Note: Descriptions are shown in the official language in which they were submitted.
W O g3/15202 ` i~ CT/US93/00601
AAMP-l, A PROTEIN WITH LOCAL HOMOLOGIES TO HIV-l ENV AND NEF PROTEINS.
~ac~JtomlD o~ ~ s~r~o~
~ield of the ~nvent ~n
The pre~ent invention relate~, in
gener~l, to AAMP-l In p~rticul~r, the present
invention rel~t-~ to ~ DNA -g~nt encoding AAHP-
l; polypept$d-s encoded by the DNA -g~ent
r co~bln~nt DNA ~olecul-s cont~lnlng the DNA
~g~ent cell~ cont~ining the r co~bin~nt DNA
~olecule a ~ethod of producing AAMP-I ~ntlbodie~
~p clflc to AAMP-l; ~nd ~ ethod of e~urlng t~e
~ount of AANP-l in ~ ~ pl-
~he ~jor ~i~toco p~tibillty co~plex
cla~ II proteins hav r-cQntly been found to
contain local ~o-ologi~ to the HrV-l nvelope
prot-in ~H. Ooldlng t al., J. EXD. Mea. 167, 914
(1988); H. Golding et al., J. Cl~n~ Inve~t. a3,
1430 ~1989): J. A. T. Youn~ g~ 332, 215
(1988)). Such ho~ologou~ re3ion~ r~y ~erve a'6
targ~t~ for antibodl-6 generat-d to HIV-l proteing
and thu~ co pro~i~e the L~une y~t-~ in AIDS.
Golding et ~1. (J,,,,E~p,,,~ed- 167, 9~4 ~1988)) bave
~dentified ~ cormon epitope loc~t~d in the c~rboxy
ter~inus of the HIV-l gp41-envelope protein and
the a~ino ter~inal portion of the beta chain of
all hu~an Hl~ cla~ lI antigen~. Although the
epitop~ 11, 5 con~ecutive id~ntitie~ or
si~il~rities, they found th~t it is an effective
exa~ple of ~olQcular ~i~icry" in that ~onoclonal
~ntibodies rai~ed aqainst synt~etic peptides fro~
each protein react interchangeably with native
~IV-l envelope and MHC cla-~ II ~olecules. One
third of HIV-l positive individuals were shown to
" ,
"' ~ - 1 -
~ . . .
WO93/15202 ~ I f.~. t~j S 7 ~ PCT/US93/~K01
have ~erum antibodle~ dlrected again~t pept~des
derived fro~ HIV-l envelope protein, the
ho~ologous peptlde from the MHC clas6 II
~olecule~, and native MHC clas~ II olecules (H.
S Golding et al. ~ Ex~. Med. 167, 914 (1988)). Two
other region~ of the HL~ cl~ss II beta chain and
another i~une related protein, interleukin-2,
also ~how li~ited ho~ology to HIV-l ~J. A. T.
Young, Nature 333, 215 (1988)).;`M.A. Vega et al.
Nature 345, 26 1990).; W. E. Relher III, et al.
PrDc. Natl. Acad. Sci. USA 83, 9188 (1986)). An
i~portant con~ideration in HIV-l vaccine
develop~ent i~ the potential existence of
additional ho~t cell ~urface protein~ wlth
ho ologies to HIV-l that ~ay cros~react with
antibodie~ directed against its peptide~.
The presènt invention relate~ to the
protein AAMP-l which has i~munoglobulin (Ig) type
do~ains that contain strong local ho~ologies to
con~erved regions of the HIV-I envelope and nef
protein~.
8~a~ O~ T~ n~rIO~
It 1B a general ob~ect of this invention
to provide AANP-l.
It i8 a 6pe~ific ob~ect of this invention
to provide a DNA segment which encodes AAMP-l, or
~egment ther~of.
It i~ a further ob~ect of the invention
to provide a polypeptide corresponding to a AAMP-
1 gene, or fragment thereof.
It i8 another object o~ the invent~on to
provide a r-co~b$nant DNA ~olecule comprising a
~ector and a DNA seg~ent encoding a AAMP-l gene.
- 2 -
W093/15202 ~ rl 3 PCT/US93/OK~1
It i~ ~ further ob~ect of t~e ~nvention
to provide ~ cell th~t contains the above-
de~cribed recombinant nolecule.
It i~ another ob~ect of the invention to
provide a nethod of producing a polypeptide
encoding a AAMP-l gene, or ~egment thereof.
It is a further ob~ect of the invention
to provide antibodies having binding affinity for
~AMP-l, or a unique portion thereof.
It i~ a further ob~ect of the invention
to provide a ~ethod of nQa~uring the a ount of
AAMP-l in ~ sa~ple.
It is another ob~ect of the invention to
provide a therapeutic dality co~pri~ing the
above-de~crib~d polypQptide~ in an a ount
effective to elicit protective antlbodies, block
har~ful auto-antibodie~, or co~pete for ~IV
binding to body cells in ~ pati-nt to tbe AIDS
viru~ and a phar ac utlcally acceptable diluent,
carrier, or excipient.
It iB a furtber ob~ct of th~ invention
~ to provide a ~ethod of preventing AIDS in a
-~ patient.
,~, .
Further ob~ect~ and advantage~ of the
pre~cnt invention will bQ clear fro~ the
de~cription t~at follows.
B~S~ D~8C%~PSS0~ 0~ TS~ D~N~8
Figure l. Nucleotide ~equence of bu an
A20S8 melano ~ cell AAMP-l cDNA ~solat~d fro~ a
la~bda gtll expre~ion library with its pr~dicted
~ino acid ~equence. The phage in~rt, AAMP-l,
w~ subcloned into Blue~cript pla~id ~Stratagene)
for production o~ double stranded cDNA for
sequencing u~ing th dldeoxynucleotide ternination
~ ~ - 3 -
,............. ..
