Note: Descriptions are shown in the official language in which they were submitted.
,~ WO 95/35376 2 1 ~ 3 5 2 6 PCT~US9S~07895
.
~d~ with
Interleulcin 8 Reoentor I tlL8Rl) Bindin~ Domains
tinn
Technical Fldd
The invention ~lal~es generally to lL8RI bintling domains. More
specifically. tne invention relates to (I) P '~1 . ' ' other than nativc n,8, compqising one
or nx re IldRI speoific binding ~lomains; and (2) ~ ' a~nsing one or more
d~ sed IL8RI ~cific bind~ng dt~mains. This inventio~ also rdates to I '~ ' '
a~coding tbe ~ '~r, - ' of the present invention. a method of using the ~ . ' and
melhod of producing the ~ , ' of the p~sent invcntion otilizing these
le ' The ~ . ' of the present invention can thus act s either
~gonis~ go ~; of IL8 foi, IL8RI or ll~R2 binding.
P ~ ' of the ~vention
C~lls ~ilize diEFusible modi~rs, cdlod cy~ines, to signd oDe Dother. A
of g~ines re tbe which iDCIodes n,8. J~. review ~ticle about
tbo chemol~e , r ~ ~litten by Miller a d., _t Rev lmm~ 12(12~: 17-46
(~992) ~md Baggiolini a al., Ad~! Immunol 55: 97-179 ~1994), haein . ' by
. ~.
SIUBSTITUTE ~ Er (RU~E~)
wo gsl3s376 2 1 9 3 5 2 6 PCT/US95/07895 ~
--2--
The ' are a group of structurally and ~ relateo
cyt~ Recent studics indicate that these proteins function in the .~. and
~n of leulcocytes Jnd other cells ~u sites of " and, therefore, appear to
be in~tant ~ ~ rnediators. S '1~, these ~nolectlles are small secrcled
pro~a that a~hibit corranon secondary proteio s~ructure and display four conaaved
cg~dne residues. The cornmon secondary s~ure of a ~' -' exhibit the foDowing
fc~es: (I) n uluno taminal loop; t2) ~ sheet in the form
of G~ Icey, nd t3) sn C~minal a~ hdix, which lies over the ~shee~ ause a
syslenutic . ' - _ for these protans has not yet been generally agreed, the proteins
Jre divided into two farnilies according to the spacing of the first two cysteine residues of
he n~ure proteins. The families are r~felred to as the CXC or CC farr~ly. In the CXC
fatnily, the ftrst two cysteine residues ale separated by an arnino acid residue; the first two
cysteine residues in the CC fan~ly are not. To date, seventeen ~ ' have beendescribe~. Six are rnembers of the CX~' family and include, pla~a factor 4 tlPF4); ~
i' , ' ' NAlP-I/L8; gro ~, ~, and r. ~-lo; rnig; ENA-78. Tbe CXC family isso hlown as the farnily. The remamulg ' are part of the CC family:
protdns tM~P-lu and MIIP-I~); monocyte ~ '
protun l/JE tMCIP-l/lE); RANlES; H~'-14; C10. and 1-309. This fartlily has also been
desigmled as the ~ farnily.
Of interest, native human IL8 acts as a ~' - for, , ' ' . and
induccs ~ ' ~aia upon systemic illjection ~nd s~in reac~on upon local injection, in
- ' u~s See ~zoni a ~. ~1991) 173: 771-774; V n D~nme. a aL J Ex~
167: 1364-1376; Ribso a al., 1 ~ . 73: 472~77 (1991). lhe rnolecule slso
~s the rele~se of ~ide nions Imd cL;cits rdcsse c~f the prinury granule
cf ~,'~ ~nclttding ~ e ndclas~se
N~ive ~n lL8 rnedi tes these biolclgic~ activitics by binding to *s rooeptor ~nd~8 ~ignal i ' 1, ~ C~dc cf rel~ctions ttltimately resulting in ~ biological
respoa~
.
SUBSTlrUT~ SHEET ~RU~E~6)
~ WO 9~513~376 2 1 9 3 5 2 6 F~ ;.,,s
-3-
Presently, two IL8 binding reeeptors have been identified and are tcnned
rlLgRI" and "IL8R~. Tbe a nino acid sequence of these pc.l~ id-" are described in
Mulphy ct ~1_ ience ~: 1280 (I,,I) ~nd Holrnes ct al."Scienee 253: 1278 (1,91),
herein , ' by rcfen~nee. Oth.er ' ' e~n eompete with ~8 to bind to the
IL8R2, ~eh u GRO~, GRO~, GROr. NAP-2 nd ENA-78 ha~e been tmplicated with
D 8R2 bin~ing by ~ :' - , with native n,g by rnasuring Ca~.
O~ers have idc~ified regions of nuive hlunan lL8 that ale i~nplia~ted in both L8RI and
~gR2 b~nding. However, at this ~ne, no ehemolcine is Icnown to cornpele ~ith nati~e IL8
fcT the lL8RI specific binding.
Disclosure of the Invention
It is one of the objiects of the present invent~on to eonfer n,8RI specific
bind~ng to 8, `.~ dc other than IL8 and, in partieular 8 I,JlJ~ Jt;~ possessing a
chemol~ine protein structure lil~e IL,8, by . ' ~ one or rnore IL8RI specific binding
dorn~ins. Tbe p~ ~ other than IL8 that posses a ehernol~ine protein structure
ineludes, for e%atnple, PF4, ~;' ' g' GROa, GRO~, GROy, IP-10, rnig,
ENA-78, MlP-la, ~DP-I,~, MCF'-IIJE, RANTES, HC-14, CIQ and 1-309. Ihe binding
domains ~re inlroduced into the l:hemo~ine protan structu~e such tbat the spacing of the
binding domains perlnit lL8RI binding.
It is n object of the present in~ention to provid~ ied L8 rnolecule tbat its binding ffinity to IL8RI is eithtr enhanced or ~edueed.
er ob~oct of the p~ent invention is to pro~ide n alte~d lL8RI
binding domsiD ~ ", ' posscssing chon~ine protein ~tre capable
of n~dDhting n*Rl pecific bi~lding ~finity. An e~mple of ~ cbe~nolcine otber than IL8
that i~ prDvidod witb ~ fmlaioo-l; cf ~8, i.c., binding to IL8RI, rnay be a
GROrp~, o that tbe lesul~ng cl~ n is n IL8JGRI~ `~,, ' Tbe al~red
domsin ~y be m de in the native n,8 or be ilnrodu~ i8 to ~er ~ . ' for
xatnp]e, tbat possesses ~ chemcil~ne protein sln~e
SU3STITUTESH~ LE26~
WO 9S/3S376 2 1 9 3 5 2 6 PCTNS9S/0789S ~,
-4-
Ya anotha object of the invenion includes providing p~ ' ' that
encode the instant desired p~lJ,A,~, vectors, and host cells that are capable ofprod tcing DUCh, '~ ' from the ~1~ ' - Furtha, rnethods of producing the
instant p~ re alDo provided.
F~ther, it is an object of the invention to provide a mahod of inhibiting or
inaeasing the biological ~ity of ra ti~e lL8 by contacting a tsrga ceD with the
of the p~sent invention.
Modes of Carlvin~ Out The InYention
The inventors herein have identified the arr~ino acid sequences of two ~8RI
specific binding do~nains within the naive IL8 p~ J,,. In r ' with the objects
of the present invention, thaefore, pul~Jt;J~,D comprising one or more IL8RI rDpecif~c
binding domains are provided, as weD as p ~ ' ' vectors and host cell containingDUch. Also provided is a rnethod of p~ucing the ~l~ ~s and a tnethod of using
them
More ~,~ii,~D)" native IL8 is l~nown, presently, to bind to two receptors,
IL8RI and lL8R2 on the surface of cert3in ceD types, such aD ~, ' The arnino acid
sequence of theDse binding dornains D~L~II~ affect the ability of native ~8 to bind to
IL8RI. The binding domains identified herein ean be linhd with otha a~nino acid
sequences to eonstr~ct ~ ~r, - ' , other than native n,8, that a~e capabb of binding to
IL8RI. Prefaably, wch other atnino Jcid sequences are effective to preclude rapid
of the r 'b. .
Prefer~bly, these binding domains ~re ~n~ed with amino ~cid sequences
daived from ~ f the , ' '~ of p~roteins e~Ded the _ ' Thus, the
IL8RI bitlding donuins can be lin~od vith f~monts daivod from o~er ~ to
const~uct I ~ r, ' ' that e~bit the c~¢anon ~econd~uy ~ res of :- '
Pul~,, ' a~.hb~dng these econd~y 8Iruaures w~ ut the l~ding domain~D) to
~ssutne a cimilar - ' as found iD rlative IL8. E~amples of _h '
âUBSTll iJTE SffEEr (Rl~LE26)
21 93526
wo ss/3s376 ~ a/u~ ,~
-5-
includc PF4, ~ JROcL, GRO~, GROy, IP-10, mig, ENA-78, MIP-ICl,
MIP-I~, MCP~ E, RANTES, HC-14, C10, and 1-309.
The arnino acid sa~uence of nalive ~8 can be dtercd within its binding
d~ins ~ iDcrease c~r deaease its L8RI binding affinity, for e~ample, by or
debdon of Imno acid residues.
The p~esent E ~1, can be dividecl into two classes:
id_., oth than Dative ILg,
~t least one L8RI specific binding dornain; nd
(2) pol~l . compIising an alxred n.8RI
spccific binding dcnnain.
