IE870319L - Vaccines against melanoma - Google Patents

Abstract

New antigenic peptides (I) comprises all or part of the amino acid sequence of the melanoma-associated p97 antigen Also claimed are recombinant viruses contg. a DNA sequence coding for (I) under control of an expression-regulating DNA sequence, and recombinant DNA vectors (plasmids) p97b, pMtp97b and pSVp97a. p97b pref. comprises a 4.4 kb insert of the p97gene (from SK-MEL28 melanoma) in pEMBL18+. Excision of this insert and insertion into the SmaI site of the eukaryotic cDNA expression vector 1995.12pUC13 yields pMRp97b, which is used to transform LMTK-cells to obtaina p97-producing transformant TKMp97-12. [CH675424A5]

Description

97 -•I 1. FIELD OF THE INVENTION The present invention is directed to vaccine formulations which can induce an immune response that selectively destroys melancma -cells in a vaecinated individual. These use a peptide or protein related to a melanoma associated antigen, obtainable in large quantities via recombinant DNA techniques and/or Dy chemical synthetic methods. This peptide or protein can be used as an immunogen in a vaccine formulation.

Where the peptide or protein related to a melanoma associated antigen is expressed Dy a recombinant virus, the recomDinant virus itself may De used as an immunogen in a vaccine formulation. The invention can utilize processes which include the use of recombinant DNA techniques as well as chemical synthetic methods that enable the production of peptides or proteins related to the melanoma associated antigen in large quantities.

The invention envisages using as immunogens peptides related to p97, a monoineric cell surface sialcglycoprotein with an apparent molecular weight of slightly less than 97,000 daltons which is a cell surface component of melanoma cells. 2. BACKGROUND OF THE INVENTION 2.1. TUMOR-ASSOCIATED ANTIGENS Work with experimental animals, particularly rodents, has shown that most tumors induced Dy oncogenic viruses express antigens encoded Dy the viral genome, and that immunization with these antigens can lead to rejection of a suDsequent cnallenge of tumor cells induced Dy the same virus. Although much of this work was done with laboratory strains of virus, such as SV40, polyoma virus / and Friend/ Moloney, or Rauscher murine leukemia viruses, horizontal and vertical transmission of oncogenic viruses in nature have Deen demonstrated; indeed a commercial vaccine against virus-induced feline leukemia and sarcoma is now available.

By contrast, a viral etiology of most human cancer has not Deen demonstrated. NotaDle exceptions are hepatitis virus (hepatcma), herpes simplex virus (cervical carcincma) , and Epstein Barr virus (nasopharyngeal carcinoma). However, during the past two decades it has Deen estaDlished that some human tumor cells express tunior antigens, i .e ., antigens that distinguish the tumor cells fran their normal cellular counterparts; some patients mount cell-mediated or humoral immune responses against these antigens (Hellstrom e_t al. 1968, Nature, 220: 1352; Morton et al., 1968, Science 162: 1279-1281; Shiku et al_., 1976, J. Exp. Med. 144: 873-881). Some of the targets of these immune responses are oncofetal or differentiation antigens encoded Dy the human genome (Hellstrom .et al.. 1970, Int. J. Cancer 6: 346-351 ).

Until recently the molecular nature of the tumor antigens was unknown, and the degree of tumor specificity of the immunological reactions was unclear. Attempts to utilize this information in developing cancer diagnostic assays or cancer therapies have Deen largely unsuccessful. Since spontaneous tumor regressions are extremely rare, one may also conclude that the immune responses demonstrated i_n vitro were ineffective iji vivo; for example, while antiDodies arid lymphocytes oDtained from a cancer patient may De effective in killing tumor cells i^n vi tro, the immune response of the same cancer patient has no effect ^n v ivo.

The introduction Dy Kohler and Milstein of the monoclonal antiDody technique (1975, Nature 256: 495-4 97 ) led to intensified searches for human tumor antigens, since it provided the means to define such antigens, Doth at the molecular level and with respect to specificity (Hellstrcm and Brown, 1979, In "The Antigens", M. Sela, ed., Academic* Press, Vol. V:l-66). Over the past several years a large numDer of tumor-associated antigens have Deen descriDed, most 10 15 20 of which have Deen defined Dy mouse monoclonal antiDodies Reisfeld and Sell, eds., Monoclonal AntiDodies and Cancer Therapy, UCLA Symposia on Molecular and Cellular Biology, New Series, Vol. 27, Alan R. Liss, Inc. New York, 1985, pp. 1-609. Although virtually all of the antiyens which have Deeu well characterized have proven to De oncofetal or differentiation antigens, and their specificity for tumors has Deen found to De quantitative rather than qualitative, several antigens are sufficiently specific for neoplastic versus normal cells (generally corresponding to a factor of 10 to 1,000 times) to De used as potential targets for identifying tumor cells and for therapy. Human monoclonal antiDodies to tumor antigens have also Deen oDtained (Cote _et al., 1983, Proc. Natl. Acad. Sci. 80: 2026-2030). This supports the previously cited evidence that some cancer patients mount an immune reaction to their tumors.

More than half of the tumor-associated cell surface antigens so far identified are proteins or glycoproteins encoded Dy the human genome (rather than Dy endogenous or exogenous viruses), with the remainder Deing glycolipids, resulting fran aDnormal expression or regulation of glyosyl transferase s . 2. 2. MELANOMA ASSOCIATED p97 ANTIGEN The p97 antigen is a tumor-associated antigen that 25 was first identified in human melanoiua Dy using monoclonal antiDodies (Brown e_t al., 1980, J. Biol Chem. 255: 4980-4 983; Dippold et al., 1980, Proc. Natl. Acad. Sci. USA 77:6114-6118; WoodDury et al_., 1980, Proc. Natl. Acad. Sci. USA 77:2183-2187). The p97 antigen has Deen studied extensively 30 with regard to its expression in normal and neoplastic tissues , and is present in most human melanaiias and in certain fetal tissues, Dut is found in only trace amounts i"h normal adult tissues (Brown e^t al., 1981, J. Immunol. 127: 539-546; Brown et al., 1981, Proc. Natl. Acad. Sci. USA 5 78: 539-543; Garrigues e t al. , 1982, Int. J. Cancer 29:511-515). p97 has Deen used as a target for diagnostic imaging of melancmas in human clinical trials (Larson et al., 1983, J. Clin. Invest. 72:2101-2114). 5 p97 is a monomeric cell surface sialcglycoprotein, with an apparent molecular weight (MW) as measured Dy sodiuni dodecylsulfate-polyacrylamide gel electropnoresis (SDS-PAGE) of slightly less than 97,000 daltons. Monoclonal antiDodies have defined three major antigenic sites which are present on 1Q a staDle 40,000 dalton tryptic fragment (Brown .et. al_., 1981, J. Immunol. 127: 539-54 6); however, the complete sequence of p97 has not Deen reported. At least two other independently characterized human melanoma-associated antigens / gp95 (Dippold ejt al., 1980, Proc. Natl. Acad. Sci. USA 77:6114-15 6118) and gp87 (Khosravi e_t al_., 1985, Int. J. Cancer 35: 73-80) appear to De identical to p97 as analyzed Dy sequential immunopr ec ipi tation.

The N-terminal amino acid sequence of p97 is homologous to transferrin, and like transferrin p97 binds 20 iron (Brown et al. / 1982, Nature/ London, 296: 171-173). Analysis of somatic cell hyDrids and in_ situ hyDridization has shown that the p97 gene, like the genes for transferrin and transferrin receptor/ is located on chromosomal region 3q21-3q29 (Plowman e_t ,al_. / 1983, Nature, London, 303: 70-72; 25 Yang et al^., 1984, Proc. Natl. Acad. Sci. USA 81: 2752-2756). These oDservations suggest that p97 plays a role in iron metaDolism. 2.3. CANCER VACCINES Studies in experimental animals, usually mice, have 30 shown that immunization with living or killed cancer cells can lead to rejection of a suDsequent challenge of viaDle cancer cells. Attempts to immunize with cell-free material" have generally Deen less successful, Dut some successes have Deen reported. (For a review see Hellstrcm and Brown, 1979, s in The Antigens, M. Sela ed. Academic Press, Vol. V:l-66). In many cases the target antigens responsible for the protective effects have been virally encoded, but in many other cases the nature of the antigen which elicits a ® protective immune response is unknown.

Studies in humans are much more difficult, and the effectiveness of cancer vaccines is disputed, in spite of some reports of success. In many cases the vaccine preparations have consisted of irradiated tumor cells or TO tumor cells killed by exposure to certain chemical agents. Because pure human tumor-associated antigens have not been available there are no reports of their use in vaccines.

A major theoretical objection to the proposed use of cancer vaccines in humans is that humans who are "15 "vaccinated", for example, with killed cancer cells or cell-free preparations, will be immunologically unresponsive because the tumor antigens that may be the targets of the immune response are present, albeit in trace amounts only, in some normal cells and will thus be perceived by the immune 20 system as "self". Most, if not all, tumor-associated antigens detected in human tumors by monoclonal antibodies are also present in some normal tissues, and there is little evidence that cancer patients respond to them effectively in vivo. There is evidence that suppressor cells play a major 25 role in down-regulating the immune response to tumor antigens (Nepom et al.. 1983, Experientia, 39:235-242). Furthermore, a suppressor cell response induced by one set of tumor antigens may prevent the induction of an effective tumor-destructive response to another set of tumor antigens, which 30 by themselves would not induce suppression (Hellstrom_et al., 1983, in Biomembranes, A. Nowotny ed., Plenum Press, pp.365-388). 2.4. RECOMBINANT DNA TECHNIQUES AND VACCINIA VIRUS The use of reconiDinant DNA technology fur the production of suDunit vaccines to protect against infections involves the molecular cloning and expression in an appropriate vector of genetic information coding for proteins which can elicit an immune response against the protein in the host animal. Recently, a novel approach has Deen described which is potentially useful in the production of suDunit vaccines (Macke tt _et , 1982, Proc. Natl. Acad. Sci. 79: 7415-7419; Mackett e_t al_., 1984, J. Virol. 49: 857-864; Panicali, D. and Paoletti, E., 1982, Proc. Natl. Acad. Sci. 79: 4927-4931). This approach involves the use of vaccinia virus as a vector to express foreign genes inserted into its genome. Upon introduction into host animals, the recomDinant vaccinia virus expresses the inserted foreign gene and thereDy elicits a host immune response to such gene products. Since live recomDinant vaccinia virus can De used as a vaccine, this approach comDines the advantages of Doth suDunit and live vaccines.

Vaccinia virus contains a linear douDle-stranded DNA genome of approximately 187 kiloDase pairs and replicates within the cytoplasm of infected cells. These viruses contain a complete transcriptional enzyme system (including capping , methylating and polyadenylating enzymes) within the virus core that are necessary for virus infectivity.

Vaccinia virus transcriptional regulatory sequences (promoters) allow for initiation of transcription Dy vaccinia RNA polymerase Dut not Dy host cell RNA polymerase.

Expression of foreign DNA in reconiDinant vaccinia viruses requires the ligation of vaccinia promoters to protein-coding DNA sequences of the foreign gene. Plasmid vectors, also called insertion vectors, have Deen constructed to insert chimeric genes into vaccinia virus. One type of insertion vector is composed of: (a) a vaccinia virus promoter including the transcriptional initiation site; (D) 8 several unique restriction endonuclease cloniig sites located downstream from the transcriptional start site for insertion of foreign DNA fragments; (c) nonessential vaccinia virus DNA (such as the TK gene) flanking the promoter and cl-oning sites 5 which direct insertion of the chimeric gene into the homologous nonessential region of the virus genome; and (d) a bacterial origin of replication and antiDiotic resistance marker for replication and selection in col i. Examples of such vectors are described Dy MacKett (Mackett e_t al ♦, 1984, 10 J. Virol. 49: 857-864).

RecomDinant vaccinia viruses are produced Dy transfection of recomDinant Dacterial insertion plasmids containing the foreign gene into cells previously infected with vaccinia virus. Homologous recomDination takes place 15 with the infected cells and results in the insertion of foreign gene into the viral genome. The infected cells can De screened using immunological techniques, DNA plaque hyDr idi zat ion, or genetic selection for recomDinant viruses which suDsequently can De isolated. These vaccinia 2q recombinants retain their essential functions and infectivity and can De constructed to accommodate approximately 35 kilobases of foreign DNA.

