CH675424A5 - Melanoma-associated antigen peptide(s) - 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.

Description

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description

The present invention is directed to antigenic peptides or proteins that can induce an immune response that can selectively destroy melanoma cells in an individual vaccinated with such peptides. Accordingly, a peptide or protein, which is related to an antigen associated with melanoma, is produced in large quantities by recombinant DNA techniques and / or by chemically synthetic methods. The peptide or protein according to the invention can be used as an immunogen in vaccine formulations. In certain embodiments, wherein the peptide or protein related to a melanoma-related antigen is expressed by a recombinant virus, the recombinant viruses themselves can be used as immunogens in vaccine formulations. The use of recombinant DNA techniques and also chemical-synthetic methods enable the production of peptides and proteins, which are related to the antigen associated with melanoma, in large quantities.

For example, the invention is illustrated using immunogenic peptides related to p97, a monomeric cell surface sialoglycoprotein with an apparent molecular weight of slightly less than 97,000 datons, which is a cell surface component of melanoma cells.

Working with experimental animals, particularly rodents, has shown that most tumors produced by oncogenic viruses express antigens encoded by the viral genome and that immunization with these antigens to reject a subsequent challenge from tumor cells that may have been induced by the same virus. Although most of this work has been done with laboratory strains of viruses such as SV40, the polyomvirus and the Friend, Moloney or Rauscher mouse leukemia viruses, the horizontal and vertical transmission of oncogenic viruses in nature has been demonstrated; in fact, a commercial vaccine against virus-induced cat leukemia and Sarcom is now available.

In contrast, a viral cause of most human cancers has not been established. Notable exceptions are the hepatitis virus (hepatoma), the herpes simplex virus (cervical carcinoma) and the Epstein-Barr virus (nasopharyngeal carcinoma). However, over the past two decades it has been recognized that some human tumor cells express tumor antigens, i.e. Antigens that can distinguish the tumor cell from corresponding normal cells; some patients show cell-mediated or humoral immune responses against these antigens (Hellstrom et 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 oncofötaie or differentiation antigens encoded by human genomes (Hellstrom et al., 1970, Int. J. Cancer, 6: 346-351).

Until recently, the molecular nature of tumor antigens was unknown and the degree of tumor specificity of immunological reactions was unclear. Attempts to use this information in the development of cancer diagnostic tests or cancer therapies have been largely unsuccessful. Since spontaneous tumor regressions are extremely rare, it can also be concluded that the immune responses demonstrated in vitro are ineffective in vivo; for example, antibodies and lymphocytes obtained from a cancer patient can be used effectively to kill tumor cells in vitro, whereas the immune response of the same cancer patient has no effect in vivo.

The introduction of the monoclonal antibody technique by Kohler and Milstein (1975, Nature, 256: 495-497) led to intensified research on human tumor antigens, since the means were available to define such antigens, both in the molecular area, as for specificity (Hellstrohm and Brown, 1979, in "The Antigens" M. Sela, ed., Academic Press, Vol. V: 1-66). A large number of tumor-associated antigens have been described in recent years, most of which have been defined by mouse monoclonal antibodies (Reisfeld and Seil, Editor, Monoclonal Antibodies 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 basically all of the antigens that have been well characterized, have proven to be oncofoietic or distinctive antigens and their specificity against tumors have been demonstrated more than quantitatively and less than qualitatively, some antigens are sufficiently specific for neoplastic against normal cells (generally a factor of 10 to 1000 times) to use as potential targets for tumor cell identification and therapy. Human monoclonal antibodies to tumor antigens have also been obtained (Cote et al., 1983, Proc. Nati. Acad. Sci. 80: 2026-2030). This supports the previously cited evidence that some cancer patients show an immune response to their tumors.

More than half of the tumor-associated surface cell antigens, as far as they are identified, are proteins or glyco-proteins that encode human genomes (less by endogenous or exogenous viruses), the rest being glyco-lipids that are characterized by abnormal expression or Regulation of glycosyl transferases originate.

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The p97 antiaeri associated with the Melamon

The p97 antigen is a tumor-associated antigen that was first identified in human melanoma using monoclonal antibodies (Brown et al, 1980, J. Biol. Chem. 255: 4980-4983; Dippold et al., 1980, Proc. Nati Acad. Sci. USA 77: 6114-6118; Woodbury et al, 1980, Proc. Natr. Acad. Sci. USA 77: 2183-2187). The p97 antigen has been extensively studied for its expression in normal and neoplastic tissues and is present in most melanomas and in certain fetal tissues, but has only been detected in trace amounts in normal adult tissue (Brown et al., 1981, J. Immunol. 127: 539-546; Brown et al., 1981, Proc. Nati. Acad. Sci. USA, 78: 539-543; Garrigues et al., 1982, Int. J. Cancer, 29: 511-515 ). p97 has been used as a target for diagnostic imaging of melanoma in human clinical trials (Larson et al, 1983, J. Clin. Invest. 72: 2101-2114).

p97 is a monomeric cell surface sialoglyeoprotein with an apparent molecular weight (MW) as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) of slightly less than 97,000 daltons. The monoclonal antibodies have defined three major antigenic centers that are present on a stable 40,000 dalton tryptic fragment (Brown et al., 1981, J. Immunol. 127: 539-546); however, the complete sequence of p97 has not been described. At least two other independently characterized antigens associated with human melanoma, namely gp95 (Dippold et al., 1980, Proc. Nati. Acad. Sci. USA, 77: 6114-6118) and gp87 (Khosravi et al., 1985, Int. J Cancer, 35: 73-80) appear to be identical to p97, as analyzed by sequential immunoprecipitation.

The N-terminal amino acid sequence of p97 is homologous with transferrin, and like transferrin, p97 binds iron (Brown et al., 1982, Nature, London, 296: 171-173). Analyzes of somatic cell hybrids and the in situ hybridization have shown that the p97 gene, like the genes for transferrin and the transferrin receptor, is present in the chromosomal region 3q21-2q29 (Plowman et al., 1983, Nature, London, 303: 70-72; Yant et al., 1984, Proc. Nati. Acad. Sci. USA, 81; 2752-2756). These observations lead to the conclusion that p97 plays a role in iron metabolism.

Cancer vaccines

Studies in experimental animals, usually in mice, have shown that immunization with live or dead cancer cells can lead to a subsequent challenge from viable cancer cells. Immunization attempts with cell-free material have generally been less successful, but some success has been reported. For an overview, see Hellstrom and Brown, 1979, in "The Antigens," M. Sela ed. Academic Press, Vol. V: 1-66. 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 causing a protective immune response is unknown.

Human studies are much more difficult and the effectiveness of cancer vaccines is controversial, despite some successes that have been reported. In some cases, the vaccine preparations consist of irradiated tumor cells or of tumor cells or killed tumor cells which have been exposed to chemicals. Since pure antigens associated with the human tumor are not available, there are no reports of their use in vaccines.

A main theoretical objection to the proposed use of cancer vaccines in humans is that people who are "vaccinated" with, for example, killed cancer cells or cell-free preparations are immunologically unreactive because the tumor antigens, which are the targets of the immune response could be present in some normal cells, but only in traces, they are recognized by the immune system as "itself". Most, if not all, of the tumor-associated antigens, which are discovered in human tumors by monoclonal antibodies, are also present in some normal tissues and, consequently, there is little prospect of cancer patients responding effectively in vivo. The suppressor cells have been shown to play a major role in down regulation in the immune response against tumor antigens (Nepon et al., 1983, Experientia, 39: 235-242). Furthermore, a suppressor cell response initiated by one set of tumor antigens can induce the induction of an effective tumor-destroying response to another set of tumor antigens that alone would not induce subversion (Hellstrom et al., 1983, in "Biomembranes", A. Nowotny ed., Plenum Press, pages 365-388).

Recombinant DNA techniques and vaccine virus

The use of recombinant DNA technology to produce subunit vaccines to protect against infection includes molecular cloning and expression in a convenient vector with genetic information that encodes proteins that encode an immune response against the protein in the host. Animal can trigger. Recently, a new route has been described that is potentially useful for the production of subunit vaccines (Mackett et al., 1982, Proc. Nati. Acad. Sei., 79: 7415-7419; Mackett et al., 1984, J. Virol., 49: 857-864; Panicali, D. and Paoletti,

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E., 1982, Proc. nat. Acad. Sci., 79: 4927-4931). This route involves the use of the vaccinia virus as a vector for the expression of foreign genes which have been introduced as an insertion into its genome. After being introduced into a host animal, the recombinant vaccinia virus expresses the inserted foreign gene, triggering an immune response in the host against such gene products. Since the live recombinant vaccinia virus can be used as a vaccine, this route combines the advantages of both subunit and live vaccines.

The vaccinia virus contains a linear double-stranded DNA genome of approximately 187 kilobase pairs and replicates in the cytoplasm of infected cells. These viruses contain a complete transcription enzyme system (including capping, methylation and polyadenylation enzymes) in the virus core, which are necessary for virus infectivity. The transcriptional regulatory sequences of the vaccine virus (promoters) allow the initiation of transcription by vaccine RNA polymerase, but not by host cell RNA polymerase.

The expression of the foreign DNA in recombinant vaccinia viruses requires the binding of vac-cinia promoters to protein-coding DNA sequences of the foreign gene. Plasmid vectors, also called insertion vectors, were constructed in order to insert a chimeric gene in the vaccinia virus. One type of insertion vector is composed of:

a) a vaccinia virus promoter containing the transcription initiation center;

b) centers cloning some individual restriction endonucleases that are downstream of the transcription start site for the insertion of foreign DNA fragments;

c) non-essential vaccinia virus DNA (such as the TK gene) flanking the promoter and cloning parts that direct the insertion of the chimeric gene into the homologous, non-essential region of the virus genome; and d) a bacterial origin for replication and an antibiotic resistance marker for replication and selection in E. coli.

Examples of such vectors have been described by MacKett (Mackett et al., 1984, J. Viral. 49: 857-864).

The recombinant vaccinia viruses are produced by transfection of recombinant bacterial insertion plasmids which contain foreign genes into cells which have previously been infected by vaccinia viruses. The homologous recombination takes place with the infected sites and causes the insertion of the foreign gene in the viral genome. The infected cells can be screened using standard immunological techniques, DNA-plaque hybridization, or genetic selection for recombinant viruses, which can then be isolated. These vaccinia recombinants have their original functions and infectivity and can be designed to take up approximately 35 kilobases of foreign DNA.

The foreign gene expression can be detected by enzymatic or immunological assays (e.g. by immunoprecipitation, radio-immunoassay or immuno-blotting). Naturally occurring membrane glycoproteins, which are produced from infected cells from recombinant vaccines, are glycosylated and can be transported to the cell surface. The high level of expression can be obtained by using strong promoters or by cloning multiple copies of a single gene.

Vaccine formulations are described that can be used to induce an immune response that selectively destroys melanoma cells in vaccinated individuals. More specifically, the vaccine formulations according to the invention contain an immunogen which induces an immune response against a melanoma-associated antigen, such as the melanoma-associated p97 antigen. A number of vaccine formulations are possible according to the invention. For example, the immunogen of a “subunit vaccine” according to the present invention contains a peptide or protein which is related to p97 and which can be formulated in a suitable adjuvant. Such peptides or proteins contain amino acid sequences derived from all or part of the amino acid sequence of p97, as essentially shown in Figure 3, and include altered amino acid sequences, in which functional equivalent amino acid residues are substituted by residues within the sequence, in one silent changes and / or modified or developed amino acid sequences, such as, for example, glycosylated amino acid sequences, phosporilated amino acid sequences, etc., or chemically modified amino acid sequences. In the following, the peptides or proteins according to the invention which are related to the melanoma-associated p97 antigen if they are changed, changed, modified or unmodified are referred to as “p97 related peptides”. If the p97-related peptide is a hapten (e.g., antigenic but not immunogenic), the haptent can be conjugated to a carrier molecule that imparts immunogenicity.

