnLeu Asn'ThrAlaGluAla AlaThr
2355 2360 2365
~i
Gly TrpGlnTh IleAspGlyLysLys TyrTyrPheAsnThr AsnThr
r
2370 2375 2380
Phe IleAlaSer ThrGlyTyrThrSer IleAsnGlyLysHis PheTyr
2385 2390 2395 2400
Phe AsnThrAsp GlyIleMetGlnIle GlyValPheLysGly ProAsn
2405 2410 2415
Gly PheGluTyr PheAlaProAlaAsn ThrAspAlaAsnAsn IleGlu
2420 2425 2430
!~ly Glnf~laIie LeuT~~rGlnAsnLys PheLeuThrLeuAsn GlyLys
2435 2440 2445
i(}
Lys 'TyrTyrPhe GlySerAspSerLys AlaValThrGlyLeu ArgThr
245C 2455 2460
Ile AspGlyLye LysTyrTyrPheAsn ThrAsnThrAlaVal AlaVal
2465 2470 2475 2480
Thr GlyTrpGln ThrIleAsnGlyLys LysTyrTyrPheAsn ThrAsn
2485 2490 2495
4() '~hr Ser Ile Ala Ser Trr Gly Tyr Thr Ile Ile Ser Gly i~ys His Phe
2500 2505 2510
'ryr Phe l~sn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro
2515 2520 2525
~i
Asp Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile
~53U 2535 2540
Glu Gly Gln Ala Ile Arg Tyr Gln Asn Arg Phe Leu Tyr Leu His Asp
J() 2545 2550 2555 2560
Asn Ile Tyr Tyr Phe Gly Asn Asn Ser Lys Ala Ala 'Thr Gly Trp Val
2565 2570 2575
>J Thr Ile App Gly Asn Arg Tyr Tyr Phe Glu Pro Asn Thr Ala Met Gly
2580 2585 2590
Ala Asn Gly Tyr Lys Thr Ile Asp Asn Lys Asn Phe Tyr Phe Arg Asn
2595 2600 2605
~>()
Gly Leu Pro Gln Ile Gly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr
2610 2615 2620
. Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile
~~J 2625 2630 2635 2640
Arg Tyr Gln Asn Arg Phe Leu His Leu Leu Gly Lys Ile 1'yr Tyr Phe
- 2645 2650 2655
CA 02296765 2000-O1-14
PCTIUS97115394
Gly Asn Asn Ser Lys Ala Val Thr Gly Trp Gln Thr Ile Asn Gly Lys
2660 2665 2670
Val Tyr Tyr Phe Met Pro Asp Thr Ala Met Ala Ala Ala Gly Gly Leu
J 2675 2680 2685
Phe Glu Ile Asp Gly Val Ile Tyr Phe Phe Gly Val Asp Gly Val Lys
2690 2695 2700
1() Ala Pro
Gly Ile
Tyr Gly
2705 2710
i:) INFORMATION
FOR SQ
ID N0:7:
(ii SEQUENCE CHARACTERISTICS:
(A> LENGTH: 811 cids
amino a
(B! TYPE: amino
acid
(C'! STRANDEDNESS: wn
unkno
!D) TOPOLOGY: unknown
~()
(i:! MOLECULE TYPE: protein
(ri) SEQUENCE DESCRIPTION:Q No:7:
~E iD
Ser Tyr Lys Ile Ile Lys HisPhe TyrPheAsnAsnAsp Gly
Asn Gly
1 5 10 15
'Ial Met Cln Leu Gly Lys GlyPro AspGlyPheGluTyr Phe
Val Phe
20 25 30
i()
F.la l'ro Ala Asn Thr Asn AsnIle GluGlyGlnAlaIle Val
Gln Asn
35 40 45
Tyr Gln Ser Lys Phe Leu AsnGly LysLysTyrTyrPhe Asp
Leu Thr
50 55 60
Asn Asn Ser Lys Ala Gly TrpArg IleIleAsnAsnGlu Lys
Val Thr
65 70 75 80
-1()Tyr Tyr Phe Asn Pro Ala IleAla AlaValGlyLeuGln Val
Asn Asn
85 90 95
Ile Asp Asn Asn L~:s Tyr T!r Phe Asn Pro Asp Thr Ala Ile Ile Ser
100 105 110
.~
i
Lys GlyTrpGln ThrValAsnGlySerArg Tyr'I'yrPheAspThr Asp
115 120 125
Thr AlaIleAla PheAsnGlyTyrLysThr IleAspGlyLysHis Phe
J() 130 135 140
Tyr PheAspSer AspCysValValLysIle GlyValPheSerThr Ser
145 150 155 160
Asn GlyPheGlu TyrPheAlaProAlaAsn ThrTyrAsnAsnAsn Ile
165 170 175
Glu GlyGlnAla IleValTyrGlnSerLys PheLeuThrLeuAsn Gly
180 185 190
Lys Lys Tyr Tyr Phe Asp Asn Asn Ser Lys Ala Val Thr Gly Leu Gln
195 200 205
Thr Ile Asp SHr Lys L.ys Tyr Tyr Phe Asn Thr Asn Thr nla Glu Ala
l)1 21U 215 220
Ala Thr Gly 'Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Thr
225 230 235 240
_ '7 j
CA 02296765 2000-O1-14
WO ~~~ PCTIUS97115394
Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys
245 250 255
Tyr Tyr Phe Asn Thr Asn Thr Ala Ile Ala Ser Thr Gly Tyr Thr Ile
260 265 27D
ile Asn Gly Lys His Phe Tyr Phe Asn Thr Asp Gly Ile Met Gln Ile
275 280 285
lO Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn
29U 295 300
Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Leu Tyr Gln Asn Glu
305 310 315 320
~i
Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys
325 330 335
Ala Val Thr Gly Trp Arg Ile Ile Asn Asn Lys Lys Tyr 'fyr Phe Asn
340 345 350
Pro Asn Asn Ala Ile Ala Ala Ile Hip Leu Cys Thr Ile Asn Asn Asp
355 360 365
~J Lys TyrT.rPhe SerTyrAspGly IleLeuGlnAsnGly'fyrIleThr
370 375 380
Ile GiuArgAsn AsnPheTyrPhe AspAlaAsnAnnGluSer LysMet
385 390 395 900
i()
Val ThrGiyVal PheLysGlyPro AsnGlyPheGluTyrPhe AlaPro
405 410 415
Ala AsnThrHis AsnAsnAsnIle GluGlyGlnAlaIleVal TyrGln
sJ 420 425 130
Asn LysPheLeu ThrLeuAsnGly LysLysT~rTyrPheAsp AsnAsp
435 940 445
~C) Ser LysAlaVal ThrGlyTrpGln ThrIleAspGlyLysLy:->'fyrTyr
450 955 960
Phe Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp
465 470 475 480
~i
Gly LysLysTyrTyr PheAsnLeuAsnThrAla GluAlaAlaThrGIy
485 490 495
Trp GInThrIleAsp GlyLysLysTyrTyrPhe AsnThrAsnThrPhe
J() 500 505 510
Ile AlaSerThrGly TyrThrSerIleAsnGly LysHisPheT;rrPhe
515 520 525
Asn ThrAspGlyIle MetGlnIleGlyValPhe LysGlyProAsnGly
530 535 590
Phe GluTyrPheAla ProAlaAsnThrAspAla AsnAsnIleGluGly
545 550 555 560
()()
Gln AlaIleLeuTyr GlnAsnLysPheLeuThr LeuAsnGiyLysLys
565 570 575
_ Tyr TyrPheGlySer AspSerLysAlaValThr GlyLeuArgThrIle
~7~ 580 5$5 590
Asp GlyLysLysTyr TyrPheRsnThrAsnThr AlaValAlaValThr
595 600 605
_ '73()
CA 02296765 2000-O1-14
PCTlUS97I15394
GIy Trp Gln Thr Ile Asn Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr
610 615 620
Ser Ile Ala Ser Thr Gly Tyr Thr Ile Ile Ser G1y Lys His Phe Tyr
7 625 630 635 640
Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro Asp
645 650 655
I() Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Iie Glu
660 665 670
ply Gln Ala Ile Arg Tyr Gln Asn Arg Phe Leu Tyr Leu His Asp Asn
675 680 685
j1
Ile Tyr Tyr Phe Gly Asn Asn Ser Lys Ala Ala Thr Gly Trp Val Thr
690 695 700
Ile Asp Gly Asn Arg Tyr Tyr Phe Glu Pro Asn Thr Ala Met Gly Ala
705 710 715 720
Asn Gly Tyr Lys Thr Ile Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly
725 730 735
~J Leu Pro Gln Ile Gly Val Phe Lys Gly Ser Asn Gly Phe Glu 'ryr Phe
740 745 750
,via Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg
755 760 765
:()
T;~r Gln Asn Arg Phe Leu His Leu Leu Gly Lys Ile Tyr Tyr Phe Gly
770 775 780
Asn Asn Ser Lys Ala Val Thr Gly Trp Gln Thr Ile Asn C;ly Lys Val
785 790 795 B00
'I'yr Tyr Phe Met Pro App Thr Ala Met Ala Ala
805 810
:') INFORMATION FOR SEQ ID NO: B:
(i) SEQUENCE CFIARACTERISTIC~:
(A) LENGTH: 91 amino acids
iH) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
!ii) MOLECULE TYPE: protein
O) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: B:
Ser Tyr Lys lle Ile Asn Gly Lys His Phe Tyr Phe Ann Ann Asp Gly
1 5 IO 15
Val Met Gln Leu Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe
20 25 30
Ala Pro Ala Asn Thr Gln Asn Asn Asn Ile Glu Gly Gln Ala Ile Val
35 40 45
(~(1
Tyr ~ln Ser Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp
50 55 60
Asn Asn Ser Lys Ala Val Thr Gly Trp Arg Ile 11~ Asn Asn Glu Lys
(~i 65 70 75 8U
Tyr Tyr Phe Asn Pro Asn Asn Ala Ile Ala Ala
85 90
- 240 -
*rB
CA 02296765 2000-O1-14
PCTIUS97115394
(2) INFORMATION FOR SEQ ID N0:9:
(i> SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7101 base pairs
i (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
IU
( i:c ) FEATURE
(A1 NAME/KEY: CDS
(B) LOCATION: 1..7098
(ii) SEQUENCE DESCRIPTION: SEQ ID N0:9:
ATG AGT TTA GTT AAT AGA AAA CAG TTA GAA AAA ATG GCA AAT GTA AGA 4B
Met Ser Leu Val Asn Arg Lys Gln Leu Glu Lys Met Ala Asn Val Arg
1 5 10 15
TTT CGT ACT CAA GAA GAT GAA TAT GTT GCA ATA TTG GAT GCT TTA GAA 96
Phe Arg Thr Gln Glu Asp Glu Tyr Val Ala Ile Leu Asp Ala Leu Glu
20 25 30
GAA TAT CAT AAT ATG TCA GAG AAT ACT GTA GTC GAA AAA TAT TTA AAA 144
Glu Tvr llis Asn Met Ser Glu Asn Thr Val Val Glu Lys Tyr Leu Lys
35 40 45
TTA AAA GAT ATA AAT AGT TTA ACA GAT ATT TAT ATA GAT ACA TAT AAA 192
~() Leu Lys Asp Ile Asn Ser Leu Thr Asp Ile Tyr Ile Asp Thr Tyr Lys
50 55 60
AAA TCT GGT AGA AAT AAA GCC TTA AAA AAA TTT AAG GAA TAT CTA GTT 240
Lys Ser Gly Arg Asn Lys Ala Leu Lys Lys Phe Lys Glu Tyr Leu Val
?J 65 70 75 80
ACA GAA GTA TTA GAG CTA AAG AAT AAT AAT TTA ACT CCA GTT GAG AAA 288
Thr Glu Val Leu Glu Leu Lys Asn Asn Asn Leu Thr Pro Val Glu Lys
85 90 95
AAT TTA CAT TTT GTT TGG ATT GGA GGT CAA ATA AAT GAC ACT GCT ATT 336
Asn Leu His Phe Val Trp Ile Gly Gly Gln Ile Asn Asp Thr Ala Ile
100 105 110
~J AAT TATATAAAT CAATGGAAAGATGTAAAT AGTGATTATAATGTT AAT 384
Asn TyrIleAsn GlnTrpLysAspValAsn SerAsp'ryrAsnVal Asn
115 120 125
GTT TTTTA1'GAT AGTAATGCATTTTTGATA AACACATTGAAAAAA ACT 432
JU Val PheTyrAsp SerAsnAlaPheLeuIle AsnThrLeuLysLys Thr
130 135 140
GTA GTAGAATCA GCAATAAATGATACACTT GAATCATTTAGAGAA AAC 480
Val ValGluSer AlaIleAsnAspThrLeu GluSerPheArgGlu Asn
JJ 145 150 155 160
TTA AATGACCCT AGATTTGACTATAATAAA TTCTTCAGAAAACGT ATG 528
Leu AsnAspPro ArgPheAspTyrAsnLys PhePheArgLysArg Met
165 170 175
V()
GAA ATAATTTAT GATAAACAGAAAAATTTC ATAAACTACTATAAA GCT 576
Glu IleIleTyr AspLysGlnLysAsnPhe IleAsnTyrTyrLys Ala
180 185 190
OJ CAA AGA GAA GAA AAT CCT GAA CTT ATA ATT GRT GAT ATT GTA AAG ACA 624
Gln Arg Glu Glu Asn Pro Glu Leu Ile Ile Asp Asp Ile Val Lys Thr
195 200 205
_ ~4 j
CA 02296765 2000-O1-14
PCTIUS97/15394
TAT CTT TCA AAT GAG TAT TCA AAG GAG ATA GAT GAA CTT AAT ACC TAT 672
Tyr Leu Ser Asn Glu Tyr Ser Lys Glu Ile Asp Glu Leu Asn Thr Tyr
310 215 220
ATT GAA GAA TCC TTA AAT AAA ATT ACA CAG AAT AGT GGA AAT GAT GTT 720
Ile Glu Glu Ser Leu Asn Lys Ile Thr Gln Asn Ser Gly Asn Asp Val
225 230 235 240
AGA AAC TTT GAA GAA TTT AAA AAT GGA GAG TCA TTC AAC TTA TAT GAA 768
I() Arg Asn Phe Glu Glu Phe Lys Asn Gly Glu Ser Phe Asn Leu Tyr Glu
245 250 255
CAA GAG TTG GTA GAA AGG TGG AAT TTA GCT GCT GCT TCT GAC ATA TTA 816
Gln Glu Leu Val Glu Arg Trp Asn Leu Ala Ala Ala Ser Asp Ile Leu
260 265 270
AGA ATA TCT GCA TTA AAA GAA ATT GGT GGT ATG TAT TTA GAT GTT GAT 864
Arg iie Ser Ala Leu Lys Glu Ile Gly Gly Met Tyr Leu Asp Val Asp
275 280 285
ATG TTA CCA GGA ATA CAA CCA GAC TTA TTT GAG TCT ATA GAG AAA CCT 912
Met Leu Pro Gly Ile Gln Pro Asp Leu Phe Glu Ser Ile Glu Lys Pro
290 295 300
AGT T:.'11GTA ACAGTGGATTTT TGGGAA ACA TTA GAA ATA 960
ATG AAG GCT
Ser Se:-Val ThrValAspPhe TrpGluMet'PhrLysLeu GluAlaIle
105 310 315 320
ATG F,FiATAC AAAGAATATATA CCAGAATATACCTCAGAA CATTTTGAC 1008
i()Met L.~sTyr LysGluTyrIle ProGluTyrThrSerGlu HisPheAsp
325 330 335
ATG T.TF;GAC GAAGAAGTTCAA AGTAGTTTTGAATCTGTT CTAGCTTCT 1056
filetLeuAsp GluGluValGln SerSerPheGluSerVal LeuAlaSer
340 395 350
AAG TChGAT AAATCAGAAATA TTCTCATCACTTGGTGAT ATGGAGGCA 1104
Lys 5erAsp LysSerGluIle PheSerSerLeuGlyAsp MetGluAla
355 360 365
-~(
)
TCA CCACTA GAAGTTAAAATT GCATTTAATAGTAAGGGT ATTATAAAT 1152
Ser ProLeu GluValLysIle AlaPheAsnSerLysGly IleIleAsn
370 375 380
CAA GGGCTA ATTTCTGTGAAA GACTCATATTGTAGCAAT TTAATAGTA 1200
Gln GlrLeu IleSerValLys AspSerTyrCysSerAsn LeuTleVal
385 390 395 400
AAA CAAATC GAGAATAGATAT AAAATATTGAATAATAGT TTAAATCCA 1248
~()Lys GlnIle GluAsnArgTyr LysIleLeuAsnAsnSer LeuAsnPro
405 410 415
GCT ATTAGC GAGGATAATGAT TTTRATACTACAACGAAT ACCTTTATT 1296
Ala IleSer GluAspAsnAsp PheAsnThrThrThrAsn ThrPheIle
~J 420 425 430
6()
GAT AGT ATA ATG GCT GAA GCT AAT GCA GAT AAT GGT AGA TTT ATG ATG 1344
Asp Ser Ile Met Ala Glu Ala Asn Ala Asp Asn Gly Arg Phe Met Met
435 440 445
GAA CTA GGA AAG TAT TTA AGA GTT GGT TTC TTC CCA GAT GTT AAA ACT 1392
Glu Leu Gly Lys Tyr Leu Arg Val Gly Phe Phe Pro Asp Val Lys Thr
450 455 460
ACT ATT AAC TTA AGT GGC CCT GAA GCA TAT GCG GCA GCT TAT CAA GAT 1490
Thr Ile Asn Leu Ser Gly Pro Glu Ala Tyr Ala Ala Ala Tyr Gln Asp
465 470 475 480
- 242 -
CA 02296765 2000-O1-14
WO 98I~540 PCT/US97115394
TTA TTA ATG TTT AAA GAA GGC AGT ATG AAT ATC CAT TTG ATA GAA GCT 1488
Leu Leu Met Phe Lys Glu Gly Ser Met Asn Ile His Leu Ile Glu Ala
485 490 495
GAT TTA AGA AAC TTT GAA ATC TCT AAA ACT AAT ATT TCT CAA TCA ACT 1536
Asp Leu Arg Asn Phe Glu Ile Ser Lys Thr Asn Ile Ser Gln Ser Thr
500 505 510
GAA CAA GAA ATG GCT AGC TTA TGG TCA TTT GAC GAT GCA AGA GCT AAA 1584
Glu Gln Glu Met Ala Ser Leu Trp Ser Phe Asp Asp Ala Arg Ala Lys
515 520 525
GCT CAA TTT GAA GAA TAT AAA AGG AAT TAT TTT GAA GGT TCT CTT GGT 1632
Ala Gln Phe Glu Glu Tyr Lys Arg Asn Tyr Phe GIu Gly Ser Leu Gly
530 535 540
?U
GAA GAT GAT AAT CTT GAT TTT TCT CAA AAT ATA GTA GTT GAC AAG GAG 1680
Glu Asp Asp Asn Leu Asp Phe Ser Gln Asn Ile Val Val Asp Lys Glu
545 550 555 560
TAT CTT TTA GAA AAA ATA TCT TCA TTA GCA AGA AGT TCA GAG AGA GGA 1728
Tyr Leu Leu Giu Lys Ile Ser Ser Leu Ala Arg Ser Ser Glu Arg Gly
565 570 575
TAT ATA CAC TAT ATT GTT CAG TTA CAA GGA GAT AAA ATT AGT TAT GAA 1776
Tyr Ile His Tyr Ile Val Gln Leu Gln Gly Asp Lys Ile Ser Tyr Glu
580 585 590
GCA GCA TGT AAC TTA TTT GCA RAG ACT CCT TAT GAT AGT GTA CTG TTT 1824
i() Ala Ala Cys Asn Leu Phe Ala Lys Thr Pro Tyr Asp Ser Val Leu Phe
595 600 605
CAG AAA AAT ATA GAA GAT TCA GAA ATT GCA TAT TAT TAT AAT CCT GGA 1872
Gln Lys Asn Ile Glu Asp Ser Glu Ile Ala Tyr Tyr Tyr Asn Pro Gly
>> 610 615 620
-)~)
GAT GGT GAA ATA CAA GAA ATA GAC AAG TAT AAA F,TT CCA AGT ATA ATT 1920
Asp Gly Glu Ile Gln Glu Ile Asp Lys Tyr Lys Ile Pro Ser Ile Ile
625 630 635 640
TCT GAT AGA CCT AF.G ATT AAA TTA ACA TTT ATT GGT CAT GGT AAA GAT 1968
Ser Asp Arg Pro Lys Ile Lys Leu Thr Phe Ile Gly His Gly Lys Asp
645 650 655
-)~ GAA TTT AAT ACT GAT ATA TTT GCA GGT TTT GAT GTA GAT TCA TTA TCC 2016
Glu Phe Asn Thr Asp Ile Phe Ala Gly Phe Asp Val App Ser Leu Ser
660 665 670
ACA GAA ATA GAA GCA GCA ATA GAT TTA GCT AAA GAG GAT ATT TCT CCT 2064
Thr Glu Ile Glu Ala Ala Ile Asp Leu Ala Lys Glu Asp Ile Ser Pro
675 680 685
AAG TCA ATA GAA ATA AAT TTA TTA GGA TGT AAT ATG TTT AGC TAC TCT 2112
Lys Ser Ile Glu Ile Asn Leu Leu Gly Cys Asn Met Phe Ser Tyr Ser
690 695 700
O()
ATC AAC GTA GAG GAG ACT TAT CCT GGA AAA TTA TTA CTT AAA GTT AAA 2160
I1e Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Lys Val Lys
705 710 715 720
GAT AAA ATA TCA GAA TTA ATG CCA TCT ATA AGT CAA GAC TCT ATT ATA 2208
Asp Lys Ile Ser Glu Leu Met Pro Ser Ile Ser Gln Asp Ser ile Ile
725 730 735
GTA AGT GCA AAT CAA TAT GAA GTT AGA ATA AAT AGT GAA GGA AGA AGA 2256
Val Ser Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly Arg Arg
740 745 750
_ ?4~ _
CA 02296765 2000-O1-14
~,p ggipg~p PCT/US97115394
GAA TTA TTG GAT CAT TCT GGT GAA TGG ATA AAT AAA GAA GAA AGT ATT 2304
Glu Leu Leu Asp His Ser Gly Glu Trp Ile Asn Lys Glu Glu Ser Ile
755 760 765
ATA GATATT TCATCA TATATATCATTTAATCCT AAAGAA 2352
AAG AAA
GAA
Ile LysAspIle SerSerLysGlu TyrIleSerPheAsnPro LyeGlu
770 775 7B0
AAT AAAATTACA GTAAAATCTAAA AATTTACCTGAGCTATCT ACATTA 2400
Asn LysIleThr ValLysSerLye AsnLeuProGluLeuSer ThrLeu
785 790 795 800
TTA CAAGAAATT AGAAATAATTCT AATTCAAGTGATATTGAA CTAGAA 2448
Leu GlnGluIle ArgAsnAsnSer AsnSerSerAspIleGlu LeuGlu
805 810 815
GAA AAAGTAATG TTAACAGAATGT GAGATAAATGTTATTTCA AATATA 2496
Glu LysValMet LeuThrGluCys GluIleAsnValIleSer AsnIle
820 825 830
'?