W093/lS202 ~ a ~ ~ PCT/US93/OO~L
~ethod (F Sanger et al Proc Natl acad Sci
74, 5463 (1977)) wit~ SequenasQ 2 0 (U S
Bioche4ical) Nucl-otide residue~ ar nu-bered
beginning at the S' end A~ino acid ~-guence
S nu~bering begin~ w$th the first ~ethionine
(underlined with ~-~) of the open reading fra~e
The a~ino ter~inal acidic region, aa26-85, i~
underlined with ~ A~ino acid region, 76-
346, co~pri-ed of potential ia~unoglobulin-like
do~ains (A F Willia~ and A N Barclay, ann
Re~ I~munol 6, 381 (1988); A F Willi~as and A
N Barclay, in ~ 9 U9 Ci~[~ - L_ T Hon~o ~t
al ~d~ (Acade~ic Pres~ Li~it~d, San Diego, CA,
1989), pp 361-387)) is underlined with econdary
structure predication~ o~ b~ta strand6, ~
and beta turns, ">~>>~, ba~ed on the ~ethod of
Chou and Fas~an (Ad~ance~ in Enz 47, 45 (1978))
Cy~teine pa~r~, 139 ~ 208, and 265 ~ 326,
predicted by i~unoglobulin do~ain homology to
~o~t likely foru disulfide bond6 are ~arked ~c ~
m. potential trans~e~brane region, aa37~-399, is
underlined, ~ _ ~ Th~ potential protein kina~e
C phosphorylation sit- at ~erine #408 iB
underlinQd with ~SSS$~ (The prot~in kinase C
phosphorylation site con~n~u~ sequ~nce (Ser/Thr-
Xaa-Lys/Arg) ~here Xaa 1~ u~ually an uncharg~d
residue ~J R Woodgett, K L Go~ld, T Hu~ter,
Eur ~ Biochem 161, 177 (1986)1, ~8 al~o found
at thr~onin~s ~238, 291f and 357)) The
polyadenylat~on ~ite ~t nucle~c acid re~id~as,
1723-1729 i8 in parentheses ~
Figure 2 Northern blot o~ human
~elanoma A2058 c~ probed with AANF-l cDNA A
~ingle 1 6~b ~and i6 seen on blots of total
cytopl~su~c (Lan- 1) and polyadenylate-enriched
~Lan~ 2) A2058 RNA Total cytoplas~ic RNA, ~1
_ ~ _
W093/1~202 PCT/USg3/~K~
~icrograms (~g), wa~ isolat~d from 6 ~illion cell~
ly-~d in Nonid~t P-~0 (0 65%), ~parat~d into an
~qu-ou~ pha~e in the pr-sence of 7M ur-a, 1%
sodiu~ dodecyl sulfate, Trls buffer, NaCl, and
EDTA, followed by phonol/chloroform extraction
RNA, 2 2 ~g, enriched for mes~cnger RNA, wa~
i~olated fro~ 16 ~illion cells with a Past Track
Xit Version 2 1 (Invitrogen Corp San Diego, CA)
aNA was denatur d in for aldehyde, electrophor-sed
in a 1% agaro-e/for-~ld hyde gel, tran ferr d to
Schlelcher ~ Sohnell Nytran nylon ~e brane and
cro~linked with ultraviolet light The 1766 bp
AAMP-l cDNA w~ labeled with (alpha-~P) dCTP (~N
R ~ear¢h Product~, ~o-ton, NA) u~ing rando~
pri ing Hybridization overnight at 65 C was
perfo Ded ac¢ording to Chur¢h and Gilbert (Proc
Natl Acad 8ci 81, 1991 (198~))
Figure 3 North rn blot of A~MP-l
expre~ion in hu an T-cell activation Hours
refer to ti-e in culture A AAMP-l 6ingle 1 6Xb
~~age B B ta-2 ~icroglobulin ~tandard
~ L~n~ 1-3~ Non-~ti~ulated hu~an CD~+ T c-ll~
(Hu~an poripberal blood ~ononuclear c~ fro~
nor-al donor~ were ~eparated by Ficoll-Hypaque
d~n~ity-gradient centrifugat~on Un~t~Julat~d
CD~+ly~phocyt~ wor~ obtained by rigorou~
i~uno~agnetic negatiYe selection with Advanced
Magn~tic Particle~ (Advanced Magnetic, Ca~bridge,
MA) or Dyn~bead~ (Dynal Inc , Fort Lee, NJ) both
bound to goat ~nti-~ouse lgG Negative 6election
was pes~or~ed as de~cribed ~K J Horgan and S
Shaw, Current Protocols in I~munology~ J E
Eoligan et al, Ed~ (Wiley Interscience, new York,
1991), p ~ 44 1 ) using a cocktail of ~onoclon~l
~ntibodi~s, ~Abs, consistin~ of ~nti-HIA cla~s II
~Ab (IVal2), CD20 ~Ab ~lF5), CD16 ~Ab (3G8), CDllb
~ - 5 -
-: ~
WOg3/15202 PCT/US93/~K~1
mAb ~NIHllb-l), CD14 ~Ab (MMA), CD8 ~Ab (~9.8),
and aAb again6t glycophorin (lOF7). Purity of tbe
i~olated cell~ wa~ greater than 98%. The selected
CD4+ T-cell6 were free of monocytes ba~ed on the
lack of prollferative response to opti~al
concentrations (1/200 dilution) of
Phytohemagglutinin (M form) ~PHA) (GIBC0, Grand
Island, NY)), lanes 1 ~nd 2 at 0 and 24 hours,
respectively, without ~itogen sti~ulation, and
lane 3 after 12 hours in the presence of the
protein synthesi6 inhibitor, cyclohexl~lde, whlc~
~as b4en freguently ob~erved to stablllze certaln
~RNA species (X. ~elly et al. P. Leder, ~çll 35,
603 (1983)). Lan ~ ~-8: CD4~T cells activat~d^
(T-cell activation a6~ays were per~or~ed uslng
6tandard techniques. Briefly, 10 million purified
CD4~ T-cell6 were cultur~d in 35 ~m flat botto~
well6 for various ti~e period6 in culture ~diu~
(RPMl 16~0 (Hazelton Biologic6 Inc. L~nexa, KS)
supple~ented with 20 ~M gluta~ine (Hazelton), 10%
heat inactivated fetal calf seru~ (~iofluid~,
Rockv~lle, ~D), 100 IU/~l of penicillin, and 100
pg/~i strepto~ycin), ither unsti~ulated or
~ti~ulat-d w~th antibodies bound to the wells. T-
cell ~tirulatory conditions were a6 described (G.A. ~an Se~ nter~ t al. Eur. J. I~unol. 21, 1711
(1991)). Honoclonal ant~bodie~ were im~obilized
on the pl~stic of the well by dilution in
pho~ph~te buf~ered saline (PES) and overnight
incubation at 4-C, followed by wa~hing with PBS.
The CD3 ~Ab, OXT3, and the CD2 mAb, 95-5-49, were
applied at 1 ~g and I0 ~g, purified~/ml
re~pectively, all in a volume of 3 ~illiliter~ per
wéll. Monoclonal antibodies were used as purified
immunoglobulin derived from asclte~ fluid; CD2 mAb
(directed again~t the Tll.l epitope): 95-5-~9,
lgGl (hybrido ~ kindly provided by Dr. R. R.
- 6 -
.
W093/15202 PCT/US93/~601
Quinones, George W~shington Univer~lty,
W~hinqton, DC); CD3 ~Ab OXT3, lgG2~ (ATCC,
Rockvllle, MD). Cyclohexi~ide, when pres~nt, w~
u~ed at ~ concentr~tion of lO ~g/~l. Lanes
4,5,6,7 ~nd 8, represent the ti~e points at l, 2,
4, 16, and 24 hour~, respectively. RNA sa~ples
were prep~red from CD4l T cell~ by the gu~nidinium
i~othiocyanate-ce~iu~ chloride ~etbod of Maniatis
et al. (T. E. Maniati~ E. F. ~ritsch, J.
Sa~brook, Y4~ C~cr1 -~ A Labor~tory Manual
(Cold Spring Narbor Laboratory, Cold Sprinq
Harbor, New York, ~d. 2, 1989), pp. 7.18-?.22.~)).
Ten ~icrogra~ of total RNA (each l~ne)
~l-ctrophoresed in a for~ldehyde/0.8% agarose gel
W~8 tr~nfiferr d to nitrocellulosQ and ~ybridized
overnight, consQcutively, ~t ~2-C to t~e (alpha-
- ~P) dCTP labeled, rando~ pri~ed probe~, AAMP-l and
beta-2 ~icroglobulin.
D~TaIL D D~8C~TI0~ 0~ s~n~Io~
In one e~bodi~ent, the pre-ent invention
relates to a DNA ~gn~nt coding for a polypeptide
co prislng an a~ino acid ~quence corresponding to
AAMP-l, or at lea~t S contiguous a~ino acid~
ther~of. In one preferrNd eobodi~ent, the DNA
segu~nt coDprises the sequence ~hown in SEQ ID
NO:l, allelic or species variation thereof, or at
least 15 contiguous nucleotides thereof
(preferably, at least 20, 30, ~0, or 50 contiguous
nucl~otides thereof). In ~ further preferred
embodi~ent, the DNA ~egment encodes t~e amino acid
~equence ~et forth in SEQ ID N0:2, allelic or
species v~ri~tion thereof, or at le~t 5
contiguous a~ino acids thereof (prefer~bly, ~t
lea~t 5, lO, l5, 20, 30 or 50 contiguous amino
acid~ thereof).