The p97-related peptides according to the invention can be produced using recombinant DNA techniques and / or chemical-synthetic methods. When p97-related peptides are chemically synthesized, such synthetic p97-related peptides may also contain those amino acid sequences derived from the regions of p97, from which

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they are believed to be antigenic (Hopp and Woods, 1981, Proc. Nati. Acad. Sei. USA, 78: 3824-3828). If the p97-related peptides according to the invention are produced using recombinant DNA techniques, an insertion of a nucleotide sequence encoding all or part of p97 is carried out in a recombinant expression vector, such as in a virus or a plasmid which in a convenient host can control the expression of a p97-related peptide that can be purified from the culture medium. The inserted nucleotide sequence is derived from all or part of the p97 sequence as shown in Figure 3, including but not limited to nucleotide sequences, in which functionally equivalent nucleotide codons are generated by codons within the sequence as a result a silent change are substituted; in other words, different codons which encode the same amino acid or functional equivalents thereof can be substituted in the sequence according to FIG. 3. If a plasmid expression vector is used, preference is given to one which is suitable for expression in eu-karyotic cells, but a prokaryotic expression vector can also be used.

In a further embodiment of the invention, in which the expression vector is a recombinant virus, a vaccine can be formulated as a viral vaccine, in which case the immunogen comprises the recombinant virus which expresses a p97-related peptide. Depending on the nature of the recombinant virus used as an immunogen, the vaccine can be formulated either as an inactivated vaccine or as a live vaccine. Appropriate immunization with vaccine formulations according to the invention can lead to the induction of an immune response, which leads to the destruction of melanoma cells in the immunized subject.

The description also indicates a system in which the vaccine formulation can be tested and how the test should be carried out. For example, vaccine formulations can be tested for their effectiveness in animal models, originally in rodents, then in non-human primates and finally in humans, preferably in patients who are in remission, but against micrometastases there is a high probability of recurrence will.

Brief description of the drawings

FIG. 1 shows an autoradiogram of the cell-free translation product of the p97 mRNA, carried out by SDS-PAGE.

Figure 1A shows in lane 1 the translation product of p97, which is enriched in mRNA, whereas lane 2 shows the translation product, which is not enriched in mRNA, each is derived from a total of 0.5 μl of translation product of 5 ng mRNA. In

Figure 1B shows lane 1 the translation product of p97 that is enriched in mRNA, whereas lane 2 shows the translation product that is not enriched with mRNA, each product being derived from 5 μl I translation product from 5 ng mRNA that is anti-enriched -p97 serum has been immunoprecipitated.

Figure 2 is a schematic representation of the structure of the 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 structure of the duplicated region of the p97 precursor (open bar) are indicated. The location of various restriction enzyme recognition sequences is indicated above the mRNA. The relative positions of four cDNA clones are given below the mRNA structure. The cDNA clone p97-3a2fl (3a2f!) Was isolated from a cDNA library, in which the cDNAs were transcribed on oligo (T) prepared p97-enriched mRNAs and cloned into pBR344; whereas cDNA clones p97-2fl (2fl), p97ljl (Ijl) and p97-10al (1 Oal) were isolated by preparing the cDNA synthesis with oligonucleoids encoding the p97 exon sequences and the resulting cDNA fragments in A.-gt10 were cloned. The

Figure 2A is a schematic representation of genomic clones B15, H17, B6.6 and E7.7 cloned in> 147.1.

FIG. 3 shows the nucleotide sequence of the human p97 precursor cDNA and its derived amino acid sequence. The N-terminal amino acid residues previously determined by protein sequence analysis were identical to those predicted from the nucleotide sequence (amino acid residues numbers 21-30). The potential glycosylation sites at amino acid residues 37, 135 and 515 (open frame) and in the membrane anchor region at the C-terminus (underlined in bold) are indicated.

A polyadenylation signal (AATAAA, indicated by the box) was found at location 3847, which is 50 base pairs upstream of the polyadenylated tract. •

Figure 4 shows a comparison of the predicted amino acid sequences of the p97 precursor and that of human serotransferrin (Yang et al., 1984, Proc. Nati. Acad. Sci. USA, 81 .: 2752-2756; Davis et al., 1985, J Mol. Bio!., 181: 111-121). The remains are in a box. Tyrosine, histidine and argin residues which are involved in the iron binding of transferrin (Metz-Boutique et al., 1984, Eur. J. Biochem., 145: 659-676) are indicated by asterisks (*).

Figure 5 is a diagrammatic representation of a two-dimensional model of the structure of p97

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based on the presence of cysteine residues obtained between the transferrin superfamily members. The three potential glycosylation sites are indicated by asterisks (*). The hydrophobic membrane anchor area can be seen at the C-terminus of p97 (COOH).

FIG. 6 is a schematic representation of the genetic structure of the p97 cDNA clones and the fragment of the genetic clone A.E7.7, which is used for the construction of the p97 expression vector; and the pSV2p97a expression vector. The following abbreviations are used: E, EcoRI: P, Pvull. Sai, Sa [l; S, Sstl; B, BamHI.

Figure 7 shows the results of gel electrophoresis for the characterization and immuno-purification of recombinant p97 proteins. A transfected CHO clone (CH03 +) and SK-MEL 28 human melanoma cells were labeled with 35S cysteine in the presence (+ TM) or in the absence (-TM) of tunicamycin (TM). The cells were placed in 100 mm 2 plates until almost confluence was reached. The medium was removed and replaced with 3 ml of cysteine-free medium, with or without the addition of 1 ng / ml tunicamycin. After 30 minutes at 37 ° C., 250 p.Ci per ml of L-35S cysteine (1016 Ci per mmol; New England Nuclear) was added for a further 6 hours. Cell harvesting, cell lysate production, immunoprecipitation and SDS-PAGE were performed as indicated above. The extracts were immunoprecipitated with p97-specific antibodies and the proteins were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

a) SDS polyacrylamide gel, stained with Coomassie blue. Lane 1, protein labeling agent; Lane 2, immunopurified p97 secreted from transferred mouse B16 cells; Lane 3, SK-MEL

28 cells + TM; Lane 4, SK-MEL 28 cell TM; Lane 5, CHOS + cells + TM; Lane 6, CHOS + time

len-TM.

b) autoradiogram of the same gel as in a)

Figure 8 shows the results of radio-immunoprecipitates of expressed p97 in transferred cells or Vp97a-NY-infected cells. BSC cells were infected overnight with a wild-type vaccine virus or with a p97 recombinant vaccine virus. These viruses or transferred cell lines CHO-p97.A were incubated with 35S-labeled methionine and cysteine with or without tunicamycin containing 2 jig / ml (Sigma). 6 hours later, the cells were lysed, precipitated with the monoclonal antibody 96.5 and separated by electrophoresis on a 10% polyacrylamide gel. The gel was subjected to autoradiography overnight. The group treated with tunocamycin is to the right of each group.

Figure 9 depicts the serum antibody titers of mice immunized with the p97 vaccine. Tp97 represents 5 mice that were immunized with 5 x 106 irradiated M2svp97a.A cells (transfected syngeneic tumor cells that express p97 on the surface) in complete Freund's adjuvant and were treated with the same number of cells in phosphate-buffered saline. p97 is a group of 5 mice immunized with 100 g purified p97 protein in Freund's complete adjuvant and post-treated with 5 ng aqueous protein. Vp97 is a group of 5 mice that were immunized with 107 plaque-forming units from VP97a-NY by tail scarification and aftertreated.

Figure 10 shows the therapeutic effect of vaccination of tumor challenged mice with a recombinant p97 vaccine virus (Vp97a-NY). The mice were challenged intravenously with 105 or 104 p97-expressing tumor cells (M2SVp97a.E). Two days later, the mice were inoculated by tail scarification by either Vp97a-NY or Vwt-NY. The weekly inoculations by tail scarification were repeated and the survival number of the mice was recorded.

The antigenic peptides or proteins according to the invention are useful for the preparation of vaccines for the prevention and treatment of melanomas. It has been found that melanomas have tumor-associated cell surface antigens, such as the p97 antigen, which is present in greater numbers in the melanoma cells than is the case in normal tissue. The peptides or proteins that are related to the melanoma-associated p97 antigen (i.e., p97-related peptides) are made using recombinant DNA techniques or by chemical-synthetic techniques. The p97-related peptides according to the invention comprise amino acid sequences which are derived in whole or in part from the amino acid sequence of p97, essentially as shown in FIG. 3. Amino acid sequences are included, which are derived from FIG. 3, in that one or more amino acid residues are replaced by another amino acid of a similar polarity and react functionally equivalent and thus bring about a silent change. Another amino acid of the same class can be used as a replacement for an amino acid in the sequence. For example, the non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and 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 glutamic acid. Furthermore, the p97-related peptides according to the invention, whether they are changed by substitution of amino acid residues or not, can be further modified or developed by glycosylation, phosphorilization etc. or by chemical changes. These p97-related peptides can be used as immunogens in vaccine formulations

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which elicit an immune response that is directed against existing melanoma cells in vaccinated patients.

Recombinant DNA techniques are preferably used to insert nucleotide sequences which encode the p97 antigen by insertion into the expression vector, which bring about the expression of the p97-related peptides in appropriate host cells. The nucleotide sequences which encode the p97 antigen comprise nucleotide sequences which are wholly or partly derived from the p97 nucleotide sequence, as essentially shown in FIG. 3. Because of 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 (ie different codons encoding the same amino acid or a functional equivalent thereof) within the p97 sequence, such as it is shown in Figure 3, are substituted, provided that the substitution leads to a silent change. Expression of vector host cell systems containing a nucleotide sequence encoding all or part of p97 can be used to produce large quantities of pure p97-related peptides in vitro, with the gene products from the cells in the Cultures can be purified and used as immunogens in subunit vaccine formulations. Purification of the p97-related peptide can be performed using a variety of biochemical methods, including immunoaffinity purification using monoclonal antibodies. In addition, the purification of the p97-related peptide can be facilitated by modifying the DNA sequences encoding p97-related peptides so that the sequences responsible for anchoring the protein in the plasma membrane are removed, while the sequences which responsible for the transport of the protein to the cell membrane are not removed, so that an amputated antigenic molecule is secreted into the culture medium by the host cell. In the event that the p97-related peptides are generated by prokaryotic cells, the lack of appropriate post-translational changes can result in an antigenically inactive product being obtained which has to be activated with appropriate chemicals or other treatments.

In certain embodiments, wherein the expression vector is a virus, the virus itself can be formulated as a vaccine. In such cases, inactive recombinant virus vaccines can be produced. If the expression vector is an infectious recombinant virus which does not cause disease in the host, either an inactivated viral vaccine or a live virus vaccine preparation which leads to substantial immunity can be formulated. A particularly effective expression active ingredient for this purpose is a recombinant vaccine virus which expresses the p97-related peptides according to the invention. To this end, a nucleotide sequence encoding all or part of the p97 antigen can be inserted as an insert into a vaccine virus vector capable of expressing the sequence in a convenient host. The invention encompasses the use of virus expression vectors other than vaccines, in particular adenoviruses.

The deduced amino acid sequence of p97 can be checked for sequences with properties, properties such as especially the hydrophilicity, which indicate the presence of this sequence on the surface of the protein molecule and its possible antigen and / or immunogen potency. These p97-related peptides can be chemically synthesized and used as immunogens in vaccine formulations.

The p97-related peptides can also be used for purposes other than vaccine production. The p97-related peptides can also be used to immunize animals to produce antisera or monoclonal antibodies which are of interest because of their specificity on melanoma cells. These can be used as components in diagnostic tests or for the affinity purification of radiolabeled drug-bound or toxin-bound antibodies in cancer therapy.

The invention is illustrated by means of examples; they describe the manufacture of the p97 vaccine, which is directed against human melanoma. The described methods and compositions are not limited to the construction of vaccines using p97, but can also be applied to other tumor-associated antigens. The invention is presented as follows, solely for the purpose of clarity of description:

a) The nucleotide and the amino acid sequence p97;

b) peptides with the antigenic properties of p97, which are produced by chemical-synthetic methods;

c) peptides with the antigenic properties of p97, which are produced by expression of vector host systems;

d) immunological characterization of the peptides with the antigenic properties p97;

e) Vaccine formulation.