0
GAT ACGCAAATT GTTGAGGAAAGG ATTGAAGAAGCTAAGAAT TTAACT 2544
Asp ThrGlnIle ValGluGluArg IleGluGluAlaLysAsn LeuThr
835 840 845
TCT GACTCTATT AATTATATAAAA GATGAATTTAAACTAATA GAATCT 2592
Ser AspSerIle Asn'I'yrIleLys AspGluPheLyeLeuIle GluSer
850 855 860
ATT TCTGATGCA CTATGTGACTTA AAACAACAGAATGAATTA GAAGAT 2640
?t) Ile SerAspAla LeuCysAspLeu LysGlnGlnAsnGluLeu GluAsp
865 870 875 880
TCT CATTTTATA TCTTTTGAGGAC ATATCAGAGACTGATGAG GGATTT 2688
Ser HisPheIle SerPheGluAsp IleSerGluThrAspGlu GlyPhe
885 890 895
AGT ATAAGATTT ATTAATAAAGAA ACTGGAGAATCTATATTT GTAGAA 2736
Ser IleArgPhe IleAsnLysGlu ThrGlyGluSerIlePhe ValGlu
900 905 910
~~)
ACT GAAAAAACA ATATTCTCTGAA TATGCTAATCATATAACT GAAGAG 2784
Thr GluLysThr IlePheSerGlu TyrAlaAsnHisIleThr GluGlu
915 920 925
ATT TCTAAGATA AAAGGTACTATA TTTGATACTGTAAATGGT AAGTTF. 2832
Ile SerLysIle LysGlyThrIle PheAspThrValAsnGIy LysLeu
930 935 940
GTA AAAAAAGTA AATTTAGATACT ACACACGAAGTAAATACT TTAAAT 2880
Val LysLysVal AsnLeuAspThr ThrHisGluValAsnThr LeuAsn
945 950 955 960
GCT GCATTTTTT ATACAATCATTA ATAGAATATAATAGTTCT AAAGAA 2928
Ala AlaPhePhe IleGlnSerLeu IleGluTyrAsnSerSer LysGlu
965 970 975
TCT CTTAGTAAT TTAAGTGTAGCA ATGAAAGTCCAAGTTTAC GCTCAA 2976
Ser LeuSerAsn LeuSerValAla MetLysValGlnValTyr AlaGln
980 985 990
O)
TTA TTTAGTACT GGTTTAAATACT ATTACAGATGCAGCCAAA GTTGTT 3024
Leu PheSerThr GlyLeuAsn1'hrIleThrAspAlaAlaLys ValVal
995 1000 1005
-244-
CA 02296765 2000-O1-14
PC1'lUS9?!15394
GAA TTA GTA TCA ACT GCA TTA GAT GAA ACT ATA GAC TTA CTT CCT ACA 3072
Glu Leu Val Ser Thr Ala Leu Asp Glu Thr I1e Asp Leu Leu Pro Thr
1010 1015 1020
TTA TCT GAA GGA TTA CCT ATA ATT GCA ACT ATT ATA GAT GGT GTA AGT 3120
Leu Ser Glu Gly Leu Pro Ile IIe Ala Thr Ile Ile Asp Gly Val Ser
1025 1030 1035 1040
TTA GGT GCA GCA ATC AAA GAG CTA AGT GAA ACG AGT GAC CCA TTA TTA 3168
Leu Gly Ala Ala Ile Lys Glu Leu Ser Glu Thr Ser Asp Pro Leu Leu
1045 1050 1055
_ AGA CAA GAA ATA GAA GCT AAG ATA GGT ATA ATG GCA GTA AAT TTA ACA 3216
Arg Gln Glu Ile Glu Ala Lys Ile Gly Ile Met Ala Val Asn Leu Thr
1060 1065 1070
~u
ACA GCT ACA ACT GCA ATC ATT ACT TCA TCT TTG GGG ATA GCT AGT GGA 3264
'I'hr Ala Thr Thr Ala Ile Ile Thr Ser Ser Leu Gly Ile Ala Ser Gly
1075 1080 1085
TTT AGT ATA CTT TTA GTT CCT TTA GCA GGA ATT TCA GCA GGT ATA CCA 3312
Phe Ser IIe Leu Leu Val Pro Leu Ala Gly lle Ser Ala Gly Ile Pro
1090 1095 1100
~J AGC TTA GTA AAC AAT GAA CTT GTA CTT CGA GAT AAG GCA ACA AAG CTT 3360
Ser Leu Val Asn Asn Glu Leu Val Leu Arg Asp Lys Ala Thr Lys Val
1105 1110 1115 1120
GTA GAT TAT TTT AAA CAT GTT TCA TTA GTT GAA ACT GAA GGA GTA TTT 3408
;!) Val Asp Tyr Phe Lys His Val Ser Leu Val Glu Thr Glu Gly Val Phe
1125 1130 1135
ACT TTA TTA GAT GAT AAA ATA ATG ATG CCA CAA GAT GAT TTA GTG ATA 3456
Thr Leu Leu Asp Asp Lys Ile Met Met Pro Gln Asp Asp Leu Val IIe
1140 1145 1150
~()
TCA GAA ATA GAT TTT AAT AAT AAT TCA ATA GTT TTA GGT AAA TGT GAA 3504
Ser Glu Ile Asp Phe Asn Asn Asn Ser Ile Val Leu Gly Lys Cys Glu
1155 1160 1165
ATC TGG AGA ATG GAA GGT GGT TCA GGT CAT ACT GTA ACT GAT GAT ATA 3552
Ile T'rp Arg Met Glu Gly Gly Ser Gly His Thr Val Thr Asp Asp Ile
1170 1175 1180
GAT Ce'1C TTC TTT TCA GCA CCA TCA ATA ACA TAT AGA GAG CCA CAC TTA 3600
Asp His Phe hhe Ser Ala Pro Ser Ile Thr Tyr Arg Glu I~ro Hi~ Leu
1185 1190 1195 1200
TCT ATA TAT GAC GTA TTG GAA GTA CAA AAA GAA GAA CTT GA'T TTG TCA 3648
Ser Ile Tyr Asp Val Leu Glu Val Gln Lys Glu Glu Leu Asp Leu Ser
1205 1210 1215
AAA GAT TTA ATG GTA TTA CCT AAT GCT CCA AAT AGA GTA TTT GCT TGG 3696
Lys Asp Leu Met Val Leu Pro Asn Ala Pro Asn Arg Val Phe Ala Trp
~J 1220 1225 1230
O)
GAA ACA GGA TGG ACA CCA GGT TTA AGA AGC TTA GAA AAT GAT GGC ACA 3744
Glu Thr Gly Trp Thr Pro Gly Leu Arg Ser Leu Glu Asn Asp Gly Thr
1235 1240 1245
AAA CTG TTA GAC CGT ATA AGA GAT AAC TAT GAA GGT GAG TTT TAT TGG 3792
Lys Leu Leu Asp Arg Ile Arg Asp Asn Tyr Glu Gly Glu Phe Tyr Trp
1250 1255 1260
AGA TAT TTT GCT TTT ATA GCT GAT GCT TTA ATA ACA ACA TTA AAA CCA 3840
Arg Tyr Phe Ala Phe Ile Ala Asp Ala Leu Ile Thr Thr Leu Lys Pro
1265 1270 1275 1280
-245-
CA 02296765 2000-O1-14
WO 98108540 PCT/US97/15394
AGA TAT GAA GAT ACT AAT ATA AGA ATA AAT TTA GAT AGT AAT ACT AGA 3888
Arg Tyr Glu Asp Thr Asn Ile Arg Ile Asn Leu Asp Ser A3n Thr Arg
' 1285 1290 1295
AGT TTT ATA GTT CCA ATA ATA ACT ACA GAA TAT ATA AGA GAA AAA TTA 3936
Ser Phe Ile Val Pro Ile Ile Thr Thr Glu Tyr Ile Arg Glu Lys Leu
1300 1305 1310
TCA TAT TCT TTC TAT GGT TCA GGA GGA ACT TAT GCA TTG TCT CTT TCT 3984
1() Ser Tyr Ser Phe Tyr Gly Ser Gly Gly Thr Tyr Ala Leu Ser Leu Ser
1315 1320 1325
CAA TAT AAT ATG GGT ATA AAT ATA GAA TTA AGT GAA AGT GAT GTT TGG 4032
Gln Tyr Asn Met Gly Ile Asn Ile Glu Leu Ser Glu Ser Asp Val Trp
1330 1335 1340
lITT ATA GAT GTT GAT AAT GTT GTG AGA GAT GTA ACT ATA GAA 'fCT GAT 4080
Ile Ile Asp Val Asp Asn Val Val Arg Asp Val Thr Ile Glu Ser Asp
1345 1350 1355 1360
AAA ATT AAA AAA GGT GAT TTA ATA GAA GGT ATT TTA TCT ACA CTA AGT 4128
Lys Ile Lys Lys Gly Asp Leu Ile Glu Gly Ile Leu Ser Thr Leu Ser
1365 1370 1375
ATT GAA GAG AAT AAA ATT ATC TTA AAT AGC CAT GAG 11TT AAT TTT TCT 4176
Ile Glu Glu Asn Lys Ile Ile Leu Asn Ser His Glu Ile Asn Phe Ser
1380 1385 1390
GGT GAG GTA AAT GGA AGT AAT GGA TTT GTT TCT TTA ACA TTT TCA ATT 4224
?() Gly Glu Val Asn Gly Ser Asn Gly Phe Val Ser Leu 'fhr Phe Ser Ile
1395 1400 1405
TTA GAA GGA ATA AAT GCA ATT ATA GAA GTT GAT TTA TTA TCT AAA TCA 4272
I,eu Glu Gly Ile Asn Ala Ile Ile Glu Val Asp Leu Leu Ser Lys Ser
1410 1415 1420
~4()
'."AT AAA TTA CTT ATT TCT GGC GAA TTA AAA ATA TTG ATG TTA AAT TCA 4320
'fyr Lys Leu Leu Ile Ser Gly Giu Leu Lys Ile Leu Met Leu Asn Ser
1425 1430 1435 1440
F,AT CAT ATT CAA CAG AAA ATA GAT TAT ATA GGA TTC AAT AGC GAA TTA 4368
Asn His Ile Gin Gln Lys Ile Asp Tyr Ile Gly Phe Ann Ser Glu Leu
1445 1450 1455
-)~ ChG AAA AAT T.TA CCA TAT AGC TTT GTA GAT AGT GAA GGA AAA GAG AAT 4416
Gln Lys Ann ile Pro Tyr Ser Phe Val Asp Ser Glu Gly Lye Glu Asn
1460 1465 1470
GGT TT'f 11TT AAT GGT TCA ACA AAA GAA GGT TTA TTT GTA TCT GAA TTA 4464
J() Cly Phe Ile Asn Gly Ser Thr Lys Glu Gly Leu Phe Val Ser Glu Leu
1475 1480 1485
CCT GAT GTA GTT CTT ATA AGT AAG GTT TAT ATG GAT GAT AGT AAG CCT 4512
Pro Asp Val Val Leu Ile Ser Lys Val Tyr Met Asp Asp Ser Lys Pro
1490 1495 1500
'fCA TTT GGA TAT TAT AGT AAT AAT TTG AAA GAT GTC AAA GTT ATA ACT 4560
Ser Plue Gly Tyr Tyr Ser Asn Asn Leu Lys Asp Val Lys Val Ile Thr
1505 1510 1515 1520
AAA GAT AATGTT AAT TTA GGT TAT TAT CTT GAT GAT ATA 4608
ATA ACA AAG
Lys Asp AsnVal Asn Leu Vly Tyr Tyr Leu Asp Asp Ile
Ile Thr Lys
1525 1530 1535
AAA ATC TCTCTT TCT ACT CAA GAT GAA AAA ATA AAG TTA 4656
TTG CTA ACT
Lys Ile SerLeu Ser Thr Gln Asp Glu Lys Ile Lys Leu
Leu Leu Thr
1540 1545 1550
CA 02296765 2000-O1-14
WO 98108540 PCT/US97l15394
AAT AGT GTG CAT TTA GAT GAA AGT GGA GTA GCT GAG ATT TTG AAG TTC 4704
Asn Ser Val His Leu Asp Glu Ser Gly Val Ala Glu Ile Leu Lys Phe
1555 1560 1565
J ATG AAT AGA AAA GGT AAT ACA AAT ACT TCA GAT TCT TTA ATG AGC TTT 4752
Met Asn Arg Lys Gly Asn Thr Asn Thr Ser Asp Ser Leu Met Ser Phe
1570 1575 1580
TTA GAA AGT ATG AAT ATA AAA AGT ATT TTC GTT AAT TTC TTA CAA TCT 4800
IO Leu Glu Ser Met Asn Ile Lys Ser Ile Phe Val Asn Phe Leu Gln Ser
1585 1590 1595 1600
AAT ATT AAG TTT ATA TTA GAT GCT AAT TTT ATA ATA AGT GGT ACT ACT 9848
Asn Ile Lys Phe Ile Leu Asp Ala Asn Phe Ile Ile Ser Gly Thr Thr
1605 1610 1615
TCT ATT GGC CAA TTT GAG TTT ATT TGT GAT GAA AAT GAT AAT ATA CAA 4896
Ser Ile Gly Gln Phe Glu Phe Ile Cys Asp Glu Asn Asp Asn Ile Gln
1620 1625 1630
CCA TAT TTC ATT AAG TTT AAT ACA CTA GAA ACT AAT TAT ACT TTA TAT 4944
Pro Tyr Phe Ile Lys Phe Asn Thr Leu Glu Thr Asn Tyr Thr Leu Tyr
1635 1640 1645
~J GTA GGA AAT AGA CAA AAT ATG ATA GTG GAA CCA AAT TAT GAT TTA GAT 4992
Val Gly Asn Arg Gln Asn Met Ile Val Glu Pro Asn Tyr Asp Leu Asp
1650 1655 1660
GAT TCT GGA GAT ATA TCT TCA ACT GTT ATC AAT TTC TCT CAA AAG TAT 5040
iU Asp Ser Gly Asp Ile Ser Ser Thr Val Ile Asn Phe Ser Gln Lys Tyr
1665 1670 1675 1680
CTT TAT GGA ATA GAC AGT TGT GTT AAT AAA GTT GTA ATT TCA CCA AAT 5088
Leu Tyr Gly Ile Asp Ser Cys Val Asn Lys Val Val Ile Ser Pro Asn
1685 1690 1695
~()
ATT TAT ACA GAT GAA ATA AAT ATA ACG CCT GTA TAT GAA ACA AAT AAT 5136
Ile 'Tyr Thr Asp Glu Ile Asn Ile Thr Pro Val Tyr Glu Thr Asn Asn
1700 1?05 1710
ACT TAT CCA GAA GTT ATT GTA TTA GAT GCA AAT TAT ATA AAT GAA AAA 5184
Thr Tyr Pro Glu Val Ile Val Leu Asp Ala Asn Tyr Ile Asn Glu Lys
1715 1720 1725
ATA AATGTT ATCAATGATCTATCT ATACGATAT TGG AGTrIAT 5232
AAT GTA
Ile AsnValAsn IleAsnAspLeuSer IleArgTyr Trp SerAsn
Val
1730 1735 1740
GAT GGTAATGF1TTTTATTCTTATGTCA ACTAGTGAA AAT AAGGTG 5280
GAA
Asp GlyAsnAsp PheIleLeuMetSer ThrSerGlu Asn LysVal
Glu
1745 1750 1755 1760
TCA CAAGTTAAA ATAAGATTCGTTAAT GTTTTTAAA AAG ACTTTG 5328
GAT
Ser GlnValLys IleArgPheValAsn ValPheLys Lys ThrLeu
Asp
1765 1770 1775
GCA AATAAGCTA TCTTTTAACTTTAGT GATAAACAA GTA CCTGTA 5376
GAT
Ala AsnLysLeu SerPheAsnPheSer AspLysGln Val ProVal
Asp
1780 1785 1790
hU
AGT GAA ATA ATC TTA TCA TTT ACA CCT TCA TAT TAT GAG GAT GGA TTG 5424
Ser Glu Ile Ile Leu Ser Phe Thr Pro Ser Tyr Tyr Glu Asp Gly Leu
1795 1800 1805
6J ATT GGC TAT GAT TTG GGT CTA GTT TCT TTA TAT AAT GAG AAA TTT TAT 5472
Ile Gly Tyr Asp Leu Gly Leu Val Ser Leu Tyr Asn Glu Lys Phe Tyr
1810 1815 1820
- 247 -
CA 02296765 2000-O1-14
PCTlUS97115394
ATT AAT AAC TTT GGA ATG ATG GTA TCT GGA TTA ATA TAT ATT AAT GAT 5520
Ile Asn Asn Phe Gly Met Met Val Ser Gly Leu Ile Tyr Ile Asn Asp
1825 1830 1835 1840
J TCA TTA TAT TAT TTT AAA CCA CCA GTA AAT AAT TTG ATA ACT GGA TTT 5568
Ser Leu Tyr Tyr Phe Lys Pro Pro Val Asn Asn Leu Ile Thr Gly Phe
1845 1850 1855
GTG ACT GTA GGC GAT GAT AAA TAC TAC TTT AAT CCA ATT AAT GGT GGA 5616
1f) Val Thr Val Gly Asp Asp Lys Tyr Tyr Phe Asn Pro Ile Asn Gly Gly
1860 1865 1870
GCT GCT TCA ATT GGA GAG ACA ATA ATT GAT GAC AAA AAT TAT TAT TTC 5664
Ala Ala Ser Ile Gly Glu Thr Ile Ile Asp Asp Lys Asn Tyr Tyr Phe
IJ 1875 1880 1885
AAC CAA AGT GGA GTG TTA CAA ACA GGT GTA TTT AGT ACA GAA GAT GGA 5712
Asn Gln Ser Gly Val Leu Gln Thr Gly Val Phe Ser Thr Glu Asp Gly
1890 1895 1900
TTT AAA TAT TTT GCC CCA GCT AAT ACA CTT GAT GAA AAC CTA GAA GGA 5760
Phe Lys Tyr Phe Ala Pro Ala Asn Thr Leu Asp Glu Asn Leu Glu Gly
1905 1910 1915 1920
GAA COCA ATT GAT TTT ACT GGA AAA TTA ATT ATT GAC GAA AAT ATT TAT 5808
Glu Ala Ile Asp Phe Thr Gly Lys Leu Ile Ile Asp Glu Asn Ile Tyr
1925 1930 1935
:AT TTT CAT GAT AAT TAT AGA GGA GCT GTA GAA TGG AAA GAA TTA GAT 5856
s() 'I'yr Phe Asp Asp Ann Tyr Arg Gly Ala Val Glu Trp Lys Glu Leu Asp
1940 1945 1950
GGT GAA ATG CAC TAT TTT AGC CCA GAA ACA GGT AAA GCT TTT AAA GGT 5904
Gly Glu Met His Tyr Phe Ser Pro Glu Thr Gly Lys Ala Phe Lys Gly
i~ 1955 1960 1965
CTA J1AT CAA ATA CGT GAT TAT AAA TAC TAT TTC AAT TCT GAT GGA GTT 5952
Leu Hen Gln Ile Gly Asp Tyr Lys Tyr Tyr F?he Asn Ser Asp Gly Val
1970 1975 1980
ATG CAi-, AAA GGA TTT GTT AGT ATA AAT GAT AAT AAA CAC TAT TTT GAT 6000
Met Gln Lys Gly Phe Val Ser Ile Asn Asp Asn Lys Fiis Ty~r Phe Asp
1985 1990 1995 2000
GAT TCT GGT GTT ATG AAA GTA GGT TAC ACT GAA ATA GAT GGC AAG CAT 6048
F,sp Ser Gly Val Met Lys Val Gly Tyr Thr Glu Ile Asp Gly Lys His
7.005 2010 2015
TTC TAC TTT GCT GAA AAC GGA GAA ATG CAA ATA GGA GTA TTT AAT ACA 6096
~f) Phe Tfr Phe Ala Glu Asn Gly Glu Met Gln Ile Gly Val Phe Asn '1'hr
2020 2025 2030
GAA GAT GGA TTT AAA TAT TTT GCT CAT CAT AAT GAA GAT TTA GGA AAT 6144
Glu Asp Gly Phe Lys Tyr Phe Ala Hi:. His Asn Glu Asp Leu Gly Asn
~J 2035 2040 2045
OU
GAA GAA GGT GAA GAA ATC TCA TAT TCT GGT ATA TTA AAT TTC AAT AAT 6192
C~lu Glu Gly Glu Glu Ile Ser Tyr Ser Gly Ile Leu Asn Phe Asn Asn
2050 2055 2060
AAA ATT TAC TAT TTT GAT GAT TCA TTT ACA GCT GTA GTT GGA TGG AAA 6240
Lys Ile Tyr Tyr Phe Asp Asp Ser Phe Thr Ala Val Val Gly Trp Lys
2065 2070 2075 2080
(o GAT TTF, GAG GAT GGT TCA AAG TAT TAT TTT GAT GAA GAT ACA GCA GAA 6288
Asp Leu Glu Asp Gly Ser Lys 'I'yr 'Tyr Phe Asp Glu lisp Thr Ala Glu
2085 2090 2095
_ ~~$ _
CA 02296765 2000-O1-14
WO 98~85A0 PCT/US97/15394
GCA TAT ATA GGT TTG TCA TTA ATA AAT GAT GGT CAA TAT TAT TTT AAT 6336
Ala Tyr Ile Gly Leu Ser Leu Ile Asn Asp Gly Gln Tyr Tyr Phe Asn
2100 2105 2110
S GAT GAT GGA ATT ATG CAA GTT GGA TTT GTC ACT ATA AAT GAT AAA GTC 6384
Asp Asp Gly Ile Met Gln Val Gly Phe Val Thr Ile Asn Asp Lys Val
2115 2120 2125
_ TTC TAC TTCTCTGAC TCTGGAATTATAGAA GGAGTACAA ATA 6432
TCT AAC
1() Phe Tyr PheSerAsp SerGlyIleIleGluSer GlyValGlnAsnIle
2130 2135 2140
GAT GAC AATTATTTC TATATAGATGATAATGGT ATAGTTCAAATTGGT 6480
Asp Asp AsnTyrPhe TyrIleAspAspAsnGly IleValGlnIleGly
2145 2150 2155 2160
GTA TTT GATACTTCA GATGGATATAAATATTTT GCACCTGCTAATACT 6528
Val Phe AspThrSer AspGlyTyrLysTyrPhe AlaProAlaAsnThr
2165 2170 2175
GTA AAT GATAATATT TACGGACAAGCAGTTGAA TATAGTGGTTTAGTT 6576
Val Asn AspAsnIle TyrGlyGlnAlaValGlu TyrSerGlyLeuVal
2180 2185 2190
~S AGA GTT GGGGAAGAT GTATATTATTTTGGAGAA ACATATACAATTGAG 6624
Arg 'galGlyGluAsp ValTyrTyrPheGlyGlu ThrTyrThr_TleGlu
~t95 2200 2205
ACT GGA TGGATATAT GATATGGAAAATGAAAGT GATAAATATTATTTC 6672
i() Thr Gly TrpIleTyr AspMetGluAsnGluSer AspLysTyrTyrPhe
2210 2215 2220
AAT CCA GAAACTAAA AAAGCATGCAAAGGTATT AATTTAATTGATGAT 6720
Asn Pro GluThrLys LysAlaCysLysGlyIle AsnLeuIleAspAsp
?J 2225 2230 2235 2240
ATA AAA TATTATTTT GATGAGAAGGGCATAATG AGAACGGGTCTTATA 6768
Ile Lys TyrTyrPhe AspGluLysGlyIleMet ArgThrGlyLeuIle
2245 2250 2255
-~()
TCA TTT GAAAATAAT AATTATTACTTTAATGAG AATGGTGAAATGCAA 6816
Ser Phe GluAsnAsn AsnTyrTyrPheAsnGlu AsnGlyGluMetGln
2260 2265 2270
TTT CGT TATATAAAT ATAGAAGATAAGATGTTC TATTTTGGTGAAGAT 6864
Phe Gly TyrIleAsn IleGluAspLysMetPhe TyrPheGlyGluAsp
275 2280 2285
GGT GTC ATGCAGATT GGAGTATTTAATACACCA GATGGATTTAAATAC 6912
SU Gly Val MetGlnIle GlyValPheAsnThrPro AspGlyPheLysTyr
2290 2295 2300
TTT GCA CATCAAAAT ACTTTGGATGAGAATTTT GAGGGAGAATCAATA 6960
Phe Ala HisGlnAsn ThrLeuAspGluAsnPhe GluGlyGluSerIle
W 2305 2310 2315 2320
(i0
AAC TAT ACT GGT TGG TTA GAT TTA GAT GAA AAG AGA TAT TAT TTT ACA 7008
Asn Tyr Thr Gly Trp Leu Asp Leu Asp Glu Lys Arg Tyr T'yr Phe Thr
2325 2330 2335
GAT GAA TAT ATT GCA GCA ACT GGT TCA GTT ATT ATT GAT GGT GAG GAG 7056
Asp Glu Tyr Ile Ala Ala Thr Gly Ser Val Ile Ile Asp Gly Glu Glu
2340 2345 2350
TAT TAT TTT GAT CCT GAT ACA GCT CAA TTA GTG ATT AGT GAA 7098
Tyr Tyr Phe Asp Pro Asp Thr Ala Gln Leu Val Ile Ser Glu
2355 2360 2365
TAG 7101
- 249
CA 02296765 2000-O1-14
PCTNS9'1115394
(2) INFORMATION FOR SEQID
NO:10:
!i) CHARACTE RISTICS:
SEQUENCE
iA)LENGTH: 2366 acids
amino
J (B)TYPE: minoacid
a
!D)TOPOLOG Y: inear
l
!ii) TYPE: otein
MOLECULE pr
(xi) DESCRIPTION:SEO ID NO:10:
SEQUENCE
Met Ser LeuValAsn ArgLysGlnLeu GluLysMetAlaAsnVdl Arg
1 5 10 15
Phe Arg ThrGlnGlu AspGluTyrVal AlaIleLeuAspAlaLeu Glu
20 25 30
Glu Tyr HisAsnMet SerGluAsnThr ValValGluLysTyrLeu Lys
35 40 45
Leu Lys AspIleAsn SerLeuThrAsp IleTyrIleAspThrTyr Lys
50 55 60
Lye Ser GlyArgAsriLysAlaLeuLys LysPheLysGluTyrLeu Val
65 70 75 80
Thr Glu ValLeuGlu LeuLysA,nAsn AsnLeuThrProValGlu Lys
85 90 95
?() Asn Leu HisPheVal TrpIleGlyG1y GlnIleAsnAspThrAla Ile
100 105 110
Asn Tyr IleAsnGln TrpLysAspVal AsnSerAppTyrAnnVal Asn
115 120 125
ii
Val Phe 'I'yrAspSPr AsnAlaPheLeu IleAsnThrLeuLyehys Thr
130 135 140
Val Val GluSerAla IleAsnAspThr LeuGluSerPheArgGlu Asn
fit)195 150 155 160
Leu Asn AspProArg PheAspTyrAsn LysPhePheArgLysArg Met
165 170 175
-IJ Glu Ile Ile'ryrAsp LysGlnLysAsn PheIleAsnTyrTyrLys Ala
180 185 190
GlriArg GluGluAsn ProGluLeuIle IleAspAspIleValLys Thr
195 200 205
J~)
Tyr Leu SerAsnGlu TyrSerLysGlu IleAspGluLeuAsnThr Tyr
21U 215 220
Ile Glu GluSerLeu AsnLysIleThr GlnAsnSerGlyAsnAsp Val
JJ 225 230 235 240
Arg Asn PheGluGlu PheLysAsriGly GluSerPheAsnLeuTyr Glu
245 250 255
O1 Gln Glu LeuValGlu ArgTrpAsnLeu AlaAlaAlaSerAspIle Leu
' 260 265 270
Arg Ile SerAlaLeu LysGluIleGly GlyMetTyrLeuAspVal Asp
275 280 285
6a
Met Leu ProGlyIle GlnProAspLeu PheGluSerIleGluLys Pro
290 295 300
Ser Ser ValThrVai AspPheTrpGlu MetThrLysLeuCluAla Ile
305 310 315 320
- 250 -
CA 02296765 2000-O1-14
WO 98108540 PCT/US97115394
Met Lys Tyr Lys Glu Tyr Ile Pro Glu Tyr Thr Ser Glu His Phe Asp
325 330 335
Met Leu Asp Glu Glu Val Gln Ser Ser Phe Glu Ser Val Leu Ala Ser
1 340 345 350
Lys Ser Asp Lys Ser Glu Ile Phe Ser Ser Leu Gly Asp Met Glu Ala
355 360 365
lU Ser Pro Leu Glu Val Lys Zle Ala Phe Asn Ser Lys Gly Ile Ile Asn
370 375 380
Gln Gly Leu Ile Ser Val Lys Asp Ser Tyr Cys Ser Asn Leu Ile Val
385 390 395 400
~J
Lys Gln Ile Glu Asn Arg Tyr Lys Ile Leu Asn Asn Ser Leu Asn Pro
405 410 415
Ala Ile Ser Glu Asp Asn Rsp Phe Asn Thr Thr Thr Asn Thr Phe Ile
420 425 430
Asp Ser Ile Met Ala Glu Ala Asn Ala Asp Asn Gly Arg Phe Met Met
435 440 445
~J Glu LeuGlyLysTyr LeuArgValGlyPhe PheProAspVal LysThr
950 955 460
Thr IleAsnLeuSer GlyProGluAlaTyr AlaAlaAlaTyr GlnAsp
465 470 475 480
i~)
Leu LeuMetPheLys GluGlySerMetAsn IleHisLeuIle GluAla
485 490 495
App LeuArgAsnPhe GluIleSerLysThr AsnIleSerGln SerThr
.iJ 500 SOS 510
Glu Gln Glu Met Ala Ser Leu Trp Ser Phe Asp Asp Ala Arg Ala Lys
515 520 525
-l0 Ala GlnPlieGluGlu TyrLysArgAsnTyr PheGluGlySerLeu Gly
530 535 540
Glu AspAspAsnLeu AspPheSerGlnAsn IleValValAspLys Glu
545 550 555 560
Tyr I.euLeuGluLys IleSerSerLeuAla ArgSerSerGluArg Gly
565 570 575
T;:r IleHisTyrIle ValGlnLeuGlnGly AspLysIleSerTyr Glu
580 585 590
Ala AlaCysAsnLeu PheAlaLysThrPro TyrAspSerValLeu Phe
595 600 605
JJ Gln LysAsnIleGlu AspSerGluIleAla TyrTyrTyrAsnPro Gly
610 615 620
Asp Gly Glu Ile Gln Glu Ile Asp Lys Tyr Lys Ile Pro Ser Ile Ile
625 630 635 640
(>()
Ser App AYg Pro Lys Ile Lys Leu Thr Phe Ile Gly His Gly Lys Asp
645 650 655
Glu Phe Asn Thr Asp Ile Phe Ala Gly Phe Asp Val Asp Ser Leu Ser
(7J 660 665 670
Thr Glu Ile Glu Ala Ala Ile Asp Leu Ala Lys Glu Asp Ile Ser Pro
675 680 685
CA 02296765 2000-O1-14
WO 98108540 PCTNS97115394
Lys Ser Ile Glu Ile Asn Leu Leu Gly Cys Asn Met Phe Ser Tyr Ser
690 695 700
Ile Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Lys Val Lys
705 710 715 720
Asp Lys Ile Ser Glu Leu Met Pro Ser Ile Ser Gln Asp Ser Ile Ile
725 730 735
1() Val Ser Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly Arg Arg
740 745 750
~i
Glu Leu Leu Asp His Ser Gly Glu Trp Ile Asn Lys Glu Glu Ser Ile
755 760 765
Ile Lys Asp Ile Ser Ser Lys Glu Tyr Ile Ser Phe Asn Pro Lys Glu
770 775 780
Asn Lys Ile Thr Val Lys Ser Lys Asn Leu Pro Glu Leu 5er Thr Leu
785 790 795 800
Leu Gln GluIleArg AsnAsnSerAsn SerSerAspIleGluLeu Glu
805 810 815
Glu Lys ValMetLeu '1'hrGluCysGlu IleAsnValIleSerAsn Ile
820 825 830
Asp Thr GlnIleVal GiuGluArgIle GluGluAlaLysAsnLeu Thr
835 840 845
i~)
Ser Asp SerIleAsn TyrIleLysAsp GluPheLysLeuIleGlu Ser
850 855 860
Ile SerAspAlaLeu CysAspLeuLys GlnGlnAsnGluLeuGlu Asp
865 870 875 880
Ser IfisPheIleSer PheGluAspIle SerGluThrAspGluGly Phe
885 890 895
Ser IleArgPheIle AsnLysGluThr GlyGluSerIlePheVal Glu
900 905 910
Thr GluLysThrIle PheSerGluTyr AlaAsnHisIleThrGlu Glu
915 920 925
.~
S
I1e SerLysIleLys GlyThrIlePhe AspThrValAsnGlyLys Leu
930 935 940
Val Lys Lys Val Asn Leu Asp Thr Thr His Glu Val Asn 'Thr Leu Asn
945 950 955 960
Ala Ala Phe Phe Ile Gln Ser Leu Ile Glu Tyr Asn Ser Ser Lys Glu
965 970 975
~J Ser Leu Ser Asn Leu Ser Val Ala Met Lys Val Gln Val Tyr Ala Gln
980 985 990
Leu Phe Ser Thr Gly Leu nsn Thr Ile Thr Asp Ala Ala Lys Val Val
995 lODO 1005
(~(?
