AU717356B2 - Cellular immunogens useful as cancer vaccines - Google Patents

Description

WO 97/25860 PCT/US97/00582 -1- "CELLULAR IMMUNOGENS USEFUL AS CANCER VACCINES" Cross-Reference to Related Application Priority from U.S. provisional patent application No. 60/010,262, filed January 19, 1996 is claimed.

Field of the Invention The invention relates to the field of cancer vaccination and immunotherapy.

Background of the Invention A current goal of cancer research is the identification of host factors that either predispose to tumor formation or serve to enhance tumor growth.

Genes that confer the ability to convert cells to a tumorigenic state are known as oncogenes. The transforming ability of a number of retroviruses has been localized in individual viral oncogenes (generally v-onc).

Cellular oncogenes (generally c-onc) present in many species are related to viral oncogenes. It is generally believed that retroviral oncogenes may represent escaped and/or partially metamorphosed cellular genes that are incorporated into the genomes of transmissible, infectious agents, the retroviruses.

Some c-one genes intrinsically lack oncogenic properties, but may be converted by mutation into oncogenes whose transforming activity reflects the acquisition of new properties, or loss of old properties. Amino acid WO 97/25860 PCT/US97/00582 -2substitution can convert a cellular proto-oncogene into an oncogene. For example, each of the members of the c-ras proto-oncogene family (H-ras, N-ras and K-ras) can give rise to a transforming oncogene by a single base mutation.

Other c-onc genes may be functionally indistinguishable from the corresponding v-onc, but are oncogenic because they are expressed in much greater amounts or in inappropriate cell types. These oncogenes are activated by events that change their expression, but which leave their coding sequence unaltered. The best characterized example of this type of proto-oncogene is cmyc. Changes in MYC protein sequence do not appear to be essential for oncogenicity. Overexpression or altered regulation is responsible for the oncogenic phenotype. Activation of c-myc appears to stem from insertion of a retroviral genome within or near the c-myc gene, or translocation to a new environment. A common feature in the translocated loci is an increase in the level of c-myc expression.

Gene amplification provides another mechanism by which oncogene expression may be increased. Many tumor cell lines have visible regions of chromosomal amplification. For example, a 20-fold c-myc amplification has been observed in certain human leukemia and lung carcinoma lines. The related oncogene N-myc is five to one thousand fold amplified in human neuroblastoma and retinoblastoma. In human acute myeloid leukemia and colon carcinoma lines, the proto-oncogene c-myb is amplified five to ten fold. While established cell lines are prone to amplify genes, the presence of known oncogenes in the amplified regions, and the consistent amplification of particular oncogenes in many independent tumors of the same type, strengthens the correlation between increased expression and tumor growth.

Immunity has been successfully induced against tumor formation by inoculation with DNA constructs containing v-onc genes, or by inoculation with v-onc proteins or peptides. A series of reports describe a form of "homologous" challenge in which an animal test subject is inoculated with either v-src oncoprotein or DNA constructs containing the v-src gene. Protective immunity was induced against tumor formation by subsequent challenge with v- WO 97/25860 PCT/US97/00582 -3src DNA or v-src-induced tumor cells. See, Kuzumaki et al., JNCI (1988), 80:959-962; Wisner et al., J. Virol. (1991), 65:7020-7024; Halpern et al., Virology (1993), 197:480-484: Taylor et al., Virology (1994), 205:569-573; Plachy et al., Immunogenetics (1994), 40:257-265. A challenge is said to be "homologous" where reactivity to the product of a targeted gene is induced by immunization with the same gene, the corresponding gene product thereof, or fragment of the gene product. A challenge is "heterologous" where reactivity to the product of a targeted gene is induced by immunization with a different gene, gene product or fragment thereof.

WO 92/14756 (1992) describes synthetic peptides and oncoprotein fragments which are capable of eliciting T cellular immunity, for use in cancer vaccines. The peptides and fragments have a point mutation or translocation as compared to the corresponding fragment of the proto-oncogene. The aim is to induce immunoreactivity against the mutated proto-oncogene, not the wild-type proto-oncogene. WO 92/14756 thus relates to a form of homologous challenge.

EP 119,702 (1984) describes synthetic peptides having an amino acid sequence corresponding to a determinant of an oncoprotein encoded by an oncogenic virus, which determinant is vicinal to an active site of the oncoprotein. The active site is a region of the oncoprotein required for oncoprotein function, catalysis of phosphorylation. The peptides may be used to immunize hosts to elicit antibodies to the oncoprotein active site. EP 119,702 is thus directed to a form of homologous challenge.

The protein product encoded by a proto-oncogene constitutes a self antigen and, depending on the pattern of its endogenous expression, would be tolerogenic at the level of T cell recognition of the self peptides of this product. Thus, vaccination against cancers which derive from proto-oncogene overexpression is problematic.

Recent attempts have been made to induce immunity in vitro or in vivo to the product of the HER-2/neu proto-oncogene. The proto-oncogene encodes a 185-kDa transmembrane protein. The HER-2/neu proto-oncogene is overexpressed in certain cancers, most notably breast cancer. In each report WO 97/25860 PCT/US97/00582 -4discussed below, the immunogen selected to induce immunity comprised a purified peptide of the p1 8 5

HER-

2 "eu protein, and not a cellular immunogen.

Disis et al., Cancer Res. (1994) 54:16-20 identified several breast cancer patients with antibody immunity and CD4+ helper/inducer T-cell immunity responses to p1 8 5

HER

2 1 ne protein. Antibodies to p1 8 5

HER-

2 ne were identified in eleven of twenty premenopausal breast cancer patients. It was assumed prior to this work that patients would be immunologically tolerant to HER-2/neu as a self-protein and that immunity would be difficult to generate.

Disis et al., Cancer Res. (1994) 54:1071-1076 constructed synthetic peptides identical to p 1 8 5

HER-

2 neu protein segments with amino acid motifs similar to the published motif for HLA-A2.1-binding peptides. Out of four peptides synthesized, two were shown to elicit peptide-specific cytotoxic T-lymphocytes by primary in vitro immunization in a culture system using peripheral blood lymphocytes from a normal individual homozygous for HLA- A2. Thus, it was concluded that the pl85HER- 2 'ne" proto-oncogene protein contains immunogenic epitopes capable of generating human CD8 cytotoxic Tlymphocytes.

The cytotoxic T cells elicited in the latter report were not, however, shown to recognize tumor cells, but only targets that bound the synthesized peptides. Other work (Dahl et al., J. Immunol. (1996), 157:239- 246) has demonstrated that cytotoxic cells may recognize targets that bind peptide but fail to recognize targets that endogenously synthesize peptide. It is thus unclear whether the cytotoxic cells elicited by Disis et al. would be capable of recognizing tumor cells. In any event, no protection against tumor growth was demonstrated by Disis et al.

Peoples et al., Proc. Acad. Sci. USA (1995), 92:432-436, report the identification of antigenic pep.uais presented on the surface of ovarian and breast cancer cells by HLA class I molecules and recognized by tumorspecific cytotoxic T lymphocytes. Both HLA-A2-restricted breast and ovarian tumor-specific cytotoxic T lymphocytes recognized shared antigenic peptides.

WO 97/25860 PCT/US97/00582 T cells sensitized against a nine-amino acid sequence of one of the peptides demonstrated significant recognition of HLA-A2 HER2/neu tumors.

It remains unclear whether Peoples et al. have successfully attacked proto-oncogene-encoded self, as the immunizing peptide which is expressed in the tumor cells contained an isoleucine at position 2, whereas the peptide expressed in normal tissue contains valine residue at this position.

Moreover, although stimulation of T cells occurred in vitro, this stimulation does not represent a true primary immune response insofar as the starting T cell population represented tumor infiltrating lymphocytes.

The research accounts of Disis et al. and Peoples et al. required a form of in vitro stimulation, either priming as described by Disis et al., or restimulation as described by Peoples et al. The in vitro protocols of Disis et al. and Peoples et al. require a mutant cell line to aid in selection of the peptide which will serve to induce reactivity. Non-mutant, peptide antigen-presenting cells have their HLA class I molecules already loaded with endogenous peptides, a phenomenon which precludes exogenous loading from without. The value of the mutant lines is that they lack the TAP genes (encoding the transporters associated with antigen presentation). Class I binding of internallyderived peptides is significantly lowered, and "empty" class I molecules are present on the cell surface and available for binding of exogenously added peptides. This availability of peptide binding sites on membrane-bound class I allows examination of whether a given peptide will even bind to class I, and (ii) function as a target in cytotoxic T cell assays. However, the need for a mutant cell line for deduction of candidate immunizing peptide sequences limits the usefulness of peptide-based immunization schemes.

Fendly et al., J. Biol. Response Modifiers (1990), 9:449-455 present an account of a polypeptide-based immunotherapy. Purified polypeptide corresponding to the extracellular domain of the pl 85 HER-2neu protein was obtained from a transfected cell line. The purified peptide was employed in the immunization of guinea pigs. The immunized animals developed a cellular immune response, as monitored by delayed-type hypersensitivity. Antisera CD/99243026.2 6 derived from immunized animals specifically inhibited the in vitro growth of human breast tumor cells overexpressing p185 H E R 2 neu There is no indication by Fendly et al. of induction of self versus non-self reactivity. It is likely that the guinea pigs were chiefly responding to non-self determinants (as defined in terms of the guinea pig host) on the human polypeptide immunogen.

The use of peptides for immunization is of necessity limited to immunization with a single haplotype. There are approximately thirty HLA types in man. In each case of peptide immunization, one must be careful to select peptides which match the host HLA type. The selected peptide must be immunogenic in the host and be capable of presentation to host immune system cells.

What is needed is an immunization method for immunizing humans and animals against self-encoded proto-oncogenes which are associated with the development of cancer, which dispenses with the need for isolating immunogenic, HLA host-matched peptides for immunization.

15 Summary of the Invention In one aspect, the invention relates to induction of reactivity to selfdeterminants of the product of an overexpressed proto-oncogene.

e In a second aspect, the invention provides a form of therapy or prophylaxis based upon the capacity to induce immune reactivity to proto-oncogene-encoded 20 self as overexpressed in tumor cells.

In a third aspect, the invention provides a cellular immunogen for use in immunization against self proto-oncogene determinants.

In a fourth aspect, the invention provides a method for vaccinating a host against disease associated with the overexpression of a proto-oncogene.

These and other aspects of the invention will be apparent from the following disclosure.

CO/99243026.2 6a A method of vaccinating a host against disease associated with the overexpression of a target proto-oncogene is provided. The method comprises:

S

a.

S S S S

S

S S S S S S

S

S

S S *5 WO 97/25860 PCT/US97/00582 -7excising cells from the host; transfecting the excised cells with at least one transgene construct.comprising at least one transgene cognate to the target proto-oncogene and a strong promoter to drive the expression of the transgene in the transfected cells, the transgene encoding a gene product which induces host immunoreactivity to host self-determinants of the product of the target proto-oncogene gene; returning the excised cells transfected with the transgene construct to the body of the host to obtain expression of the transgene in the host.

According to one principal embodiment of the invention, the transgene comprises wild-type or mutant retroviral oncogene DNA. According to another principal embodiment of the invention, the transgene comprises wildtype or mutant proto-oncogene DNA of a species different from the host species. Where the transgene comprises mutant retroviral oncogene DNA or mutant proto-oncogene DNA, the mutant DNA is preferably nontransforming.

The mutant DNA preferably comprises a deletion mutation in a region of the DNA which is essential for transformation. Preferably, the host cells are transfected with a plurality, most preferably at least five, different transgene constructs, each construct encoding a different deletion mutation.

In one preferred embodiment of the invention, the mutant DNA has at least about 75% homology, more preferably at least about homology, most preferably at least about 90% homology, with the corresponding wild-type oncogene or proto-oncogene DNA.

The invention is further directed to a cellular immunogen for immunizing a host against the effects of the product of a target proto-oncogene, the overexpression of which is associated with a cancer. The cellular WO 97/25860 PCT/US97/00581 -8immunogen comprises the host cells which have been transfected with at least one transgene construct, as described above.

The invention is also directed to a method of preparing the cellular immunogen, by excising cells from the host, and transfecting the excised cells with at least one transgene construct, as described above.

The cells transfected with the transgene are preferably rendered non-dividing prior to return to the body of the host.

The term "corresponds to" is used herein to mean that a polynucleotide sequence is homologous is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence.

The term "cognate" as used herein refers to a gene sequence that is evolutionarily and functionally related between species. For example but not limitation, in the human genome, the human c-myc gene is the cognate gene to the mouse c-myc gene, since the sequences and structures of these two genes indicate that they are highly homologous and both genes encode proteins which are functionally equivalent.

By "homology" is meant the degree of sequence similarity between two different amino acid sequences, as that degree of sequence similarity is derived by the FASTA program of Pearson and Lipman, Proc.

Natl. Acad. Sci. USA (1988), 85:2444-2448, the entire disclosure of which is incorporated herein by reference.

As used herein, the term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.

The word "transfection" is meant to have its ordinary meaning, that is, the introduction of foreign DNA into eukaryotic cells.

CD/99243026.2 9 By "transgene" is meant a foreign gene that is introduced into one or more host cells.

By transgene construct" is meant DNA containing a transgene and additional regulatory DNA, such as promoter elements, necessary for the expression of the transgene in the host cells.

The invention preferably also includes but is not limited to:- Use of a cellular immunogen in the preparation of a vaccine for immunizing a host against the effects of the product of a target proto-oncogene, the overexpression of which target proto-oncogene is associated with a cancer, comprising: excising cells from the host; transfecting the excised cells with at least one transgene construct comprising at least one transgene cognate to the target proto-oncogene and a strong promoter to drive the expression of the transgene in the transfected cells, 15 the transgene encoding a gene product which induces host immunoreactivity to host self-determinants of the product of the target proto-oncogene gene.

Use of cells transfected with a transgene construct in the preparation of a vaccine for vaccinating a host against disease associated with the overexpression of a target proto-oncogene comprising 20 excising cells from the host; •o a *e transfecting the excised cells with at least one transgene construct comprising at least one transgene cognate to the target proto-oncogene and a strong promoter to drive the expression of the transgene in the transfected CD/99243026.2 9a cells, the transgene encoding a gene product which induces host immunoreactivity to host self-determinants of the product of the target proto-oncogene gene; returning the excised cells transfected with the transgene construct to the body of the host to obtain expression of the transgene in the host.

Use of transfected cells in the preparation of a vaccine for vaccinating a host against disease associated with the overexpression of a targeted proto-oncogene comprising: excising cells from the host; transfecting the excised cells with at least one transgene construct comprising at least one transgene and a strong promoter to drive the expression of the transgene in the transfected cells, wherein the transgene comprises wild-type or mutant cognate retroviral oncogene DNA; or wild-type or mutant cognate proto-oncogene DNA of a species different from the host species; 15 returning the excised cells transfected with the transgene construct to the body of the host to obtain expression of the transgene in the host.

Description of the Figures i Fig. 1 is a plot of the mean tumor diameter over time following subcutaneous wing web inoculation of 1-day-old line TK (panel A) and line SC 20 (panel B) chickens with 100 gg of tumorigenic plasmids pcsrc527 pVSRC-C1 or pMvsrc The mean tumor diameter (mm) at a particular time point and for any one group of TK or SC line chickens inoculated CD/99243026.2 9b was computed as the sum of the diameters of the primary tumors divided by the number of chickens surviving to that point. The ratios at each time point show, for a particular group, the number o chickens bearing palpable tumors to the total number of survivors to that point (standard typeface for pcsrc527, italics for pVSRC-C1, bold typeface for pMVsrc). Error bars (unless obscured by the symbol) indicate standard error.