Foreign gene expression can De detected Dy enzymatic or immunological assays (for example, 25 immunoprecipitation, radioimmunoassay, or imiuunob lot ting ) . Naturally occurring membrane glycoproteins produced from recomDinant vaccinia infected cells are glycosylated and may be transported to the cell surface. High expression levels can be obtained by using strong promoters or by cloning 2o multiple copies of a single gene. 3. SUMMARY OF THE INVENTION Vaccine formulations are described which may be used to induce an immune response that selectively destroys melanoma cells in vaccinated indiviauals. More specifically, 9 the vaccine formulations of the present invention comprise an irnnunogen that induces an irrmune response directed against a melanoma associated antigen, viz. the melanoma associated p97 antigen or an antigenic portion thereof. According to the 5 invention, a number of vaccine formulations are possible, wherein the immunogen (e.g. in a "suDunit vaccine" of the present invention) compr ises a peptide or protein related to p97 which may De formulated with an appropriate adjuvant. Such peptides or proteins 10 comprise amino acid sequences derived frcrn all or an antigenic portion of the amino acid sequence of p97 depicted in FIG. 3 including altered amino acid sequences in which functionally equivalent amino acid residues are suDstituted for residues within the sequence 15 resulting in a silent change, and/or modified or processed amino acid sequences, as for example, glycosylated amino acid sequences, phosphorylated amino acid sequences, etc. or chemically modified amino acid sequences. Hereinafter, the peptides or proteins of the present invention which are 20 related to the melanoma associated p97 antigen whether altered, unaltered, modified or unmodified, will De referred to as "p97 related peptides". Where the p97 related peptide is a hapten ( i.e ., antigenic Dut not immunogenic) the hapten can De conjugated to a carrier molecule that confers 25 immunogenicity .

The present p97 related peptides may be produced using recomDinant DNA techniques and/or chemical synthetic methods. When p97 related peptides are chemically synthesized such synthetic p97 related peptides can catiprise 30 those amino acid sequences derived from reg ions of p97 that are. antigenic (Hopp and Woods, 19 81, Proc.

Natl. Acad. Sci. USA, 78: 3824-3 828 ). Where the p97-related peptides employed are produced Dy using recomDinant DNA techniques, a nucleotide sequence which 35 encodes the whole or a portion of p97 is inserted into a recomDinant expression vector such as a virus or a plastnid 10 which, in an appropriate host, can direct the expression of a p97 related peptide that can be purified from the culture medium. The nucleotide sequence inserted is derived from all or portions of the p97 sequence substantially as depicted in FIG. 3 including but not limited to nucleotide sequences in which functionally equivalent nucleotide codons are substituted for codons within the sequence resulting in a silent change; in other words, different codons which encode the same amino acid or its functional equivalent may be substituted within the sequence depicted in FIG. 3. When a plasmid expression vector is used, one that is suitable for expression in eukaryotic cells is preferred, but a prokaryotic expression vector may also be used.

A vaccine formulation of the invention utilizing an expression vector which is a recombinant virus may be formulated as a viral vaccine, in which case the immunogen comprises the recombinant virus that expresses a p97 related peptide. ' Depending upon the nature of the recombinant virus used as the immunogen, either an inactivated virus vaccine or a live virus vaccine may be formulated. Appropriate immunization with the vaccine formulations of the present invention can result in the induction of an immune response which leads to the destruction of melanoma cells in the immunized subject.

This disclosure describes a system in which the vaccine formulation can be tested and outlines how the test should be performed. For example, the vaccine formulations can be evaluated for efficacy in animal models, initially rodents, then in non-human primates, and finally in humans, preferably in patients who are in remission but have a high probability of recurrence due to micrometastases. 4. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 represents an autoradiograph of the cell-free translation products of p97 mRNA resolved by SDS-PAGE. 1 i In Fig. 1A, lane 1 represents the translation products of p97 enriched mRNA, whereas lane 2 represents the translation products of unenriched mRNA, each derived from 0.5pi total translation products of 5ng mRNA. In Figure IB, lane 1 5 represents the translation products of p97 enriched mRNA whereas lane 2 represents the translation products of unenriched mRNA, each derived from 5u£ translation products of 5ng mRNA immunoprecipitated with anti-p97 serum.

FIG. 2 is a diagrammatic representation of the •jq structure of p97 mRNA. The arrangement of the coding region (from the signal sequence to the anchor sequence) and the non-coding region (3'UT) as well as the duplicated domain structure of the p97 precursor (open bar) is indicated. The location of various restriction enzyme recognition sequences 15 are indicated above the mRNA. The relative positions of four cDNA clones are indicated below the mRNA structure. The cDNA clone p97-3a2fl (3a2fl) was isolated from a cDNA library in which the. cDNAs were transcribed oh oligo(T)-primed p97-enriched mRNAs and cloned in pBR322; whereas, cDNA clones 2Q p97-2fl (2fl), p97-ljl (ljl), and p97-10al (lOal) were isolated by priming cDNA synthesis with oligonucleotides that encode p97 exon sequences and cloning the resulting cDNA fragments into lambda-gtlO. Fig. 2A is a diagrammatic representation of genomic clones B15, H17, B6.6 and E7.7 25 which were cloned in lambda L47.1.

FIG. 3 represents the nucleotide sequence of human p97 precursor cDNA and its deduced amino acid sequence. The N-terminal amino acid residues determined previously by protein sequencing were identical to those predicted from the 2Q nucleotide sequence (amino acid residue numbers 21-30). The potential glycosylation sites at amino acid residues 38, 135 and 515 (open bar) and the membrane anchor region at the C-terminus (solid bar) are indicated. One polyadenylation signal (AATAAA indicated in a box) was detected at position 25 3847, which is 50 base pairs upstream of a polyadenylated tract. i 2 FIG. 4 represents a comparison of the predicted amino acid sequences of the p97 precursor and that of human serotransferrin (Yang et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:2752-2756; Davis et al., 1985, J. Mol. Biol. 5 181:111-121). Conserved residues have been boxed. Tyrosine, histidine and arginine residues implicated in iron binding of transferrin (Metz-Boutigue et al., 1984, Eur. J. Biochem. 145:659-676) are indicated by asterisks (*).

FIG. 5 is a diagrammatic representation of a two-1 o dimensional model of the structure of p97 based upon the presence of cysteine residues conserved between the transferrin superfamily members. The three potential glycosylation sites are indicated by asterisks (*). The hydrophobic membrane anchor domain is apparent at the C-15 terminus of p97 (COOH).

FIG. 6 is a diagrammatic representation of the genetic structure of: the p97 cDNA clones and fragment of genomic clone lambda E7.7 used for construction of the p97 expression vector; and the pSV2p97a expression vector. The 20 following abbreviations are used: E, EcoRI; P, PvuII; Sal, Sail; S, SstI; B, BamHI.

FIG. 7 shows the results of gel electrophoresis in the characterization and immunopurification of recombinant p97 protein. A transfected CHO clone (CH03+) and SK-MEL 28 35 25 human melanoma cells were labeled with S-cysteine in the presence (+TM) or absence (-TM) of tunicamycin (TM). Cells were seeded into 100 mm plates and allowed to reach near confluency. Medium was removed and replaced with 3 ml cysteine-free medium with or without addition of 1 ug/ml 30 tunicamycin. After 30 minutes at 37*C, 250 uCi per ml L-S-cysteine (1016 Ci per mmol; New England Nuclear) was added for a further 6 hours. Harvesting of cells, preparation of cell lysates, immunoprecipitation, and SDS-PAGE were as described supra♦ Extracts were immunopreci-35 pitated with p97-specific antibodies, and proteins were 13 analyzed by SDS-polyacrylamide gel electrophoresis (SDS- PAGE). (a), Coomassie blue stained SDS-polyacrylamide gel.

Lane 1, protein markers; lane 2, immunopurified p97 shed from transfected mouse B16 cells; lane 3, SK-MEL 28 cells +TM; 5 lane 4, SK-MEL 28 cells -TM; lane 5, CH03+ cells +TM; lane 6, CH03+ cells -TM. (b) Autoradiogram of the same gel as in (a).

FIG. 8 shows the results of radioimmunoprecipi- tation of expressed p97 in transfected cells or Vp97a-NY infected cells. BSC cells were infected overnight with wild 10 type vaccinia virus or p97 recombinant vaccinia virus. These viruses or the transfected cell line CHO-p97.A were incubated 35 with S-labeled methionine and cysteine with or without tunicamycin at 2ug/ml (Sigma). The cells were lysed six hours later, precipitated with monoclonal antibody 96.5, and 15 separated by electrophoresis on a 10% polyacrylamide gel. The gel was autoradiographed overnight. The tunicamycin treated group is on the right for each group.

FIG. 9 depicts the serum antibody titers in mice immunized with p97 vaccines. Tp97 represents five mice 20 immunized with 5 x 106 irradiated M2svp97a.A cells (syngeneic tumor cells transfected and expressing surface p97) in complete Freund's adjuvant, and boosted with the same number of cells in phosphate-buffered saline. p97 is a group of five mice immunized with lOOug purified p97 protein in 25 complete Freund's adjuvant, and boosted with 50ug aqueous protein. Vp97 is a group of five mice immunized and boosted 7 with 10 plaque forming units of Vp97a-NY by tail scarification.

FIG. 10 depicts the therapeutic effect of 30 vaccination of tumor-challenged mice with a recombinant p97 5 vaccinia virus (Vp97a-NY). Mice were challenged with 10 or 104 p97-expressing tumor cells (M2SVp97a.E) intravenously. Two days later, mice were inoculated by tail scarification either with Vp97a-NY or Vwt-NY. Weekly inoculations by tail 35 scarification were repeated, and mouse survival recorded-. 14 5. DETAILED DESCRIPTION OF THE INVENTION The present invention is concerned with vaccines for the prevention or treatment of melanomas. It is based upon the observation that melanomas have tumor-5 associated cell surface antigens, such as the p97 antigen, which are present in greater amounts in the melanoma cells than they are in normal tissues. These vaccines afford peptides or proteins related to the melanoma associated p97 antigen (i.e., p97-related peptides) jq produced using recombinant DNA techniques and/or chemical synthetic techniques. The p97-related peptides afforded by these vaccines comprise amino acid sequences derived from the whole or antigenic portions of the amino acid sequence of p97 substantially as depicted in FIG. 3. These include amino 15 acid sequences derived from FIG. 3 which have been altered by the substitution of one or more amino acid residues within the sequence by another amino acid of a similar polarity which acts as a functional equivalent thus resulting in a silent alteration. Substitutes for an amino acid within the 1 5 sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and 5 methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine, and histidine. The negatively charged (acidic) amino acids include aspartic acid and 10 glutamic acid. Moreover, the p97 related peptides herein contemplated, whether or not altered by the substitution of amino acid residues, may be further modified or processed by glycosylation, phosphorylation, etc. or by chemical modifications. These p97 related peptides can be used as immunogens in vaccine formulations that elicit an immune response directed against melanoma cells present in the vaccinated patient.

For the purposes . of the present invention, recombinant DNA techniques can be used to insert 20 nucleotide sequences encoding the p97 antigen into expression vectors that will direct the expression of the p97 related peptides in appropriate host cells. The nucleotide sequences encoding the p97 antigen comprise nucleotide sequences derived from the whole or antigenic portions of the p97 nucleotide sequence substantially as depicted in FIG. 3. Due to the degeneracy of the DNA code for amino acids, (i.e., most amino acids can be encoded by more than one codon) functionally equivalent codons (i.e., different codons which encode the same amino acid or its functional equivalent) may be substituted within 30 the p97 sequence depicted in FIG. 3 provided the substitution results in a silent change. The expression vector-host cell systems which contain a nucleotide sequence encoding all or part of p97 can be used to produce large amounts of pure p97 related peptides iji vitro in which case the gene products can 35 be purified from the cells in culture and used as I 8 inununogens in suDunit vaccine formulations. Purification of the p97 related peptide may De accomplished using a variety of Diochemical methods, including imniunoaffinity purification using monoclonal antiDodies. In addition, purification of the p97 related peptide can De facilitated by modifying the DNA sequences that encode the p97 related peptides so that the sequences res pons iDle for anchoring the protein in the plasma membrane are removed yet the sequences responsible for transporting the protein to the cell membrane are not removed, so that a truncated antigenic molecule is secreted into the culture medium Dy the host cell. In the case of p97 related peptides produced Dy prokaryotic cells, lack of appropriate postranslational modifications may result in an antigenically inactive product, which may have to De activated Dy appropriate chemical or other treatments. .

In certain formulations, where the expression vector is a virus, the virus itself can De formulated as a vaccine. In such cases inactivated recomDinant virus vaccines can De prepared. Where the expression vector is 'an infectious recomDinant virus that does not cause disease in the host either an inactivated viral vaccine or a live virus vaccine preparation which provides for substantial immunity can be formulated. A particularly useful expression vector for this purpose is a recombinant vaccinia virus which expresses p97 related peptides as contemplated herein. To this end a nucleotide sequence coding for all or part of the p97 antigen can be -inserted into a vaccinia virus vector that is capable of directing the expression of the sequence in a appropriate host. Use may be made of other virus expression vectors as vaccines, more particularly, adenoviruses .

We also contemplate the deduced amino acid sequence of p97 being examined for sequences•with properties, particularly hydrophilicity, that are predictive of the presence of that sequence at the 1 7 surface of the protein molecule ana of its probable antigenicity and/or immunogenicity . These p97 related peptides may De chemically synthesized, if in fact antigenic, for use in preparing vaccine formulations. 5 It is of interest to consider producing p97 related peptides for purposes other than vaccine production. The p97 related peptide may De used to immunize animals so as to produce antisera or monoclonal antiDodies specific for the melanoma cells of interest. ■jQ These may De used as a component in a diagnostic assay, or for the affinity purification of radiolaDeled drug-linked, or toxin-linked antiDodies to De used for cancer therapy.

