AU668134B2 - AAMP-1, a protein with local homologies to HIV-1 ENV and NEF proteins - Google Patents

Description

CORRECTED

VERSION* p pagt PcI- 3/3, Iran INTERNATIONAL APPLICATI :s 33-35, claims, replaced by new pages 33-35; pages 1/3- drawings, replaced by new pages 1/5-5/5; due to late smittal by the receiving Office ON PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) tI (51) International Patent Classification 5 (1I) International Publication Number: WO 93/15202 C12N 15/12, 5/10, A61K 37/02 Al (43) International Publication Date: 5 August 1993 (05.08.93) (21) International Application Number: PCT/US93/00601 (74) Agents: SCOTT, Watson, T. et al.; Cushman, Darby Cushman, 1100 New York Avenue, Washington, (22) International Filing Date: 29 January 1993 (29.01.93) DC 20005 (US).

Priority data: (81) Designated States: AU, CA, JP, European patent (AT, BE, 07/827,043 29 January 1992 (29.01.92) US CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE).

(71) Applicant: THE UNITED STATES OF AMERICA, represented by THE SECRETARY, DEPARTMENT OF Published HEALTH AND HUMAN SERVICES [US/US]; Na- With international search report.

tional Institutes of Health, Office of Technology Trans- Before the expiration of the time limitfor amending the fer, Box OTT., Bethesda, MD 20892 claims and to be republished in the event of the receipt of amendments.

(72) Inventors: BECKNER, Marie, E. 14225 Grand Pre Road, Silver Spring, MD 20906 LIOTTA, Lance, A.

9027 Mistwood Drive, Potomac, MD 20854 (US).

668134 (54)Title: AAMP-1, A PROTEIN WITH LOCAL HOMOLOGIES TO HIV-I ENV AND NEF PROTEINS (57) Abstract The present invention relates, in general, to AAMP-1. In particular, the present invention relates to a DNA segment encoding AAMP-I; 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-I in a sample.

(Ruerurrud to ii, PCT Gazctit No. 27/19Y13, Setion II) -4 WO 93/15202 PCT/US93/0060,1 AAMP-1, A PROTEIN WITH LOCAL HOMOLOGIES TO HIV-1 ENV AND NEF PROTEINS.

BACIGROUND OF THE IZNVZTION Field of the Invention 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 the DNA segment; recombinant DNA molecules containing the DNA segment; cells containing the recombinant DNA molecule; a method of producing AAMP-1; antibodies specific to AAMP-1; and a method of measuring the amount of AAMP-1 in a sample.

Backaround Information The major histocompatibility complex class II proteins have recently been found to contain local homologies to the HIV-1 envelope protein Golding et al., J. ExP. Med. 167, 914 (1988); H. Golding et al., J. Clin. Invest. 83, 1430 (1989); J. A. T. Young, Nature 332, 215 (1988)). Such homologous regions may serve as targets for antibodies generated to HIV-1 proteins and thus compromise the immune system in AIDS.

Golding et al. EXp. Med. 167, 914 (1988)) have identified a common epitope located in the carboxy terminus of the HIV-1 gp41-envelope protein and the amino terminal portion of the beta chain of all human HLA class II antigens. Although the epitope is small, 5 consecutive identities or similarities, they found that it is an effective example of "molecular mimicry" in that monoclonal antibodies raised against synthetic peptides from each protein react interchangeably with native HIV-1 envelope and MHC class II molecules. One third of HIV-1 positive individuals were shown to 1 WO93/1520-1 PCT/US93/00601 have serum antibodies directed against peptides derived from HIV-1 envelope protein, the homologous peptide from the MHC class II molecules, and native MHC class II molecules (H.

Golding et al. J. EX2. Med. 167, 914 (1988)). Two other regions of the HLA class II beta chain and another immune related protein, interleukin-2, also show limited homology to HIV-1 A. T.

Young, Nature 333, 215 M.A. Vega et al.

Nature 345, 26 1990).; W. E. Reiher III, et al.

Proc. Natl. Acad. Sci. USA 83, 9188 (1986)). An important consideration in HIV-1 vaccine development is the potential existence of additional host cell surface proteins with homologies to HIV-1 that may crossreact with antibodies directed against its peptides.

The present invention relates to the protein AAMP-1 which has immunoglobulin (Ig) type domains that contain strong local homologies to conserved regions of the HIV-1 envelope and nef proteins.

SUMMARY OF THE INVENTION It is a general object of this invention to provide AAMP-1.

It is a specific object of this invention to provide a DNA segment which encodes AAMP-1, or segment thereof.

It is a further object of the invention to provide a polypeptide corresponding to a AAMP- 1 gene, or fragment thereof.

It is another object of the invention to provide a recombinant DNA molecule comprising a vector and a DNA segment encoding a AAMP-1 gene.

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i, It is a further object of the invention to provide a cell that contains the above-described recombinant molecule.

It is another object of the invention to provide a method of producing a polypeptide encoding a AAMP-1 gene, or segment thereof.

It is a further object of the invention to provide antibodies having binding affinity for AAMP-1, or a unique portion thereof.

It is a further object of the invention to pro, de a method of measuring the amount of AAMP-1 in a sample.

It is another object of the invention to provide a therapeutic modality comprising the above-described polypeptides in an amount effective to elicit protective antibodies, block harmful auto-antibodies, or compete for HIV binding to body cells in a patient to the AIDS virus and a pharmaceutcially acceptable diluent, carrier, or excipient.

It is a further object of the invention to provide a method of preventing AIDS in a patient.

Further objects and advantages of the present invention will be clear from the description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Northern blot of human melanoma A2058 cells probed with AAMP-1 cDNA. A single 1.6Kb band is seen on blots of total cytoplasmic (Lane 1) and polyadenylate-enriched (Lane 2) A2058 RNA. Total cytoplasmic RNA, 41 micrograms was isolated from 6 million cells lysed in Nonidet P-40 separated into an aqueous phase in the presence of 7M urea, 1% sodium dodecyl sulfate, Tris buffer, NaCI, and EDTA, followed by phenol/chloroform extraction.

RNA, 2.2 pjg, enriched for messenger RNA, was isolated from 16 million cells with a Fast Track Kit -3i -L Version 2.1 (Invitrogen Corp. San Diego, CA). RNA was denatured in formaldehyde, electrophoresed in a 1% agarose/formaldehyde gel, transferred to Schlelcher Schnell Nytran nylon membrane and crosslinked with ultraviolet light. The 1766 bp AAMP-1 cDNA was 32 labeled with (alpa- P) dCTP (NEN Research Products, Boston, MA) using random priming. Hybridization overnight at 650C was performed according to Church and Gilbert (Proc. Natl. Acad. Sci 81, 1991 (1984)).