Glu Leu Val Ser Thr Ala Leu Asp Glu Thr Ile Asp Leu Leu Pro Thr
1010 1015 1020
Leu Ser Glu Giy Leu Pro Ile Ile Ala Thr Ile Ile Asp Gly Val Ser
(1J 1025 1030 1035 1040
Leu Gly Ala Ala Ile Lys Glu Leu Ser Glu Thr Ser Asp Pro Leu Leu
1045 1050 1055
CA 02296765 2000-O1-14
PCTIUS97/15394
Arg Gln Glu Ile Glu Ala Lys Ile Gly Ile Met Ala Val Asn Leu Thr
1060 1065 1070
Thr Ala Thr Thr Ala Ile Ile Thr Ser Ser Leu Gly Ile Ala Ser Gly
1075 1080 1085
Phe Ser Ile Leu Leu Val Pro Leu Ala Gly Ile Ser Ala Gly Ile Pro
1090 1095 1100
IU Ser Leu Val Asn Asn Glu Leu Val Leu Arg Asp Lye Ala Thr Lys Val
1105 1110 1115 1120
Val Asp Tyr Phe Lys His Val Ser Leu Val Glu Thr Glu Gly Val Phe
1125 1130 1135
li
Thr Leu Leu Asp Asp Lys Ile Met Met Pro Gln Asp Asp Leu Val Ile
1140 1145 1150
Ser Glu Ile Asp Phe Asn Asn Asn Ser Ile Val Leu Gly Lys Cys Glu
1155 1160 1165
Ile Trp Arg Met Glu Gly Gly Ser Gly His Thr Val Thr Asp App Ile
1170 1175 1180
Asp His Phe Phe Ser Ala Pro Ser Ile Thr Tyr Arg Glu Pro His Leu
1185 1190 1195 120C
Ser Ile Tyr Asp Val Leu Glu Val Cln Lys Glu Glu Leu Asp Leu Ser
1205 1210 1215
i()
Lys Asp Leu Met Val Leu Pro Asn Ala Pro Asn Arg Val Phe Ala Trp
1220 1225 1230
Glu Thr Gly Trp Thr Pro Gly Leu Arg Ser Leu Glu Asn Asp Gly Thr
1235 1240 1245
Lys LeuLeu AspArgIleArgAsp AsnTyrGluGlyGlu PheTyrTrp
1250 1255 1260
40 Arg TyrPhe AlaPheIleAlaAsp AlaLeuIleThrThr LeuLysPro
126s 1270 1275 l2so
Arg 'I'yrGlu AspThrAsnIleArg IleAsnLeuAspSer AsnThrArg
1285 1290 1295
.~
i
Ser PheIle ValProIleIleThr ThrGluTyrIleArg GluLysLeu
1300 1305 1310
Ser TyrSer PheTyrGlySerGly GlyThrTyrAlaLeu SerLeuSer
1315 1320 1325
Gln Tyr Asn Met Gly Ile Asn Ile Glu Leu Ser Glu Ser Asp Val Trp
1330 1335 1340
Ile Ile Asp Val Asp Asn Val Val Arg Asp Val Thr Ile Glu Ser Asp
1395 1350 1355 1360
Lys Ile Lys Lys Gly Asp Leu Ile Glu Gly Ile Leu Ser Thr Leu Ser
1365 1370 1375
6t)
Ile Glu G'lu Asn Lys Ile Ile Leu Asn Ser His Glu Ile Asn Phe Ser
1380 1385 1390
Gly Glu Val Asn Gly Ser Asn Gly Phe Val Ser Leu Thr Phe Ser Ile
1395 1400 1405
Leu Glu Gly Ile Asn Ala Ile Ile Glu Val Asp Leu Leu Ser Lys Ser
1410 1415 1420
j
CA 02296765 2000-O1-14
PCTII1S97I15394
Tyr Lys Leu Leu Ile Ser Gly Glu Leu Lys Ile Leu Met Leu Asn Ser
1425 1430 1435 1440
Asn His Ile Gln Gln Lys Ile Asp Tyr Ile Gly Phe Asn Ser Glu Leu
J 1495 1450 1455
Gln Lys Asn Ile Pro Tyr Ser Phe Val Asp Ser Glu Gly Lys Glu Asn
1460 1465 1470
1I) Gly Phe Ile Asn Gly Ser Thr Lys Glu Gly Leu Phe Val Ser Glu Leu
1475 1480 1485
Pro Asp Val Val Leu Ile Ser Lys Val Tyr Met Asp Asp Ser Lys Pro
1490 1495 1500
~i
Ser Phe Gly Tyr Tyr Ser Asn Asn Leu Lys Asp Val Lys Val Ile Thr
1505 1510 1515 1520
hys Asp Asn Val Asn Ile Leu Thr Gly Tyr Tyr Leu Lys Asp Asp Ile
1525 1530 1535
Lys Ile Ser Leu Ser Leu Thr Leu Gln Asp Glu Lys Thr Ile Lys Leu
1540 1545 1550
~J Asn SerValHisLeu AspGluSerGlyValAla GluIleLeuLys Phe
1555 1560 1565
Met AnnArgLysGly AsnThrAsnThrSerAsp SerLeuMetSer Phe
1570 1575 1580
j()
Leu GluSerMetAnn IleLysSerIlePheVal AsnPheLeuGln Ser
1585 1590 1595 1600
Asn IleLysPheIle LeuAspAlaAsnPheIle IleSerGlyThr Thr
?J 1605 1610 1615
Ser IleGlyGlnPhe GluPheIleCysAspGlu AsnAspAsnIle Gln
1620 1625 1630
Pro TyrPheIleLys PheAsnThrLeuGluThr AsnTyrThrLeu Tyr
1635 1640 1645
Val GlyAsnArgGln AsnMetIleValGluPro AsnTyrAspLeu Asp
1650 1655 1660
.~
i
Asp SerGlyAspIle SerSerThrValIleAsriPheSerGlnLys Tyr
1665 1670 1675 1680
Leu TyrGlyIleAsp SerCysValAsnLysVal ValIleSerPro Asn
JU 1685 1690 1695
Ile TyrThrAspGlu IleAsnIleThrProVal TyrGluThrAsn Asn
1700 1705 1710
J~ Thr TyrProGluVal IleValLeuAspAlaAsn TyrIleAsnGlu Lys
1715 1720 1725
Ile AsnValAsnIle AsnAspLeuSerIleArg TyrValTrpSer Asn
1730 1735 1740
~)~)
Asp GlyAsnAspPhe IleLeuMetSerThrSer GluGluAsnLys Val
1745 1750 1755 1760
Ser GlnValLysIle ArgPheValAsnValPhe LysAspLysThr Leu
C)S 1765 1770 1775
Ala AsnLysLeu5er PheAsnPheSerAspLys GlnAspValPro Val
1780 1785 1790
CA 02296765 2000-O1-14
WO 98108540 PCT/US97/15394
Ser Glu Ile Ile Leu Ser Phe Thr Pro Ser Tyr Tyr Glu Asp Gly Leu
1795 1800 1805
Ile Gly Tyr Asp Leu Gly Leu Val Ser Leu Tyr Asn Glu Lys Phe Tyr
J 1810 1815 1820
Ile Asn Asn Phe Gly Met Met Val Ser Gly Leu Ile Tyr Ile Asn Asp
1825 1830 1835 1840
lU Ser Leu Tyr Tyr Phe Lys Pro Pro Val Asn Asn Leu Ile Thr Gly Phe
1895 1850 1855
Val Thr Val Gly Asp Asp Lys Tyr Tyr Phe Asn Pro Ile Asn Gly Gly
1860 1865 1870
~i
Ala Ala Ser Ile Gly Glu Thr Ile Ile Asp Asp Lys Asn 'fyr Tyr Phe
1875 1880 1885
Asn Gln Ser Cly Val Leu Gln Thr Gly Val Phe Ser Thr~Glu Asp Gly
1890 1895 1900
Phe Lys Tyr Phe Ala Pro Ala Asn Thr Leu Asp Glu Asn Leu Glu Gly
1905 1910 1915 1920
Glu Ala Ile Asp Phe Thr Gly Lys Leu Ile Ile Asp Glu Asn Ile Tyr
1925 1930 1935
Tyr Phe Asp Asp Asn Tyr Arq Gly Ala Val Glu Trp Lys Glu Leu Asp
1940 1945 1950
i ()
Gly Glu Met His Tyr Phe Ser Pro Glu Thr Gly Lys Ala Phe Lys Gly
1955 1960 1965
Leu Asn Gln Ile Gly Asp 'fyr Lys Tyr Tyr Phe Asn Ser Asp Gly Val
~J 1970 1975 1980
Met GlnLys GlyPheVal SerIleAsnAspAsn LysHis'I'yrPheAsp
1985 1990 1995 2000
~1() Asp SerGly ValMetLys ValGlyTyrThrGlu IleAspGlyLysHis
2005 2010 015
Phe TyrPhe AlaGluAsn GlyGluMetGlnIle GlyValPheAsn':hr
2020 2025 2030
.~ i
Glu AspGly PheLysTyr PheAlaHisHisAsn GluAspLeuGlyAsn
2035 2040 045
Glu GluGly GluGluIle SerTyrSerGlyIle LeuAsnPheAsnAsn
J() 2050 2055 2060
Lys IleTyr TyrPheAsp AspSerPheThrAla ValValGlyTrpLys
2065 2070 2075 2080
~> Asp LeuGlu AspGlySer LysTyrTyrPheAsp GluAspThrAlaGlu
2085 2090 2095
Ala TyrIle GlyLeuSer LeuIleAsnAspGly GlnTyrTyrPheAsn
2100 2105 110
OU
Asp AspGly IleMetGln ValGlyPheValThr IJeAsnAspLysVai
2115 2120 2125
Phe TyrPhe SerAspSer GlyIleJleGIuSer ~::lyValGlnAsnIle
_ 2130 215 27.40
~)J
Asp AspAsn TyrPheTyr IleAspAspAsnGly IleValGlnIleGly
. 2145 2150 2155 2160
Val PheAsp ThrSerAsp GlyTyrLysTyrPhe AlaProAlaAsnThr
-355-
*rB
CA 02296765 2000-O1-14
gyp 9g/pg~p PCTIUS97115394
2165 2170 2175
Val AsnAsp Asn Ile Tyr Gly Gln Glu Tyr Ser Gly
Ala Val Leu Val
2180 2185 2190
j
Arg ValGly Glu Asp Val Tyr 'I'yr Glu Thr Tyr Thr
Phe Gly Iie Glu
2195 2200 2205
Thr GlyTrp Ile Tyr Asp Met Glu Ser Asp Lys Tyr
Asn Glu Tyr Phe
)U 22102215 2220
Asn ProGlu Thr Lys Lys Aia Cy~ Ile Asn.Leu Ile
Lys Gly Asp Asp
~2~5 2230 2235 2240
1J Ile LysTyr Tyr Phe Asp Glu Lys Met Arg Thr Gly
Gly Ile Leu Ile
2245 2250 2255
Ser PheGlu Asn Asn Asn Tyr Tyr Glu Asn Gly Glu
Phe Asn Met Gln
2260 2265 2270
U
Phe GlyTyr Ile Asn Ilp Glu Asp Phe Tyr Phe Gly
Lys Met Glu Asp
2275 2280 2285
Gly ValMet Gln Ile Gly Val Phe Pro Asp Gly Phe
Asn Thr Lys Tyr
22902295 2300
Phe AlaHis Gln Asn Thr Leu Asp Phe Glu Gly Glu
Glu Asn Ser Ile
?.305 2310 2315 2320
?() Asn TyrThr Gly Trp Leu Asp Leu Lys Arg Tyr Tyr
Asp Glu Phe Thr
2325 2330 X335
Asp GluTyr Ile Ala Ala Thr Gly Ile Ile Asp Gly
Ser Val Glu Glu
2340 2345 2350
ii
Tyr 'I'_,rrPhe Asp Pi-o Asp Thr Ala Val Ile Ser Glu
Gln Leu
2355 2360 2365
(:'.)INFORMATION FOR SEQ ID NO:11:
1
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
lii)MOLECULE TYPE: DNA Igenomic)
(xi)SEQUENCE DESCRIPTION: SEQ :11:
ID N0
j
()
TAGAAAAAAT
GGCAAATGT
(2) INFORMATION FOR SEQ ID N0:12:
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
O)
(ii)MOLECULE TYPE: DNA (genomic)
lxi)SEQUENCE DESCRIPTION: SEQ :1~:
ID N0
t7 TTTCATCTTG
TAGACTCAAA
G
(2) INFORMATION FOR SEQ ID N0:13:
(i)SEQUENCE CHARACTERISTICS:
) (A) LENGTH: 2~ base pairs
- z~6 -
CA 02296765 2000-O1-14
WO 981108540 PCT/US97115394
(B) TYPE: nucleic acid
(C) STR.ANDEDNESS: single
(D) TOPOLOGY: linear
> (ii) MOLECULE TYPE: DNA (genomicl
Iri) SEQUENCE DESCRIPTION: SEQ ID N0:13:
GATGCCACAA GATGATTTAG TG 22
lU
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
17 (BI TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(:ci) SEQUENCE DESCRIPTION: SEQ ID N0:14:
CTAATTGAGC TGTATCAGGA TC 22
(~) ItJFORMATION FOR SEQ ID N0:15:
i.ii SEQUENCE CHARACTERISTICS:
IA) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
?l) (C) STRANDEDNESS: single
ID) TOPOLOGY: linear
fii) MOLECULE TYPE: DNA (genomic>
(%ii SEQUENCE DESCRIPTION: SEQ ID N0:15:
CGGAATTCCT AGAAAAiAATG GCAAATG 27
(2j INFORMATION FOR SEQ ID N0:16:
':i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C1 STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(%i) SEQUENCE DESCRIPTION: SEQ ID N0:16:
JO
GCTCTAGAAT GACCATAAGC TAGCCA 26
i2) INFORMATION FOR SEQ ID N0:17:
JJ (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
6()
(ii) PIOLECULE TYPE: DNA (genomic)
(%i) SEQUENCE DESCRIPTION: SEQ ID N0:17:
CGGAATTCGA GTTGGTAGAA AGGTGGA 27
(~) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: ~7 base pairs
CA 02296765 2000-O1-14
PCTNS97115394
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
7 (ii) MOLECULE TYPE: DNA (genomic)
lx.i) SEQUENCE DESCRIPTION: N0:18:
SEQ ID
CGGAATTCGG 27
TTATTATCTT
AAGGATG
(2) INFORMATION
FOR SEQ
ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base
pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
?0
(xi) SEQUENCE DESCRIPTION: N0:19:
SEQ ID
CGGAATTCTT 28
GATAACTGGA
TTTGTGAC
(2) INFORMATION
FOR SEQ
ID N0:20:
!i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 511 amino
acids
(B) TYPE: amino acid
?() (C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
tai) MOLECULE TYPE: protein
(ii) SEQUENCE DESCRIPTION: N0:20:
SEQ ID
Leu Ile Thr Gly Phe Val GlyAspAsp Lys Tyr Tyr Phe Asn
Thr Val
1 5 10 15
Pro Ile Asn Gly Gly Ala IleGlyGlu Thr Ile Ile Asp Asp
Ala Ser
~0 25 30
Lys Asn Tyr Tyr Phe Asn GlyValLeu Gln Thr Gly Val Phe
Gln Ser
35 90 45
.~
i
Ser Thr Glu Asp Gly Phe PheAlaPro Ala Asn Thr Leu Asp
Lys Tyr
50 55 60
Glu Asn Leu Glu Gly Glu AspPheThr Gly Lys Leu Ile Ile
Ala Ile
65 70 75 BO
Asp Glu Asn Ile Tyr Tyr AspAsnTyr Arg Gly Ala Val Glu
Phe Asp
85 90 95
Trp Lys Glu Leu Asp Gly HisTyrPhe Ser Pro Glu Thr Gly
Glu Met
100 105 110
Lys Ala Phe Lys Gly Leu Asn Gln Ile Gly Asp Tyr Lys Tyr 'i'yr Phe
115 120 1<5
O)
Asn 5er Asp Gly Val Met Gln Lys Gly Phe Val Ser Ile Asn App Asn
130 135 140
Lys His Tyr Phe Asp Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu
(» 145 150 155 160
Ile Asp Gly Lys His Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile
165 170 175
~~) Gly Val Phe Asn Thr Glu Asp Gly Phe Lys Tyr Phe Ala His His Asn
_ '75g _
CA 02296765 2000-O1-14
W~ 9glpgsqp PCT/US97l15394
lao le5 190
Glu Asp Leu Gly Asn Glu Glu Gly Glu Glu Ile Ser Tyr Ser Gly Ile
195 200 205
Leu Asn Phe Asn Asn Lys Ile Tyr Tyr Phe Asp.Asp Ser Phe Thr Ala
210 215 220
_ Val Val Gly Trp Lys Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp
lU 225 230 235 240
Glu Asp Thr Ala Glu Ala Tyr Ile Gly Leu Ser Leu Ile Jisn Asp Gly
245 25' 255
1J Gln TyrTyrPheAsnAsp AspGlyIleMetGln ValGlyPheValThr
260 265 270
IIe AsnAspLysValPhe TyrPheSerAspSer GlyIle!leGluSer
275 280 285
'_'
U
Gly ValGlnAsnIleAsp AspAsnTyrPhe'TyrIleAspAspAsnGly
290 295 300
Ile ValGlnIleGlyVal PheAspThrSerAsp GlyTyrLys1'yrPhe
305 310 315 320
Ala ProAiaAsnThrVal AsnAspAsnIleTyr GlyGlnAlaVa1Glu
325 330 335
Tyr SerGlyLeuValArg ValGlyGluAspVal TyrTyrtheGlyGiu
340 345 350
Thr TyrThrIleGluThr GlyTrpIleTyrAsp MetGluAsnGluSer
355 360 365
ii
Asp LysTyrTyrPheAsn ProGluThrLysLye AlaCysLysGlyIle
370 375 380
Asn LeuIleAspAspIle LysTyrTyrPheAsp GluLysGlyIleMet
385 390 395 400
Arg ThrGlyLeuIleSer PheGluAsnAsnAsn TyrTyrPheAsnGlu
405 410 415
Asn GiyGluMetGlnPhe GlyTyrIleAsnIle GluAspL,~sMetPhe
420 425 ~13G
'ryr PheGlyGluAspGly ValMetGlnIleGiy ValPheAsnThrPro
435 440 445
i()
Asp GlyPheLysTyrPhe AlaHisGlnAsnThr LeuAspGluAsnPhe
450 455 460
Glu GlyGlu5erIleAsn TyrThrGlyTrpLeu AspLeuAspGluLys
465 470 475 480
Arg TyrTyrPheThrAsp GluTyrIleAlaAla ThrGlySerValIle
485 490 495
Ile AspGlyGluGluTyr TyrPheAspProAsp ThrAlaGlnhau
- 500 505 510
_ oj9 _
CA 02296765 2000-O1-14
WO 9810854 PCTIUS97/15394
f21 INFORMATION
FOR
SEQ
ID N0:21:
(i1 SEQUENCE CHARACTERIS TICS:
(A)LENGTH: 608 amino cids
a
lB1TYPE: minoacid
a
IC)STRANDEDNES S: nknown
u
(D1TOPOLOGY: wn
unkno
fii) MOLECULETYPE: in
l prote
()
iri) SEQUENCE DESCRIPTION: Q N0:21:
SE ID
Ser GluGluAsnLys ValSerGlnValLys IleArgPheVal AsnVal
1 5 10 15
~i
Phe LysAspLysThr LeuAIaAsnLysLeu 5erPheAsnPhe SerAsp
20 25 30
Lys GlnAspValPro ValSerGluIleIle LeuSerPheThr ProSer
35 40 45
T.yr TyrGluAspGly LeuIleGlyTyrAsp LeuGlyLeuVa1 SerLeu
SO 55 60
~J Tyr AsnGluLysPhe TyrIleAsnAsnPhe GlyMetMetVal SerGly
65 70 75 80
Leu lleTyrIleAsn AspSerLeuTyrTyr PheLysProPro ValAsn
85 90 95
i()
Asn LeuIleThrGly PheValThrValGly AspAspLysTyr TyrPhe
100 105 110
Asn ProIleAsnGly GlyAlaAlaSerIle GlyGluThrIle IleAsp
?J 115 120 125
Asp LysAsn'ryrTyr PheAsnGlnSerGly ValLeuGlnThr GlyVal
130 135 140
Phe SerThrGluAsp GlyPheLysTyrPhe AlaProAlaAsn ThrLeu
145 150 155 160
Asp GluAsnLeuGlu GlyGluAlaIleAsp PheThrGIyLye LeuIle
165 170 175
Ile AspGluAsnIle TyrTyrPheAppAsp AsnTyrA~:gGly AlaVal
180 185 190
Glu TrpLysGluLeu AspGlyGluMetHis TyrPheSerPro GluThr
J() 195 200 205
Gly LysAlaPheLys GlyLeuAsnGlnIle GlyAspTyrLys TyrTyr
210 215 220
JJ Phe AsnSerAspGly ValMetGlnLysGly PheValSerIle AsnAsp
225 230 235 240
Asn LysHisTyrPhe AspAppSerGlyVal MetLysValGly TyrThr
245 250 255
6(1
Glu IleAspGlyLys HisPheTyrPheAla GluAsnGlyGlu MetGln
260 265 270
Ile GlyValPheAsn 1'hrGluAspGlyPhe I,ysTyrPheAla HisHis
275 280 285
Asn GluAspLeuGly AsnGluGluGlyGlu GluIleSerTyr SerGly
290 :.95 300
Ile LeuAsnPheAsn AsnLysIleTyrTyr PheAspAspSer PheThr
-?6U-
CA 02296765 2000-O1-14
WO 98108540 PCTNS97/15394
305 310 315 320
Ala Val Val Gly Trp Lys Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe
325 330 335
>_
Asp Glu Asp Thr Ala Glu Ala Tyr Ile Gly Leu Ser Leu Ile Asn Asp
340 345 350
Gly Gln Tyr Tyr Phe Asn Asp Asp Gly Ile Met Gln Val Gly Phe Val
1 U 355 360 365
Thr Ile Asn Asp Lys Val Phe Tyr Phe Ser Asp Ser Gly Ile IIe Glu
370 375 380
Ser GlyVal GlnRsnIleAspAspAsn TyrPheTyrIleAsp AspAsn
385 390 395 400
Gly IleVal GlnIleGlyValPheAsp ThrSerAspGlyTyr LysTyr
405 410 415
?U
Phe AlaPro AlaAsnThrValAsnAsp AsnIleTyrGlyGln AlaVal
420 425 430
Glu TyrSer GlyLeuValArgValGly GluAspValTyrTyr PheGly
435 440 445
Glu TnrTyr ThrIleGluThrGlyTrp IleTyrAspMetGlu AsnGiu
450 455 460
?U Ser AspLys TyrTyrPheAsnProGlu ThrLysLysAlaCys LysGly
465 470 475 480
Ile AsnLeu IleAspAspIleLysTyr TyrPheAspGluLys C~lyIle
485 490 495
is
Met ArgThr GlyLeuIleSerPheGlu AsnAsnAsnTyrTyr PheAsn
500 505 510
Glu AsnGly GluMetGlnPheGlyTyr IleAsnIleGluAsp LysMet
515 520 525
Phe TyrPhe GlyGluAspGlyValMet GlnIleGlyValPhe AsnThr
530 535 540
4> Pro AspGly PheLysTyrPheAlaHip GlnAsnThrLeuAsp GluAsn
545 550 555 560
Phe GluGly GluSerIleAsnTyrThr GlyTrpLeuAspLeu AspGlu
565 570 575
(1
Lys ArgTyr TyrPheThrAspGluTyr IleAlaAlaThrGly SerVal
580 585 590
Ile IleAsp GlyGluGluTyrTyrPhe AspProAspThrAla GlnLeu
J? 595 600 605
(21 INFORMATI ON
FOR
SEQ
ID
N0:22:
(i) SEQUENCE CHARACTERISTICS :
6U (A?LENGTH: 1330 airs
base
p
(B)TYP E:
nucleic
acid
(C)STRANDEDNESS: e
doubl
(D)TOPOLOGY:
linear
~J (ii) MOLECULE TYPE: (genomic)
DNA
(ix) FEATURE:
. (A)NAM E/KEY:
CDS
(H)LOCATION:
1..1314
7U
- 2G1 -
CA 02296765 2000-O1-14
PCTIUS97I15394
(ri) SEQUENCE DESCRIPTION: SEQ ID N0:22:
ATG GCT CGT CTG CTG TCT ACC TTC ACT GAA TAC ATC AAG AAC ATC ATC 48
Met Ala Arg Leu Leu Ser Thr Phe Thr Glu Tyr Ile Lys Asn Ile Ile
O 1 5 10 15
AAT ACCTCC ATCCTGAACCTGCGC TACGAA AAT CACCTGATCGAC 96
TCC
Asn ThrSer IleLeuAsnLeuArg TyrGluSerAsn HisLeuIleAsp
2p 25 30
CTG TCTCGC TACGCTTCCAAAATC AACATCGGTTCT AAAGTTAACTTC 144
Leu SerArg TyrAlaSerLysIle AsnIleGlySer LysValAsnPhe _
35 40 4S
1~ CAT CCGATC GACAAGAATCAGATC CAGCTGTTCAAT CTGGAATCTTCC 192
Asp ProIle AspLysAsnGlnIle GlnLeuPheAsn LeuGluSerSer
SO 55 60
AAA ATCGAA GTTATCCTGAAGAAT GCTATCGTATAC AACTCTATGTAC 240
~()Lys IleGlu ValIleLeuLysAsn AlaIleValTyr AsnSerMetTyr
65 70 75 80
GAA AACTTC TCCACCTCCTTCTGG ATCCGTATCCCG AAATACTTCAAC 288
Glu AsnPhe SerThrSerPheTrp IleArgIlePro LysTyrPheAsn
85 90 ' 95
'TCC r.TCTCT CTGAACAATGAATAC ACCATCATCAAC TGCATGGAAAAC 336
Ser IieSer LeuAsnAsnGluTyr ThrIleIleAsn CysMetGluAsn
100 105 110
i~)
AAT TCTGGT TGGAAAGTATCTCTG AACTACGGTGAA ATCATCTGGACT 384
Asn SerGly TrpLyeValSerLeu AsnTyrGlyGlu IleIleTrpT'hr
115 120 125
CTG CAGGAC ACTCAGGAAATCAAA CAGCGTGTTGTA TTCAAATACTCT 432
Leu GlnAsp ThrGlnGluIleLys GlnArgValVal PheLysTyrSer
130 135 140
CAG ATGATC AACATCTCTGACTAC ATCAATCGCTGG ATCTTCGTTACC 4B0
Gln MetIle AsnIleSerAspTyr IleAsnArgTrp IlePheValThr
145 150 155 160
ATC ACCAAC AATCGTCTGAATAAC TCCAAAATCTAC ATCAACGGCCGT 528
Ile ThrAsn AsnArgLeuAsnAsn SerLysIleTyr IleAsnGlyArg
165 170 175
CTG ATCGAC CAGAAACCGATCTCC AATCTGGGTAAC ATCCACGCTTCT 576
Leu IleAsp GlnLysProIleSer AsnLeuGlyAsn IleHisAlaSer
180 185 190
j(J
AAT AACATC ATGTTCAAACTGGAC GGTTGTCGTGAC ACTCACCGCTAC 624
Asn AsnIle MetPheLysLeuAsp GlyCysArgAsp ThrHisArgTyr
195 200 205
~J ATC TGGATC AAATACTTCAATCTG TTCGACAAAGAA CTGAACGAAAAA 672
Ile TrpIle LysTyrPheAsnLeu PheAspLysGlu LeuAsnGluLys
210 215 220
GAA ATCAAA GACCTGTACGACAAC CAGTCCAATTCT GGTATCCTGAAA 720
Glu IleLys AspLeuTyrAspAsn GlnSerAsnSer GlyIleLeuLys
?.25 ~ 230 235 240
GAC TTCTGG GGTGACTACCTGCAG TACGACAAACCG TACTACATGCTG 768
Asp PheTrp GlyAspTyrLeuGln TyrAspLysPro TyrTyrMetLeu
()~ 245 250 255
AAT CTGTAC GATCCGAACAAATAC GTTGACGTCAAC AATGTAGGTATC 816
Asn LeuTyr AspProAsnLysTyr ValAspValAsn AnnValGlyIle
260 265 270
*rB
CA 02296765 2000-O1-14
WO ~~~0 PC"fIUS97115394
CGC GGTTACATG TACCTGAAA GGT CGT GGTTCTGTTATG ACTACC 864
CCG
Arg GlyTyrMet TyrLeuLys Gly Arg GlySerValMet ThrThr
Pro
275 280 285
AAC ATCTACCTG AACTCTTCC CTG CGT GGTACCAAATTC ATCATC 912
TAC
Asn ZleTyrLeu AsnSerSer Leu Arg GlyThrLysPhe IleIle
Tyr
290 295 300
AAG AAATACGCG TCTGGTAAC AAG AAT ATCGTTCGCAAC AATGAT 960
GAC
](1 Lys LyeTyrAla SerGlyAsn Lys Asn IleValArgAsn AsnAsp
Asp
305 310 315 320
CGT GTATACATC AATGTTGTA GTT AAC AAAGAATACCGT CTGGCT 1008
AAG
Arg ValTyrIle AsnValVal Val Asn LysGluTyrArg LeuAla
Lys
325 330 335
ACC AATGCTTCT CAGGCTGGT GTA AAG ATCTTGTCTGCT CTGGAA 1056
GAA
Thr AsnAlaSer GlnAlaGly Val Lys IleLeuSerAla LeuGlu
Glu
340 345 350
ATC CCGGACGTT GGTAATCTG TCT GTA GTTGTAATGAAA TCCAAG 1104
CAG
Ile ProAspVal GlyAsnLeu Ser Val ValValMetLys SerLys
Gln
355 360 365
AAC GACCAGGGT ATCACTAAC AAA AAA ATGAATCTGCAG GACAAC 115?