The invention is.described below, in more detail, with reference to the construction of a p97-based ;|5 vaccine against human melanoma.

The invention is presented as follows, solely for the purpose of clarity of description: (a) the nucleotide and amino acid sequence of p97; (d) p97 related peptides prepared Dy chemical synthetic methods; (c) p97 related peptides 20 produced Dy expression vector-host systems; (d) immunological characterization of the p97 related peptides; and (e) formulation of vaccines. 5.1. SEQUENCE ANALYSIS OF THE MELANOMA ASSOCIATED p9 7 ANTIGEN 25 The nucleotide sequence of the gene coding for p97 and its derived amino acid sequence are depicted in FIG. 3.

Functionally equivalent sequences are acceptable for the purposes of the invention. As indicated above, these include nucleotide sequences comprising all or portions of the 30 nucleotide sequence depicted in FIG. 3 which are altered by the suDstitution of different codons that encode the same or 1 8 a functionally equivalent amino acid residue thus producing a • silent change as well as amino acid sequences comprising all or portions of the amino acid sequence depicted, in FIG. 3 >. which are altered by the substitution of functionally ® equivalent amino acid residues within the sequence thus producing a silent change and derivatives thereof which are modified or processed, for example by glycosylation, phosphorylation, etc. or by other chemical modifications.

The subsections below describe the strategy that was used to determine the sequence of p97 as depicted in FIG. 3 as well as alternate techniques that could be used to determine the sequence of p97 or other tumor antigens that would be useful in vaccine formulations. 5.1.1. IDENTIFICATION AND CHARACTERIZATION 15 OF THE MELANOMA ASSOCIATED p97 ANTIGEN The activity and.amino acid sequence of the melanoma associated p97 antigen was not known; as a result, identification of the p97 antigen was accomplished using monoclonal antibodies directed against p97. A number of 20 techniques may be used to generate monoclonal antibodies specific for p97. For example, the hybridoma technique developed by Kohler and Milstein (1975, Nature 250:495-497) may be used as follows: mice or rats are immunized with human melanoma cells and lymphocytes collected from the 25 immunized animals are fused with myeloma cells; alternatively, lymphocytes from melanoma patients can be fused with myeloma cells (Cote,_et al_., 1983, Proc. Natl.

Acad. Sci. 80:2026; Haspel et^ ^1., 1985, Cancer Res. 45:3951), or the technique for producing monoclonal 30 antibodies using Epstein-Barr virus (Cole et_al., 1985, The EBV-Hybridoma Technique and its Application to Human Lung Cancer, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) can be used to generate monoclonal antibodies directed against p97. In any case, the resulting 1 0 hyDridomas are screened for the production of antiDodies that Dind to the melanoma cells Dut do not Dind to normal cells.

The monoclonal antiDodies directed against p97 descriDed aDove can De used in a nuiuDer of ways to facilitate 5 the identification, characterization, cloning and expression of nucleotide sequences which allow for the production of peptides and proteins related to the p97 antigen, in large quantities. For example, the monoclonal antiDodies may De used to further characterize the p97 antigen Dy radiolaDeling 1 q all the proteins made Dy the tumor cell, immunoprecipitating the tumor protein with the monoclonal antiDody used to identify the p97 antigen, and fractionating the immunoprecipitated proteins Dy gel electrophoresis. Protein antigens are identified as distinct Dands on the resulting 15 autoradiograph (Brown et al., 1980, J. Biol. Chem. 255:4980-4983 ). In addition, the monoclonal antiDodies directed against p97 can De used to facilitate cloning as follows: (a) to immunopurify polysomes in order to identify and'oDtain mRNA transcripts present in the iuelanoma cell which encode the p97 antigen; (D) to identify clones in a cDNA expression library that express peptides or proteins related to the p97 antigen; (c) to purify the p97 antigen in order to prepare additional monoclonal antiDodies or antisera for use in the previous two applications; or (d) to identify cells into which the gene for the p97 antigen has Deen introduced Dy tr ansfection.

The monoclonal antiDodies can also De used to facilitate structural and immunochemical characterization of the p97 antigen, so as to identify extracellular and antigenic domains of the molecule, and to purify the molecule for amino acid sequence analysis (Brown et al., 1981, Proc. Natl. Acad. Sci. USA 78: 539-543; Brown ej: al., 1982, Nature, London, 296: 171-173 ).

Further characterization of p97 involves 35 determination of cellular localization and mapping antigenic 20 25 30 20 de terminants and functional domains. SuDcellular localization can De determined Dy imiuunofluoresence microscopy and Dy cellular fractionation experiments. p97 antigens that are present on the cell 5 surface are employed for vaccine construction in the present case, as already indicated. If multiple monoclonal antiDodies are availaDle, antigenic determinants can De mapped Dy competition experiments, in which each antiDody is radiolaDeled and tested for competition with each of the 1Q other antiDodies. Domains of the molecule can De identified . Dy limited digestion with proteases followed Dy SDS-PAGE. Together these data should allow identification of regions of the molecule that are most immunogenic. If the monoclonal antiDodies were oDtained Dy immunization with intact cells, 15 then these regions of the molecule are most likely to be extracellular and to be useful for vaccine construction.

Amino acid sequence analysis allows unambiguous identification of the protein and its comparison with other proteins (Brown, e_t ^1., 1982, Nature, London, 296: 171-173). 2q If the protein comprises more than 50 amino acid residues, it may be feasiDle to determine only a part of the amino acid sequence, most often the N-terminus. Protein antigen for amino acid sequencing can De purified from cell lysates by immunoaffinity chromatography with the monoclonal antiDody, 25 followed Dy preparative SDS-PAGE. The N-terminal amino acid sequence of the purified protein is then determined by using an automatic amino acid sequencer, preferaDly a gas-phase machine for greatest sensitivity. 5.1.2. IDENTIFICATION, CLONING AND SEQUENCING OF DNA 30 CODING FOR THE MELANOMA ASSOCIATED p97 ANTIGEN Early cloning studies concentrated on aDundant proteins such as gloDin and ovalDumin, whose mRNAs often comprised 10 to 50% of total mRNA. These mRNAs could De purified to homogeneity Dy size fractionation, and pure cDNA 2 1 proDes were used to screen liDraries of a few hundred clones by colony hybridization. For proteins whose mRNAs comprise 1 of 10% of total mRNA, differential hyDridization with two cDNA proDes can De used, in which one of the cDNA proDes 5 contains the sequence of interest, and the other is a negative control. Messenger RNAs coding for low-abundance proteins, such as tumor-associated antigens, which may comprise as little as 0.01% of cellular mRNA, are much more difficult to clone, Decause tens of thousands of clones must 1Q De screened, and cDNA probes will not give a specific hyDridization signal. Both problems can De alleviated Dy enriching the mRNA for the sequence of interest.

Several approaches used to clone DNA coding for human melanoma associated p97 antigen are described below. 15 The resulting clones were analyzed in order to identify a clone or clones that spanned the entire coding region of p97. The p97 nucleotide inserts of the clones so identified can then be sequenced Dy any method known in the art. The various approaches are descriDed in more detail Delow. 20 (a) ISOLATION OF mRNA BY POLYSOME IMMUNOPURIFICATION In this technique, polysomes (which consist of mRNA, ribosomes and nascent polypeptide chains) are purified by immunoaffinity chromatography with antibodies that recognize antigenic determinants present on the nascent 25 chains. In many cases monoclonal antibodies obtained by immunization with intact cells or cell extracts recognize the antigens in their native conformations, but they may not be appropriate for polysome inununopur if icat ion, since there is a significant chance that the antigenic determinants recognized 30 may not be present in nascent chains. Since translation starts at the N-terminus of the polypeptide, epitopes close to the C-terminus are likely to De aDsent from the majority of the nascent chains. This problem is avoided either Dy using antiDodies that recognize N-terminal epitopes, or by 2 2 preparing polysomes from cells treated with a protein synthesis inhibitor that blocks termination.

A more serious problem is that mature proteins differ from nascent chains because of post translational 5 modifications. This problem is particularly acute for cell surface proteins, which are modified more extensively by removal of the signal peptides, addition of carbohydrate side chains, and formation of disulfide bridges. If a polyclonal antiserum is used for polysome immunopurification, the 10 differences in antigenicity between nascent chains and the mature protein may be of little consequence, since during immunization the rabbit or other animal is exposed not only to the native protein but also to partially or completely denatured forms, particularly if Freund's adjuvant has been 15 used. Even if these antibodies represent only a minor fraction of the antibody population there may still be enough present to bind the nascent chains. Unfortunately, preparation of a polyclonal antiserum to a low-abundance proteins may be extremely difficult. Although a monoclonal 2Q antibody may be used to purify the antigen for further immunizations, each gram of cultured cells often yields only a microgram of antigen. This is enough to immunize several mice, but barely sufficient for a single rabbit.

Another solution to the problem is to obtain a 25 monoclonal antibody that recognizes antigenic determinants present in nascent chains by using denatured p97 antigen as the immunogen used to prepare the monoclonal antibody. In the present example a panel of monoclonal antibodies that recognized three distinct epitopes on the N-20 terminal 40,000 dalton molecular weight domain of the p97 molecule were available, and we used a pool of monoclonal antibodies each with a different specificity in the hope that one or more of them would bind to nascent chains. The antibodies chosen were highly specific for p97, in that each 35 of them immunoprecipitated a single band of p97 from a 23 radiolabeled whole cell lysate, arid they had high binding affinities. For the cloning project we chose three lgG2a antibodies, one specific for each of the three epitopes. In general the chance of success may be increased by*using a 5 number of antibodies to distinct epitopes.

When using monoclonal antibodies the question remains of how one can predict whether a given monoclonal antibody or combination of antibodies will recognize the nascent chains and thus be suitable for use in polysome 10 immunopurification. One approach is to determine whether the monoclonal antibody immunoprecipitates antigen that has been translated in the reticulocyte lysate system, relying on the assumption that the in. vitro translation product, not being processed, will resemble the nascent chains. An alternative 15 approach is to proceed with the polysome immunoprecipitation on a small scale and then to use iri vitro translation to determine whether the mRNA species of interest has been enriched.

When the polysome immunopurification technique is 20 used it is important to monitor the purification by measuring mRNA activity. This can be done by translating the mRNA in a reticulocyte lysate system and analyzing the translation products by SDS-PAGE. Although the tumor-assoicated antigen may be too minor a component of the translation products of 25 unenriched mRNA to be seen among the hundreds of more abundant species, it should be detectable in the translation products derived from the enriched mRNA samples.

Alternatively the Xenopus oocyte translation system can be used, if a sensitive immunoassay is available to detect the 30 translated tumor-associated antigen. For p97 a highly sensitive double determinant immunoassay (DDIA), which employs two monoclonal antibodies specific for two different epitopes of p97, was used for this purpose.

Protein A bound to Sepharose can be used for the 35 polysome immunopurification. The protein A adsorbent has two applications in this procedure. The first is to purify the monoclonal antibodies from the crude ascites fluids, thereby removing contaminating ribonuclease activity. The protein A adsorbent is then used in conjunction with the purified 5 antibodies to immunopurify polysomes bearing the specific nascent chain.

Translation of the mRNA in a reticulocyte lysate system allows the biochemical characterization of the translation product as well as an assessment of its purity. 10 (b) OLIGONUCLEOTIDE PROBES Another method that may be used in connection with the invention to clone the cDNA coding for tumor-associated p97 antigen is to determine a. partial or complete amino acid sequence of the antigen and to synthesize an 15 oligonucleotide probe based on the nucleotide sequence deduced from the amino acid sequence. The oligonucleotide may then be used as a primer for cDNA synthesis and as a probe to screen the resulting cDNA library. Accordingly, the melanoma associated p97 protein may conveniently be purified 20 from lysates of melanoma cells by affinity chromatography with a specific monoclonal antibody (Brown, et. al., 1982, Nature, London 296:171-173). A nucleotide sequence coding for part of the determined amino acid sequence is then synthesized which can be used as a primer and/or probe. 25 Parts of the amino acid sequence containing amino acid residues coded by a single codon or two codons are most suitable for this purpose. One approach is to synthesize a longer sequence, typically 25 to 60 nucleotides, which represents the most probable coding sequence based upon the 30 known codon usage frequencies in huuans. The use of two synthetic oligonucleotides based on different parts of the amino acid sequence facilitates the screening by allowing one to identify spurious positive hybridization signals. Additionally, the use of hybridization conditions that minimize the effect of GC-content on the melting point of DNA hybrids also facilitates the screening. Once a partial cDNA clone has been obtained by this method it may be used as a probe to help obtain a full-length cDNA clone. (c) CDNA EXPRESSION LIBRARIES Cloning vectors have been developed that allow for expression of the cDNA insert in bacteria. One approach, therefore, which can be used to obtain cDNA clones for tumor-associated proteins such as p97, is to prepare a cDNA library by reverse transcribing the mRNA (enriched or unenriched) isolated from melanoma cells as described above, using oligo(T)-nucleotide primers or the synthetic oligonucleotide primers described above, and to screen such a library with a monoclonal antibody directed against the melanoma associated p97 protein. Clones that contain DNA coding for the epitopes recognized by the monoclonal antibody in the correct orientation and reading frame will express peptides or proteins related to the melanoma associated p97 protein and can be identified by transferring the proteins expressed by the clones to a nitrocellulose filter and incubating the filter with the antibody, followed by development with a labeled anti-immunoglobulin reagent.