Figure 2. Nucleotide sequence of human A2058 melanoma cell AAMP-1 cDNA isolated from a lambda gtll expression library with its predicted amino acid sequence. The phage insert, AAMP-1, was subcloned into Bluescript plasmid (Stratagene) for production of double stranded cDNA for sequencing using the dideoxynucleotide termination method Sanger et al.

Proc. Natl. Acad. Sci. USA 74, 5463 (1977)) with Sequenase 2.0 Biochemcial). Nucleotide residues are numbered beginning at the 5' end. Amimo acid sequence numbering begins with the first methionine (underlined with of the open reading frame. The amino terminal acidic region, aa26-85, is underlined with Amino acid region, 76-346, comprised of potential immunoglobulin-like domains Willims 25 and A.N. Barclay, Ann Rev. Immunol. 6, 381 (1988); A.

F. Williems and A. N Barclay, in Immunoglobulin Genes, T. Honjo et al. Eds. (Academic Press Limited, San Diego, CA, 1989), pp. 361-387)) is underlined with secondary structure predications of beta strands, and beta turns, based on the method of Chou and Fasman (Advances in Enz. 47, 45 (1978)).

Cysteine pairs, 139 208, and 265 326, predicted by immunoglobulin domain homology to most likely form disulfide bonds are marked The potential transmembrane region, aa374-399, is underlined, I 04 6 I <^j M^rt- l->hra^^L- Ml~llJ.l. c-- The potential protein kinase C phosphorylation site at serine #408 is under with (The protein kinase C phosphorylation site consensus sequence (Ser/Thr-Xaa-Lys/Arg) where Xaa is usually an uncharged residue Woodgett, K.L.Gould, T. Hunter, Eur. J. Biochem. 161, 177 (1986)), is also found at threonines #238, 291, and 357)). The polyadenlytion site at nucleic acid residues, 1723-1729 is in parentheses Figure 3. Northern blot of AAMP-1 expression in human T-cell activation. Hours refer to time in culture. A: AAMP-1 single 1.6Kb message. B: Beta-2 microglobulin standard. Lanes 1-3: Ncn-stimulated human CD4+ T cells (Human peripheral blood mononuclear cells from normal donors were separated by Ficoll-Hypaque density-gradient centrifugation.

Unstimulated CD4+lyiiLphocytes were obtained by rigorous immunomagnetic negative selection with Advanced Magnetic Particles (Advanced Magnetic, Cambridge, MA) or Dynabeads (Dynal Inc., Fort Lee, NJ) both bound to goat anti-mouse IgG. Negative selection was performed as described J. Horgan and S. Shaw, Current Protocols in Immunology, J.E. Eoligan et al, Eds.

(Wiley Interscience, New York, 1991), p. 7.44.1.) using a cocktail of anti-HLA class II mAb (IVA12), mAb (1F5), CD16 mAb (3G8), CD11b -L i WO 93/15202 PCT/US93/00601 mAb (NIHllb-1), CD14 mAb (MMA), CD8 mAb (B9.8), and mAb against glycophorin (10F7). Purity of the isolated cells was greater than 98%. The selected CD4+ T-cells were free of monocytes based on the lack of proliferative response to optimal concentrations (1/200 dilution) of Phytohemagglutinin (M form) (PHA) (GIBCO, Grand Island, lanes 1 and 2 at 0 and 24 hours, respectively, without mitogen stimulation, and lane 3 after 12 hours in the presence of the protein synthesis inhibitor, cycloheximide, which has been frequently observed to stabilize certain mRNA species Kelly et al. P. Leder, Cell 603 (1983)). Lanes 4-8: CD4+T cells activated (T-cell activation assays were performed using standard techniques. Briefly, 10 million purified CD4+ T-cells were cultured in 35 mm flat bottom wells for various time periods in culture medium (RPM1 1640 (Hazelton Biologics Inc. Lenexa, KS) supplemented with 20 mM glutamine (Hazelton), heat inactivated fetal calf serum (Biofluids, Rockville, MD), 100 IU/ml of penicillin, and 100 pg/ml streptomycin), either unstimulated or stimulated with antibodies bound to the wells. Tcell stimulatory conditions were as described (G.

A. van Seventer et al. Eur. J. Immunol. 21, 1711 (1991)). Monoclonal antibodies were immobilized on the plastic of the well by dilution in phosphate buffered saline (PBS) and overnight incubation at 4*C, followed by washing with PBS.

The CD3 mAb, OKT3, and the CD2 mAb, 95-5-49, were applied at 1 pg and 10 pg, purified lg/ml respectively, all in a volume of 3 milliliters per well. Monoclonal antibodies were used as purified immunoglobulin derived from ascites fluid; CD2 mAb (directed against the Tll.l epitope): 95-5-49, IgGl (hybridoma kindly provided by Dr. R. R.

6 L WO 93/15202 PCT/US93/00601 Quinones, George Washington University, Washington, DC); CD3 mAb OKT3, lgG2a (ATCC, Rockville, MD). Cycloheximide, when present, was used at a concentration of 10 pg/ml. Lanes 4,5,6,7 and 8, represent the time points at 1, 2, 4, 16, and 24 hours, respectively. RNA samples were prepared from CD4+ T cells by the guanidinium isothiocyanate-cesium chloride method of Maniatis et al. E. Maniatis, E. F. Fritsch, J.

Sambrook, Molecular Clonina: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, ed. 2, 1989), pp. 7.18-7.22.)).

Ten micrograms of total RNA (each lane) electrophoresed in a formaldehyde/0.8% agarose gel was transferred to nitrocellulose and hybridized overnight, consecutively, at 42*C to the (alphadCTP labeled, random primed probes, AAMP-1 and beta-2 microglobulin.

DETAILED DESCRIPTION OF THE INVENTION In one embodiment, the present invention relates to a DNA segment coding for a polypeptide comprising an amino acid sequence corresponding to AAMP-1, or at least 5 contiguous amino acids thereof. In one preferred embodiment, the DNA segment comprises the sequence shown in SEQ ID NO:1, allelic or species variation thereof, or at least 15 contiguous nucleotides thereof (preferably, at least 20, 30, 40, or 50 contiguous nucleotides thereof). In a further preferred embodiment, the DNA segment encodes the amino acid sequence set forth in SEQ ID NO:2, allelic or species variation thereof, or at least contiguous amino acids thereof (preferably, at least 5, 10, 15, 20, 30 or 50 contiguous amino acids thereof).