- 7 -
wo93~1s2o2 ~ r 1 PCT/US93/~Ko
In a ~urther embodl~ent, the pr~sent
lnventlon relat~s to a polyp~ptlde froe of
proteins witb ~hich lt i8 naturally assoclated or
a polypeptide bound to a ~olid support and
co~prlsing an a~lno acld seguence corresponding to
AAMP-l, or at least 5 cont~guous a~lno aolds
ther~of (proforably, at l-ast 5, 10, 15, 20, 30 or
50 contiguous a lno acid~ ther~of) In one
preferr d e bodl~ont, the polypeptide co~prlses
the a ino acld sequence et forth in SEQ ID N0 2,
or allelic or specles variatlon thoreo~ quival-nt
thereto (~or exa~ple, i~cunologically or
functionally, equivalent thereto), or at least 5
contiguous ~ino acids thereof (preferably, at
l-ast 5, lo, 15, 20, 30 or 50 contiguous a~ino
acids thereof)
Tn another e~bodi~ent, the prQsent
inventlon relates to a reco b$nant DNA ~olecule
co prising a vector (for ~xa~ple plas~id or viral
vector) and a DNA eg ent (as describ~d above)
coding for a polypeptide corresponding to AAMP-l,
as describ d abo~ In a preferred mbodi~ent,
the enco~ing seg~ent is pr ~-nt in the vecto~
operably linked to a pro ot r
In a ~urther ~bodi~ent, the present
inY ntion relates to a cell containing the ~bove
describ~d reco~binant DNA ~olecule ~uitable ho~t
cell~ include procaryote~ (such as bacteria,
including ~ coli) and both lower eucasyote~ (for
exa~ple yea~t) and higher eucasyotes (for exa~ple,
~a-o~lian cell6) Introduction of the reco~binant
~olecule into the cell can be effected using
~ethods known in the art
In another e~bod~oQnt, the present
invention r~lates to a ~ethod of psoducing a
polyp-ptid ha~ing an a~ino ~cid equence
cosre ponding to AAMP-l co-pri~inq culturinq the
WO93/15202 ~ 7 ~ PCT/US93/ ~ 01
above-describ~d cell under condit~on~ ~uch th~t
the DNA sega nt i8 xpre~ed and the polypeptide
thereby produced and l~olating the polypeptide
In yet ~nother embodiaent, the pr--ent
invention relates to an ~ntibody having b~nding
affinity for AaMP-l, or a unique portion thereof
In one preferred e~bodiment, AAMP-l comprise~ the
~ino acid ~equence ~et forth in 8EQ ID NO 2,
allelic or ~pecie8 vari~t~on th~reof, or at le~st
5 contiguoua a~ino acid~ thereo~ (preferably, at
lea~t 5, lO, lS, 20, 30 or 50 contiguous a~ino
acid6 thereof) In one preferr d ~bodi~ent, the
antibody is lAA3
antibodies (~onoclonal or polyclonal) can
b~ raised to aAMP-l, or unique portion~ ther of,
in lts naturally occuring for~ and in its
r co-binant for~ 8inding fragu nt~ of uch
antibodi-s are also within th~ scope of the
invention.
aA~P-l ~ay be ~oin d to other ~aterials,
particularly polyp~ptides, a~ fused or covalently
~oin~d polypeptides to b~ used a~ ir~unogen~
AAKP-l or its fragnent8 ~ay b- fu8~d or cova~l-ntly
linked to a vari-ty of i~unogens, ~uch a6 keyhole
li p t he-ocyanin, bovine s-ru~ albu~in, tetanu~
tOxoia, etc See for exa~ple, ~icrobiology,
Hodber Hedical Di~ision (Harper and Row, 1969),
L~ndsteiner, Sp~cificity of Serological Reaction~
(Dover Publication~, New York, 1962) and Williams
et al , ~ethods in I D unology and I~unocbe~istry,
Vol 1 (Academic Pre~, New York, 1967), for
descript~ons of ~othod6 of preparing polyclonal
antisera A typical ~ethod involv-~
hyp~ri~unizat~on of an ani~al w~th an antigen
m e blood of the ani~al ~8 then collected shortly
a~ter th~ r peat~d ~r~unization~ and tbe ga~ a
globulin ~ olat ~
_ g _
WOg3/15202 ~ PCT/US93/~K~I
In o~ instances, it i~ dcsirable to
prep~r- ~onoclonal antlbodics fro~ various
~a~alian hosts De~cription of techniques for
preparing such ~onoclon~l antibodie~ ~ay bc found
in Stites et al , editors, ~aslc and Clinical
I~unology, (Lange Medical Publications, Lo6
Altos, CA, Fourth edition) and r f-r nces cltcd
therein, and in partlcul~r in Xohl-r and Milstein
in Nature 256 ~95-497 (1975), which di~cu~ses one
~ethod of generating onoclonal antibodies
In another e~bodi~ent, th- pre~ nt
invention relates to a hybrido~a which produce~
~onoclonal antibody or binding frag~ent th-r o~
having binding affinity for AAKP-l In one
preferred e~bodi~ent, AAMP-l ha~ the a~ino ~cid
sequ~nce set forth in SEQ TD N0 2, allelic or
~pecie~ variation th-reof, or at least 5
contiguous a~ino acids th-reof (preferably, at
l-a~t 5, lO, 15, 20, 30 or 50 contiguous a ino
acids thereof) ~n another preferr~d e~bodi~ent,
the hybrido a co~pri~e~ laA~
In y t another e bodi~ent, the pre~ent
invention relat 8 to a diagnostic kit conpri~ing
i) at least one of the abo~e-described
~onoclonal antibodi-~, and
ii) a con~ugate co~pri-ing a bin~lng
partner of ~aid onoclonal antibody and a label
In a fur~her e~bodi~ent, the pre~ent
invention relates to a diagnostic ~it co~prising a
con~ugate coqprising
i) at lea~t one of the above-described
~onoclonal antibodies, and
ii) a label
In a further ~mbodi~ent, th~ present
invention relate~ to a ~ethod of ~aa~uring the
a~ount of AA~P-l in a sa~ple, co~pri~ing
contacting the 8~pl~ with the ~bove-descr~bod
- 10 -
. ~ .
WO g3/15202 ,~, ,.? 7 `1 PCT/US93/~K01
antlbodies ~nd ~ea~uring the a~ount of
im~unoconplexe~ for~ed between the antibodie~ and
any AAMP-l in th~ sa~ple Method~ of nea-uring
the a~ount of i~munocomplexe~ for~ed can be tho~e
S well known in the art, such as R~A, ELISA, and
direct and indirect i~unoassay~
In another bodiaent, the pr ~-nt
invention r~late6 to a therap utic ag nt ~ultable
for u~e in protecting agalnst HIV infection or
treating inflaD atory iamune or n-opla~tic
di~ord~rs co pri~ing tho abovo-identifi d
polypeptide~ in a guantity sel-ct-d d-p nding on
the route of ad~ini~tr~tion~ Although
~ubcutaneou~ or intra uscular rout-s of
ad~inistration are preferr-d, the above d~cribed
polyp~ptide~ could al~o be adnini~tered by an
intrap~ritoneal or intrav nous route One skilled
in the art will appr ciate that th a ount~ to be
ad ini~ter d for any particular tr at~ent protocol
can b~ r~adily det r~in d 8uitable a ount~ aight
be exp-cted to fall within th ran~- of l-S0
~icroaole~
~ In another ~bodi~nt, th~ pr --nt~
-~ invention relat ~ to a ~ethod of u~ing the ~bove
2S d-~crib-d polyp~ptide to prevent AID8 One
~killed in th ~rt will appr~ciat~ that the
anount~ to be admini~tered for any particular
treat~ent protocol can readily bs d~ter~ined
The present invention is describea in
further detail in the following non-li~iting
Ex~ple~
The following protocol~ and experi~ental
details are refer nced in t~e Exa~ple~ tha~
~ 35 follow
.