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Sequence analysis of the melanoma-associated p97 antiaen

The nucleotide sequence of the gene encoding p97 and the amino acid sequence derived therefrom are shown in FIG. 3. Functionally equivalent sequences fall within the definition of the present invention. These include the nucleotide sequences, which comprise all or part of the nucleotide sequence according to FIG. 3, which have been changed by substitution or which have different codons, which code the same or functionally equivalent amino acid residues and thus represent a silent change, as well as amino acid sequences 3, which contain all or part of the sequence shown in FIG. 3, which are changed by substitution or contain functionally equivalent amino acid residues and thus represent a silent change and derivatives thereof which are modified or developed, for example by glycosylation, phosphorilation, etc. or through other chemical changes.

The subsections below describe the strategy used to determine the sequence of p97, as shown in Figure 3, as well as other techniques, which are also used to determine the sequence of p97 or other tumor antigens, for vaccine formulations could be useful, can be used.

Identification and characterization of the melanoma-associated P97 antiaen

The activity and amino acid sequence of the melanoma-associated p97 antigen was not known; as a result, the identification of the p97 antigen was carried out using monoclonal antibodies directed against p97. A number of techniques can be used to produce monoclonal antibodies specific for p97. For example, the hybridoma technique developed by Kohler and Milstein (1975, Nature, 250: 495-497) can be used as follows: Mice or rats are immunized with human melanoma cells and lymphocytes which are obtained from the immunized animals are fused with myeloma cells; alternatively, lymphocytes from melanoma patients can be fused with myeloma cells (Cote et al., 1983, Proc. Nati. Acad. Sei, 80: 2026; Haspel et al., 1985, Cancer Res., 45: 3951) or es the technique can be used for the production of monoclonal antibodies using the 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., pages 77-96) for the formation of monoclonal antibodies which are directed against p97. In any case, the hybridomas obtained are screened to produce antibodies that bind to the myeloma cells and not to normal cells.

The above-described monoclonal antibodies directed against p97 can be used in many ways to facilitate the identification, characterization, cloning and expression of nucleotide sequences, which enables the production of peptides and proteins which are anti-genes P97 possess properties, allowed in large quantities. For example, the monoclonal antibodies can be used to further characterize the p97 antigen, by radiolabelling all proteins produced by the tumor cell, by immunoprecipitating the tumor protein with the monoclonal antibody used to identify the p97 antigen , and fractionation of the protein obtained by immunoprecipitation by gel electrophoresis. The protein antigens are identified as distinguishable bands on the resulting autoradiogram (Brown et al., 1980, J. Biol. Chem., 255: 4980-4983). In addition, the monoclonal antibodies directed against p97 can be used as follows to facilitate cloning:

a) immuno-purification of polysomes for the identification and extraction of mRNA transcripts which are present in the melanoma cells which code for the p97 antigen;

b) identification of clones in a cDNA expression library, which expresses peptides or proteins that are related to the p97 antigen;

c) purification of the p97 antigen to produce additional monoclonal antibodies or antisera for use in the aforementioned two applications; or d) identification of cells in which the p97 antigen has been introduced by transfection.

The monoclonal antibodies can also be used to facilitate the structural and immunochemical characterization of the p97 antigen, to identify extra-cellular and antigenic regions of the molecule and to purify the molecule for amino acid sequence analysis (Brown et al., 1981 , Proc. Nati. Acad. Sei, USA 78: 539-543; Brown et al., 1982, Nature, London, 296: 171-173).

The further characterization of p97 includes the determination of the cellular localization and mapping of the antigenic determinants and the functional areas. The subcellular localization can be determined by immunofluorescence microscopy and by cell fractionation experiments. Antigens, such as p97, that are present on the cell surface are preferred for vaccine constructions, although intracellular antigens can also be useful. If multiple

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monoclonal antibodies are available, the antigenic determinants can be mapped by competition, in which each antibody is radiolabeled and tested in competition with each of the other antibodies. Areas of the molecule can be identified by restricted digestion with proteases and subsequent SDS-PAGE. Together, these data should enable the areas with the strongest immune properties to be identified. If the monoclonal antibodies have been obtained by inoculation with intact cells, these regions of the molecule are most likely extra-cellular and useful for the construction of vaccines.

Amino acid sequence analyzes allow unambiguous identification of the protein and comparison with other proteins (Brown et al., 1982, Nature, London, 296: 171-173). If the protein contains more than 50 amino acid residues, it may be useful to determine only part of the amino acid sequence, usually the N-terminus. The protein antigen for amino acid sequencing can be purified from cell lysates by immunoaffinity chromatography with the monoclonal antibody and then by preparative SDS-PAGE. The N-terminal amino acid sequence of the purified protein is then performed using an automatic amino acid sequencer, preferably a highly sensitive gas phase machine.

ID. Cloning and seauenieruna of DNA encoding the melanoma-associated p97 antiaen

Early cionation studies focused on abundant proteins, such as globin and ovalbumin, whose mRNAs often comprised 10 to 50% of the total mRNA. These mRNAs could be purified to size homogeneity by size fractionation and the pure cDNA samples were used to screen banks of a few 100 clones by colony hybridization. For proteins whose mRNAs comprise 1 to 10% of the total mRNA, a differentiated hybridization with 2 cDNA samples can be used, one of the cDNA samples containing sequences of interest and the other representing a negative sample. The messenger hybo-nucleic acids that encode less abundant proteins, such as tumor-associated antigens, which can only comprise about 0.01% of the cellular mRNA, are much more difficult to clone because tens of thousands of clones have to be screened and the cDNA samples do not give a specific hybridization signal. Both problems can be alleviated by enriching the mRNA for the sequence of interest.

Various methods for cloning DNA encoding the p97 antigen associated with human melanoma are described below. The resulting clones were analyzed to identify one or more clones encoding the entire region of p97. The p97 nucleotide inserts of the clones identified in this way can then be sequenced by known methods. The different ways are described in detail below.

a) Isolation of mRNA by polysome immune purification

In this technique, polysomes (consisting of mRNA, ribosome and nascent polypeptide chains) are purified by immunoaffinity chromatography with antibodies that recognize the antigenic determinants present in the nascent chains. In many cases, the antibodies obtained by immunization with intact cells or cell extracts recognize the antigens in their native conformations, but are no longer useful for polysome immunopurification, since there is a noticeable change in that the recognizable antigenic determinants on the nascent chains may not be present. Since translation begins at the N-terminus of the polypeptide, epitopes that are close to the C-terminus may be absent in the majority of the nascent cells. This problem can be avoided either by using antibodies which recognize N-terminal epitopes or by producing polysomes from cells which have been treated with a protein synthesis inhibitor which blocks the termination.

A more serious problem is that mature proteins differ from nascent chains because of post-translational changes. This problem is particularly acute with surface proteins, which are more frequently changed by removing signal peptides, adding carbohydrate side chains and forming disulfide bridges. If a polyclonal antiserum is used for polysome immunopurification, the differences in antigenicity between the nascent chains and the mature protein may be of little effect since the rabbit or other animal is not only exposed to the native protein during immunization, however also partially or completely the denatured forms, especially if Freund's adjuvant was available. Even if these antibodies make up only a small fraction of the antibody population, they can still be present in sufficient quantities to form the nascent chains. Unfortunately, the production of a polyclonal antiserum against a low-level protein can be exceptionally difficult. While a monoclonal antibody can be used to purify the antigen for further immunizations, each gram of "cultured cells" often yields only one microgram of antigen. This is sufficient to immunize a few mice, but hardly enough for a single rabbit.

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Another solution to the problem is to produce a monoclonal antibody that recognizes the antigenic determinants present in the nascent chains by using a denatured p97 antigen for the production of the monoclonal antibody. In the example of the present invention, a plate with monoclonal antibodies which recognized three specific epitopes of the N-terminal 40,000 dalton / molecular region of p97 was prepared and a pool of monoclonal antibodies was used, each having a different specificity, hoping that one or more of them would tie the nascent chains. The antibodies selected were highly specific against p97, each with an individual band of p97 forming an immunoprecipitate from a radiolabeled total cell lysate and exhibiting high affinity for formation. Three IgG2a antibodies were selected for the cloning project, one specific to each of the three epitopes. In general, the chances of success can be increased by using a number of antibodies against distinguishable epitopes.

When using monoclonal antibodies, the question remains how to predict that a given monoclonal antibody or a combination of antibodies that can recognize nascent chains and is therefore useful for use in polysome immunopurification. One way to determine whether the monoclonal antibody forms antigen-immunoprecipitates that have been translated in the reticulocyte lysate system is based on the assumption that the in vitro translation product that does not respond is likely to resemble the nascent chains. An alternative way is to do the polysome immunoprecipitation on a small scale and then use the in vitro translation to determine whether the mRNA species of interest has been enriched.

When using the polysome immunopurification technique, it is important that the purification be controlled by measuring mRNA activity. This can be done by translating the mRNA in a reticu-locyte lysate system and by SDS-PAGE analysis of the translation products. Although the tumor-associated antigen may be too small a component of the translation products of the non-enriched mRNA to recognize from the hundreds of more abundant species, it should be recognizable in the translation products derived from enriched mRNA samples. Alternatively, the Xenopus Oocyt translation system can be used if a sensitive immunoassay is possible to detect the translated tumor-associated antigen. For p97, a highly specific double determinant immunoassay (DDIA) was used, which uses two monoclonal antibodies that are specific for two different epitopes of p97.

Protein A, bound to Sepharose, can be used for polysome immunopurification. Protein A adsorbent has two uses in this process. In the first, the monoclonal antibodies are separated from crude ascites fluids, removing the contaminating ribonuclease activity. The protein A adsorbent is then used in conjugation with the purified antibodies to immunopurify polysomes that carry the specific nascent chains.

The translation of the mRNA in a reticulocyte lysate system allows the biochemical characterization of the translation product as well as a determination of its purity.

b) Oligonucleotide samples

Another method according to the invention for cloning the cDNA which encodes a tumor-associated antigen, such as p97, consists in determining a partial or complete amino acid sequence of the antigen and in synthesizing an oligonucleotide sample based on the nucleotide sequence which consists of the Amino acid sequence is derived. The oligonucleotide can then be used as a primer for cDNA synthesis and then as a sample for screening the resulting cDNA library. Accordingly, the melanoma-associated p97 protein can be purified in the usual manner 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 encoding part of the determined amino acid sequence is then synthesized and can be used as a primer and / or sample. Portions of the amino acid sequence containing the amino acid residues encoded by a single codon or two codons are most suitable for this purpose. For this purpose, the synthesis of a longer sequence is typically with 25-60 nucleotides, which represent the most likely coding sequence, on the basis of known codon usage frequencies in humans. The use of two synthetic oligonucleotides based on different parts of the amino acid sequence makes screening easier by allowing one to identify apparently positive hybridization signals. In addition, the use of hybridization conditions which minimize the effect of the GC content at the melting point of the DNA hybrid also facilitates the screening. Once a partial cDNA clone has been obtained by this method, it can be used as a sample to aid in the production of a full length cDNA clone.

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c) cDNA expression libraries

Cionation vectors have been developed which enable expression of cDNA insertion in bacteria. One way to use cDNA clones for tumor-associated proteins, such as for p97, is to create a cDNA library by reverse transcribing the mRNA (enriched or non-enriched) as described above from melanoma cells, is prepared using oligo (T) nucleotide primers or synthetic oligonucleotide primers and screening such a library with a monoclonal antibody directed against the melanoma-associated p97 protein becomes. Clones, which contain the epitopes which are recognized by the monoclonal antibodies in the correct orientation and which contain the recognition frequency, express peptides or proteins which are related to the melanoma-associated p97 protein and can be identified by transferring the proteins which are expressed by the clones on a nitrocellulose filter and incubation of the filter with the antibody, followed by development with a labeled anti-immunoglobulin reagent.

