FI94836B - A method for producing an antigenic cognate peptide or protein of the melanoma-associated p97 antigen, and recombinant viruses, recombinant vectors and host cells useful therein - Google Patents

Description

1 94836

Method for Making an Antigenic Relative Peptide or Protein of the Melanoma-Related p97 Antigen, and Recombinant Viruses, Recombinant Vectors, and Host Cells Useful Therein 5 1. Field of the Invention

The invention relates to a method of producing an antigenic peptide or protein that is a related peptide or protein of the melanoma-associated p97 antigen. The invention also relates to a method of producing a recombinant virus that directs the expression of an antigenic cognate peptide or protein of the melanoma-associated p97 antigen in a bacterial or animal cell infected with the recombinant virus. In addition, the invention relates to the above.

Recombinant vectors and host cells useful in the production of 15 peptides or proteins.

Thus, in accordance with the invention, a melanoma-associated antigen-related peptide or protein is produced in large amounts by recombinant DNA methods. The peptide or protein obtainable according to the invention can be used as an immunogen in a vaccine formulation. In certain embodiments where the recombinant virus expresses a cognate peptide or protein of a melanoma-associated antigen, the recombinant virus itself may be used as an immunogen in a vaccine formulation.

The invention is illustrated by an example in which peptides closely related to the p97 antigen are used as immunogens, a monomeric cell surface sialoglycoprotein having an apparent molecular weight of slightly less than 97,000 daltons and which is a cell surface component of melanoma cells.

2. Background of the Invention 2.1 Tumor-Related Antigens

Work in experimental animals, especially rodents, has shown that most tumors caused by oncogenic viruses express antigens encoded by the viral genome and that immunization with these antigens may result in a rejection reaction upon subsequent exposure to tumor cells induced by the same virus. Although much of this work has been done with laboratory virus strains such as SV40 virus, polyoma virus, and mouse Friend, Moloney, or Rauscher leukemia viruses, horizontal and vertical transmission of naturally occurring oncogenic viruses has been demonstrated; in fact, a commercial vaccine against feline leukemia and sarcoma caused by the virus is now available.

However, the viral etiology of most human cancers has not been demonstrated. Important exceptions are hepatitis virus (liver tumor), herpes simplex virus (cervical cancer) and Epstein-Barr virus (nasopharyngeal cancer). However, over the past two decades, it has been shown that some human tumor cells have tumor antigens present, i.e., antigens that differentiate tumor cells from normal cells; some patients have cell-mediated or humoral immune responses corresponding to these antigens (Hellström et al., Nature 220 (1968) 1352, Morton et al., Science 162 (1968) 1279-1281, Shiku et al., J. Exp. Med. 144 (1976), 873-881). Some of the target antigens for these immune responses are oncofetal or differentiation antigens encoded by the human genome (Hellström et al., Int. J. Cancer 6 (1970) 346-351).

Until recently, the molecular nature of tumor antigens has not been known and the degree of tumor specificity of immunological reactions has not been clear. Attempts to • use this information to develop diagnostic methods for diagnosing cancer or treating cancer have for the most part failed. Because spontaneous tumor regressions are extremely rare, it can also be concluded that in vitro demonstrated immune responses were not affected in vivo: for example, although cancer patient antibodies and lymphocytes may effectively kill tumor cells in vitro, the same cancer patient does not have an effective immune response. in vivo.

5 The publication of methods using monoclonal antibodies by Köhler and Milstein (Nature 256 (1975) 495-497) led to an increased search for human tumor antigens, as the methods provided a means to determine these antigens both at the molecular level 10 and in terms of their specificity (Hellström and Brown). "The Antigens" (1979), edited by M. Sela, Academic Press, Vol. V, p. 166). Over the past several years, a large number of tumor-associated antigens have been described, most of which have been determined using mouse monoclonal antibodies. Reisfeld and Sell, eds., Monoclonal Antibodies and Cancer Therapy, UCLA Symposia on Molecular and Cellular Biology, New Series, Vol. 27, Alan R. Liss,

Inc., New York, 1985, p. 1-609. Although virtually all well-characterized antigens have been shown to be oncofetal or differentiation antigens and their tumor specificity has been found to be quantitative rather than qualitative, several antigens are specific enough for tumor cells compared to normal cells (usually 10-1000 times more specific). used as potential target cells in the identification of tumor cells as well as in treatment. It has also been possible to obtain human monoclonal antibodies to tumor antigens (Cote et al., Proc. Natl. Acad.

Sci. 80 (1983) 2026-2030). This supports the previously mentioned evidence that some cancer patients develop immune responses against their tumors.

More than half of the tumor-associated cell surface antigens identified to date are proteins or glycoproteins encoded by the human genome (rather than endogenous or exogenous viruses), with the remaining <94836 being glycolipids resulting from abnormal weathering of glycosyltransferases.

2.2 Melanoma-Associated p97 Antigen 5 The p97 antigen is a tumor-associated antigen that was identified from human melanoma tumor only using monoclonal antibodies (Brown et al., J.

Biol. Chem. 255 (1980) 4980-4983, Dippold et al., Proc.

Natl. Acad. Sci. USA 77 (1980) 6114-6118, Woodbury et al., 10 Proc. Natl. Acad. Sci USA 77 (1980) 2183-2187). The presence of the p97 antigen in normal tissues and tumor tissues has been extensively studied and is present in most human melanoma tumors and certain fetal tissues, but only in very small amounts in 15 normal adult human tissues (Brown et al., J. Immunol. 127 (1981) 539). 546, Brown et al., Proc. Natl.

Acad. Sci. USA 78 (1981) 539-543, Garriques et al., Int.

J. Cancer 29 (1982) 511-515). The p97 antigen has been used as a target antigen in the diagnostic imaging of melanomas in 20 human clinical trials (Larson et al., J. Clin. Invest.

72 (1983) 2101-2114).

p97 is a monomeric cell surface sialoglycoprotein with an apparent molecular weight (MP) of just under 97,000 daltons as measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Three important antigenic sites present in a stable 40,000 dalton fragment obtained by trypsin digestion have been determined using monoclonal antibodies (Brown et al., J. Immunol. 127 (1981) 539546); However, the 30 complete sequences of p95 have not yet been reported. At least two other independently characterized human melanoma-associated antigens, namely gp95 (Dippold et al., Proc. Natl. Acad. Sci. USA 77 (1980) 6114-6118) and gp87 (Khosravi et al., Int. J. Cancer 35 ( 1985) 73-80) 35 appear to be identical to p97 based on sequential immunoprecipitation analysis.

s 94836 The N-terminal amino acid sequence of p97 is homologous to transferrin and, as transferrin p97, binds iron (Brown et al., Nature (London) 296 (1982) 171-173). Analysis of somatic cell hybrids and in 5 situ hybridization has shown that the p97 gene, like the transferrin and transferrin receptor genes, is located in the chromosomal region 3q21-3q29 (Plowman et al., Nature (London) 303 (1983) 70-72; Yang et al. , Proc. Natl.

Acad. Sci. USA 81 (1984) 2752-2756). These findings suggest that p97 plays a role in iron metabolism.

2.3 Cancer vaccines

Studies in experimental animals, usually mice, have shown that immunization with live or killed cancer cells may result in a rejection reaction when exposed to live cancer cells after immunization. Attempts to immunize with cell-free material have generally not been as successful, although some successful experiments have been reported. (Overview 20 can be found in Hellström and Brown, in The Antigens, edited by M. Sela, Academic Press, Vol. V. (1979) 0 p.

1-66). In many cases, the target antigens that produce the protective effect have been encoded by viruses, but in many other cases, the nature of the antigen to elicit a protective immune response is unknown.

Studies in humans are much more difficult and the effectiveness of cancer vaccines is disputed, despite a few reports of success. In many cases, vaccine preparations have contained irradiated tumor cells or tumor cells that have been killed by exposure to certain chemicals. Because no pure human tumor-associated antigens are available, there are no reports of their use in vaccines.

An important theoretical objection to the proposed use of cancer vaccines in humans is that humans who have been "vaccinated" with 6,548,36 cancer cells or cell-free preparations, for example, do not develop an immune response because of the presence of small amounts of tumor antigens that may be the target of the immune response. in normal cells and thus the immune system recognizes them as "their own." Most, if not all, tumor-associated antigens in human tumors detected by monoclonal antibodies are also present in some normal tissues 10 and there is little evidence that cancer patients respond effectively in vivo. There is evidence that inhibitor cells play an important role in down-regulating the immune response against tumor antigens (Nepom et al., Experientia 39 (1983) 235-242). In addition, an inhibitor-cell response elicited by one group of tumor antigens may prevent the induction of an effective tumor-destroying response against another group of tumor antigens that does not cause suppression alone (Hellström et al., Biomembranes, A. Nowotny, ed.

20 Plenum Press. (1983) ss. 365-388).

2.4 Recombinant DNA Methods and Vaccine Virus The use of recombinant DNA methods in the production of vaccines for protection against inflammation from isolated components involves the molecular cloning and expression of genetic information encoding proteins that elicit an immune response against those proteins in a suitable vector. Recently, a new approach has been described that could potentially be used to produce vaccines prepared from separate components (Mackett et al., Proc. Natl. Acad. Sci. 79 (1982) 7415-7419, Mackett et al., J. Virol. 49 (1984) 857-864, Panicali, D. and Paoletti, E., Proc. Natl. Acad. Sci.

79 (1982) 4927-4931). In this approach, the vaccine virus is used as a vector to express foreign genes inserted into its genome. When the recombinant vaccine virus is introduced into host animals when it is introduced, it expresses the inserted foreign gene and thus raises the host's immune response against such gene products. Because the live combination vaccine virus can be used as a vaccine, this approach combines the advantages of both vaccines prepared from the individual components and vaccines containing live viruses.

The vaccine virus contains a linear, double-stranded DNA genome of approximately 187 kilobase pairs in length and replicates in the cytoplasm of infected cells. These viruses contain a complete system of transcriptional enzymes (including cap-forming and methyl and polyadenyl group-enhancing enzymes) necessary for its infectivity at the core of the virus.

The transcriptional regulatory sequences (promoters) of the vaccine virus allow the initiation of transcription of the vaccine-derived RNA polymerase, but not the host cell RNA polymerase.

Expression of foreign DNA in recombinant vaccine viruses requires the insertion of vaccine-derived promoters into the DNA sequences encoding the protein of the foreign gene. Plasmid vectors, also called splice vectors, have been constructed to insert chimeric genes into a vaccine virus. One type of splice vector consists of: (a) 25 vaccine virus promoters, including a transcription start site, (b) a number of unique restriction endonuclease cloning sites located downstream of the transcription start site, to add a foreign DNA fragment to the homologue, (c) to add a chimeric DNA fragment; a non-essential vaccine viral DNA flanking promoter and cloning sites targeting a non-essential region (such as the TK gene); and (d) a bacterial replication marker and an antibiotic resistance marker for replication and selection in E. coli. Mackett (Mackett et al., P. 54836 J. Virol. 49 (1984) 857-864) describes examples of such vectors.

Recombinant vaccine viruses are produced by transfecting bacterial recombinant plasmids containing a foreign gene into cells previously infected with the vaccine virus. Homologous fusion to infected cells occurs, resulting in the insertion of a foreign gene into the viral genome. Infected cells can be screened for recombinant viruses using immunological methods, DNA plaque hybridization, or genetic selection, after which they can be isolated. These vaccine combinators retain their essential functions and infectivity and can be engineered to contain about 35 kilobase pairs of foreign DNA.

Expression of a foreign gene can be detected by enzymatic or immunological assays (e.g., immunoprecipitation, radioimmunoassay, or immunoblotting). The natural membrane glycoproteins produced in cells infected with the combination vaccine are glycosylated and can be transported to the cell surface. By using strong promoters or cloning multiple copies of the same gene, abundant expression can be obtained.

3. SUMMARY OF THE INVENTION This publication describes vaccine formulations that can be used to induce an immune response that selectively destroys melanoma cells in vaccinated individuals. Vaccine formulations contain an immunogen that elicits an immune response against a melanoma-associated antigen, such as the melanoma-associated p97 antigen. Several vaccine formulations are possible. For example, the immunogen of a vaccine made from "isolated" components is a related peptide or protein of p97 and can be formulated with a suitable adjuvant. Such peptides or proteins contain amino acid sequences derived substantially or in whole or in part from the amino acid sequence of p97 depicted in Figure 3. Hereinafter, peptides or proteins closely related to the melanoma-associated p97 antigen, whether altered, unaltered, modified or unmodified, to be prepared in accordance with this invention will be referred to as "p97 related peptides". When the cognate peptide of p97 is a hapten (i.e., antigenic but not immunogenic), the hapten may be conjugated to a carrier molecule that renders it immunogenic.