Tbe, ~, ' of the preseM illvention rnay or rnay not e~hibit ~ chemolcine protcinstruc~re. The instant pol~ having similar c~r enhanced LgRI binding affinity as
cc~red to nadve Lg and can l~ompete with native Lg f~r ILgRI. Also, ~ fi~s
with decreased binding affinity to IL8RI as corr~ared to native L8 can be effec~ive
of native ILg fclr the other Tec~ptor, L8R2.
initions
The teTms as defi led herein fclrm part of he disclosure of the present
The NMR and X-~ray WJ ' " ,, , h~ , Tevealed that the three
~- ' structure of tbe ~' ' is ~ siTr~lar, heTein refared to as thc
' ' plotein ~e." lhe struaure of the T~ive hu~un 11~8 h~s boen solved and
is TllOdel f the cbanR~ine pT DteiD stn~ The stroc~re includes n ~
loc~ heet (G~ }cy), ~Ki ~buA~ . ' bdi~L
The ~ ~s ~ e to~l of tbe ~ sboct. Furlha, n tive huT~un IL8 fonDs a
h_ i hh 2-fold ~us o~' SyT~, a siA-strandod ~ sbeet v,ith a pair of belices
Iying ~p tbe ~ boet. Tne plataneDt of d~e cy~ines and tbe size of tbe ~ sbeet ~re adso
f~rs m the tbree ' ' stTucture.
SllB~lTU~E SHEEr (R~2~)
wo ssl3s376 2 1 9 3 5 2 6 r( ~ o ~
Thc present inventors have detesrnined herein the pol~.J~ regions of
r~tive IL8 that affect specific IL8Rl bir~ing Tbe dornains identified by thc inventors
belan are tbose that , - '~ ffect lL8RI binding and not IL8R2 binding These
legic~s ~re ~fcned tc~ as ~8RI Spec~lc binding domains " These domains are found in
the ' loop nd in ~nd 3 of tbe ~ sheet of na~re IL8 Though these
dc~ins nay not intenlct di~y with lL8RI, the IL8RI binding affinity of a IL8
~ .~ r ~n be drastic ally reduced ~rhen these dornains are ~placed by L ,,
dom ins fro~n otber ~ ' ' such as GROr, n IL8R2 ~gonis~ Using the binding
dc~uns of nuire human IL8 as an exa~nple, the arnino acid sequence of an IL8RI
binding domain contains a sequence Ser-Ala-Lys~lu-Leu-Arg-Cys-Gln-Cys-ne-Lys-Thr-
Tyr-Sa-L~ rP' -His, (asnino acid residues of I to 18 of SEQ ~ N0 1); rnore
prefe~ably, the arnino acid sequence corltains tbe sequence Glu-Leu-Arg-Cys-Gln-Cys-lle-
Lys-Thr-Tyr-Ser-Lys-Pro-Phe-His (resiclues 4 to 18 of SEQ ~ N0 1); even rnore
prefaably, tbe arnino acid sequence colltains tbe sequence Lys-Tbr-Tyr-Ser-Lys (residues
I l to 15 of SEQ ID N0 1) The a nincl acid sequence of an L8RI specific binding
don~iD can also contain tbe sequence Lys-Xaa-Tyr-Xaa-Lys. (SEQ ID N0 3) These
~o cid sequences are e~amples of "amino terminal" binding domains because tbe
scquences ~re based on tbe sequence o~' tbe am no tminal portion of native IL8
Anoth e~atnpb of the ~uno acid sequence of ~n IL8RI binding domain
con~ins tbe sequence Gly-Arg~lu-L1:u-Cys-Leu-~Pro (tesidues 46 to 53 of SEQ ID
NO lk tnore preferably, tbe smino ~ sequence cont~ins tbe sequence ~rg-Glu-Leu-Cys-
~ sidues 47 tn S3 of SEQ ~ N0 1) Tbe mino c~id soguence of ~tn IL8RIQocific linding domain c n contun tbe ~énce ~g-Glu-Leu-X a-X a-Xaa-Pro, (SEQ ID
N0 4) lbese ~s re eDrnples of "~ ~beet" binding domdns be~use tbe
re b~ed on tbe quonce of the ~ sheet of n~ive IL8
- lbe l~nding dom~ns of other nuive IL8, ~t s nuivle bovine IL8, porcine
1~, a~, re whilin tbe , ` of tbe present invention, nd c~n be ~ ' ' by
SIJBSTIIUTE SHEET (RU~E 26)
2 I q3526
~, WO 95t35376 P~
_7_
sequence alignrneot, for e~ample, according to the conserved cysteine residues to native
ns.
Preferably, the preient ~ ~t;J~ contain Iwo L8RI specific oind~ng
domains "spaced within the F '~, ' to perrnit lL8RI bindi~g. In this regard, thebinding dorlnains ~Ire spaced ~ L within the primary sequence of the pol~l~~4i~., wben the I ~, . ' assumes its th~e ' ^ the
Luinding dom~ins ~re positioned tl7 efficiently interact with the other portions of the
, '.,L~J~_ ~nd/or the recq7tor tl) pennit IL8RI binding. IPreferably, the preSent
,We possesses a ehemo~ Lne protein structure to rnirnic the three ~-
5" of these dornains fi7und iln the native D,8.
The a~nino acid sequence of the IL8RI specific binding dornains can be
"altered, for e~unple, by arnino acid - ' c, deletions or insertions, to eitner
increase or decrease ~8RI spcafic binding affinity. ~ , one or more sequences
of annino acids can be inserted, dldeted, or su~7stituted to truncate or e~tcise the binding
domain from the ~)IJr ~ - ~, sul-h as rlative IL8 IL8R1 17inding domains can be e~tcised
from native human IL8 and replaced ~vith amino acid seql~ence from a t -r 3
region of other ~ L_ ~ , such as GRO~. The amino acid residues t~LIat are
of pa~ticular interest fo} n.8RI specific binding have b~en identified herein as residues 11
(Lys), 13 ~ryr), 15 (Lys), 47 (~g), 48 (Glu), 49 aeu), arld 53 (Pro) of the native hurnan
~8 (SEQ ~ NO:I). lbesc Jmino acid residues are mainsained in the p~esent F 1~
to confa lL8RI spe~ific birlding affiniy or ~re altered or deleted to reduce 0 enhance
IURl ~ecific Luinding aEfiniy.
Binding of ~ to ~ r~7tor is of ~en time ~ mlata of degree.
r . ~" ~ ~d by those 1;~illed in tbe ~ t~e 7ecq~r birlding is usually ssossed
by the ~binding ~finiy" of tbe 1 '~r . - ' One rneans of ~ g binding ffiniy is
to measure lbe dliliy of the I '~, . ' to coml7ete ~rith nativc n.8 for IURI- The IC5D
i~ tbe - th t inhibits 5~ of the n~l ~r bindiPg of the
nativc IL8; the smaller tbe IC~D- the g~eatcr the binding affinity. Wore, a r ~I/r ~
SIJBSTITUT~ SHEE~ (RU~E26)
wo 95/35376 2 1 9 3 5 2 6
-8-
is considered to bind to IL8RI if, for example, its lC50 is above ' 1~ or a negative
eontrol.
lbe instant ~ ~} . - ' ean oe used to rmodulatt an IL8 receptor-mediated
bqological response.r Such biological responsts include, for e~ample, those ctUular
~vitits wbich ~re triggtred by the b~d~ng of IL~ to its receptor. ~' ' ' occurs
whtn tht instant ~ . ' eompete with tbt nativt IL8 for lL8RI and result in dtber~n ~crease or deaease of ~t bast one of these ceUular acti~rities. The na~re of thest
~ivities rnay be b ' ' or b~.vlJh~ ` ' For t~nple, a I ~} . ' modulatts an
~8 receptor-modiased response if it does not stimulate the same signal i ' as IL8
when tbe ~1~ Wc binds to an lL8 receptor. Tbe increase or deaease can be monitored
using various assays, described further b~:low, which also utilize IL8 receptor molecules as
controls.
More ~ , a cascade of b; ' reactions is tliggered when IL8
binds to its ~c~ptor. As tbe tenn is applied herein, tbe instant ~1~, ' wiU modulate
~n IL8 receptor-mediated response when it causes an increase or declease irJ any one of
these reactions. For e~ample, IL.8.receptors are G-coupled protcins which, whcn proper
signal i ac~ivity occurs, triggcrs an increase of '' ' Ca2~ and an
~ivation of ~ C. Sigral I ' can be measured by observing the
levels of inositol .' . ' (IP3) and ~ ~vl ~AG), whicb are increased due to
C activation and cyclic AI~IP (cAM). C ' ass~lys can be used to
measure tbe '' ' levds of Cs5', ]IP~, ~nd DAG to detclTnine whether tbe n.8
' ~sporise has boen rr_ ~ys for rang levds of free
cytosolic C~ re ~nown.
~ ive IL8r rcfers to ~ . . ' having ~n unino cid sogoence which
is idendc l to ~ soquence ~et ~1 ~ ource which r~raUy produoes L8, such as
humsn, b~viDe, pcie Qr other ' ~ces. N~ve IL8 may ble of ~y in lengtb
pecies to ~pecies An a~ampk of o~ive IL8 is Ihe ht~un IL:8 which has the unino
cid sequellce JS ~hown in SEQ ~ NO:I.