A possible problem is that many monoclonal antibodies cannot recognize the protein expressed by the bacterium, because in many cases only part of the cDNA is preserved in the insert and because bacteria do not protein in the same way as this is the case with eukaryotic cells. This problem is particularly acute in the case of tumor cell surface proteins, which are more strongly modified by removal of signal proteins, addition of carbohydrate side chains and formation of disulfide bridges. Accordingly, it may be necessary to generate monoclonal antibodies which are capable of recognizing the denatured antigen or which can produce polyspecific anisers by immunization with purified antigen.

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 throwing down to either identify the full length clones or a group of clones comprising the full length of the cDNA encoding p97. The identity of the cloned cDNA can be determined by sequence analysis and comparison of the derived N-terminal amino acid sequence with that which has been determined by direct amino acid sequence analysis on the p97 protein.

d) Genomic cloning

The following procedure allows DNA to be cloned using a monoclonal antibody directed against an antigenic determinant that is only present in the native protein and is not present in nascent chains or in the protein expressed in bacteria. For this purpose, a DNA derived from the human melanoma cell is introduced into mouse L-cells by transfection. Then the mouse cells expressing the melanoma-associated p97 antigen are isolated, either using the sorting of fluorescence-activated cells or by immunologically identifying colonies that produce p97-related peptides using radiolabeled monoclonal antibodies directed against p97 to detect related peptides on copies of colonies transferred to polyester cloth filters. Various subsequent rounds of transfection may be required to remove unrelated human DNA sequences. A genomic library is then prepared in an A. phage vector and screened for clones containing human repetitive sequences found in the interferons of most genes. Once a genomic clone has been identified, it can be used as a hybridization sample to identify cDNA clones containing the DNA encoding p97.

Synthesis of antiaenic fragments of the melanoma-associated p-97 antiaen and determination of the immunoenity

Synthetic peptides can be used as immunogens to elicit immune responses against the native protein and can provide protection against a number of pathogens. Such peptide sequences are selected from known amino acid sequences of the protein antigen by identifying spans of amino acids that are likely to be present on the surface of the protein molecule and exposed to the external medium. This is normally done by computer analysis of amino acid sequences, using the known hydropathy parameters for amino acids. Additional criteria such as the predicted secondary structure or flexibility can also be used.

Accordingly, peptides of the melanoma-associated p97 protein containing 5 to 50 amino acid residues can be tested for immunogenicity in experimental animals (usually mice or rabbits). Such synthetic peptides comprise the amino acid sequence as essentially shown in Figure 3; Modified sequences are further encompassed, in which functionally equivalent amino acid residues

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are substituted within the sequence in such a way that a silent change is achieved and / or changed or implemented sequences, such as glycosylated sequences, phosphorilated sequences etc. or chemically modified sequences, but the synthetic peptides are not restricted to the possibilities mentioned. These p97-related peptides are used either alone or coupled to a carrier protein, for example keyhole limp hemocyanin (KLH from diodora aspera). In any case, the use of an adjuvant is optional, but preferred. The immunized animals are vaccinated and tested for antibodies directed against the immunizing peptide. Those with antipeptide 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, e.g. by examining the delayed hypersensitivity (DTH) after the antigen-stimulated proliferation in vitro, after cytolytic T cells or after tumor rejection in a suitable model. A convenient model would be a mouse tumor that expresses the human tumor-associated antigen as a result of transvection with an appropriate cDNA expression vector construction.

The goal is to identify peptides that elicit a strong immune response against the melanoma-associated p97 antigen. Once identified, these peptides can be produced in large quantities using known chemical-synthetic methods. Alternatively, the identified peptides can be produced in large quantities by expression of the nucleotide sequences which are encoded for such peptides in expression vector host cell systems.

Production of o97-related peptides by expression vector host systems

Proteins and peptides can be produced in large quantities by inserting nucleotide coding sequences in a suitable expression vector, which is then introduced into a suitable host cell such as bacteria, yeast, insect cells and mammalian cells. Although the bacterial hosts have many advantages, they cannot develop many eukaryotic proteins appropriately and are less useful for eukaryotic cells for the expression of tumor-associated proteins. However, the recombinant proteins produced in bacteria can be useful for the introduction of T cell responses because such responses are believed to require the initial breakdown of the protein antigen.

In order to express a p97-related peptide in a vector host system, nucleotide sequences encoding the melanoma-associated p97 antigen or a part thereof are inserted into a suitable expression vector. Such nucleotide sequences include, without limitation, all or part of the DNA sequence of p97, as shown in Figure 3, including modified sequences, in which one or more codons within the sequence are replaced by a codon which is the same or a functional one encode equivalent amino acid residue, resulting in a neutral or silent change in sequence. The expression vector contains the elements necessary for the transcription and translation of the protein coding sequence used in the form of an insertion. These elements vary in their strengths and specificities. Depending on the host vector system used, one or a number of suitable transcription and translation elements can be used. For example, when cloning in mammalian cell systems, promoters which have been isolated from the genome of mammalian cells (e.g. mouse metallothionein promoter) or from viruses which grow in these cells (e.g. vaccine virus 7.5K promoter) can be used. Promoters made by recombinant DNA or synthetic techniques can also be used for the transcription of the insertion sequences.

Specific initiation signals are also required for effective translation of protein coding sequence insertions. These signals include the ATG initial codon and the adjacent sequences. In the case where either the gene or the cDNA sequence is inserted as an insert into a suitable expression vector, no additional control signals for transitions may be required. However, in cases where only part of the coding sequence is inserted, exogenous translation control signals, including the ATG codon, can be provided. The initial codon must continue to be in phase with the recognition sequence of the protein coding sequences so that the translation of the entire insertion is ensured. These exogenous translation control sequences and initial codons can be of various origins, both natural and synthetic.

Any method known to those skilled in the art of inserting DNA fragments into a vector can be used to construct expression vectors containing a chimeric gene that consists of appropriate transcription and translation control signals and protein encoding Sequences. These methods include those used in in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombinations (genetic recombinations).

Expression vectors include, without limitation, the following vectors and their derivatives: vaccinia virus, adenoviruses, insect viruses, yeast vectors, bacteriophage vectors, and plasmid DNA vectors. Cloning and expression of genes in bacterial systems is known in the art.

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mid promoters, such as the iaç promoter, the trp promoter. the recA promoter. the ribosomal RNA promoter, the Pr and PL promoter of coliphagen X and others including lacuv5. trp-lacuv5 (taci hybrid promoter, ompF. bla. Ipp and the like can be used to ensure a high degree of transcription of the adjacent DNA segments. Because of the developmental differences between the prokaryotic and eukaryotic cells, however, the p97 -related peptides are expressed in eukaryotic cells. The best established methods for expressing proteins in eukaryotic cells are:

a) the introduction of the gene into the cell, together with a drug-resistant gene and subsequent selection with the drug, preferably to achieve an enhancement such as with the dihydrofo-lat reductase methotrexate system;

b) expression of the cDNA in a plasmid vector, often based on 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.

The recombinant plasmid vectors are often used to produce cell lines that produce the protein for a long period of time, while the SV40 vectors are often used to obtain inconsistent expression. Despite the fact that mostly mammalian cells are used as hosts, insect cells and, in some cases, yeast cells can also be useful. These are described in detail below.

To construct a recombinant vaccine virus that expresses the melanoma-associated p97 antigen, the cDNA coding sequence can be linked to the 7.5K promoter of the vaccinia virus to produce a chimeric gene. This chimeric gene is bound by additional vaccinia-viral sequences which are homologous to the viral thymidine kinase gene, which is carried out on the plasmid DNA vector. The construction of the chimeric gene involves the use of both natural and synthetic control signals for the transcription and translation of the tumor-associated antigen sequence. The chimeric gene is then introduced into the vaccinia virus expression vectors by in vivo recombination between the homologous thymidine kinase region that is present in both the plasmid vector and the vaccinia virus genome. These recombinant viruses, which contain the chimeric gene, are capable of expressing p97-related peptides in an infected host and can be used as components in vaccines.

In cases where an adenovirus is used as the expression vector, the DNA sequence of interest is linked to an adenovirus transcription / translation control complex, e.g. the late promoter and the tripartite leader sequences. The chimeric gene is then inserted into the ade novirus genome by in vitro or in vivo recombination. Insertion into an insignificant region of the viral genome (e.g. region E1 or E3) leads to a recombinant virus which is viable and can express the p97-related peptides in infected hosts. There are currently two strains of adenovirus, types 4 and 7, which are used as vaccines for military personnel. They are the first candidates for the use of vectors for expression of DNA sequence insertions.

An alternative expression system that could be used to express the p97-related peptides is an insect system. In such a system, the Autoorapha californica nuclear polyhedrosis virus (AcNPV) is used as a vector for the expression of foreign genes. The virus grows in Spodoptera fruaiperda cells. The DNA sequence of interest can be cloned into non-essential regions (e.g. the polyhedrin gene) of the virus and placed under the control of an AcNPV promoter (e.g. the polyhedrin promoter). Successful insertion of the DNA sequence leads to inactivation of the polyhedrin gene and the production of a non-occluding recombinant virus (i.e. a virus lacking the protein coat encoded by the polyhedrin gene). These recombinant viruses are then used to infect Soodootera fruaioerda cells in which the gene insertion is expressed.

In addition, host cell strains can be selected which modulate or modify the expression of the sequence insert and which produce the chimeric gene products in a specific desired manner. The expression of certain promoters can be increased in the presence of certain inducers, e.g. Zinc and Kadium ions for metallothionein promoters. Accordingly, the expression of genetically engineered protein can be controlled. This is important if the protein product of the cloned gene is lethai for the host cells. Furthermore, changes, e.g. glycosylation, phosphorilation, and reactions (e.g. cleavage of protein products) important for the structure and function of the protein. Different host cells have characteristic and specific mechanisms for the processes and changes in the protein after translation. Appropriate cell lines or host systems can be selected to ensure the correct changes and reactions of the expressed foreign proteins.

In the particular embodiment for p97 described here, the p97 cDNA sequence was inserted into an expression plasmid vector which was derived from pBR322 and which contains the metallo-

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incorporated an expression plasmid vector derived from pBR322 and containing the metallothionein promoter. The entire coding sequence of p97, including the signal peptide and the membrane anchor, was inserted into the vector as an insert.

Identification of recombinant expression vectors. which have the ability to replicate and perform expression of the p97 DNA sequences

Expression vectors that have foreign gene insertions can be identified by three general methods:

a) DNA-DNA hybridization b) presence or absence of marker gene functions and c) expression of sequence insertions.

In the first way, the presence of a foreign gene which has been introduced into an expression vector can be detected by DNA-DNA hybridization using samples which contain sequences which are homologous to foreign gene insertions. In the second way, the recombinant vector / host system can be identified and selected on the basis of the presence or absence of certain “labeling” gene functions which are caused by the insertion of genes into the vector (for example by thymidine kinase activity of the system against antibiotics, Transforma -tion phenotypes, etc.). If the foreign gene e.g. inserted into the labeling sequence of the vector, recombinants containing the DNA insert can be recognized by the absence of the labeling gene function. In the third way, the recombinant expression vectors can be identified by testing the foreign gene product which is expressed by the recombinant. Such tests can be performed based on the physical, immunological, or functional properties of the gene product. If, for example, a recombinant vaccinia virus according to the invention is constructed, the chimeric gene which contains the p97-coding sequences is inserted into the thymidine kinase gene, which inactivates and forms a TK phenotype on the virus causes. Such recombinants are selected for their ability to grow in media containing 5-bromo-deoxyuridine, a nucleoside analog that is toxic to TK + cells but not toxic to TK cells. The recombinants are further identified by DNA-DNA hybridization using a cDNA sample that is sensitive to the tumor associated protein. The TK recombinant virus can be isolated by plaque purification and stocks can be made from infected cultured cells. The recombinant virus can be tested for its ability to induce the synthesis of p97-related peptides. For this purpose, infected cells can be grown in the presence of radiolabeled amino acids; then the lysates and subcellular fractions of the infected radiolabeled cells are tested by immunoprecipitation with antibodies directed against the native melanoma-associated p97 antigen. The immunoprecipitated products are then resolved by SDS-PAGE. The infected cells can also be tested by immunofluorescence using monoclonal antibodies.