A potential problem is that many monoclonal antibodies fail to recognize the protein expressed by the bacteria because, in many cases, only a part of the cDNA will be contained in the insert and bacteria do not process proteins in the manner in which eucaryotic cells do. This problem is particularly acute for tumor cell surface proteins, which are modified more extensively by removal of the signal peptides, addition of carbohydrate side chains, and formation of disulfide bridges. It may therefore be necessary to generate monoclonal antibodies that are known to recognize the denatured antigen or to prepare polyspecific antisera by immunization with purified antigen. 26 Once a recombinant virus or plasmid that is believed to contain a cDNA insert derived from a melanoma associated p97 antigen is identified, the cDNA insert can be used to screen additional libraries in order to identify 5 either full-length clones or else a group of clones that span the full length of the cDNA that codes of p97. The identity of the cDNA cloned can be established by sequence analysis and comparison of the deduced N-terminal amino acid sequence with that determined by direct amino acid sequence analysis ■jQ of the p97 protein. (d) GENOMIC CLONING The following method allows cloning of DNA using a monoclonal antibody directed against an antigenic determinant that is present only in the native protein and is not present 15 in nascent chains or in protein expressed in bacteria. To this end DNA derived from the human melanoma ceil is introduced in mouse L cells by transf ect ion. Subsequently,* mouse cells that express melanoma associated p97 antigen are isolated either by using the fluorescence-activated cell 20 sorter or by the immunological identification of colonies that produce p97 related peptides using radiolabeled monoclonal antibodies directed against p97 to detect related peptides on replicas of colonies transferred to polyester cloth filters. Several subsequent rounds of transfection may 25 be required to remove unrelated human DNA sequences. A genomic library is then prepared in a lambda phage vector and screened for clones containing human repetitive sequences which occur in the introns of most genes. Once a genomic clone is identified it can be used as a hybridization probe 30 to identify cDNA clones containing the DNA coding for p97. o n ** • 5.2. SYNTHESIS OF ANTIGENIC FRAGMENTS OF THE MELANOMA-ASSOCIATED p97 ANTIGEN AND EVALUATION OF IMM UNOGEN IC IT Y Synthetic peptides can De used as immuncgens to 5 elicit an immune response against the native protein that can provide a degree of protection against a nuinDer of pathogens. Such peptide sequences are selected from the known amino acid sequence of the protein antigen Dy identifying stretches of amino acids that are likely to De present on the surface of 10 the protein molecule, exposed to the external medium. This is most commonly achieved Dy computer analysis of the amino acid sequence using established hydropathy parameters for the amino acids. Additional criteria such as the predicted secondary structure or flexibility may also De used. 15 Accordingly, synthetic peptides comprising 5 to 50 amino acid residues of the melanaua associated p97 protein may be tested for immunogenicity in experimental animals (usually mice of rabbits). Such synthetic peptides include all or part of the amino acid sequence 20 substantially as depicted in FIG. 3 including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change and/or modified or processed sequences such as glycosylated sequences, phosphorylated 25 sequences etc., or chemically modified sequences. These p97 related peptides are used either alone or coupled to a carrier protein, such as keyhole limpet hemocyanin (KLH). In either case use of an adjuvant is optional, though preferable. The immunized animals are boosted and tested for 30 antibodies directed against the immunizing peptide. Those with anti-peptide antibodies are tested for antibodies that bind the native p97 protein. In the case of tumor-associated antigens such as p97 it is also of interest to test for a cellular immune response, for example, Dy looking for 35 delayed-type hypersensitivity (DTH ) , for an antigen 23 stimulated proliferation in. vitro, for cytolytic T-cells, or for tumor rejection in an appropriate model. An appropriate model would De a mouse tumor expressing the human tumor-associated antigen as result of transfection with an 5 appropriate cDNA expression vector construct.

The goal is to identify peptides that elicit a vigorous immune response directed against the melanoma-associated p97 antigen. Once identified, these peptides may De produced in large quantities Dy chemical synthetic methods 10 known in the art. Alternatively, the identified peptides may De produced in large quantities Dy expressing the nucleotide sequences that code for such peptides in expression vector-host cell systems. 5.3. PRODUCTION OF p97-RELATED PEPTIDES 15 BY EXPRESSION VECTOR-HOST SYSTEMS Proteins and peptides can De produced in large amounts Dy inserting nucleotide coding sequences into an appropriate expression vector, which is in turn introduced into suitaDle host cells, including, Dut not restricted to, 20 Dacteria, yeast, insect cells, and mammalian cells. Although Dacterial hosts have many advantages, they do not process many eukaryotic proteins appropriately, and they are less suitaDle than eukaryotic cells for the expression of tumor-associated proteins. However, recomDinant proteins produced 25 in Dacteria may De useful for induction of T-cell responses, since such responses are Delieved to require the initial degradation of the protein antigen.

In order to express p97 related peptides in a vector-host system, nucleotide sequences coding for the 30 melanoma associated p97 antigen or a portion thereof, are inserted into an appropriate expression vector. Such nucleotide sequences include Dut are not limited to all or part of the DNA sequence of p97 substantially as depicted in FIG. 3 including altered sequences in which one or more 2 D codons within the sequence is substituted Dy a codon which encodes the same or a functionally equivalent amino acid residue, thus, resulting in a neutral or silent change in the sequence. The expression vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. These elements vary in their strength and specificities. Depending on the host-vector system utilized, any one of a number of suitaDle transcription and translation elements may De used. For instance, when cloning in mammalian cell systems, promoters isolated from the genome of mammalian cells (e.g. mouse metallothionein promoter) or from viruses that grow in these cells (e .q . vaccinia virus 7.5K promoter) may De used. Promoters produced Dy recomDinant DNA or synthetic techniques may also De used to provide for transcription of the inserted sequences.

Specific initiation signals are also required for efficient translation of inserted protein coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where either the gene or cDNA sequence is inserted into an appropriate expression vector, no additional translational control signals may De needed. However, in cases where only a portion of the coding sequence is inserted, exogenous translational control signals, including the ATG codon may have to De provided. The initiation codon must furthermore De in phase with respect to the reading frame of the protein coding sequences to ensure translation of the entire insert. These exogenous translational control sequences and initiation codons may De of a variety of origins. Doth natural and synthetic.

Any of the methods known to those skilled in the art for the insertion of DNA fragments into a vector may De used to construct expression vectors containing a chimeric gene consisting of appropriate transcriptional and translational control signals and the protein coding 30 sequences,. These methods may include those used _in vitro recomDinant DNA techniques, synthetic techniques and in vivo recombinations (genetic recombination).

Expression vectors include, Dut are not "limited to the following vectors and their derivatives: vaccinia virus, adenoviruses, insect viruses, yeast vectors, bacteriophage vectors, and plasmid DNA vectors. The cloning and expression of genes in bacterial systems is well known in the art. For instance, when cloning in an E_^ coli, its Dacter iophages or plasmid promoters such as the lac promoter, trp promoter, recA promoter, riDosomal RNA promoter, the and PL promoters of coliphage lambda and others indluding Dut not limited to lacuv5, trp-lacuv5 (tac) hyDrid promoter, ompF, Dla, lpp and the like may De used to direct high levels of -transcription of adjacent DNA segment. However, due to the processing differences between prokaryotic and eukaryotic cells, it may be preferable to express the p97 related peptides used for the invention in eukaryotic cells. The best established methods of expressing proteins in eukaryotic cells are (a) introduction of the gene into the cell together with a drug resistance gene followed by selection with drug, preferably obtaining amplification as with the dihydrofolate reductase-methotrexate system; (D) expression of cDNA in a plasmid vector, often based upon pBR322, using a strong eukaryotic promoter and other regulatory sequences; (c) expression of cDNA in a viral vector, often derived from SV40, again using strong promoters, in this case an SV40 promoter. Recombinant plasiuid vectors are often used to produce cell lines that produce the protein over a long period of time, whereas SV40 vectors are often used to obtain transient expression. Although mammalian cells have most often been used as hosts, insect cells, and in cone cases yeast cells, may also be suitable. Some are described in more detail below. 31 10 15 20 25 30 35 In order to construct a recombinant vaccinia virus expressing the melanoma associated p97 antigen, the cDNA coding sequence can be ligated to the 7.5K promoter of vaccinia virus to form a chimeric gene. This chimeric gene is flanked by additional vaccinia viral sequence homologous to the viral thymidine kinase gene, which is carried on the plasmid DNA vector. The construction of the chimeric gene involves the use of both natural and synthetic control signals for transcription and translation of the tumor-associated antigen sequence. The chimeric gene is then introduced into vaccinia virus expression vectors through in vivo recombination between the homologous thymidine kinase region present on both the plasmid vector and the vaccinia virus genome. These recombinant viruses containing the chimeric gene are capable of directing the expression of p97 related peptides in an infected host and can be used as components of a vaccine.

In cases where an adenovirus is used as an expression vector, the DNA sequence of interest is ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequences.

This chimeric gene is then inserted in the adenovirus genome by _in vitro or jji vivo recombination. Insertion in a nonessential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the p97 related peptide in infected hosts. Presently, there are two strains of adenovirus (types 4 and 7) approved and used as vaccines for military personnel.

They are prime candidates for use as vectors to express the inserted DNA sequence.

An alternative expression system which could be used to express the p97 related peptides is an insect system. In one such system, Autoqrapha californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera fruqiperda 32 cells. The DNA sequence of interest can De cloned into nonessential regions (for example the polyhedrin £ene) of the virus and are placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful insertion of 5 the DNA sequence will result in inactivation of the polyhedrin gene and production of non-occluded recomDinant virus (i .e. , virus lacking the proteinaceous coat coded for Dy the polyhedrin gene). These recomDinant viruses are then used to infect Spodoptera fruq iperda cells in which the 10 inserted gene is expressed.

In addition, host cell strains iuay De chosen which modulate the expression of the inserted sequences, or modify and process the chimeric gene product in a specific fashion desired. Expression from certain promoters can De elevated 15 in the presence of certain inducers (e .q . zinc and cadmium ions for metallothionein promoters). Therefore, expression of the genetically engineered protein may be controlled.

This is. important if the protein product of the cloned gene is lethal to the host cells. Furthermore, modifications 20 ^ e .q ., g lycosylat ion, phosphorylation, etc.) and processing (e.q ., cleavage) of protein products are important for the structure and function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. 25 Appropriate cell lines or host systems can De chosen to ensure the correct modification and processing of the foreign protein expressed.

In the particular emDodiment descriDed herein for p97, we ligated the p97 cDNA sequence into an expression 30 plasmid vector derived from pBR322 which contains the metallothionein promoter. The entire coding sequence of p97 including the signal peptide and the membrane anchor was inserted into the vector. 5.3.1. IDENTIFICATION OF RECOMBINANT EXPRESSION VECTORS CAPABLE OF REPLICATING AND DIRECTING THE EXPRESSION OF THE p97 DNA SEQUENCES Expression vectors containing foreign gene inserts can De identified Dy three general approaches: (a) DNA-DNA hybridization, (D) presence or aDsence or marker gene functions, and (c) expression of inserted sequences. In the first approach, the presence of a foreign gene inserted in an expression vector can De detected Dy DNA-DNA hyDridization using proDes comprising sequences that are homologous to the foreign gene insert. In the second approach, the recombinant vector/host system can De identified and selected based upon the presence or absence of certain "marker" gene functions caused by the insertion of genes in the vector (e .q. thymidine kinase activity, resistance to antibiotics, transformation phenotype ,e tc .) . For example, if the foreign gene is inserted within the marker gene sequence of the vector, recombinants containing the DNA insert can be identified by the absence of the marker gene function. In the third approach, recombinant expression vectors can be identified by assaying the foreign gene product expressed by the recombinant. Such assays can be based on the physical, immunological, or functional properties of the gene product.

For example, when constructing a recombinant vaccinia virus for the purposes of this invention, the chimeric gene containing the p97 coding sequences is inserted into the thymidine kinase gene, thereby inactivating and endowing on the virus a TK phenotype. Such recombinants are selected by their ability to grow in media containing 5-bromo-deoxyuridine a nucleoside analog that is lethal to TK+ cells but not TK~ cells. The recombinants are further identified by DNA-DNA hybridization, usiny a cDNA probe specific for the tumor-associated protein. TK recombinant, virus can be isolated by plaque-purification and stocks are prepared from infected cultured cells. The recombinant virus 3 4 can De tested for its aDility to induce synthesis of the p97 related peptides. To this end, infected cells can be grown in the presence of radiolabeled amino acids; then lysates and subcellular fractions of the infected radiolabeled cells are 5 tested by immunoprecipitation with antibodies directed against the native melanoma associated p97 antigen. The immunoprecipitated products are resolved by SDS-PAGE.