SUBSTITVTE SHEET (RU~E26)
~ ` 2 1 93526
~ WO 95135376 PCT/USg5/07895
_9_
T~te terrn "DL8 reeeptor," as usai herein refers to any of the several
vertebrate DL8 rec~,rs, or fraglrtents thereof which are c~tpable of bindirtg to DL8. For
e~r~le, hutnart IL8RI and IL8]R2 ~re . , ~t by d~is t0n.
l'rte ~errrt "e -' " refers to a . ' '~ of na~rally oceurnrtg
p~çins, whictt ~ diflusible rna~ors that eells use to signal otte rtoth. ~ review
~cle by Miller ct al., loe. d~., describes ~he ehetno~ine ~ . lbe ' are
rels,~ed. Reeent studies irtdicnte ~tat dlese potéirts function in
~te utd tctivation oi~ leui~ocytes arid other eells at sites of ~ artd,
therefore, ~pear to be irriponant " ~ rrtediators. S~ , these r~tolecules
~re srriall seereteo proteins that ~isplay four conserved eysteine residues. To date. about
seventeert different _ have beert described. They include plssdet faetor 4 (PF4);
NAP-I/IL8; gro a, ~, and y, IP-10; rriig; ENA-78; .' _
' ~ y proteins (MIP-I artd MIP-I~); rnOnOcyte ' protein-l/lE
(MCP-I/JE); RANlES; HC-14; C10, and 1-309. Other ' ean be idendfied by
tbeir arriino acid horrtology to dte Irnown ' ' und by their sirnilarity in seeondary
protein structures and biologicil activities to the ~rtown i' ' -
A " ' ' _ rtrnount" of dhe present pctl.~l . refers to dhe u~ountooeded to erihartce or reduce dlle DL8 reeeptor rnediated biological response of a eell
~oducing L8 ~eceptor. Such l~iologieil responses ean be rnoriitored by dhc assays
describ~d below.
An ~inbibiting alrtount" of dle preseM r ~ refers to dhe aTnount
noodod 0 irthibit IL8 binding OD ttte IL8 ~ce~s. ~agb DL8 birtding rnay not be
a~lely ~ _ i loss DL8 will be bDand OD its ~tors in ~hc prescnce of rt
inbibitu~g ~t ~Df tbe p~se~lt pOl~r, - ' tbart in dle ~bxn~
E~unpla of ~furi~ctiorul; ' - ~ o~ rtn altered or unalter~d ttirtding
don~in or ~ - ' irtcl~e roceptor bi~ling ~fi~ty, ability 00 tliggcr ~ biologicalre~sc, 5ig~ r, ~ - ~!tC. Thtl~ Dl~;ROrc~imcrac~ tits tbe s~ne i~nctional
~ts IL8 if dte chirrtcra hits the sune t~ceptor binding ~Iffinity, for c~arnple.
âUBSTITUTE SHEE~ (Rl~E2~
WO 9~/3~376 2 1 9 3 5 2 6 ~ v . s
-10-
A , conta~ining A is " ' "~ free of' B when at least 85%
by weight of the total A+B in the . is A. P~fably, A compriises at least
bont 90* by weight of the total of A~]B in the . - more preferably at least
~t 9~ or even 999~ by weight.
A kornoter" is a DNA sequence that initiates and regulates thc
. of coding sequence when the promoter is operably lin~eo to the coding
prwnoter is - ~ n to the codiog sequence when the pr'omoter is not
~Iy lin~ed to the codiing sequence iin nature. In contrast, a "oative" or ~
~moter is opcrably lin~ed to the coding sequence in nahlre.
An "origin of replication" is a DNA sequence that iioitiates and regulates
~ic~or~ of pol~ ' ' such as arl expression vector. The origin of replication
behsYes as an unit of ~ ' replicatiion withiin a cell, capable of
replica~ion ~nder its owrl control. With certain origins of r 1' " , an expression vector
c n be reproduced at a high copy nurnbl~r in the presence of the -rr ~ ' proteins within
tbe ceL~ Enamples of such origins are the 2~1 and ~ '~ replicating sequences,
wbich Jre effective in yeast; nd the ~ral T-antigen, effective iin COS-7 cells. O~er
origins of replicalion are l~nown in the alrt and can be utilized in the ~,.,. host.
An n . vector" is a p~l~ ' ' that comprises ~1~ ' ' ~
d~t regulate the expressiion of a coding sequence and incl~ies, for example, a promoter, a
tamin;llor and an origin of . -- -
Host cells capable of Ino iucing the F~sent ~ ' are cultured "underoonditions iindDcing , ~ " Soch ~ onditions allow i , uld tr~nslation of the
ng the F '~,, lbose conditions include cultivation i
~en - rDdia . , - pH, ~. For example, ~f the ~p prornoter is
~d iin tbe e~ession V# lor, the r~ w~l 1~ tryptophan to t~iigger the pr~noter and
~IDce i . 'IhC ex~ct canditiorls w~l v~ry f~n host ceJI to host cell and from
c~siorl vector to expression vector.
SUBSrlTllTE ~HEET (RU~E26J
21 93526
~ WO 95135376 PCT/US95/07895
-11-
B. Gcneral Mcthod
P of thc Amino Acid Seau0ce of L8Rl Sr~ccific Bindin~ Domains
'llte present inventors have determined the F '~,, ' regions of nalivc L8
that ffect specific IL8RI binding. Tbe domains identified hcrein ~rc thoK that
'1~ ffect IL~RI binding ~md r~ IL8R2 binding. Thus, an . ' - of thc
p esent invention, tbc ~bility to bi~ld IL8RI is com~rcd on ~tny pol~l . ' by '
u least one L8RI binding domai~l, tbus producing an antagonist of D,8 binding to IL8RI.
In a preferred . t, dte ~ 1~ of tnc Fresent invention contains nvo binding
domains. One domain is selected from the group of amino taminal binding dom~ins and
the other domain is selected from tne group of ~ sneet domains ss desciibed i.~ greatcr
detail below.
The following amino acid sequences are e~amples of amino termiT~ binding
domains. The group was thusly named because dte sequences are bas~d on the amino acid
~equence of tbe amulo tenninal pt~rtion of nalive D~8. Using the binding domains of
nstive human IL8 as n escample, the amino acid soquence of an n,8RI binding domain is
Sa-Ala-Lys-Glu-Leu-Arg~ys~ln~s De-Lys-Thr-Tyr-Sa-L~ Ib~, P~ , (amino acid
residues of I to 18 of SEQ 11) N(~.l); rnore preferably, the amino acid sequence is Glu-
Leu-Arg-Cy~Gln Cys-D~-Lys-l~-Tyr-Ser-Lys-Pro-Phe-His (residues 4 to 18 of SEQ IDNO:I); even more p~efaably, the amino acid sequence is Lys-Thr-Tyr-Ser-Lys (residues 11
to 1~ of SEQ ID NO:l). The ~nino cid sequence of Jn lL8RI specific binding domain
can ~150 be Lys-xut-Tyr-xsa-Lys (SEQ ID NO:3), whae ~sa represents ny amino acid~ue.
htotbcr group of IL8Rl binding dom ins is tbe group of ~ sbeet binding
domuns. lhe mino cid #quel~ces of dtete domains Are bascd on the ~oguence of the~tird ~and of d~e ~ sbeet of Dsi ve IUI. ht e~ntple of ~e ~no cid sequatce of such
~tn IL8RI specific binding domaut is Gly-h~Glu-l~L~u-~Pn~ (residues 46 to ~3
of SEQ lD NO:l); more prefaablly, Ihe ~no cid ~e is Arg-Glu-Leu-Cys-Leu-
SUB'~TITlJT~ SHEET (RU~E ~6)
WO 95135376 2 1 9 3 5 2 6 PCTIUS95/07895
-12-
Asp-Pro ~residues 47 to 53 of SEQ ID NO: 1). The amino acid sequence of an IL8R1specific binding domain can also be Arg-Glu-Leu-Xaa-Xaa-Xaa-Pro (SEQ ID NO:4).
The binding domains of other native IL8, such as native bovine IL8, can be
determined by sequence alignment of the native human IL8 to other native IL8.
The presence of any one of the binding domains above may be necessary by
not be optimal to influence receptor binding, ~ i(,uhuly when placed in the context of a
non-IL8 poly~ Lille. In a preferred . ~ Y : of the present invention, therefore, a
polyl,~Lide comprising IL8R1 binding ~lomains possess a chemokine protein structure so
that the IL8R1 specific bmdmg domains may assume a ~"..ri~"..,.l;.", similar to the one in
native IL8. Once the primary sequence of the binding dornain is determined to be used in
the present polyy~lid~.~, in one c...l~ll;,.,. .1l of the mverltion, the domains are spaced
within the ~olyl.~Lide to permit IL8R1 binding.
A poly~Li~ exhibiting a chemokine protein structure and comprising one
or more IL8R1 specific binding domains can be ~ (l by the following formula:
A - B - C;
where B represents an IL8R1 specific binding domam. Optionally, B can represent a
sequence containing more than one IL8R1 specific bmding domain, such as ,~ i by
the formula -b, - X - b2 - where bl and b2 each represent an IL8R1 specific binding
domain and X represents one or more amino acid residues. Preferably, the amino acid
sequence of b~ is selected from a group of amino terminal bindmg domains, and the
amino acid sequence of b2 is selected from the group of ~ sheet bmding domains as
disclosed above. Also, preferably, togelher A - B - C exhibit the secondary structural
features of a rhPmnkin.o
The poly~ Lid~ of the present invention exhibiting a chemokine protein
strQCture comprise four conserved cysteine residues when properly aligned with other
chemokine ~u~.r~ily members. The, l....,..1~;... - can be aligned utilizing typical
sequence alignment programs. An example of am alignment of the . l....,~i.~. ~ is shown in
Miller et al., Crit Rev Tmmun 12(1.2):17-46 (19g2). The corlserved cysteines form
~UBSTITU~E SHE~ (RU~E26~
~ wo ssl3s3~6 2 ~ 9 3 5 2 6 r~ 0 ~
disulfide oonds that aid thc formation a che~nolcine protein s~ucturc. A pol~ .lc of the
present invendon e7~hioiting a chano~ine protein struc~re, ~referably, thaefore, comprises
n mino temlinal portion, ~hich includes ~ loop; ~ i . ' ' ,~ sheet in the fo~n of a
G~dc ~y; and C-tenr~nal a he]hl~ that lies over tbe ~ sheet
lbe three stranded 1~ sheet of the I ~ ~, ' of the preseM inYaltion is
~enbly of ~rdlar size to those found in ~ Por e~am,ole, the strands of the ,B
d~et re about 12 to 3 ~nino acicl residues in bngth; more p~ferably, fro n ~DOUt 10 to 3
~no cid residues; most pref~ly, 7 to 3 mino cid residues. The amino acid
equence af the ,~ sheet ~8RI spl~cific domains are preferably , ~ into this
r,econdary strucb~re; rnore preferat~ly, the sequence of the do~nain is placed in the third
~and of the ~ sheet.