Cells into which a plasmid vector has been introduced by transfection can easily be identified by FACS analysis or by attempts to bind copies of cell colonies to polyester fabric. The amount of p97-related peptide present can be determined by a quantitative radio dioimmunoassay and its subcellular localization can be determined by cellular fractionation and by immunofluorescence microscopy. The structure of the expressed p97-related peptide can be determined by SDS-PAGE and by amino acid sequence analysis.

Purification of the p97-related peptide from expression vector host systems

Many tumor associated antigens, such as p97, are cell surface glycoproteins and include an N-terminal signal peptide and a C-terminal anchor peptide (Davis et al., 1985, J. Mol. Biol., 181: 111-121). When expressed in an appropriate vector, the protein is expected to be transported to the cell surface. To facilitate the purification of the protein, it may be preferred to delete the DNA sequence encoding the membrane anchor region so that the mature protein is released into the culture medium.

The p97-related peptides can be purified from these host cells by detergent lysis and then affinity chromatography using monoclonal antibodies. If a trimmed protein has to be purified from the culture medium, a serum-free medium is preferably used, which can then be used for affinity chromatography with monoclonal antibodies. It is important that the antigen can be eluted from the antibody adsorbent without reducing the antigenic activity or denaturing it. This can be achieved by raising or lowering the pH or by using a chaotrope. It may be necessary to select a monoclonal antibody that contains the antigen

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releases relatively mild conditions. The affinity-purified antigen can be further purified by HPLC.

The immunological characterization of p97-related peptides

The ability of the synthetic or recombinant antigens to trigger an anti-tumor reaction can first be demonstrated in experimental animals. This is achieved by building a model system in which the human melanoma-associated p97 protein is expressed in cells from strains with convenient hereditary properties of the experimental type. The animals are then immunized with the p97-related peptide according to the invention by various methods and then tested for the development of antibodies which are directed against the melanoma-associated p97 antigen, for cell-mediated immunity, such as delayed hypersensitivity to the p97 antigen and to their ability to reject a challenge from viable, syngeneic tumor cells expressing the p97 anti-gene. In addition, in vitro tests for cellular immunity can be performed by measuring the proliferation of the lymphocytes in response to the p97-related peptide and determining the ability of the lymphocytes of immunized animals or human melanoma patients to determine tumor cells that contain the p97 -Antigen express, kill. Furthermore, by immunizing mice with mouse p97, one is able to determine the extent to which it is possible to induce an immune response against an antigen that is present in trace amounts in normal tissues.

Non-human primates can be used for the safety of p97-related peptides of the present invention. To this end, the animals can be immunized using experimental conditions, which are also ethically responsible for human cancer patients, and then tested as described above, except that the tumor translation experiments are not feasible as they make use of them of inherited tribes. The safety of the immunization procedure is carried out by observing the effect of the immunization on the general health of the immunized animal (weight changes, fever, appetite, behavior, etc.) and by observing pathological changes during the autopsy.

Finally, the p97-related peptide according to the invention can be tested in human cancer patients. After the introductory phase, testing advanced cancer patients to determine the lack of toxicity, cancer patients with decreasing disease symptoms but with a high probability of recurrence could be tested. Your immune response can be performed as described above for the non-human primates, except that the effect of the treatment is tested on the condition being tested or on the frequency of the disease's subsidence. In the case of the melanoma antigen p97 benign nevi (moles), which expresses the antigen, will also be checked.

Formulation of the vaccine

Synthetic or recombinant DNA techniques produce a synthetic peptide, a purified protein, or a recombinant virus that can be used to treat existing conditions as an immunogen and as a vaccine to protect cancer patients with a high risk of flare-up of their condition finally prophylactically vaccinating high-risk individuals. In fact, the synthetic or recombinant melanoma-associated p97 antigen can be used in combination with other immunogens to produce multivalent vaccines for the prevention of melanoma and other cancers. Examples of different vaccine formulations are discussed below.

Viral vaccine formulations

If a p97-related peptide according to the invention is produced by a recombinant virus, it can be formulated either as a living recombinant viral vaccine or as an inactivated recombinant viral vaccine. The choice depends on the nature of the recombinant virus used, which expresses the p97-related peptide. If the recombinant virus is infectious to the host to be immunized but does not cause disease, a live vaccine is preferred as multiplication in the host results in an extended stimulus of a similar type and the size is comparable to that seen in natural subclinical infections and consequently leads to substantial long-term immunity. When introduced into a host, the infectious recombinant viruses can express the p97-related peptide from its chimeric gene and thereby stimulate an immune response. The living recombinant virus itself can be used as a preventive vaccination against melanoma. The production of such a recombinant virus, which can be used in these formulations, can include both in vitro systems (e.g. tissue culture cells) and in vivo systems (e.g. natural host animals such as cows). Usual methods for the production and formulation of smallpox vaccinations can be adapted for the formulation of live recombinant virus vaccines.

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Multivalent live virus vaccines can be made from single or a few infectious recombinant viruses that develop a number of antigens against various tumor or cancer cells. For example, a vaccinia virus (which can take up about 35 kilobases of foreign DNA) can be manipulated to contain coding sequences for other epitopes; such a recombinant virus itself can be used as an immunogen in a multivaltene vaccine. Alternatively, a mixture of vaccines and / or other agents that are capable of expressing the expression of direct genes encoding different epitopes can be formulated in a multivalent vaccine.

Regardless of whether the recombinant virus is infectious to the virus to be immunized or not, an inactivated vaccine formulation can be prepared. Inactivated agents are "dead" in the sense that their infectivity has been destroyed, usually by treatment with formaldehyde. Ideally, the infectivity of the virus is destroyed without affecting the capsid or the envelope protein, which carries the immunogenicity of the virus. In order to produce inactivated vaccines, large amounts of the recombinant virus have to be cultivated in cultures in order to provide the necessary quantity of the relevant antigen. A mixture of inactivated viruses that express different epitopes can be used to formulate "multivalent" vaccines. In some cases, this may be preferred over live vaccine formulations because of possible difficulties due to mutual interactions between the co-administered viruses. In any event, the inactivated recombinant virus or the mixture of viruses should be formulated with an appropriate adjuvant to increase the immune response to their antigen. Useful adjuvants include, without limitation, mineral gels such as aluminum hydroxide; surfactants such as lysolecithin; Pluron polyols; Polyanions; Peptides; and oil emulsions.

Many methods can be used to introduce the vaccine formulations described above; these include, without limitation, the intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, and intranasal routes. If a live recombinant virus-vaccine formulation is used, it can be by the natural route of infection with the original wild-type virus that was used to produce the recombinant virus in the vaccine formulation.

Subunit vaccine formulation

In an alternative to the viral agents, the p97-related peptides themselves can be used as an immunogen in subunit vaccine formulations. Subunit vaccines include only the relevant immunogenic material necessary to immunize the host. Accordingly, the p97-related peptides can be purified from the recombinants which express the peptides. Such recombinants include all previously described virus-infected culture cells, bacterial transformants or yeast transformants. The p97-related peptides or proteins can also be synthesized chemically. Regardless of whether the p97-related peptides have been purified from the recombinants or have been chemically synthesized, the end product can be adjusted to the appropriate concentration and formulated with an appropriate vaccine adjuvant and packaged for use. Appropriate adjuvants include, without limitation, mineral gels, e.g. Aluminum hydroxide, surface-active substances such as lysolecithin, pluron polyols, polyanions, peptides and oil emulsions. The p97-related peptide can also be incorporated into liposomes or conjugated to polysaccharides and / or polymers for use in vaccine formulations.

In those cases where the p97-related peptide is a hapten, i.e. a molecule which is antigenic in that it can react selectively with conjugated antibodies but is not immunogenic so that it cannot elicit an immune response, the hapten can be covalently linked to a carrier or an immunogenic molecule and the hapten Vehicle can be formulated for use as a vaccine; for example, a large protein, such as a protein serum albumin, can impart immunogenicity to the coupled hapten.

EXAMPLE: Melanoma Associated p97 Antiaen

In the example described below, cDNA clones which were derived from different regions of the p97 mRNA were assembled and inserted as an insert in an expression vector which was directed against the expression of a p97-related peptide. The p97-related peptides produced by the expression of the vector host cell can be formulated as a vaccine.

Purification of p97 mRNA

Polysomes of SK-MEL28 melanoma cells were prepared by magnesium precipitation (Carey et al., 1976, Proc. Nati. Acad. Sci. USA, 73: 3270-3282). Polyso-

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me, which had nascent p97 chains, by incubation with: 3 IgG2a monoclonal antibodies (96.5, 118.1, 133.2) which are specific for certain epitopes of p97 (Brown et al., 1980, J. Biol. Chem., 255: 4980-4983; Brown et al., 1981, J. Immunol., 127: 539-546; Brown et al., 1981, Proc. Nati. Acad. Sei., USA, 78: 539-543 ; Plowman et al., 1983, Nature, London, 303: 70-72), followed by 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 a typical experiment, 150 E.260 units of polysomes yielded 260 ng of p97-enriched mRNA, which was 0.23% of the total mRNA. When translating into Xenopus in oocytes and testing for p97 as described (Brown et al., 1981, Proc. Nati. Acad. Sei. USA, 78: 539-543; Plowman et al., 1983, Nature, Londen, 303: 70-72), the p97-enriched mRNA gave 80 pg p97 per ng mRNA, while the p97-non-enriched mRNA gave only 0.44 pg p97 per ng mRNA, which shows that the p97 mRNA activity enriched about 180-fold has been. The yield of p97 mRNA activity was 42%. Translation in the reticulocyte lysate system (Pelham and Jackson, 1976, Eur. J. Biochem., 67: 247-256) showed that the p97-enriched mRNA encoded a major poly-peptide which had an apparent molecular weight of 84,000 daltons. as analysis by SDS-PAGE revealed, which was undetectable, was immunoprecipitated in the translation products of non-enriched mRNA and in the antiserum specific for p97 (Figure 1). It was concluded that this was the non-glycosylated precursor of p97.

Production and construction of cDNA clones

The two techniques described below were transcribed to construct cDNA clones from the mRNA matrices isolated as described above.

Construction of cDNA clones by Primino with OliggCn

The p97-enriched mRNA prepared as above was used as a matrix (template) for cDNA synthesis using Oligo (T) as a primer. The cDNA was cloned in pBR322 as follows: p97-enriched mRNA, the four dNTPs and oligo (T) (Collaborative Research, Walthma, MA) were incubated with reverse transcriptase (Molecular Genetic Resources) for the synthesis of the first cDNA strand. The second strand was synthesized by incubation with the large fragment of E. coli DNA polymerase (Bethesda Research Labs., Bethesda MD) and the double-stranded cDNA was obtained with SI nuclease (obtained from D. Durnam of "The Fred Hutchinsen Cancer Research Center », Seattie, WA). The cDNA was then dC-tailed with terminal deoxynucleotide transferase (Bethesda Research Labs., Bethesda, MD), with PstI digested. provided with dG-tailed pBR322 (Bethesda Research Labs., Bethesda MD) (Villa-Komaroff et al., 1978, Proc. Nati. Acad. Sei. USA, 75: 3727-3731) and for the transformation of CaCl2-treated E coli-RRI used. The DNA from colonies of transformed bacteria was bound on paper (Taub and Thompson, 1982, Anal. Biochem., 126: 222-230) and by differential hybridization with cDNA samples which were enriched for p97 and non-enriched mRNA matrices were synthesized, subjected to screening.