Relative peptides of p97 can be produced by recombinant DNA methods and / or by chemical synthesis. When p97 related peptides are chemically synthesized, such synthetic p97 related peptides may contain 15 amino acid sequences derived from putative antigenic regions of p97 (Hopp and Woods,

Proc. Natl. Acad. Sci. USA 78 (1981) 3824-3828). When p97 related peptides are produced in accordance with this invention using recombinant DNA methods, a nucleotide sequence encoding all or part of p97 is inserted into a recombinant expression vector, such as a virus or plasmid, which in a suitable host can direct p97: n expression of a related peptide, which peptide can be purified from the culture medium. The added nucleotide sequence is derived from substantially all or part of the p97 sequence depicted in Figure 3. When a plasmid expression vector is used, a vector suitable for expression in eukaryotic cells is preferred, but a prokaryotic expression vector may also be used.

In another embodiment of the invention, wherein the expression vector is a recombinant virus, the vaccine may be formulated as a viral vaccine, wherein the immunogen is a recombinant virus that expresses a related peptide of p97. Depending on the nature of the recombinant virus used as the immunogen, either an inactivated viral vaccine or a vaccine containing live ίο 94836 viruses may be formulated. Proper immunization with ro-vaccine formulations may lead to the induction of an immune response, which in turn leads to the destruction of melanooro cells in the immunized person.

This publication also describes a system in which a vaccine formulation can be tested and outlines how testing should be performed. For example, the efficacy of vaccine formulations can be determined in animal models, first in rodents, then in monkeys, and finally in humans, preferably in patients in remission, but with a high likelihood of recurrence due to micro metastases.

4. Brief description of the figures

Figure 1 shows an autoradiogram of translation products separated by SDS-PAGE without p97-15 mRNA cells. In Figure 1A, lane 1 shows the translation products of p97 enriched mRNA, while lane 2 shows the translation products of unenriched mRNA, both obtained from 0.5 ml of a total of 5 ng of mRNA translation products. In Figure 1B, lane 1 shows translation products of p97-enriched mRNA, while lane 2 shows translation products of unenriched mRNA, both obtained from 5 ml of 5 ng mRNA translation products immunoprecipitated with anti-p97 serum.

Figure 2 shows in diagrammatic form the structure of p97 mRNA. The order of the coding region (from the signal sequence to the anchor sequence) and the uncoded region (3'UT) as well as the structure of the repeat region of the p97 precursor (open column) are marked. The recognition sequences of the various restriction enzymes are labeled above the mRNA. The relative positions of the four cDNA clones are labeled below the mRNA structure. cDNA clone p97-3a2fl (3a2fl) was isolated from a cDNA library in which cDNAs had been copied from oligo (T) sequence-initiated enriched mRNAs of p97 and cloned into pBR322; while cDNA clones p97-2fl (2fl), U 94836 p97-ljl (ljl) and p97-10al (10al) were isolated by initiating cDNA synthesis with oligonucleotides encoding p97 exon sequences and cloning the resulting cDNA fragments into lambda-gt10: C. Figure 2A shows in diagrammatic form the gen-5 clones B15, H17, B6.6 and E7.7 cloned in lambda-L47.1.

Figure 3 shows the nucleotide sequence of the human p97 precursor cDNA and the deduced amino acid sequence. The N-terminal amino acid residues previously determined by protein sequencing were identical to the residues predicted from the nucleotide sequence (amino acid residues Nos. 21-30). Possible glycosylation sites at amino acid residues 38, 135, and 515 (open column) and the region of attachment to the membrane at the C-terminus (packed column) are indicated. One polyadenylation signal (framed AATAAA) was detected at position 3847, located 50 base pairs upstream of the polyadenylated region.

Figure 4 shows the predicted amino acid sequences of the p97 precursor and the amino acid-20 subsequence of human serotransferrin (Yang et al., Proc. Natl. Acad. Sci. USA 81 (1984) 2752-2756, Davis et al., J. Mol. Biol. 181 (1985) 111-121) for comparison. The surviving remains are framed. Tyrosine, histidine, and arginine residues involved in iron binding of transferrin (Metz-Boutique et al., 25 Eur. J. Biochem. 145 (1984) 659-676) are marked with asterisks (*) ·

Figure 5 is a diagrammatic two-dimensional model of the structure of p97 based on the presence of conserved cysteine residues 30 among members of the transferrin large family. Three possible glycosylation sites are marked with asterisks (*). The hydrophobic membrane attachment region is shown at the C-terminus (COOH) of p97.

Figure 6 shows in diagrammatic form the following genetic structures: lambda E7.7 of genomic clone 12 S4836 used to generate the pDNA clones of p97 and the ex-35 expression vector of p97. fragment structure and pSV2p97a. The structure of the expression vector. The following abbreviations are used: E,

EcoRI: P, PvuII; Sai, Sali: S, SstI: B, BamHI.

Figure 7 shows the results of gel electrophoresis in the characterization and immunological purification of yh-5 recombinant p97 protein. The transfected CHO clone (CHO 3 +) and human melanoma SK-MEL 28 cells were labeled with 35S-cysteine in the presence (+ TM) or absence (-TM) of tunicamycin (TM). Cells were seeded in 10 mm 2 plates and allowed to grow to a nearly uniform cell mass. The medium was removed and replaced with 3 ml of cysteine-free medium with or without the addition of 1 μg / ml tunicamycin. After incubation for 30 minutes at 37 ° C, 250 μL / ml L-35S-cysteine (1016 Ci / mmol, New England Nuclear) was added for an additional 6 hours. Cell recovery, preparation of cell lysates, immunoprecipitation, and SDS-PAGE were performed as described above. Extracts were immunoprecipitated with antibodies specific for p97 and proteins were analyzed using SDS-polyacrylamine-20 gel electrophoresis (SDS-PAGE), (a) Coomassie

Blue-stained SDS-polyacrylamide gel. Lane 1, marker proteins; lane 2, p97 immunoprecipitated from transfected mouse B16 cells; lane 3, SK-MEL 28 cells + TM; lane 4, SK-MEL 28 - cells -TM; lane 5, CHO 3 + 25 cells + TM, lane 6, CH 3 O + cells -TM. (b) the same gel as in (a) autoradiogram.

Figure 8 shows radioimmunoprecipitation of p97 expressed in transfected cells or cells infected with Bp97a-NY. BSCs were infected overnight with wild-type vaccine virus or p97 combination vaccine virus. These viruses or the transfected cell line CHO-p97. A was incubated with 35S-labeled methionine and cysteine with or without 2 μg / ml tunicamycin (Sigma). After six hours, the cells were lysed, precipitated with monoclonal antibody 96.5 and separated by electrophoresis on a 10% polyacrylamide gel. Gel autoradiography was performed overnight. The group treated with tunicamycin is the rightmost in each group.

Figure 9 depicts serum antibody titers in mice immunized with p97 vaccines. Tp97 shows a group of five mice immunized with 5 x 10 6 irradiated M2svp97a.A cells (transfected tumor cells expressing surface p97 antigen) in Complete 10 Freund's adjuvant adjuvant and received the same number of cells as booster in phosphate buffered saline. saline. p97 is a group of five mice immunized with 100 μg of purified p97 protein in Complete Freund's adjuvant adjuvant and the 15 booster doses received contained 50 pq of aqueous protein solution. Vp97 is a group of five mice that were both immunized with 107 plaque-forming units of Vp97a-NY and received the same booster dose using the tail-cutting method.

Figure 10 depicts the therapeutic effect of vaccination with recombinant p97 vaccine virus (Vp97a-NY) in tumor-exposed mice. Mice were challenged intravenously with 105 or 104 p97-expressing tumor cells (M2SVp97a.E). After two days, mice were vaccinated with either Bp97a-NY or Vwt-NY with a tail-25 truncation method. Weekly vaccinations by the tail-cutting method were repeated and the survival of the mice was recorded.

5. DETAILED DESCRIPTION OF THE INVENTION The invention relates to a method for producing an antigenic peptide or protein that is a related peptide or protein of the melanoma-associated p97 antigen and has the amino acid sequence of amino acid residues 20-738 shown in Figure 3. The method of the invention is characterized in that a bacterial 35 or animal cell containing a nucleotide sequence encoding an antigenic peptide or 14,54336 protein is cultured under the control of another nucleotide sequence that regulates gene expression such that the cultured bacterial or animal cell expresses said peptide or protein. pep-5 tide or protein is recovered from the culture.

The invention relates to the production of vaccines for the prevention or treatment of melanoma. It is based on the finding that melanomas contain tumor-associated cell surface antigens, such as the p97 antigen, which are present in higher amounts in melanoma cells than in normal tissues. According to the present invention, melanoma-associated p97 antigen related peptides or proteins (i.e., p97 related peptides) are produced using recombinant DNA methods. According to the present invention, the p97 related peptides contain amino acid sequences derived from either substantially all or a portion of the p97 amino acid sequence shown in Figure 3. These include amino acid sequences derived from the sequences shown in Figure 3 that have been altered by substituting one or more amino acid residues in the sequence with another amino acid of similar polarity that is a functionally equivalent amino acid and thus results in an ineffective change. Amino acids that replace an in-sequence amino acid may be selected from other members of the class to which the amino acid belongs. Examples of non-polar (hydrophobic) amino acids are alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Polar, neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. In addition, the p97 related peptides obtainable according to the present invention, whether altered by amino acid residue substitutions or not by S4836, can be further modified or processed by glycosylation, phosphorylation, etc., or chemical modifications. These p97 related peptides can be used as immunogens in vaccine formulations that elicit an immune response against melanoma cells in a vaccinated patient.

According to one embodiment of the present invention, recombinant DNA methods are used to insert nucleotide sequences encoding the p97 antigen into expression vectors that direct the expression of p97 related peptides in appropriate host cells. The nucleotide sequences encoding the p97 antigen include nucleotide sequences derived from all or part of the p97 nucleotide sequence shown substantially in Figure 3.

Due to the degeneracy of the DNA code of amino acids (this means that most amino acids can be encoded by more than one codon), functionally equivalent codons (this means different codons encoding the same amino acid or an amino acid functionally equivalent to it) can be used. replace in the sequence of p97 shown in Figure 3 with the proviso that the substitution results in an ineffective change. Expression vector host cell systems containing the nucleotide sequence encoding all or part of the p97 antigen can be used to produce large amounts of pure cognate peptides of p97 in vitro, allowing the gene products to be purified from culture cells and used as immunogens in isolated components. Relative peptides of p97 can be purified using a variety of biochemical methods, including immunoaffinity purification using monoclonal antibodies. In addition, purification of p97 cognate peptides can be facilitated by modifying the DNA sequences encoding the p97 cognate peptides so that the 35 sequences responsible for attaching the protein to the 16 94836 plasma membrane are deleted without deleting those sequences responsible for transporting the protein to the cell membrane. to the culture medium of the antigenic molecule. In the case where p97 related peptides are produced in prokaryotic cells, the absence of appropriate post-translational modifications may result in an antigenically inactive product that may need to be activated using appropriate chemical or other treatments.

In certain embodiments where the expression vector is a virus, the virus itself may be formulated as a vaccine. In such cases, inactivated combination virus vaccines can be prepared. When the expression vector is formed from an infectious recombinant virus that does not cause disease in the host, either an inactivated viral vaccine or a vaccine containing live viruses that confers substantial immunity can be prepared. A particularly useful expression vector for this purpose is a recombinant vaccine virus that expresses related peptides of p97. In this case, a nucleotide sequence encoding all or part of the p97 antigen can be inserted into a vaccine viral vector capable of directing the expression of the sequence in a suitable host. The invention includes the use of other viral expression vectors as vaccines, more particularly the use of adenoviruses as vaccines.

Thus, the invention also relates to a method of producing a recombinant virus which directs the expression of an antigenic cognate peptide or protein of the melanoma-associated p97 antigen in a bacterial or animal cell infected with the recombinant virus. This method of the invention is characterized in that (a) the bacterial or animal cell is infected with a recombinant virus whose genome contains a nucleotide sequence encoding an antigenic cognate peptide or protein of the melanoma-related p97 antigen having the second nucleotide sequence shown in Figure 3; under the control of a nucleotide sequence that regulates gene expression, and (b) the recombinant virus is produced by propagating the infected cell.