Fig. 2 is a plot of the growth of challenge (wing web) tumors in test and control line TK chickens under conditions of priming and homologous challenge with plasmid pcsrc527 (panel A: test; control), or (ii) priming and homologous challenge with plasmid pVSRC-C1 (panel B: test; control). Test chickens were primed at 1 day posthatch with 100gg of construct; test and control chickens were challenged at five weeks posthatch with 200pg of construct, the mean challenge diameter was computed as in Fig. 1. At each time point the ratio of chickens bearing palpable challenge tumors to total number of survivors to that point is indicated (standard typeface for control group, bold i typeface for test group). The statistical comparison between the mean challenge tumor diameters of the test versus the control group at particular time point was made using a two-tailed student's t test, (p<0.001). The statistical 0 WO 97/25860 PCT/US97/00582 comparison between the ratios of chickens bearing palpable challenge tumors to total number of survivors of the test versus the control group at a particular time point was made using a chi-squared test; the paired ratios are underlined for only those time points where p<0.05. Error bars indicate standard error.

Figs. 3A and 3B are plots of the growth of challenge (wing web) tumors in TK chickens under conditions of priming with plasmid pVSRC-C1 and heterologous challenge with plasmid pcsrc527 (Fig. 3A: test; control) or (ii) priming with pcsrc527 and heterologous challenge with pVSRC-C1 (Fig. 3B: test; control). Test chickens were primed at 1 day posthatch with 100 Ag of construct; test and control chickens were challenged at five weeks posthatch with 200 1g of construct. The mean challenge tumor diameter was computed as in Figs. 1A and lB. At each time point the ratio of chickens bearing palpable challenge tumors to total number of survivors to that point is indicated (standard typeface for control group, bold typeface for test group). Statistical comparisons were made between test and control groups at a particular time point as described for Figs. 2A and 2B.

for the student's t test], and the paired ratios are underlined for only those time points where, in the chi-squared test, p <0.05. Error bars indicate standard error.

Detailed Description of the Invention A vaccination strategy is provided to prevent development of cancers. The vaccination method may be carried out on a subject at risk for a particular cancer, but before the development of the cancer. The practice of the invention may serve for the immunoprevention of prevalent human cancers, such as colon carcinoma, breast carcinoma, and various lymphomas whose progress is accompanied by the overexpression of a cellular proto-oncogene.

The vaccination strategy of the present invention relies on the induction of an immune response that targets tumor cells by virtue of the recognition of the proto-oncogene-specific antigenicity. The aim of the vaccine protocol is to induce reactivity to self-determinants of an overexpressed proto- SUBSTITUTE SHEET (RULE 26) WO 97/25860 PCT/US97/00582 11 oncogene product. The strategy exploits the structural relatedness between the product of the cellular proto-oncogene and that of the product of genes cognate to the target proto-oncogene. The cognate gene may comprise a wild-type or mutant cognate retroviral oncogene or a wild-type or mutant proto-oncogene of a species different from the host species. The starting point of the vaccine strategy is the high degree of primary sequence homology that exists between the protein product of a targeted proto-oncogene and that of its cognate retroviral oncogene, or between the proto-oncogene product and the product of a cognate proto-oncogene from a different species. However, in contrast to other proposed vaccine strategies, the present invention is not based on the immune recognition of a determinant defined by a cancer specific mutation.

For those tumors showing proto-oncogene overexpression, this sequence homology permits application of the following strategy, which can be employed either prophylactically or therapeutically under conditions of cellsurface expression, or other forms of adjuvanicity, as chosen to enhance immunogenicity: immunization of host biopsied cells with a DNA construct comprising a transgene cognate to the target proto-oncogene, which transgene encodes a gene product which induces host immunoreactivity to host selfdeterminants of the product of the target proto-oncogene; return of the transfected cells to the body of the host to obtain expression of the transgene in the host, and thus immunity against the proto-oncogene product. The invention relies on the targeting of a self-determinant found on an overexpressed or overabundant proto-oncogene-encoded product. The foreign peptide elements of the immunizing oncogene product will trigger peripheral lymphocytes exhibiting a weak cross reactivity for the self peptides of the targeted protooncogene product. Although such self peptides would be present in normal cells expressing the proto-oncogene, targeting of the tumor cells is favored in view of their overexpression of the proto-oncogene.

The immune strategy exploits the antigenicity of two alternative types of determinants: tumor-associated antigenic determinant(s) induced as a consequence of the activity of the oncogene product, an enzymatic WO 97/25860 PCT/US97/00582 12modification of a cellular protein effected by the oncogene product, or tumor associated antigenic determinant(s) intrinsic to the oncogene-encoded product itself. The difficulty in exploiting the first alternative by traditional means, i.e., antigen purification, is that at present little or no systematic information exists bearing on the properties of an antigen that, though oncogene-induced, is not oncogene-encoded. This situation makes purification of any such antigen problematic. However, this problem is obviated from the outset by the present invention which utilizes biopsied cells which, as transfected in culture by the cognate retroviral oncogene, would express the relevant antigenicity.

In terms of exploiting the second alternative, that of an antigenicity intrinsic to the proto-oncogene product, a relevant consideration is that the protocol of immunization according to the present invention primes the host to determinants of the oncogene product itself. A consequence of this immunization is induction of T-cell reactivity to the divergent, i.e foreign, peptide determinants of the retroviral oncogene product, those peptide determinants that show sequence differences with the positionally homologous determinants of the cellular proto-oncogene product. The induction of this reactivity does not in itself have vaccine potential, since the foreign determinants specific to the retroviral oncogene product are normally absent from the cellular proto-oncogene product. Nevertheless, the foreign peptide elements, notably those that differ by only a single amino acid from the positionally homologous self peptides, trigger peripheral T-lymphocytes exhibiting a weak cross-reactivity for the self peptides. Although such self peptides are present in normal cells expressing the proto-oncogene, targeting of the tumor cells is favored in view of their overexpression of the proto-oncogene.

It is possible that many tumor-associated and overexpressed proto-oncogenes might possess mutations. In some cases, overexpression may very well arise as a direct consequence of one or more of the mutations.

However, the present vaccination method does not have as its object the deliberate targeting of non-self determinants generated by proto-oncogene mutations. Unlike prior vaccination methods designed to target such mutation- WO 97/25860 PCT/US97/00582 13 driven non-self determinants, it is the aim of the present invention to induce reactivity for self-determinants in the overexpressed product of tumor associated and overexpressed proto-oncogenes.

Prior efforts attempting to elicit reactivity to proto-oncogene self determinants have relied on in vitro protocols utilizing mutant cell lines to identify individual self peptide immunogens (Disis et al., Cancer Res. (1994) 54:1071-1076; Peoples et al., Proc. Natl. Acad. Sci USA (1995), 92:432-436).

According to the present invention, the host immune system is presented with the full array of naturally-derived class I binding peptides. The vaccine strategy of the present invention obviates the need for any a priori assessment of the immunogenicity of individual peptides.

While the cellular immunogens of the invention display self peptides, non-self peptides would also be presented which may serve as more effective tolerance breakers. The value of a non-self, but closely related to self, peptide is that it may more readily activate those T cells that have both a weak cross reactivity for the cognate self peptide and an activation threshold (determined by the tightness of binding to the T cell receptor) too high to be triggered by the self peptide. Moreover, cognate non-self is inductive of a good immune response, simply because it does in fact constitute nonself. The nonself immune response is expected to predispose the induction of the inevitably weaker response to the self determinants on the same protein product, since the resultant cytokine release provides local help to initiate the weaker anti-self response.

As hereinafter exemplified in a model of src-oncogene-based tumor formation, immunization with cells transfected with a transgene construct expressing the v-src oncogene product induces reactivity to the product of the c-src proto-oncogene, thereby conferring protection against the growth of tumors displaying overexpression of the c-src proto-oncogene.

WO 97/25860 PCT/US97/00582 14- Target Proto-Oncogenes According to the present invention, patients with a family history of a cancer characterized by the overexpression of a particular proto-oncogene are selected for immunization, Alternatively, patients whose tumors can be shown to overexpress the proto-oncogene are selected. Overexpression of a proto-oncogene may derive from an increase over a basal level of transcription.

Overexpression may also derive from gene amplification, that is, an increase in gene copy number, coupled with a basal or elevated level of transcription.

Proto-oncogene overexpression may be assayed by conventional probing techniques, such as described in Molecular Cloning: A Laboratory Manual J.

Sambrook et al., eds., Cold Spring Harbor Laboratory Press, 2nd ed. 1989.

The level of target proto-oncogene expression may be determined by probing total cellular RNA from patient cells with a complementary probe for the relevant mRNA. Total RNA from the patient cells is fractionated in a glyoxal/agarose gel, transferred to nylon and hybridized to an appropriately labelled nucleic acid probe for the target mRNA. The number of relevant mRNA transcripts found in the patient cells is compared to that found in cells taken from the same tissue of a normal control subject.

As an alternative to measuring mRNA transcripts, the expression level of a target proto-oncogene may be assessed by assaying the amount of encoded protein which is formed. Western blotting is a standard protocol in routine use for the determination of protein levels. See Molecular Cloning, supra, Chapter 18, incorporated herein by reference. Accordingly, a cell lysate or other cell fraction containing protein is electrophoresed on a polyacrylamide gel, followed by protein transfer to nitrocellulose, and probing of the gel with an antibody specific for the protein in question. The probe step permits resolution of the desired protein from all other proteins in the starting mixture.

The bound antibody may be prelabeled, by a radioisotope such as 1251, so as to permit its detection on the gel. Alternatively, a secondary reagent (usually an anti-immunoglobin or protein A) may be radiolabeled or covalently coupled WO 97/25860 PCT/US97/00582 15 to an enzyme such as horseradish peroxidase or alkaline phosphatase. The strength of the signal is proportional to the amount of the target protein. The strength of the signal is compared with the signal from a sample analyzed in the same manner, but taken from normal as opposed to tumor tissue.

A description of the methodology and use of Western blotting to determine the levels of the c-src-encoded protein pp60 c rc in adenomatous polyps (colonic epithelia) is provided by Cartwright et al., Proc. Natl. Acad. Sci. USA (1990), 87:558-562, the entire disclosure of which is incorporated herein by reference.

An at least about eight-fold increase in that gene's expression in the patient cells compared to expression in normal control cells from the same tissue would indicate candidacy for vaccination.

Table 1 includes a partial list of representative proto-oncogenes, the overexpression of which has been associated with one or more malignancies.

Each listed proto-oncogene is a target proto-oncogene according to the present invention. The corresponding oncogene, of which the target proto-oncogene is the normal cellular homolog, is also identified. This list of target protooncogenes is intended to be representative, and not a complete list.

Table 1 Representative List of Target Proto-Oncogenes Proto- Oncogene Tumor Comments/References AKT-2 ovarian v-Akt is the oncogene of the AKT8 virus, which induces lymphomas in mice.

1. Bellacosa et al., (1995) Int. J. Cancer 64(4):280-5: Southern-blot analysis has shown AKT-2 amplification in 12.1% of ovarian carcinomas, while Northern bot analysis has WO 97/25860 PCT/US97/00582 16revealed overexpression of AKT-2 in 3 of fresh ovarian carcinomas which were negative for AKT-2 amplification.

2. Cheng et al., (1996) Proc. Natl. Acad. Sci.

USA 89(19): 9267-71): Amplification of AKT-2 has been detected in 10% of pancreatic carcinomas.

AKT-2 pancreatic Cheng et al., (1996) Proc. Natl. Acad. Sci. USA 93(8):3636-41: Amplification of AKT-2 has been detected in 10% of pancreatic carcinomas.

c-erbB-2 bladder c-ErbB-2 is also known as HER2/neu. V-erbB is the oncogene of the avian erythroblastosis virus.

1. Underwood et al., (1995) Cancer Res.

55(11):2422-30: Protein overexpression was observed in 45% of patients with non-recurrent disease and 50% of patients with recurrent disease; 9% of bladder tumors analyzed shoed gene amplification.

2. Coombs et al., (1993) Pathology 169(1):35- 42: c-ErbB-2 gene amplification was observed in 14% of bladder tumors analyzed.

3. Gardiner et al., (1992) Urolog. Res. 20(2): 17- Nineteen percent of primary transitional cell bladder carcinomas showed c-erbB-2 gene amplification.

c-erbB-2 breast 1. Molina et al., (1966) Anticancer Research 16(4B):2295-300: Abnormal c-erbB-2 levels were found in 9.2% of patients with locoregional breast WO 97/25860 PCTIUS97/00582 17 carcinoma, and in 45.4% of patients with advanced disease. 2. DePotter et al., (1995) VirchowsArch. 426(2):107-15: Overexpression of the oncoprotein is observed in about 20% of invasive duct cell carcinomas of the breast. 3.

Bandyopadhyay et al., (1994) Acta Oncol.

33(5):493-8: 35.4% of breast tumors showed cerbB-2 overexpression; 17.4% showed gene amplification. 4. Fontana et al., (1994) Anticancer Res. 14(5B):2099-104: 26% of samples showed c-erbB-2 amplification. 5. Press et al., (1993) Cancer Research 53(20):4960-70: Amplified overexpression was identified in 38% of primary breast cancers. 6. Berns et al., (1992) Cancer Res. 52(5):1107-13: 23% of primary breast cancer tissues exhibited amplification. 7. Delvenne et al., (1992) Eur. J.

of Cancer 28(2-3):700-5: c-erbB-2 mRNA was overexpressed in 34% of breast tumor samples.

8. Inglehart, (1990) Cancer Res. 50(20):6701-7: Two to thirty-two-fold gene amplification was found in multiple stages of tumor progression. 9.

Slamon et al., (1989) Science 244:707-12: A 28% incidence of amplification of c-erbB-2 was found in 189 primary breast cancers. 10. Kraus et al., (1987) EMBO J. 6(3):605-10: Eight cell lines demonstrated c-erbB-2 mRNA levels ranging from 4 to 128-fold overexpression. 60% of all tumors analyzed showed elevated levels of c-erbB- 2 mRNA.

WO 97/25860 PCT/US97/00582.

18c-erbB-2 lung 1. Osaki et al., (1995) Chest 108(1):157-62: Lung tissue overexpression of c-erbB-2 was discovered in 42.5% of samples. 2. Lorenz et al., (1994) Clin. Invest. 72(2):156-63: A 64-fold increase in the amount of c-erbB-2 mRNA was observed; 33% of lung tumors showed overexpression of c-erbB-2.

c-erbB-2 ovarian 1. Katsaros et al., (1995) Anticancer Res.

15(4): 1501-10: Abnormally high expression of cerbB-2 was found in 31% of tumor samples. 2.

Felip etal., (1995) Cancer 75(8):2147-52: 21.7% of ovarian tumors showed overexpression of cerbB-2. 3. Fan et al., (1994) Chin. Med. J.

107(8):589-93: c-erbB-2 amplification was found in 30.8% (8 of 26) of human ovarian cancers. 4.

vanDam et al., (1994) J. of Clin. Path.

47(10):914-9: 24% of ovarian tumors showed cerbB-2 overexpression. 5. Csokay et al., (1993) Eur. J. of Surg. Oncology 19(6):593-9: c-erbB-2 amplification was found in 34% of fresh ovarian tumor samples. 6. McKenzie et al., (1993) Cancer 71(12):3942-5: 30% of ovarian tumor samples indicated c-erbB-2 overexpression. 7.

Hung et al., (1992) Cancer Letters 61(2):95-103: A 100-fold c-erbB-2 overexpression was discovered in one human cell line. Two to fourfold amplification was also discovered.

MDM-2 leukemia MDM-2 is the murine double minute-2 oncogene.