A 243 base pair (bp) clone, p97-3a2fl, was identified which hybridized with p97-enriched cDNA but not with non-enriched cDNA and also selected p97 mRNA in hybrid selection-translation experiments. There was a polyadenylation signal (AATAA) and a poly (A) tract at the 3 'end of the cDNA (see FIG. 2). p97-3a2fl, which was nick-translated, hybridized 100 times more strongly with p97-enriched mRNA than with non-enriched melanoma mRNA and undetectably with fibroblast mRNA. Northern blot analysis with the cloned cDNA as a sample identified an approximately 4 kilobase mRNA which was present in SK-MEL28 melanoma cells and was absent in fibroblasts.

Genomic clonino of d97 and use of synthetic oliaonucleotides for the primina of cDNA synthesis

Attempts to obtain cDNA clones extending more than 1 kb from the polyadenylation center have been unsuccessful, probably due to an area of high GC content (greater than 80%) with an extensive secondary structure. To overcome this problem, genomic cloning was used. Four overlapping genomic clones were isolated from banks of A.L47.1, which contained size-fractionated SK-MEL28 DNA, which was enriched in a specific p97 restriction fragment. These four genomic clones spanned 28 kb and contained the entire coding region of p97, including the regulatory region of the gene. The genomic clones sequentially arranged from 5 'to 3' were: XB15, XH17, XB6.6 and XE7.7. The nomenclature consists of a letter which refers to the restriction enzyme used to form the fragment and the numbering indicates the kilobase size of the fragment which was cloned with AL47.1. Starting from the 5 'terminus, the X clone B15 accordingly contains a 15 kb BamHI p97 fragment; the \ clone H17 contains a 17kb Hindill p97 fragment; and X-Clon B6.6 contains a 6.6kb Barn Hi p97 Frag

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ment and the X-clone E7.7 contains 7.7 kb EcoRI p97 fragment (cf. FIG. 2A). The restriction fragments of the clones which hybridized with 4kb p97 mRNA on Northern blots were sequenced and the p97 exons were identified by a computer-assisted homology search between the predicted coding sequences and the amino acid sequences of the human and chicken transferrin (Yang et al., 1984, Proc. Nati. Acad. Sei., USA, 81; 2752-2756; McGillivray et al., 1982, Proc. Nati. Acad. Sei., USA, 79: 2504-2508; Jetsch and Chambon, 1982, Eur. J. Biochem., 122: 291-295). Three synthetic oligonucleotides, the sequences of which were based on the p97 genomic exon sequences, were used to prime the cDNA synthesis to SK-MEL28 mRNA and the resulting cDNA was cloned with an X-gt10 as follows: the p97cDNA was cloned with a dG Tail and linked with a bridge oligonucleotide (AATTCCCCCCCCCCCC) and X-gt10, which were restricted with EcoRI. The bridge oligonucleotides allowed the insertion and ligation of the cDNA sequence with the dG tails at the EœRI center of the X-gt10. The X phage was coupled (Grosveld et al., 1981, Gene, 13: 227-237) and coated on E. coli c600-rK_mK + hfi. The cDNA library in A. gt10 was screened for p97 insertion by plaque hybridization (Benton and. Davis, 1977, Science, 196: 180) with genomic exon fragments as samples. The samples were radiolabeled with 32P-TTP (New England Nuclear, 3200 Ci / mmole) by nick translation to a specific activity of 5-10 x 108 cpm / ng. Three overlapping cDNA clones (10 al, 1jl, 2fl), which spanned 2368 nucleotides of the p97 mRNA and encompass the entire coding region, were identified as samples using p97 exon-specific fragments (FIG. 2).

DNA sequence analysis of d97

CDNA insertions were cut out and subcloned into the plasmid vector pEMBL18 + (Dente et al., 1983, Nucleic Acids Res., H: 1645-1644) and then propagated in E. coli and a restriction map was created. The cDNA was also subcloned in the M13mp18 phage cloning vector (Yanish-Perrone et al., 1985, Gene, 33: 103-119) and using the Sanger dideoxy method (Sanger et al., 1977, Proc. Nati. Acad. Sci. USA, 74: 5463-5467). The M13 clones, which contained large inserts, were deleted by forming deletions using DNAse 1 (Hong, 1983, J. Mol. Biol., 158: 539-549) or exonuclease III (Henikoff, 1984, Gene, 28: 351-359) and sequenced using synthetic 21-mer oligonucleotide primers.

The p97 cDNA sequence is shown in Figure 3. An open recognition frequency of 2214 nucleotides stretches from the first ATG to the sequence in the vicinity, which matches the initial sequence determined by Kozak (Kozak, 1980, Nucleic Acids Res., 8: 127-142) to the TGA in Position 2215. Most 5 'cDNA clones contain an additional 60 nucleotides upstream of the initial 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 1667 nucleotides. Residues 20-32 of the predicted amino acid sequence are identical to the known N-terminal amino acid sequence from p97 (Brown et al., 1982, Nature, London, 296: 171-173), which further proves the identity with the cloned cDNA predicted molecular weight of precursor 80196 daltons, which is in good agreement with the determined molecular weight of the in vitro translation product.

Construction of a recombinant expression plasmid. which contains the p97 coding sequence

The large size of the p97 gene required the joining of the cDNA clones obtained by specifically priming the melanoma mRNA with reverse transcriptase. The three cDNA-X.-gt10 clones (1 Oal, Ijl and Ifl; see FIG. 2) which encode the coding region from the signal peptide to the membrane anchor sequence were used. The p97 insert of clone 10al was excised by digestion with EcoRI and the oligo (dG) sequence at 5 'of cDNA-10al was removed by digestion with exonuclease III to form clone 10alb with a HindIII site . 30 bp upstream of the starting methionine of the p97 preprotein. The p97 insertions of the three cDNA clones 10alb, 1jl and 2fi and the genomic clones E7.7 were linked together at the Pvull, Sstl and EcoRI restriction enzyme sites and at the Hindlll-EcoRI sites as an insert in the plasmid Vector pEMBL18 + inserted (Dente et al., 1983, Nuc. Acid Res., H: 1645-1655) as shown in Figure 2. The final construction, p97b, comprises 4.4 kb p97 insertion in the plasmid vector pEMBL18 +, which was used to transform E. coli HB101. The insert in p97b contains 30 bp of the 5'-untranslated region of the p97 mRNA, the entire coding sequence and the 3'-untranslated region which is bound by a 5'HindIIi center and which contains the 3'EcoRI site.

The 4.4 kilobase p97 insert was cut out of p97b with HindIII and EcoRI and the ends were filled using the Klenow fragment of E. coli DNA polymerase. The blunt-ended fragment was inserted as the insertion on the single Smal stelie of the eukaryotic cDNA expression vector 1995.12 pUCI3, a derivative; of the vector mThGH-112 (Palmiter et al., 1983, Science, 222: 809-814), which was developed by Dr. Richard Palmiter (University of Washington, Seattle,

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Washington) was made available. This vector uses the mouse metallothionein promoter to express foreign genes in eukaryotic cells. The construction with the p97 insertion in the correct orientation was identified by restriction analysis and designated pMTp97b.

The recombinant plasmid was transfected into LMTK ~ cells and the transfectants were selected by growth in HAT medium. The clones obtained from the transfection plate were spread in 96-well microtest plates and provided with culture medium, and the cell lysate from contact plates were tested for p97 by a two-site immuno-radiometric test. The subclones were spread out and tested again. The clone TKMp97-12, which expresses approximately 4,000,000 molecules of p97 per cell, was grown, induced with cadmium and used as a source of p97 for the immunization.

Immunization of mice with p97-related peptides

The TKMp97-12 cells were grown, induced with cadmium and (14.4 g) were incubated for 10 min on ice with 70 ml TNEN (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40) lysed. The lysate was ultracentrifuged at 200,000 g for 45 min at 4 ° C and half of the lysate was placed on a 1 ml immunoaffinity column which was specific for p97 (Fab fragment of antibody 96.5 of Sepharose was coupled). The immuno-adsorbent was washed intensively, first with TNEN and then finally with 20 mM Tris-HCl, pH 6.8.

For immunization, 0.5 ml of the adsorbed immunoaffinity column, prepared as described above, was mixed with 0.5 ml of 20 mM Tris-HCl, pH 6.8, and cultured with 1 ml of Freund's complete adjuvant. Four BALB / c mice each received 0.4 ml of the emulsion intraperitoneally. Three weeks later, the mice were treated with a quarter of this amount of the antigen in incomplete Freund's adjuvant. The control mice were immunized with an immunoaffinity column with an antibody which was not related to p97 and were otherwise treated identically. Blood was withdrawn from the four p97-immunized mice and the two control mice 1 week after the immunization was boosted. The sera were tested for antibodies against p97 by immunoprecipitation with radio-iodinated SK-MEL melanoma cells and subjected to SDS-PAGE. The results showed that the sera of the four mice immunized with p97 had immunoprecipitated p97, while the control sera were negative. The sera were also tested for the presence of antibodies directed against p97 using an ELISA assay with glutaraldehyde-fixed SK-MEL27 melanoma cells (20,000 cells per microtest hole). The fixed cells were incubated with 0.05 ml serum diluted 1/10000 for 1 hour at room temperature, washed and then for 1 hour at room temperature with 0.05 ml horseradish peroxidase-conjugated goat antismic acid IgG (Southern Biotech) incubated. The optical densities (read at 490 nm) of the sera of the p97-immunized mice were 0.350, 0.243, 0.343, 0.200, while the optical densities of the control sera were 0.036 and 0.057.

Characterization of o97. Structure of p97

The structure of p97 was determined from the amino acid sequence of the p97 precursor, which has four structural regions. Because residue 20 of the precursor sequence corresponds to the N-terminus of mature p97, amino acid residues 1-19 may represent a signal peptide, an assumption supported by the length and hydrophobic nature. Amino acids 20-361 and 362-713 encompass two homologous regions of 342-352 amino acids. Possible N-linked glycosylation sites are located at positions 38 and 135 in the N-terrrial region and in position 515 in the C-terminal region. Finally, we assume that amino acids 714-718 an area of predominantly uncharged and hydrophobic residues, p97, are anchored in the cell membrane (Davis et al., 1985, J. Mol biol, 181: 111-121) and themselves expand into the cytoplasm.

The region structure of p97 is supported by protease digestion experiments. Digestion of p97 with trypsin, papain (Brown et al., 1981, J. Immunol., 127: 539-546) or thrombin results in a glycosylated, antigenic fragment with a molecular weight of approximately 40,000 daltons. The fragment was then purified from a thrombin digestion of p97 metabolically labeled with 35S methionine or 35S cysteine and sequenced as described (Brown et al., 1982, Nature, London, 296: 171-173). The cysteine residues were identified at sites 7 and 17 and the methionine residues at sites 2-20. Identical results were obtained with intact p97 in full agreement with the N-terminal sequence of p97, which was predicted from the cDNA sequence. We conclude that the proteinase-resistant fragment with a molecular weight of 40,000 daltons corresponds to the N-terminal region of p97. It was not possible to isolate the C-terminal region of p97, probably because of its protease sensitivity.

Homology of p97 with transferrin

A search on the amino acid sequence library of the Protein Identification Resource (Release 5.0; Dayhoff et al., 1981, Nature, London, 290: 8) showed that p97 is impressively homologous with three members

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the transferrin superfamily is: human serum transferrin, human lacto-transferrin and chicken transferrin (37-39% homology, see FIG. 3). Since human and chicken transferrin show 50% homology, p97 must have been derived from serum transferrin about 3,000,000 years ago. p97 has 14 cysteine residues, which are homologous in each area. The human transferrin contains all of these cysteines in homologous positions in both areas, while the human lacto-transferrin and the chicken transferrin only lack two of these cysteine residues (in their C-terminal areas). In contrast to p97, these proteins contain 4-7 additional cysteines in their C-terminal areas and have no corresponding member in the N-terminal area. The human transferrin contains two additional cysteine units in its N-terminal region. The positions of most disulfides in human transferrin, lacto-transferrin and chicken transferrin were determined directly (McGillivray et al., 1982, Proc. Nati. Acad. Sei, US, 79: 2504-2508; Metz-Boutique et al., 1984, Eur. J. Biochem., 145: 659-676; Mazurier et al., 1983, Experientia (Basel), 39: 135-141; MacGilliv-ray et al., 1983, J. Biol. Chem., 258 : 3453-3553; Williams et al., 1982, Eur. J. Biochem., 122: 297-303; Williams, 1974, Biochem J., 141: 745-752). The presence of 7 disulfide bonds in each region of p97 can be predicted (see FIG. 5).