TGC
Asn AspGlnGly IleThrAsn Lys Lys MetAsnLeuGln AppAsn
Cys
370 375 380
AAT GGTAACGAT ATCGGTTTC ATC TTC CACCAGTTCAAC AATAT'C 1200
GGT
o() Asn GlyAsnAsp IleGlyPhe Ile Phe HisGlnPheAsn AsnIie
Gly
385 390 395 400
GCT AAACTGGTT GCTTCCAAC TGG AAT CGTCAGATCGi,ACGTTCC 1248
TAC
Al.a LyeLeuVal AlaSerAsn Trp Asn ArgGlnIleGlu ArgSer
Tyr
i~ 405 410 415
TCT CGCACTCTG GGTTGCTCT TGG TTC ATCCCGGTTGAT GACGGT 1296
GAG
Ser ArgThrLeu GlyCysSer Trp Phe IleProValAsp AspGly
Glu
420 425 930
TGG GGTGAACGT CCGCTGTAACCCGGGAAAGCTT 1330
Trp GlyGluArg ProLeu
435
(2) INFORMATION FORSEOID N0:23:
(i) CHARACTERISTICS :
SEQUENCE
(A ) : 438 acids
LENGTH amino
(B ) amino
TYPE: acid
(D )
TOPOLOGY:
linear
( ii) TYPE: protein
MOLECULE
( xi)SEQUENCEDESCRIPTION: N0:23:
SEQ ID
ij
Met AlaArgLeu LeuSerThr Phe TyrIleLysAsn IieIle
Thr Glu
1 5 10 15
Asn ThrSerIle LeuAsnLeu Arg SerAsnHisLeu IleAsp
Tyr Glu
(7(1 20 25 30
Leu SerArgTyr AlaSerLys Ile GlySerLysVal AsnPhe
Asn Ile
35 40 45
( Asp ProIleAsp LysAsn PheAsn Glu Ser
Gln Leu Ser
Ile
Gln
Leu
50 55 60
Lys Ile Glu Val Ile Leu Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr
65 70 75 80
- 2G3 -
CA 02296765 2000-O1-14
~rp yglpgsqp PCT/US97115394
Glu Asn Phe Ser Thr Ser Phe Trp Ile Arg Ile Pro Lys Tyr Phe Asn
85 90 95
Ser IleSerLeuAsn AsnGluTyrThrIle IleAsnCysMetGlu Asn
100 105 110
Asn SerGlyTrpLys ValSerLeuAsnTyr GlyGluIleIleTrp Thr
115 120 125
Leu GlnAspThrGln GluIleLysGlnArg ValValPheLysTyr Ser
130 135 140
Gln MetIleAsnIle SerAspTyrIleAsn ArgTrpIlePheVal Thr
145 150 155 160
~i
Ile ThrAsnAsnArg LeuAsnAsnSerLys IleTyrIleAsnGly Arg
165 170 175
Leu IleAspGlnLys ProIleSerAsnLeu GlyAsnIleHisAla Ser
180 185 190
Asn AsnIleMetPhe LysLeuAspGlyCys ArgAspThrHisArg Tyr
195 200 ?.U5
Ile Trp Ile Lys Tyr Phe Asn Leu Phe Asp Lys Glu Leu Asn Glu Lys
alU 215 220
Glu Ile Lys Asp Leu Tyr Asp Asn Gln Ser Asn Ser Gly Ile Leu Lys
~25 230 235 40
i0 '
Asp Phe Trp Gly Asp Tyr Leu Gln 'Tyr Asp Lys Pro Tyr Tyr Met Leu
245 250 255
Asn Leu Tyr Asp Pro Asn Lys Tyr Val Asp Val Asn Asn Val Gly Ile
260 265 270
Arg Gly Tyr Met Tyr Leu Lys Gly Pro Arg Gly Ser Val Met Thr Thr
'75 280 285
Asn Ile 1'~~r Leu Asn Ser Ser Leu Tyr Arg Gly Thr Lys Phe Ile Ile
290 295 300
Lys Lys Tyr Ala Ser Gly Asn Lys Asp Asn Ile Val Arg Asn Asn Asp
.105 310 315 320
.~ S
rlrg Val Tyr Iie Asn Val Val Val Lys Asn Lys Glu Tyr Arg Leu Ala
325 330 335
Thr Asn Ala Ser Gln Ala Gly Val Glu Lys Ile Leu Ser Ala Leu Glu
340 345 350
Ile Pro Asp Val Gly Asn Leu Ser Gln Val Val Val Met Lys Ser Lys
355 360 365
Asn Asp Gln Gly Ile Thr Asn Lys Cys Lys Met Asn Leu Gln Asp Asn
370 375 380
Asn Gly Asn Asp Ile G1y Phe Ile Gly Phe His Gln Phe Asn Asn Ile
3B5 390 395 4U0
(O)
A1a Lys L~u Val Ala Ser Asn Trp Tyr Asn Arg Gln Ile Glu Arg Ser
405 410 415
Ser Arg Thr Leu Gly Cys Ser Tro Glu Phe ile Pro Val Asp Asp Gly
(» 420 425 430
Trp Gly Glu Arg Pro Leu
435
I2) INFORMATION FOR SEQ ID N0:24:
_ 7(~4 -
CA 02296765 2000-O1-14
WO gg/~qp PCTIUS9711S394
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
lxi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
Met Gly His His His His His His His His His Fiis Ser Ser Gly His
1 5 10 15
Ile Glu Gly Arg His Met Ala
(2) INFORMATION FOR SEQ ID N0:25:
(i1 SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1402 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA Igenomic)
( i r, l FEATURE
(A) NAME/KEY: CDS
(B) LOCATION: 1..1386
i~)
lxi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
ATG GGC CAT CAT CAT CAT CAT CAT CAT CAT CAT CAC AGC AGC GGC CAT 48
Met G1_r~ His His His His His His His His His Hip Ser Ser Gly His
ij 1 5 10 15
ATC GAA GGT CGT CAT ATG GCT AGC ATG GCT CGT CTG CTG TCT ACC TTC 96
Ile Glu Gly Arg His Met Ala Ser Met Ala Arg Leu Leu Ser Thr Phe
20 25 30
ACT GAA TAC ATC AAG AAC ATC ATC AAT ACC TCC ATC CTG AAC CTG CGC 144
Thr Glu Tyr Ile Lys Asn Ile Ile Asn Thr Ser Ile Leu Asn Leu Arg
35 40 45
TAC GAA TCC AAT CAC CTG ATC GAC CTG TCT CGC TAC GCT TCC AAA ATC 192
Tyr Glu Ser Asn His Leu Ile Asp Leu Ser Arg Tyr Ala Ser Lys Ile
50 55 60
AAC ATC GGT TCT AAA GTT AAC TTC GAT CCG ATC GAC AAG AAT CAG ATC 240
Asn Ile Gly Ser Lys Val Asn Phe Asp Pro Ile Asp Lys Asn Gln Ile
65 70 75 80
CAG CTG TTC AAT CTG GAA TCT TCC AAA ATC GAA GTT ATC CTG AAG AAT 288
Gln Leu Phe Asn Leu Glu Ser Ser Lys Ile Glu Val Ile Leu Lys Asn
85 90 95
GCT ATC GTA TAC AAC TCT ATG TAC GAA AAC TTC TCC ACC TCC TTC TGG 336
Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser Phe Trp
100 105 110
ATC CGT ATC CCG AAA TAC TTC AAC TCC ATC TCT CTG AAC AAT GAA TAC 384
Ile Arg Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu Asn Asn Glu T~_.~r
115 120 125
OJ ACC ATC ATC AAC TGC ATG GAA AAC AAT TCT GGT TGG AAA GTA TCT CTG 432
Thr Ile Ile Asn Cys Met Glu Asn Asn Ser Gly Trp Lys Val Ser Leu
130 135 140
AAC TAC GGT GAA ATC ATC TGG ACT CTG CAG GAC ACT CAG GAA ATC AAA 480
~() Asn Tyr Gly Glu Ile Ile 'frp Thr Leu Gln Asp Thr Gln Glu Ile Lys
-2G5-
CA 02296765 2000-O1-14
PCTNS97I15394
145 150 155 160
CAG CGT GTT GTA TTC AAA TAC TCT CAG ATG ATC AAC ATC TCT GAC TAC 528
Gln Arg Val Val Phe Lys Tyr Ser Gln Met Ile Asn Ile Ser Asp T'yr
J 165 170 175
ATC AAT CGC TGG ATC TTC GTT ACC ATC ACC AAC AAT CGT CTG AAT AAC 576
Ile Asn Arg Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu Asn Asn
180 185 190
TCC AAA ATC TAC ATC AAC GGC CGT CTG ATC GAC CAG RAA CCG ATC TCC 624
Ser Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gln Lys Pro IZe Ser
195 200 205
AAT CTGGGTAAC ATCCACGCTTCT AATAAC TTC AAACTGGAC 672
ATC
ATG
Asn LeuGlyAsn IleHisAlaSer AsnAsnIleMetPhe LysLeuAsp
.10 215 220
GGT TGTCGTGAC ACTCACCGCTAC ATCTGGATCAAATAC TTCAATCTG 720
Gly CysArgAsp ThrHisArgTyr IleTrpIleLysTyr PheAsnLeu
.''.25 230 235 240
TTC GACAAAGAA CTGAACGAAAAA GAAATCAAAGACCTG TACGACAAC 768
Phe AspLysGlu LeuAsnGluLys GluIleLysAspLeu TyrAspAsn
245 250 255
CAG TCCAATTCT GGTATCCTGAAA GACTTCTGGGGTGAC TACCTGCAG 816
Gln SerAsnSer GlyIleLeuLys AspPheTrpGlyAsp 1'yrLeuGln
260 265 270
i()
TAC GACAAACCG TACTACATGCTG AATCTGTACGATCCG AACAAATAC 864
'I'yrAspLysPro TyrTyrMetLeu AsnLeuTyrAspPro AsnLyeTyr
275 280 285
GTT CACGTCAAC AATGTAGGTATC CGCGGTTACATGTAC CTGAAAGGT 9i2
Val AspValAsn AsnValGlyIle ArgGlyTyrMetTyr LeuLyeGly
290 295 300
CCG CGTGGTTCT GTTATGACTACC AACATCTACCTGAAC TCTTCCCTG 960
4t) Pro ArgGlySer ValMetThrThr AsnIleTyrLeuAsn SerSerLeu
305 310 315 320
TAC CGTGGTACC AAATTCATCATC AAGAAATACGCGTCT GGTAACAAG 1008
Tyr ArgGlyThr LysPheIleIle LysLysTyrAlaSer GlyAsnLys
325 330 335
GAC AATATCGTT CGCAACAATGAT CGTGTATACATCAAT GTTGTAGTT 1056
Asp AsnIleVal ArgAsnAsnAsp ArgValTyrIleAnn ValValVal
390 345 350
JU
AAG AACAAAGAA TACCGTCTGGCT ACCAATGCTTCTCAG GCTGGTGTA 1104
Lys AsnLysGlu TyrArgLeuAla ThrAsnAlaSerGln AlaGlyVal
35 5 360 365
JJ GAA AAGATCTTG TCTGCTCTGGAA ATCCCGGACGTTGGT AATCTGTCT 1152
Glu LysIleLeu SerAlaLeuGlu IleProAspValGly AsnLeuSe.
370 375 380
CAG GTAGTTGTA ATGAAATCCAAG AACGACCAGGGTATC ACTAACAAA 1200
6l) Gln ValValVal MetLysSerLys AsnAppGlnGlyIle ThrAsnLye
385 ' 390 395 400
TGC AAAATGAAT CTGCAGGACAAC AATGGTAACGATATC GGTTTCATC 1248 -
Cys LyeMetAsn LeuGlnAspAsn AsnGlyAsnAspIle GlyPheIle
()~ 905 410 415
- 2GG -
*rB
CA 02296765 2000-O1-14
PCTIUS97/15394
GGT TTCCACCAG TTC ATCGCT GTTGCT TCC TGG 1296
AAC AAA AAC
AAT CTG
Gly PheHisGln PheAsnAsn IleAlaLysLeuValAla SerAsnTrp
420 425 430
TAC AATCGTCAG ATCGAACGT TCCTCTCGCACTCTGGGT TGCTCTTGG 1344
Tyr AsnArgGln IleGluArg SerSerArgThrLeuGly CysSerTrp
435 440 945
- GAG TTCATC~CCGGTTGATGAC GGTTGGGGTGAACGTCCG CTG 1386
Glu PheIlePro ValAspAsp GlyTrpGlyGluArgPro Leu
450 455 460
- TAACCCGGGA 1402
AAGCTT
IJ (2) INFORMATION FORSEQID
N0:26:
(i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 462 amino
acids
(B) TYPE:
amino
acid
(D) TOPOLOGY:
linear
( ii)MOLECULE TYPE:
protein
( r.i)SEQUENCE DESCRIPTION: SEQ N0:26:
ID
_~s
Met GlyHisHis HisHisHis HisHisHisHisHisSer SerGlyHis
1 5 10 15
Ile GluGlyArg HipMetAla SerMetAlaArgLeuLeu SerThrPhe
i() 20 25 30
Thr GluTyrIle LysAsnIle IleAsnThrSerIleLeu AsnLeuArg
35 40 45
ij Tyr GluSerAsn HisLeuIle AspLeuSerArgTyrAla ScrLysIie
50 55 60
Asn IleGlySer LysValAsn PheAspProIleAspLys AsnG1nIle
65 70 75 80
Gln LeuPheAsn LeuGluSer SerLysIleGluValIle LeuLysAsn
85 90 95
Ala IleValTyr AsnSerMet TyrGluAsnPheSerThr SertheTrp
W 100 105 110
Ile ArgIlePro LysTyrPhe AsnSerIleSerLeuAsn AsnGluTyr
115 120 125
JU Thr IleIleAsn CysMetGlu AsnAsnSerGlyTrpLys ValperLeu
130 135 140
Asn TyrGlyGlu IleIleTrp ThrLeuGlnAspThrGln GluIleLys
145 150 155 160
Gln ArgValVal PheLysTyr SerGlnMetIleAsnIle SerAspTyr
165 170 175
Ile AsnArgTrp liePheVal ThrIleThrAsnAsnArg LeuAsnAsn
()( ) 180 185 19U
7_
CA 02296765 2000-O1-14
WO 98/08540 PCT/US97/15394
Ser Lys Ile Tyr Ile Asn Gly Arg Leu ile Asp Gln Lys Pro Ile Ser
195 200 205
Asn Leu GlyAsnIleHis AlaSerAsnAsnIle MetPheLysLeuAsp
210 215 220
Cly Cys ArgAspThrHis ArgTyrIleTrpIle LysTyrPheAsnLeu
225 230 235 240
I() Phe Asp LysGluLeuAsn GluLysGluIleLys AspLeuTyrAspAsn
245 250 255
Gln Ser AsnSerGlyIle LeuLysAspPheTrp GlyAspTyrLeuGln
260 265 270
T;~rAsp LysProTyrTyr MetLeuAsnLeuTyr AspProAsnLysTyr
275 280 285
~.'alAsp ValAsnAsnVal GlyIleArgGlyTyr MetTyrLeuLysGly
29U 295 300
Pro Arg GlySerValMet ThrThrAsnIleTyr LeuAsnSerSerLeu
305 310 315 320
~> 'T.~rllrgGlyT'hrLysPhe IleIleLysLysTyr AlaSerC;lyAsnLye
325 330 335
Asp Ann IleValArgAsn AsnAspArgValTyr IleAsnValValVal
340 345 350
?()
Lys Asn LysGluTyrArg LeuAlaThrAsnAla SerGlnAlaGlyVal
355 360 365
Glu Lys IleLeuSerAla LeuGluIleProAsp ValGlyAsnLeuSer
370 375 380
Gln Val ValVa.lMetLys SerLysAsnAspGln GlyIleThrAsnLys
385 390 395 400
Lys MetAsnLeuGln AspAsnAsnGlyAsn AspIleGlyPheIle
405 410 415
GlyrPhe HisGlnPheAsn AsnIleAlaLysLeu ValAlaSerAsnTrp
420 425 430
-)
;
T_.rAsn ArgGlnIleGlu ArgSerSerArgThr LeuGlyCy:SerTrp
435 440 495
Glu Phe ileProValAsp AspGlyTrpGlyGlu ArgProLeu
?() 450 455 460
(~) INFORMATION FORSEQ ID N0:27:
(i ) ISTICS:
SEQUENCE
CHARACTER
~J (A) basepairs
LENGTFF:
3891
(F3) nuc leicacid
TYPE:
(C) double
STRANDEDNESS:
( D) linear
TOPOLOGY:
(t0 (ii ) LECULE YPE:DNA(genomic)
MO T
- 268 -
CA 02296765 2000-O1-14
PCTIUS97115394
(ix) FEATURE:
(A) NAME/KEY: CDS
(8) LOCATION: 1..3888
(xi)SEQUENCE ID
DESCRIPTION: N0:27:
SEQ
ATG CAATTTGTTAAT AAACAATTTAATTAT AAAGATCCTGTAAAT GGT 48
Met GlnPheValAsn LysGlnPheAsnTyr LyslispProValAsn Gly
. 1 5 10 15
IU
GTT GATATTGCTTAT ATAAAAATTCCAAAT GTAGGACAAATGCAA CCA 96
Val AspIleAlaTyr IleLysIleProAsn ValGlyGlnMetG3.nPro
2p 25 30
GTA A.r,AGCTTTTAAA ATTCATAATAAAATA TGGGTTATTCCAGAA AGA 144
Val LysAlaPheLys IleHisAsnLysIle TrpValIleProGlu Arg
35 90 45
GAT ACATTTACAAAT CCTGAAGAAGGAGAT TTAAATCCACCACCA GAA 192
Asp ThrPheThrAsn ProGluGluGlyAsp LeuAsnProProPro Glu
50 55 60
GCA AAACAAGTTCCA GTTTCATATTATGAT TCAACATATTTAAGT ACA 240
Ala LysGlnValPro ValSerTyrTyrAsp SerThrTyrLeuSer Thr
~J 65 70 75 80
GAT T,ATGAAAAAGAT AATTATTTAAAGGGA GTTACAAAATTATTT GAG 288
11~p AsnGluLysAsp AsnTyrLeuLysGly ValThrLysLeuPhe Glu
85 90 95
i~)
AGA F~TTTATTCAACT GATCTTGGAAGAATG TTGTTAACATCAATA GTA 336
Arg IIeTyrSerThr AspLeuGlyArgMet LeuLeuThrSerIle Val
100 105 110
AGG GGAATACCATTT TGGGGTGGAAGTACA ATAGATACAGAATTA AAA 384
Arg GlyIleProPhe 'frpGlyGlySerThr IleAspThrGluLeu Lys
115 120 125
CTT ATTGATACTAAT TGTATTAATGTGATA CAACCAGATGGTAGT TAT 432
~l(1 Val IleAspThrAsn CysIleAsnValIle GinProAspGlySer Tyr
130 135 140
AGA TCAGAAGAACTT AATCTAGTAATAATA GGACCCTCAGCTGAT ATT 480
Arg SerGluGluLeu AsnLeuValIleIle GlyProSerAlaAsp Ile
~I~ 145 150 155 160
ATl1 CAGTTTGAATCT AAAAGCTTTGGACAT GAAGTTTTGAATCTT ACG 528
Ile G~nPheGluCys LysSerPheGlyHis GluValLeuAsnLeu Thr
165 170 175
CGA AATGGTTATGGC TCTACTCAATACATT AGATTTAGCCCAGAT TTT 576
Arg AsnGlyTyrGly SerThrGlnTyrIle ArgPheSerPr'oAsp Phe
180 185 190
~J ACA TTTGGTTTTGAG GAGTCACTTGAAGTT GATACAAATCCTCTT TTA 624
Thr PheGlyPheGlu GluSerLeuGluVal AspThrAsnProLeu Leu
195 200 205
GGT G.CAGGCAAATTT GCTACAGATCCAGCA GTAACATTAGCACAT GAA 672
O) Gly AlaGlyLysPhe AlaThrAspProAla ValThrLeuAlaHis Glu
21 0 215 220
CTT ATACATGCTGGA CATAGATTATATGGA ATAGCAATTAATCCA AAT 720
- Leu IIeHisAlaGly HisArgLeuTyrGly IleAlaIleAsnPro Asn
225 230 235 240
AGG GTTTTTAAAGTA AATACTAATGCCTAT TATGAAATGAGTGGG TTA 768
Arg ValPheLysVal AsnThrAsnAlaTyr TyrGluMetSerGly Leu
245 250 255
7U
CA 02296765 2000-O1-14
PCTNS97I15394
GAA TTTGAG GAA CAT GAT 816
GTA CTT GCA AAG
AGC AGA
ACA
TTT
GGG
GGA
Glu ValSerPheGlu GluLeu Thr Phe His Asp Lys
Arg Gly Ala
Gly
260 265 270
TTT ATAGATAGTTTA CAGGAAAACGAA TTTCGT TAT TAT AAT 864
CTA TAT
Phe IleAspSerLeu GlnGluAsnGlu PheArg Tyr 'I'yr Asn
Leu Tyr
275 280 285
AAG TTTAAAGATATA GCAAGTACACTT AATAAA AAA TCA GTA 912
GCT ATA
j~) Lys PheLysAspIle AlaSerThrLeu AsnLys Lys Ser Val
Ala Ile
290 295 300
GGT ACTACTGCTTCA TTACAGTATATG AAAAAT TTT AAA AAA 960
GTT GAG
Gly ThrThrAlaSer LeuGlnTyrMet LysAsn Phe Lys Lys
Val Glu
305 310 315 320
TAT CTCCTATCTGAA GATACATCTGGA AAATTT GTA GAT TTA 1008
TCG AAA
Tvr LeuLeuSerGlu AspThrSerGly LysPhe Val Asp Leu
Ser Lys
325 330 335
AAA TTTGATAAGTTA TACAAAATGTTA ACAGAG TAC ACA GAT 1056
ATT GAG
Lys PheAspLysLeu TyrLysMetLeu ThrGlu Tyr Thr Asp
Ile Glu
340 345 350
AAT TTTGTTAAGTTT TTTAAAGTACTT AACAGA ACA TAT AAT 1104
AAA TTG
I;sn PheValLysPhe PheLysValLeu AsnArg Thr Tyr Asn
Lye Leu
355 360 365
TTT GATAAAGCCGTA TTTAAGATAAAT ATAGTA AAG GTA TAC 1152
CCT AAT
i() Phe lispLysAlaVal PheLysI1eAsn IleVal Lys Val Tyr
Pro Asn
370 375 380
ACA ATATATGATGGA TTTAATTTAAGA AATACA TTA GCA AAC 1200
AAT GCA
Thr !leTyrAspGly PheAsnLeuArg AsnThr Leu Ala Asn
Asn Ala
W 385 390 395 400
TTT AATGGTCAAAAT ACAGAAATTAAT AATATG TT"T ACT CTA 1248
AAT AAA
Phe AsnGlyGlnAsn ThrGluIleAsn AsnMet Phe Thr Leu
Asn Lye
405 410 415
a
AAA AATTTTACTGGA TTGTTTGAATTT TATAAG CTA TGT AGA 1296
TTG GTA
hys AsnPhe1'hrGly LeuPheG1uPhe TyrLys Leu Cys Arg
Leu Val
420 425 430
-)~ GGG ATAATAACTTCT AAAACTAAATCA TTAGAT GGA ThC AAG 1344
AAA AAT
fly IleIleThrSer LysThrLysSer LeuAsp Gly Tyr Lye
Lys Asn
435 440 445
GCA TTAAATGATTTA TGTATCAAAGTT AATAAT GAC TTG TTT 1392
TGG TTT
J() Ala LeuAsnAspLeu Cy~IleLysVal AsnAsn Asp Leu Phe
Trp Fhe
450 455 460
AGT CCTTCAGAAGAT AATTTTACTAAT GATCTA AAA GGA GAA 1440
AAT GAA
Ser ProSerGluAsp AsnPheThrAsn AspLeu Lys Gly Glu
Ann Glu
>> 465 470 475 480
ATT ACA GATACT AATATAGAA GCAGAA TTA 1488
TCT GCA GAA
AAT
ATT
AGT
Ile ThrSerAspThr AsnIleGluAla AlaGlu Asn Ile Leu
Glu Ser
985 490 495
6()
GAT TTA CAACAA TAT TTA TTT AATTTTGATAATGAA CC.T 1536
ATA TAT ACC
Asp LeuIleGlnGln Tyr LeuThrPileAsnPlneAspAsnGlu Pro
Tyr
500 505 510
()J GAA AATATTTCAATA GAA CTTTCAAGT GACATTATAGGCCAA TTA 1584
AAT
Glu AsnIleSerIle Glu LeuSerSer AspIleIleGlyGln Leu
Asn
515 520 525
GAA CTTATGCCTAAT ATA AGATTTCCT AATGGAAAAAAGTAT GAG 1632
GAA
Glu LeuMetProAsn Ile ArgPhePro AsnGlyLysLysTyr Glu
Glu
CA 02296765 2000-O1-14
WO 98/08540 PCTIUS97/15394
530 535 540
TTA GAT AAA TAT ACT ATG TTC CAT TAT CTT CGT GCT CAA GAA TTT GAA 1680
Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu
J 545 550 555 560
CAT GGT AAA TCT AGG ATT GCT TTA ACA AAT TCT GTT AAC GAA GCA TTA 1728
His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu
565 570 575
TTA AAT CCT AGT CGT GTT TAT ACA TTT TTT T.CT TCA GAC TAT GTA AAG 1776
Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys
- 580 585 590
IJ AAA GTT AAT AAA GCT ACG GAG GCA GCT ATG TTT TTA GGC TGG GTA GAA 1824
Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu
595 600 605
CAA TTA GTA TAT GAT TTT ACC GAT GAA ACT AGC GAA GTA AGT ACT ACG 1872
Gln Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr
610 615 620
GAT AAA ATT GCG GAT ATA ACT ATA ATT ATT CCA TAT ATA GGA CCT GCT 1920
Asp Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala
625 630 635 64U
TTA AAT ATA GGT AAT ATG TTA TAT ARA GAT GAT TTT GTA GGT GCT TTA 1968
Leu Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu
645 650 655
i(1
ATA TTT TCA GGA GCT GTT ATT CTG TTA GAA TTT ATA CCA GAG ATT GCA 2016
lle Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala
660 665 670
.iJ ATA CCT TTAGGT GCACTT TCA GCGAATAAG 2064
GTA ACT GTA TAT
TTT ATT
Ile Pro ValLeuGlyThrPhe AlaLeu SerT'yrIleAlaAsnLys
Val
675 680 685
GTT CTA ACCGTTCAAACAATA GATAAT T'TAAGT AAAAGAAATGAA 2112
GCT
Val Leu ThrValGlnThrIle AspAsn LeuSer LysArgAsnGlu
Ala
690 695 700
AAA TGG GATGAGGTCTATAAA TATATA ACAAAT TGGTTAGCAAAG 2160
GTA
Lys Trp AppGluValTyrLys TyrIle ThrAsn TrpLeuAlaLys
Val
705 710 715 '20
GTT AAT ACACAGATTGATCTA ATAAGA AAAATG AAAGAAGCTTTA 2208
AAA
Val Asn ThrGlnIleAspLeu IleArg LysMet LysGluAlaLeu
Lys
725 730 735
GAA AAT CAAGCAGAAGCAACA AAGGCT ATAAAC TATCAGTATAAT 2256
ATA
Glu Asn GlnAlaGluAlaThr LysAla IleAsn TyrGlnTyrAsn
Ile
740 795 750
CAA TAT ACTGAGGAAGAGAAA AATAAT AATTTT AATATTGATGAT 2304
ATT
Gln Tyr ThrGluGluGluLys AsnAsn AsnPhe AsnIleAspAsp
Ile
755 760 765
TTA AGT TCGAAACTTAATGAG TCTATA AAAGCT ATGATTAATATA 2352
AAT
Leu Ser SerLysLeuAsnGlu SerIle LysAla MetIleAsnIle
Asn
77 0 775 780
AAT AAA TTTTTGAATCAATGC TCTGTT TATTTA ATGAATTCTATG 2400
TCA
Asn Lys PheLeuAsnGlnCys SerVal TyrLeu MetAsnSerMet
Ser
G1 785 790 795 800
ATC CCT TATGGTGTTARACGG TTAGAA TTTGAT GCTAGTCTTAAA 2448
GAT
- Ile Pro TyrGlyValLysArg LeuGlu PheAsp AlaSerLeuLys
Asp
805 810 815
70
- 27I -
*rB
CA 02296765 2000-O1-14
WO 98105540 PCTIUS97I15394
GAT GCA TTA TTA AAG TAT ATA TAT GAT AAT AGA GGA ACT TTA ATT GGT 2496
Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly
820 825 830
CAA GTA GAT AGA TTA AAA GAT AAA GTT AAT AAT ACA CTT AGT ACA GAT 2544
Gln Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp
835 840 845
ATA CCT TTT CAG CTT TCC AAA TAC GTA GAT AAT CAA AGA TTA TTA TCT 2592
lU Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser
850 855 B60
ACA TTT ACT GAA TAT ATT AAG AAT ATT ATT AAT ACT TCT ATA TTG AAT 2640
Thr Phe Thr Glu Tyr Ile Lys Asn Ile Ile Asn Thr Ser Ile Leu Asn
~J 865 870 875 880
TTA AGA TAT GAA AGT AAT CAT TTA ATA GAC TTA TCT AGG TAT GCA TCA 2688
Leu Arg Tyr Glu 5er Asn His Leu Ile Asp Leu Ser Arg Tyr Ala Ser
885 890 895
AAA ATA AAT ATT GGT AGT AAA GTA AAT TTT GAT CCA ATA GAT AAA AAT 2736
Lys Ile Asn Ile Gly Ser Lys Val Asn Phe Asp Pro Ile Asp Lys Asn
900 905 910
CAA ATT CAA TTA TTT AAT TTA GAA AGT AGT AAA ATT GAG GTA ATT TTA 2784
Gln Ile Gln Leu Phe Asn Leu Glu Ser Ser Lys I1e Glu Val Ile Leu
915 920 925
AAA AAT GCT ATT GTA TAT AAT AGT ATG TAT GAA AAT TTT AGT ACT AGC 2832
i(l Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser
930 935 940
TTT TGG ATA AGA ATT CCT AAG TAT TTT AAC AGT ATA AGT CTA AAT AAT 2880
Phe Trp Ile Arg Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu Asn Asn
945 950 955 960
GAA TAT ACA ATA ATA AAT TGT ATG GAA AAT AAT TCA GGA TGG AAA GTA 2928
Glu Tyr Thr Ile Ile Asn Cys Met Glu Asn Asn Ser Gly Trp Lys Val
965 970 975
TCA CTT AAT TAT GGT GAA ATA ATC TGG ACT TTA CAG GAT ACT CAG GAA 2976
Ser Leu Asn Tyr Gly Glu Ile Ile Trp Thr Leu Gln Asp Thr Gln Glu
980 985 990
ATA AAA CAA AGA GTA GTT TTT AAA TAC AGT CAA ATG ATT AAT ATA TCA 3024
Ile Lys Gln Arg Val Val Phe Lys Tyr Ser Gln Met Ile Asn Ile Ser
995 1000 1005
GAT TAT ATA AAC AGA TGG ATT TTT GTA ACT ATC ACT AAT AAT AGA TTA 3072
>U Asp Tyr Ile Asn Arg 'rrp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu
1010 1015 1020
AAT AAC TCT AAA ATT TAT ATA AAT GGA AGA TTA ATA GAT CAA AAA CCA 3120
Asn Asn Ser Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gln Lys Pro
JJ 1025 1030 1035 1040
hU
ATT TCA AAT TTA GGT AAT ATT CAT GCT AGT AAT AAT ATA ATG TTT AAA 3168
Ile Ser Asn Leu Gly Asn Ile His Ala Ser Asn Asn Ile Met Phe Lys
1045 1050 1055
TTA GAT GGT TGT AGA GAT ACA CAT AGA TAT ATT TGG ATA AAA TAT TTT 3216
Leu Asp Gly Cys Arg Asp Thr His Arg Tyr Ile Trp Ile Lys Tyr Phe
1060 1065 1070
AAT CTT TTT GAT AAG GAA TTA AAT GAA AAA GAA ATC AAA GAT TTA TAT 3264
Asn Leu Phe Asp Lys Glu Leu Asn Glu Lys Glu Ile Lys Asp Leu Tyr
1075 1080 1085
GAT AAT CAA TCA AAT TCA GGT ATT TTA AAA GAC TTT TGG GGT GAT TAT 331
70 Asp Asn Gln Ser Asn Ser Gly Ile Leu Lys Asp Phe Trp Gly Asp Tyr
CA 02296765 2000-O1-14
WO 98108540 PCT/US97115394
1090 1095 1100
TTA CAATATGATAAA TACTAT TTA AAT TTA GAT CCAAAT 3360
CCA ATG TAT
Leu GlnTyrAspLys TyrTyr Leu Asn Leu Asp ProAsn
Pro Met Tyr
1105 1110 1115 1120
AAA TATGTCGATGTA AATGTA ATT AGA GGT ATG TATCTT 3408
AAT GGT TAT
Lys TyrValAspVal AsnVal Ile Arg Gly Met TyrLeu
Asn Gly Tyr
1125 1130 1135
~ ()
AAA GGGCCTAGAGGT GTAATG ACA AAC ATT TTA AATTCA 3456
AGC ACT TAT
Lys GlyProArgGly ValMet Thr Asn Ile Leu AsnSer
Ser Thr Tyr
1140 1145 1150
AGT TTGTATAGGGGG AAATTT ATA AAA AAA GCT TCTGGA 3504
ACA ATT TAT
Ser LeuTyrArgGly LysPhe Ile Lys Lys Ala SerGly
Thr Ile Tyr
1155 1160 1165
AAT AAA GAT AAT ATT GTT AGA AAT AAT GAT CGT GTA TAT ATT AAT GTA 3552
Asn Lys Asp Asn Ile Val Arg Asn Asn Asp Arg Val Tyr IIe Asn Val
1170 1175 1180
GTA GTT AAA AAT AAA GAA TAT AGG TTA GCT ACT AAT GCA TCA CAG GCA 3600
Val Val Lys Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gln Ala
~J 1185 1190 1195 1200
GGC GTA GAA AAA ATA CTA AGT GCA TTA GAA ATA CCT GAT GTA GGA AAT 3648
Gly Val Glu Lys Ile Leu Ser Ala Leu Glu Ile Pro Asp Val Gly Asn
1205 1210 1215
>l)
CTA AGTCAA GTAGTA GTAATGAAG TCAAAAAATGATCAAGGA ATAACA 3696
Leu SerGln ValVal ValMetLys SerLysAsnAspGlnGly IleThr
1220 1225 1230
>> AAT AAATGC AAAATG AATTTACAA GATAATAATGGGAATGAT ATAGGC 3744
Asn LysCys LysMet AnnLeuGln AspAsnAsnGlyAsnAsp IleGly
1235 1240 1245
TTT ATAGGA TTTCAT CAGTTTAAT AATATAGCTAAACTAGTA GCAAGT 3792
Phe IleGly PheHis GlnPheAsn AsnIleAlaLysLeuVal AlaSer
1250 1255 1260
AAT TGGTAT AATAGA CAAATAGAA AGATCTAGTAGGACTTTG GGTTGC 3840
Asn TrpTyr AsnArg GlnIleGlu ArgSerSerArgThrLeu GlyCys
~J 1265 1270 1275 1280
TCA TGGGAA TTTATT CCTGTAGAT GATGGATGGGGAGAAAGG CCTVCTG 3888
Ser TrpGlu PheIle ProValAsp AspGlyTrpGlyGluArg ProLeu
1285 1290 1295
TAA 3891
(2) INFORMATION SEQ ID N0:28:
FOR
~J (i) SEQUENCECHARACTERISTICS:
(A) acids
LENGTH:
1296
amino
(B)
TYPE:
amino
acid
(D)
TOPOLOGY:
linear
O) (ii) MOLECULETYPE: protein
(xi) SEQUENCEDESCRIPTION: ID N0:28:
SEQ
_ Met Gln Val Lys Gln Phe TyrLys Pro Val Asn
Phe Asn Asn Asp Gly
1 5 10 15
Val Asp Ala Ile Lys Ile AsnVal Gln Met Gln
Ile Tyr Pro Gly Pro
20 25 30
7() Val Lys Phe Ile His Asn IleTrp Ile Pro Glu
Ala Lys Lys Vai Arg
- 273 -
CA 02296765 2000-O1-14
WO 98/OS540 PCTIUS97/15394
35 40 45
Asp Thr PheThrAsn ProGluGluGlyAspLeu AsnProProProGlu
50 55 60
J
Ala Lys GlnValPro ValSerTyrTyrAspSer ThrTyrLeuSerThr
65 70 75 80
Asp Asn GluLysAsp AsnTyrLeuLysGlyVal ThrLysLeuPheGlu
1 85 90 95
()
Arg Ile TyrSerThr AspLeuGlyArgMetLeu LeuThrSerIZeVal
100 105 110
Arg Gly IleProPhe TrpGlyGlySerThrIle AspThrGluLeuLys
115 120 125
Val Ile AspThrAsn CysIleAsnValIleGln ProAspGlySerTyr
130 135 140
?0
Arg Ser GluGluLeu AsnLeuValIleIleGly ProSerAlaAspIle
145 150 155 160
Ile Gln PheGluCys LysSerPheGlyHisGlu ValLeuAsnLeuThr
165 170 175
Arg Asn GlyTyrGly SerThrGlnTyrIleArg PheSerProAspPhe
180 185 190
?() Thr Phe GlyPheGlu GluSerLeuGluValAsp ThrAsnProLeuLeu
195 200 205
Gly Ala GlyLysPhe AlaThrAspProAlaVal ThrLeuAlaHisGlu
'~10 215 220
i7
Leu Ile HisAlaGly HisArgLeuTyrGlyIle AlaIleAsnProAsn
225 230 235 240
Arg Val PheLysVaI AsnThrAsnAlaTyrTyr GluMetSerGlyLeu
4~) 295 250 255
Glu Val SerPheGlu GluLeuArgThrPheGly GlyHisAspAlaLy.,
260 265 270
Phe Ile AspSerLeu GlnGluAsnGluPheArg LeuTyrTyrTyrAsn
275 280 285
Lys Phe LysAspIle AlaSerThrLeuAsnLys AlaLyeSerIleVal
290 295 300
1
()
Gly Thr ThrAlaSer LeuGln'ryrMetLysAsn ValPheLysGluLys
305 310 315 320
Tyr Leu LeuSerGlu AspThrSerGlyLysPhe SerValAspLysLeu
325 330 335
Lys Phe AspLysLeu TyrLysMetLeuThrGlu IleTyrThrGluAsp
340 345 350
W) Asn Phe ValLysPhe PheLysValLeuAsnArg LysThr'ryrLeuAsn
355 360 365
Phe Asp LysAlaVal PheLysIleAsnIleVal ProLysValAsnTyr
370 375 380
~)
J
Thr Ile TyrAspGly PheAsnLeuArgAsnThr AsnLeuAlaAlaAsn
385 390 395 400
Phe Asn GlyGlnAsn ThrGluIleAsnAsnMet AsnPheThrLysLeu
405 410 415
-274-
CA 02296765 2000-O1-14
WO 98108540 PCTIUS97/15394
Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg
420 425 430
Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly Tyr Asn Lys
J 435 440 445
Ala Leu AsnAspLeuCys IleLysValAsnAsnTrp AspLeuPhePhe
450 455 460
Ser Pro SerGluAspAsn PheThrAsnAspLeuAsn LysGlyGluGlu
465 470 475 480
Ile Thr SerAspThrAsn IleGluAlaAlaGluGlu AsnIleSerLeu
485 490 495
1~
Asp Leu IleGlnGlnTyr TyrLeuThrPheAsnPhe AspAsnGluPro
500 505 510
Glu Asn IleSerIleGlu AnnLeuSerSerAspIle IleGlyGlnLeu
515 520 525
Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu
530 535 540
Leu AspLys TyrThrMetPheHis TyrLeuArgAlaGln GluPheGlu
545 550 555 560
His GlyLys SerArgIleAlaLeu ThrAsnSerValAsn GluAlaLeu
565 570 575
Leu AsnPro SerArgValTyrThr PhePheSerSerAsp TyrValLys
580 585 590
Lys ValAsn LysAlaThrGluAla AlaMetPheLeuGly TrpValGlu
iJ 595 600 605
Gln LeuVal TyrAspPheThrAsp GluThrSerGluVal SerThrThr
610 615 620
40 Asp LysIle AlaAspIleThrIle IleIleProTyrIle GiyProAla
625 630 635 640
Leu AsnIle GlyAsnMetLeuTyr LysAspAspPheVal GlyAlaLeu
645 650 655
~j
Ile PheSex GlyAlaValIleLeu LeuGluPheIlePro GluIleAla
660 665 670
Ile ProVal LeuGlyThrPheAla LeuValSerTyrIle AlaAsnLys
JU 675 680 685
Val LeuThr ValGlnThrIleAsp AsnAlaLeuSerLys ArgAsnGlu
690 695 700
JJ Lys TrpAsp GluValTyrLysTyr IleValThrAsnTrp LeuAlaLys
705 710 715 720
Val Asn Thr Gln Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala Leu
725 730 735
~)U
Glu Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Ann Tyr Gln Tyr Asn
740 745 750
Gln Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp
GJ 755 760 765
Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile
770 775 780
7() Asn Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met
_~7~_
CA 02296765 2000-O1-14
PCTIUS97115394
785 790 795 800
Ile ProTyrGly ValLysArgLeuGlu AspPheAspAla SerLeuLys
805 810 815
J
Asp AlaLeuLeu LysTyrIleTyrAsp AsnArgGlyThr LeuIleGly
820 825 830
Gln ValAspArg LeuLysAspLysVal AsnAsnThrLeu SerThrAsp
~~) 835 840 845
ile ProPheGln LeuSerLysTyrVal AspAsnGlnArg LeuLeuSer
850 855 860
Thr PheThrGlu TyrIleLysAsnIle IleAsnThrSer IleLeuAsn
865 870 875 880
Leu ArgTyrGlu SerAsnHisLeuIle AspLeuSerArg TyrAlaSer
885 890 995
Lys IleAsnIle GlySerLysValAsn PheAspProIle AspLysAsn
900 905 910
Gln IleGlnLeu PheAsnLeuGluSer SerLyeIleGlu ValIleLeu
~J 915 920 925
L,ys AsnAlaZle ValTyrAsnSerMet TyrGluAsnPhe SerThrSer
930 935 940
iU Phe TrpIleArg IIeProLyETyrPhe AsnSerIleSer LeuAsnAsn
945 950 955 960
Glu TyrThrIle IleAsnCysMetGlu AsnAsnSerGly TrpLysVal
965 970 975
.i
i
Ser LeuAsnTyr GlyGluIleIleTrp ThrLeuGlnAsp ThrGlnGlu
980 985 990
Ile LysGlnArg ValValPheLysTyr SerGlnMetIle AsnIlaSer
995 1000 1005
rasp TyrIleAsriArgTrpIlePheVal ThrIleThrAsn AsnArgLeu
1010 1015 1020
ksn AsnSerLys IleTyrI1eAsnGly ArgLeuIleAsp GlnLysPro
.025 1030 1035 1040
Ile SerAsnLeu GlyAsnIleHisAla SerAsnAsnIle MetPheLys
1045 1050 1055
JO
Leu AspGlyCys ArgAspThrHisArg TyrIleTrpIle LyeTyrPhe
1060 1065 1070
Asn LeuPheAsp LysGluLeuAsnGlu LysGluIleLys AspLeuTyr
1075 1080 1085
Asp AsnGlnSer AsnSerGlyIleLeu LysAspPheTrp GlyAspTyr
1090 1095 1100
OU Leu GlnTyrAsp LysProTyrTyrMet LeuAsnLeuTyr AspProAsn
110 5 ~ 1110 1115 1120
Lys TyrValAsp ValAsnAsnValGly IleArgGlyTyr MetTyrLeu
1125 1130 1135
()
1
Lys GlyProArg GlySerValMetThr ThrAsnIleTyr LeuAsnSer
1140 114 5 1150
Ser LeuTyrArg GlyThrLy~PheIle IleLysLysTyr AlaSerGly
1155 1160 116 5
77() -
CA 02296765 2000-O1-14
PCT/US97115394
Asn Lys Asp Asn Ile Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val
1170 1175 1180
Val Val Lys Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gln Ala
J 1185 1190 1195 1200
Gly Val Glu Lys Ile Leu Ser Ala Leu Glu Ile Pro Asp Val Gly Asn
1205 1210 1215
Leu Ser Gln Val Val Val Met Lys Ser Lys Asn Asp Gln Gly Ile Thr
1220 1225 1230
Rsn Lys Cys Lys Met Asn Leu Gln Asp Asn Asn Gly Asn Asp Ile Gly
1235 1240 1245
~i
Phe Ile Gly Phe His Gln Phe Asn Asn Ile Ala Lys Leu Val Ala Ser
1250 1255 1260
Asn Trp Tyr Asn Arg Gln Ile Glu Arg Ser Ser Arg Thr Leu Gly Cys
1265 1270 1275 1280
Ser Trp Glu Phe Ile Pro Val Asp Asp Gly Trp Gly Glu Arg Pro Leu
1285 1290 1295
~J (?.) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
.~() (C) STRANDEDNESS: single
tDl TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA"
J7
(r.i) SEQUENCE DESCRIPTION: SEQ ID N0:29:
CGCCATGGCT AGATTATTAT CTACATTTAC 30
(2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
tA) LENGTH: 26 base pairs
(S1 TYPE: nucleic acid
4~ (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
;ii) MOLECULE TYPE: other nucleic acid
(A1 DESCRIPTION: jdesc = "DNA"
~0
tr.i) SEQUENCE DESCRIPTION: SEQ ID N0:30:
GCAAGCTTCT TGACAGACTC ATGTAG 26
JJ (2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1596 base pairs
tBl TYPE: nucleic acid
tC) STRANDEDNESS: double
-(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(fi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
AGATCTCGAT CCCGCGAAAT TAATACGACT CACTATAGGG GAATTGTGAG CGGATAACAA 60
TTCCCCTCTA GAAATAATTT TGTTTAACTT TAAGAAGGAG ATATACCATG GGCCATCATC 120
CA 02296765 2000-O1-14
PCTIUS97I15394
ATCATCATCA TCATCATCATCACAGCAGCG GCCATATCGAAGGTCGTCATATGGCTAGCA180
TGGCTAGATT ATTATCTACATTTACTGAAT ATATTAAGAATATTATTAATACTTCTATAT240
S TGAATTTAAG ATATGAAAGTAATCATTTAA TAGACTTATCTAGGTATGCATCAAAAATAA300
ATATTGGTAG TAAAGTAAAT TTTGATCCAA TAGATAAAAA TCAAATTCAA TTATTTAATT 360
TAGAAAGTAG TAAAATTGAG GTAATTTTAA AAAATGCTAT TGTATATAAT AGTATGTATG 420
~ ()
AAAATTTTAG TACTAGCTTT TGGATAAGAA TTCCTAAGTA TTTTAACAGT ATAAGTCTAA 480
ATAATGAATA TACAATAATA AATTGTATGG AAAATAATTC AGGATGGAAA GTATCACTTA 540
ATTATGGTGA AATAATCTGG ACTTTACAGG ATACTCAGGA AATAAAACAA AGAGTAGTTT 600
TTAAATACAG TCAAATGATT AATATATCAG ATTATATAAA CAGATGGATT TTTGTAACTA 660
TCACTAATAA TAGATTAAAT TTTATATAAA TGGAAGATTAATAGATCAAA 720
AACTCTAAAA
?()