1. Bueso-Ramos et al., (1993) Blood 82(9):2617- WO 97/25860 PCT/US97/00582.

19- 23: 53% of cases showed overexpression of MDM-2 mRNA. The level of MDM-2 mRNA overexpression in some cases of leukemias was comparable to that observed in some sarcomas, which demonstrate more than 50-fold MDM-2 gene amplification. No evidence of gene amplification was observed. 2. Watanabe et al., (1994) Blood 84(9):3158-65: 28% of patients with B-cell chronic lymphocytic leukemia or non- Hodgkin's lymphoma had 10-fold higher levels of MDM-2 gene expression. MDM-2 overexpression was found more frequently in patients at advanced clinical stages, c-myb colon V-myb is the oncogene of the avian myeloblastoma virus. 1. Ramsay et al., (1992) Cell Growth and Diff. 3(10):723-30: c-myb levels were always higher in colon cancer samples than normal tissue. 2. Alitalo et al., (1984) Proc.

Natl. Acad. Sci. 81(14):4534-8: c-myb levels were always higher in colon cancer samples than normal tissue.

c-myc breast V-myc is the oncogene of the avian myelocytoma virus. 1. Lonn et al., (1995) Cancer 75(11):2681-7: Amplification of c-myb occurs in 16% of patients with breast cancer. 2. Hehir et al., (1993) J. of Surg. Oncology 54(4):207-9: cmyc overexpression was found in 60% of breast carcinoma samples. 3. Kreipe et al., (1993) Cancer Research 53(8):1956-61: Amplification of WO 97/25860 PCTIUS97/00582 c-myc was found in 52.6% of samples that displayed a Ki-S1 labelling index exceeding 4. Watson et al., (1993) J. Nat. Cancer Inst.

85(11):902-7: Amplification of c-myc occurs in up to 20 30% of breast cancers. 5. Berns et al., (1992) Cancer Research 52(5):1107-13: Amplification was found in 20% of primary breast cancer patients; the range was 3-14 gene copies.

6. Watanabe et al., (1992) Cancer Research 52(19):5178-82: Expression of c-myc was increased by c-myc gastric/ 1. Rigas, (1990) Clin. Gastroent. 12(5):494-9: colorectal Overexpression of c-myc is found in 80 of colon cancers. 2. Erisman et al., (1988) Oncogene 2(4):367-78: Adenocarcinoma cell lines express 5-10-fold elevated levels of c-myc mRNA. Eight to thirty-seven-fold higher levels of c-myc protein was found in tumor cell lines compared to normal cells. 3. Sikora et al., (1987) Cancer 59(7):1289-95: Up to 32-fold overexpression of c-myc mRNA was observed in 12 to 15 tumors.

4. Tsuboi et al., (1987) Biochem. and Biophys.

Res. Comm. 146(2):705-10: Gastric Cancer: A 2-3-fold overexpression was observed in gastric cancer. A 2-10-fold overexpression was observed in colorectal cancer.

c-myc lung 1. Lorenz et al., (1994) Clin. Invest. 72(2):156- 63: A 57-fold increase in c-myc mRNA levels was observed. 23% of samples indicated strong WO 97/25860 PCT/US97/00582 -21 expression of c-myc. 2. Kato et al., (1993) Jap.

J. of Cancer Res. 84(4):355-9: Liver tissue metastases from human small cell lung carcinoma revealed 30-fold amplification of c-myc.

c-myc naso- Porter et al., (1994) Acta Oto-Laryng. 114(1): pharn- 1105-9: 22% of samples showed intense staining geal for c-myc.

c-mvc ovarian 1. Bian et al., (1995) Chin. J. of Ob. Gyn.

30(7):406-9: 50% of samples showed amplification of c-myc. 2. Katsaros et al., (1995) Anticancer Res. 15(4):1501-10: 26% of samples exhibited c-myc amplification. 3. van Dam et al., (1994) J. Clin. Path. 47(10):914-9: Overexpression of c-myc was found in 35% of ovarian carcinomas. 4. Xin et al., (1993) Chin.

J. of Ob. Gyn. 28(7):405-7: 54.5% of samples showed amplification of c-myc. 5. Tashiro et al., (1992) Int. J. of Cancer 50(5):828-33: Overexpression was found in 63.5% of all serous adenocarcinoma tissues and 37.3% of all ovarian carcinoma tissues. Significant overexpression of c-myc was observed at Stage III compared with other stages.

c-nmc prostate Nag et al., (1989) Prostate 15(2):115-22: A fold amplification of c-myc was observed. Fiftyfold higher levels of mRNA transcripts of c-myc were found.

WO 97/25860 PCT/US97/00582 -22c-ras lung Ras oncogenes were first recognized as the transforming genes of Harvey and Kirsten murine sarcoma viruses. Lorenz et al., (1994) Clin.

Invest. 72(2):156-63: a 13-fold increase in overexpression of c-Ki-ras was observed. 18% of tumors displayed strong overexpression of c-Kiras.

c-ras ovarian 1. Katsaros et al., (1995) Anticancer Res.

15(4):1501-10: Higher levels of ras protein than in normal or benign ovarian tumors were found in of. tumor samples. 2. vanDam et al., (1994) J. of Clin. Path. 47(10):914-9: 20% of ovarian tumors exhibited c-ras overexpression.

The levels of expression of c-ras were much higher in tumors of patients with recurrent or persistent disease after chemotherapy, than in the tumors of patients at initial presentation.

c-src breast V-src is the oncogene of the Rous sarcoma virus, which induces sarcomas in chickens.

Muthuswamy et al., (1994) Mol. and Cell. Biol 14(1):735-43: c-erbB-2-induced mammary tumors possessed 6-8-fold higher c-src kinase activity than adjacent epithelium.

c-src colon/ 1. Cartwright et al., (1994) J. of Clin. Invest.

colorectal 93(2):509-15: c-src activity is 6-10-fold higher in mildly dysplastic ulcerative colitis (a chromic inflammatory disease of the colon with a high on incidence of colon cancer) than in non-dysplastic WO 97/25860 PCT/US97/00582 -23epithelia. This data suggests that activation of csrc is an early event in the genesis of UC colon cancer. 2. Talamonti et al., (1993) J. of Clin.

Invest. 91(1):53-60: High level of c-src activity from colorectal cancer is found in liver metastases. 3. Termuhlen et al., (1993) J. of Surg. Res. 54(4):293-8: Colon carcinoma metastases to the liver had significantly increased activity of c-src with an average 2.2-fold increase.

Extrahepatic colorectal metastases demonstrated an average 12.7-fold increase in c-src activity over normal mucosa.

c-yes colon V-yes is the oncogene of two avian sarcoma viruses, Esh sarcoma virus and Y73. 1. Pena et al., (1995) Gastroent. 108(1):117-24: Twelve to fourteen-fold higher expression of c-yes was found in colonic transforming oncogene adenomas compared to normal mucosa. Activity of c-yes was elevated in adenomas that are at greatest risk for developing cancer. 2. Park et al., (1993) Oncogene 8(10):2627-35: A ten to 20-fold higher than normal activity of c-yes was observed in 3 out of 5 colon carcinoma cell lines. A higher than normal activity was found in 10 out of 21 primary colon cancers, compared to normal colonic cells.

WO 97/25860 PCT/US97/00582 -24- Selection of Cognate Transgene for Preparation of Cellular Immunogen According to the present invention, a transgene construct is engineered comprising a transgene which is cognate to the target proto-oncogene (hereinafter "cognate transgene" or The transgene is selected such that it encodes a gene product which induces host immunoreactivity to host selfdeterminants of the product of the target proto-oncogene. The transgene should be expressed to very high levels in the transfectants. Thus, the construct should contain a strong promoter.

The product encoded by the cognate gene must have a high degree of sequence homology with the product of the target proto-oncogene, but also must display some amino acid differences with the target proto-oncogene product. Thus, there must be a subset of one or more amino acid differences between the target proto-oncogene and its cognate in order to provide immunogenic stimulus. Two classes of genes that satisfy these criteria are retroviral oncogenes and xenogenic proto-oncogenes. The word "xenogenic" is intended to have its normal biological meaning, that is, a property or characteristic referring or relating to a different species. Thus, a xenogenic proto-oncogene is meant to include the a homologous proto-oncogene of a species other than the host organism species. It may be appreciated that in the case of a target proto-oncogene, e.g. MDM2, for which no retroviral homolog is yet known, a xenogenic homologue is advantageously utilized as the source of the DNA for the cognate transgene.

In principle, a more effective immunogenic stimulus would depend on the particular sequence, and not on the distinction between a retroviral oncogene and a xenogenic proto-oncogene in terms of their relative transforming capacity. Thus, in certain cases, a retroviral oncogene may be better at providing a tolerance-breaking immunogenic stimulus, and in other cases, a xenogenic proto-oncogene may be more effective.

The retroviral oncogene or xenogenic proto-oncogene

DNA

forming the CTG may comprise the wild type oncogene or proto-oncogene DNA. More preferably, a mutant DNA is utilized, which is engineered so as WO 97/25860 PCT/US97/00582 to be non-transforming in the host. The DNA is mutated to include one or more nucleotide insertions, deletions or substitutions which will encode an oncogene product which is nontransforming in the host, but retains the requisite degree of sequence homology with respect to the target proto-oncogene. A cognate transgene deletion mutant (hereinafter "dCTG") is preferred.

A protein sequence is generally considered "cognate" with respect to the target proto-oncogene-encoded protein if it is evolutionarily and functionally related between species. A more precise view of cognation is based upon the following sequence comparison carried out utilizing the FASTA program of Pearson and Lipman, Proc. Natl. Acad. Sci. USA (1988), 85:2444- 2448, the entire disclosure of which is incorporated herein by reference.

Cognation is attained upon satisfying two criteria imposed by FASTA; (i) alignment of segments corresponding to at least 75% of the target protooncogene's encoded amino acid sequence; (ii) at least 80% amino acid identity within the aligned sequences. The segments of the target proto-oncogene protein sequence and protein test sequence satisfying the two criteria are referred to as "homology regions". Accordingly, at least 75% of the target proto-oncogene protein sequence is alignable with the test sequence. The alignable segments or homology regions may, however, represent less than of the total test polypeptide chain for the case of test sequences that may significantly exceed the target proto-oncogene protein in length.

One skilled in the art, armed with the FASTA program, may survey existing sequence data bases (either protein sequences or DNA sequences, insofar as the amino acid sequence is determined by FASTA for all reading frames) for test sequences which are cognate with respect to the target proto-oncogene. At the same time, one can isolate and then sequence what are very likely to be cognate test sequences feline MDM-2, as likely to be cognate to human MDM-2) and use FASTA to verify the presumed cognation.

according to the criteria set above. One may obtain the sequences of presumptive cognate proto-oncogenes from a large number of mammalian WO 97/25860 PCT/US97/00582 -26sequences and screen these sequences with FASTA according to the aforesaid formulation of cognation.

Because the product encoded by a CTG differs at a small number of amino acid positions from the product encoded by the target proto-oncogene, an immunogenic stimulus is provided that is directed against the foreign protein and (ii) with a lower probability, induce an anti-self response. The CTG is selected such that the gene product will yield the greatest immunogenic stimulus to induce anti-self reactivity. Provided that overall sequence homology .(preferably greater than about 75%) is maintained, the presence of scattered amino acid differences is desired, since any one residue would likely have a relatively low probability of inducing self-reactivity. Moreover, the greatest number of residue differences would be advantageous, consistent with maintaining the requisite degree of general sequence homology.

The selection of amino acid modifications for the CTG may be facilitated by resort to available computer-based models used to identify immunogenic peptide fragments of polypeptides. These models could be employed to select CTGs which would possess the maximum number of immunogenic peptides for a given HLA haplotype.

Screening Procedure for CTG Selection Notwithstanding the availability of computer-based algorithms which have some predictive value, it is desirable to design CTGs with resort to a screening procedure based on an actual experimental assay that can be HLAhaplotype specific. Accordingly, cells are biopsied from a normal volunteer of particular haplotype. The cells are transfected with a CTG construct, preferably a dCTG construct, satisfying the criteria set for cognition. More preferably, the cells are transfected with multiple dCTGs, preferably at least five dCTGs, satisfying the criteria for cognition. The at least five dCTGs are selected to display amino acid differences that essentially extend throughout the polypeptide chains of the encoded sequences. The transfected cells are then used to immunize the volunteer in accordance with the immunization method of the WO 97/25860 PCT/US97/00582 -27present invention. After immunization, the human subject is tested in a standard delayed hypersensitivity (DH) reaction with 104-106 irradiated, autologous fibroblasts, as transfected with the same dCTG (or series of dCTGs) as used for the immunizing preparation. A positive DH reaction (induration) would verify the induction of reactivity. The induction of reactivity in this assay is readily demonstrable because of the priming to the non-self determinants on the dCTGencoded protein and the readout in the DH reaction of the same nonself determinants. Once DH reactivity is demonstrated in a DH reaction that directly tests the antigenicity of the non-self determinants encoded by the dCTG priming with a non-self construct, DH testing with the same non-self construct), the subject can be then tested in a DH reaction based on testing with the autologous cells transfected with a dCTG derived from the human protooncogene itself priming with a non-self construct, testing with the human self construct). Testing of a battery of human volunteers will lead to a catalogue of HLA-matched dCTGs, such that, for individuals of the same HLA haplotype, the use of the particular dCTG would be inductive of reactivity to proto-oncogene-encoded self. Different CTGs may thus be tested so as to correlate maximal secondary stimulation with a particular HLA haplotype.

At the same time, this procedure may be used with patients undergoing tumor resection (if post-operative immuno-suppressive protocols are not mandatory), such that prior to resection, a course of immunization would have been initiated, the endpoint of which would represent the development of a DH reaction.

Any given amino acid difference between the CTG-encoded product and the proto-oncogene-encoded product has a low probability of being a "tolerance-breaker". Thus, it is preferable to transfect the host cells with a mixture of multiple different CTGs, preferably dCTGs. The number of different dCTGs is preferably five or more. Moreover, it is preferred that, among themselves, the multiple dCTGs show amino acid differences that essentially extend throughout the polypeptide chains of the encoded sequences.

The dCTGs would be selected to maximize amino acid differences and, at the WO 97/25860 PCT/US97/00582 -28same time, make sure that differences are found all along the polypeptide chain.

It would thus not be preferable to select a battery of deletions all from within the same domain of the polypeptide chain.

According to a protocol which utilizes 107 irradiated cells for immunization containing five separate dCTGs, five groups of 2 X 10 6 cells are included in one inoculate, each group of 2 X 10 6 having been transfected with a separate dCTG from the total set of five CTGs that are cognate to a particular -proto-oncogene.

Selection of Non-Transforming Cognate Transgenes Non-transforming cognate transgene variants are most advantageously derived via deletion of a sequence essential for transformation.

Unlike point mutations which are potentially reversible due to back mutations, deletion mutations are irreversible. Furthermore, deletion mutations do not possess the inherent disadvantage attaching to point mutations, namely, even though the requirement for generation of an acceptable cognate transgene is for a qualitative difference with the wild type, non-transforming versus transforming, any given point mutation may be neutral or else quantitative in its effect, that is, the mutation may reduce but not totally eliminate transformability. Thus, according to a preferred embodiment of the invention, a deletion is created in a region of the cognate transgene which encodes an amino acid sequence required for transformation. Consonant with nontransformability, the smallest deletion possible so as to leave intact the bulk of the antigenicity of the transgene product is selected.

The engineering of a cognate transgene deletion mutant that satisfies these criteria is facilitated by reports of structure-function relationship in oncogene-encoded proteins. Such reports serve to identify regions of oncoproteins that are essential for transformation, as opposed to regions which are either neutral or serve merely to modulate transformability. Although such reports are usually based on in vitro transformation assays, and are therefore independent of immune effects, these studies can be exploited to aid in the WO 97/25860 PCT/US97/00582 -29construction of non-transforming dCTGs for use in the practice of the present invention.

The deletion mutant is engineered to include at least a part of the region identified as critical for transformation. In those cases where essential amino acids have been identified, the deletion will span these residues. The engineering of any desired deletion can be readily accomplished by polymerase chain reaction (PCR) according to conventional PCR techniques, based upon the known nucleotide sequence of the unmutated cognate transgene.

