CA2128879A1 - Aamp-1, a protein with local homologies to hiv-1 env and nef proteins - Google Patents

Abstract

The present invention relates, in general, to AAMP-1. In particular, the present invention relates to a DNA segment encoding AAMP-1; polypeptides encoded by said DNA segment; recombinant DNA
molecules containing the DNA segment; cells containing the recombinant DNA molecule; a method of producing an AAMP-1 polypeptide;
antibodies specific to AAMP-1; and a method of measuring the amount of AAMP-1 in a sample.

Description

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 -~ . . .

2 ~ 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.

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.

.

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).

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 . ~ .

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 .

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 ;:

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 :

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

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 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.

~, 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 , .

Gly A~p Glu Glu Tle Ile Glu Val Val Glu Leu Asp Pro Gly Pro Pro :~ 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 , " "

. ,~

WOg3/15202 i 12~ 79 PCT/US93/00601 Glu Gly Val V~l Gly Ser ~et Glu Gly Pro Asp Asp Ser Glu V~l Thr 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 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 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 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 ~.

Glu Glu Val Trp Ser Ph~ Glu Ala Gly A~p Lsu Glu Trp Met Glu Trp ' 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 :
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 ;
~ .

~g~

,g ~

, ~. , ~ , . ,, WO93/15202 ,,;~ PCT/USg3/~K~

Asn Cys Pro Ala Thr Cy6 Gly Arg Val Leu Pro A~p Gly Ly6 Arg Ala Val Val Gly Tyr Glu Asp Gly Thr Ile Arg Ile Trp Asp Leu Lys Gln 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 Leu T~r Cy~ Val Ala Ala A~n Gln A~p Gly Ser Leu Il~ Leu T~r Gly 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 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 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 .

Ile Tyr Thr Trp Leu Ar~ Arg Leu Leu Gly Ile Ser Val Ser Thr Ser 3~5 350 355 "
"

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 Pro Ala Ala Trp Met Al~ Ser Cy~ Ala Ser Gly T~r Pro Gly Pro Ala 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 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 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 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 ~: 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 ':'`.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 Leu Glu Trp Met Glu Trp H~ Pro Arg Ala Pro Val Leu Leu Ala Gly Thr Ala A~p Gly A~n Thr Trp Met Trp Ly~ Val Pro A~n Gly a~p Cys Lys T,hr Phe Gln Gly Pro A~n Cy~ Pro Ala Thr Cy~ Gly Arg Val Leu Pro Asp Gly Ly6 Arg Ala Val V~l Gly Tyr Glu A~p Gly T~r Ile Arg ' Ile Trp Asp Leu Ly~ Gln Gly Ser Pro Ile Hl~ Val Leu Ly~ Gly Thr Glu Gly Hi~ Gln Gly Pro LQu $hr Cy~ Val Al~ Ala A8n Gln A~p Gly 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 Ser Gln ~ro Ser Leu Gly Glu Gly Glu Glu Ser GlU 5er Asn Ser Val Glu Ser Leu Gly Phe C~8 Ser Val Met Pro Leu Ala Ala Val Gly Tyr ~ , ~, .~

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 Ile Ser V~l Ser T~r S~r Arg Al~ Ser Cy8 S-r Cy~ Cy- Gly Arg Gln 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 L4u Arg S~r Trp mr L~u Pro Ser Ala Ly~ Met Pro Pro Trp Trp ~ ~OS ~.~0 ~15 '~':

~::
,,~ ,.
:;

~, , ~
.;, '" ~h ~ 32 -

Claims (21)

WHAT IS CLAIMED IS:

1. A DNA segment coding for a polypeptide comprising an amino acid sequence corresponding to AAMP-1, or at least 5 contiguous amino acids thereof.

2. The DNA segment according to claim 1, wherein said DNA segment comprises the sequence shown in SEQ ID NO:1, allelic or species variation thereof, or at least is contiguous nucleotides thereof.

3. The DNA segment according to claim 2, wherein said DNA segment has the sequence shown in SEQ ID NO:1, allelic or species variation thereof.

4. The DNA segment according to claim 3, wherein said DNA segment has the sequence shown in SEQ ID NO:1.

5. The DNA segment according to claim 1, wherein said DNA segment encodes the amino-acid sequence set forth in SEQ ID NO:2, allelic or species variation thereof, or at least 5 contiguous amino acids thereof.

6. The DNA segment according to claim 5, wherein said DNA segment encodes the amino acid sequence set forth in SEQ ID NO:2, allelic or species variation thereof.

7. The DNA segment according to claim 6, wherein said DNA segment encodes the amino acid sequence set forth in SEQ ID NO:2.

8. A polypeptide free of proteins with which it is naturally associated and comprising an amino acid sequence corresponding to AAMP-1, or at least 5 contiguous amino acids thereof.

9. The polypeptide according to claim 8, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2, allelic or species variation thereof, or at least 5 contiguous amino acids thereof.

10. The polypeptide according to claim 9, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2, allelic or species variation thereof.

11. The polypeptide according to claim 10, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.

12. A polypeptide bound to a solid support and comprising an amino acid sequence corresponding to AAMP-1.

13. The polypeptide according to claim 12, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2, allelic or species variation thereof, or at least 5 contiguous amino acids thereof.

14. The polypeptide according to claim 13, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2, allelic or species variation thereof.

15. The polypeptide according to claim 14, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.

16. A recombinant DNA molecule comprising a vector and the DNA segment according to claim 1.

17. A cell that contains the recombinant DNA
molecule according to claim 16.

18. A method of producing a polypeptide having an amino acid sequence corresponding to AAMP-1 comprising culturing the cell according to claim 17 under conditions such that said DNA
segment is expressed and said polypeptide thereby produced and isolating said polypeptide.

19. A vaccine comprising the polypeptide according to claim 8 in an amount effective to elicit protective antibodies in a patient to HIV
and a pharmaceutically acceptable diluent, carrier, or excipient.

20. A method of preventing AIDS in a patient comprising administering to said patient the polypeptide according to claim 8 under conditions such that HIV infection is prevented.

21. A therapeutic modality useful in the treatment of inflammatory, immune, or neoplastic disorders in a patient comprising administering to said patient an effective amount of the polypeptide according to claim 8.