AACCAATTTC AAATTTAGGTAATATTCATGCTAGTAATAA TATAATGTTTAAATTAGATG 780
GTTGTAGAGA TACACATAGATATATTTGGATAAAATATTT TAATCTTTTTGATAAGGAAT B40
~J TAAATGAAAA AGAAATCAAAGATTTATATGATAATCAATC AAAT'TCAGGTATTTTAAAAG 900
ACTTTTGGGG TGATTATTTACAATATGATAAACCATACTA TATGTTAAATTTATATGATC 960
CAAATAAATA TGTCGATGTA AATAATGTAG GTATTAGAGGTTATATGTAT CTTAAAGGGC
1020
i
t)
CTAGAGGTAG CGTAATGACT ACAAACATTT ATTTAAATTCAAGTTTGTAT AGGGGGACAA
1080
AATTTATTAT AAAAAAATAT GCTTCTGGAA ATAAAGATAATATTGTTAGA AATAATGATC
1140
.i~ GTGTATATAT TAATGTAGTA GTTAAAAATA AAGAATATAGGTTAGCTACT AATGCATCAC
1200
AGGCAGGCGT AGAAAAAATA CTAAGTGCAT TAGAAATACCTGATGTAGGA AATCTAAGTC
1260
AAGTAGTAGT AATGAAGTCA AAAAATGATC AAGGAATAACAAATAAATGC AAAATGAATT
1320
TACAAGATAA TAATGGGAAT GATATAGGCT TTATAGGATTTCATCAGTTT AATAATATAG
1380
CTAAACTAGT AGCAAGTAAT TGGTATAATA GACAAATAGAAAGATCTAGT AGGACTTTGG
1440
GTTGCTCATG GGAATTTATT CCTGTAGATG ATGGATGGGGAGAAAGGCCA CTGTAATTAA
1500
TCTCAAACTA CATGAGTCTG TCAAGAAGCT TGCGGCCGCACTCGAG 1546
(2) INFORMATION FOR SEQ ID N0:32:
1()
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
!B) TYPE: amino acid
lC) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
!xi) SEQUENCE DESCRIPTION: SEQ ID
N0:32:
(o)
Met Ais His His His His His Met Ala
1 5
(2) INFORMATION FOR SEQ ID N0:33:
O7
li) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
!H) TYPE: nucleic acid
!C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- 278 -
CA 02296765 2000-O1-14
WO 98108540 PCT/US97I15394
(ii)MOLECULE TYPE: other
nucleic acid
(A) DESCRIPTION: /descDNA"
= "
(xi)SEQUENCE DESCRIPTION: :33:
SEQ ID N0
_1
TATGCATC AC CATCACCATC A 21
(2) INFORMATION
FOR
SEQ
ID
N0:34:
' 10 (i)SEQUENCE CHARACTERISTICS:
(A) LENGTH: ~3 base
pairs
(B) TYPE: nucleic acid
. (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
IS
(ii)MOLECULE TYPE: other id
nucleic ac
(A) DESCRIPTION: /desc
= "DNA"
(xi)SEQUENCE DESCRIPTION:
SEQ ID N0:34:
?0
CATGTGATGG 23
TGATGGTGAT
GCA
f2.) INFORMATION
FOR
SEQ
ID
N0:35:
~S li)SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1351 base
pairs
(B) TYPE: nucleic acid
iC) STRANDEDNESS: double
(D) TOPOLOGY: linear
i ()
(ii)MOLECULE TYPE: other
nucleic acid
(A) DESCRIPTION: /desc"DNA"
=
(ix)FEATURE:
iS (A) NAME/KEY: CDS
(B) LOCATION: 1..1335
(xi)SEQUENCE DESCRIPTION:
SEQ ID N0:35:
ATG CATCAC CAT CAC CAT CAC CGT CTGCTGTCTACC TTCACT 48
ATG GCT
Met HisHis His His His His Arg LeuLeuSerThr PheThr
Met Ala
1 5 10 15
GAA TACATC AAG AAC ATC ATC TCC ATCCTGAACCTG CGCTAC 96
AAT ACC
Glu TyrIle Lys Asn Ile Ile Ser IleLeuAsnLeu ArgTyr
Asri Thr
20 25 30
GAA TCCAAT CAC CTG ATC GAC CGC TACGCTTCCAAA ATCAAC 144
CTG TCT
Glu SerAsn His Leu Ile Asp Arg TyrAlaSerLys IleAsn
Leu Ser
SU 35 40 45
ATC GGTTCT AAA GTT AAC TTC ATC GACAAGAATCAG ATCCAG 192
GAT CCG
Ile GlySer Lys Val Asn Phe Ile AspLysAsnGln IleGln
Asp Pro
50 55 60
SS
CTG TTCAAT CTG GAA TCT TCC GAA GTTATCCTGAAG AATGCT 240
AAA ATC
Leu PheAsn Leu Glu Ser Ser Glu ValIleLeuLys AsnAla
Lys Ile
65 70 75 80
6U ATC GTATAC AAC TCT ATG TAC TTC TCCACCTCCTTC TGGATC 288
GAA AAC
Ile ValTyr Asn Ser Met Tyr Phe SerThrSerPhe TrpIle
Glu Asn
85 90 95
CGT ATCCCG AAA TAC TTC AAC TCT CTGAACAATGAA TACACC 336
TCC ATC
OJ Arg IlePro Lys Tyr Phe Asn Ser LeuAsnAsnGlu TyrThr
Ser Ile
100 105 110
ATC ATCAAC TGC ATG GAA AAC GGT TGGAAAGTATCT CTGAAC 384
AAT TCT
Ile IleAsn Cys Met Glu Asn Gly TrpLysValSer LeuAsn
Asn Ser
115 120 125
_~7y_
CA 02296765 2000-O1-14
~rp gg~pg~p PCTIUS97115394
TAC GGT GAA ATC ATC TGG ACT CTG CAG GAC ACT CAG GAA ATC AAA CAG 432
Tyr Gly Glu Ile Ile Trp Thr Leu Gln Asp Thr Gln Glu Ile Lys Gln
130 135 140
CGT GTT GTA TTC AAA TAC TCT CAG ATG ATC AAC ATC TCT GAC TAC ATC 480
Arg Val Val Phe Lys Tyr Ser Gln Met Ile Asn Ile Ser Asp Tyr Ile
145 150 155 160
AAT CGC TGG ATC TTC GTT ACC ATC ACC AAC AAT CGT CTG AAT AAC TCC 528
1l) Asn Arg Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu Asn Asn Ser
165 170 175
:AAA ATC TAC ATC AAC CGC CGT CTG ATC GAC CAG AAA CCG ATC TCC AAT 576
Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gln Lys Pro Ile Ser Asn
1J 180 185 190
CTG GGT AAC ATC CAC GCT TCT AAT AAC ATC ATG TTC AAA CTG GAC GGT 624
Leu Gly Asn Ile His Ala Ser Asn Asn Ile Met Phe Lys Leu Asp Gly
195 200 205
'_' 0
TGT CGT GAC ACT CAC CGC TAC ATC TGG ATC AAA TAC TTC AAT CTG TTC 672
Cys Arg Asp Thr Hip Arg Tyr Ile Trp Ile Lys Tyr Phe Asn Leu Phe
210 215 220
~J GlIC AAA GAA CTG AAC GAA AAA GAA ATC AAA GAC CTG TAC GAC AAC CAG 720
Asp Lys Glu Leu Asn Glu Lys Glu Ile Lys Asp Leu Tyr Asp Asn Gln
~25 230 235 240
TCC AAT TCT GGT ATC CTG AAA GAC TTC TGG GGT GAC TAC CTG CAG TAC 768
.i~) Ser Asn Ser Gly Ile Leu Lys Asp Phe Trp Gly Asp Tyr Leu Gln Tyr
245 250 255
GAC AAA CCG TAC TAC ATG CTG AAT CTG TAC GAT CCG AAC AAA TAC GTT 816
Asp Lys Pro Tyr Tyr Met Leu Asn Leu Tyr Asp Pro Asn Lys Tyr Val
iJ 260 265 270
GAC GTC AAC AAT GTA GGT ATC CGC GGT TAC ATG TAC CTG AAA GGT CCG 864
Asp Val Asn Asn Val Gly Ile Arg Gly Tyr Met Tyr Leu Lys Gly Pro
275 280 285
CGT GGTTCTGTT ATGACTACCAACATC TACCTGAACTCTTCC CTGTAC 912
Arg GlySerVal MetThrThrAsnIle TyrLeuAsnSerSer Leu1'yr
290 295 300
-)J C;~T GGTACCAAA TTCATCATCAAGAAA TACGCGTCTGGTAAC AAGGAC 960
Arg GlyThrLys PheIleIleLysLys TyrAlaSerGlyA.sn.LysAsp
305 310 315 320
AAT ATCGTTCGC AACAATGATCGTGTA TACATCAATGTTGTA GTTAAG 1008
J~ A3n ileValArg AsnAsnAspArgVal TyrIleAsnValVal ValLys
325 330 335
AAC AAAGAATAC CGTCTGGCTACCAAT GCTTCTCAGGCTGGT GTAGAA 1056
Asn LysGluTyr ArgLeuAlaThrAsn AlaSerGlnAlaGly ValGlu
390 395 350
AAG ATCTTGTCT GCTCTGGAAATCCCG GACGTTGGTAATCTG TCTCAG 1104
Lys IleLeuSer AlaLeuGluIlePro AspValGlyAsnLeu SerGln
355 360 365
GTA GTTGTAATG AAATCCAAGAACGAC CAGGGTATCACTAAC AAATGC 1152
Val 'JalValMet LysSerLyeAsnAsp GlnGlyIleThrAsn LysCys
370 375 380
W AAA ATGAATCTG CAGGACAACAATGGT AACGATATCGGTTTC ATCGGT 1200
Lys MetAsnLeu GlnAspAsnAsnGly AsnAspIleGlyPhe IleGly
385 390 395 400
TTC CACCAGTTC AACAATATCGCTAAA CTGGTTGCTTCCAAC TGGTAC 1248
Phe HisGlnPhe AsnAsnIleAlaLys LeuValAiaSerAsn TrpTyr
- 28U -
i
CA 02296765 2000-O1-14
PCTIUS97115394
q05 410 415
AAT CGT ATC GAACGT TCC TCT ACTCTGGGTTGC TCTTGG GAG 1296
CAG CGC
Asn Arg Ile GluArg Ser Ser ThrLeuGlyCys SerTrp Glu
Gln Arg
420 425 430
TTC ATC GTT GATGAC GGT TGG GAACGTCCGCTG TAACCCGGGA
CCG GGT 1345
Phe Ile Val AspAsp Gly Trp GluArgProLeu
Pro Gly
435 440 445
AAGCTT 1351
(2) INFORMATION FORSEQ ID N0:36:
1J (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH:
445
amino
acids
(B) TYPE:
amino
acid
(D) TOPOLOGY:
linear
?U (ii) MOLECULE TYPE: protein
(ii) SEQUENCE DESCRIPTION: N0:36:
SEQ ID
Met His His HisHis His Met ArgLeuLeuSer ThrPhe Thr
His Ala
1 5 10 15
Glu Tyr Ile Lys Asn Ile Ile Asn Thr Ser Ile Leu Asn Leu Arg '1'yr
20 25 30
?() Glu Ser Asn His Leu Ile Asp Leu Ser Arg Tyr Ala Ser Lys Ile Asn
35 40 45
Ile Gly 5er Lys Val Asn Phe Asp Pro Ile Asp Lys Asn Gln Ile Gin
50 55 60
i~
Leu Phe Asn Leu Glu Ser Ser Lys Ile Glu Val Ile Leu Lys Asn Ala
65 70 75 80
Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser Phe Trp Ile
85 90 95
Arg Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu Asn Asn Glu Tyr Thr
100 105 110
ile IleAsnCys MetGluAsnAsn Ser TrpLysValSer LeuAsn
Gly
115 120 125
Tyr GiyGluIle IleTrpThrLeu Gln ThrGlnGluIle LysGln
Asp
130 135 140
i()
Arg ValValPhe LysTyrSerGln Met AsnIleSerAsp TyrIle
Ile
145 150 155 160
Asn ArgTrpIle PheValThrIle Thr AsnArgLeuAsn AsnSer
Asn
JS 165 170 175
Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gln Lys Pro Ile Ser Asn
180 185 190
Leu Gly Asn Ile His Ala Ser Asn Asn Ile Met Phe Lys Leu Asp Gly
195 200 205
Cys Arg Asp Thr His Arg Tyr Ile Trp Ile Lys Tyr Phe Asn Leu Phe
~10 215 220
' G~
Asp Lys Glu Leu Asn Glu Lys Glu Ile Lys Asp Leu Tyr Asp Asn Gln
225 230 235 240
Ser Asn Ser Gly Ile Leu Lys Asp Phe Trp Gly Asp Tyr Leu Gln Tyr
245 250 255
_ ~gl _
CA 02296765 2000-O1-14
PCT/US97l15394
Asp Lys Pro Tyr Tyr Met Leu Asn Leu Tyr Asp Pro Asn Lys Tyr Val
260 265 270
Asp Val Asn Asn Val Gly Ile Arg Gly Tyr Met Tyr Leu Lys Gly Pro
J 275 280 285
Arg Gly Ser Val Met Thr Thr Asn Ile Tyr Leu Asn Ser Ser Leu Tyr
290 295 300
I() Arg Gly Thr Lys Phe Ile Ile Lys Lys Tyr Ala Ser Gly Asn Lys Asp
305 310 315 320
Asn Ile Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val Val Val Lys
325 330 335
~i
Asn Lys Glu Tyr Arg Leu Ala 'Thr Asn Ala Ser Gln Ala Gly Val Glu
340 345 350
Lys Ile Leu Ser Ala Leu Glu Ile Pro Asp Val Gly Asn Leu Ser Gln
355 360 365
Val Val Val Met Lys Ser Lys Asn Asp Gln Gly Ile Thr Asn Lys Cys
370 375 3B0
~J I,ys Met Asn Leu Gln Asp Asn Asn App Gly Phe Ile Gly
Asn Giy Ile
385 390 395 400
Phe )Iis Glr~ Phe Asn Asn Leu Val Ser Asn Trp Tyr
Ile Ala Lys Ala
405 410 415
i
()
Asn Arg Gln Ile Glu Arg Ser Thr Leu Cys Ser Trp Glu
Ser Arg Gly
420 425 430
Phe lle Pro Val Asp Asp Gly Glu Arg Leu
Trp Gly Pro
435 440 445
;2) INFORMATION FOR SEQ ID
N0:37:
(i) SEQUENCE CHARACTERISTICS:
~lU (A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
tii) MOLECULE TYPE: otheric acid
nucle
LA) DESCRIPTION: ide~c "DNA"
=
(r,i) SEQUENCE DESCRIPTIOId:ID N0:37:
SEQ
CGCATATGAA 27
TATTCGTCCA
TTGCATG
(2) INFORMATION FOR SEQ ID
N0:38:
(i) SEQUENCE CHARACTERISTICS:
JJ tA) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
O() (ii) MOLECULE TYPE: otheric acid
nucle
'(A) DESCRIPTION: /desc "DNA"
=
(ii) SEQUENCE DESCRIPTION:ID N0:38:
SEQ
GGAAGCTTGC 27
AGGGCAATTA
CATCATG
(<) INFORMATION FOR SEQ ID
N0:39:
(i) SEQUENCE CHARACTERISTICS:
7() (A) LENGTH: 3876 base
pairs
-282-
CA 02296765 2000-O1-14
PCTIUS97/13394
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
_ (B) LOCATION: 1..3873
~ l)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:39:
ATG CCA GTT ACA ATA AAT AAT TTT AAT TAT AAT GAT CCT ATT GAT AAT 48
Met Pro Val 'Thr Ile Asn Asn Phe Asn Tyr Asn Asp Pro Ile Asp Asn
1 5 10 15
GAC AAT ATT ATT ATG ATG GAA CCT CCA TTT GCA AGG GGT ACG GGG AGA 96
Asp Asn Ile Ile Met Met Glu Pro Pro Phe Ala Arg Gly Thr Gly Arg
20 25 30
?U
TAT TAT AAA GCT TTT AAA ATC ACA GAT CGT ATT TGG ATA ATA CCC GAA 144
Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Ile Pro Glu
35 40 45
AGA TI,T ACT TTT GGA TAT AAA CCT GAG GAT TTT AAT AAA AGT TCC GGT 192
Arg '1'yr Thr Phe Gly Tyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly
50 55 60
ATT TTT AAT AGA GAT GTT TGT GAA TAT TAT GAT CCA GAT TAC TTA AAT 240
s() Ile Phe Asn Arg Asp Val Cys Glu Tyr Tyr Asp Pro Asp Tyr Leu Asn
65 70 75 80
ACC AAT GAT AAA AAG AAT ATA TTT TTC CAA ACA TTG ATC AAG TTA TTT 288
Thr Asn Asp Lys Lys Asn Ile Phe Phe Gln Thr Leu Ile Lys Leu Phe
85 90 95
AAT AGA ATC AAA TCA AAA CCA TTG GGT GAA AAG TTA TTA GAG ATG ATT 336
Asn Arg Ile Lys Ser Lys Pro Leu Gly Glu Lys Leu Leu Glu Met Ile
100 105 110
ATA AAT GGT ATA CCT TAT CTT GGA GAT AGA CGT GTT CCA CTC GAA GAG 384
Ile Asn Gly Ile Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu Glu Glu
115 120 125
TTT AAC ACA AAC ATT GCT AGT GTA ACT GTT AAT AAA TTA ATT AGT AAT 432
Phe Ann Thr Asn Ile Ala Ser Val Thr Val Asn Lys Leu Ile Ser Asn
130 135 140
CCA GGA GAA GTG GAG CGA AAA AAA GGT ATT TTC GCA AAT TTA ATA ATA 480
Pro Gly Glu Val Glu Arg Lys Lys Gly Ile Phe Ala Asn Leu Ile Ile
145 150 155 160
TTT GGA CCT GGG CCA GTT TTA AAT GAA AAT GAG ACT ATA GAT ATA GGT 528
Phe Gly Pro Gly Pro Val Leu Asn Glu Asn Glu Thr Ile Asp Ile Gly
JJ 165 170 175
ATA CAA AAT CAT TTT GCA TCA AGG GAA GGC TTT GGG GGT ATA ATG CAA 576
Ile Gln Asn His Phe Ala Ser Arg Glu Gly Phe Gly Gly Ile Met Gln
180 185 190
ATG AAA TTT TGT CCA GAA TAT GTA AGC GTA TTT AAT AF,T GTT CAA GAA 624
Met Lys Phe Cys Pro Glu Tyr Val Ser Val Phe Asn Asn Val Gln GIu
195 200 205
AAC AAA GGC GCA AGT ATA TTT AAT AGA CGT GGA TAT TTT TCA GAT CCA 672
Asn Lys Gly Ala Ser Ile Phe Asn Arg Arg Gly Tyr Phe Ser Asp Pro
210 215 220
GCC TTG ATA TTA ATG CAT GAA CTT ATA CAT GTT TTG CAT GGA TTA TAT 720
Ala Leu Ile Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr
- 283 -
CA 02296765 2000-O1-14
WO 98/08540 PCTNS97115394
225 230 235 240
GGC ATTAAAGTA GATGATTTACCAATT GTACCAAATGAAAAAAAA TTT 768
Gly IleLysVal AspAspLeuProIle ValProAsnGluLysLys Phe
J 245 250 255
TTT ATGCAATCT ACAGATACTATACAG GCAGAAGAACTATATACA TTT 816
Phe MetGlnSer ThrAsp1'hrIleGln AlaGluGluLeuTyrThr Phe
_
~0 260 265 270
GGA GGACAAGAT CCCAGCATCATATCT CCTTCTACAGATAAAAGT ATC 864
Gly GlyGlnAsp ProSerIleIleSer ProSerThrAspLysher Ile
275 280 285
TAT GATAAAGTT TTGCAAAATTTTAGG GGGATAGTTGATAGACTT AAC 912
Tyr AspLysVal LeuGlnAsnPheArg GlyIleVaIAspArgLeu Asn
290 295 300
AAG GTTTTAGTT TGCATATCAGATCCT AACATTAACATTAATATA TAT 960
Lys ValLeuVal CysIleSerAspPro AsnIleAsnIleAsnIle Tyr
305 310 315 320
AAA AATAAATTT AAAGATAAATATAAA TTCGTTGAAGATTCTGAA GGA 1008
Lye AsnLysPhe LysAspLysTyrLys PheValGIuAspSerGlu Gly
~J 325 330 335
AAA TATAGTATA GATGTAGAAAGTTTC AATAAATTATATAAAAGC TTA 1056
Lys TyrSerIle AspValGluSerPhe AsnLysLeuTyrLysSer Leu
340 345 350
i(1
ATG TTAGGTTTT ACAGAAATTAATATA GCAGAAAATTATAAAATA AAA 1104
Met LeuGlyPhe ThrGluIleAsnIle AlaGluAsnTyrLysIle Lys
355 360 365
ACT AGAGCTTCT TATTTTAGTGATTCC TTACCACCAGTAAAAATA AAA 1152
Thr ArgAlaSer TyrPheSerAspSer LeuProProValLysIle Lys
370 375 380
AAT TTATTAGAT AATGAAATCTATACT ATAGAGGAAGGGTTTAAT ATA 1200
40 Asn LeuLeuAsp AsnGluIleTyrThr IleGluGluGlyPheAsn Ile
385 390 395 900
TCT GATAAAAAT ATGGGAAAAGAATAT AGGGGTCAGAATAAAGCT ATA 1248
Ser AspLysAsn MetGlyLysGluTyr ArgGlyGlnAsnLysAla Ile
405 410 415
AAT AAACAAGCT TATGAAGAAATCAGC AAGGRGCATTTGGCTGTA TAT 1296
Asn LysGlnAla TyrGluGluZleSer LysGluHisLeuAlaVal Tyr
420 425 430
i(1
AAG ATACAAATG TGTAAAAGTGTTAAA GTTCCAGGAATATGTATT GAT 1344
Lys IleGlnMet CysLysSerValLys ValProGlyIleCysIle Asp
435 440 495
JJ GTC GATAATGAA AATTTGTTCTTTATA GCTGATAAAAATAGTTTT TCA 1392
Val AspAsnGlu AsnLeuPhePheIle AlaAspLysAsnSerPhe Ser
450 455 460
GAT GATTTATCT AAAAATGAAAGAGTA GAATATAATACACAGAAT AAT 1440
Asp AspLeuSer LysAsnGluArgVal GluTyrAsnThrGlnAsn Asn
465 ' 470 475 480
TAT ATAGGAAAT GACTTTCCTATAAAT GAATTAATTTTAGATACT GAT 1488
Tyr IleGlyAsn AspPheProIleAsn GluLeuIleLeuAspThr Asp
485 490 495
TTA ATAAGTAAA ATAGAATTACCAAGT GAAAATACAGAATCACTT ACT 1536
Leu IIeSerLys ZleGluLeuProSer GluAsnThrGluSerLeu Thr
500 505 510
_?g4_
CA 02296765 2000-O1-14
WO 98108540 PCT/US97115394
GAT TTT AAT GTA GAT GTT CCA GTA TAT GAA AAA CAA CCC GCT ATA AAA 1584
Asp Phe Asn Val Asp Val Pro Val Tyr Glu Lys Gln Pro Ala Ile Lys
515 520 525
7 AAA GTT TTT ACA GAT GAA AAT ACC ATC TTT CAA TAT TTA TAC TCT CAG 1632
Lys Val Phe Thr Asp Glu Asn Thr Ile Phe Gln Tyr Leu Tyr Ser Gln
530 535 590
_ ACA TTT CCT CTA AAT ATA AGA GAT ATA AGT TTA ACA TCT TCA TTT GAT 1680
1() Thr Phe Pro Leu Asn Ile Arg Asp Ile Ser Leu Thr Ser Ser Phe Asp
545 550 555 560
GAT GCA TTA TTA GTT TCT AGC AAA GTT TAT TCA TTT TTT TCT ATG GAT 1728
Asp Ala Leu Leu Val Ser Ser Lys Val Tyr Ser Phe Phe Ser Met Asp
jj 565 570 575
TAT ATT AAA ACT GCT AAT AAA GTA GTA GAA GCA GGA TTA TTT GCA GGT 1776