The following describes a representative protocol for deriving a non-transforming dCTG of the smallest possible deletion, for use in the practice of the present invention. A test dCTG, engineered on the basis of known or ascertained transformation-specific domains, and driven by the strongest possible promoter, is used to transfect murine 3T3 cells. A sister culture of 3T3 cells is also transfected, with non-deleted CTG. Each CTG or dCTG cell culture is inoculated into nude mice, in the absence of any treatment to render the cells non-dividing. Those dCTGs which do not yield tumors in the mice even after prolonged observation are then utilized as transgenes for the biopsied human cells which, upon transfection with the transgene, will serve as a cellular vaccine according to the practice of the present invention. The dCTGs are selected with the smallest deletion mutant consonant with non-transformability.

Some CTGs representing xenogenic proto-oncogenes may not be tumorigenic in the 3T3/nude mouse assay. For any such non-transforming CTG, it is not essential to generate a dCTG. However, even given nontumorigenicity in nude mice, it may be desirable to opt for generation of a deletion mutant when the transgene is based upon a xenogenic proto-oncogene.

In such cases, the deletion would be engineered so as to remove the homologous region to that deleted in the particular dCTG that corresponds to the deletion in the corresponding retroviral oncogene dCTG.

Even though the transgene construct may comprise mutant oncogene or proto-oncogene DNA which is nontransforming, it is nevertheless preferable, as a safety measure, to treat the transfected cells to render them non- WO 97/25860 PCT/US97I00582 30 dividing before inoculation back into the host. The cells are irradiated with a radiation dosage sufficient to render them non-dividing.

Oncogenicity Assay of Connate Transgenes As a further safety measure, the oncogenicity of a given dCTG is preferably thoroughly tested prior to infection of the human host cells which are used as cellular immunogens according to the practice of the present invention. For example, an oncogenicity testing regimen may take the form of three separate assays: dCTG transfection of NIH 3T3 cells, followed by inoculation into nude mice; (ii) dCTG transfection of human fibroblasts, followed by inoculation into nude mice; and (iii) dCTG transfection of human fibroblasts, followed by an in vitro test of anchorage-dependent growth. In principle, all three should be negative to validate the use of any given dCTG in the vaccination method of the present invention.

According to the oncogenicity assay after stable transfection of NIH 3T3 cells with the test dCTG, the transfectants are inoculated into nude mice. Tumorigenicity of the transfectants in the mice is then evaluated according to standard protocols.

According to oncogenicity assay human fibroblasts are transfected with the test dCTG as proposed in the above human immunization protocol. After stable dCTG transfection of human fibroblasts, however, rather than carrying out X-irradiation of the transfectants to render them non-dividing, followed by inoculation of the irradiated transfectants back into the human host, the transfectants are directly inoculated into nude mice as a direct test of tumorigenicity. Given the greater susceptibility of murine 3T3 cells to oncogenic transformation, vis a vis primary human or murine transfectants fibroblasts, assay (ii) is probably much less sensitive than assay but does have the advantage of offering a direct test of dCTG oncogenicity in human cells.

According to oncogenicity assay (iii), non-irradiated dCTGtransfected human fibroblasts are assayed for anchorage-dependent growth, i.e.

WO 97/25860 PCT/US97/00582 -31 colony formation in soft agar, as a test of dCTG transforming potential in human cells. Anchorage independence, as defined by the ability of cells to grow when suspended in semisolid medium, is a common phenotype acquired by human tumor cells, particularly those tumor cells of mesenchymal origin, such as fibrosarcomas. While assay (iii) has no in vivo readout, it offers an independent test of the critical issue of dCTG oncogenicity in human cells.

The oncogenicity assays are performed according to published protocols. Assay comprising dCTG transfection of NIH 3T3 cells followed by inoculation into nude mice, may be performed according to the protocol of Stevens et al., Proc. Natl. Acad. Sci. USA (1988), 85:3875-3879, including DNA transfection by the calcium phosphate coprecipitation method of Manohaven et al., Carcinogenesis (1985), 6:1295-1301. Accordingly, NIH 3T3 cells (7.5 X 105 cells per 100-mm dish) are exposed to a calcium phosphate- DNA coprecipitate (40 /g of genomic DNA plus 3 Ag of pSV2neo per dish) for 4 hours. Two days later, each dish is trypsinized and reseeded into a 175-cm 2 flask. For the next 10 days, cultures are selected in G418 (400 /tg/ml), and the flasks are then trypsinized and cells are replated in the same flask to disperse the G418-resistant colonies into a diffuse lawn of cells. Two days later, the cells are harvested and washed with serum-free medium prior to injection. One injection of 5 X 106 cells into the right flank and one injection of 1 X 10 7 cells into the left flank, each in a volume of 200 p are done on each nude mouse.

Injection sites are monitored at 3- or 4-day intervals for 100 days. The sites are scored for the number of tumors induced per injection site.

Oncogenicity assay whereby dCTG transfection of human fibroblasts followed by inoculation into nude mice, is carried out in the same manner as assay except that for assay (ii) the human fibroblast transfectants are substituted for the murine 3T3 transfectants.

Assay (iii), involves a test of the in vitro anchorage-dependent growth of dCTG-transfected human fibroblasts. The assay is carried out as described in Stevens et al., J. Cancer Res. and Clin. Oncol. 1989, 115:118- 128. 1 x 10' cells are seeded per 60-mm dish into 0.33% Noble agar over a WO 97/25860 PCT/US97/00582 -32 6-ml 0.5% agar base layer in Hams F10 supplemented with 6% fetal bovine serum. A portion of the agar suspension is diluted with Hams F10 plus 6% fetal calf serum to 200 cells/5 ml to determine the cloning efficiency of these cells when seeded into plastic 60-mm dishes. Agar dishes are fed with 1 ml Hams F10 supplemented with 6% fetal bovine serum on the 1st and 15th day after seeding. Four weeks after seeding, all agar colonies >75 /tm in diameter are counted and the colony counts are normalized to the plating efficiencies which aliquots of the initially seeded cells showed on plastic. This comparison, or normalization, of the agar colony counts to the plastic dish colony counts is useful in identifying and correcting for any mechanical artifacts which might result from the seeding into agar of dead cells that had persisted from the initial transfection treatment or from heat-induced cell death, which might have occurred while suspending cells in molten agar during the process of seeding the agar dishes.

The following is a partial list of various deletions which, based upon published accounts of experiments with human or animal cells, are believed to render the identified CTG non-tumorigenic.

WO 97/25860 WO 9725860PCTIUS97/00582 33 Table 2 Deletion Mutations Rendering Indicated Gene Non-Transforming CTG Genbank Number Amino acids References accession of deleted, number for amino rendering sequence acids in CTG nongene transforming Akt-2 (c-akt) M95936; 480 148-234 Bellacosa et (mouse) SEQ ID al., Science NO:3 (Mus (1991), musculus 254:274serine/threon 278; ine kinase) Bellacosa et al., Onco gene (1993), 8(3):745-54.

WO 97/25860 WO 9725860PCTIUS97/00582.

34 CTG Genbank Number Amino acids References accession of deleted, number for amino rendering sequence acids in CTG nongene transforming c-neu M11730; 1255 1-731 Bargmann erbB-2) (rat) SEQ ID et al., NO:4

EMBO

(human (1988), tyrosine 7(7) :2043kinase-type 52; receptor Bernards et (HER2) gene al., Proc.

Natl. A cad.

Sci. USA (1987), 84(19):6854 -8.

WO 97/25860 WO 9725860PCTIUS97100582 35 CTG Genbank Number Amino acids References accession of deleted, number for amino rendering sequence acids in CTG nongene transforming mdm-2 (human) U33199; SEQ ID (human mdm2-A mRNA); U33200; SEQ ID NO:6 (human mdm2-B mRNA); U33201; SEQ ID NO:7 (human mdm2-C mRNA); U33202; SEQ ID NO:8 (human mdm2-D mRNA); U33203; 489 9-155 Dubs- Poterszman, Onco gene (1995), 11(1 1):2445 I j J WO 97/25860 WO 9725860PCT/US97/00582 36 CTG Genbank Number Amino acids References accession of deleted, number for amino rendering sequence acids in CTG nongene transforming c-myb J02012; SEQ 640 275-327 Kalkbrenner (human) ID NO: 10 et al., (proviral Onco gene oncogene v- (1990), mzyb) 5(5):657-61.

c-,nyc X00364; 439 129-144 Sarid et at., (human) SEQ ID Proc. Natl.

NO:11 Acad. Sci.

(human c- USA (1987), myc 84(1): 170-3.

oncogene) v-ras M77193; 189 32-44 Zhang et (Harvey SEQ ID at., Science Murine NO: 12 (Rat (1990), Sarcoma sarcoma 249:162-5 Virus) virus v-ras (1990) oncogene) WO 97/25860 PCT/US97/00582 -37- CTG Genbank Number Amino acids References accession of deleted, number for amino rendering sequence acids in CTG nongene transforming v-src (Rous U41728; 526 430-433 Bryant et Sarcoma SEQ ID al., Mol.

Virus) NO:13 (RSV Cell. Bio.

Schmidt- (1984), Ruppin A 4(5):862-6.

clone SRA- V; v-src gene) c-yes D00333; 541 438-441 Zheng et (chicken) SEQ ID al.; NO: 14 Oncogene (human c- (1989), yes-2 gene) 4(1):99-104.

Engineering of Vectors for Host Cell Transfection The engineering of vectors for expression of a particular CTG, preferably a dCTG, is based on standard methods of recombinant DNA technology, i.e. insertion of the dCTG via the polylinker of standard or commercially available expression vectors. The dCTG is operably linked to a strong promoter. Generally speaking, a "strong" promoter is a promoter which achieves constitutively high expression of the dCTG in the transfected cells.

Each promoter should include all of the signals necessary for initiating transcription of the relevant downstream sequence. These conditions are fulfilled, for example, by the pBK-CMV expression vector available from Stratagene Cloning Systems, La Jolla, CA (catalog no. 212209). The pBK- WO 97/25860 PCTIUS97/00582- -38- CMV vector contains the cytomegalovirus (CMV) immediate early promoter.

dCTGs xenogenic with respect to a particular target proto-oncogene may be isolated by conventional nucleic acid probing techniques, given the availability of a highly homologous probe represented by the cognate retroviral oncogene and/or the human proto-oncogene itself.

Collection of Host Cells for Transfection The host cells which may be transfected to derive the cellular immunogens of the present invention must express class I MHC and be susceptible to isolation and culture. Fibroblasts express class I MHC and may be cultured. Accordingly, punch biopsies of host human skin are performed to harvest fibroblasts. Punch biopsies can be performed by a competent physician as a standard clinical procedure. Each biopsy yields a starting population of 1-2 X 10 7 cells that would proliferate in culture. Methods for the preparation of tissue cultures of human fibroblasts are well developed and widely used. See, Cristofalo and Carpenter, J. Tissue Culture Methods (1980), 6:117-121, the entire disclosure of which is incorporated herein by reference. Essentially, skin obtained by punch biopsy is washed using an appropriate wash medium, finely minced and cultured in a suitable culture medium, such as Dulbecco's Modified Eagle Medium (DMEM), under CO 2 at 37°C. The cells are trypsinized with a trypsin solution and transferred to a larger vessel and incubated at 37 0 C in culture fluid.

Host Cell Transfection The expression vector carrying the dCTG is used to transfect biopsied host cells according to conventional transfection methods. One method of transfection involves the addition of DEAE-dextran to increase the uptake of the naked DNA molecules by a recipient cell. See McCutchin and Pagano, J.

Natl. Cancer Inst. (1968) 41:351-7. Another method of transfection is the calcium phosphate precipitation technique which depends upon the addition of Ca+ to a phosphate-containing DNA solution. The resulting precipitate WO 97/25860 PCT/US97/00582 39 apparently includes DNA in association with calcium phosphate crystals. These crystals settle onto a cell monolayer; the resulting apposition of crystals and cell surface appears to lead to uptake of the DNA. A small proportion of the DNA taken up becomes expressed in a transfectant, as well as in its clonal descendants. See Graham et al., Virology (1973), 52:456-467 and Virology (1974), 54:536-539.

Preferably, transfection is carried out by cationic phospholipidmediated delivery. In particular, polycationic liposomes can be formed from N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) or related liposome-forming materials. See Felgner et al., Proc. Natl. Acad.

Sci. USA (1987) 84:7413-7417 (DNA-transfection); Malone et al., Proc. Natl.

Acad. Sci. USA (1989), 86:6077-6081) (RNA-transfection). One preferred technique utilizes the LipofectAMINE" Reagent (Cat. No. 18324-012, Life Technologies, Inc., Gaithersburg, MD) which is a 3:1 liposome formulation of the polycationic lipid 2,3-dioleyloxy-N- [2(sperminecarboxamido)ethyl-N, N-dimethyl-1 -propanaminium trifluoroacetate (DOSPA) (Chemical Abstracts Registry name: aminopropyl)amino]- 1-oxypentyl}amino)ethyl]-N,N-dimethyl-2,3-bis(9octadecenyloxy)-l-propanaminium trifluoroacetate), and the neutral lipid dioleoyl phosphatidylethanolamine (DOPE) in membrane filtered water.

Transfection utilizing the LipofectAMINE" Reagent is carried out according to the manufacturer's published protocol. The protocol (for Cat. No. 18324-012) provides for either transient or stable transfection, as desired.

The advantage of transient expression is its rapidity, i.e. there is no requirement for cellular proliferation to select for stable integration events.

This rapidity could conceivably be of major clinical importance, in cases of an already metastatic tumor burden, wherein the weeks required for selection of stable transfectants may simply not be available to the clinician.

There are, nonetheless, two general disadvantages to the use of transient transfection. The first is that expression usually peters out after a few days, in contrast to the continual expression in the case of stable transfection.

WO 97/25860 PCT/US97/00582 This is not particularly crippling in terms of our immunization protocol. The inoculated, irradiated cells used for immunization would likely not survive in vivo for more than 4 or 5 days, in any case. Thus the nominal advantage accruing to stable transfection, that of a long-duration expression by the progeny of the parental inoculated cell, is not of particular relevance in the case of the immunizing regime described herein, which is based on the use of non-dividing, probably short-lived cells.

A second disadvantage of transient transfection resides in the fact that it yields a cell population, only a subset of which has actually been transfected and thus expresses the protein encoded by the transgene. This problem is obviated in the case of stable transfection, wherein over time one can develop a pure population of transfectants via selection for a resistance marker, such as neo, under conditions of clonal proliferation of the initial stable transfectants, i.e. daughter cells of transiently transfected cells lack the transgene, in contrast to the case with stable transfectants. In the situation where there is sufficient time to effect immunization based on stably transfected cells, the progeny of all transfected clones would be utilized, not just the progeny of a single clone, as is sometimes done for detailed biochemical and molecular analyses of gene expression. Clearly the more clones utilized, the more quickly one can arrive at the requisite number of cells to be used for immunization.

Percentage of Cells Exhibiting dCTG Expression The percentage of cells exhibiting dCTG expression may be determined by an immunohistology assay. In this procedure, a small number of cells 500) from the harvested pellet following centrifugation of transfected cells are deposited on a cover slip and fixed with cold acetone. At this point, a standard immunohistological assay is carried out with the cells on the cover slip, i.e. addition of a primary monoclonal antibody reactive to the dCTGencoded protein, followed by the addition of a developing antibody, e.g. a fluorescent tagged antibody reactive to the primary monoclonal antibody.

WO 97/25860 PCT/US97/00582 -41 Measurement of the percentage of cells scoring as dCTG-positive in the fluorescent assay allows a determination of the number of positive transfectants in the starting culture, and thus the number of total cells to be used for immunization to arrive at the desired number of dCTG-positive cells to be inoculated in the patient.