7 it WO 93/15202 PCT/US93/00601 In a further embodiment, the present invention relates to a polypeptide free of proteins with which it is naturally associated or a polypeptide bound to a solid support and comprising an amino acid sequence corresponding to AAMP-1, or at least 5 contiguous amino acids thereof (preferably, at least 5, 10, 15, 20, 30 or contiguous amino acids thereof). In one preferred embodiment, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2, or allelic or species variation thereof equivalent thereto (for example, immunologically or functionally, equivalent thereto), or at least uontiguoue amino acids thereof (preferably, at least 5, 10, 15, 20, 30 -r 50 contiguous amino acids thereof).

In another embodiment, the present invention relates to a re'..mbinant DNA molecule comprising a vector (for example plasmid or viral vector) and a DNA segment (as described above) coding for a polypeptide corresponding to AAMP-1, as described above. In a preferred embodiment, the encoding segment is present in the vector operably linked to a promoter.

In a further embodiment, the present invention relates to a cell containing the above described recombinant DNA molecule. Suitable host cells include procaryotes (such as bacteria, including E. goi) and both lower eucaryotes (for example yeast) and higher eucaryotes (for example, mammalian cells). Introduction of the recombinant molecule into the cell can be effected using methods known in the art.

In another embodiment, the present invention relates to a method of producing a polypeptide having an amino acid sequence corresponding to AAMP-1 comprising culturing the 8 i I tf WO 93/15202 PCT/US93/00601 above-described cell under conditions such that the DNA segment is expressed and the polypeptide thereby produced and isolating the polypeptide.

In yet another embodiment, the present invention relates to an antibody having binding affinity for AAMP-1, or a unique portion thereof.

In one preferred embodiment, AAMP-1 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 (preferably, at least 5, 10, 15, 20, 30 or 50 contiguous amino acids thereof). In one preferred embodiment, the antibody is 1AA3.

Antibodies (monoclonal or polyclonal) can be raised to AAMP-1, or unique portions thereof, in its naturally occuring form and in its recombinant form. Binding fragments of such antibodies are also within the scope of the invention.

AAMP-1 may be joined to other materials, particularly polypeptides, as fused or covalently joined polypeptides to be used as immunogens.

AAMP-1 or its fragments may be fused or covalently linked to a variety of immunogens, such as keyhole limpet hemocyanin, bovine serum albumin, tetanus toxoid, etc. See for example, Microbiology, Hoeber Medical Division (Harper and Row, 1969), Landsteiner, Specificity of Serological Reactions (Dover Publications, New York, 1962) and Williams et al., Methods in Immunology and Immunochemistry, Vol. 1 (Academic Press, New York, 1967), for descriptions of methods of preparing polyclonal antisera. A typical method involves hyperimmunization of an animal with an antigen.

The blood of the animal is then collected shortly after thp repeated immunizations and the gamma globulin is isolated.

9 tI i- r: i i WO 93/15202 PCT/US93/00601 In some instances, it is desirable to prepare monoclonal antibodies from various mammalian hosts. Description of techniques for preparing such monoclonal antibodies may be found in Stites et al., editors, Basic and Clinical Immunology, (Lange Medical Publications, Los Altos, CA, Fourth edition) and references cited therein, and in particular in Rohler and Milstein in Nature 256:495-497 (1975), which discusses one method of generating monoclonal antibodies.

In another embodiment. ~he present invention relates to a hybridoma which produces a monoclonal antibody or binding fragment thereof having binding affinity for AAMP-1. In one preferred embodiment, AAMP-1 has the amino acid sequence set forth ii. SEQ ID NO:2, allelic or species variation thereof, or at least contiguous amino acids thereof (preferably, at least 5, 10, 15, 20, 30 or 50 contiguous amino acids thereof). In another preferred embodiment, the hybric.ima comprises 1AA3.

In yet another embodiment, the present invention relates to a diagnostic kit comprising: i) at least one of the above-described monoclonal antibodies, and ii) a conjugate comprising a binding partner of said monoclonal antibody and a label.

In a further embodiment, the present invention relates to a diagnostic kit comprising a conjugate comprising: i) at least one of the above-described monoclonal antibodies, and ii) a label.

In a further embodiment, the present invention relates to a method of measuring the amount of AAMP-1 in a sample, comprising contacting the sample with the above-described 10 Signatory's Name RICE CO PATENT ATTORNEYS FBR/9-93/P1 WO 93/15202 PC/US93/00601 antibodies and measuring the amount of immunocomplexes formed between the antibodies and any AAMP-1 in the sample. Methods of measuring the amount of immunocomplexes formed can be those well known in the art, such as RIA, ELISA, and direct and indirect immunoassays.

In another embodiment, the present invention relates to a therapeutic agent suitable for use in protecting against HIV infection or treating inflammatory immune or neoplastic disorders comprising the above-identified polypeptides in a quantity selected depending on the route of administration. Although subcutaneous or intramuscular routes of administration are preferred, the above described polypeptides could also be administered by an intraperitoneal or intravenous route. One skilled in the art will appreciate that the amounts to be administered for any particular treatment protocol can be readily determined. Suitable amounts might be expected to fall within the range of 1-50 micromoles.

In another embodiment, the present invention relates to a method of using the above described polypeptide to prevent AIDS. One skilled in the art will appreciate that the amounts to be administered for any particular treatment protocol can readily be determined.

The present invention is described in further detail in the following non-limiting Examples.

EXAMPLES

The following protocols and experimental details are referenced in the Examples that follow: 11 WA. =AU.Lpienr_ i -1 WO 93/15202 PCT/US93/00601 Cells. Human peripheral blood mononuclear cells (PBMC) from normal donors were separated by Ficoll-Hypaque density-gradient centrifugation.

Resting CD4+ T lymphocytes were subsequently obtained by rigorous immunomagnetic negative selection with Advanced Magnetic Particles (Advanced Magnetic, Cambridge, MA) or Dynabeads (Dynal Inc., Fort Lee, NJ) both bound to goat anti-mouse IgG. Negative selection was performed as described (Horgan, K.J. and Shaw, Immunomagnetic negative selection of lymphocyte subsets in Coligan, J.E. et al. (Eds.) Current Protocols in Immunology, Wiley Interscience, New York (1991) p. using a cocktail of mAbs consisting of anti-HLA class II mAb (IVA12), CD20 mAb CD16 mAb (3G8) CDllb mAb (NIHllb-1), CD14 mAb (MMA), CD8 mAb and mAb against glycophorin (10F7). Purity of the isolated cells was more than 98%. The selected CD4+ T-cells were free of monocytes based on the criterion that there be no proliferative response to optimal concentrations (1/200 dilution) of Phytohemagglutinin (M form) (PHA) (GIBCO, Grand Island, NY) (Davis, and P.E. Lipsky (1986) J. Immunol. 136:3588).