Infected cells can also tested by immunofluorescence using monoclonal antibody. 1Q Cells into which a plasmid vector has been introduced by transfection can readily be identified by FACS analysis or by binding assays of replicas of cell colonies on polyester cloth. The amount of p97-related peptide present can be determined by a quantitative radioimmunoassay, and its 15 subcellular localization can be determined by cellular fractionation and by immunofluorescence microscopy. The structure of the p97 related peptide expressed can be determined by SDS-PAGE and by amino acid sequence analysis. 5.3.2. PURIFICATION OF THE p97—RELATED PEPTIDE 20 FROM EXPRESSION VECTOR-HOST SYSTEMS Many tumor-associated antigens such as p97 are cell-surface glycoproteins and contain an N-terminal signal peptide and a C-terminal anchor peptide (Davis _e_t a_l., 1985 , J. Mol. Biol. 181: 111-121). When expressed in an 25 appropriate vector it is expected that the protein will be translocated to the cell surface. To facilitate purification of the protein it may be preferable to delete the DNA sequence coding for the membrane anchor region, so that the mature protein is released into the culture medium. 30 The p97 related peptide can be purified from that host cells by detergent lysis followed by affinity chromatography using monoclonal antibodies. If a truncated-protein is to be purified from the culture medium it is preferable to use serum-free medium, and then to use affinity chromatography with monoclonal antiDodies. It is important that the antigen can De eluted from the antiDody adsorDent without either reducing its antigenicity or denaturing it. This may De achieved Dy raising or lowering the pH or Dy using a chaotrope. It may De necessary to select a monoclonal antiDody that will release the antigen under relatively mild conditions. The affinity-purified antigen may De purified further Dy HPLC. 5.4. IMMUNOLOGICAL CHARACTERIZATION OF p97-RELATED PEPTIDES The aDility of the synthetic or recomDinant antigen to elicit an antitumor response can De evaluated initially in experimental animals. This is achieved Dy constructing a model system in which the human melanoma-associated p97 protein is expressed in cells of the appropriate inDred strain of the experimental species. Animals are then immunized with the p97-related peptide used for the invention Dy various protocols and then tested for the development of antiDodies directed against the melanonta-associated p97 antigen, for cell-mediated immunity such as delayed-type hypersensitivity to the p97 antigen, and for their aDility to reject a challenge of viaDle, syngeneic tumor cells expressing the p97 antigen. In addition, in vitro assays of cellular immunity can De done to measure proliferation of lymphocytes in response to the p97 related peptide and the aDility of lymphocytes from immunized animals, or human melanoma patients, to kill tumor cells expressing the p97 antigen. Moreover, Dy immunizing mice with mouse p97, one is aDle to determine the extent to which it is possiDle to induce an immune response to an antigen that is present in trace amounts in normal tissues.

Non-human primates may De used to estaDlish the safety of the p97-related peptides used for the invention. n £» O U To this end the animals can De immunized using protocols that could ethically De applied to human cancer patients, and then tested as descriDed aDove, except that the tumor transplantation experiments will not De feasiDle, as these 5 require the use of inDred strains. The safety of the immunization procedure is determined Dy looking for the effect of the immunization on the general health of the. immunized animals (weight changes, fever, appetite, Dehavior , etc.) and looking for pathological changes on autopsy. 10 Finally the p97-related peptide used for the invention can De tested in human cancer patients. After initial phase I testing in advanced cancer patients, to estaDlish lack of toxicity, cancer patients in remission Dut with a high proDaDility of recurrence could De tested. Their 15 immune response would De evaluated as descriDed aDove for non-human primates, except that the effect of the treatment on established disease or on the frequency of recurrence will De examined. In the case of the melanoma antigen p97 Denign nevi (moles), which express the antigen, will also De 20 examined. 5.5. FORMULATION OF A VACCINE In this case of this invention, it is our purpose to produce, Dy synthetic or recomDinant DNA techniques, a peptide or a protein, or a recomDinant virus 25 that may be used as an immunogen and a vaccine to protect cancer patients at high risk of recurrence of their disease, to treat estaDlished disease, and ultimately to vaccinate high-risk individuals prophylactically• In fact, the synthetic or recomDinant me lanoma-asscciated p97 antigen may 30 De used in combination with other iiumunogens to prepare multivalent vaccines for the prevention of melanoma and other cancers. Examples of various vaccine formulations are discussed below. 5.5.1. VIRAL VACCINE FORMULATIONS When the p97-related peptide used for this invention is produced by a recomDinant virus, either a live recomDinant viral vaccine or an inactivated recomDinant viral 5 vaccine can De formulated. The choice depends upon the nature of the recomDinant virus used to express the p97 related peptide. Where the recomDinant virus is infectious to the host to De immunized Dut does not cause disease, a live vaccine is preferaDle Decause multiplication in the host <10 leads to a prolonged stimulus of similar kind and magnitude to that occurring in natural suDclinical infections and, therefore, confers suDstantial long-lasting immunity. The infectious recomDinant virus, upon introduction into a host, can express the p97-related peptide -frcstn its chimeric gene 15 and thereDy stimulate an immune response. The live recomDinant virus Dy itself may be used as a preventative vaccine against melanoma. Production of such recomDinant virus to De' used in these formulations may involve both in vitro (e .q . tissue culture cells) and in^ vivo (e .q . natural 20 host animal such as cows) systems. Conventional methods for the preparation and formulation of smallpox vaccine may De adapted for the formulation of live recomDinant virus vaccine.

Multivalent live virus vaccines can De prepared 25 from a single or few infectious recomDinant viruses that express a variety of antigens of different tumor or cancer cells. For example, a vaccinia virus (which can accommodate approximately 35 kiloDases of foreign DNA) can De engineered to contain coding sequences for other epitopes; such a 20 recomDinant virus itself can De used as the iuuuunogen in a multivalent vaccine. Alternatively, a mixture of vaccinia and/or other viruses, each capaDle of directing the expression of a different gene coding for different epitopes can De formulated in a multivalent vaccine . 38 Whether or not the recomDinant virus is infectious to the host to De immunized, an inactivated vaccine formulation may De prepared. Inactivated vaccines are "dead" in the sense that their irifectivity has Deen destroyed, 5 usually Dy treatment with formaldehyde. Ideally, the infectivity of the virus is destroyed without affectiiq the capsid or envelope proteins which carry the inimungenicity of the virus. In order to prepare inactivated vaccines, large quantities of the recomDinant virus must De grown in culture 10 in order to provide the necessary quantity of relevant antigens. A mixture of inactivated viruses which express different epitopes may De used for the formulation of "multivalent" vaccines. In some instances this may be preferable to live vaccine formulations because of potential 15 difficulties with mutual interference of live viruses administered together. In either case, the inactivated recombinant virus or mixture of viruses should be formulated with a suitable' adjuvant in order to enhance the immunological response to their antigens. Suitable adjuvants 20 include, but are not limited to, mineral gels, e .q . aluminum hydroxide; surface active substances such as lysolecithin; pluronic polyols; polyanions; peptides; and oil emulsions.

Many methods may De used to introduce the vaccine formulations descriDed aDove; these include Dut are not 25 limited to intradermal, intramuscular, intraperitoneal, intravenous, suDcutaneous and intranasal routes. When a live recomDinant virus vaccine formulation is used, it may De introduced via the natural route of infection of the parent wild type virus which was used to make the recombinant virus 30 in the vaccine formulation. 5.5.2. SUB UNIT VACCINE FORMULATIONS In an alternative to viral vaccines, the p97-related peptide itself may De used as an inununoyen in suDunit vaccine formulations. SuDunit vaccines comprise solely the 39 relevant immunogenic material necessary to immunize a host. Accordingly, the p97-related peptide may De purified from recomDinants that express the peptide. Such recomDinants include any of the previously descriDed virus-infected 5 cultured cells, Dacterial transformants, or yeast transformants . Alternatively, however, the p97-related peptides or proteins may De chemically synthesized.

Whether the p97-related peptides are purified from 10 recomDinants or chemically synthesized, the final product may De adjusted to an appropriate concentration and formulated with any suitaDle vaccine adjuvant and packaged for use . SuitaDle adjuvants include, Dut are not limited to: Mineral gels, e .q ., aluminum hydroxide; surface active suDstances 15 such as lysolecthin; pluronic polyols; polyanions; peptides; and oil emulsions. The p97-related peptide may also De incorporated into liposomes, or conjugated to polysaccharides and/or other polymers for use in a vaccine formulation.

In instances where the p97-related peptide is a 20 hapten, i .e., a molecule that is antigenic in that it can react selectively with cognate antiDodies, Dut not immunogenic in that it cannot elicit an immune response, the hapten may De covalently Dound to a carrier or immunogenic molecule and the hapteri-carr ier may De formulated for use as 25 a vaccine; for instance, a large protein such as protein serum alDumin will confer immunogenicity to the hapten coupled to it. 6. EXAMPLE: MELANOMA ASSOCIATED p97 ANTIGEN In the example descriDed Delow, cDNA clones derived 30 frctn various regions of p97 mRNA were pieced together and inserted into an expression vector which directs the expression of a peptide related to p97. The p97-relateo peptides produced Dy the expression vector-host cell may De formulated in a vaccine. 4 0 6.1. PURIFICATION OF p97 mRNA Polysomes were prepared from SK-MEL28 melanoma cells (Carey et 1976, Proc. Natl. Acad. Sci. USA 73:3270-3282) Dy magnesium precipitation. From this 5 preparation polysomes bearing p97 nascent chains were purified by incubation with 3 IgG2a monoclonal antibodies (96.5, 118.1. 133.2) specific for distinct epitopes of p97 (Brown et al^., 1980, J. Biol. Chem. 255: 4980-4983; Brown et al., 1981, J. Immunol. 127:539-546; Brown e_t _al., 1981, Proc. 10 Natl. Acad. Sci. USA 78: 539-543; Plowman e_t al., 1983, Nature, London 303:70-72 ) followed Dy affinity chromatography on protein A sepharose. The p97-enriched mRNA was eluted using EDTA and purified by affinity chromatography on oligo(T)-cellulose (Bethesda Research Labs, Bethesda MD). In 15 a typical experiment, 150 E^gQ units of polysomes yielded 260 ng p97-enriched mRNA, which represents 0.23% of the total mRNA. When translated in Xenopus oocytes and assayed for p97 as described (Brown et al..1981, Proc. Natl. Acad. Sci. USA 78: 539-543; Plowman et a_l., 1983, Nature, London 303: 70-72), 20 p97-enriched mRNA yielded 80 pg p97 per ng mRNA, whereas p97-unenriched mRNA yielded only 0.44 pg p97 per ng mRNA, showing that p97 mRNA activity had been enriched 180-fold. The yield of p97 mRNA activity was 42%. Translation in the reticulocyte lysate system (Pelham & Jackson, 1976, Eur. J. 25 Biochem. 67: 247-256) showed that p97-enriched mRNA coded for a major polypeptide with an apparent molecular weight of 84,000 daltons as analyzed by SDS-PAGE which was not detectable in the translation products of unenriched mRNA, and was immunoprecipitated by antiserum specific for p97 20 (FIG. 1). We concluded that this was the unglycosylated precursor of p97. d 1 6.2. PREPARATION AND CONSTRUCTION OF cDNA CLONES Two techniques described below were used to construct cDNA clones transcribed from the mRNA templates isolated above. 5 6.2.1. CONSTRUCTION OF cDNA CLONES PRIMED BY OLIGO(T) The p97-enriched mRNA prepared aDove was used as template for oligo(T )-primed cDNA synthesis. The cDNA was cloned in pBR322 as follows: for first strand cDNA synthesis, p97-enriched mRNA, the four dNTPs and oligo(T) 10 (CollaDorative Research, Walthma, MA) were incuDated with reverse transcriptase (Molecular Genetic Resources). The second strand was synthesized Dy incubation with the large fragment of E_^ coli DNA polymerase (Bethesda Research LaDs, Bethesda MD), and the double stranded cDNA was digested with 15 SI nuclease (gift from D. Durnam of The Fred Hutchinsen Cancer Research Center., Seattle, WA). The cDNA was then detailed with terminal deoxynucleotidyl transferase (Bethesda Research Labs, Bethesda, MD), annealed with Pst I-digested, dG-tailed pBR322 (Bethesda Research Labs, Bethesda, MD) 20 (Villa-Komaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731) and used to transform CaC^"treated E^ coli RR1. DNA from colonies of transformed bacteria was bound to paper (Taub & Thompson, 1982, Anal. Biocheiu. 126: 222-230) and screened by differential hybridization with cDNA proDes 25 synthesized on p97-enriched and unenriched mRNA templates.