.~
W093/15202 ,' 1 2 ~ PCT/US93/~601
Cell~ Human peripheral blood ~ononuclear cells
(PBMC) fro~ nor~al donors were eparated by
Ficoll-Hypaque dcnsity-gradient centrifugation
R sting CW~ T ly~pbocyt-s wer- ~ub-equ-ntly
S obtained by r~gorous i~uno~agnetic negative
select~on with Advanced ~agnetic Particl-s
(Advanced Magnetic, C~ bridge, MA) or Dynabeads
(Dynal Inc , Fort L-e, NJ) bo~h bound to goat
anti-~ouse IgG Negative selection wa~ pcrfor~ed
as described (Horgan, ~ J and Shaw, S , I~uno-
~agnetic negative ~election of ly~phocyt- ub~-t6
in Coligan, J E et al (Ed~ ) Current Proto~ol~
in I~unology, Wiley Interscience, N~w York ~1991)
p 7 4 1 ) u~ing a cocktail of aAbs con~i~ting of
anti-HL~ cla~s II ~Ab (rVA12), CD20 ~Ab (lF5),
CD16 ~Ab (3G8) CDllb ~Ab (NIHllb-l), CD14 ~Ab
(MHA), CD8 ~Ab (B9 8), and ~Ab again~t glycophorin
llOF7) Purity of th i~olat d cell~ wa~ ~ore
than 98~ The s~lected CD4+ T-cell~ were free of
~on~cytes ba~d on the criterion that there be no
proliferative r ~pon~e to opti al concentration~
- (1/200 dilution) o~ Phytohe~agglutinin (M for~)
(PHA) (GIBC0, Grand I-land, NY) (Davi~, L , and
P E Lip~ky (1986) J I~unol 136 3588)
,
T-cell act~vation a~av6 T-cell activation
as~ays were perfor~ed using standard techniques
Briefly lOxlO' purified CD~ T-cell~ were cultured
in 35m~ flat bottom wells for variou~ ti~e periods
in culture ~edium tRPMI 1640 (Hazleton Biologic~
Inc ~enexa, XS) ~upple~ented with 20 mM gluta~ine
(~azleton), 10% h~at in~ctivated FCS (Biofluids,
Rockville, ~D), 100 m/~l of penicillin, and 100
~g/~l strepto~ycin)], either unstimulated or
sti~ulat~d with antibodies bound to the well6 T-
cell ~tinulatory oon~itions ~re a~ de~cribed (~an
So~enter, G A et al (1991) Eur J I~ uol
- 12 -
.
W093~1~202 2 ~ 8 7 9 PCT/US93/~K~1
21:1711). ~Abo were immob~lized on the plastic of
the well by dilutlon in PBS and o~ernight
incubation at 4-C, followed by washing with PBS.
The CD3 ~Ab OKT3 and the CD2 Ab 95-5-49 were
s applied at 1 ~g and 10 ~q purified Ig/~l
respQctively, all in a volu~e of 3 ~l/well.
Monoclonal following antibodiQs were usQd as
purified i unoglobulin derived fro~ a-cites
fluid; CD2 Ab (directed again-t th- Tll.l
epitope): 95-5-49, ~gGl (hybrido~a kindly
provided by Dr. R.R. Quinones, George ~ashington
Univer~ity, Wa~hington, D.C.); CD3 ~Ab OKT3, IgG2a
(ATCC, Rockville, MD). Cyclohexi~ide, when
pre~nt, was used ~t a concentration of 10 ~g/~l.
~5 CD4~ T cell RNA DreDaration. RNA sa~ple~ were
prepared fro~ CD4+ T cell6 by the guanidiniu~
i~othiocy~nate-C~Cl ~ethod of Maniatis et al.
(Maniatis, T.E. et al. (1989) Molecular cloning:
a laboratory ~anual. 2nd Edition. Cold Sprinq
Harbor Laboratory,` Cold Spring Harbor, N.Y.). 10
~g of total RNA was resolved for each condition on
a for ald~hyde 0.8~ agaro~e gel, tran~ferred to
nitroc*llulo~e, and hybridized at 42-C to ~P-
labelQd purified AAMP-l cDNA insert prepared by
randon pri~ing.
Ant1L~gy_PTeDarstion. The adaptive pa~ive
tran~fer technique in 8~lb/c ~ice utilizing whole
cells from the human ~elanoma A2058 cell line as
antigen wa~ u~ed to generate hybridomas with
~yelo~a cell~. Selection of tbe lAA3 clone was
ba6ed on it6 inhibition of motility when a~ayed
in ~odlfied Boyden chambers described previously
(Stracke, M.L. et al. ~1987) Biochem. BioDhYs.
~. Co-n. 146, 339-345) uslng gelatin coated
filter and ~ariou~ che~oattractant~ (collagen
- 13 -
;:
WO93/l5202 ~ r~OrJ~ PCT/US93/~K01
type IV, la~inin, autocrlne ~otlllty ractor~
fibronectln, ~nd in-ulin-lik~ growt~ factor I.
The clone lAA3 wa~ rQcloned by li~iting dllution
to produce the 1AA3AA clone.
cDNA Librarv and Screenina. The hu~an aelano~a
A2058 cDNA expression library wa~ con-tructed in
the l~ bda gtll vector by Clontech L~boratori-s,
Inc. Y1090 Escherichia coli lnfect-d by the phage
were plated and blott~d onto nltrocellulo-e
filters (Schleicher ~ Schnell) for inaunoa~ay
with lAA3AA antibody. Reactive plaques were
detected u~inq peroxidase-coupled antibody
specific for ~ou~e IgG.
Northern Blotting. Preparation of A2058 hu~an
~elano a RNA enriched for ~essenger RNA w~
i~olated with a Fa~t Track Xit Ver6ion 2.1
I m itrogen Corp). Total cytopla~ic RNA wa6
isolated according to a publi6hed ethod (Gough,
N.M. (1988) Anal. Biochem. 173, 93-95) by
~u~pending 4~1 cell~ on ic- with 0.8~1 chilled
Solution A (10 ~M Tris Cl, pH 7.5, 0.15 MNaCl, 1.5
~M Mgcl" and 0.65% Nonidet P-~0). The ~upernate,
obtained after vortexing and centrifuging (800 x
G, 5 ~in, 4-C) wa~ ~ixed with 0.8 ~1 Solution B
(7M Urea, 1~ 8DS, 0.35 M NaCl, 10 ~ EDTA, pH 8.0,
and 10 ~M Tris Cl, p~ 7.5) and 1.6 ~1. Solution C
(phenol: chloroform: i~oa~yl alcohol (50:50:1).
RNA wa~ removed in the aqueou~ phase and et~anol
precip~tated.
RNA was denatured in formaldehyde,
~eparated on a 1% a~aro~e/formaldehyde gel
(Sa~brook, J. et al. (1989) ~olecular Cloning: A
Laboratory Manual, Cold Spring Harbor L~boratory,
Cold Spring Harbor, NY) and tran~ferred o~erniqht
to S~S Nytran (Schle~oher ~ Schnell) and
:
, .. . . . . . . .. . . . . . ... .... . . .