The deduced amino acid sequence of p97 obtained according to the invention can also be studied to find sequences whose properties, in particular hydrophilicity, predict the presence of that sequence on the surface of the protein-10 molecule and its possible antigenicity and / or immunogenicity. These p97 related peptides can be chemically synthesized and used as immunogens in a vaccine formulation.

Relative peptides of p97 can also be used for purposes other than vaccine production. The p97 cognate peptide can be used to immunize animals to produce a specific antiserum or specific monoclonal antibodies against the melanoma cells of interest. These can be used as a component in a diagnostic assay or in the purification of affinity chromatography of radiolabeled drug or toxin-labeled antibodies that will be used in the treatment of cancer.

The invention is illustrated by examples illustrating the construction of a vaccine based on the p97 antigen against human melanoma. However, the methods and compositions described herein are not limited to the preparation of vaccines using p97, but can be applied to other tumor-associated antigens.

The invention is presented as follows for the sole purpose of clarifying the description: (a) the nucleotide and amino acid sequence of p97, (b) chemically synthesized p97 related peptides, (c) p97 related peptides produced in expression vector host systems, (d) immunological characterization of p97 related peptides of p97; and (e) formulation of vaccines.

5.1 Sequencing of the melanoma-associated p97 antigen The nucleotide sequence of the gene encoding the p97 antigen and the deduced amino acid sequence are depicted in Figure 3. Functionally equivalent sequences are within the scope of this invention. These include, but are not limited to, nucleotide sequences that contain all or part of the nucleotide sequence shown in Figure 10 3 and that have been modified by substitution with different codons encoding the same or functionally equivalent amino acid residue, resulting in an ineffective change, as well as amino acid sequences containing 3 of the amino acid sequence shown in whole or in part and which have been modified by functionally substituting the corresponding amino acid residues within the sequence to produce a non-active change, and modified, for example, by glycosylation, phosphorylation, etc., or by other chemical modifications.

The sections below describe the strategy used to determine the sequence of p97 shown in Figure 3, as well as alternative methods that could be used to sequence p97 or other tumor antigens useful in vaccine formulations.

5.1.1 Identification and Characterization of Melanoma-Associated p97 Antigen The activity and amino acid sequence of melanoma-associated p97 antigen are unknown, and therefore identification of p97 antigen was performed using a monoclonal antibody raised against p97. Several methods can be used to produce monoclonal antibodies specific for p97. For example, the hybridoma technique developed by Köhler 19 94836 and Milstein (Nature 250 (1975) 495-497) can be used as follows: mice or rats are immunized with human melanoma cells and lymphocytes recovered from immunized animals are fused with 5 myeloma cells, alternatively melanoma cells, melanoma cells Cote et al., Proc. Natl. Acad. Sci. 80 (1983) 2026, Has-pel et al., Cancer Res. 45 (1985) 3951) or a method for producing monoclonal antibodies using Epstein-Barr. virus, (Cole et al., The EBV-Hybridoma Technique and its Application to Human Lung Cancer, in Monoclonal Antibodies and Cancer Therapy, Alan R.

Liss. Inc. (1985) ss. 77-96) can be used to produce monoclonal antibodies raised against p97. In each case, the resulting hybridomas are screened to produce antibodies that bind to melanoma cells but do not bind to normal cells.

The monoclonal antibodies raised against p97 described above can be used in a number of ways to facilitate the identification, characterization, cloning, and expression of their nucleotide sequence to produce large amounts of p97 antigen-related peptides and proteins. For example, monoclonal antibodies can be used to further characterize the p97 antigen by radiolabeling all proteins produced by the tumor cell, immunologically precipitating the tumor protein with the monoclonal antibody, and fractionating the immunologically precipitated proteins by gel electrophoresis. Protein antigens are identified in the autoradiogram obtained as distinct bands (Brown et al., J. Biol. Chem. 255 (1980) 4980-3983). Furthermore, monoclonal antibodies raised against p97 can be used to facilitate cloning by: (a) immunological purification of polysomes to identify and recover mRNA transcripts encoding the p97 antigen in a melanoma cell; to identify clones expressing related peptides or proteins from a cDNA library, (c) to purify the p97 antigen for preparation of novel monoclonal antibodies, or an antiserum for use in the preceding two applications, or (d) to identify cells to which the p97 antigen has been exported transfected-10 racket.

Monoclonal antibodies can also be used to facilitate structural and immunochemical characterization of the p97 antigen, such as to identify extracellular and antigenic regions of the molecule and to purify the molecule for amino acid sequence analysis (Brown et al., Proc. Natl. Acad. Sci. USA 78 (1981) 539-543, Brown et al., Nature, London, 296 (1982) 171-173).

Upon further characterization of p97, its location in the cell is determined and antigenic determinants and functional regions are mapped. The intracellular location can be determined by immunofluorescence microscopy and cell fractionation experiments. Cell surface antigens, such as p97, are preferred for vaccine formation, although intracellular antigens may also be used. If multiple monoclonal antibodies are available, antigenic determinants can be mapped by competition experiments in which each antibody is radiolabeled and tested for 30 determinants of the determinant against each other antibody. Regions of the molecule can be identified using limited protease digestion followed by SDS-PAGE. All of this information should allow the most immunogenic regions of the molecule to be identified. If monoclonal antibodies are obtained by immunization with intact cells, these regions of the molecule are most likely extracellular and useful in vaccine formation.

Amino acid sequence analysis allows unambiguous identification of the protein and comparison of the protein with other proteins (Brown et al., Nature, London, 296 (1982) 171-173). If a protein contains more than 50 amino acid residues, it may be possible to determine only a portion of the amino acid sequence, most often the N-terminal amino acid sequence. For amino acid sequencing, the protein-10 antigen can be purified from cell lysates by immunoaffinity chromatography using monoclonal antibodies, followed by preparative SDS-PAGE. The N-terminal amino acid sequence of the purified protein is then determined using an automated amino acid sequencer, preferably a gas phase apparatus, to obtain maximum sensitivity.

5.1.2 Identification, cloning and sequencing of DNA encoding melanoma-associated p97 antigen

The first cloning studies focused on common proteins such as globin and ovalbumin, whose mRNA often comprised 10-50% of the total mRNA.

These mRNAs could be purified to homogeneity by size fractionation, and pure cDNA probes were used to screen libraries of a few hundred clones using colony hybridization. For proteins whose mRNA contains 1-10% of the total mRNA, differential hybridization can be used using two cCNA probes, in which method one cDNA probe contains the sequence of interest and the other is a negative control. Proteins present in small amounts, such as messenger RNAs encoding tumor-associated antigens, which may account for only 0.01% of cellular mRNA, are much more difficult to clone because there are tens of thousands of clones to be screened and no cDNA probes. give a specific hybridization signal. Both 22 9 4 8 3 6 problems can be reduced by enriching the mRNA of the sequence of interest.

Several methods used to clone DNA encoding the human melanoma-associated p97 antigen are described below. The resulting clones were analyzed to identify a clone or clones that contained the entire p97 coding region. The p97 nucleotide inserts of the clones thus identified can then be sequenced in any manner known in the art. These different approaches are described in more detail in private-10.

(a) Isolation of mRNA by immunological purification of the polysome In this method, polysomes (containing mRNA, ribosomes, and emerging polypeptide chains) are purified by immunoaffinity chromatography using antibodies that recognize antigenic determinants in the emerging chains. In many cases, monoclonal antibodies obtained by immunization with intact cells or cell extracts recognize antigens in their native conformations, but may not be suitable for immunological purification of polysomes, as it is very possible that the identified antigenic determinants are not present in the resulting chains. Because translation begins at the N-terminus of the polypeptide, epitopes close to the C-terminus are likely to lack most of the chains. This problem is avoided by either using antibodies that recognize N-terminal epitopes or by preparing polysomes from cells treated with an inhibitor of termination-inhibiting protein synthesis.

A major problem is that the mature proteins differ from the resulting chains due to post-translational modifications. This problem is particularly acute for cell surface proteins, which are more fully modified through the removal of signal peptides, the addition of carbohydrate side chains, and the formation of disulfide bridges. If a polyclonal antiserum is used for the immunological purification of polysomes, the differences in antigenicity between the chains formed and the mature protein may be minor, as during immunization the rabbit or other animal is exposed not only to the native protein but also to partially or completely denatured forms, especially if Freud's adjuvant is used. adjuvant. Although these antibodies represent only a small fraction of the antibody population, they may be present enough to bind the chains formed. Unfortunately, it is very difficult to prepare polyclonal antiserum for proteins present in small amounts. Although monoclonal antibodies can be used to purify antigen for other immunizations, only one microgram of antigen is often obtained per gram of cultured cells. This amount is sufficient to immunize multiple mice, but it is also sufficient to immunize a single rabbit.

Another solution to the problem is to produce a monoclonal antibody that recognizes antigenic determinants in the resulting chains, using the denatured p97 antigen as an immunogen in the preparation of a monoclonal antibody. In the example of this invention, a panel of monoclonal antibodies recognizing three distinct epitopes from the N-terminal region of the 40,000 dalton molecular weight molecule was used, and a mixture of monoclonal antibodies of varying specificity was used in the hope that one or more of them would bind to the resulting chains. The selected antibodies were highly p97-specific, with each of them immunologically precipitating p97 as a single band from a radiolabeled whole cell lysate, and had high binding affinities. Three IgG2a antibodies were selected for cloning project 24,94836, one specific for each of the three epitopes. In general, the chance of success can be increased by using multiple antibodies to distinct epitopes.

5 When using monoclonal antibodies, the question remains how to predict whether a particular monoclonal antibody or combination of antibodies will recognize the chains formed and thus be suitable for immunological purification of polysomes. One way to determine whether a monoclonal antibody 10 precipitates an antigen copied in a reticulocyte lysate system is based on the assumption that the in vitro translation product, unprocessed, resembles the chains formed. An alternative approach is to proceed with the immunological precipitation of polysomes on a small scale and then use in vitro translation to determine whether the mRNA species of interest is enriched.

When using the immunological purification method for polysomes, it is important to monitor purification by measuring mRNA activities. This can be done by copying the mRNA in a reticulocyte lysate system and analyzing the translation products by SDS-PAGE. Although the tumor-associated antigen may be too small a component of the unenriched mRNA translation products to be distinguished from the hundreds of more common species, it should be possible to detect it among the translation products derived from the enriched mRNA samples. Alternatively, the Xenopus early ovum system may be used if a sensitive immunoassay method is available to detect the replicated tumor-associated antigen. For p97, a highly sensitive dual determinant immunoassay (DDIA) assay was used for this purpose, utilizing two monoclonal antibodies specific for two different epitopes of p97.

25 9 4 8 3 6

Sepharose gel-bound protein A can be used for immunological purification of polysomes. The Protein A adsorbent has two applications in this method. A first application is to use it to purify monoclonal antibodies from crude ascites fluids, thereby removing contaminating ribonuclease activity. The protein A absorbent was then used with purified antibodies to immunologically purify polysomes carrying specific forming chains.

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

(b) Oligonucleotide Probes Another method that can be used in accordance with the invention to clone a cDNA encoding a tumor-associated antigen, such as p97, is to determine the partial or entire amino acid sequence of the antigen and synthesize an oligonucleotide based on the nucleotide sequence derived from the amino acid sequence. The oligonucleotide can then be used as a primer and probe for cDNA synthesis in screening the resulting cDNA library. Thus, melanoma-associated p97 protein can be readily purified from melanoma cell lysates by affinity chromatography using specific monoclonal antibodies (Brown et al. Nature, London, 296, (1982) 171-173). A nucleotide sequence encoding a portion of the determined amino acid sequence is then synthesized and used as a primer and / or probe. Most suitable for this purpose are portions of an amino acid sequence containing 30 amino acid residues encoded by one or two codons. One way is to synthesize a longer sequence, typically 25-60 nucleotides, that represents the most probable coding sequence as estimated from known codon frequencies in humans. The use of two synthetic oligonucleotides based on different parts of the amino acid sequence facilitates screening by identifying false positive hybridization signals. In addition, the use of hybridization conditions that minimize the effect of GC content on the melting point of the DNA hybrid also facilitates screening. Once one partial cDNA clone has been obtained by this method, it can be used as a probe to help obtain a full-length cDNA clone.