A 243-Dase pair (bp) clone, p97-3a2fl, was identified, which hybridized to p97-enriched cDNA but not detectaDly to unenriched cDNA and also selected p97 mRNA in hybrid-selected translation experiments. A polyadenylation 30 signal (AATAA) and a poly(A) tract were present at the 3' end of the cDNA (see FIG. 2). Nick translated p97-3a2fl hybridized 100-fold more strongly to p97-enriched mRNA than to unenriched melanoma mRNA, and not detectably to fibroblast mRNA. Northern blot analysis with the cloned cDNA as a probe 4 2 identified an mRNA of approximately 4 kilobases (kb), which was present in SK-MEL 28 melanoma cells and absent from f ibroblasts. 6.2.2. GENOMIC CLONING OF p97 AND THE USE OF SYNTHETIC 5 OLIGONUCLEOTIDES TO PRIME cDNA SYNTHESIS Attempts to obtain cDNA clones extending more than 1 kb from the polyadenylation site were unsuccessful, possibly due to a region of high GC content (greater than 80%) with extensive secondary structure. Genomic cloning was 10 used to circumvent this problem. Four overlapping genomic clones were isolated from.libraries of lambda L47.1 containing size-fractionated SK-MEL 28 DNA enriched for a specific p97 restriction fragment. These four genomic clones span 28kb and contain the entire coding region of p97 15 including the regulatory region of the gene. The genomic clones as arranged sequentially from 5' to 31 are: lambda B15, lambda H17, lambda B6.6, and lambda E7.7. The nomenclature consists of a letter which refers -to the restriction enzyme used to generate the fragment and the 20 numeral indicating the kilobase size of the fragment which was cloned into lambda L47.1. Thus, starting from the 5' terminus, lambda clone B15 contains a 15kb BamHI p97 fragment; lambda clone H17 contains a 17kb Hindi!I p97 fragment; lambda clone B6.6 contains a 6.6kb BamHI p97 25 fragment; and lambda clone E7.7 contains a 7.7kb EcoRI p97 fragment (see FIG. 2A). Restriction fragments of the clones that hybridized to the 4 kb p97 mRNA on Northern blots were sequenced and p97 exons were identified by a computer assisted homology search between the predicted coding 30 sequences and the amino acid sequence of human and chicken transferrin (Yang et^ al., 1984, Proc. Natl. Acad. Sci. USA 81:2752-2756; McGillivray et al., 1982, Proc. Natl Acad. Sci. USA 79:2504-2508; Jetsch & Chambon, 1982, Eur. J. Biochem. 122:291-295). 4 3 Three synthetic oligonucleotides, the sequences of which were based on p97 genomic exon sequences, were used to prime cDNA synthesis on SK-MEL 28 mRNA and the resulting cDNA was cloned into lambda-gtlO as follows: the p97 cDNA was dG-5 tailed and ligated with a bridger oligonucleotide (AATTCCCCCCCCCCCC) and lambda-gt10 which had been restricted with EcoRI. The bridger oligonucleotide permitted insertion and ligation of the dG-tailed cDNA sequence into the EcoRI site of lambda gtlO. The lambda phage was packaged (Grosveld 10 .££ al., 1981, Gene 13:227-237), and plated on E. coli Cg0Q rK~" mK*hf1. The cDNA libraries in lambda-gtlO were screened for the p97 insert by plaque hybridization (Benton & Davis, 1977, Science 196:180) with genomic exon fragments as probes. 32 Probes were radiolabeled with P-TTP (New England Nuclear, 15 3200 Ci/mmole) by nick-translation to a specific activity of Q 5-10 X 10 cpm/ug. Three overlapping cDNA clones (lOal, ljl, 2fl) spanning 2,368 nucleotides of the p97 mRNA, including the entire coding region, were identified, by using p97 exon specific fragments as probes (FIG* 2). 20 6.3. DNA SEQUENCE ANALYSIS OF p97 cDNA inserts were excised and subcloned into plasmid vector pEMBL18+ (Dente jet'al«r 1983, Nucleic Acids Res. 11:1645-1655) in Ej_ Coli for subsequent propagation and restriction mapping. cDNA was also subcloned into M13mpl8 25 phage cloning vector (Yanish-Perrone et al^., 1985, Gene 33:103-119) and sequenced using the dideoxy method of Sanger (Sanger et al^., 1977, Proc. Natl. Acad. Sci. USA 74:5463-5467). M13 clones containing large inserts were sequenced by generating deletions using DNAse I (Hong, 1982, J. Mol. Biol. 30 158:539-549) or exonuclease III (Henikoff, 1984, Gene 28:351-359), and by using synthetic 21-mer oligonucleotide primers.

The p97 cDNA sequence is shown in Fig. 3. An open reading frame of 2,214 nucleotides extends from the first 4 4 ATG, the sequence around which conforms with the consensus initiation sequence determined by Kozak (Kozak, 1980. Nucleic Acids Res. 8:127-142), to the TGA at position 2,215. The most 5' cDNA clone contains an additional 60 nucleotides 5 upstream of the initiating ATG. The 3' non-coding region of p97 mRNA, which was not obtained as a cDNA clone, was identified as a single genomic exon containing 1,667 nucleotides. Residues 20-32 of the predicted amino acid sequence are identical to the known N-terminal amino acid 10 sequence of p97 (Brown et al., 1982, Nature, London 296:171-173), proving the identity of the cloned cDNA. Furthermore, the predicted molecular weight of the precursor is 80,196 daltons, in good agreement with the observed molecular weight of the rn vitro translation product. 15 6.4. CONSTRUCTION OF A RECOMBINANT EXPRESSION PLASMID CONTAINING THE p97 CODING SEQUENCE The large size of the p97 gene necessitated piecing together the cDNA clones that were obtained by specific priming of melanoma mRNA with reverse transcriptase. The 20 three cDNA lambda gtlO clones (lOal, ljl, and lfl; see FIG. 2) that encompassed the coding region from the signal peptide through the membrane anchor sequence were used. The p97 insert of clone lOal was excised by digestion with EcoRI and the oligo(dG) sequence at the 5' end of the cDNA lOal was 25 removed by digestion with exonuclease III, generating clone lOalb, with a HindiII site 30 bp upstream from the initiating methionine of the p97 preprotein. The p97 inserts of the three cDNA clones lOalb, ljl, and 2fl, and genomic clone E7.7 were ligated together at PvuII, SstI and EcoRI restriction 30 enzyme sites and inserted into the Hindlll-EcoRI sites of the plasmid vector pEMBLl8+ (Dente, et al. 1983, Nuc. Acid Res. 11:1645-1655) as shown in Figure 2. The final construct, p97b, contains the 4.4 kb p97 insert in plasmid vector pEMBLl8+ which was used to transform E^ coli 4 5 10 HB101. The insert in p97D contains 30 Op of the 5' untranslated region of p97 mRNA, the entire coding sequence, and the 3' untranslated region, bounded Dy a 5 ' HindllI site and a 3' EcoRI site.

The 4.4 kiloDase p97 insert was excised from p97D with Hindlll and EcoRI, and the ends were filled in using tne Klenow fragment of E^_ coli DNA polymerase . The Dlunt-ended fragment was inserted into the unique Smal site in the eukaryotic cDNA expression vector 1995. 12 pUC13, a derivative of vector mThGH-112, (Palmiter _et al., 1983 , Science 222: 809-14), which was oDtained from Dr. Richard Palmiter (University of Washington, Seattle, Washington). This vector uses the mouse metallothionein promoter to express foreign genes in eukaryotic cells. The construct with the p97 insert 15 in the correct orientation was identified Dy restriction analysis and designated pMTp97o.

The recomDinant plasmid was transfected into LMTK cells, and transfectants were selected Dy growth in HAT medium. Clones picked from the transfected dish were expanded in 96-well microtest plates, and spent culture medium and cell lysates from replicate plates were assayed for p97 Dy a two-site immunoradiometric assay. Subclones were expanded and retested. Clone TKMp97-12 which expresses approximately 4, 000,000 molecules of p97 per cell was grown up, induced with cadmium, and used as a source of p97 for immun ization. 20 25 6.5. IMMUNIZATION OF MICE WITH p97 RELATED PEPTIDES The TKMp97-12 cells were grown up, induce with cadmium and (14.4 g) were lysed Dy incubation for 10 minutes 30 on ice with 70 ml TNEN (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40 ) . The lysate was ultracentrif ug ed at 200, 000 x g for 45 minutes at 4°C, and half of the lysate was passed over a 1 ml immunoaffinity column specific for p97 (FaD fragment of antiDody 96.5 coupled to Sepharose*) . The 35 *(Trade Mark) 4 6 immunoadsorbent was washed extensively, first with TNEN, and finally with 20 mM Tris-HCl, pH 6.8.

For the immunization, 0.5 mi of the adsorbed immunoaffinity column prepared as descriDed aDove was mixed 5 with 0.5 ml 20 mM Tris-HCl, pH 6.8, arid emulsified with 1 ml complete Freund's adjuvant. Four BALB/c mice each received 0.4 ml of the emulsion intraperitoneally. Three weeks later the mice were boosted with one fourth of this amount of antigen in incomplete Freund's adjuvant. Control mice were 1Q immunized with an immunoaffinity column of an antiDody unrelated to p97 that had Deen otherwise treated identically. Four of the p97-immunized mice and two control mice were Died one week after the Doost. The sera were tested for antiDodies to p97 by inununoprecipitation from radioiodinated 15 SK-MEL meloma cells followed by SDS-PAGE. The results showed that sera from the four p97-immunized mice immunoprecipitated p97, whereas the control sera were negative. The sera were also tested for the presence of antibodies directed against p97 using an ELISA assay on glutaraldehyde-fixed SK-MEL 28 2q melanoma cells (20,000 cells per microtest well). The fixed cells were incubated with 0.05 ml serum diluted 1/10,000 for 1 hour at room temperature, washed, and then incubated for 1 hour at rocxn temperature with 0.05 ml horseradish peroxidase-conjugated goat anti-mouse IgG (Southern Biotech). 25 The optical densities (read at 490 nm) of sera from tne p97-immunized mice were 0.350, 0.243, 0.343, 0.200, whereas the optical density of sera from controls were 0.036 and 0.05 7. 6.6. CHARACTERIZATION OF p97 6.6.1. STRUCTURE OF p97 30 The structure of p97 was determined from the amino acid sequence of the p97 precursor which comprises four structural domains. Since residue 20 of the precursor sequence corresponds to the N-terminus of mature p97, amino 4 7 acid residues 1-19 probably constitute a signal peptide, a conclusion that is supported by its length and hydrophobic nature. Amino acids 20-361 and 362-713 comprise two homologous domains of 342 and 352 amino acids. Potential N-5 linked glycosylation sites occur at positions 38 and 135 in the N-terminal domain and position 515 in the C-terminal domain. Finally, we believe that amino acids 714-738, a region of predominately uncharged and hydrophobic residues, anchor p97 in the cell membrane (Davis et al., 1985, J. Mol. 1 q Biol. 181:111-121) and may extend into the cytoplasm.

The domain structure of p97 is supported by the protease digestion experiments. Digestion of p97 with trypsin, papain (Brown e: al., 1981, J. Immunol. 127:53 9-546) or thrombin produced a glycosylated , ^antigenic fragment of 15 approximately 4 0,000 daltons in molecular weight. The fragment was purified from a thrombin digest of p97 that had 35 35 been metabolically labelled with S-methionine or S- cysteine and sequenced as described (Brown et al., 1982, Nature, London 296:171-173). Cysteine residues were identified at positions 7 and 17, and methionine residues at positions 2 and 20. Identical results were obtained with intact p97, and are in complete agreement with the N-teriuinal sequence of p97 predicted frcm the cDNA sequence. We conclude that the 40,000 dalton molecular weight protease- resistant fragment corresponds to the N-terminal domain of p97. We have been unable to isolate the C-terminal domain of p97, possibly because it is protease-sensitive. 6.6.2. HOMOLOGY OF p97 WITH TRANSFERRIN A search of the amino acid sequence library of the 30 Protein Identification Resource (Release 5.0; Day ho f f et al . , 1981, Nature, London 290: 8) showed that p97 is strikingly homologous to three members of the transferrin superfamily: human serum transferrin, human lactotr ansf err in and chicken transferrin (37%-39% homology, see Fig. 4). Since human and 20 25 4 8 chicken transferrin show 50% homology to each other, p97 must have diverged from serum transferrin more than 300 million years ago. p97 has 14 cysteine residues located in homologous positions in each domain. Human transferrin 5 contains all of these cysteines in homologous positions in both domains, while human lactotransferrin. and chicken transferrin lack only two of these cysteine residues (in their C-terminal domains). Unlike p97, these proteins contain 4-7 additional cysteines in their C-terminal domains 1 q which have no corresponding member in the N-terminal domain. Human transferrin also contains 2 extra cysteines unique to its N-terminal domain. The positions of most of the disulfides in human serum transferrin, lactotransferrin and chicken transferrin have been determined directly 15 (McGillivray et _al., 1982, Proc. Natl. Acad. Sci. USA 7 9: 2504-2508; Me tz-Boutig ue ^t al^, 1984, Eur. J. Biochem 145:659-676; Mazurier et al., 1983, Experientia (Basel) 39:135-141; MacGillivray et al., 1983, J. Biol. Chem. 25 8:3 54 3-3 553; Williams et al.-, 1982, Eur. J. Biochem. 2q 122: 297-303; Williams, 1974 , Biochem J. 141: 74 5-752 ). One can thus predict the presence of 7 disulfide bonds in each domain of p97 (see Fig. 5).