W093/l5202 , ;~3i~ A ~_~ PCT/US93/~K01
cro~slinked to it wlth ultravlol-t llght ln tbe
Stratalinker apparatus (Stratagene) ~he 1766 bp
cDNA lnsert label-d with (~-~P)dCTP (NEN ~e~earch
Products) with the Random Pri~r DNA Labeling
Sy~ten (~ethesda Research Laboratori, Life
Technologies, Inc ) was used as probe
Northern blots for total A2058 ~elano~a
cytoplas~ic RNA and RNA enriched for nessenger RNA
were perfor-ed wlth the Church protocol (Church,
G and Gilbert, W (1984) Proc Natl Acad Scl
81, 1991) The fllter was wa~hed at 65 ~ for 20
ninut-s wlth wash buffer (lt sodiun dodecyl
~ulfate, ~0 ~M Na~,P0" 1 ~M EDTA) three ti es ~nd
then autoradiographed at -70 C
D~ seauencin~ Positive phage in~ert6 were
~ubcloned into Blue~cript plas~id (phage~id)
(Strataqene) for production of DNA for ~equencinq
Doubl--~trandea cDNA was equ~nced using the
dideoxynucleotiae chain termination ~ethod with
Sequena~e (Unit d States ~ioche~ical) Sequence
obtained with the SK pri~er (Stratagene) specific
for the ad~ac~nt Bluescript ~ector region waF
deter~ined first Sub~eguent ~guencing utilized
pri ers prepared on ~ite based on previously
obtained ~eguence for both strand~ co~pletely
~ç~çnce Data An~ly~i6. GenBank (Intelligenetic~,
Inc ) wa~ searched with the progra~ and further
analy~es of the ~equence was acco~pli~hed with
RAOARGOS, PESTFIND, PROSITE, AACLUST, RERN~T
NALIGN, PALIGN, REPEATS, SEQIN, TRANSL, AND DIAPR0
progra~ in PC/Gene (Intelligenetlc~, Inc )~ The
NBRF protein ~-quence data ba-e from the Protein
Identification Re~ource National Biomedical
Re~earch Foundatlon (NBRF) was ~earched with the
P~S, XQS, and m progra~ and other progra~ were
- 15 -
:
W093/15202 i~ 2 ~ ~ 7~ PCT/US93/~K~I
u~ed for sequence analy~e~ In t~e AIIGN program,
~equenc~s are ~atched with a bias and gap penalty,
6cored in a ~trlx, ~cra~bled ~nd re~cor~d ~any
ti~o~ to yield a ~ean b~t rando~ core and
standard deviation (SD) The score for the real
seguence~ is expr~ssed as the number of SD unit6
away ~ro~ the random ~ean score (Dayhoff, M 0 et
al (1983) ~th EnzY~ 91, 52~-545) All of our
align~ents were done with the Mutation Data Matrix
(250 PAM6), ~d, a bia~ of 6, a gap penalt~ of 6
and 150 rando~ run~ (Willia~s, A F and ~arclay,
a N (1988) ~Dn Rev I~munol 6, 381-405)
FXA~
Characterization of AAMP-l
AAHP-l anti~odie6 Th- ~onoclonal antibody
produc d again~t AA~F~ of the IgG-I subtype
It cryoprecipitate~ and lo~e~ acti~ity with
fre-zing and purification ~ethod~ that reguire
pr cipitation Initial re~ult~ indicated that
thi- antibody inhibited adhe~ion and Jotility of
A2058 ~lano~ c-ll~ in cbe~oattractant ~say~
perfor~ed witb th~ ~odifl-d Boyden cha~ber
How ~er, tbe inbibition occurred in an all or none
fa~bion witbout a reli~ble do~e response curve and
steric hindrance due to ~elf aggregation of the
antibody cannot be ruled out at this time
Char~cterization of the Proteins Identif~ed bY
lAA3AA Antibodv A2058 ~el~noma cell surface
i~munofluore~cent ~taining has been seen with
lAA3AA lt identifie~ a prota~n on A2058 whole
cell lysate i~unoblot~ with a ~olecular weight of
approxi~ately 95kD that ~how~ an apparent slight
incr~a~e with reduction to 105kD
: .
~; - 16 -
.,
: :.
WO93/15202 ;`' `~ ?~ PCT/US93/~K~1
The betagalactosidase fusion protein
shows positive stalning with the lAA3Aa antlbody
on i~unoblots. The predicted AAMP-l protein i8
described below. ~t6 ~olecular ~eight and
glycosylation potential ~re not consi~tent wltb
the protein ldentified by lAA3Aa de~cribed above.
solation of lAA3Aa Po~itive cDNa Clones. Inltlal
~creQn~ng of ph~ge plaque~ yi-ld~d thr-e po~itlve
clones si~ilar ln ~lze, identlficd aB LAA34A,
lAA335A, and AAMP-l. They all cro-s hybridi~ed
~ith each other on dot blot~. AAMP-l was slightly
larger (le~s than lObp different) ~nd was cho-en
for ~eguencing. -
~ort~ern Blot of A2058 Melano a Total CytoDlasmic
and Polvadenylate Enriched RNa. ~hen all three
po~itive clone~ were u~ed to probe a blot of total
cytopla~ic A2058 RNA they hybridized with only
one band. A ~ingle band at 1.6kb i8 ~een on a
blot of both total cytopla~mic and polyadenylate
enriched A2058 RNA probed with AA~P-l in Figure 1.
~ucleotide Seauenoe. The AAMP-l cDNA has 1766bp
with the longe~t open reading fra~e ~1245bp)
occurring in the second reading fra~e of tbe
~equence (Figure 2; SEQ ID N0:1). 67% of the
~eguence excluding the poly A tail is involved
with repeats that include 7 or ~ore nucleotides
each. The largest direct repeat is A G G A G G A
A G A G at nucleotides #201 and ~168S. Its
soguence overlaps with that of a ten me~ber repeat
at nucleotides #197 and #1428. Anot~er ten membex
direct repeat occurs at positions ~948 and ~1508
and a third 10 ~e~ber repoat is at #1111 and
~1171. Se~eral pal1ndro~es exist in tbe seguence.
The longest palindro~e G G G T T C T A G A A C C C
- 17 -
W093/15202 2 1 2 '~ ,~ 7 ~3 PCT/US93/0060l
occurs at nucleotide #228 Ten n~b-r pal~ndromee
also occur at nucleotld~ #11~9 and ~13~0 ~lg~t
~ember pallndro~es are present ~t nucleotldes
#228, ~1119, #1515, and #1710 The la~t 25
nucleotides of the 1766bp ~equenc~ co~prl-e the
polyadenyl~ted nucleotlde tall and the con~en~u6
sequence A A T A A A A that co~only precQdes
poly A tail is pr sent at nucl~otlde ~1723
.
Predicted A~ino Ac~d Se~uence The 12~5bp open
reading fra~e in AA~P-l cDNA predlcts a protein
with a ~olecular weight of at least 4~ kilodaltons
when the first ~ethionine ln the equence (coded
by th~ tweltth codon) i- assun~d to be the
initiating ~ethionine ~Figure 2; S~Q ID N0 2)
The predicted protein contain~ ~ultiple
iJ unoglobulin-llke do~ins gualifying it as a
~e~ber of the i_unoglobulin (Ig) superfa~ily It
contain~ two potential trans~ brane r gion~ and
several serine/threonine phosphorylation sites
An acidic amino ter~inal region is also pre~ent
Nucleic acid
~-~ si~ilarity with CD4, an iD unoglobulin superfa~ily
bor, proapted a ~earch for Ig do~ain~ in AAMP-
1 proteln Fourt-on cy~t~ine~ are pr 6-nt with
~ight pr-s-nt on th a~ino ter~inal ~ide of the
potential trans~Qmbrane region~ (TMR) Seven
cy~teine pairs have 57 - 78 intervening a~ino
acids Tbese ~izes are consi6tent with t~ose
found in Ig dom~in6 I~munoglobulin V type
do~ains u~ually ha~e 65 - 75 intervening a~ino
acids ~nd C type do~ain~ have 55 - 60 Additional
cysteine pairs with ~3 and ~4 intervening a~ino
acids were ~l~o evaluated to find all po6sible
doD~ins with signific~nt ho~ology to t~e Ig
do~ains of thQ ~uperfa~ily ~ber~ Several of
,'' -
'~ ~
wo93/1s2o2 ,;~ 7 ~3 PCT/US93/~K~
these potential domalns are o~erlapping ~o t~at
fewer do~ains (1-3) are actually po~ible.
Significant ho~ology wa~ interpreted a~
align~ent~ yieldinq ALIGN scor-s of greater than
3.0 SD u~ing the AIIGN progra~ available in the
NBRF data base package (Protein Identification
Re~ource (1991) Proteln Seguence Database,
National Bio~edical Res-arch Foundation,
Washington, DC). The appropriate para et~r~ for
detecting ir~unoglobulin-like do~ains a~ ~pecified
by Willia~s an~ Barclay were used and are llsted
in the Method~. Score~ of 3.1, 4.3, and 5.2 SD
unit~ indicate chance probabilitie~ of 10-', 10-',
and 10-7, respectively, for aligned ~egu-nces to
show si~ilarities unles~ they are related.