(c) cDNA Expression Libraries Cloning vectors have been developed that allow expression of a cDNA insert in bacteria. Thus, one approach that can be used to obtain cDNA clones of tumor-associated proteins such as p97 is to prepare a cDNA library as described above by reverse transcribing mRNA isolated from melanoma cells (enriched or unenriched) to the oligo (T) nucleotides described above. using primers or synthetic oligonucleotide primers and screening such a library with a monoclonal antibody raised against melanoma-associated p97 protein. Clones containing DNA encoding epitopes recognized by a monoclonal antibody in the correct orientation and reading frame expression express melanoma-associated p97 protein-related peptides or proteins and can be identified by transferring the antibodies expressed by the clones to the protein expressed by the clones. after which the filter is developed with a labeled anti-immunoglobulin reagent.

One potential problem is that many monoclonal antibodies do not recognize the protein expressed by bacteria, because in many cases the insert contains only a portion of the cDNA and the bacteria do not process the proteins like eukaryotic cells. This problem is particularly acute with tumor cell surface proteins that have been substantially modified to remove signal peptides by adding carbohydrate side chains and forming disulfide bridges. Thus, it may be necessary to generate monoclonal antibodies known to recognize a denatured antigen or to prepare a poly-5 specific antiserum by immunopurification with the antigen.

Once a recombinant virus or plasmid believed to contain a cDNA insert derived from the melanoma-associated p97 antigen has been identified, the cDNA insert 10 can be used to screen other libraries to either identify full-length clones or otherwise identify a group of clones spanning p97. full-length cDNA. The cloned cDNA can be identified by sequence analysis and by comparing the deduced N-terminal amino acid sequence to the amino acid sequence determined by direct analysis of the amino acid sequence of the p97 protein.

(d) Genome cloning

The following method allows DNA to be cloned using a monoclonal antibody raised only against an antigenic determinant present in the native protein, which determinant is not present in the resulting chains or in the bacteria in the expressed protein. In this case, DNA from human melanoma cell 25 is introduced into mouse L cells by transfection. Mouse cells expressing melanoma-associated p97 antigen are then isolated either using a fluorescence-activated cell sorter or by immunologically identifying colonies that produce p97 related peptides using radiolabeled monoclonal anti-p97 anti-p97 monoclonal antibodies. copies of colonies transferred on top of polyester fabric filters. Several consecutive rounds of transfection may be required to remove irrelevant human DNA sequences. The genomic library is then prepared in a lambda phage vector and screened for clones that contain human repeat sequences present in the introns of most genes. Once a genomic clone has been identified, it can be used as a hybridization probe to identify cDNA clones containing DNA encoding p97.

5.2 Synthesis and Determination of Immunogenicity of Melanoma-Associated P97 Antigenic Fragments Synthetic peptides can be used as immunogens to elicit an immune response against a native protein that can provide some protection against a variety of atogens. These amino acid sequences are selected from the known amino acid sequence of the protein antigen by identifying those amino acid sequences that are likely to be present on the surface of the protein molecule facing the external medium. This is most commonly accomplished by computer analysis of the amino acid sequence using the determined hydropathy parameters 20 of the amino acids. Additional criteria, such as predicted secondary structure or flexibility, may also be used.

Thus, synthetic peptides containing 5-50 amino acid residues of the melanoma-associated p97 protein can be tested for their immunogenicity in experimental animals (usually mice or rabbits). Such synthetic peptides include, but are not limited to, substantially all or part of the amino acid sequence shown in Figure 3, including altered sequences in which residues of the sequence have been functionally replaced with corresponding amino acid residues to give an inactive transformation and / or modified, modified or percentated sequences, etc. or chemically modified sequences. These p97 su-35 vesicle peptides are used either alone or coupled to a carrier protein such as volcanic conifer hemocyanin (KLH). In both cases, the use of an adjuvant is optional, although preferred. The immunized animals are given a booster dose and tested for antibodies raised against the peptide used for immunization. Animals with anti-peptide antibodies are tested for antibodies that bind native p97 protein. In the case of tumor-associated antigens, such as p97, it is also interesting to test a cell-mediated immune response, for example, by looking for delayed hypersensitivity (DTH), antigen-stimulated proliferation in vitro, cytolytic T cells, or tumor rejection in a suitable model. A suitable model could be a mouse tumor that expresses a human tumor-associated antisense as a result of transfection with an appropriate cDNA expression vector construct.

The goal is to identify peptides that elicit a strong immune response against the melanoma-associated p97 antigen. Once these peptides have been identified, they can be produced in large quantities by chemical synthetic methods known in the art. Alternatively, identified peptides can be produced in large quantities by expressing the nucleotide sequences encoding these peptides in expression vector host cell syntheses.

5.3 Generation of p97 related peptides using an expression vector host system

Proteins and peptides can be produced in large quantities by inserting the nucleotide sequences to be encoded into a suitable expression vector, which in turn is introduced into appropriate host cells, including, but not limited to, bacteria, yeast, insect cells, and mammalian cells. Although bacterial hosts have many advantages, they do not process many eukaryotic proteins properly and are not as suitable for the expression of tumor-associated proteins Express-35 as eukaryotic cells. However, recombinant proteins produced in bacteria can be used to induce T cell responses, as these responses are believed to require initial degradation of the protein antigen.

To express p97 related peptides in a vector host system, nucleotide sequences encoding the melanoma-associated p97 antigen or a portion thereof are inserted into a suitable expression vector. These nucleotide sequences include, but are not limited to, substantially all or part of the DNA sequence shown in Figure 3 10, including modified sequences in which one or more intronecond codons are replaced with a codon encoding the same or a functionally equivalent amino acid residue, resulting in a neutral or non-active 15 tos sequence. The expression vector contains the elements required for transcription and translation of the inserted protein coding sequence. The intensity and specificities of these elements vary. Depending on the host vector system used, any of a number of suitable transcriptional and translational elements may be used. For example, when cloning in mammalian cell systems, promoters isolated from the genome of mammalian cells (e.g., the mouse metallothionein promoter) or promoters isolated from viruses growing in these cells (e.g., the vaccine virus 7.5K promoter) can be used. Promoters produced by recombinant DNA methods or synthetic methods can also be used to effect transcription of added sequences.

Specific initiation signals are also required for efficient translation of the added protein coding sequences. These signals include the ATG start codon and adjacent sequences. When either a gene or a cDNA sequence is inserted into a suitable expression vector, but translation control signals are not necessarily required. However, in cases where only a portion of the 3i 94836 coding sequence is added, the ATG codon may need to be added, including external translation control sequences. In addition, the start codon must be in step with the reading step of the protein coding sequences to ensure translation of the entire insert.

These external translation control sequences and start codons can be derived from a variety of both natural and synthetic sources.

Any method known to those skilled in the art for inserting DNA fragments into a vector can be used to construct expression vectors containing a chimeric gene consisting of appropriate transcriptional and translational control signals and protein coding sequences. These methods may include in vitro recombinant DNA methods, synthetic methods, and methods used in in vivo recombination (genetic recombination).

Expression vectors include, but are not limited to, the following vectors and derivatives thereof: vaccine virus, adenoviruses, insect viruses, yeast vectors, bacteriophage vectors, and plasmid DNA vectors. Cloning and expression of genes in bacterial systems is well known in the art. For example, when cloning Escherichia coli, its bacteriophage or plasmid promoters such as the Lac promoter, the trp promoter, the recA promoter, the ribosomal RNA promoter, the coliform phage lambda PR and PL promoters, and other promoters can be used. but not limited to, lacuv5, trp-lacuv5 ftae) hybrid promoter, ompF. bla. Ipp and 30 correspond to the large-scale transcription of the adjacent DNA segment. However, due to processing differences between prokaryotic and eukaryotic cells, it may be advantageous to express c97 related peptides in eukaryotic cells. The best known methods for expressing proteins in eukaryotic cells include (a) introducing 32,94836 genes into a cell together with a drug resistance gene and subsequently selecting for the drug, preferably to effect amplification such as in a dihydrofolate reductase-methotrexate system, (b) 5 cD: in a plasmid divector using a strong eukaryotic promoter and other regulatory sequences, (c) frequent expression of cDNA in an SV40-derived viral vector again using strong promoters, in this case the SV40-10 promoter. Recombinant plasmid vectors are often used to produce cell lines that produce protein over a long period of time, while SV40 vectors are often used to produce transient expression. Although mammalian cells are most commonly used as hosts, insect cells and, in some cases, yeast cells may also be suitable. Some are described in more detail below.

To generate a recombinant vaccine virus expressing the melanoma-associated p79 antigen, the cDNA-20 sequence encoding can be ligated to the vaccine virus 7.5K promoter to form a chimeric gene. This chimeric gene is flanked by another vaccine virus sequence homologous to the viral thymidine kinase gene carried by the plasmid DNA vector. Generation of the chimeric gene: 25 involves the use of both natural and synthetic tumor-associated antigen sequence transcription and translation control signals. The chimeric gene is then introduced into vaccine virus expression vectors in both a plasmid vector and by in vivo recombination between a homologous thymidine kinase region in the vaccine virus genome. These recombinant viruses containing the chimeric gene are capable of directing the expression of related peptides of p97 in an infected host and can be used as components of a vaccine.

33 9 4 8 3 6

When an adenovirus is used as an expression vector, the DNA sequence of interest is ligated into an adenovirus transcription / translation control complex, e.g., the late promoter and Tripartite lea-5 derequence. This chimeric gene is then introduced into the adenovirus genome by in vitro or in vivo recombination. Addition to a non-essential region of the viral genome (e.g., region E1 or E3) results in a recombinant virus that is viable and capable of expressing the p97 cognate peptide in infected hosts. There are currently two adenovirus strains (types 4 and 7) that have been approved and used as vaccines for military personnel. They are the primary candidates for use as vectors for expression of the inserted DNA sequence.

An alternative expression system that could be used to express cognate peptides of p97 is an insect system. In one such system, Autographa californica muclear polyhedrosis virus (AnNPV) is used to express foreign genes in the vector. Vi rus grows in Spodoptera frugiperda cells. The DNA sequence of interest can be cloned into non-essential regions of the virus (e.g., the polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., the poly - ** 25 hadrin promoter). Successful addition of the DNA sequence results in inactivation of the polyhedrin gene and production of free recombinant virus (i.e., a virus lacking the protein envelope encoded by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed.

In addition, host cell strains can be selected that modulate the expression of the inserted sequences or modify and process the product of the chimeric gene in the desired specific manner. Expression from certain promoters 34,94836 may be increased in the presence of certain inducing agents (e.g., in the case of metallothionein promoters of zinc and cadmium ions). Thus, the expression of the genetically engineered protein can be controlled. This is important if the product of the cloned gene is to be killed on host cells. In addition, modifications (e.g., glycolysis, phosphorylation, etc.) and processing (e.g., cleavage) of protein products are important for protein structure and function. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins. Suitable cell lines or host systems can be selected to ensure proper modification and processing of the expressed foreign protein.

In this specific embodiment described for p97, the DNA sequence of p97 was ligated into an expression plasmid vector derived from pBR322 containing a metallothionein promoter. The entire coding sequence of p97, including the signal peptide and the membrane attachment anchor, was inserted into the vector.

5.3.1 Identification of recombinant expression vectors capable of replicating and directing the expression of p97 DNA sequences

Expression vectors containing foreign gene inserts can be identified using three general methods: (a) DNA-DNA hybridization, (b) presence or absence of marker gene functions, and (c) expression of inserted sequences. Using the first method, the presence of a foreign gene inserted into an expression vector can be detected by DNA-30 DNA hybridization with a foreign gene insert using probes containing sequences homologous. In another approach, a recombinant vector / host system can be identified and selected based on the presence or absence of certain "marker gene" functions (e.g., thymidine kinase activity, antibiotic resistance, phenotypic change, etc.) caused by the insertion of genes into the vector. For example, if a foreign gene is added to the marker gene sequence of a vector, recombinants containing a DNA insert can be identified by the absence of function of the marker gene. Using the third method, recombinant expression vectors can be identified by determining the foreign gene product expressed by the recombinant. These assays may be based on the physical, immunological or functional properties of the gene product.

For example, when constructing a recombinant vaccine virus in accordance with the present invention, a chimeric gene containing sequences encoding p97 is inserted into the thymidine kinase gene to inactivate the viral TK + phenotype and provide the virus with the TK_ phenotype. These recombinants are selected based on their ability to grow on TK + cells, but not on TK_ cells, in lethal media containing a 5-bromideoxyuridine nucleoside analog. Recombinants are further identified by DNA-DNA hybridization using a cDNA probe specific for the tumor-associated protein. TK recombinant virus can be isolated by plaque purification and strains are prepared from infected cultured cells. The recombinant virus can be tested for its ability to induce the synthesis of “25 p97 related peptides. In this case, infected cells can be grown in the presence of radiolabeled amino acids; then lysates and intracellular fractions of infected, radiolabeled cells are tested by immunological mixing with antibodies raised against native melanoma-associated p97 antigen. Immunologically precipitated products are separated by SDS-PAGE. Infected cells can also be tested by immunofluorescence using a monoclonal antibody.