The amino acid homology between domains of p97 (46% - achieved by insertion of 7 gaps of 9 residues) is more 25 striking than that seen in human transferrin (43% - 16 gaps, 45 residues) or chicken transferrin (35% - 12 gaps, 49 residues). Given the extensive sequence homology between p97 and transferrin, and the apparently similar folding patterns, based upon the conservation of cysteines, we believe that if 20 the present low-resolution X-ray structure of transferrin (Gorinsky_et .al.., 1979, Nature, London 281: 157-158) can be refined it may be possible to deduce the three-dimensional structure of p97. 4 9 6.6.3. FUNCTION OF p97 Its membership in the transferrin superfamily,- its ability to bind iron (Brown et al., 1982, Nature London 296:171-173), and its common chromosomal localization with transferrin and the transferrin receptor (Plowman et al., 1983, Nature, London, 303:70-72; Yang et al., 1984, Proc. Natl. Acad. Sci. USA 81:2752-2756) all support a role of p97 in iron transport. The iron binding pocket of transferrin is thought to contain 2-3 tyrosines, 1-2 histidines and a single bicarbonate-binding arginine (Metz-Boutigue et al., 1984, Eur. J. Biochem. 145:659-676). Conservation of these amino acids in p97 support its proposed role in iron metabolism (see FIG. 4). Since p97 is a membrane bound transferrin-like molecule and has no homology with the transferrin receptor (Schneider et al., 1984, Nature, London, 311:675-678), its role in cellular iron metabolism may differ from that provided by circulating serum transferrin and the cellular receptor for transferrin. Expression of the cloned p97 cDNA in eukaryotic cells will allow experimental testing of its functional properties. 6.6.4 CONCLUSION Based on these data it is clear that cDNA constructs for the melanoma-associated p97 have been obtained and that these can be expressed efficiently in mammalian cells to produce large amounts of antigenic p97. 7. EXPRESSION OF CLONED p97 AND VACCINE TESTING The experiments detailed herein describe the expression of the cloned p97 protein and its vaccine testing. Expression of p97 protein in a secreted form (by the transfected mouse cell clone B16svp97a.l4) enabled purification of milligram quantities of the full length p97 protein. The protein in purified form was used for in vitro testing of induction of cellular immunity and for testing its 50 potential as a subvmit vaccine. The p97 gene product was also expressed on the cell surface of metastatic murine . melanoma cells, providing a model for testing the efficacy of vaccines in preventing tumor growth in a syngeneic system.

The p97 gene was inserted into a live vaccinia recombinant virus for use as a vaccine formulation capable of generating effective cellular immunity. The recombinant vaccinia virus, Vp97a-NY, was evaluated for its ability to generate immunity in mice, using a variety of assays for demonstrating humoral and cellular immunity. Using the syngeneic murine tumor model described supra, the Vp97a-NY recombinant virus vaccine was demonstrated to provide a protective effect from tumor cell challenge. The vaccine also provided a therapeutic effect in mice with existing growing lung metastases, a property which is analogous to the proposed use of the vaccine for generating an immunotherapeutic anti-tumor response in human melanoma patients with existing tumor.

In addition to the mouse studies, where there is only 91% homology between human p97 and the mouse homologous protein (over the regions examined thus far), the Vp97a-NY vaccine has also been tested in non-human primates. There is a much closer homology between human p97 and the monkey form of the protein (as revealed by the cross-reactivity at the monoclonal antibody level). Because of the potential difficulty in generating an immune response against a "self" protein, the closely related Macaque monkey was used to test the immunogenicity of the Vp97a-NY vaccine. The recombinant vaccinia vaccine was tested in monkeys and shown to induce humoral immunity directed against the p97 protein. Thus far the monkeys have exhibited no noticeable symptoms of deleterious side effects from exposure to the vaccine, after having received two inoculations of the live recombinant vaccinia virus, over a period of six weeks. 7.1. PLASMID EXPRESSION The expression plasmid driven by the SV-40 early promoter sv2 was constructed from the cDNA plasmid clone p97a, which is similar to plasmid p97b, except that the j. entire 3'UT region is utilized (FIG. 6). All cDNA clones were originally isolated from lambda gtlO libraries with synthetic EcoRI-dG (9-17) linkers as previously described. Inserts were excised by EcoRI and subcloned into pEMBL18+ for subsequent propagation and characterization. Clone lOal was subcloned into M13mpl8 and an RF form was digested with BamHI and SphI, treated briefly with Exonuclease III, blunted with SI nuclease, treated with Klenow, and religated. Several plaques were isolated and sequenced, one of which had removed the dG tail and retained 33 bp of the p97 5' untranslated 15 region inserted into the Hindlll site of M13mpl8. An RF of this subclone (lOala) was used for generating the intact p97 cDNA; otherwise, all fragments were isolated from the plasmid subclones. The 550 bp Hindlll-PvuII fragment from lOala and the 735 bp PvuII-Sall fragment from ljl were isolated from 20 IMP agarose gels and ligated into pEMBL18+ at the Sail and Hindlll sites, generating p5'p97. E7.7 genomic clone in pEMBL18+ was digested to completion with EcoRI and digested partially with SstI, and the 4.5 kb fragment was separated by fractionation through 0.8% LMP agarose. This 4.5 kb 3' 25 fragment was ligated with the 404 bp SstI fragment from 2fl and the 535 bp BamHI-SstI fragment from ljl into pEMBL18+ at the Sail and EcoRI sites, generating p3'p97. The 1285 bp Hindlll-Sall fragment of p5'p97 was then ligated into p3'p97, generating pp97a. The EcoRI-partial Hindlll fragment from 30 this clone was inserted into pSV2neo (Southern, et al., 1982, J. Mol. App. Genet. 1:327-341) at the Hindlll and EcoRI sites, eliminating the neomycin coding region and SV4 0 splice/polyA sequences while retaining the SV40 early promoter and 72 bp enhancer, 33 bp p97 5'UTR, the entire p97 ^ o o ^ coding region, 3' UTR and 1.4 kb 3' flanking DNA. The resulting plasmid was termed pSVp97a.

The sv2 driven plasmid was transfected by calcium phosphate precipitation into a variety of eukaryotic cell 5 lines, and expressing cells were cloned and selected using co-transfection of a dominant selectable marker. To accomplish this, Chinese hamster ovary (CHO) cells were cultured in Hank's F1 medium containing 15% fetal calf serum (FCS), 4mM L-glutamine, 1.3 mM proline, and antibiotics. B16 TO cells were cultured in RPMI medium containing 0.15% bicarbonate and 1735.cells in DMEM medium (Gibco) both supplemented with 15% FCS, and antibiotics. Cells were transfected by a modified calcium phosphate technique (Wigler M., et al., 1978, Cell 14:725-731) with 20 ug per plate of 15 pSV2p97a plasmid DNA and 0.5 ug of either pSV2DHFR or pSV2neo respectively. All plasmids were linearized at the EcoRI site. Stable tranfectants were selected using hypoxanthine negative (HAT-) medium for CHO cells or 0.5 ug/ml Geneticin (G418, Gibco) for the B16 and 1735 cells. Surviving cells 20 began forming visible colonies 7 days after transfection and were overlaid with sterile polyester filters held in place by glass beads. The filters remained in place for five days, allowing the cells to grow up into the polyester matrix, generating a replica of the colonies on the plate. The 25 filters were then removed and used for a live cell binding assay with iodinated anti-p97 monoclonal antibody. Ten micrograms of labeled monoclonal antbody were incubated with up to 20 filters in 10 ml FCS at 4°C for one hour. The filters were washed extensively in phosphate-buffered saline 30 (PBS), dried and exposed to XAR-5 film overnight at -70"C.

Filters were subsequently stained with 7% methylene blue to visualize the cell colonies. In all the cell lines used except the B16 mouse line, the expressing cells contained antigenic p97 protein on their cell surfaces. 5 In the B16-transfected cells, p97 was released into the medium, a finding unique to this cell type. The secretion of p97 enabled purification of full length p97 protein from the medium of the cells. Clone B16SVp97a.l4 g expressed approximately 4 ug/ml of p97 in spent medium.

Recombinant p97 was purified from spent medium of transfected clone B16SVp97a.14, which shed large quantities of p97 antigen into the medium. Cells were maintained at near 9 . 2 confluency (10 cells) in 850 cm roller bottles by 10 continually adding small amounts of fresh medium. They continued to shed antigen without detaching for weeks, allowing continual harvest and freezing down of spent medium.

The purification of p97 was accomplished by immunoaffinity chromatography, using sepharose linked to Fab fragments of 15 monoclonal antibody 96.5. To this end, three liters of spent medium was run over a series of three 30 ml columns. The first contained 15 ml of G-25 superfine Sephadex* (Pharmacia), the second contained 20 ml of Sepharose 4b (Pharmacia), and the third contained 8 ml of cyanogen bromide-activated 20 Sepharose (Sigma) conjugated to Fab fragments of monoclonal antibody 96.5 (10 mg protein/ml of Sepharose). Subsequently, the affinity column was washed extensively with cold PBS and the antigen was eluted with 30 ml of 0.1 M citrate, pH 5 and 30 ml of 0.1 M citrate, pH 4. These conditions were shown 25 not to alter the immunoreactivity of the antigen, while accomplishing complete elution of the antigen from monoclonal antibody 96.5. The two elutions were neutralized with 3.0 ml and 4.5 ml, respectively, of 2 M Tris, pH 8. The purified eluate was concentrated using an Amicori* apparatus with a PM10 30 filter, and washed with two 10 ml volumes of PBS, leaving a final yield of 4.95 mg in 4.5 ml as determined by Bradford assay (Bioracf) . Fifteen ug of the product was run on SDS- PAGE (FIG. 7) and visualized both by Coomassie Blue and silver stain. A double determinant immunoassay (DDIA) 35 (Brown, et al. 1981, Proc. Natl. Acad. Sci. 78:539) provided *(Trade Mark) 5 4 an independent confirmation of the molar quantity of purified protein. Control preparations were performed in parallel with spent medium from the parental B16 cell line, and showed no detectable protein. In a subsequent preparation 30 mg of g 95% pure p97 protein was purified from 300 mg of monoclonal antibody 96.5 Fab fragments conjugated to Sepharose. The purified p97 protein was immunogenic producing a strong antibody response in mice immunized with the protein as described in Section 7.3. infra. 10 7.2. CONSTRUCTION AND EXPRESSION OF RECOMBINANT p97 VACCINIA VIRUS The coding region of p97 was inserted by cutting p97a with Hindlll, converting the ends to blunt ends, and ligating into the vaccinia insertion vector pGS-20 (Mackett 15 et al^., 1984, J. Virol. 49:857-864) which had been opened at the Smal site. The PGS-20 vector utilizes the 7.5 K promoter, and contains flanking sequences from the vaccinia thymidine kinase (TK) gene. A recombinant virus was generated by the method or Mackett et al_., supra, and Vp97a-NY 20 was isolated which causes infected cells to express p97 protein of the correct size and glycosylation (FIG. 8). Surface expression of p97 was also confirmed in cells infected with the recombinant p97 virus (Table I) below.

TABLE I SURFACE p97 EXPRESSION OF TRANSFECTED MOUSE CELLS AND CELLS INFECTED WITH RECOMBINANT VACCINIA VIRUS1 Molecules of Cell Type Virus Used to Infect Cell p97 Expressed per Cell M2SVp97a.A None 3,210,000 M2svp97a.E/F2 M2 parent BSC BSC BSC None None None Vwt-NY vaccinia Vp97a-NY vaccinia 434,000 2,000-5,590 5,220 1,140,000 Cells were trypsinized briefly, washed and aliguoted into tubes containing 10 , to 10 or 10 cells. Non-expressing carrier cells were added to tubes with lower cell numbers so that a total of 10 cells were used per tube. 1 x 10 cpm of iodinated monoclonal antibody 96.5 (123 ng) was incubated, in a total volume of 50 ul, with the cells for 60 minutes on ice. Cells were washed and spun 4 times in PBS + 10% fetal calf serum, then resuspended and counted in a Micromedic 4/600plus gamma counter. (-) signifies less than. 7.3. RECOMBINANT p97 VACCINIA VIRUS IS IMMUNOGENIC IN MICE Inoculation of mice with Vp97a-NY generated a strong humoral antibody response. Mice were immunized once, boosted once at four weeks, then bled at five weeks. Titers were assessed by ELISA, using antigen-coated plates and a Protein A conjugated to horseradish peroxidase as detection reagent. Data were converted to monoclonal antibody equivalents by comparison to a standard curve for ELISA binding generated using anti-p97 monoclonal antibody 133.2. The results showed a strong induction of serum antibody (FIG. 9). Cellular immunity was detected using an in vitro proliferation assay with purified p97 protein as the stimulating antigen (Table II). 5S TABLE II PROLIFERATION ASSAY OF MURINE SPLEEN CELLS Stimulating 5 Antigen Con A (10 ug/ml) p97 Protein (3 ug/ml) 10 (10 ug/ml) (20 ug/ml) (50 microgr/ml) UV-Inactivated Vaccinia Virus 15 (10 pfu/ml) P97-Transfected Irradiated Syngeneic Tumor Cells (10 ) Parental Irradiated 20 Tumor Cells (10 ) Proliferative Index of vp97 Recombinant Immune Spleen Cells 71 27 43 56 44 Proliferative Index of Control Spleen Cells 50 2 2 2 3 91 86 Spleen cells were isolated from mice inoculated by tail scarification with 10 pfu Vp97a-NY recombinant virus, boosted one month later with the same dose and killed one 25 week subsequently. Naive spleen^cells were used for controls in this experiment. 10 cells were cultured per well in 96-well round bottom plates in 0.22 ml RPMI supplemented with 0.5% normal mouse serum, penicillin/streptomycin, glutamine, bicarbonate, and 2.5 x 30 10 M 2-mercaptoethanol. Cultures were pulse-labeled for six hours on day four, with 25 uCi/well tritiated thymidine (New England Nuclear), harvested with a PHD cell harvester, and counted using Optifluor in a Beckman*LS 3801 counter. Proliferation indices were calculated by 35 dividing cpm averages of quadruplicate wells stimulated with each antigen by the average cpm average of control (media).