Significant align~ent scores were found
for both V and C2 typ~ overlapping i~ unoglobulin
do~ains in AAMP-l. Cysteine t265 ~hows
~ignificant ho~olo~y either aB the first cy~teine
for~ing a disulfide bond or a~ the second cysteine
in an overlapping domain. When V type
i~unoglobulin do~ain~ are ~atched against the
putative AAMP-l do ain includinq cy~teine~ #,265
and #326 for~inq the predicted disulfide bond,
significant AIIGN scores can be obtained with
i~ounoglobulln dom~in~ from se~er~l Ig ~uperfamily
~ember~. Typical i Dunoglobulin domains are
defined a8 beginning 20 ~mino acids before the
first cysteine and ending 20 a ino acids after the
second cysteine of the disulfide bond. A
truncated vercion of the putative do~ain, a~252-
326, al80 ~atched again6t several V type do~ains
resulting in enhanced AIIGN 6core~ co~parQd to
those as~ociated with the complete do~ain and
rev~aled additional significant ho ologi~s when
~atched witb everal nore ~e~bers of the Tg
:
"
WO93/15202 PCT/US93/~U~1
superfa~ily, tbus, incre~ing the probability th~t
this i8 a Ig donain
Four Ig do~ains ~howed significant
homology witb tbe overlapping AAMP-l region
involving cysteines #208 and #265, aal88-285 A
larger, overlapping and inclu~ive region,
involving cysteines #208 and #282, region a~l88-
302, yield-d 1-~8 ~gnificant AI$GN ~cor 8
overall
Another region of AAMP-l involving
cy~teine #139 shows Ig do ain ho~ology of a les~er
degree Putative Ig do~ain~ with C#139 as either
the first or the second cysteine for~ing a
di-ulfide bond show significant rg do~ain
homology The putative donain of AAMP-l including
the aall9-228 reqion utilizes cysteine #139 as the
fir~t cysteine in the predicted disulfide bond
Its align~ nt i~ ~ignificant with at lQast three
other Ig do~ain~ at the pre~ent ti~e li~ted in
Table I and the half do ain ~atches with
additional half do~ains (myelin ba~ic
glycoprotein, HAG(IV), 3 gSSD: NCAM (V), 3 64SD;
and polyIgR~II), 3 11) Th~ other pos~ible Ig
do~ain involve~ cysteine #139 a~ the second
cysteine in a disulfide bond and includes the
region, aa79-159
Other ~horter ~ignificant align~ents were
found while searching thè Ig ~uperfamily and its
relatives for homology with AAMP-l One involves
a 17 amino acid region of CD4 (RWHUT4) which is
~lightly proximal to it~ hu~an i~munodeficiency
viru~ I (HIV-I) binding ~ite ~atching the region
including AAMP-l cysteine ~265 with an ALIGN score
of S 03SD There are two reqions in the ~IV-l
gpl20 envelope protein that ar si~ilar and one
region in the HIV-l nef proteln that i~ ~i~ilar to
,,
~ - 20 -
:~
~; .
WO 93/15202 ~ , r, ;~ ~ PCI`/US93/00601
Other Ig ~uperfa~ily ~e~bers ~ound ln the
PIR databa~e who~e Ig doaain~ were included in the
~earches for ~ignificant homology wlth AAMP-l
6howed le~s ~onology and are l~-ted a~ follow6:
S MUC18 (PIR3:A3~507) do~ain~ I-V, huaan ~elanoaa
cell surface glycoprotein: CD8 tRWHUT8) V region
do~ain, huaan T cell ~urface glycoprotein CD8
precur~or; Po protein (PIR3:JQ0622) V region
doaain, rat peripheral ~yelin protein O precur~or;
Ig la~bda chain C region; Ig beavy chain V
(GlHUNM) V region do ain, bu~an imaunoglobulin
heavy chain V-II region; CEA (PIR3:A36319) doaains
I, IV, and V, hu~an carcinoe~bryonic antigen
(clone co~CEAl); TcR-beta V (RWHUVY) V region
do~ain, ~uaan T-cell receptor beta chain precur~or
V region; Thy-l (TDHU) V region domain, human Thy-
1 ~ brane glycoprotein precur~or; MRC OX-2
(TDRTOX ) V and C region do~ain~, rat OX-2
~e~brane glycoprotein precursor; CD3 ep~ilon
(PIR2:A25769) C r~gion do~ain, human T cell
surface glycoprotein CD3 epsilon chain; ICAM
(PIR2:S00573) do~ains I-V, hu~an intercellular
adhesion ~olecule ls Ig X~ppa C (~3HU) C reg~on
:~ do~ain, hu~an i~unoglobulin kappa cha$n C region;
alpha-l-beta glycoprotein (OMHUlB) domains I and
II~ hu~an alph-l-beta-glycoprotein; MHC-II beta
(HLHU3D) non Ig type and C region domain6, human
~a~or histoco~patib~lity antigen, claæs II, beta
chain; amalga~ (PIR3:A31923) domains I-III,
Drosophila melanogaster; platelet derived growth
factor receptor (PDGF-R) domain (V), and human
ly~phocyte surface antigen precuræor CDW44
(PIR3:A32376~.
AAMP-l Internal Homolooy. Significant internal
- 35 ho~ology i~ al~o seen within AAMP-l, predominantly
involving the putative Ig do~ain6. The region,
~ 21 ~
:
.
W093/15202 . ~ ' PCT/US93/00601
a~ll9-169, including cy~telne ~139 shows ho~ology
with ~t le~st three other regions containinq
cysteines that m~y or may not be invol~ed witb
disulfide bond~. The~e ~re l~sted:
S cysteine ~a reg~on ~LIGN ~core
#265 245-295 6.29 SD
#216 196-246 4.11 SD
#96 76-126 4.02 SD
Another significant al~gnment i~ obtained
by match~ng the putative domain~, region aal88-
285 with region 76-lS9 yielding ~n ALIGN ~core of
6.63 SD.
Secondary Structure Prediction~ for Putative
Im~unoglobulin Domains in AAMP-l. The AAMP-l
region, aa76-346, enco~pas~ing all of the
potential Ig do~ain~ has predicted ~condary
structure characteri~tic~ consi~tent wit~ wh~t i6
found in Ig do~ain~. The Ig fold consi~tc of two
beta 6heet~ each containing 3-~ anti-parallel beta
str~nd~ (or ~heet~. The reg~on, ~24S-346,
cont~ins 4-8 beta ~h~at~ sep~r~ted by 10 potent~al
bet~ turns. The region, a~ll9-228, cont~in~ 8-9
beta heet~ s~parate~ ~y 10 potenti~l b~ta turn~
The other two region~, ~a76-159 and aal88-228 ~re
~i~ilar. Th~e pr~dictions are from the PIR
CHOFAS-Protein Secondsry Structure Prediction
Progra~.
Potent~al Trans~embrane Region~. There are two
potential transmembr~ne region~ predicted
according to the ~ethod of Rao and Argo6. meir
~thod uee~, a conformational preference parameter
- 22 -
W093/1520t PCT/US93/~K~l
for ~e~brane-buried h~llcos call-d the ~buried-
helix para eter~ ba~ed on hydratlon potentlal,
free energy of tran~fer fro~ aqueous helix to
nonpolar helix, polarity, bulk confor~ation~l
S preference, and turn confor~atlon~l pre~erence
expressed as a ~um of value6 for ach a lno acld
(Rao) One 24 a~lno acid reglon that uet their
criteria included aa323-347 wlth a-partic acid
#338 having the higbest ~buri-d hellx par~eter~
of 1 216 Tbe other potential trans~ brane
region, aa374-399, ha~ a peak value of 1 181 and
contain~ no charged residue6 Co~pari~ons were
~ade wlth other Ig superfa~ily De~bers'
trans~brane reglon~ (CD2, $cRa alpha, CD4, Poly
~5 Ig R, ~AG, and NCAM) No ~lgnificant align~ent6
were found for the ntire predicted tran~embrane
reglons but an align-ent ~core of 3 15 SD was
obtalned for a 19 anino acid region (aa379-397) of
the second AANP-l tran~ne brane region ~entioned
above wlth CD4's trans~e~brane region
Potential Phos~horylation Sites On the a~ino
~; ter inal side of the AAMP-l T~R~ there are five
site~ tbat have tbe con~-n~us pattern for
potential casein kina~e II pho~phorylation ~ite
(S,T)-x-x-(E,D) ~he~e in~olve serines at
po~itions #3, ~122, and #308 and tbreonines at
positions #109 and #165
Four potential protein k~na~e C
pbo~phorylation sites are al~o pre~ent with tbe
consensus pattern of (S,T)-x-(R,X). The~e include
two tbreonines at positions #238 and ~291 on tbe
~mino terminal side of tbe TMR and a threonine at
pos$tion ~35~ and a serine at position S308 on the
carboxy ter inal side of the TMR
~;~ 35 *
'~
~ - 23 -
WOg3/15202 PCT/US93/~K01
' 7 3
All public~tions mentioned herein~bove
are hereby incorpor~ted in their entirety by
reference.