36 94836

Cells into which the plasmid vector has been introduced by transfection can be readily identified by FACS analysis or by determining the binding of copies of the cell colonies to the polyester fabric. The amount of p97 su-5 peptide present can be determined by a quantitative radioimmunoassay method, and its intracellular location can be determined by cell fractionation and immunofluorescence microscopy. The structure of the expressed p97 related peptide can be determined by SDS-PAGE and by analyzing the amino acid sequence.

5.3.2 Purification of 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., J. Mol. Biol. 181 (1985) 111-121). When expressed in a suitable vector, the protein is expected to be transferred to the cell surface. To facilitate purification of the protein, it may be advantageous to delete the DNA sequence encoding the region of attachment to the membrane 20, whereby the mature protein is released into the culture medium.

The p97 cognate peptide can be purified from host cells by lysing the cells with a detergent and then performing affinity chromatography using monoclonal antibodies. If the protein to be purified from the culture medium is a blunt protein, it is preferable to use a serum-free medium and then to use affinity chromatography with monoclonal antibodies. It is important that the antigen can be eluted from the antibody adsorbent either without reducing its antigenicity or without denaturing it. This can be accomplished by raising or lowering the pH or using a chaotropic agent. It may be necessary to select a monoclonal antibody that releases the antigen under relatively mild conditions. The affinity purified antigen can be further purified by HPLC.

37 94836 5.4 Immunological characterization of related peptides of p97

The ability of a synthetic antigen or recombinant antigen to elevate an antitumor response can be assessed initially in experimental lives. This can be done by establishing a model system in which the human melanoma-associated p97 protein is expressed in cells of a suitable inbred strain of an experimental animal species. The animals are then immunized with the p97 cognate peptide using various immunization protocols, followed by testing for the development of antibodies to the melanoma-associated p97 antigen, the development of cell-mediated immunity against p97, such as delayed hypersensitivity, and their ability to repel p97. upon exposure to syngeneic tumor cells. In addition, in vitro cellular immunity assays can measure lymphocyte proliferation in response to a p97 cognate peptide and the ability of lymphocytes obtained from immunized animals or melanoma patients to kill tumor cells expressing p97 antigen. Further, by immunizing mice with mouse p97, one can determine the extent to which it is possible to induce an immune response to an antigen present in small amounts in normal tissues.

Monkeys can be used to determine the safety of p97 related peptides obtained in accordance with this invention. In this case, the animals can be immunized using immunization protocols that could be ethically applied to cancer patients, after which they can be tested as described above except that tumor transplants cannot be used, as these require the use of inbred strains. The safety of the immunization method is determined by observing the effect of immunization on the general health of the immunized animals (weight changes, fever, appetite, behavior, etc.) and observing 35 pathological changes at necropsy.

38 94836

Finally, the p97 related peptide obtained according to the present invention can be tested in cancer patients. Once the initial phase I testing has been performed in patients with advanced cancer to determine non-toxicity, patients in the re-5 mission phase with a high probability of disease recurrence could be tested. Their immune responses would be assessed in monkeys as described above except that the effect of treatment on pre-existing disease or recurrence rate 10 is examined. In the case of the melanoma antigen p97, benign eyelids (birthmarks) expressing the antigen are also examined.

5.5 Vaccine formation

It is an object of this embodiment of the invention to produce synthetically or by recombinant DNA methods a synthetic peptide, purified protein or recombinant virus that can be used as an immunogen and vaccine to protect cancer patients at high risk of recurrence, to treat existing disease and ultimately to treat individuals. with a high risk of developing the disease, as a preventive measure. In fact, the synthetic or recombinant melanoma-associated p97 antigen can be used in combination with other immunogens to prepare multifunctional vaccines for the prevention of melanoma and other cancers. Examples of different vaccine formulations are described below.

5.1.1 Viral vaccine formulations

When the p97 cognate peptide-30 di of the present invention is produced by a recombinant virus, either a recombinant virus vaccine containing live viruses or an inactivated recombinant virus vaccine can be formulated. The choice depends on the northwest of the recombinant virus used to express the p97 cognate peptide. When the recombinant virus is infectious to the 35 hosts to be immunized but does not cause the disease, a vaccine containing live viruses is preferred because amplification in the host results in a prolonged stimulus of the same type and magnitude as those produced by natural, asymptomatic infections. therefore considerable long-term immunity. When introduced into a host, an infectious recombinant virus can express a p97 cognate peptide from its chimeric gene and thus stimulate an immune response. The live recombinant virus as such can be used as a vaccine to prevent melanoma. The production of such a recombinant virus for use in these formulations may involve both in vitro (e.g., tissue culture cells) and in vivo (e.g., a natural host animal such as cows) systems. Conventional methods of preparing and formulating a smallpox vaccine can be applied to the formulation of a recombinant virus vaccine containing animal viruses.

Multifunctional live virus vaccines can be prepared together or from a few infectious recombinant viruses that express several antigens from 20 different tumor or cancer cells. For example, a vaccine virus (which may contain about 35 kilobase pairs of foreign DNA) can be made to contain sequences encoding other epitopes; such a recombinant virus as such can be used as an immunogen in a multistage vaccine. Alternatively, a mixture containing vaccine viruses and / or other viruses, each capable of directing the expression of a different gene encoded by different epitopes, may be formulated as a multi-acting vaccine.

Whether or not the recombinant virus is infectious to the host to be immunized, an inactivated vaccine formulation can be prepared. Inactivated vaccines are "dead" in the sense that their infectivity is usually destroyed by formaldehyde treatment. Ideally, the infectivity of the virus will be destroyed without affecting the capsular or envelope proteins carrying the immunogenicity of the virus. To prepare inactivated vaccines, large amounts of recombinant virus must be grown in culture to obtain the required amount of appropriate antigen. Mixture-5 containing various epitopes expressing, inactivated and viruses can be used in the formulation of "multi-acting" vaccines. In some cases, this may be advantageous over vaccine formulations containing live viruses due to the potential difficulties of interacting with co-administered live viruses. In either case, the inactivated recombinant virus or mixture of viruses should be formulated with a suitable adjuvant to enhance the immunological response to their antigens. Suitable adjuvants include, but are not limited to, mineral gels, e.g., aluminum hydroxide, surfactants such as lysolecithin, pluron polyols, polyanions, peptides, and oil emulsions.

Many methods can be used to administer the vaccine formulations described above. These include, but are not limited to, dermal, intramuscular, abdominal, intravenous, subcutaneous, or nasal routes of administration. When a recombinant virus vaccine formulation containing live viruses is used, it can be administered using the natural route of infection of the wild-type carrier virus used to prepare the recombinant virus in the vaccine formulation.

5.5.2 Vaccine Formulations Prepared from Isolated Components As an alternative to viral vaccines, the cognate peptide p97 as such can be used as an immunogen in vaccine formulations prepared from isolated components. Vaccines prepared from isolated components contain only the relevant immunogenic material required to immunize the host. So p97's su-! L 'IH? · Herring I I T in! The 4i 94836 peptide can be purified from recombinants expressing the peptide. These recombinants are any of the previously described virus-infected cultured cells, bacterial transformants, or yeast trans-5 formants.

Whether the p97 cognate peptides are purified from recombinants or chemically synthesized, the final product can be adjusted to an appropriate concentration and formulated with any suitable vaccine adjuvant and packaged for use. Suitable adjuvants include, but are not limited to, mineral gels, e.g., aluminum hydroxide, surfactants such as lysolecithin, pluron polyols, polyanions, peptides, and oil moles. The related peptide of p97 can also be combined with liposomes or conjugated to polysaccharides and / or other polymers for use in a vaccine formulation.

When the cognate peptide of p97 is a hapten, this means a molecule that is antigenic so that it can react selectively with related antibodies, but is not immunogenic because it is unable to elicit an immune response, the hapten can be covalently bound to a carrier, or an immunogenic molecule and the halene carrier molecule can be formulated for use as a vaccine; for example, a large protein, such as a serum albumin protein, renders the hapten coupled thereto immunogenic.

6. Example: Melanoma-associated p97 antigen

In the following example, cDNA clones from different regions of p97 were ligated together and inserted into an expression vector that directs expression of a related peptide of p97. Relative peptides of p97 produced by the expression vector host cell system can be formulated as a vaccine.

42 94836 6.1 Purification of p97 mRNA

Polysomes were prepared from SK-MEL28 melanoma cells (Carey et al., Proc. Natl. Acad. Sci. USA 73 (1976) 3270-3281) by magnesium precipitation. Polysomes carrying p97 chains formed from this preparation were purified by incubation on three separate epitopes of p97 (Brown et al., J. Biol. Chem. 255 (1980) 4980-4983, Brown et al., J. Immunol 127 (1981) 539-546, Brown et al., Proc. Natl.

Acad. Sci. USA 78 (1981) 539-543, Plowman et al., Nature, 10 London, 303, (1983) 70-72) with a specific IgG2a monoclonal antibody (96.5, 118.1, 133.2) and then subjected to affinity chromatography Protein A - Using Sepharose gel. p97-enriched mRNA was eluted with EDTA and purified by affinity chromatography using oligo (T) -15 cellulose (Bethesda Research Labs, Bethesda, MD). In a typical experiment, 150 E 2 units of polysomes produced 260 mg of p97 enriched mRNA, which accounts for 0.23% of total ismRNA. When p97-enriched mRNA was replicated in Xenopus early oocytes and determined for p97 as described (Brown et al., Proc. Natl. Acad.

Sci. USA 78 (1981) 539-543, Plowman et al., Nature, London, 303 (1983) 70-72), it produced 80 pg of p97 per ng of mRNA, while the unenriched mRNA of p97 produced only 0.44 pg of p97 per ng of mRNA, indicating that the mRNA activity of 25 p97 was enriched 180-fold.

The yield of p97 mRNA activity was 42%. Translation in the reticulocyte lysate system (Pelham & Jackson, Eur. J. Biochem. 67 (1976) 247-256) showed that the enriched mRNA of p97 encoded an important polypeptide with an apparent Mole-30 cubic weight of 84,000 daltons by SDS-PAGE. analyzed, which could not be detected from the translation products of the unenriched mRNA and which immunoprecipitated with antiserum specific for p97 (Fig. 1). It was concluded that this was a non-glycosylated precursor of p97.

43 94836 6.2 Preparation and generation of cDNA clones

The two methods described below were used to generate cDNA clones copied from the mRNA templates isolated above.

5 6.2.1 Generation of cDNA clones initiated with the oligo (T) sequence

The p97 enriched mRNA prepared above was used as a template in the synthesis of cDNA initiated with the oligo (T) sequence. The cDNA was cloned into pBR322 as follows: To synthesize the first 10 cDNA strands, p97 enriched mRNA, four dNTPs, and an oligo (T) sequence (Collaborative Research, Walthma, MA) were incubated with a reverse transcriptase enzyme (Molecular Genetic Resources). The second strand was synthesized by incubating a large fragment of E. coli DNA polymerase (Bethesda Research Labs,

Bethesda, MD) and the double-stranded cDNA was digested

S1 nuclease (gift from D. Durnam, The Fred Hutchinse Cancer Research Center, Seattle, WA). The cDNArha was then added using the dC-tail using terminal deoxynucleotidyltransferase-20 (Bethesda Research Labs, Bethesda, MK), ligated into PstI digested cG-tailed pBR322 (Bethesda Research Labs, Bethesda, MD) (Villa-Komaroff. Proc. Natl. Acad. Sci. USA 75 (1978) 3727-3731) and used to transform CaCl2-treated E.

25 coli RR1. DNA from colonies of transformed bacteria was fixed on paper (Taub & Thompson, Anal. Biochem. 126 (1982) 222-230) and screened by differential hybridization using cDNA probes synthesized from enriched and unenriched mRNA templates of p97.

A 243 bp (bp) clone, p97-3a2f1, was identified that hybridized to p97 enriched cDNA but not detectably with unenriched cDNA and that also selects for p97 mRNA in hybrid-selected translation experiments. (hybrid-selected transla- 35 tion experiments). The polyadenylation signal (AATAA) and the 44,94836 poly (A) region were located at the 3 'end of the cDNA (see Figure 2). Nick-translated p97-3a2fl hybridized 100-fold more strongly to p97 enriched mRNA than to unenriched melanoma mRNA and did not hybridize to detectable amounts of fibroblast mRNA. Northern blot analysis using cloned cDNA as a probe identified approximately 4 kilobases (ke) of mRNA present in SK-MEL28 melanoma cells but not in fibroblasts.