T-cell activation assays. T-cell activation assays were performed using standard techniques.

Briefly 10x10' purified CD4+ T-cells were cultured in 35mm flat bottom wells for various time periods in culture medium [RPMI 1640 (Hazleton Biologics Inc. Lenexa, KS) supplemented with 20 mM glutamine (Hazleton), 10% heat inactivated FCS (Biofluids, Rockville, MD), 100 IU/ml of penicillin, and 100 pg/ml streptomycin)], either unstimulated or stimulated with antibodies bound to the wells. Tcell stimulatory conditions were as described (van Seventer, G.A. et al. (1991) Eur. J. Immuol.

12 WO 93/15202 PCT/US93/00601 21:1711). mAbs were immobilized on the plastic of the well by dilution in PBS and overnight incubation at 4*C, followed by washing with PBS.

The CD3 mAb OKT3 and the CD2 uAb 95-5-49 were applied at 1 pg and 10 pg purified Ig/ml respectively, all in a volume of 3 al/well.

Monoclonal following antibodies were used as purified immunoglobulin derived from ascites fluid; CD2 mAb (directed against the Tl1.1 epitope): 95-5-49, IgG1 (hybridoma kindly provided by Dr. R.R. Quinones, George Washington University, Washington, CD3 mAb OKT3, IgG2a (ATCC, Rockville, MD). Cycloheximide, when present, was used at a concentration of 10 pg/ml.

CD4+ T cell RNA preparation. RNA samples were prepared from CD4+ T cells by the guanidinium isothiocyanate-CsCl method of Maniatis et al.

(Maniatis, T.E. et al. (1989) Molecular cloning: a laboratory manual. 2nd Edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, pg of total RNA was resolved for each condition on a formaldehyde 0.8% agarose gel, transferred to nitrocellulose, and hybridized at 42*C to "Plabeled purified AAMP-1 cDNA insert prepared by random priming.

Antibody Preparation. The adaptive passive transfer technique in Balb/c mice utilizing whole cells from the human melanoma A2058 cell line as antigen was used to generate hybridomas with myeloma cells. Selection of the 1AA3 clone was based on its inhibition of motility when assayed in modified Boyden chambers described previously (Stracke, M.L. et al. (1987) Biochem. Biophvs.

Res. Comm. 146, 339-345) using gelatin coated filters and various chemoattractants (collagen 13 (Rlrrred to ill P(*T CivettW No. 271193 Sectlon II) L

C

WO 93/15202 PCT/US93/00601 type IV, laminin, autocrine motility factor, fibronectin, and insulin-like growth factor I.

The clone 1AA3 was recloned by limiting dilution to produce the 1AA3AA clone.

cDNA Library and Screenina. The human melanoma A2058 cDNA expression library was constructed in the lambda gtll vector by Clontech Laboratories, Inc. Y1090 Escherichia coli infected by the phage were plated and blotted onto nitrocellulose filters (Schleicher Schnell) for immunoassay with 1AA3AA antibody. Reactive plaques were detected using peroxidase-coupled antibody specific for mouse IgG.

Northern Blotting, Preparation of A2058 human melanoma RNA enriched for messenger RNA was isolated with a Fast Track Kit Version 2.1 (Invitrogen Corp). Total cytoplasmic RNA was isolated according to a published method (Gough, N.M. (1988) Anal. Biochem. 173, 93-95) by suspending 4ml cells on ice with 0.8ml chilled Solution A (10 mM Tris Cl, pH 7.5, 0.15 MNaCl, mM Mgcl,, and 0.65% Nonidet P-40). The supernate, obtained after vortexing and centrifuging (800 x G, 5 min, 4'C) was mixed with 0.8 ml Solution B (7M Urea, 1% SDS, 0.35 M NaCI, 10 mM EDTA, pH and 10 mM Tris Cl, pH 7.5) and 1.6 ml. Solution C (phenol: chloroform: isoamyl alcohol (50:50:1).

RNA was removed in the aqueous phase and ethanol precipitated.

RNA was denatured in formaldehyde, separated on a 1% agarose/formaldehyde gel (Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) and transferred overnight to S&S Nytran (Schleicher Schnell) and 14 1 WO 93/15202 PCT/US93/0060 crosslinked to it with ultraviolet light in the Stratalinker apparatus (Stratagene). The 1766 bp cDNA insert labeled with (a-"P)dCTP (NEN Research Products) with the Random Primer DNA Labeling System (Bethesda Research Laboratories, Life Technologies, Inc.) was used as probe.

Northern blots for total A2058 melanoma cytoplasmic RNA and RNA enriched for messenger RNA were performed with the Church protocol (Church, G. and Gilbert, W. (1984) Proc. Natl Acad. Sci 81, 1991). The filter was washed at 65*C for minutes with wash buffer sodium dodecyl sulfate, 40 mM NaHPO,, 1 mM EDTA) three times and then autoradiographed at DNA Sequencing. Positive phage inserts were subcloned into Bluescript plasmid (phagemid) (Stratagene) for production of DNA for sequencing.

Double-stranded cDNA was sequenced using the dideoxynucleotide chain termination method with Sequenase (United States Biochemical). Sequence obtained with the SK primer (Stratagene) specific for the adjacent Bluescript vector region was determined first. Subsequent sequencing utilized primers prepared on site based on previously obtained sequence for both strands completely.

Sequence Data Analysis. GenBank (Intelligenetics, Inc.) was searched with the program and further analyses of the sequence was accomplished with RAOARGOS, PESTFIND, PROSITE, AACLUST, KERMIT NALIGN, PALIGN, REPEATS, SEQIN, TRANSL, AND DIAPRO programs in PC/Gene (Intelligenetics, Inc.). The NBRF protein sequence data base from the Protein Identification Resource National Biomedical Research Foundation (NBRF) was searched with the PQS, XQS, and NEW programs and other programs were 15 r 2j I WO 93/15202 PCT/US93/00601 used for sequence analyses. In the ALIGN program, sequences are matched with a bias and gap penalty, scored in a matrix, scrambled and rescored many times to yield a mean best random score and standard deviation The score for the real sequences is expressed as the number of SD units away from the random mean score (Dayhoff, M.O. et al. (1983) Meth. Enzvm. 91, 524-545). All of our alignments were done with the Mutation Data Matrix (250 PAMs), md, a bias of 6, a gap penalty of 6 and 150 random runs (Williams, A.F. and Barclay, A.N. (1988) Ann. Rev. Immunol. 6, 381-405).