If, as would be almost certain, the percentage of cells scoring as dCTG-positive is less than one hundred percent, one can simply increase the number of cells to be used for immunization, so as to include the desired number of transfectants. The non-transfected cells in the immunizing population would simply represent x-irradiated, autologous fibroblasts that would constitute no danger to the patient.

Transfectant Irradiation Prior to return to the host, the transfected cells are preferably irradiated. The transfectants are irradiated with a radiation dose sufficient to render them non-dividing, such as a dose of 25 By or 2500R. The cells are then counted by trypan blue exclusion, and about 2 X 10 7 irradiated transfectants are resuspended in a volume of 0.2-0.4 ml of Hanks Balanced Salt Solution.

Vaccination Procedure The transfected cells are returned to the host to achieve vaccination. The cells may be reimplanted at the same body site from which they were originally harvested, or may be restored to a different site.

It is the object of the present invention to generate a systemic tumor immune response, so as to fight metastasis formation wherever any metastases are found. Accordingly, there is no reason to inject the transfected cells at the same body site from which they were taken. Intramuscular or subcutaneous inoculation at a distal site would suffice to yield a systemic response. Thus, patients are preferably vaccinated by subcutaneous inoculation of the transfected cells.

WO 97/25860 PCT/US97/00582.

-42- For s-crc overexpression associated with colon carcinoma, partial venous inoculation is preferred, as the liver is a frequent site of metastases. For vaccinating against breast cancers and lymphomas, systemic immunization is preferred.

As a general rule, it is desirable to generate the strongest immune response consistent with clinical monitoring of no adverse side effects, i.e.

multiple rounds of inoculation with, for example 107 cells, at each round. The number of rounds of inoculation is selected accordingly. The efficacy of the inoculation schedule may be monitored by a delayed hypersensitivity reaction administered to the patient. A course of about up to 10 inoculations, at 2-3 week intervals, may be utilized. It may be appreciated that the inoculation schedule may be modified in view of the immunologic response of the individual patient, as determined with resort to the delayed-type hypersensitivity (DTH) reaction.

Patient Response Monitoring by Delayed-type Hypersensitivity Reaction Patients are assessed for reactivity to the irradiated transfectants by a test of skin reactivity in a DTH reaction. DTH has been used clinically (Chang et al. (1993), Cancer Research 53:1043-1050). To measure reactivity to the autologous irradiated transfectants, 104 10 6 cells in a volume of 0.1 ml Hanks buffered saline solution (HBSS) are inoculated intradermally into the host. Induration is measured 48 hours later, as an average of two perpendicular diameters (responses of greater than 222 mm is considered positive).

One advantage to the DTH assay is that it can independently assess the induction of T cell reactivity to the transfectants used for immunization the set of 5 or more dCTGs chosen for immunization purposes, each containing non-self determinants) and (ii) transfectants, as transfected with the human dCTG itself containing only self determinants.

Thus, the induction of reactivity to the transfectants used for immunization establishes that the immunizing transfectants are in fact immunogenic, that is, the patient has not exhibiting a much weakened capacity for immune response.

WO 97/25860 PCT/US97/00582 -43 If the patient is demonstrably capable of response to the immunizing transfectants, then skin testing with the dCTG (human) transfectants would establish whether or not reactivity to the human proto-oncogene encoded product had been induced. According to the practice of the invention, inoculation of the immunizing transfectants would continue for at least as long as the induction of reactivity to the human proto-oncogene-encoded protein occurs.

The practice of the invention is illustrated by the following nonlimiting examples.

Example 1 Immunization of Chickens Against c-src(527)-Induced Tumors By Vaccination with v-src DNA A. Genes The oncogene c-src(527) is an activated form of chicken c-src.

Its protein product pp60 c src( 52 7 differs from the protein product of c-src, pp60 c src, by only a single amino acid substitution, phenylalanine for tyrosine at residue 527 (Kmiecik and Shalloway, (1987) Cell 49, 65-73). This substitution eliminates the negative regulatory influence exerted on pp60 csr phosphokinase activity by the enzymatic phosphorylation of the position 527 tyrosine. The protein product of v-src, pp60 rc shows a number of sequence differences with pp60-src (Takeya and Hanafusa, (1983) Cell 32, 881-890), including scattered single amino acid substitutions within the first 514 residues and a novel C terminus of 12 amino acids (residues 515-526), in place of the nineteen C terminal amino acids of pp60" s rc (residues 515-533). Both the v-src-positive plasmid, pMvsrc, and the c-src(527)-positive plasmid, pcsrc527, were originally shown (Kmiecik and Shalloway, (1987) Cell 49, 65-73) to transform murine NIH 3T3 cells in culture. However, the v-src-induced transformants exhibited a more rapid or more extensive colony growth in soft agarose than the csrc(527)-induced transformants, as well as a usually shorter latency of tumor formation in nude mice WO 97/25860 PCTfUS97/00582 -44- B. Plasmids 1. pvSRC-C1 The pVSRC-CI plasmid was prepared as described by Halpern et al., (1991) Virology 180, 857-86. Essentially, the plasmid was derived from the pRL'-src plasmid (Halpern et al., (1990) Virology 175, 328-331) by subcloning the v-src(+) XhoI-EcoRI fragment of the latter into the multiple cloning sequence of pSP65 (Melton et al., (1984) Nucleic Acids Res. 12, 7035- 7056) which had been cleaved with Sall and EcoRI; since ligation of the XhoI overhang at the Sall site destroys both recognition sequences, subsequent removal of the v-src(+) insert from the vector was achieved by digestion with EcoRI and with HindIII, which cleaves at a position in the multiple cloning sequence adjacent to the Sall site. The pVSRC-C1 plasmid was restricted with EcoRI and HindlII, so as to liberate the tumorigenic insert. This insert included the v-src oncogene of the subgroup A strain of Prague RSV, as flanked downstream by a portion of the long terminal repeat (LTR) of RSV (from the start of the LTR, to the single EcoRI site).

2. pMvsrc The pMvsrc plasmid was generously provided by Dr. David :Shalloway, Cornell University, Ithaca, NY. The plasmid is prepared according to Johnson et al., (1985) Mol. Cell. Biol. 5, 1073-1083. Briefly, the 3.1-kb BamHI-Bg/II Schmidt Ruppin A v-src fragment from plasmid pN4 (Iba et al., (1984) Proc. Nat. Acad. Sci. USA 81, 4424-4428) is inserted into the pEVX plasmid (Kriegler et al., (1984) Cell 38,483-491) at a Bg/II site lying between two Moloney murine leukemia virus (MoMLV) long terminal repeats (LTRs).

This fragment contains 276 bp of pBR322 DNA from the pBR322 BamHI to Sall sites followed by 2.8 kb of Rous sarcoma virus (RSV) DNA from the Sall site that is about 750 bp upstream of the env termination codon down to the NruI site that is about 90 bp downstream of the v-src termination codon. (The WO 97/25860 PCT/US97/00582 NruI site is converted to a Bg/II site in the construction of pN4.) Ligation is performed by using a 10:1 insert-vector DNA fragment molar ratio.

The pMvsrc plasmid was restricted with NheI, so as to liberate a tumorigenic fragment. The fragment included the v-src oncogene of the subgroup A strain of Schmidt-Ruppin RSV, as flanked upstream by most of the Moloney murine leukemia virus (MoMLV) LTR (from the NheI site near the start of the LTR, to the 3' end of this LTR) and downstream by a small portion of the MoMLV LTR (from the 5' start to the Nhel site).

3. pcsrc527 The pcsrc527 plasmid is prepared according to Kmiecik and Shalloway, (1987) Cell 49, 65-73. Briefly, a plasmid is constructed by cleaving expression vector pEVX (Kriegler et al., (1984) Cell 38,483-491 at its unique BgIII site lying between two MoMLV LTRs and inserting the 3.2 kilobase (kb) pair BamHI-BgIII hybrid src fragment from plasmid pHB5 in the proper orientation. This fragment contains sequences from pBR322, the SRA env 3' region, SRA v-src, src from recovered ASV, and chicken c-src. The BgIII site is generated by insertion of a linker at the SacI site about 20 bp downstream from the c-src termination codon. The restriction map of pMHB5 contains the MoMLV splice donor about 60 bp downstream from the 3'end of the upstream LTR and the v-src splice acceptor about 75 bp upstream from the src ATG.

Plasmid pMHB5527 is constructed by inserting the synthetic double-stranded DNA oligomer CCAGTTCCAGCCTGGAGAGAACCTATA (SEQ ID NO:1) 3' 3' TCGGGGTCAAGGTCGGACCTCTCTTGGATATCTAG (SEQ ID NO:2) into pMHB5 between the BanII site at c-src codon 524 and the downstream unique BgIII site. This alters the TAC Tyr 527 codon to a TTC Phe codon while preserving the remaining c-src coding region. Equimolar amounts of the double-stranded oligomer and three gel-purified tandem restriction fragments from pMHB5 are ligated in one reaction, which contains the following: the WO 97/25860 PCT/US97/00582 -46oligomer with BanlI and BgIII complementary ends, the 3 kb BgIII-BgII (BgII in the pEVX ampicillin resistance gene) partial digest fragment, the adjacent 6.1 kb BgI-BgII (downstream BgII in c-src) fragment, and the 0.38 kb BgII-BanII (BanlI at c-src codon 524) fragment.

Plasmid pcsrc527 is constructed by replacing the 2 kb Sall (in env)-MluI (in c-src) fragment in plasmid pMHB5527, with the homologous fragment from plasmid p5H. This fragment contains the coding sequence for the c-src amino region (codons 1 to 257) that have been isolated by molecular cloning of a c-src provirus and previously shown by sequencing to contain authentic c-src sequence without the mutation at codon 63 (Levy et al., (1986) Proc. Natl. Acad. Sci. USA 83, 4228-4232). Equimolar amounts of complementary gel-purified Sall-MluI fragments from p5H and the other plasmids are ligated.

The pcsrc527 plasmid was restricted with NheI, so as to liberate a tumorigenic fragment. The tumorigenic fragment included the c-src(527) oncogene, as flanked by the same LTR complement as in pMvsrc.

C. Animals Chickens of two closed lines, SC and TK, were utilized. These lines differ at the major histocompatibility complex (B2/B 2 for the SC line,

B'S/B

2 1 for the TK line). Embryonated eggs were obtained from Hyline International (Dallas Center, IA). All chickens were hatched at the University of New Hampshire Poultry Research Farm and housed in isolation.

D. Tumor Induction by Plasmid DNA Tumors were induced by subcutaneous inoculation in the wing web of a src-positive plasmid according to the technique described by Fung et al. (1983) Proc. Natl. Acad. Sci. USA 80, 353-357 and Halpern et al., (1990) Virology 175, 328-331. Of the three tumorigenic plasmids utilized here, all were adjusted, prior to inoculation, to a concentration of 100 /g of enzymerestricted DNA per 100 Il of phosphate-buffered saline. The conditions of WO 97/25860 PCT/US97/00582 -47inoculation used for particular experiments (age of chicken at time of inoculation, amount of plasmid, etc.) are indicated below.

E. Growth of Primary (wing web) Tumors in TK or SC Chickens Inoculated with pVSRC-C1, pMvsrc or pcsrc527 Individual 1-day-old chickens of line TK or of line SC were inoculated with 100 ig of either pVSRC-C1, pMvsrc or pcsrc527. The mean tumor diameter (mm) at a particular time point and for any one group of TK or SC line chickens inoculated with an individual src-positive construct was computed as the sum of the diameters of the primary tumors divided by the number of chickens surviving to that point. The results are shown in Fig. 1A (line TK) and Fig. 1B (line SC). The ratios at each time point show, for a particular group, the number of chickens bearing palpable tumors to the total number of survivors to that point (standard typeface for pcsrc527, italics for pVSRC-C1, bold typeface for pMVsrc). Error bars (unless obscured by the symbol) indicate standard error.

F. Growth of Challenge (wing web) Tumors in Test and Control Line TK Chickens Under Conditions of Priming and Homologous Challenge with pcsrc527. or Priming and Homologous Challenge with pVSRC-Cl Growth of challenge (wing web) tumors in test and control line TK chickens was determined under conditions of priming and homologous challenge with pcsrc527, or (ii) priming and homologous challenge with pVSRC-C1. Test chickens were primed at 1 day posthatch with 100 Ig of construct; test and control chickens were challenged at five weeks posthatch with 200 ig of construct. The mean challenge tumor diameter was computed as described in the preceding section. At each time point the ratio of chickens bearing palpable challenge tumors to total number of survivors to that point is indicated for priming and homologous challenge with pcsrc527 (Fig. 2A) and priming and homologous challenge with pVSRC-Cl (Fig. 2B) SUBSTITUTE SHEET (RULE 26) WO 97/25860 PCT/US97/00582 -48- (standard typeface for control group, bold typeface for test group). The statistical comparison between the mean challenge tumor diameters of the test versus the control group at a particular time point was made using a two-tailed student's t test, The statistical comparison between the ratios of chickens bearing palpable challenge tumors to total number of survivors of the test versus the control group at a particular time point was made using a chi-squared test; the paired ratios are underlined for only those time points where p 0.05. Error bars indicate standard error.

G. Growth of Challenge (wing web) Tumors in Test and Control line TK chickens under Conditions of Priming with pVSRC-C1 and Heterologous Challenge with pcsrc527, or Priming with pcsrc527 and Heterologous Challenge with pVSRC-Cl Growth of challenge (wing web) tumors in test and control line TK chickens, was determined under conditions of priming with pVSRC-C1 and heterologous challenge with pcsrc527, or (ii) priming with pcsrc527 and heterologous challenge with pVSRC-C1. Test chickens were primed at 1 day posthatch with 100 1g of construct; test and control chickens were challenged at five weeks posthatch with 200 /4g of construct. The mean challenge tumor diameter was computed as described in Section E. At each time point the ratio of chickens bearing palpable challenge tumors to total number of survivors to that point is indicated for priming with pVSRC-C1 and heterologous challenge with pcsrc527 (Fig. 3A) and priming with pcsrc527 and heterologous challenge with pVSRC-CI (Fig. 3B) (standard typeface for control group, bold typeface for test group). Statistical comparisons were made between test and control groups at a particular time point as described in the preceding section for the student's t test], and the paired ratios are underlined for only those time points where, in the chi-squared test, p <0.05. Error bars indicate standard error.

SUBSTITUTE SHEET (RULE 26) WO 97/25860 PCT/US97/00582 -49- H. Discussion In a direct comparison of the growth of tumors induced in line TK by either pMvsrc or pVSRC-C1, a similar pattern of relatively rapid regression was observed. This result established that the difference in LTR complement between these two v-src positive constructs did not exert a major influence on the tumor growth pattern in the TK line (Fig. 1A). By contrast, much more extensive and persistent tumor growth resulted from inoculation of TK chickens with the pcsrc527 construct (Fig. 1A). The relatively greater growth capacity of tumors induced by this construct indicated that in the TK line, the c-src(527) oncogene is much more highly tumorigenic than the v-src oncogene. This difference did not, however, generalize to the SC line (Fig.

1B). The SC line was chosen for comparison with the TK line on the basis of earlier observations (Halpern et al., (1993) Virology 197, 480-484) that v-src DNA-induced tumors engender a much weaker tumor immune response in line SC than in line TK. Whereas the growth of pcsrc527-induced primary tumors was virtually indistinguishable in the two lines, the growth of the v-src-induced tumors was considerably greater in the SC than in the TK line (Figs. 1A and 1B). Thus v-src, but not c-src(527), gives rise to primary tumors whose growth patterns differ in the two lines analyzed here.

Only minimal protection against homologous challenge was observed under conditions of priming to c-src(527) DNA, indicative of the induction of a relatively weak tumor immune response (Fig. 2A; a statistically significant lowering of challenge tumor growth in the test versus the control chickens was observed at only one time point). By contrast, the v-src DNAprimed chickens showed excellent protection against the homologous tumor challenge (Fig. 2B).