While the foregoinq invention ha6 been
S de~cribed in ~o~e detail for purpo8eo of clarity
~nd underst~nding, it will be appr ciated by one
skilled in the ~rt from ~ reading of thio
di~closure that variou~ changes ln forn ~nd det~il
can be ~ade w~thout departing fro~ the true scope
of the invention ~nd appended clai~s.
24 -
~,
WO g3/15202 -~ 3 7 ^ PCr/USg3/00601
' SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Beckner, Marie E.
Llotta, L~nce A.
(ii) TITLE OF INVENTION: AAMP-l
(iii) NUMBER OF SEQUENCES: 2
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: CVSHMAN, DARBY ~ CUSHMAN
(B? SIPEP~: El-v nt~ Floor, 1615 L. Str~t, N.W.
(C) CITY: Wash~ngton
(D) STATE: D.C.
(E) COUNTRY: USA
(F) ZIP: 20036
(v) oo~PUT8R READABLE FORM:
(A) MEDTUM TYPE: Floppy disk
(8) COMPUTER: IBM PC co~p~tible
.:,:-, ~ ~ .
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOF$WARE: ~tentIn R-le~e ~1.0, Ver~ion #1.25
(vi) CURRENT APPLS,CATION DATA:
, (A) APP~ICATION NUMBER:
(B) FILI~G DATE:
; (C~ CLASSIFICATION:
x) TELECOHHUNICATION INFORMATIO~:
(A) TELEPHONE: (202~86I-3000
(B) TELEFAX: (202) 822-0944
, ,,
b"'~
~ 25 -
WO g3/15202 ~ 7 ., PCr~USg3/00601
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1766 ba~e p~irs
(B) TYPE: nucleic acld
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/XEY: CDS
(B) LOCATION: 35..1279
(xi) SEQUENCE DESC~IPTlON: SEQ ID NO:l:
GGGCcAGaGA AGTGGAATCC GCCGCTSGCG CCGC ATG GAG TCC GAA TCG GAA ~ 52
Met Glu Ser Glu Ser Glu
~ -;
AGC GGG GCT GCT GCT GAC ACC CCC CCA C~G GAG ACC CTA AGC TTC CAT 100
Ser Gly Ala Ala Ala Asp Thr Pro Pro L~u Glu Thr ~u Ser Phe His
, .
10 15 20
GGT GAT GAA GAG ATT ATC GAG GTG GTA GAA CTT GAT CCC GGT CCG CCG 148
Gly A~p Glu Glu Tle Ile Glu Val Val Glu Leu Asp Pro Gly Pro Pro
25 30 35
:~ GAC CCA GAT GAC CTG GCC CAG GAG ATG GAA GAT GTG GAC m GAG GAA 196
:~ A~p Pro Asp Asp Leu Ala Gln Glu Met Glu Asp Val Asp Phe Glu Glu
40 45 5~ .
GAA GAG GAG GAA GAG GGC AAC;GAA GAG GGC TGG GTT CrA GAA CCC CAG 244
Glu Glu Glu Glu Glu Gly A~n Glu Glu Gly Trp Val Leu Glu Pro Gln
55 60 65 70
, " "
- 26 -
. ,~
WOg3/15202 i 12~ 79 PCT/US93/00601
GAA GGG GTG GTC GGC AGC ATG GAG GGC CCC GAC GAT ACC GAG GTC ACC 292
Glu Gly Val V~l Gly Ser ~et Glu Gly Pro Asp Asp Ser Glu V~l Thr
75 80 85
TTT GCA TTG CAC TCA GCA TCT GTG m TGT GTG AGC CTG GAC CCC AAG 3 ~ o
Phe Ala Leu His Ser Al~ Ser Val P~e Cys Val Ser Leu Asp Pro Lys
90 95 100
acc AAT ACC TTG GCA GTG ACC GGG GGT GAA GAT GAC AAa GCC TTC GTA 388
Thr Asn Thr Leu A~la V~l Thr Gly Gly Glu A p Asp Lys Ala Phe Val
105 110 115
TGG CGG CTC AGC GAT GGG GAC CTG CTC T~T GAG TGT GCA GGC CAT AAA ~36
Trp Arg L~u Ser Asp Gly Glu L~u Leu Phe Glu Cys Ala Gly Nls Lys
120 125 130
GAC TCT GTG ACT TGT GCT GCT TTC AGC CAT GAC TCC ACT CTA GTG GCC ~84
A p S~r V~l T~r Cys Ala Gly Ph~ S~r His Asp Ser Thr L u V~l Al~
135 1~0 1~5 150
ACA GGG GAC ATG AG~ GGC CTC TTG AAA GTG TGG CAG GTG GAC ACT AAG 532
Thr Gly Asp M~t Ser Gly Lsu L~u Lys V~l Trp Gln V~l Asp Thr Lys
155 160 165
~.
GAG GAG GTC TGG TCC TTT GAA GCG GGA GAC CTG GAG TGG ATG GAG TGG 580
Glu Glu Val Trp Ser Ph~ Glu Ala Gly A~p Lsu Glu Trp Met Glu Trp '
170 175 180
CAT CCT CGG GCA CCT GTC CTG TTG GCG GGC aCA GCT GAC GGC AAC ACC 628
His Pro Arg Ala Pro Val L~u Leu Ala Gly Thr Ala Asp Gly Asn Thr
185 190 195
:
TGG:ATG TGG AAA GTC CCG AAT GGT GAC TGC AAG ACC TTC CAG GGT CCC 676
; Trp Met ~ Lys V~l Pro Asn Gly Asp Cys Lys Thr Phe Gln Gly Pro
200 205 210 ;
~ .
27 -
~g~
,g ~
, ~. , ~ ,
. ,,
WO93/15202 ,,;~ PCT/USg3/~K~
AAC TGC CCA GCC ACC TGT GGC CGA GTC CTC CCT GAT GGG AAG AGA GCT724
Asn Cys Pro Ala Thr Cy6 Gly Arg Val Leu Pro A~p Gly Ly6 Arg Ala
215 220 225 230
GTG GTA GGC TAT GAA GAT GGG ACC ATC AGG ATT TGG GAC CTG AAG CAG772-
Val Val Gly Tyr Glu Asp Gly Thr Ile Arg Ile Trp Asp Leu Lys Gln
235 240 245
GGA AGC C aTc CAT GTA CTG ;AA G~G ACT GAG GCT CAC CAG GGC CCA 820
Gly Ser Pro Ile Hls Val ~eu ~y6 Gly Thr Glu Gly Hl~ Gln Gly Pro
250 255 260
C ACC TGT GTT GCT GCC AAC CAG GAT GGC AGC TTG ATC CTA ACT GGC 868
Leu T~r Cy~ Val Ala Ala A~n Gln A~p Gly Ser Leu Il~ Leu T~r Gly
265 270 275
TCT GTG GAC TGC CAG GCC AAG CTG GTC AGT GCC ACC acc GGC AAG GTG916
S-r Val A p Cys Gln Ala Lys Lsu Val Ser Ala Thr T~r Gly Ly~ Val
280 285 290
GTG GGT GTT m AGA CCT GAG ACT GTG GCC TCC CAG CCC AGC CTG GGA 96
Val Gly Val Ph~ Arg Pro Glu T~r V~l Ala Ser Gln Pro Ser Leu Gly
295 300 305 310
GAA GGG GAG GAG AGT GAG TCC AAC TCG GTG GAG TCC TTG GGC TTC TGC ~ 1012
Glu Gly Glu Glu Ser Glu S-r A~n Ssr Val Glu Ser Leu Gly Phe Cys '
: 31S 320 325
AGT GTG ATG CCC CTG GCA GCT GT$ GGC TAC CTG GAT GGG ACC TTG GCC1060
Ser Val Met Pro Leu Ala Ala Val Gly Tyr Leu A~p Gly T~r Leu Ala
~: 330 335 340
.