6.2.2 Cloning of the p97 genome and use of synthetic oligo-10 nucleotides to initiate cDNA synthesis

Attempts to obtain cDNA clones spanning more than 1 fence from the polyadenylation site were unsuccessful, possibly due to a region rich in (more than 80%) GC groups and having a remarkably large second-15 yard structure. To circumvent this problem, genome cloning was used. Four overlapping genomic clones were isolated from lambda-L47.1 libraries and contained size-fractionated SK-MEL28 DNA enriched for the specific p97 restriction fragment. These four genomic clones span 28 kb and contain the entire coding region of p97, including the regulatory region of the gene. The genomic clones arranged sequentially from 5 'to 3' are: lambda B15, lambda H17, lambda B6.6 and lambda E7.7. The name consists of • 25 letters referring to the restriction enzyme used to generate the fragment and a number indicating the size of the fragment cloned into lambda L47.1 in kilobases.

Thus, starting at the 5 'end, lambda clone B15 contains a 15 kb BamHI-p97 fragment. lambda clone H17 contains a 17 kb HindIII-p97 fragment, lambda clone B6.6 contains a 6.6 kb BamHI-p97 fragment and lambda clone E7.7 contains a 7.7 kb EcpRI-p97 fragment (see Figure 2A). Restriction fragments of clones hybridized to 4 kb p97-mRNA in Northern blot analysis were sequenced and p97-ek-35 sons were identified using a computer-aided predicted coding sequence and the amino acid sequence of human and chicken transferrin ( Yang et al., Proc. Natl. Acad. Sci. USA 81 (1984) 2752-2756 McGillivray et al., Proc. Natl. Acad.

5 Sci. USA 79 (1982) 2504-2508, Jetsch & Chambon, Eur. J.

Biochem. 122 (1982) 291-295).

Three synthetic oligonucleotides, the sequences of which were based on the exon sequences of the p97 genome, were used to initiate cDNA synthesis from SK-MEL28 mRNA, and the resulting cDNA was cloned into lambda-gt10 as follows: p97 cDNA was ligated to the dG and ligated to its oligonucleotide bridge (AATTCCCCCCCCCCCC) and lambda-gt10 treated with EcoRI restriction enzyme. The oligonucleotide bridges allowed the insertion and insertion of a cDNA sequence with a dG tail into the EcoRI site of lambda-gt10. Lambda phage were packaged (Grosveld et al., Gene 13 (1981) 227-237) and plated into E. coli strain CK-mK + hf1. The cDNA libraries in lambda-gt10 were screened for the p97 insert by plaque hybridization (Benton & Davis, Science 196 (1977) 180) using exon fragments of the genome as probes. The probes were labeled with 32 P-radioactive TTP (New England Nuclear, 3200 Ci / mmol) by nick translation to give a specific activity of 5-10 x 108 cpm / μg. Three partially overlapping cDNA clones (10α1, 11j, 2fl) spanning 2,368 nucleotides of p97 mRNA, including the entire coding region, were identified using exon-specific fragments of p97 as probes (Figure 2).

6.3 DNA Sequence Analysis of p97 The cDNA inserts were excised and subcloned into the plasmid vector pEMBL18 + (Dente et al., Nucleic Acids Res.

11 (1983) 1645-1655) in E. coli for subsequent propagation and restriction mapping. The cDNA was also subcloned into the cloning vector of 35 M13mp18 phages (Yanish-Perrone et al., «94836

Gene 33 (1985) 103-119) and sequenced using the Sanger dideoxy method (Sanger et al., Proc. Natl. Acad.

Sci. USA 74 (1977) 5463-5467). M13 clones containing large inserts were sequenced by making deletions using DNase I (Hong, J. Mol. Biol. 185 (1982) 539-549) or exonuclease III (Henikoff, Gene 28 (1984) 351-359) and using synthetic, 21-mer oligonucleotide primers.

The cDNA sequence of p97 is shown in Figure 3. An open reading frame of 10,214 nucleotides extends from the first ATG codon surrounded by a sequence consistent with the Consensus initiation sequence defined by Kozak (Kozak, Nucleic Acids Res. 8 (1980) 127-142). , To the TGA codon at position 2 215. The cDNA clone 15 at the 5 'end contains a further 60 nucleotides upstream of the ATG start codon. The non-coding region of the 3 'end of the p97 mRNA, which was not obtained as a cDNA clone, was identified as a simple genomic exon containing 1667 nucleotides. Residues 20-32 of the predicted amino acid sequence are identical to the known N-terminal amino acid sequence of 20 p97 (Brown et al., Nature, London, 296 (1982) 171-173), which identifies the cloned cDNA. In addition, the predicted molecular weight of the precursor is 80,196 daltons, which is in good agreement with the observed molecular weight of the in vitro translation product.

6.4 Construction of a recombinant expression plasmid containing the p97 coding sequence The large size of the p97 gene necessitated the ligation of the cDNA clones obtained by initiating the transcription of melanoma mRNA specifically by the reverse transcriptase enzyme. Three lambda-gt10 clones of the cDNA (10a1, lj1 and lfl; see Figure 2) were used, which contained a regulatory region from the signal peptide to the membrane attachment sequence. The p97 insert of clone 10α1 was cleaved by digestion with EcoRI and the oligo (dG) sequence at 47 '486 of the cDNA-10α1 was removed by digestion with exonuclease 111 to form clone 10alb with a HindIII site of 30 bp upstream of the starting methionine of the j> 97 preprotein. The p97 inserts of three cDNA clones, lnalb, ljl 5 and 2fl, and genomic clone E7.7 were ligated together into PvuII. SstI and EcoRI restriction sites and the plasmid vector pEMBL18 + (Dente et al., Nuc.

Acid Res. 11 (1983) 1645-1655) to HindIII-EcoRI sites as shown in Figure 2. The final construct, p97b, 10 contains a 4.4 kb p97 insert in the plasmid vector pEMBL18 +, which was used to transform E. coli HB101. The insert in p97b contains a 30 bp sequence of the 5 'non-replicated region of the p97 mRNA, the entire coding sequence, and a 3' untranslated region flanked by a 5 'HindIII site and a 3' EcoRI site. .

The 4.4 kilobase p97 insert was excised from p97b using HindIII and EcoRI and the ends were filled in using the E. coli DNA polymerase K1 enow fragment. The blunt-ended fragment was inserted into a single SmaI site in the eukaryotic 20 cDNA expression vector 1995.12 pUC13, a derivative of the vector mThGH-112 (Palmiter et al., Science 222 (1983) 809-14) obtained from Dr. Richard Palmiter (University of Washington, Seattle, Washington). This plasmid uses the mouse metallothionein promoter to express foreign genes in eukaryotic cells. The construct with the p97 insert in the correct orientation was identified by restriction analysis and named pMTp97b.

The recombinant plasmid was transfected into LMTK cells 30 and transfectants were selected in HAT medium based on growth. Clones picked from the transfected bat were allowed to propagate in 96-well microtiter plates, and the culture medium used and cell lysates of replica plate colonies were determined for p97 35 by a two-site immunoradiometric assay. The subclones were allowed to multiply and retested. Clone TKMp97-12, which expresses approximately 4,000,000 p97 molecules per cell, was grown, induced with cadmium, and used as a source of 5 p97 used for immunization.

6.5 Immunization of mice with p97 related peptides TKMp97-12 cells were grown, induced with cadmium and (14.4 g) lysed by incubation for 10 minutes on ice in 70 ml of TNEN buffer (20 mmol / l Tris-10 HCl, pH 8, 0.100 mmol / l NaCl, 1 mmol / l EDTA, 0.5% NP-40). The lysate was ultracentrifuged 20 0000 x g in 45 minutes at 4 ° C and half of the lysate was passed through p97 for specific, 1 ml of the immunoaffinity-finiteettipylvääseen (96.5 Fab fragment coupled to Sepharose 15 antibody gel). The immunoadsorbent was washed thoroughly first with TNEN buffer and finally with 20 mmol / l Tris-HCl buffer, pH 6.8.

For immunization, 0.5 ml of the immunoaffinity column material prepared as described above was mixed with 0.5 ml of 20 mmol / l Tris-HCl buffer, pH 6.8, and emulsified with 1 ml of Complete Freund's adjuvant. with. Each of the four BALB / c mice received 0.4 ml of peritoneal emulsion. After three weeks, the mice received a booster dose of a quarter of this amount of antigen in the Incomplete Freund's adjuvant. Control mice were immunized with immunoaffinity column material containing non-p97 antibody and otherwise treated in an identical manner. The blood of four mice immunized with p97 and two control mice was collected one week after the booster dose. Sera were tested for antibodies to p97 by immunoassaying with radioiodine-labeled SK-MEL melanoma cells followed by SDS-PAGE. The results 35 showed that the sera of four mice immunized with p97 immunoprecipitated p97, while the control sera were negative. Sera were also tested for antibodies to p97 using an ELISA assay using glutaraldehyde-fixed SK-MEL28 melanoma-5 cells (20,000 cells per microtest well). The fixed cells were incubated with 0.05 ml of 1 / 10,000 diluted serum for one hour at room temperature, washed and then incubated for one hour at room temperature with 0.05 ml of 10 horseradish peroxidase-conjugated goat anti-mouse cells.

With IgG (Southern Biotech). The sera of mice immunized with p97 (read at 490 nm) were 0.350, 0.243, 0.343 and 0 # 200, while the sera of the control sera were 0.036 and 0.057.

6.6 Characterization of p97 6.6.1 Structure of p97 The structure of p97 was determined from the amino acid sequence of the p97 precursor containing four structural readings.

Because residue 20 of the precursor sequence corresponds to the N-20 terminus of mature p97, amino acid residues 1-19 are likely to form a signal peptide, a conclusion supported by the length and hydrophobic nature of the peptide. Amino acids 20-361 and 362-713 form two homologous regions of 342 and 352 amino acids. Possible N-linked glycosylation sites 25 are located at positions 38 and 135 in the N-terminal region and at position 515 in the C-terminal region. Finally, it is believed that amino acids 714-738, a region containing predominantly uncharged or hydrophobic residues, attaches p97 to the cell membrane (Davis et al., J. Moll. Biol. 181 (1985) 30 111-121) and may extend into the cytoplasm.

Degradation experiments with protease support the regional structure of p97. Digestion of p97 with trypsin, papain (Brown et al., J. Immunol. 127 (1981) 539-546) or thrombin yielded a glycosylated, antigenic fragment with a Mole-35 having a basis weight of about 40,000 daltons. The fragment was purified to 94836 from a thrombin-digested mixture of p97 metabolically labeled with 35S-methionine or 3Scysteine and sequenced as described (Brown et al., Nature, London, 296 (1982) 171-713). Cysteine residues 5 were identified at positions 7 and 17 and methionine residues at positions 2 and 20. Identical results were obtained with intact p97 and the results obtained are in complete agreement with the N-terminal sequence of p97 predicted from the cDNA sequence. It is concluded that the protease resistant fragment with a molecular weight of 40,000 daltons corresponds to the N-terminal region of p97. We have not been able to isolate the C-terminal region of p97, possibly because it is sensitive to protease.

6.6.2 Homology of p97 with transferrin A search of the amino acid sequence library of 15 Protein Identification Resource (Release 5.0; Dayhoff et al., Nature, London, 290 (1981)) showed that p97 is remarkably homologous to three members of the transferrin large family. namely with human serum transferrin, 20 human lactotransferrin and chicken transferrin (37-39% homology, see Figure 4). Because human and chicken transferrin are 50% homologous to each other, p97 must have differed from human serum transferrin more than 300 million years ago. p97 25 has 14 cysteine residues located at homologous positions in each region. Human transferrin contains all of these cysteines at homologous positions in both regions, whereas human lactotransferrin and chicken transferrin lack only two of these cysteine residues (their C-terminal regions). Unlike p97, these proteins additionally contain 4-7 cysteines in their C-terminal regions that do not have a corresponding member in the N-terminal region. Human transferrin also contains 2 additional cysteines unique to its N-terminal region. The positions at which most di- 5i 94836 sulfides are present in human serum transferrin, lactotransferrin, and chicken transferrin have been determined directly (McGillivray et al., Proc. Natl. Acad.

Sci. USA 79 (1981) 2504-2508, MetzBoutique et al., Eur. J.