The C-tenninal a helil~ of the pbl~, , ' o~ the present invention having a
chanoldne pratein strucbrre lies aver the ~ sheet. The kng~h of the belh~ is not critical
nd may or ~nay not ovahang the edge of the ~ shee~ Usually, the length of the a-heli~
is from at~out g to 25 residues; m~re usuaDy, from at~out 1~ to 22; evan mare usuaUy 15 to
bout 19 residues. Typically, the hdk is an ~ .-, helil~ that rnay t~e positivdy 0
negatively charged. Most che~nol;ine hdices are positivdy chargd. The charge of the
heli~ can t~e chosal depanding if similar ar dissimilar biological activity is desired.
The amino taminal panion cont3ins an tail ~hich ~ains no panicular
s~cture and an loop. Preferably, mino acid sequence of lhe unino tenr~nal IL8RI
binding domains ~re , ~ in the loop panion. The en~re pcrlion including tail and
loop is from bout 25 t,D 14 ulinD icid residu~; more p~Dly, f~om ~bout 22 to about
18 mino ~ roddoas. lbe loop con~ises frorn bout IS tD bout 6 mino cid
~iduc~ more prd~bly bout 12 to bout 8 annno cid r~sidocs. In ~ion, preferably.
he tail af the mino t0nin~ p~rtian con~es Ihe ~nnno; cid soguence Glu-Lc~Arg
This sequence is ~ ~Le far IL8 seceptor binding.
SUE~STiTUTE SHEE~ 6~
WO95/35376 21 ~ 3 5 2 6 PCT/US95107895 ~
-14-
ln addition, the present ~ s , ' ' herein rnay contain
sequeDces that are not specific for ~81~1 binding but are sequences that are non-specific.
These xquences, lite Glu-Leu-Arg C&~ affect binding of dth IL8 receytors.
More ~ ~ ~ q~, ~ a c.*im-ric chemol~ne is one means of
` _ a l '~, . ' of the present invention bav~ing binding doma~ns ~ 's~
to perrDit Il.8RI biDding. Por e~mpl~, the n.8RI specific binding domains can spaced to
pe~Tni~ ~8RI hinding by ' ~ ti~e don~uDs for the ' ' ~ regions in the
GROr y ~ , the C-terminal a helis of GRO~ c&n be subsituted into
the Dative hurnan IL8 ~ ,t;d~. Therefore, the binding dorna}ns retain their native
5, In such nn . ~ ' t, the ~IS.,, ' can eJ~hibit non-naive lL8
iiological ~vity due to the preseDce of the GROr c bclbu Thusl iD one . ' " of
the present invention, the bindiDg domain of IL8Rl can be placed in & pol.~ t;~i~ having
chemol~De protein structure so as to displaced tbe i , " native chemol ine
sequences. Thus, conferring IL8R1 bi~nding activity to that p~ while
the chemol~ine protein structure.
In an altaDative ~ , it rnay be desirable to i~nsert an L8RI binding
donuin in a pGI~l . ' without removal of any sequences. TDe tecbniques for insation,
deletion ~nd ' of amino &cicl residues by &Itering pol~ ' ' sequences
er~Dg the ~1~, . ' to be altered are ._ ' iD the &r
ID addition, f~ments cf the &nUDO &~id sequences of the :' ' can
be ~anbled togetber to conslruct & p ~!}, ' of tl~e preseDt invention. For e~;~uDple,
the pracnt p ~ . ' may possess ti~e nnno ~i sequeuce of dle YIUDo td of
n tive DtmuD IL8, tbe f~t two stand; of tbe ,~ sheet ~e of N~P-2, the third strand
of t'he ~ sboet of IL8, ~i the a hdi~ of GRO~ Tbe unino ~cid equences to be utilized
tD ~ct tbe, ~r . - ' of the preseDt illVelltiC~D do Dot bave to be idcntical to the
~ces fouDd i~ tbe ~ ' to e~ubit Ihe clesi~d socondary strua~e features. For
aun~lc, tbe IIIIDO cid soqu~ces n~ly oe r~nts or fusions of thc ~xnces fouDd in
tbe ~' '
SUBSTITLITE ~HEEr ~RULE 26)
~ WO 95135376 2 1 9 3 5 2 6 PCI/lJS95/07895
-15-
Mutants of the ' ' c~n be ~ by rnil~ing w..s~
arnino cid ' - of such. lhe following are e~amples of ~ ~dti.
Gly ~ Ala; Val ~ nc ~ L~; Asp ~ Glu; Lys ~ .4rg; Asn ~ Gln; and
Pbe ~ Trp ~ Syr. Also, insa~ions and ddetions can be mdde to the amino acid
soquences of tbe ' ' p~n ided ~at the chenioldne prDtein s~ure is
Tbe choice of u~ o cid sequence for tbe p3esent ~ - ' can also be
~osen iFor tbe~r bility to confcr i~unctional ' Por e~unple, the heli
~ce of GRC~ rn~y be chosal for tbe p~sent ~ '~, . ' to confa a biological
~ivity of GROy. Further. it is ~ the sequences of the pr~esent ~ ;d~ cdn
be altcred to reduce or enhance t}le biological activities. Por exan~le, the amino acid
~ce a GROplL8 chimerd alhibiting lL8RI specific binding can be altered to reducerls ability to trigga IL8RI signal ~ '
The binding dornains can be altered to inaease or decrease the binding
sffirdty of the present ~ . to IUR I . Such ~ , with altered binding
affini~es can be used as agorlsts or ~ _ of IL8 as desired. Mutants of the binding
don~ins can be _ i, for l~ample, by mal~ing lunino acid ' thdt rnaintain
or enbznce or reduce the binding affinity of the ~I~ to IL8R1. Otba altaed
binding donuins can be rnade by deleting or inserting residues to the arnino acid sequence
of the ~Itcrcd bir~ding domains so as to alter tbe binding affinity of the ~
~dditional amino ~cid residues can be . ' ~t the N- or C~minus. In particular,
so~ne or dl of tbe amino acid re!;idues of the binding domun clm be eJ~cis~d to decrease
be lim}ing dlinity of the p~eser~a ~ ~r ir' '
lhe ino cid re~;dues tbat re of p~r ime~st for IL8RI spociEic
binding ~re l li~.' ' in the bi~Dding domains bd~:
Ser-Al~L~u-Leu-Arg Cys GI~'' !, Ibr-l~r-Ser-L~P~Pne-His,
(a~nino ~d residl~es of I to 18 of SI~Q II) NO.I);
Gh~A~II ~L~hr-l'yr-S~r-L~Pa~P~Hi$
- (residues 4 to 18 of SEQ ~) NO:l);
~UE~STITUTE ~llEE~ (~ULE26~
r
WO 95/35376 2 1 9 3 5 2 6 PCTNS95/07895
-16-
L~7hr-l`yr-Ser-L~, (residues 11 so 15 of SEQ ID NO:I);
L~X~T~r-X a-L~, (SEQ ID NO:3);
Gly-~Gl~La~L,_ ~ ,. (~sidues 46 so S3 of SEQ ID NO:I);
J~r~Gh-L~~ sp P~ idues 47 so 53 of SEQ ~ NO:I); ~nd
A~GI~Le~X~X~Xaa-Pro, (SEQ IO NO:4).
The higblighted umno id ~dues cone~Dpond so ~Didues 11, 13, 15, 47, 4g, 49, and 53
of SEQ n) NO:I. Prcfersbly, ~ese anmw acid residues in she binding dorNuns are altered
s~y . dele~on, or inse rdon of ~nosher ;~mino acid residue so enhance os da rease
she bind~ng a~inity of she dornain.
Construc~nr P.d~ - ' Encodin~ the r~l~,~vt;~D. EsDression Vectors. and Host
Cells
Once she ancQo acid sequence of she presenS ~ Jt;d~ is
then pol~ ' encoding the ~ D can be c The F~
soquences can be isolaled fmJn }nown librarics. The ~ , sequences can be ligatedtogether to produce a c#hng sequence. Known linl~ers or restrictions sites can be used to
construct the varions ~nents. These ~equences can be altercd us~ng I ~ chain
~on (PCR) or Qte specific . _ ~Ite~nadve, the I '~ ' sequence c2n
be syntbesized with a '1~ available,
Tbe ~ ' encoding the present ~ r -~ " ~ csn be used to
cons~uct n e~ession vector to produc~ tbe F ~,, ' At the nunun~ an e~F,ression
voc~r w~D con~in ~ promo~r ~vhich iD opable in tbe host cell nd ~lporably hn~d to the
} ~ ~ptbep~sent~ '!,,, ' E~ion-voc~snuy lsoinclude
signsl _, I - lectable mar}ers, origins of ,' nd ~equences
' _ ~D boDt ~1 oguences for ~urposes of integ~a~ion into tbe bost genome. These
~ditiond ~ ts re aption~l b~t c~n be includod tD opdmize
A promoter is a DNA se~loence ~srn or 5' to the I ~
encoding tbe present ~ ~,, ' The ~pron~a wiD initiate and regulate e~rpression of tbe
SUBSTITUTE S~IEET (RULE ~6)
21 9352~
wo s~/3~376
-17-
coding sequence in the desircd host cell. To initiate expression, promoter sequences bind
RNA ~ ~ arld initiate the ' . (3') i . ' of a coding sequence (e.g.