The amino acid homology between the regions of p97 (46% - achieved by inserting 7 loops of 9 residues) is more impressive than that in human transferrin (43% - 16 loops, 45 residues) or in chicken transferrin (35% , 12 loops, 49 remnants) can be determined. Given the extensive sequence homology between p97 and transferrin, the apparently similar fold pattern based on the conservation of cysteines, it is believed that if the present low resolution x-ray structure of transferrin (Gorinsky et al, 1979, Nature, London, 281: 157 -158), the three-dimensional structure of p97 may be derived.

Function of d97

His membership in the transferrin superfamily, his ability to bind iron (Brown et al., 1982, Nature, London, 296: 171-173) and his common chromosomal localization with transferrin and the transferrin receptor (Plowman et al., 1983 , Nature, London, 303: 70-72; Yang et al., 1984, Proc. Nati. Acad. Sei, USA, §1: 2752-2756) all support the role of p97 in iron transport. The iron binding pocket of transferrin is believed to contain 2-3 tyrosines, 1-2 histidines and a single bicarbonate binding arginine (Metz-Boutique et al., 1984, Eur. J. Biochem., 145: 659-676). The conservation of these amino acids in p97 supports its 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 which is formed by circulation of the serum trans-ferrin and the cellular receptor for transferrin. The expression of the cloned p97 cDNA in eukaryotic cells will allow experimental tests on the functional properties.

Enough

Based on this data, it is clear that cDNA constructions for melanoma-associated p97 have been obtained and that they can be expressed efficiently in mammalian cells to obtain large amounts of the antigenic p97.

Expression of aeclontem d97 and vaccine tests

The detailed experiments described here describe the expression of the cloned p97 protein and corresponding vaccine tests. The expression of p97 protein in a secreted form (by the transferred mouse cell clone B16svp97a.14) allowed the purification in milligram amounts of the full length of the p97 protein. The purified protein was used for in vitro tests for the induction of cellular immunity and for testing its potential as a subunit vaccine. The p97 gene product was also expressed on the cell surface of metastatic mouse melanoma cells and represents a model for testing the efficacy of vaccines for the prevention of tumor growth in an oxygenetic system.

The p97 gene was inserted into a live recombinant vaccinia virus as an insert for use as a vaccine formulation capable of conferring effective cellular immunity. The recombinant vaccinia virus Vp97a-NY was selected for its ability to generate immunity in mice using a variety of experiments to demonstrate humoral and cellular immunity. Using the syngeneic mouse tumor model as described above, the recombinant virus vaccine Vp97a-NY showed the conferment of a protective effect from a tumor cell challenge. The vaccine also showed a therapeutic effect in mice with existing, growing lung metastases, a property which is analogous to the proposed use of the vaccine for the formation of an immunotherapeutic anti-mor response in human melanoma patients with an existing tumor.

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molog mouse protein exists (in the areas tested so far), the Vp97a-NY vaccine was also tested in non-human primates. There is much greater homology between human p97 and the monkey form of the protein (as demonstrated by cross-reactivity at the monoclonal antibody level). Because of possible difficulties in generating an immune response against a "self" protein, the most closely related macaquenaffe was used to test the immunogenicity of the Vp97a-NY vaccine. The recombinant vaccinia vaccine has been tested in monkeys and has been shown to induce humoral immunity to the .p97 protein. So far, the monkeys showed no noticeable symptoms of harmful side effects from the vaccine after being given two inoculations of the live recombinant vaccinia virus for a period of 6 weeks.

Plasmid expression

The expression plasmid, which is driven by the SV2 early promoter sv2, was constructed from the cDNA plasmid clone p97, which is similar to plasmid p97b, except that the entire 3'UT region is used (Figure 6). All cDNA clones were originally isolated from X-gt10 libraries with synthetic EcoRI-dG (9-17) links as previously described. The inserts were excised by EcoRI and subcloned into pEMBL18 + for subsequent propagation and characterization. The clone 10al was subcloned into M13mp18 and an RF form was digested with BamHI and Sphl, briefly treated with exonuclease III, blunted with S1 nuclease, treated with Klenow and reconnected. Various plaques were isolated and sequenced, one of which had the dG tail removed and 33 bp of the p97 5'-untranslated region inserted at the HindIII site of M13mp18 as an insert. An RF from this subclone (10ala) was used to generate the intact p97 cDNA; on the other hand, all fragments were isolated from the plasmid subclones. The 550 bp Hindlll-Pvull fragment from 10ala and the 735 bp Pvull-Sall fragment from 1jl were isolated from LMP agarose gels and incorporated into pEMBL18 + at the Sa [l and Hindlll sites to form p5'p97. The genomic clone E7.7 in pEMBLI 8+ was completely digested with EcoRI and partially digested with Sstl and the 4.5 kb fragment was separated by fractionation through 0.8% LMP agarose. This 4.5 kb3 'fragment was bound with the 404 bp Sstl fragment from 2 ml and the 535 bp BamHl Sstl fragment from 1 ml into the pEMBLI8 + at the Sali and EcoRI positions. whereby p3'p97 was formed. The 1285 bp HindIII-SalI fragment of p5'p97 was then incorporated into p3'p97-forming-pp97a. The EcoRI partial HindIII fragment from this clone was inserted as an insert in pSV2neo (Southern et al., 1982, J. Mol. App. Genet., 1: 327-341) at the Hindill and EcoRI sites. the neomycin coding region and the SV40 splicing / polyA sequences were removed, with the early SV40 promoter and 72 bp enhancer, 33 bp p975'UTR, the entire p37 coding region, 3'UTS and the 1st , 4 kb of 3 'flanking DNA remained. The resulting plasmid was named pSV2p97a.

The sv2-driven plasmid was transfected by calcium phosphate precipitation in a number of eukaryotic cells and the expressed cells were cloned and selected using a co-transfection of a dominant selectable marker. Chinese hamster ovary cells (CHO) were cultured in Hank-Fl medium containing 15% fetal calf serum (FCS), 4 mM L-glutamine, 1.3 mM proline and antibiotics. The B16 lines were cultivated in RPMI medium containing 0.15% bicarbonate and 1735 cells in DMEM medium (Gibco), each with 15% FCS and containing antibiotics. The cells were transfected by a modified calcium phosphate technique (Wigler M., et al., 1978, Cell, 14: 725-731), with 20 (ig per plate pSV2p97a plasmid DNA and 0.5 ng either pSV2DHFR or pSV2neo. All plasmids were linearized at the EcoRI site. Stable transfection agents were selected using hypoxanthine negative (HAT -) medium for CHO cells or 0.5 g / ml Geneticin (G418, Gibco) for the B16 and 1735 cells The surviving cells began to form visible colonies 7 days after transfection and were covered with sterile polyester filters held in place by glass beads, and the filters remained in place for 5 days with the "Cells in the polyester matrix were allowed to grow, making a copy of the colonies on the plate. The riters were then removed and used to test for living cells with an iodinated anti-p97 monoclonal antibody." Microgram labeled monoclonal antibody was incubated with up to 20 filters in 10 ml FCS at 4 ° C for 1 hour. The filters were washed extensively in phosphate buffered saline (PBS), dried and left on an XAR-5 film overnight at -70 ° C. The filters were then stained with 7% methylene blue in order to make the cell colonies visible. In all cell lines used, with the exception of the B16 mouse line, the expression cells contained the antigenic p97 protein on their surface.

In the B16-transfected cells, p97 was released into the medium, which was a unique find in this cell type. The secretion of p97 made it possible to purify the p97 protein from the cell medium to its full length. Clone B16SVp97a.14 expressed about 4 ng / ml p97 in the used medium. The recombinant p97 was purified from the used medium of the transferred clone B16SVp97a.14, which released large amounts of p97 antigen into the medium. The cells were kept near confluence (109 cells) in 850 cm2 roller bottles, being continuous

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Cells were kept near confluence (109 cells) in 850 cm2 roller bottles, with small amounts of fresh medium being added continuously. They continued pouring out antigen without detachment for weeks, allowing continuous harvesting and freezing of the used medium. Purification of p97 was performed by immunoaffinity chromatography using Sepharose bound to Fab fragments of monoclonal antibody 96.5. For this purpose, three liters of the used medium were placed on a series of three 30 ml columns. The first contained 15 ml G-25 superfine Sephadex (Pharmacia), the second contained 20 ml Sepharose 4b (Pharmacia) and the third contained 8 ml cyanogen bromide activated Sepharose (Sigma), which was attached to Fab fragments of monoclonal antibody 96, 5 was bound (10 mg protein / ml Sepharose). The affinity columns were then washed extensively with cold PBS and the antigen was eluted with 30 ml 0.1 M citrate, pH 5 and 30 ml 0.1 M citrate, pH 4. It has been shown that these conditions do not change the immunoreactivity of the antigen while performing the entire elution of the antigen from the monoclonal antibodies 96.5. The two elutions were neutralized with 3.0 ml or 4.5 ml, 2 M-Tris, pH8. The purified eluate was concentrated using an Amicon apparatus with a PM10 filter and washed with two 10 ml volumes of PBS to give a final yield of 4.95 mg in 4.5 ml as described by the Bradford assay ( Biorad) was determined. 15 ng of the product were placed on SDS-PAGE (Figure 7) and both stained with Coomassie blue and silver dye. A double determinant immunoassay (DDIA) (Brown et al., 1981, Proc. Nati. Acad. Sci., 78: 539) provided independent confirmation of the molar quantity of the purified protein. In parallel, control preparations were prepared in the used medium of the B16 stem cell line, which contained no detectable protein. In a subsequent preparation, 30 mg 95% pure p97 protein was purified from 300 mg monoclonal antibody 96.5 Fab fragments conjugated with Sepharose. The purified p97 protein was immunogenic and produced a strong anti-body response in mice which were immunized with the protein as described below.

Construction and expression of the recombinant p97 vaccinia virus

The coding region of p97 was inserted by cutting p97a with HindIII, converting the ends to blunt ends and into the vaccinia insertion vector pGS-20 (Mackett et al., 1984, J. Viral. 49: 857- 864) was inserted, which was open at the Smal site. The pGS-20 vector uses the 7.5 K promoter and contains flanking sequences of the vaccinia thymidine kinase (TK) gene. A recombinant virus was generated by the method of Macket et al, suora, and Vp97a-NY was isolated, which causes infected cells to express and glycosilize p97 protein of the correct size (Figure 8). The surface expression of p97 was also confirmed in cells infected with the recombinant p97 virus below (Table I).

Table I

p97 surface expression on transferred mouse cells and cells which were infected with the recombinant vaccinia virus1

Cell type Virus used Expressed p97 molecules / cell

Cell infection

M2SVp97a.A

no

3,210,000

M2svp97a.E / F2

no

434,000

M2 strain none

2,000-

BSC

no

5590

BSC

Vwt-NY Vaccinia

5220

BSC

Vp97a-NY Vaccinia

1,140,000

1 The cells were briefly trypsinized, washed and placed in aliquots in tubes containing 103 to 104 or 105 cells. Non-expressing carrier cells were placed in the tubes with the lower number of cells so that there were a total of 105 cells per tube. 1 x 106 cpm of iodinated monoclonal antibody 96.5 (123 ng) was incubated, in a total volume of 50 ml, with the cells on ice for 60 min. The cells were washed and swirled four times in PBS + 10% fetal calf serum and then resuspended and counted in a Micromedic 4/600 pIus y counter (-) means less than.