EXAMPLE 1 Characterization of AAMP-1 AAMP-1 Antibodies. The monoclonal antibody produced against AAMP-1 is of the IgG-I subtype.

It cryoprecipitates and loses activity with freezing and purification methods that require precipitation. Initial results indicated that this antibody inhibited adhesion and motility of A2058 melanoma cells in chemoattractant assays performed with the modified Boyden chamber.

However, the inhibition occurred in an all or none fashion without a reliable dose response curve and steric hindrance due to self aggregation of the antibody cannot be ruled out at this time.

Characterization of the Proteins Identified by 1AA3AA Antibody. A2058 melanoma cell surface immunofluorescent staining has been seen with 1AA3AA. It identifies a protein on A2058 whole cell lysate immunoblots with a molecular weight of approximately 95kD that shows an apparent slight increase with reduction to 105kD.

16 r i~ 3 WO 93/15202 PCT/US93/00601 The betagalactosidase fusion protein shows positive staining with the 1AA3AA antibody on immunoblots. The predicted AAMP-1 protein is described below. Its molecular weight and glycosylation potential are not consistent with the protein identified by 1AA3AA described above.

Isolation of 1AA3AA Positive cDNA Clones. Initial screening of phage plaques yielded three positive clones similar in size, identified as 1AA34A, 1AA335A, and AAMP-1. They all cross hybridized with each other on dot blots. AAMP-1. was slightly larger (less than 10bp different) and was chosen for sequencing.

Northern Blot of A2058 Melanoma Total Cvtoplasmic and Polvadenylate Enriched RNA. When all three positive clones were used to probe a blot of total cytoplasmic A2058 RNA they hybridized with only one band. A single band at 1.6kb is seen on a blot of both total cytoplasmic and polyadenylate enriched A2058 RNA probed with AAMP-1 in Figure 1.

Nucleotide Seauence. The AAMP-1 cDNA has 1766bp with the longest open reading frame (1245bp) occurring in the second reading frame of the sequence (Figure 2; SEQ ID NO:1). 67% of the sequence excluding the poly A tail is involved with repeats that include 7 or more nucleotides each. The largest direct repeat is A G G A G G A A G A G at nucleotides #201 and #1685. Its sequence overlaps with that of a ten member repeat at nucleotides #197 and #1428. Another ten member direct repeat occurs at positions #948 and #1508 and a third 10 member repeat is at #1111 and #1171. Several palindromes exist in the sequence.

The longest palindrome G G G T T C T A G A A C C C -17- 4- :i WO 93/15202 PCT/US93/00601 occurs at nucleotide #228. Ten member palindromes also occur at nucleotides #1149 and #1340. Eight member palindromes are present at nucleotides #228, #1119, #1515, and #1710. The last nucleotides of the 1766bp sequence comprise the polyadenylated nucleotide tail and the consensus sequence A A T A A A A that commonly precedes a poly A tail is present at nucleotide #1723.

Predicted Amino Acid Sequence. The 1245bp open reading frame in AAMP-1 cDNA predicts a protein with a molecular weight of at least 44 kilodaltons when the first methionine in the sequence (coded by the twelfth codon) is assumed to be the initiating methionine (Figure 2; SEQ ID NO:2).

The predicted protein contains multiple immunoglobulin-like domains qualifying it as a member of the immunoglobulin (Ig) superfamily. It contains two potential transmembrane regions and several serine/threonine phosphorylation sites.

An acidic amino terminal region is also present.

Immunoalobulin Superfamilv HomoloMv. Nucleic acid similarity with CD4, an immunoglobulin superfamily member, prompted a search for Ig domains in AAMP- 1 protein. Fourteen cysteines are present with eight present on the amino terminal side of the potential transmembrane regions (TMR). Seven cysteine pairs have 57 78 intervening amino acids. These sizes are consistent with those found in Ig domains. Immunoglobulin V type domains usually have 65 75 intervening amino acids and C type domains have 55 60. Additional cysteine pairs with 43 and 44 intervening amino acids were also evaluated to find all possible domains with significant homology to the Ig domains of the superfamily members. Several of 18 r WO 93/15202 PCT/US93/00601 these potential domains are overlapping so that fewer domains are actually possible.

Significant homology was interpreted as alignments yielding ALIGN scores of greater than 3.0 SD using the ALIGN program available in the NBRF data base package (Protein Identification Resource (1991) Protein Sequence Database, National Biomedical Research Foundation, Washington, DC). The appropriate parameters for detecting immunoglobulin-like domains as specified by Williams and Barclay were used and are listed in the Methods. Scores of 3.1, 4.3, and 5.2 SD units indicate chance probabilities of and 10", respectively, for aligned sequences to show similarities unless they are related.

Significant alignment scores were found for both V and C2 type overlapping immunoglobulin domains ir AAMP-1. Cysteine #265 shows significant homology either as the first cysteine forming a disulfide bond or as the second cysteine in an overlapping domain. When V type immunoglobulin domains are matched against the putative AAMP-1 domain including cysteines #265 and #326 forming the predicted disulfide bond, significant ALIGN scores can be obtained with immunoglobulin domains from several Ig superfamily members. Typical immunoglobulin domains are defined as beginning 20 amino acids before the first cysteine and ending 20 amino acids after the second cysteine of the disulfide bond. A truncated version of the putative domain, aa252- 326, also matched against several V type domains resulting in enhanced ALIGN scores compared to those associated with the complete domain and revealed additional significant homologies when matched with several more members of the Ig 19 -6- WO 93/15202 PCT/US93/00601 superfamily, thus, increasing the probability that this is a Ig domain.

Four Ig domains showed significant homology with the overlapping AAMP-1 region involving cysteines #208 and #265, aal88-285. A larger, overlapping and inclusive region, involving cysteines #208 and #282, region aal88- 302, yielded less significant ALIGN scores overall.