~wal ,,ene) into rnRNA. A prorno~r may Iso bave DNA sequences that }cgulate the
ntc of e~cpression by er~hancing or ~ - q~ irlducirlg t)r Tcpressir~g i , These
~queDces t an overla.D the sequallxs that initiue expressior,. Most host t dl sys~ems
include regul tory ~cxs withirl tne prornoter sequerPxs. For eT~rnplc, when a
repressor protcin binds to the lac operon, an E. c~l~i rcgula~Ty prmnoter set~uence,
. of the ~ . gene is inhibited. Another exa~nple is the ycast alcohol
~' h~ " promoter, which has an u,Dslream at~vuor sequence (UAS) that modulates
ex,Dression in the ~bscncc of a readily available source of vlucose. ~ " some
viral crlhancers not only arnplify l~ut also regulate e~prcssit~n ir~ ' txlls. These
enhancers can be ~ ' intD " promoter sequences, nd the promoter will
become ac~ve only in the prcsentc of an inducer, such as a hormone or enzyrne substrate
(Sassone-Corsi and Borelli (1986~1 Trcnds Cenet. 2:215; MEniads c~ al. (198~) Science
236:1237).
Functional ' promot~rs may also be uscd, for e~ample, synthetic
prorno~rs based on a t onsensus ~equence of different u~urflotcrs. Also, effective
prornoters can t ontain a ~ revion ~n~ed with a ~ v e~Lpression initiation
revion. Exarnples of hyb id pror~ers re tbe E. co~i lac operator lin~ed to the E. t~o~i tac
, - ~ivation revion; thl: yeast alt ohol ~ S~ ' (ADH) revula~ory sequence
hn~ed to tbe yeast gl~ (GAPDH) L '. '
ctivatiorl revion (U.S. P~t Nos. 4,876,197 nd 4,880,734, . ' bercin by
~x); nd the _,; ...L_ (CMV) enhanccr lin~ed to the SV40 (simian virus)
l~r.
Ibe ~ er~VP the present ~ ~, . ' rnay Iso be ~ed in
~g f~me to ~ Siv~ ~cc lbe sivru~ qu~x f~ment ~fpically eDcodes a
peptide cornp~ixd of ~, "' ' annno cids which tlirects the prescnt ~ ~,, ' to the
~xll rnernb~ane. Preferably, there are processing sites er coded between the leader fragment
~IBSTITU~E SH~Er (RUl E26~
wo ss/3s376 2 1 9 3 5 2 6 PCT/USgS/07X9S
-18-
and the gene or fragment thaeof that call be cleaved either in vivo or in v~o DNA
encoding s~itable signal sequences can be derived frorn genes for secre~ed ' " host
ceO protdns, soch s the yeast invertase gene tEP 12 873; rP o2,096,080), the A-factor
tene (U S P~t No 4,588,o84), interf~ron dgnal sequence (EP oO 057)
A prefemd class of seaaion leadas, for ycast ~, are those that
~nploy ~ ~ragrnen~ of the yeast ~ ~ r gene, which contains both a "pre" signal
equence, nd a "pro" region The types of ~ rtnr fragrnents that can be employed
include the fuD-length pre-pro alpha factor leader tabout 83 unino acid rcsidues) as weO as
truncated ~ ~ ~gr1nr leaders (typicaDy ~bout 25 to about 50 amino acid residues) tU S
Pa~t Nos 4,540,083 and 4,870,008, ' herein by reference; EP 324 274)
~dditional leaders ernploying an - ~ r ' leaoer fragment that pro~ides for secretion
ir~clude hybrid ~ ~; - sr1llr leaders made with a I , of a first yeast signal
scquence, but a pro-region from a second yeast ~ - 'grtnr (See e.g., PCP WO
89102463 )
TypicaDy, i are regulatory sequences, such as I ~h~l~Jla~ l and
sequences, located 3' or ~ of the stop codon of the
F ~ encoding the present Q ~ I~,~. L UsuaOy, the terminator of rlative host
cell proteins are operable when attached 3' of the I ',~ ' ~ ' encoding the preseM
E~nples re the S~ enswe ~ ~ terminator and the
' .~ termtnator Further, viral i are also cperable in certain host ceOs;
fRr instance, the SV40 temunator is fum tional irl CHO cells
For ~ xeleclable rn~s, an angin of , ' nd
5~l ' ,, ho~t cells ~qoences rna9 qptior~y be incloded in n aqpresdRn vectDr A
~e rn~er c n be uxd to xlseeD for host ceOs that poteDdaOy cRrlt~n the
~e#ion ~ar SDch m~rs rn~y r~ ~ the host cell m~nme to drugs wch ~s
~lin. '' , 1, _~1 , uoornyan, nd ~ Iso, rr~ers may
be ~, ~' ~ genes, uch ~s those in 1he his~ ne~ r ,' and leucine pathways
~UBSTITUTE SHEEr (RULE26)
~ W095135376 21 9 3526 Pcrn~ssS/a7sss
-19-
1'nus, vhen kuc;iine iis absent Som l~e mediia, for e~ample, only thc ceDis with a
; ., ~ ' Bene iin the lalcine pa~hwsy will wrviive.
jig~n of ~pL~iion may be neodod for the a~ssion V# lor hereiin to
~te iiD the bost celL (~'in oruins of ~pl'iic~iion enablc n a~prcss'iion vector to be
~oduc~ ~t ~ hiigh copy n~nber iin lhe plesence of tDe, . , pmc~'ins ~ithin thecdL e~mpks ~ Qnigins th~t an ~e usod Daa'in ~re t'De 2p and ' ',~ Icplicstiing
~peoces, ~hich re cffeAiive in ~c~; u~d the vi~l T~ e~eco'ive in ~IOS-7 ceJ~s.
r, veC~s he~D nuy be iiD~ted iir~to tDe DO~ cdl genorne or
~m ~ ~iin thecell. ~1~ ' ' sequer~es~- ' ,, to soquences
~Sin the host cell genorne may b~ ncaled iirl the eJ~pressiioL veaor to iintegrate the
a~sion assette. ~literna~ive, t~e ~ ' _ soquences ~re not lin}ed to the
e}sion vector. For e~ample, e~sion vectors can inteerate into the CHO genome vian ~ched ~' ~ ' ' ' red~se gene. In ycsst, it is rLore ' _ ff the
' _ sequences fl~ the a~pression cassc~ yeast
geDome ~ces ~re those diisclosed in PCI WO9O/01800~ ~d the HIS4 gene sequences,
de~d iD Genbanl~, accession n3. 101331.
lbe choiice of prom~ter, taminator, and otber optioD I demcnts of arl
e~iion vt~ ~11 slso dq~nd on the host cell chosl. Tbe inveDtion is not dcpendent
on tD~ host cell kc~d. ~ d the kvcl of prol~ iin eqlression w~l diictate the
op~ bost cell. ~ variety of hos~s for C~ssioD heleiin &re ~nown m the rt nd
rni~e from the Am~ican l~pe Culmre C~ion (~TC~:). B~l bosts suitable for
_tDcpacntr ~,, ' iii~lode, ~t}imiD~ion r , ~ - ,~,
q ~ ~ " ~b~, r, r ~ 1~ - , aDd S ~ - Yst hosts
the fdb~ ~ msy b~ , r~
_, , S ., . ~d Y~ 1 - '- ' '- ~ost cdls
1~t c~ be ~od be~ ib~ t re not linbtd to CHiO ~, HeL~ cdls, b~y h~ns~er
~q ~HR) alls, mo~ ~idD~ alk (OOS), h~Nn ' . ~ ~ c~ cdls
. Slop G2). u~d odler cdl line:s. A oumber of in~.t ~ Di hos~ re diso avdl ble for
SUBSTI~UTE SHe~T (Rl~L~26)
WO 95135376 2 1 9 3 5 2 6 r~l,L~ ~ ~
-20-
e~pression of ~ A ~-lr~e,,-, proteins: Aedes a~gypti, Bon~y~ mori. Drosophla
, ~nd .C.r ', frugiperd'a as desc~ibed in PCI W0 89~146699; Carbonell
a al., (1985) J. Virol. 56:153; Wright (~1986) Nature 321:718; Srruth et al., (1983) Mol.
ÇeD. BiQI. 3:2156; and see gene~ally, P~aser, a al. (1989) in Yirro CeD. Dev. Biol. 25:225.
. -
~ fter ~rector - ~ the e~pression vector con~ising a p
encoding the p~esent ~1~1, ' is insested into the host ceD. Many i
techniques e~ist in th~ aTt for inse~ting ~pression vectors into bacterial, yeast, insect, and
cdls. Tbe j e, Flrocedure to introduce the e cpression vector
d~pends upon tbe host to be
Methods of . ' ~ ogeDous DNA into bacterial hosts, for e~anrple,
re ~reD-~nown in the ~, and typicaDy protocol includes dther treating the ba~ria with
ÇaCI~ or other aLents, such as divalent cations and DMSO. DNA can Plso be introduced
into bæ~ial ~lls by .~ r viral infection. r '- p~ocedures usually
vary with the bactcrial species to be I ' ' as desaibed in ~ g., ~lasson a al. (1989)
F~MS Microbiol. Lett. 60 273; P~va ~t al. (1982) Proc. Natl. Acad. Sci. USA 79:5582;
EP Publ. Nos. 036 259 and 063 953; PCI WO 84~4541, Bacil~), ~r a al. (1988)
Proc. Nad. ~cad. Sci. 85:856; Wang a al. (Isso) ~. Bac~riol. 172:949, C' ,, ~
(Cohen a al. (1973) Proc. Nad. Ac~d. Sci. 69:2110; Dowa a a~ (1988) Nucleic Acids
Res. 16:6127; R~sbna (1978) "~ roved n~hod for ~ r - of r ' ' - coli
~ritb CoEl-~hived pl~ds in ~çnaiç~jneaine: Proce4din~ ~ ~e 1 ~ - -
rr~. on Ga~c Eneineaiq~eds. H.W. Boya nd S. Nicosia); Mandel ~t al.