Priming with v-src DNA engenders a relatively greater degree of protection against challenge with c-src(527) DNA, than that afforded by priming with c-src(527) DNA itself (Fig. 3A). The degree of protection was weaker than that determined (Fig. 2B) for the case of priming and homologous challenge with v-src DNA. Only marginal protection was SUBSTITUTE SHEET (RULE 26) WO 97/25860 PCT/US97/00582 observed, however, when the heterologous challenge protocol was carried out in the reverse order (Fig. 3B). These results demonstrate that induction of reactivity to an antigenicity specified in tumor cells by an overexpressed protooncogene can confers tumor immunity.

Example 2 Vaccination Protocol The following is a representative vaccination protocol according to the present invention.

A. Skin Punch Biopsy A punch biopsy of skin is obtained by a trained physician following standard medical practice.

B. Preparation of Primary Fibroblast Culture Under sterile conditions, the skin obtained by punch biopsy is put in a tube with 10 ml of the following wash medium: Dulbecco's Modified Eagle Medium (DMEM), containing sodium bicarbonate (30 ml/liter of a 5.6% solution) and penicillin/streptomycin (2 ml/liter of a pen-strep stock solution containing 5000 units penicillin and 5000 ig of streptomycin/ml, pH In a sterile hood, the skin biopsy is added to a Petri dish, and then transferred several times to new Petri dishes containing the same wash medium. The biopsy is then finely minced with two scalpels, and 2-4 pieces mm 3 of the minced biopsied are placed in the middle part of one or more T25 flasks. The flask is placed in a tissue culture incubator at 37"C for one half hour with the cap firmly closed, then opened for 10 minutes. The following culture medium is prepared: DMEM containing sodium bicarbonate; antibiotics; and 10% fetal calf serum containing 2.5 A/g/ml fungizone, 40 iAg/ml gentamicin, and 1% glutamine( 3 Two ml of the culture medium is then added to the flask, and the flask is incubated at 37 0 C CO2), with the cap lightly unscrewed.

The flask is left for three days without moving so as to obtain adhesion of the SUBSTITUTE SHEET (RULE 26) WO 97/25860 PCT/US97/00582 -51 separate pieces of skin to the plastic. Afterwards, the medium is changed two times per week over a 3-4 week period always adding 2-3 ml of medium. To trypsinize the skin cell culture, one needs zones of confluence. After aspirating the culture medium, 5 ml of the Puck's Saline A/EDTA solution (0.4 g EDTA to 1 liter of Puck's Solution A) is added and immediately aspirated. Then 1 ml of trypsin solution (0.05/0.02% trypsin in PBS, without Ca++ or Mg+ is added and incubated for 5 min at 37 0 C, at which time 2 ml of culture fluid is added to stop the action of the trypsin. The cells are then transferred to a larger flask (T75) and incubated at 37°C in 15 ml of culture fluid, which is changed every 2 days.

C. Fibroblast Transfection The fibroblasts (2 X 105 cells) are washed twice in DMEM without serum or antibiotics. A LipofectAMINE'"-DNA solution is prepared by mixing in tube #1 mix 400/zl DMEM and 10/l of dCTG vector DNA (ltg/ul). In tube 400 pl DMEM and 25 Ml of LipofectAMINE Reagent (Life Technologies, cat. no. 18324-012) are mixed. The contents of tube #1 and #2 are mixed together and are then left sitting at room temperature for hours. Then, 3.2 ml of the LipofectAMINE'"-DNA solution is added to the cells. The cells are incubated for six hours at 37°C, washed once with Hank's Balanced Salt Solution, and then refed with growth medium and incubated for an additional 24 hours at 37 0

C

D. Transfectant Irradiation Transfectants are irradiated to a dose of 25 By or 2500R. the cells are then counted by trypan blue exclusion. 2 X 10 7 irradiated transfectants are resuspended in a volume of 0.2-0.4 ml of Hanks Balanced Salt Solution.

E. Vaccination Patients are vaccinated by subcutaneous inoculation of 2 X 10 7 irradiated cells at 2-3 week intervals. A shorter or longer regimen is used, WO 97/25860 PCT/US97/00582 -52depending upon the results of delayed type hypersensitivity (DTH) reaction monitoring (described below).

F. Patient Assessment by DTH Monitoring Patients are assessed for reactivity to the irradiated transfectants by a test of skin reactivity in a DTH reaction, as described by Chang et al.

(1993), Cancer Research 53:1043-1050. To measure reactivity to the autologous irradiated transfectants, 104 10 6 transfected irradiated cells in a volume of 0.1 ml HBSS are inoculated intradermally. Induration is measured 48 hours later, as an average of two perpendicular diameters. Responses of greater than 2 mm are considered positive.

Example 3 v-mvc Transfection of Murine Fibroblasts A. Vector Preparation The v-myc retroviral oncogene of avian myelocytomatosis virus MC29 (Land et al. (1983), Nature 304:596-602) was obtained from the American Type Culture Collection, Rockville, MD, 20852, as the pSVv-myc vector (ATCC No. 45014). The v-myc-positive EcoRI-KpnI fragment of pSVvmyc was ligated into the polylinker sites of the pBK-CMV plasmid (Stratagene Cloning Systems, La Jolla, CA).

B. Cell Transfection Stable transfection using the pBK-CMV-v-myc vector was carried out on a line of A31 fibroblasts (Balb/c origin), obtained from the ATCC. 2 X 105 cells were seeded in a 100 mm/dish and allowed to grow for 18-20 h (RPMI 1640 medium and 10% fetal bovine serum), at which time the cells reached 50-70% confluence. The cells were then washed twice in Dulbecco's Modified Eagles Medium (without serum or antibiotics). A LipofectAMINE"- DNA solution was prepared according to Example with the pBK-CMV-v- CD/99243026.2 53 myc vector DNA, and 3.2 ml of the LipofectAMINE

TM

-DNA solution added to the cells. The cells were then incubated for 6 hours at 37°C, washed once with Hank's Balanced Salt Solution, and then refed with the growth medium and incubated for an additional 24 hour at 37 0 C. Thereafter, the cells were fed once every two days with growth medium containing 250 gg/ml geneticin (G418; Gibco BRL cat. no.

11811) as the selective marker. Within two weeks, colonies were picked and expanded into permanent cell lines. The cells were then washed and collected by centrifugation.

It should be noted that the procedure for transient transfection is the same, through the point of incubation with the Lipofectamine T M -DNA solution. Thereafter, the cells are washed and incubated for 72 hours in growth medium.

All references cited with respect to synthetic, preparative and analytical procedures are incorporated herein by reference.

The present invention may be embodied in other specific forms without 15 departing from the spirit or essential attributes thereof and, accordingly, reference should be made to the appended claims, rather than to the foregoing specification, as indication the scope of the invention.

It will be understood that the term "comprises" or its grammatical variants as used herein is equivalent to the term "includes" and is not to be taken as excluding the 20 presence of other elements or features.

0** 09 0 o o WO 97/25860 PCTIUS97/00582 -54- SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Allegheny University of the Health Sciences Halpern, Michael S.

England, James M.

(ii) TITLE OF INVENTION: CANCER VACCINE (iii) NUMBER OF SEQUENCES: 14 CORRESPONDENCE

ADDRESS:

ADDRESSEE: Seidel, Gonda, Lavorgna Monaco, P.C.

STREET: Suite 1800, Two Penn Center Plaza CITY: Philadelphia STATE: PA COUNTRY: USA ZIP: 19102 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 60/010,262 FILING DATE: 19-JAN-1996 (viii) ATTORNEY/AGENT INFORMATION: NAME: Monaco, Daniel A.

REGISTRATION NUMBER: 30,480 REFERENCE/DOCKET NUMBER: 7933-33 PC (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: (215) 568-8383 TELEFAX: (215) 568-5549 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CCAGTTCCAG CCTGGAGAGA ACCTATA 27 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS:.

LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 97/25860 WO 9725860PCTfUS97/00582.

55 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GATCTATAGG TTCTCTCCAG GCTGGAACTG GGGCT INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 1599 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GAGACTGTGC CCTGTCCACG GTGCCTCCTG CATGTCCTGC

TAGGTGACAG

CTCCACAAGC

GACGGCTCCT

TTAAACAACT

ACCTTTGTCA

TCTCCAGACG

CAGCGGGCCC

ACGACTGAGG

TTCGACTATC

AAGGCCACTG

GATGAAGTCG

CTCACTGCGC

GCCAACGGGG

GCCCGGTTTT

GTATACCGCG

ACTGACTTTG

GGGACCCCGG

GACTGGTGGG

AACCAGGACC

ACGCTCAGCC

AGGCTTGGTG

ATCAACTGGC

TCCGAGGTCG

CCCCCTGACC

CGTACCACGC

GTGGTGAATA

TCATTGGGTA

TCTCCGTAGC

TACGCTGCCT

AGAGGGAGGA

CAGGCGAGGA

AGATGGAAGT

TCAAACTCCT

GCCGCTACTA

CTCACACAGT

TGAAGTATGC

GTGAGCTGTT

ATGGTGCAGA

ACATCAAGCT

GCCTCTGCA.A

AGTACCTGGC

GGCTGGGTGT

ACGAGCGCCT

CCGAGGCCAA

GGGGGCCCAG

AGGACGTGGT

ACACAAGGTA

GCTATGACAG

TGCCACCATG

CATCAAGACC

CAAGGAGAGG

AGAATGCCAG

GCAGTGGACC

GTGGATGCGG

CCCCATGGAC

GGCGGTCAGC

TGGCAAGGGA

CGCCATGAAG

CACCGAGAGC

CTTCCAGACC

CTTCCACCTG

GATTGTCTCG

GGAAAACCTC

AGAGGGCATC

GCCTGAGGTG

GGTCATGTAC

CTTCGAGCTC

GTCCCTGCTT

CGATGCCAAG

CCAGAAGAAG

CTTCGATGAT

CCTGGGCTTA

AATGAGGTGT

TGGAGGCCAC

CCCGAGGCCC

CTGATGAAGA

ACAGTCATCG

GCCATCCAGA

TACAAGTGTG

AAGGCACGGG

ACCTTTGGCA

ATCCTGCGAA

CGGGTCCTCC

CACGACCGCC

TCCCGGGAGC

GCTCTTG.AGT

ATGCTGGAC.A

AGTGACGGGG

CTGGAGGACA

GAGATGATGT

ATCCTCATGG

GCTGGGCTGC

GAGGTCATGG

CTCCTGCCAC

GAATTTACCG

CTGGAGCTGG

TGCCCTGAGC

CTGTCATCAA

GGTACTTCCT

CTGATCAGAC

CCGAGAGGCC

AGAGGACCTT

TGGTCGCCAA

GCTCCCCCAG

CTAAAGTGAC

AAGTCATCCT

AGGAAGTCAT

AGAACACCAG

TGTGCTTTGT

GTGTCTTCAC

ACTTGCACTC

AAGATGGCCA

CCACCATGAA

ATGACTATGG

GCGGCCGCCT

AAGAGATCCG

TTAAGAAGGA

AGCACAGGTT

CCTTCAAACC

CCCAGTCCAT

ACCAGCGGAC

TGTCCCGAGC

AGAAGGCTGG

GCTGAAGAGC

TCTACCCCCC

GCGACCCAAC

CCACGTGGAT

CAGCCTCAAG

TGACTCCTCC

CATGAATGAC

GGTGCGGGAG

CATTGCCAAG

GCACCCGTTC

GATGGAGTAT

AGAGGAGCGG

GCGGGACGTG

CATCAAGATC

AACCTTCTGT

CCGGGCCGTG

GCCCTTCTAC

CTTCCCGCGC

CCCCAAGCAG

CTTCCTCAGC

TCAGGTCACG

CACAATCACA

CCACTTCCCC

120 18~a0 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 WO 97/25860 WO 9725860PCTIUS97/00582 56 CAGTTCTCCT ACTCGGCCAG CATCcGCGAG TGAGCAGTCT GCCCACGCAG AGGACGCACG CTCGCTGCCA TCACCGCTGG GTGGTTTTTT ACCCCTGCC INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 4530 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: AATTCTCGAG CTCGTCGACC GGTCGACGAG CTCGAGGGTC GACGAGCTCG AGGGCGCGCG CCCGGCCCCC ACCCCTCGCA GCACCCCGCG CCCCGCGCCC TCCCAGCCGG GTCCAGCCGG AGCCATGGGG CCGGAGCCGC AGTGAGCACC ATGGAGCTGG CGGCCTTGTG CCGCTGGGGG CTCCTCCTCG CCCTCTTGCC CCCCGGAGCC GCGAGCACCC AAGTGTGCAC CGGCACAGAC ATGAAGCTGC GGCTCCCTGC CAGTCCCGAG ACCCACCTGG ACATGCTCCG CCACCTCTAC CAGGGCTGCC AGGTGGTGCA GGGAAACCTG GAACTCACCT ACCTGCCCAC CAATGCCAGC CTGTCCTTCC TGCAGGATAT CCAGGAGGTG CAGGGCTACG TGCTCATCGC TCACAACCAA GTGAGGCAGG TCCCACTGCA GAGGCTGCGG ATTGTGCGAG GCACCCAGCT CTTTGAGGAC AACTATGCCC TGGCCGTGCT AGACAATGGA GACCCGCTGA ACAATACCAC CCCTGTCACA GGGGCCTCCC CAGGAGGCCT GCGGGAGCTG CAGCTTCGAA. GCCTCACAGA GATCTTGAAA GGAGGGGTCT TGATCCAGCG GAACCCCCAG CTCTGCTACC AGGACACGAT TTTGTGGAAG GACATCTTCC ACAAGAACAA CCAGCTGGCT CTCACACTGA TAGACACCAA CCGCTCTCGG GCCTGCCACC CCTGTTCTCC GATGTGTAAG GGCTCCCGCT GCTGGGGAGA GAGTTCTGAG GATTGTCAGA GCCTGACGCG CACTGTCTGT GCCGGTGGCT GTGCCCGCTG CAAGGGGCCA CTGCCCACTG ACTGCTGCCA TGAGCAGTGT GCTGCCGGCT GCACGGGCCC CAAGCACTCT GACTGCCTGG CCTGCCTCCA CTTCAACCAC AGTGGCATCT GTGAGCTGCA CTGCCCAGCC CTGGTCA.CCT ACAACACAGA CACGTTTGAG TCCATGCCCA ATCCCGAGGG CCGGTATACA TTCGGCGCCA GCTGTGTGAC TGCCTGTCCC TACAACTACC TTTCTACGGA CGTGGGATCC TGCA.CCCTCG TCTGCCCCCT GCACAACCAA GAGGTGACAG CAGAGGATGG AACACAGCGG TGTGAGAAGT GCAGCAAGCC CTGTGCCCGA. GTGTGCTATG GTCTGGGCAT GGAGCACTTG CGAGAGGTGA GGGCAGTTAC CAGTGCCAAT ATCCAGGAGT TTGCTGGCTG CAAGAAGATC TTTGGGAGCC TGGCATTTCT GCCGGAGAGC TTTGATGGGG ACCCAGCCTC CAACACTGCC CCGCTCCAGC CAGAGCAGCT CCAAGTGTTT GAGACTCTGG AAGAGATCAC AGGTTACCTA TACATCTCAG CATGGCCGGA CAGCCTGCCT GACCTCAGCG TCTTCCAGAA CCTGCAAGTA ATCCGGGGAC GAATTCTGCA CAATGGCGCC TACTCGCTGA CCTC4CAAGGC GCTGflCATC 1560 1599 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 WO 97/25860 WO 9725860PCT1US97/00582 57 AGCTGGCTGG GGCTGCGCTC ACTGAGGGAA CTGGGCAGTG