Another region of AAMP-1 involving cysteine #139 shows Ig domain homology of a lesser degree. Putative Ig domains with C#139 as either the first or the second cysteine forming a disulfide bond show significant Ig domain homology. The putative domain of AAMP-1 including the aall9-228 region utilizes cysteine #139 as the first cysteine in the predicted disulfide bond.

Its alignment is significant with at least three other Ig domains at the present time listed in Table I and the half domain matches with additional half domains (myelin basic glycoprotein, MAG(IV), 3.95SD; NCAM 3.64SD; and polyIgR(II), 3.11). The other possible Ig domain involves cysteine #139 as the second cysteine in a disulfide bond and includes the region, aa79-159.

Other shorter significant alignments were found while searching the Ig superfamily and its relatives for homology with AAMP-1. One involves a 17 amino acid region of CD4 (RWHUT4) which is slightly proximal to its human immunodeficiency virus I (HIV-I) binding site matching the region including AAMP-1 cysteine #265 with an ALIGN score of 5.03SD. There are two regions in the HIV-1 gpl20 envelope protein that are similar and one region in the HIV-1 nef protein that is similar to AAMP-1.

i 20 iiII WO 93/15202 PCT/US93/00601 Other Ig superfamily members found in the PIR database whose Ig domains were included in the searches for significant homology with AAMP-1 showed less homology and are listed as follows: MUC18 (PIR3:A34507) domains I-V, human melanoma cell surface glycoprotein; CD8 (RWHUTS) V region domain, human T cell surface glycoprotein CD8 precursor; Po protein (PIR3:JQ0622) V region domain, rat peripheral myelin protein 0 precursor; Ig lambda chain C region; Ig heavy chain V (G1HUNM) V region domain, human immunoglobulin heavy chain V-II region; CEA (PIR3:A36319) domains I, IV, and V, human carcinoembryonic antigen (clone cosCEAl); TcR-beta V (RWHUVY) V region domain, human T-cell receptor beta chain precursor V region; Thy-1 (TDHU) V region domain, human Thy- 1 membrane glycoprotein precursor; MRC OX-2 (TDRTOX V and C region domains, rat OX-2 membrane glycoprotein precursor; CD3 epsilon (PIR2:A25769) C region domain, human T cell surface glycoprotein CD3 epsilon chain; ICAM (PIR2:S00573) domains I-V, human intercellular adhesion molecule 1; Ig kappa C (K3HU) C region domain, human immuncglobulin kappa chain C region; alpha-l-beta glycoprotein (OMHU1B) domains I and II, human alph-l-beta-glycoprotein; MHC-II beta (HLHU3D) non Ig type and C region domains, human major histocompatibility antigen, class II, beta chain; amalgam (PIR3:A31923) domains I-III, Drosophila melanogaster; platelet derived growth factor receptor (PDGF-R) domain and human lymphocyte surface antigen precursor CDW44 (PIR3:A32376).

AAMP-1 Internal Homology. Significant internal homology is also seen within AAMP-1, predominantly involving the putative Ig domains. The region, 21 i 8 .i *I WO 93/15202 PCT/US93/00601 aall9-169, including cysteine #139 shows homology with at least three other regions containing cysteines that may or may not be involved with disulfide bonds. These are listed: Cysteine aa region ALIGN score #265 245-295 6.29 SD #216 196-246 4.11 SD #96 76-126 4.02 SD Another significant alignment is obtained by matching the putative domains, region al188- 285 with region 76-159 yielding an ALIGN score of 6.63 SD.

Secondary Structure Predictions for Putative Immunoalobulin Domains in AAMP-1. The AAMP-1 region, aa76-346, encompassing all of the potential Ig domains has predicted secondary structure characteristics consistent with what is found in Ig domains. The Ig fold consists of two beta sheets each containing 3-4 anti-parallel beta strands (or sheets). The region, aa245-346, contains 4-8 beta sheets separated by 10 potential beta turns. The region, aai19-228, contains 8-9 beta sheets separated by 10 potential beta turns.

The other two regions, aa76-159 and aa188-228 are similar. These predictions are from the PIR CHOFAS-Protein Secondary Structure Prediction Program.

Potential Transmembrane Reaions. There are two potential transmembrane regions predicted according to the method of Rao and Argos. Their method uses a conformational preference parameter i 22 i- I 9 f WO 93/15202 PCT/US93/00601 for membrane-buried helices called the "bu!-tdhelix parameter" based on hydration potential, free energy of transfer from aqueous helix to nonpolar helix, polarity, bulk conformational preference, and turn conformational preference expressed as a sum of values for each amino acid.

(Rao) One 24 amino acid region that met their criteria included aa323-347 with aspartic acid #338 having the highest "buried helix parameter" of 1.216. The other pu.-ential transmembrane region, aa374-399, has a peak value of 1.181 and contains no charged residues. Comparisons were made with other Ig superfamily members' transmembrane regions (CD2, TcRa alpha, CD4, Poly Ig R, MAG, and NCAM). No significant alignments were found for the entire predicted transmembran* regions but an alignment score of 3.15 SD was obtained for a 19 amino acid region (aa379-397) of the second AAMP-1 transmembrane region mentioned above with CD4's transmembrane region.

Potential Phosphorvlation Sites. On the amino terminal side of the AAMP-1 TMRs there are five sites that have the consensus pattern for potential casein kinase II phosphorylation sites, These involve serines at positions #122, and #308 and threonines at positions #109 and #16!.

Four potential protein kinase C phosphorylation sites are also present with the consensus pattern o; These include two threonines at positions #238 and #291 on the amino terminal side of the TMR and a threOnine at position #357 and a serine at position $308 on the carboxy terminal side of the TMR.

23 10 WO 93/15202 PCT/US93!00601 All publications mentioned hereinabove are hereby incorporated in their entirety by reference.

While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention and appended claims.

24 I 1 11 WO 93/15202 PCT/US93/00601 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Beckner, Marie E.

Liotta, Lance A.

(ii) TITLE OF INVENTION: AAMP-1 (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: CUSHMAN, DARBY CUSHMAN STREET: Eleventh Floor, 1615 L. Street, N.W.

CITY: Washington STATE: D.C.