5 Biochem. 145 (1984) 659-676, Mazurier et al., Experientia (Basel) 39 (1983) 135-141, MacGillivray et al., J. Biol.

Chem. 258 (1983) 3543-3553, Williams et al., Eur. J. Biochem. 122 (1982) 297-303, Williams et al., Biochem. J. 141 (1974) 745-752). Thus, 10 disulfide bonds in each region of p97 can be predicted (see Figure 5).

The amino acid homology between regions of p97 (46% obtained by adding 9 residues to 7 holes) is more striking than the amino acid homology observed in human transferrin (43% 16 holes, 45 residues) or chicken transferrin (35% 12 holes, 49 15 residues). Based on extensive sequence homology between p97 and transferrin and cysteine conservation-based, seemingly similar compression patterns, we believe that if the current X-raya-20 knee of transferrin is poorly resolved (Gorinsky et al., 28, Nature, 197, London, 1979). ) can be made finer, it may be possible to deduce the three-dimensional structure of p97.

6.6.3 Function of p97 The membership of p97 in the transferrin large family, its ability to bind iron (Brown et al., Nature, London, 296 (1982) 171-173) and its common chromosomal location with transferrin and transferrin receptor (Plowman et al. , Nature, London, 303 (1983) 70-72, Yang et al.,

Proc. Natl. Acad. Sci. USA 81 (1984) 2752-2756) support all of its roles in iron transport. The iron binding pocket of transferrin is thought to contain 2-3 tyrosine, 1-2 histidine and one bicarbonate binding Arginine (Metz-Boutique et al., Eur. J. Biochem. 145 (1984) 659-676). The preservation of these amino acids in p97 supports 35 of its proposed role in iron metabolism (see Figure 52 94836). Because p97 is a membrane-bound, transferrin-like molecule and is not homologous to the transferrin receptor (Schneider et al., Nature, London, 311 (1984) 675-678), its role in cellular iron metabolism may differ from that of circulating serum transferrin and the role of the cellular transferrin receptor. Expression of the cloned p97 cDNA in eukaryotic cells allows testing of its functional properties.

6.6.4 Conclusion 10 From these data, it is clear that melanoma-associated p97 cDNA assemblies have been successfully obtained and can be efficiently expressed in mammalian cells to produce large amounts of antigenic p97.

15 7. Expression of cloned p97 and vaccine testing These detailed experiments describe the expression of cloned p97 protein and its vaccine testing. Expression of the p97 protein in secreted form (using the transfected mouse cell clone B16svp97a.14) allowed purification of the full-length p97 protein in milligrams. The protein in purified form was used to test for the induction of cellular immunity in vitro and to test its potential as a vaccine prepared from isolated components. The product of the p97 gene was also expressed on the cell surface of metastatic mouse melanoma cells, providing a model for testing the efficacy of vaccines to inhibit tumor growth in a syngeneic system.

The p97 gene was added to live vaccine recombinant virus for use as a vaccine formulation capable of generating effective cellular immunity. The ability of the recombinant vaccine virus Vp97a-NY to generate immunity in mice was assessed using several assays to demonstrate humoral and cellular immunity.

53,94836 Using the syngeneic mouse tumor model described above, the Vp97a-NY combination virus vaccine was shown to provide a protective effect on tumor cell exposure. The vaccine also had a therapeutic effect in mice with growing lung-5 colonies, a property analogous to the proposed use of the vaccine to elicit an immunotherapeutic anti-tumor response in melanoma patients already with tumors.

In addition to mouse studies, with only 91% homo-10 logs between human p97 and mouse homologous protein (for the regions studied so far), the Vp97a-NY vaccine has also been tested in monkeys. There is much closer homology between human p97 and the monkey form of the protein (inferred from cross-reactivity at the level of monoclonal antibodies). Due to the potential difficulty of developing an immune response against the "own" protein, a closely related Macaque monkey was used to test the immunogenicity of the Vp97a-NY vaccine. The combination vaccine virus was tested in monkeys and was shown to induce humoral immunity against p97. To date, monkeys have not shown any detectable symptoms of adverse side effects when exposed to the vaccine after receiving two vaccinations with a combination vaccine containing live viruses for six to 25 weeks.

7.1 Plasmid Expression The expression plasmid under the control of the SV2 40 early promoter sv2 was constructed from the cDNA plasmid clone p97a, which is similar to plasmid p97b except that the entire 3'UT region is in use (Figure 6). All cDNA clones were originally isolated from lambda-gt10 libraries using synthetic EcoRI-dG (9-17) linkers as previously described. The inserts were digested with EcoRI and subcloned into pEMBL18 + plasmid for subsequent propagation and characterization. Clone 10α1 was subcloned into M13mp18 and the 54,94836 RF form was digested with BamHI and Sghr, briefly treated with exonuclease III, blunt-ended with S1 nuclease, treated with the K1enow fragment, and reassembled. Several plaques were isolated and sequenced, one of which had removed the dG tail and retained the unreadable region of the 5 'end of p97 containing 33 bp added to the HindIII site of M13mp18. The RF of this subclone (10ala) was used to generate intact p97 cDNA; otherwise, all fragments were isolated from plasmid midaclones. The 550 bp HindiII-PvuII fragment of 10ala and the 735 bp PvuII-SalI fragment of ljl were isolated from LMP agarose gels and ligated into plasmid pEMBL18 + at the SalI and HindIII sites to give p5'p97.

The genomic clone E7.7 of the pEMBL18 + plasmid was completely digested with EcoRI and partially digested with SstI, and the 4.5 kb fragment was isolated by fractionation with 0.8% LMP-agarose. This 4.5 kb 3 'fragment was ligated into a 2 μl 404 bp SstI fragment and a 1 μl 535 bp BamHI-SstI fragment into the pEMBL18 + plasmid at the SalI and EcoRI sites to give p3'p97. The 1285 bp HindIII-SalI fragment of p5'p97 was then ligated into p3'p97 to give pp97a. The EcoRI partial HindIII fragment of this clone was inserted into the HindIII and EcoRI sites of pSV2neo plasmid (Southern et al., J. Mol. Appl. Genet. 1 (1982) 327-341). wherein the neomycin coding region and SV40-Splice / polyA sequences are eliminated, while the SV40 early promoter and 72 bp enhancer, 33 bp p97-5'UTR, p97 coding region completely, 3'UTR and 1.4 kb of 3 'end reu-30 DNA. The resulting plasmid was named pSVp97a.

The Sv2-directed plasmid was transfected into a number of eukaryotic cell lines by the calcium phosphate precipitation method, and the expressing cells were cloned and selected by co-transfecting a dominant, selectable marker. To perform this test, Chinese hamster (CHO) ovarian cells were cultured in Hank's F1 medium containing 5% fetal calf serum (FCS), 4 mmol / l L-glutamine, 1.3 mmol / 1 proline and antibiotics. B16 cells were cultured in RPMI medium containing 0.15% bicarbonate and 1735 cells in DMEM medium (Gibco), both supplemented with 15% FCS and antibiotics.

Cells were transfected using the modified calcium phosphate method (Wigler, M. et al., Cell 14 (1978) 725-731) 10 and using 20 Mg of either pSV2DHFR or pSV2neo plasmid, respectively, per plate. All plasmids were linearized at the EcoRI site. Persistent transfectants were selected using hypoxanthine-free (HAT) medium for CHOs cells or 0.5 μq / ml Geneticin (G418, Gibco) for B16-15 and 1735 cells. Surviving cells began to form visible colonies 7 days after transfection and were placed on sterile polyester filters attached with glass beads. The filters were kept in place for five days to allow the cells to grow into polyester mat-20 rice to form a copy of the colonies on the plate. The filters were then removed and used in a live cell binding assay using an iodine-labeled monoclonal antibody against p97. Ten milli-25 grams of labeled monoclonal antibody was incubated with up to 20 filters in 10 ml of FCS at 4 ° C for one hour. The filters were washed thoroughly in phosphate buffered saline (PBS), dried and exposed to XAR-5 film overnight at 30-70 ° C. The filters were then stained with 7% methylene blue to visualize cell colonies. In all cell lines used except cells expressing the mouse B16 cell line, the antigenic p97 protein contained their cell surfaces.

56 In cells transfected with B16, p97 was released into the medium, a finding characteristic of this cell type. Secretion of p97 allowed full-length purification of p97 protein from cellular medium. Clone B16SVp97a.

5 14 expresses approximately 4 μ9 / ιη1 p97 in the medium used.

Recombinant p97 was purified from the used medium of the transfected B16SVp97a.14 clone producing large amounts of p97 antigen in the medium. Cells were maintained at near confluency (109 cells) in 10,850 cm 2 spinner flasks by continuously adding small amounts of fresh medium. They continued to produce antigen without running out for weeks, allowing the spent medium to be continuously recovered and frozen. Purification of p97 was performed by immunoaffinity chromatography using 96.5 Fab fragments of monoclonal antibody mounted on Sepharose gel. In this case, three liters of spent medium were passed through three 30 ml columns connected in series. The first contained 15 ml of Sephadex-G25 (superfine grade, Pharmacia) gel, the second contained 20 ml of 20 Sepharose 4b gel (Pharmacia) and the third contained 8 ml of cyanogen bromide activated Sepharose gel (Sigma) conjugated to a monoclonal antibody. 96.5 Fab fragments (10 mg protein / ml Sepharose). The affinity column was then washed thoroughly with cold PBS and the antigen was eluted with 30 ml of 0.1 mol / l citrate, pH 5, and with 30 ml of 0.1 mol / l citrate, pH 4. These conditions were not observed. altering the immunoreactivity of the antigen, but the elution of the antigen from monoclonal antibody 96.5 was complete. The two eluates were neutralized with 3.0 ml and 4.5 ml, respectively, in 2 mol / L Tris, pH 8. The purified eluate was concentrated using an Amicon apparatus and a PM10 filter and washed with two 10 ml volumes of PBS: the final yield was 4.95 mg in 4.5 ml as determined by the Bradford Assay (Biorad). Of the 15 μg of product, 57,94836 was run on SDS-PAGE (Figure 7) and the gram was stained with both Co-massie Blue and silver staining. A dual-determinant immunoassay (DDIA) assay (Brown et al., Proc. Natl. Acad. Sci. 78 (1981) 539) confirmed the molar amount of pendant-5 independently purified protein. Control preparations were run simultaneously from the medium used for the B16 stem cell line and no protein was detected. In a subsequent preparation step, 30 mg of 95% pure p97 protein was purified using 300 to 10 mg of 96.5 Fab fragments of monoclonal antibody conjugated to Sepharose. The purified p97 protein was immunogenic and elicited a strong antibody response in mice immunized with the protein described in section 7.3 below.

7.2 Generation and expression of recombinant p79 vaccine virus The p97 coding region was added by cleavage of p97a with HindIII. by blunting the ends and inserting it into the vaccine insertion vector pGS-20 (Mackett et al., J. Virol. 49 20 (1984) 857-864) opened at the SmaI site ♦ The PGS-20 vector uses the 7.5 K promoter and contains flanked by the sequences of the vaccine thymidine kinase (TK) gene. The recombinant virus was generated by the above-mentioned method of Mackett et al., And Vp97a-NY, which causes infected cells to express the correct size and glycosylation protein p97 (Figure 8), was isolated. Cell surface expression of p97 was also confirmed in cells infected with recombinant p97 virus (Table I) below.

58 94836

Table I

Expression of p97 on the surface of transfected mouse cells as well as 5 cells infected with the combination vaccine virus1

expressed

For infection of cells p97 molecules Cell type__Virus used per cell_M2SVp97a.A No virus 3 210 000 10 M2svp97a.E / F2 No virus 434 000 M2 stem cell No virus 2 000- BSC No virus 5 590 BSC Vwt-NY vaccine virus 5 220 BSC Vp97 NY vaccine virus 1,140,000 15 'Cells were briefly trypsinized, washed and divided into tubes containing 103 to 104-10 s of cells. Unexpressed carrier cells were added to tubes containing smaller numbers of cells so that the total number of cells per tube was 105 cells. 1 x 106 cmp of iodine-labeled monoclonal antibody 96.5 (123 ng) was incubated in a total volume of 50 μΐ with cells for 60 minutes on ice. The cells were washed and centrifuged 4 times in a solution containing PBS and 10% fetal calf serum, after which the cells were resuspended and counted in a Micromedic 4 / 600plus gamma counter.