Tyr Ile Lys Thr Ala Asn Lys Val Val Glu Ala Gly Leu Phe Ala Gly
580 585 590
TGG GTG AAA CAG ATA GTA GAT GAT TTT GTA ATC GAA GCT AAT AAA AGC 1824
Trp Val Lys Gln Ile Val Asp Asp Phe Val Ile Glu Ala Asn Lys Ser
595 600 605
AGT RCT ATG GAT AAA ATT GCA GAT ATA TCT CTA ATT GTT CCT TAT ATA 1872
Ser Thr Met Asp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr Ile
610 615 620
GGA TTA GCT TTA AAT GTA GGA GAT GAA ACA GCT AAA GGA AAT TTT GAA 1920
Gly Leu Ala Leu Asn Val Gly Asp Glu Thr Ala Lys Gly Asn Phe Glu
625 630 635 640
AGT GCT TTT GAG ATT GCA GGA TCC AGT ATT TTA CTA GAA TTT ATA CCA 1968
Ser Ala Phe Glu Ile Ala Gly Ser Ser Ile Leu Leu Glu Phe Ile Pro
j1 645 650 655
GAA CTT TTA ATA CCT GTR GTT GGA GTC TTT TTA TTA GAA TCA TAT ATT 2016
Glu Leu Leu Ile Pro Val Val Gly Val Phe Leu Leu Glu Ser Tyr Ile
660 665 670
GAC RAT AAA AAT AAA ATT ATT AAA ACA ATA GAT AAT GCT TTA ACT AAA 2064
Asp Asn Lys Asn Lys Ile Ile Lys Thr Ile Asp Asn Ala Leu Thr Lys
675 680 685
AGA GTG GAA AAA TGG ATT GAT ATG TAC GGA TTA ATA GTA GCG CAA TGG 2112
Arg Val Glu Lys Trp Ile Asp Met Tyr Gly Leu Ile Val Ala Gln Trp
690 695 700
CTC TCA ACA GTT AAT ACT CAA TTT TAT ACA ATA AAA GAG GGA ATG TAT 2160
JO Leu Ser Thr Val Asn Thr Gln Phe Tyr Thr Ile Lys Glu Gly Met Tyr
705 710 715 720
AAG GCT TTA AAT TAT CAA GCA CAA GCA TTG GAA GAA ATA ATA AAA TAC 2208
Lys Ala Leu Asn Tyr Gln Ala Gln Ala Leu Glu Glu Ile Ile Lys Tyr
~J 725 730 735
6()
AAA TAT AAT ATA TAT TCT GAA GAG GAA AAG TCA AAT ATT AAC ATC AAT 2256
Lys Tyr Asn Ile Tyr Ser Glu Glu Glu Lys Ser Asn Ile Asn Ile Asn
740 745 750
TTT AAT GAT ATA AAT TCT AAA CTT AAT GAT GGT ATT AAC CAA GCT ATG 2304
Phe Asn Asp Ile Asn Ser Lys Leu Asn Asp Gly Ile Asn Gln Ala Met
755 760 765
C7J GAT AAT ATA AAT GAT TTT ATA AAT GAA TGT TCT GTA TCA TAT TTA ATG 2352
Asp Asn Ile Asn Asp Phe Iie Asn Glu Cys Ser Val Ser Tyr Leu Met
770 775 780
AAA AAA ATG ATT CCA TTA GCT GTA AAA AAA TTR CTA GAC TTT GAT AAT 2400
70 Lys Lys Met Ile Pro Leu Ala Val Lys Lys Leu Leu Asp Phe Asp Asn
- 285 -
CA 02296765 2000-O1-14
WO 98108540 PCT/US97/15394
785 790 795 800
ACT CTC AAAAAAAATTTA TTAAATTATATAGATGAA AATAAATTATAT 2448
Thr Leu LysLysAsnLeu LeuAsnTyrIleAspGlu AsnLysLeuTyr
805 810 815
TTA ATT GGAAGTGTAGAA GATGAAAAATCAAAAGTA GATAAATACTTG 2496
Leu Ile GlySerValGlu AspGluLysSerLysVal AspLysTyrLeu
820 825 830
IU
AAA ACC ATTATACCATTT GATCTTTCAACGTATTCT AATATTGAAATA 2544
Lys Thr IleIleProPhe AspLeuSerThrTyrSer AsnIlefluIle
835 840 845
IJ CTA ATA AAAATATTTAAT AAATATAATAGCGAAATT TTAAATARTATT 2592
Leu Ile LysIlePheAsn LysTyrAsnSerGluIle LeuAsnAsnIle
850 855 860
ATC TTA AATTTAAGATAT AGAGATAATAATTTAATA GATTTATCAGGA 2640
Ile Leu AsnLeuArgTyr ArgAspAsnAsnLeuIle AspLeuSerGly
865 870 875 880
TAT GGA GCAAAGGTAGAG GTATATGATGGGGTCAAG CTTAATGATAAA 2688
Tyr Gly AlaLysValGlu ValTyrAspGlyValLys LeuAsnAppLys
885 890 895
AAT CRA TTTAAATTAACT AGTTCAGCAGATRGTAAG RTTAGAGTCACT 2736
Asn Gln PheLysLeuThr SerSerAlaAspSerLys IleArgValThr
900 905 910
.i
(1
CAA AAT CACAATATTATA TTTAATAGTATGTTCCTT GATTTTAGCGTT 2784
c:,lnTsn GinAsnIleIle PheAsnSerMetPheLeu AppPheSerVal
915 920 925
?> AGC TTT TGGATAAGGATA CCTAAATATAGGAATGAT GATATACAAAAT 2832
ser hhe TrpIleArgIle ProLysTyrArgAsnAsp AspIleGlnAsn
930 935 940
TRT ATT CATAATGAATAT ACGATAATTRATTGTATG AAAAATAATTCA 2880
-Il)Tyr t_e HisAsnG1u'I'yrThrIleIleAsnCysMet LysAsnAsnSer
945 950 955 960
GGC TGG AAAATATCTATT AGGGGTAATAGGATAATA TGGACCTTAATT 2928
Gly Trp LysIleSerIle ArgGlyAsnArgIleIle 'I'rpThrLeuIle
'13 965 970 975
GAT ATR RATGGAAAAACC AAATCAGTATTTTTTGAA TATAACATAAGA 2976
App Ile AsnGlyLysThr LysSerValPhePheGlu TyrAsnIleArg
980 985 990
~U
GAA GRT ATATCAGAGTAT ATAAATAGATGGTTTTTT GTAACTATTACT 3024
Glu Asp ileSerGluTyr IleAsnArgTrpPhePhe ValThrIleThr
995 1000 1005
JJ AAT AAT TTGGATAATGCT AAAATTTATATTRATGGC ACGTTAGAATCA 3072
Asn Asn LeuAspAsnAla LysIleTyrIleAsnGly ThrLeuGluSer
1010 1015 1020
AAT ATG GATATTAAAGAT ATAGGAGAAGTTATTGTT AATGGTGAAATA 3120
Ann Mec AspIleLysAsp IleGlyGluValIleVal llsnGlyGluIle
1025 ' 1030 1035 1040
ACA T'~TRAATTAGATGGT GATGTAGATAGARCACAA TTTATTTGGATG 3168
Thr Phe LysLeuAspGly AspValAspArgThrGln PheIleTrpMet
1045 1050 1055
AAA TrITTTTAGTATTTTT AATACGCAATTAAATCAA TCAAATATTAAA 3216
Lys T,'rPheSerIlePhe AsnThrGlnLeuAsnGln SerAsnIleLys
1060 1065 1070
- 286 -
CA 02296765 2000-O1-14
WO 98108540 PCTNS97/15394
GAG ATA TAT AAA ATT CAA TCA TAT AGC GAA TAC TTA AAA GAT TTT TGG 3264
Glu Ile Tyr Lys Ile Gln Ser Tyr Ser Glu Tyr Leu Lys Asp Phe Trp
1075 1080 1085
GGA AAT CCT TTA ATG TAT AAT AAA GAA TAT TAT ATG TTT AAT GCG GGG 331
Gly Asn Pro Leu Met Tyr Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly
1090 1095 1100
AAT AAA AAT TCA TAT ATT AAA CTA GTG AAA GAT TCA TCT GTA GGT GAA 3360
- ~~1 Asn Lys Asn Ser Tyr Ile Lys Leu Val Lys Asp Ser Ser Val Gly Glu
1105 1110 1115 1120
ATA TTA ATA CGT AGC AAA TAT AAT CAG AAT TCC AAT TAT ATA AAT TAT 3408
Ile Leu Ile Arg Ser Lys Tyr Asn Gln Asn Ser Asn Tyr Ile Asn Tyr
1? 1125 1130 1135
AGA AAT TTA TAT ATT GGA GAA AAA TTT ATT ATA AGA AGA GAG TCA AAT 3456
Arg Asn Leu Tyr Ile Gly Glu Lys Phe~Ile Ile Arg Arg Glu Ser Asn
1140 1145 1150
TCT CAA TCT ATA AAT GAT GAT ATA GTT AGA AAA GAA GAT TAT ATA CAT 3504
Ser Gln Ser Ile Asn Jlsp Asp Ile Val Arg Lys Glu Asp Tyr Ile His
1155 1160 1165
CTA GAT TTG GTA CTT CAC CAT GAA GAG TGG AGA GTA TAT GCC TAT AAA 3552
Leu hsp Leu Val Leu His His Glu Glu Trp Arg Val Tyr Ala Tyr Lys
1170 1175 1180
TAT TTT AAG GAA CAG GAA GAA AAA TTG TTT TTA TCT ATT ATA AGT GAT 3600
?0 'tyr Phe Lys Glu Gln Glu Glu Lys Leu Phe Leu Ser Ile Ile Ser Asp
1185 1190 1195 1200
TCT AAT GAA TTT TAT AAG ACT ATA GAA ATA AAA GAA TAT GAT GAA CAG 3648
Ser Asn Glu Phe Tyr Lys Thr Ile Glu Ile Lys Glu Tyr Asp GIu Gln
1205 1210 1215
CCA TCA TAT AGT TGT CAG TTG CTT TTT AAA AAA GAT GAA GAA AGT ACT 3696
Pro Ser Tyr Ser Cys Gln Leu Leu Phe Lys Lys Asp Glu Glu Ser Thr
1220 1225 1230
GAT GAT ATA GGA TTG ATT GGT ATT CAT CGT TTC TnC GAA TCT GGA CTT 3799
Asp Asp .le Gly Leu Ile Gly Ile His Arg Phe Tyr Glu Ser Gly Val
1235 1240 1245
TTA CGT AF~A AAG TAT AAA GAT TAT TTT TGT ATA AGT AAA TGG TAC TTTi 379
I,eu Arg Lys Lys Tyr Lys Asp Tyr Phe Cys Ile Ser Lys Trp Tyr Leu
1250 1255 1260
AAA GAG GTA AAA AGG AAA CCA TAT AAG TCA AAT TTG GGA TGT AAT TGG 38~10
hys Glu 'Jal Lys Arg Lys Pro Tyr Lys Ser Asn Leu Gly Cys Asn Trp
1265 1270 1275 1280
CAG TTT ATT CCT AAA GAT GAA ACT GAA TAA 3876
GGG TGG
Gln Phe Ile Pro Lys Asp Glu Thr Glu
Gly Trp
1285 1290
i2) INFORMATION FOR SEQ ID
N0:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1291 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
b~
(ri) SEQUENCE DESCRIPTION:
SEQ ID N0:40:
Met Pro Val Thr Ile Asn Asn Tyr Asn Asp Pro Ile Asp Asn
Phe Asn
1 5 10 15
CA 02296765 2000-O1-14
WO 98108540 PCTIUS97115394
Asp Asn Ile Ile Met Met Glu Pro Pro Phe Ala Arg Gly Thr Gly Arg
20 25 30
'i'yr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Ile Pro Glu
J 35 40 45
Arg Tyr Thr Phe Gly Tyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly
50 55 60
lle Phe Asn Arg Asp Val Cys Glu Tyr Tyr Asp Pro Asp Tyr Leu Asn
65 70 75 80
Thr Asn Asp Lys Lys Asn Ile Phe Phe Gln Thr Leu Ile Lys Lcu Phe
85 90 95
1~
Asn ArgIle LysSerLysProLeuGly GluLysLeuLeu GluMetIle
100 105 110
lle AsnGly IleProTyrLeuGlyAsp ArgArgValPro LeuGluGlu
115 120 125
Phe AsnThr AsnIleAlaSerValThr ValAsnLysLeu IleSerAsn
130 135 140
Pro GlyGlu ValGluArgLysLysGly IlePheAlaAsn LeuIleIle
115 150 155 160
Phe GlyYro GlyProValLeuAsnGlu AsnGluThrIle AspIleGly
165 170 175
.i()
pie GlnAnn HisPheAlaSerArgGlu GlyPheGlyGly IleMetGln
180 185 190
Met LysPhe CysProGluTyrValSer ValPheAsnAsn ValGlnGlu
195 200 205
Asn LysGly AlaSerIlePheAsnArg ArgGlyTyrPhe SerAspPro
210 215 220
Ala LeuIle LeuMetHisGluLeuIle HisValLeuHis GlyLeuTyr
225 230 235 290
Gly IleLys ValAspAspLeuProIle ValProAsnGlu LysLysPhe
245 250 255
.~
i
Phe matGln SerThrAsp'I'hrlle~ln AlaGluGluLeu TyrTlnrPhe
260 265 270
Gly GlyGln AspProSerIleIleSer ProSerThrAsp LysSerIle
~1) 275 280 285
Tyr AspLys ValLeuGlnAsnPheArg GlyIleValAsp ArgLeuAsn
290 295 300
J~ Lys ValLeu ValCy~IleSerAspPro AsnIleAsnIle AsnIleTyr
305 310 315 320
Lys AsnLys PheLysAspLys'ryrLys PheValGluAsp SerGluGly
325 330 335
Lys TyrSer IleAspValGluSerPhe AsnLysLeuTyr LysSerheu
340 345 350
Met LeuGly PheThrGluIleAsnIle AlaGluAsnTyr LysIleLye
(W 355 360 365
Thr ArgAla SerTyrPheSerAspSer LeuProProVal LysIleLys
370 375 380
70 Asn LeuLeu AspAsnGluIleTyrThr IleGluGluGly PheAsnIle
_ ?gg _
CA 02296765 2000-O1-14
WO 98108540 PCTIUS97/15394
385 390 395 400
Ser Asp Lys Asn Met Gly Lys Glu Tyr Arg Gly Gln Asn Lys Ala Ile
405 410 415
j
Asn LysGlnAlaTyr GluGluIleSerLys GluHisLeuAlaVal Tyr
420 425 430
_ Lys IleGlnMetCys LysSerValLysVal ProGlyIleCysIle Asp
435 440 495
Val AspAsnGluAsn LeuPhePheIleAla AspLysAsnSerP.heSer
450 455 460
1~ Asp AspLeuSerLys AsnGluArgValGlu TyrAnnThrGlnAsn Asn
465 470 475 480
Tyr IieGlyAsnAsp PheProIleAsnGlu LeuIleLeuAspThr Asp
485 490 495
?U
Leu IleSerLysIle GluLeuProSerGlu Asn'ThrGluSerLeu Thr
500 505 510
lasp PheAsnValAsp ValProValTyrGlu LysGlnProAlaIle Lys
~
'J 515 520 525
~ys ValPhe'I'hrAsp GluAsnThrIlePhe GlnTyrLeuTyrSer C~ln
530 535 540
:U 'Phr PheProLeuAsn IleArgAspIleSer LeuThrSerSerPhe Asp
545 550 555 560
Asp AlaLeuLeuVal SerSerLysValTyr SerPhePheSerMet Asp
565 570 575
ii
Tyr IleLysThrAla AsnLysValValGlu AlaGlyLeuPheAia Gly
580 585 590
Trp ValLysGlnIle ValAspAspPheVal IleGluAlaAsnLys Ser
S95 600 605
Ser ThrMetAspLys IleAlaAspIleSer LeuIleValProT'yrIle
610 615 620
-)~ Gly LeuAlaLeuAsn ValGlyA,pGluThr AlaLysGlyAsnPhe Glu
G25 630 635 640
Ser AlaPheGluIle AlaGlySerSerIle LeuLeuCluPheIle Pro
645 650 655
fit)
Glu LeuLeuIlePro ValValGlyValPhe LeuLeuGluSerTyr Ile
660 665 670
Asp AsnLysAsnLys IleIleLysThrIle AspAsnAlaLeuThr Lys
675 680 685
Arg ValGluLysTrp IleAspMet'I'yrGly LeuIleValAlaGln Trp
690 695 700
O) Leu SerThrValAsn ThrGlnPheTyrThr IleLysGluGlyMet Tyr
705 710 715 720
Lys AlaLeuAsnTyr GlnAlaGlnAlaLeu GluGluIleIleLys Tyr
725 730 735
-
fi
Lys TyrAsnIleTyr SerGluGluGluLys SerAsnIleAsnIle Asn
790 745 750
Phe AsnAspIleAsn SerLysLeuAsnAsp GlyIleAsnGlnAla Met
755 760 765
_ egg _
CA 02296765 2000-O1-14
WO 98/08540 PCTIUS97/15394
Asp Asn Ile Asn Asp Phe Ile Asn Glu Cys Ser Val Ser Tyr Leu Met
770 775 780
Lys Lys Met Ile Pro Leu Ala Val Lys Lys Leu Leu Asp Phe Asp Asn
785 790 795 800
Thr Leu Lys Lys Asn Leu Leu Asn Tyr Ile Asp Glu Asn Lys Leu Tyr
805 810 815
1() Leu Ile Gly Ser Val Glu Asp Glu Lys Ser Lys Val Asp Lys Tyr Leu
820 825 830
Lys ':'hr Ile Ile Pro Phe Asp Leu Ser Thr Tyr Ser A sn Ile Glu Ile
835 840 845
l~
Leu IleLysIlePheAsn LysTyrAsnSerGluIle LeuAsnAsnIle
850 855 860
Ile LeuAsnLeuArgTyr ArgAspAsnAsnLeuIle AspLeuSerGly
865 870 875 880
Tyr GlyAlaLysValGIu ValTyrAspGlyValLys LeuAsnAspLys
885 890 895
:-,sn GlnPheLysLeuThr SerSerAlaAspSerLyy IleArgValThr
900 9U5 910
Gln AsnGlnAsnIleIle PheAsnSerMetPheLeu AspPheSerVal
y15 92U 925
()
Ser PheTrpIleArgIle ProLysTyrArgAnnAsp AspIleGlnAsn
930 935 940
'I'yr IleHisAsnGluTyr 'I'hrIleIleAsnCysMet LysAsnAsnSer
945 950 955 960
Gly 1'rpL,,sIleSerIle ArgGlyAsnArgIleIle TrpThrLeuIle
965 970 975
4(~nsp IleAsnGlyLysThr LysSerValPhePheGlu TyrAsnIleArg
980 985 990
Glu AspI1eSerGluTyr IleAsnArgTrpPhePhe ValThrIlPThr
995 1000 1005
..l
i
:a AsnLeuAspAsnAla LysIleTyrIieAsnGly ThrLeuc;luSer
n
.010 1015 1020
Asn MetTsspIleLysAsp IleGlyGluValIleVal AsnGlyGluIle
~()1025 , 1030 1035 1090
Thr PheLysLeuAspGly AspValAspArgThrGln PheIleTrpMet
1045 1050 1055
~J Lys T.yrPheSerIlePhe AsnThrGlnLeuAsnGln SerAsnIleLys
1060 1065 1070
Glu IleTyrLyeIleGln SerTyrSerGluTyrLeu LysAspPheTrp
1075 1080 1085
W)
Gly AsnProLeuMetTyr AsnLysGluTyrTyrMet PheAsnAluGly
1090 1095 1100
Asn LysAsnSerTyrIle LysLeuValLysAspSer SerValGlyGlu
1105 1110 1115 1120
Ile LeuIleArgSerLys TyrAsnGlnAsnSerAsn TyrIleAsnTyr
1125 1130 1135
70 Arg AsnLeuTyrIleGly GluLyePheIleIleArg ArgGluSerAsn
-2~)0-
CA 02296765 2000-O1-14
WO 98108540 PCTIUS97/15394
1140 1145 1150
Ser Gln Ser Ile AspAspIle ArgLysGluAsp TyrIleHis
Asn Val
1155 1160 1165
i
Leu Asp Leu Val HisHisGlu TrpArgValTyr AlaTyrLys
Leu Glu
1170 1175 1180
T'yr Phe Lys Glu GluGluLys PheLeuSerIle IleSerAsp
Gln Leu
1185 1190 1195 1206
Ser Asn Glu Phe LysThrIle IleLysGluTyr AspGluGln
Tyr Glu
1205 1210 1215
Pro Ser Tyr Ser GlnLeuLeu LysLysAspGlu GluSerThr
Cys Phe
1220 122 5 1230
nsp Asp Ile Gly IleGlyIle ArgPheTyrGlu SerGlyVal
Leu His
1235 1240 1245
Leu Arg Lys Lys LysAspTyr CysIleSerLys TrpTyrLeu
Tyr Phe
1250 1255 1260
Lys Glu Val Lys LysProTyr SerAsnLeuGly CysAsnTrp
Arg Lys
1265 1270 1275 1280
~~ln Phe Iie Pro AspGluGly ThrGlu
Lys Trp
1285 1290
?() f2) INFORMATION SEQID
FOR N0:41:
(i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH:
3876
base
Dairs
(8) TYPE:nucleic acid
>> (C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii)MOLECULE DNA(genomic)
TYPE:
( FEATURE
~
r.
)
(A) NAME/KEY: CDS
IB) LOCATION: 1..3873
li) SEQUENCE ID
DESCRIPTION: N0:41:
SEQ
:~ 1
hTG CCF.GTT ACA AATAATTTT TATAATGATCCT ATTGATAAT 48
ATA AAT
h9et Pro val Thr AsnAsnPhe TyrAsnAspPro IleAspAsn
Ile Asn
1 5 10 15
AAT AAT ATT ATT ATGGAGCCT TTTGCGAGAGGT ACGG:~GAGA 96
ATG CCA
Asn Asn Ile Ile MetGluPro PheAlaArgGly ThrGlyArg
Met Pro
20 25 30
TAT TAT AAA GCT AAAATCACA CGTATTTGGAT'AATACCGGAA 144
TTT GAT
Tyr Tyr Lys Ala LysIleThr ArgIleTrpIle IleProGlu
Phe Asp
35 40 45
AGA TAT ACT TTT TATAAACCT GATTTTAATAAA AGTTCCGGT 19~
GGA GAG
Arg Tyr Thr Phe TyrLysPro AspPheAsnLys SerSerGly
Gly Glu
SO 55 60
ATT TTT AAT AGA GTTTGTGAA TATGATCCAGAT TACTTAAAT 240
GAT TAT
Ile Phe Asn Arg ValCysGlu TyrAspProAsp TyrLeuAsn
Asp Tyr
65 70 75 80
() i
ACT AAT GAT AAA AATATATTT CAAACAATGATC AAGTTATTT 28B
AAG TTA
Thr Asn Asp Lys AsnIlePhe GlnThrMetIle LysLeuPhe
Lys Leu
85 90 95
AAT AGA ATC AAA AAACCATTG GAAAAGTTATTA GAGATGATT 336
TCA GGT
_ 7 c) j _
CA 02296765 2000-O1-14
WO 98/08540 PCT/US97/15394
Asn Arg Ile Lys Ser Lys Pro Leu Gly Glu Lys Leu Leu Glu Met Ile
100 105 110
ATA AAT GGT ATA CCT TAT CTT GGA GAT AGA CGT GTT CCA CTC GAA GAG 384
Ile Asn Gly Ile Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu Glu Glu
115 120 125
TTT AACACA ATTGCTAGT GTAACTGTTAAT TTA ATCAGTAAT 432
AAC AAA
Phe AsnThrAsn IleAlaSer ValThrValAsnLysLeu IleSerAsn
130 135 190
CCA GGAGAAGTG GAGCGAAAA AAAGGTATTTTCGCAAAT TTAATAATA 480
L~ro GlyGluVal GluArgLy, LysGlyIlePheAlaAsn LeuIleIle
145 150 155 160
TTT GGACCTGGG CCAGTTTTA AATGAAAATGAGACTATA GATATAGGT 528
Phe flyProGly ProValLeu AsnGluAsnGluThrIle AspIleGly
165 170 175
ATA CAAAATC11TTTTGCATCA AGGGAAGGCTTCGGGGGT ATAATGCAA 576
Ile GlnAsnHis PheAlaSer ArgGluGlyPheGlyGly IleMetGln
180 185 190
ATG AAGTTTTGC CCAGAATAT GTAAGCGTATTTAATAAT GTTCAAGAA 624
Met LysPheCys ProGluTyr ValSerValPheAsnAsn ValGlnGlu
195 200 205
AAC AAhGGCGCA AGTATATTT AATAGACGTGGATATTTT TCAGATCCA 67?