COUNTRY: USA ZIP: 20036 COMPUTER READABLE FORM: A) MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (202)861-3000 TELEFAX: (202) 822-0944 25

L

WO 93/15202 PCr/US93/00601 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 1766 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ix) FEATURE: NAME/KEY: CDS LOCATION: 35. .1279 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: GGGCCAGAGA AGTGGAATCC GCCGCTI'GCG CCGC ATG GAG TCC GAA TCG GAA Met Glu Ser Glu Ser Glu

AGC

Ser GGG GCT Gly Ala GAT GAA Asp Giu

GCT

Al a

GCT

Ala GAC ACC Asp Thr CCC CCA Pro Pro 15

CTG

Leu GAG ACC Glu Thr CTA AGC Leu Ser TTC CAT Phe His 100

GGT

Gly GAG ATT Glu Ile ATC GAG GTG Ile Glu Val 30 GTA GAA CTr GAT Val Glu Loeu Asp

CCC

Pro GGT CCG CCG Gly Pro Pro 148

GAC

Asp

GAA

GiU

CCA

Pro

GAG

Glu

GAT

Asp

GAG

Glu GAC CTG Asp Leu GCC CAG Ala Gin 45 GAG ATG Glu Met

GAA

Glu

GGC

Gly GAT GTG Asp Val so GAC TTT GAG GAA Asp Phe Glu Glu CTA GAA CCC CAG Leu Glu Pro Gin 196 4

GAA

GlU

GAG

Glu

GGC

Gly 60

AAC

Asn

GAA

Glu

GAG

Glu

TGG

Trp, 65

GT'

Val1 26 .1.3 WO 93/15202 PCT/US93/00601

GAA

Glu

GGG

Gly

GTG

Val GTC GGC Val Gly AGC ATG GAG Ser met Glu GGC CCC Gly Pro 80

GAC

Asp GAT AGC Asp Ser GAG GtC Glu Val

ACC

Thr 292

TTT

Phe

GCA

Ala

TTG

Leu

CAC

His

TCA

Ser GCA TCT GTG Ala Ser Val Phe 95

TGT

Cys

GTG

Val AGC CTG Ser Leu

GAC

Asp 100

GCC

Ala CCC AAG Pro Lys 340

ACC

Thr

AAT

Asn

ACC

Thr 105

CTC

Leu ?I'G GCA Leu Ala GTG ACC Val Thr

GG

Gly 110

CTG

Lou GGT GAA Gly Glu

GAT

Asp GAC AAA Asp Lys 115 TTC GTA Phe Val

TGG

Trp

CGG

Arg 120

TCT

Ser AGC GAT Ser Asp GGG GAG Gly Glu 125 CTC TiTT Lou Phe GAG TGT Giu Cys 130 GCA GGC Ala Gly CAT AAA His Lys 436

GAC

Asp 135

ACA

Thr

GTG

Val ACT TGT Thr Cys

GCT

Ala 140

GGT

Gly '1TC AGC CAT Phe Ser His

GAC

Asp 145

TCC

Ser ACT CTA Thr Lou GTG GCC Val Ala 150 ACT AAG Thr Lys 165 484

GGG

Gly

GAC

Asp ATG AGT Met Ser 155 GGC CTC T'TG AAA Gly Leu Lou Lys

GTG

Val 160

GAC

Asp TGG CAG Trp Gin GTG GAC Val Asp 532

GAG

Glu

GAG

Glu

GTC

Val

TGG

Trp 170

TCC

Ser TTT GMA Phe Glu GCG GGA Ala Gly 175 CTG GAG Lou Glu TGG ATG GAG TGG Trp Met Glu Trp 180 580

CAT

His

CCT

Pro

CGG

Arg 185

TGG

Trp GCA CCT Ala Pro GTC CTG TTG Val Lou Lou 190 GCG GGC Ala Gly ACA GCT Thr Ala

GAC

Asp 195 GGC MAC ACC Gly Asn Thr 628

TGG

Trp,

ATG

Met 200 AAA GTC Lys Val CCG MAT Pro Asn 205 GGT GAC TGC Gly Asp Cys AAG ACC Lys Thr 210 TTC CAG GGT CCC Phe Gin Gly Pro 676 -27 L WO 93/15202 PCrT/US93/00601

AAC

Asn 215

GTG

Val

GGA

Gly

CTC

Loeu

TCT

Ser

GTG

Val 295

GAA

Glu

TGC

Cys

CCA

Pro GCC ACC TGT GGC CGA GTC CTC CCT GAT GGG AAG Ala Thr Cys 220 Gly Arg Val Leu Pro 225

ATT

Ile Asp Gly Lys AdA Arq

OCT

Ala 230 724 GTA GGC Val Gly AGC CCT Ser Pro ACC TOT Thr Cys 265 GTG GAC Val Asp 280 GOT GTT Gly Val 000 GAG Gly 0Th OTO ATO Val Met TAT GAA Tyr Olu 235

ATC

Ile 250

CAT

His GAT 000 Asp Gly GTA CTO Val Leu GCC AAC Ala Asn 0CC AAO Ala Lys 285 ACC ATC AGO Thr Ile Arg 240 'AA 000 _y Gly 255 OTT OCT Val Ala TOC CAG Cys Gin TTT AGA Phe Arg GAG AOT Oiu Ser 315

CAG

Gin 270

GAT

Asp ACT GAG Thr Giu OOC AGC Oly Ser AGT 0CC Ser Ala GCC TCC Ala Ser 305

TOO

Trp

GOT

Gly

GAC

Asp

CAC

His

CCT

Pro 300

GAG

Glu CTO GTC Leu Val ACT GTO Thr Val AAC TCO Asn Ser GTT GOC Val Gly 335 TTO ATC Leu Ile 275 ACC ACC Thr Thr 290 CAG CCC 01n Pro TCC TTO Ser Leu OAT 000 Asp Gly CTG AAO CAG IAU Lys Gin 245 CAG GGC CCA Gin Gly Pro 260 CTA ACT GGC Leu Thr Gly GOC AAO GTG Gly Lys Val AOC CTG GGA Ser Leu Gly 310 GGC TTC TOC Gly Phe Cys 325 772 820 868 916 964 1012 1060 GAG TCC Glu Ser GCA OCT Ala Ala

OTO

Val 320

GAG

Glu

AGT

Ser

CCC

Pro 330

CTO

Leu TAC CTO Tyr Leu

ACC

Thr 340 TTG 0CC Leu Ala ATC TAT Ile Tyr

ACC

Thr 345 TGG CTA CGC AGA Trp Leu Arg Arg

CTC

Leu 350 TTA GOC ATC AGT Leu Gly I'.s Ser

GTC

Val 355 AGC ACC ACT Ser Thr Ser 1108 28 9 WO 93/15202 CGG GCA TCC Arg Ala Sez 360 CCT GCA GCC Pro Ala Ala 375 GCC TGC TVr Ala Cys Let CCC TCA GCJ Pro Ser Ala