7.3 Recombinant p97 vaccine virus is immunogenic in mice Vaccination of mice with Vp97a-NY produced a strong humoral antibody response. Mice were immunized once, received a booster dose after four weeks, and their blood was collected after five weeks. Titers were determined by ELISA using antigen-coated plates and horseradish peroxidized conjugated Protein Aita as the detection reagent. The data were modified to correspond to the concentration of the monoclonal antibody by comparing them to a standard ELISA binding curve obtained using monoclonal antibody 5 against p97 133.2. The results showed that serum antibodies were strongly induced (Figure 9). Cellular immunity was determined using an in vitro proliferation assay using purified p97 protein as a stimulating antigen (Table II).

10

Table II

Proliferation Assay of Mouse Spleen Cells1 2 3 vp97 Recombinant Immune Per- Proliferation of Control Spleen Cell Proline Cells

Stimulating ferritic initerative antigen__exex__index_

Con A (10 mg / ml) 71 50 p97 protein 20 (3 mg / ml) 27 2 (10 mg / ml) 43 2 (20 mg / ml) 56 2 (50 mg / ml) 44 3 UV-inactivated 25 vaccine virus (10 pfu / ml) 91 2 p97 transfected irradiated syngeneic tumor cells (104) 86 2 30.

Irradiated stem tumor cells (104) 3 1 35

Spleen cells were isolated from mice vaccinated by the tail-tailing method using 107 pfu of Vp97a-NY recombinant viruses, which received the same booster dose after 60,94836 months and were sacrificed one week later. Native spleen cells were used as control cells in this experiment. 105 cells per well were cultured in 96-well round-bottom plates in 0.22 ml of RPMI medium supplemented with 0.5% normal mouse serum, penicillin / streptomycin, glutamine, bicarbonate and 2 .5 x 105 mol / l 2-mercaptoethanolil. Cultures were pulsed to be radiolabeled for six hours for four days with 25 μΟΙ / ^ ορρβ tritiated thymidine (New England Nuclear), harvested with a PHD cell harvester, and counted using an Optifluor on a Beckman LS 3801 counter. Proliferative indices were calculated by dividing the means of the cmp readings of the four parallel wells stimulated with each antigen by the mean of the cpm readings of the control (medium).

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

To determine whether the recombinant virus in the spleen cells of immunized mice also stimulated helper cells, the cells were stimulated in vitro and the supernatant was assayed for interleukin-2 (IL-2), a helper T cell factor. Spleen cells previously obtained from mice immunized twice with the recombinant Vp97a-NY vaccine or the parent vaccine were cultured. 105 cells were incubated for 48 hours in 0.2 ml of medium used in proliferation assays in 96-well round-bottom plates with or without stimulating antigen. Supernatants were collected, supernatants from four wells were pooled and frozen prior to IL-2 assay, 104 previously IL-2-free mouse T cell line CTLL cells were used in the IL-2 assay and incubated in each well with Click's Medium. with different dilutions of the supernatants of the assay method diluted in medium in triplicate. A standard curve was generated using recombinant IL-98036 2 from Genentech, CA. CTLL cells were labeled with radioactive thymidine according to the standard proliferation assay method during the last six hours of the 24-hour incubation period, after which they were harvested and counted as described in the proliferation assay methods. The results shown in Table III show that p97 stimulates IL-2 production in spleen cells of mice immunized with recombinant p97 in vitro.

Table III

Stimulation of IL-2 production by spleen cells immune to p97

Unit-produced immunogen In vitro stimulated IL-2_ 15 Vp97a-NY p97 protein 4.4 (20 μ9 / πι1)

Vp97a-Ny Medium 0.25

Vwt-NY p97 protein 0.25 * (20 μq / ml)

Vwt-NY Medium 0.25 1 2 3 4 5 6 7 8 9 10 11

Delayed hypersensitivity response 2 was further measured using the foot-pad 3 swelling assay in mice vaccinated with Vp97a-NY. A group of five 4 mice (C3H / Hen strain) were vaccinated using the tail-cutting method and a recombinant or strain vaccine virus.

6

Six days later, the hind paws of each mouse were exposed to 7 by inoculating 20 μΐ PBS or 20 μΐ cells in 8 PBS (5 x 10 5 cells per mouse). Foot paths 9 were measured after 24 hours by a double-blind method using a Fowler micrometer. The thickness of the footprint of the foot injected with PBS was subtracted from the thickness measured on the test foot in each mouse, and the biscuit values for the increase in swelling of the footprint as well as the standard deviations were calculated. The results shown in Table IV show that the p97-specific delayed hypersensitivity response was induced in mice immunized with the recombinant p97 vaccine virus.

Table IV

5 Antigen-specific swelling of the footpad in mice immunized with p97

Vaccination Foot swelling used for vaccination Exposure curve immunogenized antigen __ (mm x 10'2) _ 10 Vp97a-NY p97 transfected 40.3 (± 6.8) fed syngeneic tumor cells

Vp97a-NY syngeneic 3.0 (± 2.8) tumor cells

Sgenic tumor cells transfected with Vwt-NY p97 were 1.5 (± 2.3)

Vwt-NY syngeneic strain 5.5 (± 5.0) tumor cells 7.4 Protection and treatment with p97 vaccine virus in a rodent-20 tumor model

To determine the efficacy of vaccination, mice were vaccinated using different vaccination protocols and recombinant p97 vaccine virus prepared according to the invention, followed by exposure to a p97-transfected syngeneic tumor cell (M2SVp97a.2E). Mice were then immunized with live Vp97a-NY recombinant vaccine virus or strain virus (Vwt-NY) using the tail cleavage method or 100 μg of purified p97 protein or 5 x 10 6 irradiated M2-K1735 tumor cells with complete Freund's 30 adjuvant-adjuvanted. Intravenous tumor cell challenge was performed two weeks after the last injection of M2SVp97a.2E, a metastatic tumor clone prepared from M2-K1735 (mouse melanoma malal-35 li) by transfecting tumor cells with an early promoter of SV40-promoted SV40. human 63,94836 with its p97 coding sequence. Several expressing clones were selected and the tumor clone M2SVp97a.2E used to express a medium amount of p97, about 400,000 molecules per cell, i.e., as much as 5 human melanoma p97 antigen is expressed. Two intravenous tumor exposures were used with 5 x 10 5 or 1 x 10 5 cells injected into the tail vein of syngeneic C3H / Hen mice. Mice were sacrificed 16 days after tumor challenge and the lungs were removed. A mouse was judged to be positive if larvae stained with the naked eye were present in the lungs stained with India ink. The results are shown in Table V.

Table V 15

Exposure of vaccinated mice to syngeneic p97-transfected melanoma cells

Clear Pulmonary Exposure

Reading vaccine of cell mice administered in immune-stimulated tones__number dose__amount_

Vp97a-NY 2 5 X 105 1/5

Vp87a-NY 1 5 X 105 2/4 Irradiated 2 5 x 105 0/4 25 syngeneic melanoma cells

Vwt-NY 2 5 X 10s 9/10 p97 protein 2 5 x 105 3/3 ni 3 ° Vp97a-NY 1 x 10s 0/1

Vp97a-NY 1 1 x 10s 0/4 Treated 0 1 x 105 5/6 nond 35 The results shown in Table V show that the two Vp97a.NY immunizations had a significant protective effect on 64 94836, although no vaccine containing purified p97 was observed. protective effect (despite very high antibody titers). The protective tumor immunity of the recombinant virus may be due to its ability to enhance cellular immunity.

5 In a treatment experiment, mice were vaccinated with a low dose of p97-expressing tumor cells, followed two days later by a combination virus vaccine. Mice were challenged with 105 or 104 p97-expressing tumor cells (M2SVp97a.E) administered intravenously. Two 10 days later, mice were vaccinated with either Vp97a-NY or Vwt-NY using the tail truncation method. Weekly vaccinations using the tail-cutting method were repeated and mouse survival was recorded. The results shown in Figure 10 show that recombinant p97 vaccine-15 vaccination has a therapeutic effect in mice with lung metastases.

7.5 The recombinant p97 vaccine virus is immunogenic in Macaque monkeys

Two Macaca Fasicularis (macaque) monkeys were scratched with either 2 x 10 8 plaque forming units (pfu) of the Vp97a-NY combination vaccine or the same dose of carrier vaccine. After two weeks, serum vaccine and p97 titers were tested by ELISA. The results shown in Table VI show that humoral antibodies against p97 could be detected two weeks after a single Vp97a-NY vaccination.

65 94836

Table VI

Serum antibody titers in vaccine-injected mice 5 Anti-p97 titers

Anti-vaccine titer (/ xg / ml mono-ter (serum clonal vas-Immunogen / dilution with two substances per week in serum O) valent_

Vp97a-NY / week 0 1/20 0.54 10 Vp97a-NY / week 2 1/2000 6.54

Vwt-NY / week 0 1/20 0.50

Vwt-NY / week 2 1/2000 0.34 8. Deposit of microorganisms 15 The following E. coli strain carrying said plasmid has been deposited with the ATCC Depository, Rockville, MD, and has been assigned the following accession number: E. coli strains_Plasmid_Deposit number E. coli HB101 p97b 53 403 20 The following combination vaccine virus has been deposited with the ATCC Depository, Rockville, MD, and has been assigned the following deposit number:

Virus_Talletusnumero

Vp97a-NY VR 2159 The following cell lines carrying said plasmids have been deposited with the ATCC Depository, Rockville, MD, and have been assigned the following accession numbers:

Cellular Plasmid_Deposit Number TKMp97-12 PMTp97b CRL 8985 30 (mouse cell) P16SVp97a.14 pSVp97a CRL 9304 (mouse melanoma cell)

Deposited micro-organisms and cells is not intended to limit the scope of the present invention, since tal-35 letettu embodiment is intended only for one side 66 94 836 illustrate the invention and any microorganisms, or cells that are functionally equivalent are within the scope of this invention. In fact, in addition to the modifications presented and described herein, many modifications of the invention will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. These modifications are intended to fall within the scope of the attached requirements.

It is also understood that all exnase pairs of nucleotide 10 sizes are zoomed and have been used for imaging purposes.

il. uu.t miu lii * ·

Claims (21)

A method of producing an antigenic peptide or an antigenic protein derived from the melanoma-associated antigen p97 and having the amino acid sequence formed in FIGS. 3, characterized in that a bacterium or animal cell is cultured which contains a nucleotide sequence encoding the antigenic peptide or protein under control of another nucleotide sequence that regulates gene expression such that the cultured bacterial or animal cell expresses said peptide or protein, and the antigenic peptide or the antigenic protein is utilized from the culture. 15 2. A method according to claim 1, characterized in that the cultured cell is a bacterium. Method according to claim 2, characterized in that the bacterium is E. coli deposited at the depository ATCC with deposit number 53403. Method according to claim 1, characterized in that the cultured cell is an animal cell line. '25 5. A method according to claim 4, characterized in that the animal cell line is the mouse cell line TKMp97-12 deposited with the deposit institution ATCC with the deposit number CRL 8985. A method of producing a recombinant virus which controls the expression of an antigenic peptide or antigenic protein derived from the melanoma-associated antigen p97 in a bacterial or animal cell infected with said recombinant virus. (a) a bacterial or animal cell is infected with a recombinant virus whose genome contains a nucleotide sequence encoding the antigenic peptide or protein derived from the melanoma-associated antigen p97 and having the nucleotide sequence indicated in Figure 3; under the control of another nucleotide sequence that regulates gene expression, and (b) recombinant virus is produced by expression of the infected cell. 10 7. A method according to claim 6, characterized in that the virus is a envelope virus. Method according to claim 7, characterized in that the virus is a vaccinia virus. 9. A method according to claim 6, characterized in that the virus is a naked virus. Method according to claim 9, characterized in that the virus is an adenovirus. Method according to claim 6, characterized in that the virus is a baculovirus. 20 Method according to claim 11, characterized in that the virus is a nuclear polyhed-rosis virus. A method according to claim 6, characterized in that the virus is a bacteriophage. 25 Method according to claim 13, characterized in that the bacteriophage is a lambda phage. 15. Recombinant DNA vector, characterized in that it contains p97b, which has the structure shown in Figure 2. 16. Escherichia coli, which has been deposited with the depository ATCC with deposit number 53403. 17. Recombinant DNA vector, characterized in that it contains pMT97b which has been incorporated into the deposited cell line CRL 8985. 72 94836 18. The cell line TKMp97-12, which is deposited at the ATCC deposition institution with deposit number CRL 8985. 19. Recombinant DNA vector, characterized in that it contains pSVp97a, which has the structure shown in Figure 6. 20. Cell line B16SVp97a.14, deposited with the depository ATCC with deposit number CRL 9304. 21. Virus Vp97a-NY, deposited at the ATCC deposition institution with deposit number VR 2159.