(1970) J. MoL Biol. ~:159, S~eto (1988) Biochim Biol~bvs ~ 949:318; r
a~ agg7) FEMS MicrobioL Lc~ 4~:173 r~ ie~n~r ct al. (~988)
An~L Bio~l~n 17038, P~ ~, (Aug~ ct ol. (1990) FE~S Mi~oiol. LetL
.203, ' ~ ), (B~ny a ai' (~sso) J. B~ioL 144:698, H~ (i987)
~1~ r - of ~ s by d~_t~ in Su Genetics (ed.
SVBSTIITUTE SHEET ~RULE26)
21 93~
WO 9S/35376 PCT/[~S9S/0789S
-21-
J. Fenetti and R. Curtiss m): Perry et al. (1981) lnfec. Ilrunun. ~:1295; Powcll et al.
(1988) A~DI. EnYiron. Microbiol. i4:655; Sornlcuti a al. (1987) Proc. 4dh Evr. Con~.
E- ~ . IA12,.' ., ).
'- rnetlhods f0 yeast hosts are also well-~nown in dhe ar~ and
t,Ypically include dtber tbe L ' - of, .' or of irnact yeast cells t~ated
witS l}ali c~ions Fl - . is ulother mams f0 i ' ~ st hosts. lhese
rnahods re desaibeJ m, f~r c~ ple, Med ods in r . . ~1 _.. Volwne 194, 1991,
Yiuide to Ycast G~ics and Mo]lec~r Biology." T ~ procodures usuaDy
Yaty with the yeast spocies to be i ' i, e.g., Kurtz a al. (1986) Mol. Cell. Biol.
6:142 ano Kunze a al;. (1985) J. ]3asic Microbiol. 25:141; f0 Cantida; Gleeson et al.
(1986) ~. Gen. Microbiol. 132:34~i9 ano P ~_ ' . et al. (1986) Mol. Gen. Gena.
202:302 f0 lu ~ Das a al.. (1984) ~. Bactcriol. 158:1165, and De L ..JC et
al. (1983) J. Bac~rioL 154:1~65, Van den Berg et ~1. (1990) Bio/Technoloe~ 8:135, for
~1 ~., .. . Cregg a al. (19&S) Mol. CeD. Biol. S:337~; and Kunze ct al. (1985)
Basic Mic~obiol. 25:141, and U.S. Patent Nos. 4,837,148 a~ld 4,929,555; f0 Picha;
E~innen a al. (1978) P~c Nadl. ~cad. Sci. USA ~;1929, lmd Ito a al. (1983) J. Bacteriol.
153:163 f0 r ~ . Bea~:h and Nurse (1981) Na~e 300-.706 for Schzo-
., . DaYidow a al. (1985) Curr. Gena. 10:3g, ~no Gaillardin a al. (1985)
Curr. Genet. 10:49 for ra~.
Me~ods for " e - ~ into ' cells
re hlown in Ihe ut aDd include Yi~al inf~ i0~ ' - calciurn
pho~hatel 1, polyb~ene rncdiated i ' p~plastfusion, :l ~. -
of tbe, ~, ' ' ) in lipo~ ~d d~ect 0f tbe DNA
mto ~
Ibe med~ot f~r _ ~ of n e~cssion vector f0 i ' of
insoct cdls for e~e~ioo of . ' be~in is slightly differeDt d~n tbat genlllly
~pplic~e to lhe - ~ of b~l aq~sion veatlr, ye st aq~ssi0n voct0, 0
~ ' e~;ion voaD~r. In an ' ' of tbc prescnt invention, a; .. ~
SlJBSTlTUTE SHEEr ~RIJLE 26)
W0 95/35376 2 1 9 3 5 2 6 ~ o ~ ~
-22-
vaxor is -~ ~ iti, . with te~hniques that are l~own in thc an, for example,
~s describe~i in Kitts a al., BioT~chniaues 14: 81~817 (1993), Sr~tith et al., Mol CeD
B~DI. 3: 2156 (1983), and L~ow an~i Summer, Virol. 17: 31 (1989). In one
Rf Ihe present invendon, a t ' ~ , expression Yector is ~ in
to Su~rs an~i Smtth, Tex~ls Agrictdtural r, Station Bulietin No.
IS5S (1987). Moreover, m~ials and methotls for L .~1 .r ' t ell expression
~ystctns ~re ~ n~ailable in }it form, for e~arnple, the MaxBacX }it frotn
Inv~rogen (San Diego, CA).
Also, tne~hods for ~r~ i lt g DNA into an inselx host cell are
hlown in the ~IrL For exatnple, an inselx celi can be infectet~ ~tth a virus containing an
- ' enctxing the present ~1~, ' When the vilUs is replicadng in the
infe~i ceD, the pres~ent ~1.~ iD be expressed if operably lin~e~i to a suitable
promoter. A variety of suitable inselx ceDs and ~iruses are l~nown and incltlde foDowing
~ritho~t limitadon.
Inse(x cells frotn any Rrd~er of the Class Inse~xa can be grown in the media
of tbis invendon. The orders Diptera alld l :. ' . are preferred. ExaJnple of inse~x
~pecies are }isted jn Weiss a al., "CeD C ulture Methot~is for l~e-Scale r , . of
r ~b~r in GTanados a ~1. (eds.), The Biolo~v of r .~. Vol. ~ Prac~cai
~ -- - for Insect Cont~ol. pp. 63-8~ at p. 64 (198~). Insec~ cell lines detiveti from
the following insects that can be nse~i herein Ime exernpl~y: C'~ / . p
~~lbly, cell fine CP-128); Tt ' . " n~ (p~ably, cdl ;iine lN-368); A~uogr~ph
'o, .. fiwata ~bly, cdl ~De Sfg); L~ d~spar, Mamcstra
bra}, ~cs ~opic~s, Org~na ~ ~ . N~rlo se~ cdcs acgypti;
liltol~, Bl~clla g~ar~, Drn . ' ' ~ , Hdiothis zar; ~ c~gua;
~, ' '~ o~c, Plo ha i ~ / '' Am5aaa moorc~ is c-r~grwn. .
ora~ A~rLrtis s~gc~, Bomb~ mon; ~ r~as c~o~h
~sia g_. ' r, Aparacks I ' ' A~a caja; and Ponhfi disp~.
SUBSrlTllTE SHEEr (RULE26-)
~ wo ss/3s376 2 ~ 9 3 5 ~ ~ PCr/US9~0789~
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Preferred insect cell lines are froml .~p - ', u frugiperda, and especially prcfened is cell
line Sfg. lbe Sfg cell line can be used in herein and obtained from Max D. Summers
(Te~as A & M Uni~e~sity, Colleg~ Sta~ion, Te~s, 77843, U.S~) Other S.frYgip~rda cell
lines, such as IPL,Sf-21AE m, ~ dcscribed in Vaughn a al., in vifro 13: 213-217 (Ig77).
lbe insect cell lines of this inYention are sui~ble for the ~ of
numerous ~ viruses such ~s ~ .o..,~, po~ vinreD,; ' .~D and
, of which ' , '~: ' ViTUSe5 (NPV) nd granulosis ~/iTuses (GV)
fTom the group of b~ ~D are prefened. Furth preferred are NPV viTUses such as
those fTom AutoOrapha spp., ~r- ~, Spp., T ' ,' D~p., r ~ ~ Spp.. Gallerai
~pp., and Lymlantria spp. More pqrefe~red are ~ ' .h. D strain ~ ~,., ' - californira
NPV (AcNPV), r 7 oY NPV, Gallcria n~ellonella NPV, and any plaque purified
~ains of AcNPV, such as E2, R51, Sl, M3, .~ A ~ and desaribod by Smith et a~
ViTol ~0: 828-838 (1979); Smith l!t al., 1 ViTOI ~: 311-319 (1980); and Smlith et aL, Virol
89: ~17-~27 (15~78).
Typically, insect cells ~r ~o~ ufrugipcrda type 9 (SFg) sre infected with
' .h~ D strain ~ O. . ' - cal'ifornica NPV (AcNPV) containing a ~ '.~ '
encoding the preDen~ p '.~, ' Such ~ ~ is p~roduce~ by ' ' ,,
' between a transfeT vectoT containir~g the co~ng sequence nd ~ .D
se~uences and a genomic ' . DNA. P~ably, the genonuc L ' ...~ DNA is
linc uized and contains a ~ ^ ' cssential genc lb~ transfer vec~, p~feTably,
~ins the nuck~ide sequences, Dooded ~o raton~ the d~ ~ ' gone uld a
' . p~lyho~in promoter u~ _T cpesably lin}Od tO ~
encoding Ihe prese~ ~ ,, ' s de~ibod in Soe Kilt6 c~ al., ~io~. ' . 14(0:
810-817 (1993).