ATC TAT ACC TGG CTA CGC AGA CTC TTA GGC ATC AGT GTC AGC ACC AGT 1108
Ile Tyr Thr Trp Leu Ar~ Arg Leu Leu Gly Ile Ser Val Ser Thr Ser
3~5 350 355
28 -
"
"
WOg3/15202 f I ~. ù `~ 7 .~ PCT/US93/~
CGG GCA TCG TGC AGC TGC TGT GGG AGG CAG GCA CTG CCG TGG TAT ATA . 115
Arg Al~ Ser Cys Ser Cy~ Cy~ Gly Arg Gln Al~ L~u Pro Trp Tyr Ile
360 365 370
CCT GCA GCC TGG ATG GCA TCG TGC GCC TCT GGG ACG CCC GGA CCG GCC 1204
Pro Ala Ala Trp Met Al~ Ser Cy~ Ala Ser Gly T~r Pro Gly Pro Ala
375 380 385 390
GCC TGC TTA CTG ACT ACC GGC GCC ACA CGG CTC aGA TCC TGG ACT TTG 12S2
ala Cys Leu Leu Thr T~r Gly Ala Thr ar~ L u arg S-r Trp Thr Leu
395 400 405
CCC TCA GCA Aa~ ATG CCT CCC TGG TGG T`GACCAQGTC AGGAGACCAC 1299
Pro S~r Al~ Ly- ~ t Pro Pro Trp Trp
~10 415
AAAGcGAAaG TAT51TGIGr C Q AGGCCT GAcccTTAaT GCCTGCAGCC CCTGCCTGTG 1359
IGICIGCTGT 5GAGCCCACG AAGCGACCCC TGCCCCTGTC TGccaGcaGA GGCAGTAGGG 1~19
CACAGAGGGA AGAGGaGGGT GGGGCCCTGG ATGACTTTCC AGccrcTTcA ACTGACTTGC 1~79
; ~ TCCCCTCTCC ITISCIICSC TTTAGAGACC CAGCCCAGGC CCCTCCCa,CC CTTGCCCAGA lS39
~ CCTGGTGGGC CCTTCAG~GG GAGGGCTGGA CCSGISICTC TTTCACTTTC AIITGCTGGT, 1599
:: . .
: GTGaGccATG GGGTGTGTAT TTGTATGTGG GG~GTAGGTG TTTGAGGTTC CCGTTCTTTC 1659
: CCTTCCCAAG TCTCTGGGGG TGGAAAGGaG GAAGaGATAc TAGTTAAAGA m TAAAAAT 1719
; ~ GIAAATAAAA TATACTTCCC AC ~u~AA~ AAAAAAAAAA AAAAAAA 1766
,: ~
~ 29 -
,-,
,.~
WO 93/15202 PCT/US93/~K~1 ~
~ ~ 2
(2) INFORHATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 415 a~ino ~cids
(B) TYPE: a~ino ~cid
(D) TOPO~OGY: llne~r
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESC~IPT~ON: SEQ ID NO:2:
Met Glu S~r Glu Ser Glu S~r Gly Ala Al~ Al~ Asp Thr Pro Pro Leu
1 5 10 15
Glu T~r Leu Ser P~e Hi~ Gly A p Glu Glu Ile Ile Glu V~l V~l Glu
` 20 2S 30
: L-u A~p Pro Gly Pro Pro A p Pro Asp Asp L~u Al~ Gln Glu Mct Glu
~ 35 40 ~5
-~ A~p V~l A p Ph~ Glu Glu Glu Glu Glu Glu Glu Gly A n Glu Glu Gly
SO SS 60
Trp Val Leu Glu Pro Gln Glu Gly Val Val Gly Ser Met Glu Gly Pro
70~ 75 ~ 80
A-p A p Ser Glu Val Thr Phe Al~ L~u Hi~ Ser Ala Ser Val Phe Cy~
Val Ser Leu Afip Pro Lys Thr Asn Thr Leu Al~ Val T~r Gly Gly Glu
100 105 110
~: Asp A~p Ly~ Ala Phe Val Trp~ Arg L~u Ser Asp Gly Glu Leu Leu Phe
~-- 115 120 125
Glu Cys Ala Gly Hi~ Ly~ Asp S-r Val Thr Cy8 Al~ Gly Phe Ser His
130 . 135 1~0
30 -
':'`.3~
WO 93/15202 PCl/US93/00601
A~p Ser T~r Leu Val Ala ll~r Gly Asp Met Ser Gly L:~u Leu Lys Val
145 150 155 $60
Trp Gln Val Asp ~rhr Ly6 Glu Glu V~l Trp Ser Phe Glu Ala Gly Asp
165 170 175
Leu Glu Trp Met Glu Trp H~ Pro Arg Ala Pro Val Leu Leu Ala Gly
180 185 190
Thr Ala A~p Gly A~n Thr Trp Met Trp Ly~ Val Pro A~n Gly a~p Cys
195 200 205
Lys T,hr Phe Gln Gly Pro A~n Cy~ Pro Ala Thr Cy~ Gly Arg Val Leu
210 215 220
Pro Asp Gly Ly6 Arg Ala Val V~l Gly Tyr Glu A~p Gly T~r Ile Arg
225 230 235 240
'
Ile Trp Asp Leu Ly~ Gln Gly Ser Pro Ile Hl~ Val Leu Ly~ Gly Thr
245 2S0 255
Glu Gly Hi~ Gln Gly Pro LQu $hr Cy~ Val Al~ Ala A8n Gln A~p Gly
- 260 265 270
Ser Leu Ile Leu Thr Gly Ser V~l A~p Cy~ Gln Al~ Lys Leu Val Ser
275 280 28~
Ala Thr Thr Gly Lys Val Val Gly V~l P~e Arg Pro Glu Thr Val Ala
290 295 300
Ser Gln ~ro Ser Leu Gly Glu Gly Glu Glu Ser GlU 5er Asn Ser Val
305 310 315 320
Glu Ser Leu Gly Phe C~8 Ser Val Met Pro Leu Ala Ala Val Gly Tyr
325 330 335
~ ,
-- -- 31 --
~,
.~
WO 93/15202 PCT/US93/~K~l
21.. `~`.. `:, ).7~ `
Leu Asp Gly T~r ~u Ala Ile Tyr T~r ~rp Leu ~rg Arg Leu Leu Gly
340 345 350
Ile Ser V~l Ser T~r S~r Arg Al~ Ser Cy8 S-r Cy~ Cy- Gly Arg Gln
3S5 360 365
Ala L~u Pro Trp Tyr Il~ Pro Ala Ala Trp Met Al~ Ser Cy¢ Ala Ser
370 375 ` 380
Gly Thr Pro Gly Pro Al~ Al~ Cy~ LQu Leu Thr T~r Gly Al~ Thr Arg
385 390 395 400
L4u Arg S~r Trp mr L~u Pro Ser Ala Ly~ Met Pro Pro Trp Trp
~ ~OS ~.~0 ~15
'~':
~::
,,~ ,.
:;
~,
, ~
.;,
'" ~h
~ 32 -