Asn LysGlyAla SerIlePhe AsnArgArgGlyTyrPhe SerAspPro
:10 215 220
GCC TTGATATTA ATGCATGAA CTTATACATGTTTTACTiTGGATTATAT 720
f~la LeuIleLeu MetHisGlu LeuIleHisValLeuHis GlyLeuTyr
~25 230 235 24p
i>
GGC ATTAAAGTA GATGATTTA CCAATTGTACCAAATGAA AAAAAATTT 768
<;ly IleLysVal AspAspLeu ProIleValProAsnGlu LysLysPhe
295 250 255
~()TTT ATGCAATCT ACAGATGCT ATACAGGCAGAAGAACTA TATACATTT 816
Phe MetGlnSer ThrAspAla IleGlnAlaGluGluLeu TyrThrPhe
260 265 270
GGA GGACAAGAT CCCAGCATC ATAACTCCTTCTACGGAT AAAAGTATC 864
Gly GlyGlnAsp ProSerIle lleT'hrProSerThrAsp LysSerIle
275 280 285
TAT GATAAAGTT TTGCAAAAT TTTAGAGGGATAGTTGAT AGACT'"AAC 912
Tyr AspLysVal LeuGlnAsn PheArgGlyIleValAsp ArgLeuAsn
>~1 290 295 300
AAG GTTTTAGTT TGCATATCA GATCCTAACATTAATATT AATATATAT 960
Lys ValLeuVal CysIleSer AspProAsnIleAsnIle AsnIleTyr
305 310 315 320
Ji
AAA AATAAATTT AAAGATAAA TATAAATTCGTTGAAGAT TCTGAGGGA 1008
Lys AsnLysPhe LysAspLye TyrLysPheValGluAsp SerGluGly
325 330 335
(O)AAA TATAGTATA GATGTAGAA AGTTTTGATAAATTATAT AAAAGCTTA 1056
Lys TyrSerIle AspValGlu SerPheAspLysLeuTyr LysSerLeu
340 345 350
ATG TTTGGTTTT ACAGAAACT AATATAGCAGAAAATTAT AAAATAAAA 1104
OJ Met PheGlyPhe ThrGluThr AsnIleAlaGluAsnTyr LysIleLys
355 360 365
ACT AGAGCTTCT TATTTTAGT GATTCCTTACCACCAGTA AAAATAAAA 1152
Thr ArgAlaSer TyrPheSer AspSerLeuProProVal LysIleLys
370 375 380
- 292 -
CA 02296765 2000-O1-14
WO 98108540 PC'fILJS97/15394
AAT TTA TTA GAT AAT GAA ATC TAT ACT ATA GAG GAA GGG TTT AAT ATA 1200
Asn Leu Leu Asp Asn Glu Ile Tyr Thr Ile Glu Glu Gly Phe Asn Ile
385 390 395 400
J TCT GAT AAA GAT ATG GAA AAA GAA TAT AGA GGT CAG AAT AAA GCT ATA 1248
Ser Asp Lys Asp Met Glu Lys Glu Tyr Arg Gly Gln Asn Lys Ala Ile
405 410 415
AAT AAA CAA GCT TAT GAA GAA ATT AGC AAG GAG CAT TTG GCT GTA TAT 1296
ll) Asn Lys Gln Ala Tyr Glu Glu Ile Ser Lys Glu His Leu Ala Val Tyr
420 425 430
AAG ATA CAA ATG TGT AAA AGT GTT AAA GCT CCA GGA ATA TGT ATT GAT 1344
Lys Ile Gln Met Cys Lys Ser Val Lys Ala Pro Gly Ile Cys Ile Asp
435 440 445
'_'U
GTT GAT AAT GAA GAT TTG TTC TTT ATA GCT GAT AAA AAT AGT TTT TCA 1392
Val Asp Asn Glu Asp Leu Phe Phe Ile Ala Asp Lys Asn Ser Phe Ser
450 455 460
GAT GAT TTA TCT AAA AAC GAA AGA ATA GAA TAT AAT ACA CAC AGT AAT 1440
Asp Asp Leu Ser Lys Asn Glu Arg Ile Glu Tyr Asn Thr Gln Ser Asn
465 470 475 480
TAT ATA GAA AAT GAC TTC CCT ATA AAT GAA TTA ATT TTA GAT ACT GAT 1488
Tyr lle Glu Asn Asp Phe Pro Ile Asn Glu Leu Ile Leu Asp Thr Asp
485 490 495
TTA ATA AGT AAA ATA GAA TTA CCA AGT GAA AAT ACA GAA TCA CTT ACT 1536
?~) Leu Ile Ser Lys Ile Glu Leu Pro Ser Glu Asn Thr Glu Ser Leu Thr
500 505 510
GAT TTT AAT GTA GAT GTT CCA GTA TAT GAA AAA CAA CCC GCT ATA AAA 1584
Asp Phe Asn Val Asp Val Pro Val Tyr Glu Lys Gln Pro Ala Ile Lys
iJ 515 520 525
AAA ATT TTT ACA GAT GAA AAT ACC ATC TTT CAA TAT TTA TAC TCT CAG 1632
C.ys Ile Phe Thr Asp Glu Asn Thr Ile Phe Gln Tyr Leu Tyr Ser Gln
530 535 540
ACA TTT CTC TTA GAT ATA AGA GAT ATA AGT TTA ACA TCT TCT~ TTT GAT 1680
Thr Phe Leu Leu Asp Ile Arg Asp Iie Ser Leu Thr Ser Ser Phe Asp
595 550 555 560
GAT GCATTA TTATTTTCTAAC AAA TATTCATTTTTT TCTA'rGGAT 1728
GTT
Asp AlaLeu LeuPheSerAsn LysValTyrSerPiiePhe SerMetAsp
565 570 575
TAT ATTAAA ACTGCTAATAAA GTGGTAGAAGCAGGATTA TTTGCAGGT 1776
JU Tyr IleLys ThrAlaAsnLys ValValGluAlaGlyLeu PheAlaGly
580 585 590
TGG GTGAAA CAGATAGTAAAT GATTTTGTAATCGAAGCT AATAAAAGC 1824
Trp ValLys GlnIleValAsn AspPheValIleGluAla AsnLysSer
595 600 605
AAT ACTATG GATAAAATTGCA GATATATCTCTAATTGTT CCTTATATA 1872
Asn ThrMet AspLysIleAla AspIleSerLeuIl.eVal ProTyrIle
610 615 620
GGA TTAGCT TTAAATGTAGGA AATGAAACAGCTAAAGGA AATTTTGAA 1920
Gly LeuAla LeuAsnValGly AsnGluThrAlaLysGly AsnPheGlu
625 630 635 640
(7J AAT GCTTTT GAGATTGCAGGA GCCAGTATTCTACTAGAA TTTATACCA 1968
Asn AlaPhe GluIleAlaGly AlaSerIleLeuLeuGlu PheIlePro
645 650 655
GAA CTTTTA ATACCTGTAGTT GGAGCCTTTTTATTAGAA TCATATATT 2016
Glu LeuLeu IleProValVal GlyAlaPheLeuLeuGlu SerTyrIle
;_
CA 02296765 2000-O1-14
WO 981'08540 PCT/US97115394
660 665 670
GAC AAT AAAAATAAAATT ATT ACA ATA AAT GCTTTAACTAAA 2064
AAA GAT
Asp Asn LysAsnLysIle Ile Thr Ile Asn AlaLeuThrLys
Lys Asp
J 675 680 685
AGA AAT GAAAAATGGAGT GAT TAC GGA ATA GTAGCGCAATGG 2112
ATG TTA
Rrg Asn GluLysTrpSer Asp Tyr Gly Ile ValAlaGlnTrp _
Met Leu
690 695 700
CTC TCA ACAGTTAATACT CAA TAT ACA AAA GAGGGAATGTAT 2160
TTT ATA
Leu Ser ThrValAsnT'hrGln Tyr Thr Lys GluGlyMetTyr
Phe Ile
SOS 710 715 720
:1AGGCT TTAAATTATCAA GCA GCA TTG GAA ATAATAAAATAC 22pg
CAA GAA
Lys Ala LeuAsnTyrGln Ala Ala Leu Glu IleileLysTyr
Gln Glu
725 730 735
AGA TAT AATATATATTCT GAA GAA AAG AAT ATTAACATCGAT 2256
AAA TCA
T~rgTyr AsnIleTyrSer Glu Glu Lys Asn IleAsnIleAsp
Lys Ser
790 745 750
TTT AAT GAT ATA AAT TCT AAA CTT AAT GAG GGT ATT AAC CAA GCT ATA 2304
Phe Asn Asp Ile Asn Ser Lys Leu Asn Glu Gly Ile Asn Gln Ala Ile
755 760 765
C'~F,TT~sTATAAATAATTTT AATGGATGT GTATC~~TATTTAATG '
ATA TCT 3
5:
i.spRsn IleAsnAsnhhe IleAsnGlyCys ValSerTyrLeuMet
Ser
770 775 780
i()
F,AAAT,AATGATTCCATTA GCTGTAGAAAAA CTAGACTTTGATAAT 2400
TTA
L;s Lys MetIleProLeu AlaValGluLys LeuAspPheAspAsn
Leu
785 790 795 800
.-.-",CTCTC ARAAAAAATTTG TTAAATTATATA GAAAATAAATTATAT 2448
GAT
Thr Leu LysLysAsnLeu LeuAsnTyrIle GluAsnLyeLeuTyr
Asp
805 810 815
TTG ATT GGAAGTGCAGAA TATGAAAAATCA GTAAATAAATACTTG 2496
AAA
.,euIle GlySerAlaGlu TyrGluLysSer ValAsnLysTyrLeu
Lys
820 825 830
AAA ACC ATTATGCCGTTT GATCTTTCAATA ACCAATGATACAATA 2544
TAT
Lys Thr IleMetProPhe AspLeuSerIle ThrAsnAspThrIle
Tyr
N35 890 845
'Tr1ATA GhAATCTTT.AAT AAATATAATRGC ATTT'~AAATAATATT 2592
GAA
Leu Ile GluMetPheAsn LysTyrAsnSer IleLeuAsnAsnIle
Glu
85U 855 860
J
()
ATC TTA AATTTAAGATAT AAGGATAATAAT ATAGATTTATCAGGA 2640
TTA
Ile Leu AsnLeuArgTyr LysAspAsnAsn IleAspLeuSerGly
Leu
865 870 875 880
TAT GGG GCAAAGGTAGAG GTATATGATGGA GAGCTTAATGATAAA 2688
GTC
Tyr Gly AlaLysValGlu ValTyrAspGly GluLeuAsnAspLys
Val
885 890 895
AAT CAA TTTAAATTAACT AGTTCAGCA AGT AAGATTAGAGTG ACT 2736
AAT
TssnGln PheLysLeuThr SeiSerAlaAsnSer LysIleArgVal Thr
900 905 910
CAA AAT CRGAATATCATA TTTAATAGTGTGTTC CTTGATTTTAGC GTT 2784
Gin Asn GlnAsnIleIle PheAsnSerValPhe LeuAspPheSer Val
915 920 925
AGC TTT TGGATAAGAATA CCTAAATATAAGAAT GATGGTATACAA AAT 2832
Ser Phe TrpIleArgIle ProLysTyrLysAsn AspGlyIleGln Asn
930 935 940
- 294 -
CA 02296765 2000-O1-14
WO 98108540 PCTIUS97115394
TAT ATT CAT AAT GAA TAT ACA ATA ATT AAT TGT ATG AAA AAT AAT TCG 2880
Tyr Ile His Asn Glu Tyr Thr Ile Ile Asn Cys Met Lys Asn Asn Ser
945 950 955 960
J GGC TGG AAA ATA TCT ATT AGG GGT AAT AGG ATA ATA TGG ACT TTA ATT 2928
Gly Trp Lys Ile Ser Ile Arg Gly Asn Arg Ile Ile Trp Thr Leu Ile
965 970 975
GAT ATA AAT GGA AAA ACC AAA TCG GTA TTT TTT GAA TAT AAC ATA AGA 2976
Asp Ile Asn Gly Lys Thr Lys Ser Val Phe Phe Glu Tyr Asn Ile Arg
980 985 990
GAA GAT ATA TCA GAG TAT ATA AAT AGA TGG TTT TTT GTA ACT ATT ACT 3024
Glu Asp Ile Ser Glu Tyr Ile Asn Arg Trp Phe Phe Val Thr Ile Thr
995 1000 1005
AAT AAT TTG RAT AAC GCT AAA ATT TAT ATT AAT GGT AAG CTA GAA TCA 3072
Asn Asn Leu Asn Asn Ala Lys Ile Tyr Ile Asn Gly Lys Leu Glu Ser
1010 1015 1020
AAT ACA GAT ATT AAA GAT ATA AGA GAA GTT ATT GCT AAT GGT GAA ATA 3120
Asn Thr Asp Ile Lys Asp Ile Arg Glu Val Ile Ala Asn Gly Glu Ile
1025 1030 1035 1040
ATA TTT AAA TTA GAT GGT GAT ATA GAT AGA RCA CAA TTT ATT TGG ATG 3168
Iie Phe L_.rs Leu Asp Gly Asp Ile Asp Arg Thr Gln Phe Ile Trp Met
1045 1050 1055
:,AA TAT TTC AGT ATT TTT AAT ACG GAA TTA AGT CAA TCA AAT ATT GAA 3216
.i~) Lys Tyr Phe Ser Ile Phe Asn Thr Glu Leu Ser Gln Ser Asn Ile Glu
1060 1065 1070
GAA AGA TAT AAA ATT CAA TCA TAT AGC GAA TAT TTR AAA GAT TTT TGG 3269
Glu Arg Tyr Lys Ile Gin Ser Tyr Ser Glu Tyr Leu Lys Asp the Trp
~J 1075 1080 1085
GGA AAT CCT TTA ATG TAC AAT AAA GAA TAT TAT ATG TTT AAT GCG GGG 3312
Gly Asn Pro Leu Met Tyr Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly
1090 1095 1100
~~)
AAT AAA AAT TCA TAT ATT AAA CTA AAG AAA GAT TCA CCT GTA GGT GAA 3360
Asn Lys Asn Ser Tyr Ile Lys Leu Lys Lys Asp Ser Pro Val Gly Glu
1105 1110 1115 110
aTT TTA ACA CGT AGC AAA TAT AAT CAA AAT TCT AAA TAT A':A AAT TAT 3408
tle Leu 'ihr Arg Ser Lys Tyr Asn Gln Asn Ser Lys Tyr Ile Asn Tyr
1125 1130 1135
AGA GAT TTA TAT ATT GGA GAA AAA TTT ATT ATA AGA AGA AAG TCA AAT 3456
~(J Arg Asp Leu Tyr Ile Gly Glu Lys Phe Ile Ile Arg Arg Lys Ser Asn
1140 1145 1150
TCT CAA TCT ATA AAT GAT GAT ATA GTT AGA AAA GAA GAT TAT ATA TAT 3504
Ser Gln Ser Ile Asn Asp Asp Ile Val Arg Lys Glu Asp Tyr Ile Tyr
1155 1160 1165
CTA GAT TTT TTT AAT TTA AAT CAA GAG TGG AGA GTA TAT ACC TAT AAA 3552
Leu Asp Phe Phe Asn Leu Asn Gln Glu Trp Arg Val Tyr Thr Tyr Lys
1170 1175 1180
(~(1
TAT TTT AAG AAA GAG GAA GAA AAA TTG TTT TTA GCT CCT ATA AGT GAT 3600
Tyr Phe Lys Lys Clu Glu Glu Lys Leu Phe Leu Ala Pro Ile Ser Rsp
1185 1190 1195 1200
OJ TCT GAT GAG TTT TAC AAT ACT ATA CAA ATA AAA GAA TAT GAT GAA CAG 3648
Ser Asp Glu Phe Tyr Asn Thr Ile Gln Ile Lys Glu Tyr Asp Glu Gln
1205 1210 1215
$-
CA 02296765 2000-O1-14
WD 9g~g~p PCTIIJS97115394
CCA ACA TAT AGT TGT CAG TTG CTT TTT AAA AAA GAT GAA GAA AGT ACT 3696
Pro Thr Tyr Ser Cys Gln Leu Leu Phe Lys Lys Asp Glu Glu Ser Thr
1220 1225 1230
GAT GAG ATA GGA TTG ATT GGT ATT CAT CGT TTC TAC GAA TCT GGA ATT 3744
Asp Glu Ile Gly Leu Ile Gly Ile His Arg Phe Tyr Glu Ser Gly Ile
1235 1240 1245
GTA TTT GAA GAG TAT AAA GAT TAT TTT TGT ATA AGT AAA TGG TAC TTA 3792
11) Val Phe Glu Glu Tyr Lys Asp Tyr Phe Cys Ile Ser Lys Trp Tyr Leu
1250 1255 1260
AAA GAG GTA AAA AGG AAA CCA TAT AAT TTA AAA TTG GGA TGT AAT TGG 3840
Lys Glu Val Lys Arg Lys Pro Tyr Asn Leu Lys Leu Gly Cys Asn Trp
1265 1270 1275 1280
CAG TTT ATT CCT AAA GAT GAA GGG TGG ACT GAA TAA 3876
Gln Phe Ile 1'ro Lys Asp Glu Gly Trp Thr Glu
1285 1290
y)
(2) INFORMATION 5EQ ID N0:42:
FOR
(i) SEQUENCECHARACTERISTICS:
(A) acids
LENGTH:
1291
amino
~J (D)
TYPE:
amino
acid
(D1
TOPOLOGY:
linear
iii) MOLECULETYPE: protein
?t) (xi) SEQUENCEDESCRIPTION: IDN0:42:
SEQ
Met Yro Thr Asn Asn Phe TyrAsn ProIleAsp
VW Ile Asn Asp Asn
I 5 10 15
Asn Asn Ile Met Glu Pro PheAla GlyThrGly
Ile Met Pro Arg llrg
20 25 30
Tyr Tyr Ala Lys Ile Thr ArgIle IleIlePro
Lys Phe Asp Trp Glu
35 40 45
Arg Tyr Thr Phe G.ly Tyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Cly
50 55 60
Ile Phe Asn Arg Asp Val Cys Glu Tyr Tyr Asp Pro Asp T'yr Leu Asn
GS 70 75 80
'rhr Asn Asp Lys Lys Asn Ile Phe Leu Gln Thr Met Ile Lys Leu the
85 90 95
Asn Arg Ile Lys Ser Lys Pro Leu Gly Glu Lys Leu Leu Glu Met Ile
100 105 110
Ile Asn Gly Ile Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu Glu Glu
115 120 125
ji
Phe Asn 1'hr Asn Ile Ala Ser Val Thr Val Asn Lys Leu Ile Ser Asn
130 135 140
Pro Gly Glu Val Giu Arg Lys Lys Gly Ile Phe Ala Asn Leu Ile Ile
145 150 155 160
CA 02296765 2000-O1-14
WO 9810854 PCT/US97/l5394
~Phe Gly Pro Gly Pro Val Leu Asn Glu Asn Glu Thr Ile Asp Ile Gly
165 170 175
Ile Gln Asn His Phe Ala Ser Arg Glu Gly Phe Gly Gly Ile Met Gln
J 1$0 185 190
Met Lys Phe Cys Pro Glu Tyr Val Ser Val Phe Asn Asn Val Gln Glu
195 200 205
Asn Lys Gly Ala Ser Ile Phe Asn Arg Arg Gly Tyr Phe Ser Asp Pro
210 215 220
Ala Leu IleLeuMetHis GluLeuIleHisVal LeuHisGlyLeu'Cyr
X25 230 235 240
li
Gly Ile LysValAspAsp LeuProIleValPro AsnGluLysLysPhe
295 250 255
Phe Met GlnSerThrAsp AlaIleGlnAlaGlu GluLeuTyrThrPhe
260 265 270
Gly Gly GlnAspProSer IleIleThrProSer ThrAspLysSerIle
275 280 285
~J ~'yrAsp LysValLeuGln AsnPheArgGlyIle ValAspArgLeuAsn
290 295 300
L;:sVal LeuValCysIle SerAspP=-oAsnIle AsnIleAsnIleT~r
305 310 315 320
i
t)
L.rsAsn LysPheLysAsp LysTyrLysPheVal GluAspSerGluGly
325 330 335
Lys Tv_rSerIleAspVal GluSerPheAspLys LeuTyrLysSerLeu
?? 340 345 350
Met Phe GlyPheThrClu ThrAsnIleAlaGlu AsnTyrLysIleLys
355 360 365
Thr Arg AlaSerTyrPhe SerAspSerLeuPro ProValLysIleLys
370 375 380
Asn Leu LeuAspAsnGlu IleTyrThrIleGlu GluGlyPheAsnlle
385 390 395 400
.~
i
Ser Asp LysAspMetGlu LysGluTyrArgGly GlnAsnLysAlalle
405 410 41!i
F~snLys GlnAlaTyrGlu GluIieSerLysGlu HisLeuA1aValTyr
J~) 420 425 930
Lys Ile GlnMetCysLys SerValLysAlaPro GlyIleCysIleAsp
435 440 445
~J Val Asp AsnGluAspLeu PhePheIleAlaAsp LysAsnSerPheSer
450 455 460
Asp Asp LeuSerLysAsn GluArgIleGluTyr AsnThrGlnSerAsn
465 470 975 480
Tyr Ile Giu Asn Asp Phe Pro Ile Asn Glu Leu Ile Leu Asp Thr Asp
485 490 495
Leu Ile Ser Lys Ile Glu Leu Pro Ser Glu Asn Thr Glu Ser Leu Thr
UJ 500 505 S10
Asp Phe Asn Val Asp Val Pro Val Tyr Glu Lys Gln Pro Ala Ile Lys
515 520 525
7l) Lys Ile Phe Thr Asp Glu Asn Thr Ile Phe Gln Tyr Leu Tyr Ser Gln
- 297 -
CA 02296765 2000-O1-14
~rp 9g~p PCTIUS97/15394
530 535 540
Thr Phe Leu Leu Asp Ile Arg Asp Ile Ser Leu Thr Ser Ser Phe Aso
545 550 555 560
i
Asp Ala Leu Leu Phe Ser Asn Lys Val Tyr Ser Phe Phe Ser Met Asp
565 570 575
Tyr Ile Lys Thr Ala Asn Lys Val Val Glu Ala Gly Leu Phe Ala Gly
) t) 580 585 590
Trp Val Lys Gln Ile Val Asn Asp Phe VaI Ile Glu Ala Asn hys Ser
595 600 605
Asn Thr Met Asp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro ~ryr Ile
610 615 620
Gly Leu Ala Leu Asn Val Gly Asn Glu Thr Ala Lys Gly Asn Phe Glu
625 630 635 640
Asn Ala Phe Glu Ile Ala Gly AIa Ser Ile Leu Leu Glu Phe Ile Pro
645 650 655
Glu Leu Leu Ile Pro Val Val Gly Ala Phe Leu Leu Glu Ser Tyr Ile
660 665 670
l~sp Asn Lys Asn Lys Ile Ile Lys Thr Ile Asp Asn Ala Leu Thr Lys
675 580 685
!() F.rc7 Ann Vlu Lys Trp Ser Asp Met Tyr Gly Leu Ile Val Ala Gln Trp
59U 695 700
I.eu Sar Thr Val Asn Thr Gln Phe Tyr Thr Ile Lys Glu Gly Met Tyr
7D5 710 715 720
i~
Lys AIa Leu Asn T~~r Gln Ala Gln Ala Leu Glu Glu Ile Ile Lys Tyr
725 730 735
Arg 'I':~r Asn Ile Tyr Ser Glu Lys Glu Lys Ser Asn Ile Ann Ile Asp
'14U 745 750
Phe Asn App IIe Asn Ser Lys Leu Asn Glu Gly Iie T.sn Gln Ala lle
755 760 765
Asp Asn Ile Asn Asn Phe Ile Asn Gly Cys Ser Val Ser Tyr Leu Met
770 775 780
Lys Lys Met Ile Pro Leu Ala Val Glu Lys Leu Leu Asp Phe Asp Asn
785 790 795 800
J (~)
Thr Leu Lys Lys Asn Leu Leu Asn Tyr Ile Asp Glu Asn Lys Leu Tyr
805 B10 815
i,eu Ile Gly Ser Ala Glu Tyr Glu Lys Ser Lys Val Asn Lys Tyr Leu
JJ 820 825 830
Lys ThrIle MetProPheAsp LeuSerIleTyrThr AsnAsp Ile
Thr
835 84U 845
Leu IleGlu MetPheAsnLys TyrAsnSerG1uIle LeuAsnAsnIle
850 855 860
Ile LeuAsn LeuArgTyrLys AppAsnAsnLeuIle AspLeuSerGly
865 870 875 880
~t
Tyr GlyAla LysValGluVal TyrAspGlyValGlu LeuAsnAspLye
885 890 895
Asn GlnPhe LysLeuThrSer SerAlaAsnSerLys IleArQValThr
900 9D5 910
CA 02296765 2000-O1-14
WO 98/8540 PCT/US9711S394
Gln Asn Gln Asn Ile Ile Phe Asn Ser Val Phe Leu Asp Phe Ser Val
915 920 925
Ser Phe Trp Ile Arg Ile Pro Lys Tyr Lys Asn Asp Gly Ile Gln Asn
930 935 940
Tyr Ile His Asn Glu Tyr Thr Ile Ile Asn Cys Met Lys Asn Asn Ser
945 950 955 960
I() Gly Trp Lys Ile Ser Ile Arg Gly Asn Arg Ile Ile Trp Thr Leu Ile
965 970 975
Asp Ile Asn Gly Lys Thr Lys Ser Val Phe Phe Glu Tyr Asn Ile Arg
9A0 9B5 990
J1
Glu Asp Ile Ser Glu Tyr Ile Asn Arg Trp Phe Phe Val Thr Ile Thr
995 1000 1005
Asn Asn AsnAsnAla Lys Tyr IleAsnGly Leu GluSer
Leu Ile Lys
- 1010 1015 1020
Jlsn Thr IleLysAsp Ile Glu ValIleAla Gly GluIle
Asp Arg Asn
1025 1030 1035 1040
Ile Fine LeuAspGly Asp Asp ArgThrGln Ile TrpMet
Lys Ile Phe
1045 1050 1055
Lys Ty: SerIlePhe Asn Glu LeuSerGln Asn IleGlu
Phe Thr Ser
1060 1065
la7o
Glu Arg Tyr Lye Ile Gln Ser Tyr Ser Glu Tyr Leu Lys Asp Phe Trp
1075 1080
1085
Gly A>n Pro Leu Met Tyr Asn Lys Glu 'fyr Tyr Met Phe Asn A1a Gly
1090 1095 1100
Asn Lys Asn TyrIle LysLeuhysLysAspSer ProValGly
Ser Glu
110 5 111 0
1115 1120
Ile Leu 1'hrArgSerLys TyrAsnGlnAsnSerLys .TyrIleAsn
'rya
1125 1130
1135
Arg Asp LeuTyrIleGly GluLysPheIleIleArg ArgLysSer
Asn
1140 1145 1150
Ser Gln SerIleAsnAsp AspIleValArgLysGlu rlspTy_~Ile
Tvr
1155 1160 1165
Leu Asp thePheAsnLeu AsnGlnGluTrpArgVal TyrT'hrTyr
Lys
1170 1175 1180
Tyr Phe LysLysGluGlu GluLysLeuPheLeuAla ProIleSer
Asp
1185 1190 1195 1200
Ser Asp GluPheTyrAsn ThrIleGlnIleLysGlu TyrAspGlu
Gln
1205 1210 1.x.15
Pro Thr TyrSerCysGln LeuLeuPheLysLysAsp GluGluSer
Thr
1220 1225 1230
Asp Glu IieGlyLeuIle GlyIleHisArgPheTyr GluSerGly
Ile
1235 1240 1245
Val Phe GluGluTyrLys AspTyrPheCysIleSer LysTrpTyr
Leu
1250
1255 1260
Lys Glu ValLysArgLys ProTyrAsnLeuLysLeu Gl.yCy~Asn
Trp
1.265 1270 1275
1280
Gln Phe IleProLysAsp GluGlyTrpThrGlu
CA 02296765 2000-O1-14
yy0 gg~qp PCT/US97/15394
1285 1290
(2) INFORMATION FOR SEQ ID N0:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1526 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D> TOPOLOGY: linear
IU
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "DNA"
i ir.l FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 108..1523
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:43:
AGATCTCGAT CCCGCGAAAT TAATACGACT CACTATAGGG GRATTGTGAG CGGATAACAA 60
TTCCCCTCTA GAAATAATTT TGTTTAACTT TAAGAAGGAG ATATACC ATG GGC CAT 116
Met Gly His
1
~i
C~'~T CAT CF1T CAT C'.AT CAT CAT CAT CAC AGC AGC GGC CAT ATC GAA GGT 164
His tiffs His His His His His His His Ser Ser Gly His Ile Glu Gly
10 15
~() CGT CAfATGGCTAGC ATGGCTGATACAATA CTAATAGAA TTTAAT 212
ATG
Arg flipMetAlaSer MetAlaAspThrIle LeuIleGluMetPheAsn
~0 25 30 35
AAA TATAATAGCGAA ATTTTAAATAATATT ATCTTAAATTTAAGATAT 260
?J Lys TyrAsnSerGlu IleLeuAsnAsnIle IleLeuAsnLeuArgTyr
40 45 50
AGA GATAATAATTTA ATAGATTTATCAGGA TATGGAGCAAAGGTAGAG 308
Arg AppAsnAsnLeu IleAspLeuSerGly TyrGlyAlaLy>ValGlu
55 60 65
C;TA TT1TGATGGGGTC AAGCTTAATGATAAA AATCAATTTAAATTAACT_ 356
Val TyrAspGlyVal LysLeuAsnAspLys AsnGlnPheLysLeuThr
70 75 80
AGT TCAGCAGATAGT AAGATTAGAGTCACT CAAAATCAGAATATTATA 404
Ser SerAlaAspSer LysIleArgValThr GlnAnnGlnAsnIleIle
85 90 95
TTT AATAGTATGTTC CTTGATTTTAGCGTT AGCTTTTGGATAAGGATA 452
Phe AsnSerMetPhe LeuAspPheSerVal SerPheTrpIleArgIle
~
100 105 110 115
CCT AAATATAGGAAT GATGATATACAAAAT TATATTCATAATGAATAT 500
J~ Pro LysTyrArgAsn AspAspIleGlnAsn TyrIleHisAsnGluTyr
120 125 130
ACG ATAATTAATTGT ATGAAAAATAATTCA GGCTGGAAAATATCTATT. 548
Thr IleIleAsnCys MetLysAsnAnnSer GlyTrpLysIleSerIle
135 140 145
AGG GGTAATAGGATA ATATGGACCTTAATT GATATAAATGGAAAAACC 596
Arg GlyAsnArgIle IleTrpThrLeuIle AspIleAsnClyLysThr
150 155 160
(~
i
AAA TCAGTATTTTTT GAATATAACATAAGA GAAGATATATCAGAGTAT 644
Lys SerValPhePhe GluTyrAsnIleArg GluAspIleSerGluTyr
165 170 175
ATA AATAGATGGTTT TTTGTAACTATTACT AATAATTTGGATAATGCT 692
-3UU-
CA 02296765 2000-O1-14
...: .
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