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The recombinant p97 vaccinia virus is immununoaen in mice

Inoculation of mice with Vp97a-NY produced a strong humoral antibody response. The mice were immunized once and aftertreated once after 4 weeks, and blood was drawn after 5 weeks. The titers were determined by ELISA, using antigen-coated plates and a protein A, which was conjugated with horseradish peroxidase, as the detection reagent. The data were converted to monoclonal antibody equivalents by comparing to a standard curve for ELISA binding formed using Antip97 monoclonal antibodies 133.2. The results showed a strong induction of serum antibodies (Figure 9). Cellular immunity was demonstrated using an in vitro proliferation assay with purified p97 protein as the stimulation antigen (Table II).

Table II

Proliferation of mouse spleen cells1

Stimulating antigen proliferative. Index of vp97-re-proliferative index of combined immune spleen cells from control spleen cells

Con A

(10ng / ml) 71 50

p97 protein

(3ng / ml)

27th

2nd

(10 | ig / ml)

43

2nd

(20 ng / ml)

56

2nd

(50 ng / ml)

44

3rd

UV-inactivated vaccinia virus (107 pfu / ml)

91

2nd

p97 transfected irradiated syngeneic

86

2nd

Tumor cells (104)

Originally irradiated tumor cells (104)

3rd

1

1 Spleen cells were isolated from mice that were inoculated by tail scarification with 107 pfu Vp97a-NY-re-combinant virus and aftertreated with the same amount 1 month later and killed a week later. Native spleen cells were used to control the experiment. 105 cells were cultured per well in 96-well round bottom plates in 0.22 ml RPMI, to which 0.5% normal mouse serum, penicillin / streptomycin, glutamine, bicarbonate and 2.5 x 105 M2 mercaptoethanol were added. Cultures were butt-tagged with 25 (iCi / well ground thymidine (New Englang Nuclear) for 6 hours a day, harvested with a PHD cell meter and counted using Opti-fluor in a Beckman LS 3801 counter. The proliferation -Indices were calculated by dividing the mean of the number of pulses per minute of the four-fold holes stimulated with each antigen by the average of the number of pulses per minute of the control (medium).

The results, which are shown in Table II, show that the T cells proliferate in response to the p97 protein antigen.

To ensure that auxiliary cells are also stimulated by the recombinant viruses in spleen cells from immunized mice, the cells were stimulated in vitro and the supernatants tested for the production of interleukin-2 (IL-2), an auxiliary T cell factor. The spleen cells were cultured from mice which had previously been immunized twice with the recombinant Vp97aNY vaccinia or the original vaccinia. 105 cells were incubated for 48 hours in 0.2 ml medium identical to that used for the proliferation experiments in 96-well round-bottom plates in the presence or absence of the stimulation antigen. The supernatants were collected, pooled from four holes and frozen before the IL-2 test. The IL-2 test uses 104 mouse T cell line CTLL cells previously depleted of IL-2, with each well made up to three times the amount of click medium with different dilutions of the supernatant to be tested. A standard curve with recombinant IL-2 obtained from Genentech, CA was recorded. The CTLL cells were impact-labeled with thymidine according to the usual proliferation test techniques in the last 6 hours of the 24-hour incubation, then harvested and counted as described in the proliferation test procedures. The results, shown in Table III, show that IL-2 production on spleen cells from mice immunized with the recombinant p97 vaccinia virus is stimulated in vitro by p97.

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Table IH

Stimulation of the IL-2 product by p97-immune spleen lines

vaccine immunogen

In vitro stimulus

Manufactured IL-2,

units

Vp97a-NY

p97 protein (20 (ig / ml)

4.4

Vp97a-NY

medium

0.25

Vwt-NY

p97 protein (20 (ig / ml)

0.25

Vwt-NY

medium

0.25

Table IV

In addition, a delayed hypersensitivity response was measured using the footpad swell test in mice inoculated with Vp97aNY. Five mice (C3H / Hen strain) per group were inoculated by tail scarification with a recombinant or parental vaccinia virus strain.

6 days later, the hind paws of each mouse were challenged by inoculating 20 ixl PBS or 20 jxl cells in PBS (5 x 105 cells per mouse). The balls of the feet were measured 24 hours later with a double-blind sample using a Fowler micrometer. The foot pad thickness of the PBS-injected paws was subtracted from the measured thickness of the experimentally treated foot foot of each mouse, and means that the standard deviations of the increase in foot pad swelling were also calculated. The results are shown in Table IV and show the induction of a p97-specific delayed hypersensitivity response in mice which were immunized with the recombinant p97 vaccinia virus.

Table IV

Antigen-specific footpad swelling in p97-immune mice

Vaccine Challenges the Antigen Ball of Foot Swelling

Immunogen (mmx 10 ~ 2)

Vp97a-NY

p97-transfected syngeneic tumor cells

40.3 (+/- 6.8)

Vp97a-NY

original syngeneic tumor cells

3.0 (+/- 2.8)

Vwt-NY

p97-transfected syngeneic tumor lines

1.5 (+/- 2.3)

Vwt-NY

original syngeneic tumor cells

5.5 (+ 7-5.0)

Protection and Theraoie with D97 Vaccinia Vaccine in a Mouse Tumor Model

In order to determine the vaccination effectiveness, mice were vaccinated by various methods using the recombinant p97 vaccinia virus according to the invention and then challenged with a p97-transfected syngeneic tumor cell (M2SVp97a.2E). To this end, mice were immunized with Vp97a-NY recombinant live vaccinia virus or the original strain (Vwt-NY) by tail scarification; or immunized intraperitoneally in complete Freund's adjuvant with 100 ng of purified p97 protein or with 5 x 106 irradiated M2-K1735 tumor cells. An intravenous tumor cell challenge was created 2 weeks after the last vaccination by injection of M2SVp97a.2E, a metastatic tumor clone derived from M2-K1735 (a mouse melanoma model) by transfection with the human p97 coding sequence, which was expressed in an expression plasmid contained, which is driven by the early SV-40 promoter. A variety of expression clones were selected and the one used for a tumor challenge, the clone M2SVp97a.2E expresses a medium level of p97, about 400,000 molecules per cell, or an equivalent to human melanoma p97 antigen density . Two doses for intravenous tumor challenge were used, 5 x 105 or 1 x 105 cells, which were injected into the tail vein of C3H / Hen syngeneic mice. The mice were slaughtered 16 days after the tumor challenge and the lungs removed. A mouse was rated positive if tumors were visible on the lungs which were stained with India ink. The results are shown in Table V.

24th

5

10th

15

20th

25th

30th

35

40

45

50

55

CH 675 424 A5

Table V

Challenge of vaccinated mice with syngeneic p97-transfected melanoma cells Vaccine number Challenge number of mice with open

Immunizations Cell dose of visible lung metastasis

Vp97a-NY

2nd

5 x 105

1/5

Vp97a-NY

1

5x10s

2/4

Irradiated syngenetic

2nd

5 x 105

0/4

Melanoma cells

Vwt-NY

2nd

5x10®

9/10

p97 protein

2nd

5 X105

3/3

Vp97a-NY

2nd

1 x 105

0/1

Vp97a-NY

1

1 x 105

0/4

impartial

0

1 x 105

5/6

The results presented in Table V show that with two immunizations with Vp97a-NY there is a considerable protective effect, although no protective effect could be found with the purified p97 protein vaccine (despite the extremely high antibody titer evoked). The ability of the recombinant virus to elicit cellular immunity may account for its protective tumor immunity.

In a therapy experiment, mice were inoculated with a low dose of p97-expressing tumor cells and inoculated with the recombinant vaccinia vaccine 2 days later. The mice were challenged with 105 or 10 * p97 expressing tumor cells (M2SVp97a.E) intravenously. Two days later, the mice were inoculated by tail scarification with either Vp97a-NY or Vwt-NY. The weekly inoculations by tail scarification were repeated and the surviving mice recorded. The result is shown in FIG. 10 and shows the therapeutic effect of the recombinant p97 vaccinia virus vaccination in mice with existing lung metastasis.

The recombinant p97 vaccinia virus is immunogenic in Macau monkeys

Two Macaca fasicularis (Macaquen) monkeys were scarified either with 2 x 108 plaque forming units (pfu) or with Vp97a-NY recombinant vaccinia or with the same dose of the original Vacci-nia strain. Two weeks later, the sera were tested by ELISA for titer of vaccinia and p97. The results are shown in Table VI and show that the humoral antibodies against p97 were detectable 2 weeks after a single inoculation with Vp97a-NY.

Table VI

Serum antibody kills in vaccinia-inoculated monkeys

Immunogen / week anti-vaccinia titer (serum ver Arrti p97 trter (ng / ml monoclonal thinning with double background) antibody 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

Vwt-NY / week 2

1/2000

0.34

Deposit of microorganisms

The following E. coli strain. which carries the plasmid mentioned was deposited on December 27, 1985 with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, 20852-US, and bears the following deposit number:

E. coli strains

Plasmid

Deposit no.

E. coli HB101

p97b

53 403

25th

5

10th

15

20th

25th

30th

35

40

45

50

55

CH 675 424 A5

The following recombinant vaccinia virus was deposited with ATCC, Rockvilie, MD, on January 6, 1987 and received the following deposit number:

virus

Filing No.

Vp97a-NY

VR 2159

The following cell lines carry the plasmids listed and were also deposited with ATCC, Rock-ville, MD, and received the following accession numbers:

Cell line

Plasmid

Filing No.

deposited on

TKMp97-12 (mouse cells)

PMTp97b

CRL 8985

12/27/1985

B16SVp97a.14

pSVp97a

CRL 9304

6.1.1987

(Mouse melanoma cells)

The present invention is not restricted to the deposited microorganisms and cells, since the deposited embodiment only serves to explain one aspect of the present invention and all microorganisms or cells which are functionally equivalent are covered by the present invention. Indeed, various changes to the invention are possible, in addition to those shown and described herein, which is obvious to those skilled in the art. Such changes also belong to the invention, which is defined in the appended claims.

It is expressly pointed out that all base pair sizes which have been given for the nucleotides represent approximate values and have been given only for the purpose of the description.

Claims (11)

Claims 1. An essentially pure antigenic peptide or protein with the antigenic properties of the antigen associated with melanoma p97, characterized in that it comprises an amino acid sequence essentially according to Rgur 3 or an antigenic part thereof. 2. Recombinant virus, characterized in that its genome comprises a nucleotide sequence encoding an antigenic peptide or protein according to claim 1. 3. Recombinant virus according to claim 2, characterized in that it contains the vaccinia virus Vp97a-NY with the ATCC deposit no. VR 2159. 4. subunit vaccine formulation, characterized in that it contains as an immunogen an effective dose of a peptide or protein according to claim 1 in a mixture with a pharmaceutical carrier. 5. Vaccine formulation with inactivated virus, characterized in that it contains an effective dose of a recombinant virus according to claim 2 or 3 in the non-infectious state in a mixture with a pharmaceutical carrier. 6. vaccine preparation with live virus, characterized in that it contains the live virus according to claim 2 or 3 in a harmless dose. 7. Recombinant DNA vector for the production of a peptide or protein according to claim 1, characterized in that it contains p97b, deposited in E. coli under the ATCC no. 53403, pMTp97b, where p97b in E. coli under ATCC no. 53403 is deposited or comprises pSV2p97a with the restriction map according to marg. 6. 8. Bacterium Escherichia coli, characterized in that it contains the recombinant DNA vector according to claim 7, which comprises p97b, with the accession number ATCC 53403. 9. cell lines, characterized in that they contain the recombinant DNA vector pMTp97b or pSV2p97a according to claim 7. 10. Cell line according to claim 9, characterized in that it represents TKMp97-12 or B16SVp97a.14 and has the deposit number CRL 8985 or CRL 9304. 11. A method for producing a peptide or protein with the antigenic properties of the antigen associated with melanoma p97 according to claim 1, characterized in that all or part of the DNA sequence of p97 according to FIG. 3 is inserted into a vector and with this Vector host cells are transformed and the peptide or protein is obtained from the cultured cells which express the peptide or protein. 26