7he t~nd ~roctor and li~rd; ' . genome Te mm~d into
SF9 i~uect c~lls, urd the ra~ es ~y co~ng ~ r ~ - a coding
the preseM ~ ., - Withollt a funaional esDential 8ene the; ' .. ~ genome
SUBISrlTUTE SHEET (~ULE26)
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cannot p~ducc a viable virus Thus, th~ viable viruses from the ~ most lil~ely
contain the ~ '~ ' ' encoding the present POI.~J, ~ ' snd the needed essential gene
soquences from the transfcr vector. FunhG, I c~ of occlusion bodies in the ir~ected ceUs
~re ano~ ~ ~ that the pol~ ' encoding the present ~ ' was
inh~ the ~ ~ genome.
Tne essential gene and thi: polyhe~hin gene flan} e~ch other in the
b culovirus genome. 'Ihe codi~ng qu0ce in the trsnsfer ve~r is flan~ed ~t its S' with
the e~eni~ gene soquences and the polyhedrin pro~notcr nd u its 3- vnth the polyhedlin
temunalDr. lllus, when the dd ' event occurs the ~
encoding the present pol~JtiJ~ displaces the L ' .~.w polyhed~in gene. Such
. ,.~ without a polyhedrin gene wiU not produce occlusion bodies in the infected~lls Of cou~se, another mesns for t~ if coding sequence wss , ' into
the ~ ~ genorne is to sequence the ' ' ' ~ genomic DNA.
A' .~}~r, e~nession of the present ~ ,~th~ by cells infec~d with the
baculovirus is nother ~wiG.~... means.
Isol;~ion of the ~ ~vti~i,
B~sed on tbe physical ~' of the plesent ~ ~ti~. weU
b~own mahods can be selected to pu~ify the ~ , of the present invention. Such
physicd - include h, . - ~. isoelectric poim, ~e, Lolubility,
3 . ac. r~ , D~lly ocQ~ing n.8 ~re found s dimcr of identicsl
~ .
S~tio~ q~ c~ be choscn for ~ nd . ~ A
dn~le n~hod m y ~ or ~: of techliques may be needed to punfy the
~t i 1~ lo d~e des~ed pmity. l~e ~tion ~e s-wected is not critica~
he inva~L Many l~cbni~es re ~sv~ Por gle, the following ~re
~oo tecb~es ~ ~ e ~e: di~llysi4 u' ~ w filtaltion. and SDS
- gw ~ L .' ~ .', separatesdifferent
SU~STIl UTE SHEEr (RU~26)
21 S35~
W0 95135376 I
-25-
dec~ically charged ~ , Antibodies to the p~sent POIJ~ S carl 21so be used
in affinity ~ ' _, h~ to sep;uate the desired ~l~r ~ ' ~ ~ frorn i" "y
dissimilarprotèms u v!r_I bighI liquid ' ~ is a separation
n~hod basod on ~ t in h, .- ' ~.
Rece:F~r Binding Assays
Receptor binding assays haein may uilize c~lls that naturally produce the
lL8RI or IL8R2 receptors, such 25 human , ~ ~1 .d~, a pol~ ~ ~
encoding dther he ~8RI or IL8R2 can be introduced ?nto a cell to ~duce ~he desired
receptor. For the assay, dther w~lole cells or ' cm be used to deternnine
receptor binding. Typically, the ~l5say for receptor bin~ing is pelforrned by ~ g if
the present ~ , ' can compete with r~dioactive, native IL8 for binding to L8RI.Tbe less the ~.it~ me2sur~ d the less the naive L8 was binding to the receptor.
ii Biolog~csl Actn~ity Assllys
Signal T ' Assays
One means of rneasurrng the biological ctivity of the present pulJ~t;~e is
by a signs~ ssay. 1'ypicd signal i ' ~says rneDe Ca~, ]P~? and
DAG leYds ~5 desaibed in ?nore det?~il below.
Most ~u?iar CP ions re , ~ in ff~ tr
~mn, noolhcr_, "! YCSiC~ but ?i~g of ~esent r 1~ '- totheL8RI
~I tnggor ~ in~e~e of free CP ions in the cytoplasn. Wlth ^ dYes, soch as
,fira-2, the -' of ~oe ~CII?~ c~n be m~ Tbe eaer of ~ 2 is ?sdded to
tbe ad~e maiia of the host cdls e~essng IL8RI or ILgR2 ra~tor prl~,, ' The
e#er of.fi~2 is lipaph~ic u~ d~ses ~ss d~e n~ne. Once inside the ceil, the
f~2 ~r is hydro~yzod by cyu~so1ic a;,~ases lt~ its ~ forrn. snd then the
dye c~nnot diffilse bac~ out of thle cell. The 'i~ , ' ' fonn of fi:~-2 wiD fluoresce
~UE~STITUTE SHEET ~RU~E26)
W09513!i376 2 1 93526
-26-
whcn it binds to the frec CaZ~ ions, whi~,h arc rclcascd after binding of a ligand to the IL8
receptor. The ~ can bc mcasured without Iysing thc cclls at an exciution
~rum of 340 rlm or 380 nrn nd at " ~n of 500 rlrn. S~urai a al.,
EP 480 381 and Adachi a a~., PEBS L~ tt 311(2): 17~183 (1992) describc somc c~amples
of ~ys rncas~ng frcc i " ' C~'
The risc of free cytosolic Ca~ ' is precedcd by thc hydrolysis
of I . ~ ' 4~1 ' . ' . Hydrolysis of this I - , U, ~ by the phs na-
rncmb~ane en~,Ymc I' ,' 'i, C yidds 1,2^ ' yl~~~/l (DAG), which remains in thc
rnanbranc, and thc water-solublc inositol 1,4,5 ', ' . ' ~3). Binding of IL8 or IL8
gonists will increase the . ' of DAG and lP,. Thus, signal i ' ' activity
Qn bc mcas~ed by I " ' e the ' of thesc hydrolysts prooucts.
To rneasure thc IP3 , ' .l~ hbeDed 3H-inositol is
ddcd to thc rnedia of host cells e~pres.~ng IL8RI or ~8~2. The ~H-inositol tal~cn up by
the ceDs and after stimulation of thc ce]ls with the prcscnt pol~,, ' ', thc rcsulting
mositol i ' . ' is separated f~om Ihe mono and di, ' .' forms and mcasured.
S~urai a al., EP 480 381 describes onc cxampic of rncasuring inositol i ' . ' Icvels.
, ~rsham provides an irlositol 1,4,5~, '. ' . ' ssay system. With this
~em ~n provides trit,Ylated ~oositol 1,4,5; '. ' , ' ' and a receptor capable of~" ~, ' ' ' ~ the n ' '.~, inositol ~Dm other inositol I - ~ With these rcagents
an effecliYe and ~ate . ' ' assay csn be perfo~mcd to determine the inositol
i',' .' Icvels.
~Y
, ~, ' (MPD) ~uy is n aumple of another rnethod for
rnc~ring the biological cdvity of IL8RI Kdi~d biologic l activily. r
aiveMIP-2 ~ ,, " can timl~late Da~ph~ tg ~e" '
MPO is r~sod aDd can be rne~d accordiDg to the procod~es described irl Suzu~i a
aL, n~l Biochem 132: 34~3S2 (1983).
SUBSTITUTE SHEET ~RIJLE~6~
W~9~/3~5376 2 1 93526 PCT~/~JS95/07895
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- Assays
is another IL8RI mediated biological activity that
can ~e mea herein. Tne ~SQIys can be perf~med on, `1~ labeled
es-eDtially s described in DeForgc cf al., l~nQnol 148: ~133-2141 (1992).
C E~mDles
~ be a~mples prese~ted below re providod 8S fur~er g~de to ~e
of o~dinary sl~ll in the art, snd are not to be coDsm#d 8S limiting the
invention in any way.
E~amDle 1: ~8 Mutants - Alt~in~ the B Sheet Bindin~ Domain
r~
The amino acid secluence of the ~ J~S d~picted below is 8S foQnd in
NO: ~ eJ~cept as follows:
SllBSTITUTE SHEET (Rl~LE2B)
.
WO 95/35376 2 1 9 3 5 2 6 PCTIU595107895
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~mino J4id 46 47 48 49 50 51 52 53
Po~on
N~iveHm~an ~8 G R E L C L D P
M~nt I K
Mol ot2 K K
M~nt 3 K
Mt~nt 4 A
M~antS K A
Mu~ant6 K E A
Mutant 7 K E V
Mulant 1~ K N
Mutant2~ K R N
MutDnt3~ K N
M~nt 4~ A N
Mutant 5~ K A N
Mulant 6a K E A N
Mutant 7~ K E V N
HostCdl: Yeast: ~ , ccrc~s~c ~r
B~: r
p~ f~ I of tbe p~t ~ r . ill set fdl bdow.
1 F 5t F~ow S ~
~nd S~ odi~n ~oc ~ S.4. La~d 30 Sû mLs of ya~t , t, pH ~dju~d to
S5. ~n S~ mL plastic sc~w cap tube u d lldd 40D pL o~ lesin sluny. Rocl~ tubc o~ ght
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~ WO9S/3~376 21935~ "" ~ ~
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u 4C Centr~uge tube at 3,000 rpm at 4C for twenty minutes. Pour off . t,
unbound materiaL
To nsh the rcsin, t~r~ the ~stn pella to an 1.5 rAL Eppendorf tube.
Wash out S0 mL mbe wrth about I mL of 50 mM sodium acetate pH SA. Ccntrifuge
~dcIf ~ube at 2,000 rpm for l~en r~unutcs. Remove thc ~r, Add about I mL
of S0 mM sodium cet~Uc to mbe, V~ tube to Iqend peUa. Repeat wash steps.
To dute the preseDIt ~ , dd 10~ p L of S0 mM HEPES pH 8.3,
1.0 M Naa Roc~ tube U 4~ fi~r twa~ty nunutes. Centrifuge the tube at 2,00 rprn for
ten ~r~iuutes. Rcmove , to Eppendor~ labeleo 1.0 M NaCI. Rcpeat clution steps.