*(Registered Trade Mark) 5 7 The results shovm in Table II indicate that T cells are proliferating in response to the p97 protein antigen.

In order to ascertain whether helper cells were also stimulated by the recombinant virus in spleen cells from immunized mice, the cells were stimulated in vitro and the supernatants assayed for production of interleukin-2 (IL-2), a helper T cell factor. Spleen cells were cultured from mice immunized twice previously with recombinant Vp97a-NY vaccinia or parental vaccinia. 10 cells were incubated for 48 hours in 0.2 ml of medium identical to that used for proliferation assays, in 96-well round bottom plates, in the presence or absence of the stimulation antigen. Supernatants were collected, pooled from four wells, and frozen prior to IL-2 4 assay. The IL-2 assay utilized 10 previously IL-2 starved mouse T cell line CTLL cells incubated in each well with varying dilutions of assay supernatant with Click's Medium, in triplicate. A standard curve was constructed with recombinant IL-2 obtained from Genentech, CA. CTLL cells were pulse-labeled with thymidine according to the usual proliferation assay technique in the last six hours of 24 hour incubation, then harvested and counted as described for the proliferation assay methods. The results shown in Table III indicate that IL-2 production from spleen cells of mice immunized with recombinant p97 vaccinia virus is stimulated in vitro by p97.

TABLE III STIMULATION OF IL-2 PRODUCTION BY p97 IMMUNE SPLEEN CELLS Vaccination Immunogen In vitro Stimulus Units IL-2 Produced Vp97a—NY p97 Protein 4.4 (20 ug/ml) Vp97a-NY Medium 0.25 Vwt-NY p97 Protein 0.25 (20 ug/ml) Vwt-NY Medium 0.25 In addition, a delayed type hypersensitivity response was measured using the foot-pad swelling assay in Vp97a-NY inoculated mice. Five mice (C3H/Hen strain) per group were inoculated by tail scarification with recombinant or parental strain vaccinia virus. Six days later, the hind paws of each mouse were challenged by inoculation of 20 ul PBS or 20 ul cells in PBS (5 X 10 cells per mouse). The foot pads were measured 24 hours later in a double blind manner, using a Fowler micrometer. The foot pad thickness of the PBS-injected foot pad was substracted from the measured thickness of the experimental foot from each mouse, and means as well as standard deviations of the incremental foot pad swelling were calculated. The results shown in Table IV show the induction of a p97-specific delayed type hypersensitivity response in mouse immunized with recombinant p97 vaccinia virus. 5 y TABLE IV ANTIGEN-SPECIFIC FOOTPAD SWELLING IN p97 IMMUNE MICE Vaccination Footpad Swelling Immunogen Challenging Antigen (mm X 10 ) 5 Vp97a-NY p97-transfected syngeneic 40.3 (+/- 6.8) tumor cells Vp97a-NY parental syngeneic 3.0 (+/- 2.8) tumor cells Vwt-NY p97-transfected syngeneic 1.5 (+/- 2.3) 10 tumor cells Vwt-NY parental syngeneic 5.5 (+/- 5.0) tumor cells 7.4. PROTECTION AND THERAPY WITH p97 VACCINIA VIRUS IN A MURINE TUMOR MODEL 15 In order to assess vaccination efficacy, mice were vaccinated with various protocols using the present recombinant p9 7 vaccinia virus and then challenged with a p97-transfected syngeneic tumor cell (M2SVp97a.2E). To this end, mice were immunized with Vp97a-NY recombinant live 20 vaccinia virus or the parental strain (Vwt-NY) by tail scarification; or with 100 ug purified p97 protein or 5 X 106 irradiated M2-K1735 tumor cells intraperitoneally in complete Freund's adjuvant. An intravenous tumor cell challenge was given two weeks following the last vaccination by injection 25 of M2SVp97a.2E, a metastatic tumor clone prepared from M2- K1735 (a murine melanoma model) by transfection with the human p97 coding sequence contained in an expression plasmid driven by the SV40 early promoter. A variety of expressing clones were selected, and the one used for tumor challenge, 30 clone M2SVp97a.2E, expresses a medium level of p97, about 400,000 molecules per cell or equivalent to human melanoma p97 antigen density. Two doses of intravenous tumor 5 5 challenge were used, 5 X 10 or 1 X 10 cells which were injected into the tail vein of syngeneic C3H/Hen mice. The 35 mice were sacrificed 16 days after tumor challenge and lungs were removed. A mouse was scored as positive if there were tumors visible to the naked eye on lungs stained with India ink. The results are shown in Table V. 6 0 TABLE V CHALLENGE OF VACCINATED MICE WITH SYNGENEIC p97 TRANSFECTED MELANOMA CELLS Vaccine Vp97a-NY ii 1 o Irradiated Syngeneic Melanoma Cells Vwt-NY 15 p97 Protein Vp97a-NY Naive No. of Immunizations Challenge Cell Dose .5 2 1 2 5 X 10" 2 2 2 1 0 n 1 X 10" No. of Mice with Obvious Lung Metastasis 1/5 2/4 0/4 9/10 3/3 0/1 0/4 5/6 The results presented in Table V demonstrate that 20 there was a considerable protective effect with two immunizations of Vp97a.NY although no protective effect was seen with the purified p97 protein vaccine (despite the extremely high antibody titers elicited). The ability of the 6 1 recombinant virus to elicit cellular immunity may be responsible for its protective tumor immunity.

In a therapy experiment, mice were inoculated with a low dose of p97-expressing tumor cells and then two days later, inoculated with the recombinant vaccinia vaccine. 5 4 Mice were challenged with 10 or 10 p97-expressing tumor cells (M2SVp97a.E) intravenously. Two days later, mice were inoculated by tail scarification either with Vp97a-NY or Vwt-NY. Weekly inoculations by tail scarification were repeated, and mouse survival recorded. The results shown in Figure 10 indicate the therapeutic effect of recombinant p97 vaccinia virus vaccination in mice with existing lung metastasis. 7.5. RECOMBINANT p97 VACCINIA VIRUS 15 IS IMMUNOGENIC IN MACAQUE MONKEYS Two Macaca fasicularis (macaque) monkeys were Q scarified with either 2 X 10 plaque forming units (pfu) of Vp97a-NY recombinant vaccinia, or the same dose of parental strain vaccinia. Two weeks later the sera were tested by 20 ELISA for titers to vaccinia and p97. The results shown in Table VI demonstrate that humoral antibodies to p97 were detectable two weeks after the single inoculation with Vp97a-NY. 5 10 6 2 TABLE VI SERUM ANTIBODY TITERS IN VACCINIA INOCULATED MONKEYS Anti-p97 Titer Anti-Vaccinia Titer (ug/ml monoclonal 5 (serum dilution with antibody Immunogen/Week twice background) equivalents) Vp97a-NY/Week 0 1/20 0.54 Vp97a-NY/Week 2 1/2000 6.54 Vwt-NY/Week 0 1/20 0.50 10 Vwt-NY/Week 2 1/2000 0.34 8. DEPOSIT OF MICROORGANISMS The following E. coli strain carrying the listed plasmid has been deposited with the ATCC, Rockville, MD and has been assigned the following accession number; 15 E. coli Strains Plasmid Accession Number3 E. coli HB101 p97b 53,403 The following recombinant vaccinia virus has been deposited with the ATCC, Rockville, MD, and has been assigned the following accession number: 20 Virus Accession Number13 Vp97a—NY VR 2159 The following cell lines carrying the listed plasmids have been deposited with the ATCC, Rockville, MD, and have been assigned the following accession numbers: a Deposit Date : 27 December 1985 b Deposit Date : 6 January 1987 63 Cell Line TKMp97-12 (Mouse cell) Plasmid PMTp97b Accession Number CRL 8985^ B16SVp97a.14 (mouse melanoma ^SVp97a CRL 9304d 5 cell) The present invention is not limited to the use of the microorganisms and cells deposited ; the deposited entities merely illustrate the practicing which are functionally equivalent are usable for this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the 15 foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. sizes given for nucleotides are approximate and are used for 20 purpose of description. c Deposit Date : 27 December 1985 d Deposit Date : 6 January 1987 10 of the invention,and any microorganisms or cells It is also to be understood that all base pair

Claims (9)

1. CLAIMS: 1A live virus vaccine formulation comprising a recombinant virus, the genome of which comprises a nucleotide sequence encoding an antigenic peptide or protein related to the melanoma-associated p97 antigen or an antigenic portion thereof which is under the control of a second nucleotide sequence that regulates gene expression so that a peptide or protein related to the melanoma-associated p97 antigen or an antigenic portion thereof (respectively) is expressed in a host infected with the virus, the virus being infectious without causing disease in a host to be vaccinated.

2. The live virus vaccine formulation of claim 1 in which the recombinant virus comprises an enveloped virus. 3. The live virus vaccine formulation of claim 2, in which the enveloped virus comprises a vaccinia virus. 4. The live virus vaccine formulation of claim l in which the recombinant virus comprises a naked virus. 5. The live virus vaccine formulation of claim 4 in which the naked virus comprises an adenovirus. 5. An inactivated virus vaccine formulation comprising an effective dose of a recombinant virus, the genome of which comprises a nucleotide sequence encoding an antigenic peptide or protein related to the melanoma-associated p97 antigen or an antigenic portion thereof which is under the control of a second nucleotide sequence that regulates gene expression so that a peptide or protein related to the melanoma-associated p97 antigen or an antigenic portion thereof (respectively) is expressed in a host infected with the virus, the said recombinant virus being in a noninfectious state, and being mixed with a pharmaceutical carrier. 65 7. A formulation according to claim 6 wherein the said virus comprises an enveloped virus.

3. A formulation according to claim 7 wherein the said virus comprises a vaccinia virus. 5 9. A formulation according to claim 6 wherein the said virus comprises a naked virus. 10. A formulation according to claim 9 wherein the said virus comprises an adenovirus. 11. A formulation according to claim 6 wherein 10 the said virus comprises a nuclear polyhedrosis virus. 12. A formulation according to claim 11 wherein the said virus comprises a baculovirus. 13. A formulation according to claim 6 wherein the said virus comprises a bacteriophage. 15 1

4. A formulation according to claim 13 wherein the said virus comprises a lambda phage. 1

5. A subunit vaccine formulation in which the immunogen comprises an effective dose of a substantially pure antigenic peptide or protein related to the melanoma- 20 associated p97 antigen or an antigenic portion thereof, produced either (a) by recombinant DNA technology, that is to say, a procedure wherein the peptide or protein specified was purified from a cultured cell containing a nucleotide sequence encoding the peptide or protein which 25 is under the control of a second nucleotide sequence that regulates gene expression so that the peptide or protein is expressed by the cultured cell, or (b) by chemical synthesis, mixed with a pharmaceutical carrier. 1

6. A formulation according to claim 15 in the case of 30 which the mode of production employed was that specified at "(a)11/ and the cultured cell specified comprised a microorganism. 1

7. A formulation according to claim 16 in the case of which the microorganism specified comprised a bacterium. 1

8. A formulation according to claim 16 in the case of which the microorganism specified comprised a yeast. 1

9. A formulation according to claim 15 in the case of which the mode of production employed was that specified at "(a)", and the cultured cell specified comprised an animal cell line. 20. A formulation according to claim 15 in the case of which the mode of production employed was that specified at "(a)", and the cultured cell specified comprised an insect cell line. 21. A live virus vaccine formulation according to claim 1, substantially as hereinbefore described. 22. An inactivated virus vaccine formulation according to claim 6, substantially as hereinbefore described. 23. A subunit vaccine formulation according to claim 15, substantially as hereinbefore described. F. R. KELLY & CO., AGENTS FOR THE APPLICANTS.