AACACCCACC

CAAGCTCTGC

TGCCACCAGC

TGCAGCCAGT

CCCAGGGAGT

AATGGCTCAG

AAGGACCCTC

ATGCCCATCT

ACCCACTCCT

CTGACGTCCA

TTTGGGATCC

CTGCAGGAAA

CAGATGCGGA

TTTGGCACAG

GCCATCAAAG

GCATACGTGA

ACATCCACGG

CGGGAAAACC

AAGGGGATGA

GTGCTGGTCA

GACATTGACG

CTGGAGTCCA

ACTGTGTGGG

ATCCCTGACC

GTCTACATGA

GAGTTGGTGT

AATGAGGACT

GACGATGACA

TTCTGTCCAG

TCTACCAGGA

CCCAGGTCTC

TCTGCTTCGT

TCCACACTGC

TGTGCGCCCG

TCCTTCGGGG

ATGTGAATGC

TGACCTGTTT

CCTTCTGCGT

GGAAGTTTCC

GTGTGGACCT

TCGTCTCTGC

TCATCAAGCG

CGGAGCTGGT

TCCTGAAAGA

TCTACAAGGG

TGTTGAGGGA

TGGCTGGTGT

TGCAGCTGGT

GCGGACGCCT

GCTACCTGGA

AGAGTCCCAA

AGACAGAGTA

TTCTCCGCCG

AGCTGATGAC

TGCTGGAAAA

TCATGGTCAA

CTGAATTCTC

TGGGCCCAGC

TGGGGGACCT

ACCCTGCCCC

GTGGCGGTGG

CACTGGCACC

GCACACGGTG

CAACCGGCCA

AGGGCACTGC

CCAGGAGTGC

CAGGCACTGT

TGGACCGGAG

GGCCCGCTGC

AGATGAGGAG

GGATGACAAG

GGTGGTTGGC

ACGGCAGCAG

GGAGCCGCTG

GACGGAGCTG

CATCTGGATC

AAACACATCC

GGGCTCCCCA

GACACAGCTT

GGGCTCCCAG

GGATGTGCGG

CCATGTCAAA

CCATGCAGAT

GCGGTTCACC

TTTTGGGGCC

GGGGGAGCGG

ATGTTGGATG

CCGCATGGCC

CAGTCCCTTG

GGTGGATGCT

GGGCGCTGGG

GGACCTGACA

CTCCGAAGGG

CCCTGGGACC

GAGGACGAGT

TGGGGTCCAG

GTGGAGGAAT

TTGCCGTGCC

GCTGACCAGT

CCCAGCGGTG

GGCGCATGCC

GGCTGCCCCG

ATTCTGCTGG

AAGATCCGGA

ACACCTAGCG

AGGAAGGTGA

CCTGATGGGG

CCCAAAGCCA

TATGTCTCCC

ATGCCCTATG

GACCTGCTGA

CTCGTACACA

ATTACAGACT

GGGGGCAAGG

CACCAGAGTG

AAACCTTACG

CTGCCCCAGC

ATTGACTCTG

AGGGACCCCC

GACAGCACCT

GAGGAGTATC

GGCATGGTCC

CTAGGGCTGG

GCTGGCTCCG

GACTGGCCCT

AGCTCTTTCG

GTGTGGGCGA

GGCCCACCCA

GCCGAGTACT

ACCCTGAGTG

GTGTGGCCTG

TGAAACCTGA

AGCCTTGCCC

CCGAGCAGAG

TCGTGGTCTT

AGTACACGAT

GAGCGATGCC

AGGTGCTTGG

AGAATGTGAA

ACAAAGAAAT

GCCTTCTGG

GCTGCCTCTT

ACTGGTGTAT

GGGACTTGGC

TCGGGCTGGC

TGCCCATCAA

ATGTGTGGAG

ATGGGATCCC

CCCCCATCTG

AATGTCGGCC

AGCGCTTTGT

TCTACCGCTC

TGGTACCCCA

ACCACAGGCA

AGCCCTCTGA

ATGTATTTGA

CATCCACCAT

GAACCCGCAC

GGGCCTGGCC

GTGTGTCAAC

GCAGGGGCTC

TCAGCCCCAG

TGCCCACTAT

CCTCTCCTAC

CATCAACTGC

AGCCAGCCCT

GGGGGTGGTC

GCGGAGACTG

CAACCAGGCG

ATCTGGCGCT

AATTCCAGTG

CTTAGACGAA

CATCTGCCTG

AGACCATGTC

GCAGATTGCC

CGCTCGGAAC

TCGGCTGCTG

GTGGATGGCG

TTATGGTGTG

AGCCCGGGAG

CACCATTGAT

AAGATTCCGG

GGTCATCCAG

ACTGCTGGAG

GCAGGGCTTC

CCGCAGCTCA

AGAGGAGGCC

TGGTGACCTG

1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 WO 97/25860 WO 9 725860PCTJUS97/00582 58

GGAATGGGGG

CGGTACAGTG

CTGACCTGCA

TCGCCCCGAG

AAGACTCTCT

GTGGAGAACC

CCTGCCTTCA

GGGGCTCCAC

CTGGACGTGC

GAGCAGGGAA

CAGGGGAACC

CAGATGGCTG

GATTCTGAGG

CCCTTTCCTT

CGGCCCTAAG

AGTACTGCCC

TGCTTTTCTG

CAGGGGAGAA

TTGTCCATTT

CAGCCAAGGG

AGGACCCCAC

GCCCCCAGCC

AGGGCCCTCT

CCCCAGGGAA

CCGAGTACTT

GCCCAGCCTT

CCAGCACCTT

CAGTGTGAAC

GGCCTGACTT

TGCCATGCCA

GAAGGGGTCC

CCCTGCCCAA

CCAGATCCTG

GGAGTGTCTA

CCCATGAGGA

TTTAGTTTTT

TGGGTGTTGT

GCAAATATAT

GCTGCAAAGC

AGTACCCCTG

TGAATATGTG

GCCTGCTGCC

GAATGGGGTC

GACACCCCAG

CGACAACCTC

CAAAGGGACA

CAGAAGGCCA

CTGCTGGCAT

GGAACCTGTC

AGCCTCGTTG

TGAGACTCTA

GGTACTGAAA

AGAACAAAAG

AGGAACAGCA

ACTTTTTTTG

ATGGGGAGGC

TTTGGAAAAC

CTCCCCACAC

CCCTCTGAGA

AACCAGCCAG

CGACCTGCTG

GTCAAAGACG

GGAGGAGCTG

TATTACTGGG

CCTACGGCAG

AGTCCGCAGA

CAAGAGGTGG

CTAAGGAACC

GAAGAGGAAC

GGGTCCAGTG

GCCTTAGGGA

CGACCCATTC

ATGGTGTCAG

TTTTGTTTTT

AAGTGTGGGG

ATGACCCCAG CCCTCTACAG

CTGATGGCTA

ATGTTCGGCC

GTGCCACTCT

TTTTTGCCTT

CCCCTCAGCC

ACCAGGACCC

AGAACCCAGA

AGCCCTGATG

GAGGGCCCTC

TTCCTTCCTG

AGCACTGGGG

GATGCCACAG

AGCTGGCCTG

AGAGACTGTC

TATCCAGGCT

TTAAAGACGA

GGTCCTTCTC

CGTTGCCCCC

CCAGCCCCCT

GGAAAGGGCC

TGGGGGTGCC

CCACCCTCCT

ACCAGAGCGG

GTACCTGGGT

TGTCCTCAGG

CGACCACTTC

CTTGAGTTCC

AGTCTTTGTG

CCCAGCTTGG

AGAGGGGAAG

CCTGAAACCT

TTGTACAGAG

AATAAAGACC

CACACCCACT

3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4530 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 891 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID ATGTGCAATA CCAACATGTC TGTACCTACT GATGGTGCTG GCTTCGGAAC AAGAGACCCT GGATCTTGAT GCTGGTGTAA TTGGATCAGG ATTCAGTTTC AGATCAGTTT AGTGTAGAAT TCAGAAGATT ATAGCCTTAG TGAAGAAGGA CAAGAACTCT TATCAAGTTA CTGTGTATCA GGCAGGGGAG AGTGATACAG GAAATTTCCT TAGCTGACTA TTGGAAATGC ACTTCATGCA CCATCACATT GCAACAGATG TTGGGCCCTT CGTGAGAATT AAAGATAAAG GGGAAATCTC TGAGAAAGCC AAACTGGAAA

TAACCACCTC

GTGAACATTC

TTGAAGTTGA

CAGATGAAGA

ATTCATTTGA

ATGAAATGAA

GGCTTCCTGA

ACTCAACACA

ACAGATTCCA

AGGTGATTGG

ATCTCTCGAC

TGATGAGGTA

AGAAGATCCT

TCCCCCCCTT

AGATAAAGGG

AGCTGAAGAG

120 180 240 300 360 420 480 WO 97/25860 WO 9725860PCTIUS97/00582 59 GGCTTTGATG TTCCTGATTG TAAAAAACT

ATAGTGAATG

GAGGAI4AATG ATGATAAAAT TACACAAGCT

TCACAATCAC

CAGCCATCAA CTTCTAGTAG CATTATTTAT

AGCAGCCAAG

AGGGAAGAAA CCCAAGACA AGAAGAGAGT

GTGGAATCTA

GAACCTTGTG TGATTTGTCA AGGTCGACCT

AAAAATGGTT

GGACATCTTA TGGCCTGCTT TACATGTGCA

AAGAAGCTAA

CCAGTATGTA GACAACCAAT TCAAATGATT

GTGCTAACTT

INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 657 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: ATTCCAGAGA

GTCATGTGTT

AAGAAAGTGA

AGACTATTCT

AAGATGTGAA AGAGTTTGAA GTTTGCCCCT

TAATGCCATT

GCATTGTCCA

TGGCAAAACA

AGAAAAGGAA

TAAGCCCTGC

ATTTCCCCTA G ATGTGCAATA

CCAACATGTC

TGTACCTACT

GATGGTGCTG

GCTTCGGAAC

CCCCTTCCAT

AAAGGGAAAG

GAAGAGGGCT

TGTGTTGAGG

TATTCTCAGC

TTTGAAAGGG

GCCATTGAAC

AAAACAGGAC

A-AGAGACCCT

CACATTGCAA

ATAAAGGGGA

TTGATGTTCC

AAAATGATGA

CATCAACTTC

AAGAAACCCA

CTTGTGTGAT

ATCTTATGGC

GGACTATTGG

CAGATGTTGG

AATCTCTGAG

TGATTGTAAA

TAAAATTACA

TAGTAGCATT

AGACAAAGAA

TTGTCAAGGT

CTGCTTTACA

AA.ATGCACTT

GCCCTTCGTG

AAAGCCAAAC

AAAACTATAG

CAAGCTTCAC

ATTTATAGCA

GAGAGTGTGG

CGACCTAAAA

TGTGCAAAG.A

TAACCACCTC

CATGCAATGA

AGAATTGGCT

TGGAAAACTC

TGAATGATTC

AATCACAAGA

GCCAAGAAGA

AATCTAGTTT

ATGGTTGCAT

ACAGATTCCA

AATGAATCCC

TCCTGAAGAT

AACACAAGCT

CAGAGAGTCA

AAGTGAAGAC

TGTGAAAGAG

GCCCCTTAAT

TGTCCATGGC

540 600 660 720 780 840 891 120 180 240 300 360 420 480 540 600 657 120 180 240 CCCTGCCCAG TATGTAGACA ACCAATTCAA ATGATTGTGC TAACTTATTT

CCCCTAG

INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 966 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: ATGTGCAATA CCAACATGTC TGTACCTACT GATGGTGCTG TAACCACCTC

ACAGATTCCA

GCTTCGGAA~C AAGAGACCCT GGTTAGACCA AAGCCATTGC TTTTGAAGTT

ATTAAAGTCT

GTTGGTGCAC AAAAAGACAC TTATACTATG AAAGAGGATC TTGATGCTGG

TGTAAGTGAA

CATTCAGGTG ATTGGTTGGA TCAGGATTCA GTTTCAGATC AGTTTAGTGT

AGAATTTGAA

WO 97/25860 WO 9725860PCTIUS97/00582 60

GTTGAATCTC

GAAGATGATG

TTTGAAGAAG

ATGAATCCCC

CCTGAAGATA

ACACAAGCTG

AGAGAGTCAT

AGTGAAGACT

GTGAAAGAGT

CCCCTTAATG

GTCCATGGCA

TCGACTCAGA

AGGTATATCA

ATCCTGAAAT

CCCTTCCATC

AAGGGAAAGA

AAGAGGGCTT

GTGTTGAGGA

ATTCTCAGCC

TTGAAAGGGA

CCATTGAACC

AAACAGGACA

AGATTATAGC

AGTTACTGTG

TTCCTTAGCT

ACATTGCAAC

TAAAGGGGAA

TGATGTTCCT

AAATGATGAT

ATCAACTTCT

AGAAACCCAA

TTGTGTGATT

TCTTATGGCC

CTTAGTGAAG

TATCAGGCAG

GACTATTGGA

AGATGTTGGG

ATCTCTGAGA

GATTGTAAAA

AAAATTACAC

AGTAGCATTA

GACAAAGAAG

TGTCAAGGTC

TGCTTTACAT

CCAATTCAAA

AAGGACAAGA

GGGAGAGTGA

AATGCACTTC

CCCTTCGTGA

AAGCCAAACT

AAACTATAGT

AAGCTTCACA

TTTATAGCAG

AGAGTGTGGA

GACCTAAAAA

GTGCAAAGAA

TGATTGTGCT

ACTCTCAGAT

TACAGATTCA

ATGCAATGAA

GAATTGGCTT

GGAAAACTCA

GAATGATTCC

ATCACAAGAA

CCAAGAAGAT

ATCTAGTTTG

TGGTTGCATT

GCTAAAGAAA

AACTTATTTC

300 360 420 480 540 600 660 720 780 840 900 960 966 AGGAATAAGC CCTGCCCAGT ATGTAGACAA

CCCTAG

INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 399 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: ATGTGCAATA CCAACATGTC TGTACCTACT GATGGTGCTG TAACCACCTC ACAGATTCCA GCTTCGGAAC AAGAGACCCT GGTTAGACAA GAAAGTGAAG ACTATTCTCA GCCATCAACT TCTAGTAGCA-TTATTTATAG CAGCCAAGAA GATGTGAAAG AGTTTGAAAG GGAAGAAACC CAAGACAAAG AAGAGAGTGT GGAATCTAGT TTGCCCCTTA ATGCCATTGA ACCTTGTGTG ATTTGTCAAG GTCGACCTAA AAATGGTTGC ATTGTCCATG GCAAAACAGG ACATCTTATG GCCTGCTTTA CATGTGCAAA GAAGCTAAAG AAAAGGAATA AGCCCTGCCC AGTATGTAGA CAACCAATTC AAATGATTGT GCTAACTTAT TTCCCCTAG INFORMATION FOP 'QID NO: 9: SEQUENCE ?iCTERISTICS: LENGT:- 309 base pairs TYPE: acid STRANDEiJNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: ATGTGCAATA. CCAACATGTC TGTACCTACT GATGGTGCTG, TAACCACCTC ACAGATTCCA GCTTCGGAAC AAGAGACCCT GGTTAGACCA AAGCCATTGC TTTTGAAGTT ATTAAAGTCT 120 180 240 300 360 399 120 WO 97/25860 WO 9725860PCTIUS97/00582 61 GTTGGTGCAC AAAAAGACAC TTATACTATG AAAGAGGTTC TTTTTTATCT TGGCCAGTAT ATTATGACTA AACGATTATA TGATGAGAAG CAACAACATA TTGTAA.ATGA TTGTGCTAAC TTATTTCCCC TAGTTGACCT GTCTATAAGA GAATTATATA TTTCTAACTA TATAACCCTA