Receptor Binding Assay:
Below is a rnethod for tcs~ing tne binding affinity of tne preseM
r,
Tcst for present pol~ d~ abiliq to bind lL8R2 rcceptors as mcasured
by ~ , ' ' ~itb '251-lL,8.
DNA cncoding tbe receptor was isolated fro~n hurnan genomic DNA by
PCR using . 'i" ' ' ' prirr~ers bascd on pubL~shed sequenccs, as described in Murphy
a aL, ~ 253: 1280 (1991), nd Holmes a a~, ,Science 253: 1278 (1991). DG44.
a~uese hams~ onry (CHO) c~ls wae t~ with either IL8RI or ndR2 cDNA
~kr thc con~ of Ihc ~ ' .' irn~nodiatc auly promota and enhancer. A
~andsrd c lciurn pbosphatc pr~c~ol was uxd. Tbe ~ssion voctor includod the
~' S; ~ ' - ~e 8a~ n~ tJ e ceUs were ~od in h~ ~ and thyrn idine
ddi~t medirmL aones e~a~sing Ihe rocq~tor wae ideu~ied by ~ - Id~ding
C~r~e roceptor c~essing ~ were to c~lr~ce in K-well r .~ ~r~,D
pl tes f tbe rccep~r linding u~y. Pl te ~s t 1-2 st ~05 ccDs pa cm2 in SO~
DDlb~cco's Morlified Esgle's M~dirJm a~MEM), S09~ Hun'~ P12, ~,, ''
thyn~dine, 10* di-l~zed fetal c~f ~um (dFCS). Ne~a, is~cubate ~ at
SUE3STITUTE SHEEr ~RUI E~6~
WO 95/35376 2 1 9 3 5 2 6 PCT/IJS9S/0789S ~
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room ~ r for three hours with C.2 rnL Hepes-BSA binding buff containing 0.2
nM ~1-lL,8 and the wanted of the present pcl~l,..J-,~. 'Ihe binding
buffer 25 mM Hepes, pH 7.0, 150 n~ N~CI; S mM C~CI2; 5 mM MgCI2: I mg/mL
BSA. Measure , ' binding in the p~sence of I ~g~rL of tullabtJed ~8. Wash
cells o~tce w th Hepe~BSA binding bufFer. De~nine bound ~ ~8 with a garruna
counter.
NeLtrophil C Assay:
Test for present POI~J p ' ' ' ability to induce c' of r,, ' '
lhe assays essentially as described in D~eForge ct al., J lmmunol 148: 2133-2141 (1992).
For ~his assay, freshly isc~late neutrophils from whole blood using Neulrophil
Isolation Media M), I ' ~ by Cardinal Associates, Santa Fe, New Mexico.
Isolate the cells essentially as described by Cardinal Associates. Add 17 ml, of NIM and
30 rrL of blood to 50 rnL tubes. Centrifuge tube at 500 g for 50 minutes at room~ emoYc _ ~ai blood cells by Iysis in ice cold water.
Label the purified ~ ' ' by incubation with 2~7~;S (2~O~A~
5ll ' ~) L.u,.y~ ' fl ester, r ~ by Molecular Pmbes,
Eugene, Oregon. The procsdure as described in DeForgc a al., J Immunol 148: 2133-
2141 (1992). Suspend the 1l ,' ' at 2 ~ ~o6 celL~/nL in PBS without calcium and
0.1~ BSA. Add labd to the above ceDs ~t 8 final - of 2 ,uM.
Incuhte ceDs 30 minute incuhtion u .~7C Wash the labeled . ' twice with PBS
~t calci~m uld _ Rccuspeltd the cdl pcDet in H~s B~nced S lt
~ol~ion with 0.1~ BS~
For the: - u~y,~e~ ~dl ' chur~er
~hh ~10 pm d~ 3 ~m pore, bonded r ~ To tbe bottotn of the
~lls, ~ 30 pL of the Hu~s, Q~9~ BSA bm~cr ~ith the w nted ~tnt of the p~r.sent
- To the top Qf tbe wdls, ~dd suspen~ion 50 pL of labded cdls U a
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~ WO 95/35376 2 1 9 3 5 2 6 PCT/US95107895
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of 5 x 106 cells/~nL. Incus~ate the cells at 37C for 25 mtnutes. Quantify
ophil ugration by I ~ading of the filter.
Use to de~ct for ~ , 485 nm e~citation and 530 n~n esnission
detection filtess. F~Ma-l~Phe ,u 100 DM I r J by Sipu~ SL Louis, Missoun,
s positive control for ma~imal s~nal on each . .
E~le 2: IL8 Mutants - ~te~inrJ the Amino Tem~nal Bindin~ Domain
~, '
The amino actd s~luence of the ~ ~t;~, as found in SEQ ID NO: l
e~cept as follows:
A~nino Acid 11 12 13 14 15
Position of
SEQ ID NO: I
NativeHuman IL8 K T Y S K
Mutant 8 Q Y S
Mutant g K M A
Mutant 10 K M Q
Mutant 11 K L A
Mutant 12 Q M Q
Mutant 13 Q L A
Mtst nt 14 Q H S
Mut nt15 N L Q
Msn~t 16 N M A
~n~o ~dd 11 ~2 13 14 15
Po~itio~ of
SEQ n~ NO I
N~ltive~usnan IL8 K T Y S K
~BSTITUT~ SHEEr (RU~E26'
2 ~ 93526
WO 95135376 PCTNS9S/07895
-32-
Mutant 8a Q Y S Q
M~nt9a K :M A Q
Mol~nt 101l R M Q Q
Mo~-nt lla K L A Q
Mu~-nt 12a Q M Q Q
Mul-nt 13a Q `L ~ Q
Mu~n 14a Q H S Q
Mulant ISa N L Q Q
Molant 16a N M A Q
Amino Aad 11 12 13 14 IS
Posi~on of
SEQ II) NO: I
NabYeH= IL8 K T Y S K
Munmt & Q Y S N
Mulant 9a K M A N
Mul~nt lOa K M Q N
Mulnnt lla K . L A N
Mul-nt 12a Q M Q N
Mu~nt 13a Q L ~ N
Mu~nt 14a Q H S N
Mu~nt 15a N L Q N
Mllant 16a N M ~ N
Host Cdl: ~ in E~e I
n ~ ~ m E~nple l
R~ Binding Assay: As in E~mp!le I
SUBSTITl~E ~HEEr (RU~E 26)
~ WO 95135376 2 1 9 3 ~ 2 6 1 ~ Ya/u ~ ~
-33 -
Neu~ophil ~ Assay: As in E~ le 1
E~e 3: GR0~8RI Bind:in~ D~nnsin C~ineras
11~ ~nno ~cid so~ce i~ desdbed in PCI appln. no. W09V00326,
1~ , - by ~fe~nce. lbe u~no ~cid seqoenoe of d~e pOl~r, ' as found in
SEQ lD N~.2 c~ ~s follows:
SUBISTITUTE ~EEr (~ULE26~
WO 9S/35376 21 9 3 5 2 6 PCT/~S9S/0789S
34 ~
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~UBSTITUTE SHEET (RU~E26
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WO 95135376 36 PCTIUS9S/07895
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SUBSTITUTE SHEET (Rl~26~
WO 95/35376 PCT/[~S95/07895
21 93526
10-
Host Cdls: As in E~csmple 1
P~,, ~ h~on: ~s ~n E~mple I
Recop~r Binding Assay~ lc 1
Nu~hil r ~5say: ~.s in ~le I
SUBSTl rlJTE SH~ET (RU~E 26)
~ WO 95135376 2 1 9 3 5 2 6 PCT/US95/07895
41-
srQ~NcF ~T8~G
aPPLIC~S: ~-CIl~ori P~trlc' ~
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SUBSTIITUTE SHEET (RULE 26)
WO 95/35376 2 1 q 3 5 2 6 PCTIUS95/07895
-42- --
8~1CI~ 8~Q ID ~lo:l:
8 r ~1~ LY. 01U LL ~ C5r- ~ C~r. Il- LY. 5~r Syr 8 r Ly- Pro
ro LY P~ S1~ LY. ~ ~U ~r~ V l Sl- ~lu 8-r 01y Pro
20 ~5 30
al L ~ 5hr Olu S1~l Sl V l LY. ~U 8 r a~ aly arsr alu
~5 ~0 ~5
L SL q~O L~ a~ rro LY. C1~L ~ ~ ~ O~ ar~ V l V l Olu LY~
LL L~ r~ >1- 01~ ~l 8 r
C5 70
(2~ ~ ~CR 8~Q SD 170 Z
A I ~IIGT}I 73 ~o
P ~ ~PB ~luo c ~d
C~ Jl-
Dl ~OPOLOt;~r l~Lr
17~L~Cm~ ~P~ ~rot lu
8 QlJ~ I~Q D 170 2
l~ 8 s ~1 V~l 5hr Olu L U ar~ Cy~ O1SL q~ L ~L O1~L ~r L U e~
5 10 15
~ly Sl- 111~ L U LY. a~ S1- O1U 8 r V~l a~ V~L1 ~ 8 s Pso ely
20 ~5 30
P~ ~. ~ al. ~ ~LS ~U V~L1 S1-- al~ *Is L U LY. ~ o~y LY.
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LY~ a1-- ~ L U ~ TrO U-- ~S PrO 11--t ~1 ~J1SI LY~ Sl 1 Olu
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LY. S~ L IL a SL LY~ Oly 8 1r 7hr
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SUeSTlTUT~ SHET(RULE26)
Wo95135376 21 935~ ""~
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~D~ ~rOPOLO~ u~r
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