AAAGCGAAAG

TGTCTGGTGT

CACAGAGGGA

TCCCCTCTCC

CCTGGTGGGC

GTGAGCCATG

CCTTCCCAAG

GTAAATAAAA

PCT/US93/00601 1 TGC AGC Cys Ser

TGC

cys TGG ATG GCA Trp Met Ala 380 CTG ACT ACC Laeu Thr Thr 395

TGT

Cys 365

TCG

Ser

GGG

Gly

GGG

Gly

TGC

Cys

GCC

Ala

TGG

Trp

AGG

Arg

GCC

Ala

ACA

Thr CAG GCA Gin Ala TCT CGG Ser Gly 385

CTG

Lau 370

ACG

Thr

CCG

Pro

CCC

Pro

TGG

Trp

GGA

Gly TAT ATA Tyr Ile

CCG

Pro

GCC

Ala 390 Leu 1156 1204 1252 CGG CTG AGA TCC TGG ACT Arg Leu Arg Ser Trp Thr 400 405 ~AAG ATG CC Lys Met P~ 410 TAT1?ITGTGT

TGAGGGGACG

AGAGGAGGGT

TTTTCTC

CCTTCAGAGG

GGGTGTGTAT

TCTCTGGGQG

TATACTTCCC

.T CCC ro Pro

CCAAAGGCCT

AAGGGACCCC

GGGGCCCTGG

TTTAGAGACC

GAGGGGTGGA

TTGTATGTGG

TGGAAAGGAG

AGAAAAAAAA

TGG TGACCACC rrp 415

GACCGTTAAT

TGCCCCTGTC

ATGACI'TCC

CAGCCCAGGG

CCTGTI'TCTC

GGAGTAGGTG

GAAGAGATAC

;TC AGGAGACCAC 1299

GGCTGCAGCC

TGCCAGCAGA

AGCCTCTTCA

CCCTCCCACC

TTT1CACTTTC

TTTGAGGTTC

TAGTTAAAGA

AAAAAAA

CCTGCCTGTG

GGCAGTAGGG

ACTGACTTGC

CTTGCCCAGA

ATI'TGCTGGT

CCGTTCTTTC

TTTTAAAAAT

1359 1419 1479 1539 1599 1659 1719 1766 29 WO 93/15202 WO 9315202PCTr/US93/00601 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 415 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Giu Ser Glu Ser Glu Ser Gly Ala Ala Ala Asp Thr Pro 1 Glu 5 Phe 10 Glu Pro Leu Thr Leu Ser His Gly Asp Glu 25 Asp Ile Ile Glu Val Val Giu Glu Met Glu Leu Asp Pro Asp Val Asp Gly Pro Pro Asp Pro 40 Glu Asp Leu Ala Gin Asn Phe Glu Glu Try Val GlU 55 Glu Glu Glu Glu Gly Ser Giu Glu Gly Leu Glu Pro Asp Gin 70 Thr Gly Val Val Gly 75 Ser Met Glu Gly Pro 8o Asp Ser Glu Val Pro Phe Ala Leu His 90 Leu Ala Ser Val Phe Cys Val Ser Leu Asp Asp Lys 115 Asp 100 Ala Lys Thr Asn Thr 105 Leu Ala Val Thr Phe Val Trp Arg 120 Ser Asp Gly Glu 125 Gly Gly Gly Glu 110 Leu Leu Phe Phe Ser His Glu Cys Ala Gly His Lys Asp Ser Val Thr Cys Ala 140 130 135 30 WO 93/15202 WO 9315202PC/US93/00601 Asp 145 Trp Ser Thr Lou Val Gin Val Asp Thr 165 Ala 150 Thr Gly Asp Met Ser 155 Gly Lau Lou Lys Val1 160 Lys Glu Giu Val Trp 170 Ser Phe Giu Ala Gly Asp 175 Leu Giu Trp Thr Ala Asp 195 Met 180 Giu Trp His Pro Arg 185 Ala Pro Vai Lou Lou Ala Giy 190 Gly Asp Cys Gly Asna Thr Trp Met 200 Trp Lys Val Pro Asn 205 Lys Thr 210 Phe Gin Gly Pro Asn 215 Cys Pro Ala Thr Cys Gly Arg Val 220 Asp Gly Thr Ile Leu Pro 225 Asp Gly Lys Arg Ala 230 Val Val Gly Tyr Giu 235 Arg 240 Ile Trp Asp Lou Lys 245 Gin Gly Ser Pro Ile 250 His Val Lou Lys Gly Thr 255 Glu Gly His Ser Lou Ile 275 Gin 260 Gly Pro Lou Thr Cys 265 Val Ala Ala Asn Gin Asp Gly 270 Leu Vai Ser Lou Thr Gly Ser Vai 280 Asp Cys Gin Ala Lys 285 Ala Thr Thr Gly Lys Vol 290 Val 295 Gly Vol Phe Arg Pro 300 Glu Thr Vol Ala Ser 305 Gin Pro Ser Lou Gly 310 Giu Gly Giu Giu Ser 315 Glu Ser Asn Ser Val 320 Giu Ser Lou Gly Phe 325 Cys Ser Vol Met Pro 330 Leu Ala Ala Vol Gly Tyr 335 -31 WO 93/15202 Lou Asp Gly Ile Ser Val 355 Thr 340 Lou Ala Ile Tyr Thr 345 Trp Lou Arg Arg PCI'/US93/0060 I Lou Lou Gly 350 Gly Arg Gin Ser Thr Ser Arg Ala 360 Ser Cys Ser Cys Cys 365 Aia Lou 370 Pro Trp Tyr Ile Pro 375 Ala Ala Trp Met Ala Ser Cys Ala 380 Thr Gly Ala Thr Ser Giy 385 Thr Pro Gly Pro Ala 390 Ala Cys Lou Lou Thr 395 Arg 400 Lou Arg Ser Trp Thr 405 Lou Pro Ser Ala Lys 410 Met Pro Pro Trp Trp 415 32

Claims (17)

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:l, allelic or species variation thereof, or at least 15 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. 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 contiguous amino acids thereof.

6. The DNA segment according to claim 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. 33 SUBSTITUTE SHEET i I WO 93/15202 PCr/IS93/00601

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 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 contiguous amino acids thereof. 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 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 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. 34 SUBSTITUTE SHEET c WO 93/15202 PCT/US93/00601 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 polypept4i3 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. 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. 35 I SUBSTITUTE SHEET F