GGAATTTAG

INFORMATION FOR SEQ ID NO:l0: SEQUENCE CHARACTERISTICS: LENGTH: 1897 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID CACAGATAAG GTTATTTGGG TACCCTCTCG AAAAGTTAAA CCGGACATCG CCCAAAAGGA

TGAGGTGACT

GAGAATTATC

TCATTAGAAT

ACTTAACAAA

ATACGGTCCA

GTGCAGGGAG

AGAGGAAGAT

TGCAAAGTTG

GCGCCGGAAG

GGCAACCACC

AGGCCCGCTC

TGCTGAGCCA

TATCAATGTT

TGAGAAAGAA

GAAAGGGCAG

CACCACGGTT

GGAACATCAC

TGCGTCCCCC

TCTCTTAGAA

TCACGAGAAT

GATGTCTGTT

CGGCGGAGCA

AAGAAAGATG

ACTCTACATT

CGGACAGATG

GGTCCATGGA

AAGCGCTGGT

AGGTGGCACA

AGAATTATTT

CTGCCTGGAC

GTCGAGCAGG

GGCTTCCAGA

CCGGGGGCCG,

CAAAATGTCC

CCTCAGCCAG

AAACGAATAA

CAGGCATTAC

GCTGACAATA

CACTGTACTC

GCACGGTGCA

TTTGCAGAAA

CTGAACCTGG

ACCACCCCAT

GTCTAGAGCT

AGGCGAGCCC

CATCTTTCTC

TTCAGTGCCA

CTAAAGAGGA

CGGACATTGC

ACCATCTGAA

ACCAGGCACA

GGACTGATAA

AGGGTTACCC

AGAGCAGCCA

GCCAGGCCCC

CTGGTCAGAT

CTGCTGCAGC

AGGAATTAGA

CAACACAGAA

COAGGACCAG

CATCTCCACC

TGATTGTTCA

CACTCCAGTT

ACAACCCTGC

TCCACAAGGA

CAATTATAAT

TCTTTTTGCA

AAAGATTAAT

GCACCGGTGG

GGATCAAAGG

TAAGCATTTG

TCCAGAAGTG

CAAGAGACTG

CGCTGTCAAG

GCAGGAGTCC

TCTGATGGCC

TCTGGGCAGT

CCCATATCCA

TATTCAGAGA

GTTGCTACTT

CCACACAGCA

TGGTGACAAT

AGTGGATCAT

CCAGAGCAAC

AATAGACTCC

ACTAACCTCC

CCAGACTTTC

AATCTTGCGA

GGCTGGAGGC

CTACTTGTGT

CAGAAAGTAT

GTAATAGAAC

AAGGGAAGGA

AAGAAAACCT

GGAAACAGAT

AACCACTGGA

TCCAAAGCCG

TTTGCCCACA

GACTACCCCT

GTAGCACTGC

CACTATACTG

ATGTCGACTG

AACTACCCCG

GCGCtTGTTT

GGTTGCTTAC

ATCCTGGATA

TTCTTAAACA

ACGCCAGTGT

ACTGAATACA

ATCGGGCTGT

ACATAGATAA

GTTTTATATT

TAAACCCAGA

ACGTGCAGAA

TTGGAAAACA

CCTGGACAGA

GGGCAGAAAT

ATTCCACCAT

GCCCGCCCTC

ACCCACCTGC

ACTACCACAT

ATATAAATAT

ATGAAGACCC

AGAATGAACT

GCTGGCACAG

CCTGTTTGGG

CTGAGGAAAG

ATGTTAAGAA

CATCGTCCAA

GTGGCCACAA

GGAAGATGCA

AACGGGGCAA

180 240 300 309 12.0 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 WO 97/25860 WO 9725860PCT/US97/00582.

62 GGCTTGACCG AGGGGACTAT AACATGTATA GGCGAAAAGC CTTAGGAAGT CCCCTCGAGG TATGGCAGAT ATGCTTTTGC TCTTAATCGT AGGTTAACAT GTATATTACC AAATAAGGGA AGGTATTATA TGATCCCATT GGTGGTGAAG GAGCGACCTG GAACTGTCTG TCCTTGCGTC ATTCCTCATC GGATCATGTA TAACAGGATG GCACCATTCA TCGTGGCGCA. TGCTGATTGG ACCCACGAAT GTACTTAAGC TTGTAGTTGC TAACAATAAA ACATTGGTGT GCACCTGGGT TGATGGCCGG ACCGTCGATT TGAATGAAGC TGAAGGCTTC AGGTACCCTT ACTTGAT IN'FORMATION FOR SEQ ID NO:l1: SEQUENCE CHARACTERISTICS: LENGTH: 8082 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

GGGGTCTCGG

ATAGGGAGGG

ATCGCCTGAT

AGGGCATATG

CGCGGCAGAG

TGCGACTAAG

GTGCCATTCT

CCCTGACGAC

TTGTAACGCG

GGAAATGTAG

GCACCAAATA

GGCGTTAACA

TATGATTGGA

GAGTTGTGTA

ACCTCTCACC

TGCGAACACC

1440 1500 1560 1620 1680 1740 1800 1860 1897 AGCTTGTTTG GCCGTTTTAG GGTTTGTTGG AATTTTTTTT TCGTCTATGT ACTTGTGAAT

TATTTCACGT

GTTAATTTTC

GTAACTCCTC

GGAAACTTGT

CTCCTGCTCC

GCTGCCAGTA

AGAGGGGGCG

CGCGTTTGCG

CAGTGCATCG

TCAACGGGTA

AATTGTTTTC

GTGAGGGACC

TAAAAAATCC

AAATCATGTG

GTTTGCGGGT

ACTAGGAAAT

CCCCAAAAAA

TTGCCATTAC

ACCATCTCTT

TTTCTTCGGA

TTTAAGGAAC

TGCCCCCACC

GAGGGCACAC

GAGGGAAAGA.

GCAAAGGCCT

GATTTGGAAG

ATAACCCATC

TCTTTTGGAG

AAGGATGAGA

CGAGGGAATA

TGGGGCTGGG

TACATACAGT

TAATGCCTGG

AGGCACGGAA

CGGTTCTCCA

ATGCGGTTGA

CCTTCTGCAG

CGCCTGTCCT

TGACCCCCGC

TTACTTTACT

CGCTTTGCAG

GGAGGCAGGA

CTACTATATT

TTGAACAGCG

GTGGTGGAGG

AGAATGTTTT

TACATTATAT

CAACTAGCTG

GCACTTTCAC

AAGGCAGCCA

GTAATACTCC

TAGGGTGATG

ATAGTCACCT

CCAACCTGAA

TCCCCCGCTG

CCTCGTTGAC

TTCGCAAACC

CAAAATCCAG

GTAATTTGCA

CACTTAACAC

TACATGCTAT

GAGAGAAUAAG

TTGTTTTTCA

ATTAAATATA

AGTCGAAGCG

TAGTATTCAG

AATTTTAATT

TCTCCTCTTC

TTCATTAGCA

CTGAACCACT

AGAATAACAA

GAAACCTTGC

ATCCAGGCGC

TGAACGCGGG

CATAGCGATT

ATCCTTAAAG

TTGAA.CGCTG

ACACACACCC

TTTACTTAAA

TGCCGTGGAA

GATCATTTCA

TAAATAAAAT

AAAAAATTGT

AGCTCAAGAC

TTTGATCAGA

GTGGTGATAG

TTTTCCTCCA

GGAGGTGGCT

ACCTCGGACG

GATGATCTCT

TGCTGCCCAG

GGTTGCTCCC

CTGAATTGTG

AGCTGCAAAC

CTTTCCCCCG

ATGCCTTTGG

TAACACAAAA

GGGAGCAAAC

GTGAATACAC

GAGTCAGTGA

TCCCCCCCCC

ATCGATGCAT

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 WO 97/25860 WO 9725860PCTIUS97/00582 63

TTTTTGTGCA

AACGTCCGGT

ACTCCCCCGG

GTTCGGGTAC

CCCGGCAGCC

CTGAAACCTG

GTACAGACTG

AGACACATCT

GCGGGGCAGG

GGAAGGTATC

CAAATGCAAT

GGGTTATTTT

TCTACACTAA

TCCCTGGGAC

CCTGGTACGC

GTTCCGCCTG

TACGGAGGAG

GCGCGTAGTT

GCTGCCCGGC

CCTCCCGGGT

TCTCCGCCCA

GCTGCTCGCG

AGGGCAGGGC

ATAAAAGCCG

GGGAGCGAGC

CGTCCTGGGA

CTGATCCCCC

GCGGGCGGGC

GCGGGGAGGC

CTGAAAGGCT

GGTAAGCACC

GAGTCGAATG

TGACCGCATT

TTGTCCGGGG

CTCGGTCCAC

CCCCTGCCCC

CGAGACTGTT

GCTGAGAAAT

GCAGAGAGCA

CAGGGCTAAA

AGGGGCGGTA

CAATCCAGAT

GGGAGTTTAT

AATCATTCTA

CATCCCACGC

TCTTGATCAA

GCGTGGCGTG

CGATGATTTA

CAGCAGAGAA

AATTCATGCG

TGAGTCTCCT

TCCCAAAGCA

CCGGCCCTTT

GCCGCCACCG

TTCTCAGAGG

GTTTTCGGGG

GGGCGGCCGG

AGGGAGATCC

AGCCAGCGGT

ACTTTGCACT

TATTCTGCCC

CTCCTTGCAG

GAAGTCCACT

CCTAAATAGG

TCCAATAATA

AGGAAAGAGT

AAGCTCTCCA

TCCCATATTC

GCAAACCGGC

TGGGAACTCC

GGCAACCTCC

CAGACGCCTC

TCTGCTGCTT

AGCTGTGCAT

TCATAACGCG

GGCATCGTTT

TCTGAACGCG

AGCGCGGCCC

GCGGTGGGCG

TACTCACAGG

AGGGAGAGGG

GCTCTCTTAC

CCCCACCTTC

GAGGGCGTGG

ATAATGCGAG

CCGGGCCCCG

CTTGGCGGGA

CTTTATCTAA

CTAGGGTGGA

GGAGCGAATA

CCGCAACCCT

GGAACTTACA

ATTTGGGGAC

CTGCTTAGAC

TGCCTTTTA.A

GTGTCTTTTC

AAAGGGGAAA

TAACGGTTTT

CTTGCCCCTT

TCCCGTCTAG

GCCACAGGGC

GTGTGGGAGG

CTCTCGCCCT

CCGCACGGGG

TGGCAGCAAA

ACATAATGCA

CTCTCCAAGT

TCCTCCTTAT

CGCCCATTAA

TTTCCCCAGC

CGCAGTGCGT

ACAAGGATGC

TTTGAGAGGG

TCTGTTTACA

CCCACCCTCC

GGGAAAAGAA

GGTCTGGACG

GCCGTCCCTG

AAAAGAACGG

CTCGCTGTAG

AGAGCCGGGC

GGGGGCTTCG

TGCCGCATCC

ACACCCGAGC

ACTTCCCCGC

GCTGGATTTT

TTTATTTTTT

TCCCATTCCT

GAGGACCTGG

TTTCACAAGG

TTAGGAAGTC

CACCTTTGAT

GCAAAGGGGA

CGTGGGGGTG

AGCCCAGCTC

CCCCACGGAA

TTGGGGGACT

TAATACATGA

ATACGTGGCA

GCCTCTATCA

TACCCTTCTT

CTTAGCGAGG

TCTCTGTGTG

GGTTTGTCAA

AGCAAAAGAA

TCCTAGAGCT

CCACCCTCCC

AAAAGATCCT

GCTGAGGACC

GCTCCCCTCC

AGGGAGGGAT

TAATTCCAGC

GAGCAGAGCT

CCTCTGGCCC

ACGAAACTTT

AAGGACGCGA

CGCTGCCAGG

TTTCGGGTAG

TATCACTTTA

GCGCTATTGA

AAAGGAATTA

GTCTCTGCTG

CGGTCCCGCG

TTCTCCCAAA

TTTGTCTCTT

GGACGGTGGG

TGGAACAGGC

GCCTGAGCAG

CAGTCTGGGT

CTCCCCCCAA

ATGCGTTGCT

TTCCTCCCTA

TCCTCCACTC

CGCCCTGCAG

GAGGGCAGCT

ACAGTACTGC

AATGGTAGGC

AGAGTGCTCG

CATA.AGCGCC

CTCTCGCTAA

CCCGAGCTGT

TGCCTCGAGA

CGCGCTGAGT

GAGAGGCAGA

GCGCTGCGGG

AGCCCTCCCG

GCCCATAGCA

CTCTCCCGAC

ACCCGCTTCT

TGGAAAACCA

ATGCTGAGAT

CACTTTTCTC

1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 WO 97/25860 WO 9725860PCTIVS97/00582 64

AGAGTAGTTA

GTGACTTGTC

ACCCTTTCGA

TCCTGCTTTA

GGGCAGAAAG

AGCCAGATCG

GCTCACCGCA

CGCGGCGATT

CAATAATA-CA

CAGGCAGGGG

AAACCAGGGC

CCTCGCGCCC

CTGCTACCCT

GCATCTCCGT

TTTATGGAGG

AGAAAAGCTG

TGGAGAGGGA

CGAGTGGGAA

GCGCCCAGGC

GGCAAATTGT

CGATTCCTCT

CGGGGGCTCG

ACTGTCCAAA

TTTAGGGGAT

CCAGGTTTCC

TCCAGCAGCC

TCGACTACGA

AGCAGCAGCA

AGCTGCTGCC

ACGTTGCGGT

CCACGGCCGA

GTTTCATCTG

TGGTAACTGG

AAGATGGGAG

GATTTCTGCC

TTGTGTTAAT

CCCCTTGCAT

CTCCGCAGCC

TTTCTGACAG

CCAACCCGCC

ATTTAAAACC

AAAAGGGAGG

GAATCTCCGC

GAGATGCGGA

TGGTGGGGTG

ATTGAGTGCG

GGTGTTAAAG

GCAAAAGGAG

AGGTTGGGAG

CAGCCGCAGC

GCCTCTCGCC

TTTCCTCACC

GCCGCTGCGG

GCGGGCACCA

GGGGGTGAAA

AGCTCTGCAA

GCACCAAGAC

TCCCGCGACG

CTCGGTGCAG

GAGCGAGCTG

CACCCCGCCC

CACACCCTTC

CCAGCTGGAG

CGACCCGGAC

GGCTGGGGTG

AGGAGAAGGC

TTATGAATAT

TGCTCTCTGG

CCTGAGCTCC

GCTGACTTGT

CCGGAGACGG

CTGATCCTTT

TGGGTCTCTA

CGAGGATGTG

ACCCAGCCCT

GGAACTGCGA

GCTCCGGGGG

AAGGGAGGTG

CCCGCGGCTG

TGTTGGACGG

GGGCTGCGGT

GGAGGGGCCC

TTCTCCTTCA

GCCACCTCCC

GGCCGACTCC

AGCCGCTGGT

GGGTGCTCCC

GGGGAGAGGT

CCCTTTAACT

ATGCCCCTCA

CCGTATTTCT

CAGCCCCCGG

CTGTCCCCTA

TCCCTTCGGG

ATGGTGACCG

GACGAGACCT

GGGGGTAATC

AGAGGGAAAA

ATTCACGCTG

GTTTTGGGGG

TTGGAGTAGG

CCCCGTCTCC

ACACTGCGGC

TAP.GAAGTTG

GAGGTGTTAG

TCCGATTCTC

GACTCCCCTG

GGAGCGGGGC

AGGTATCGCA

CCCCTATTAT

AGCTCGCCAC

GGGCGGTACT

GCCGGCGGGG

CGGCGCGGAG

GGTGGCGCAA

GCGGCTTCTT

CGGGCTTTGC

TCACTAAGTG

TTTATTCCCC

TCGGGACTGT

CAAGACTGCC

ACGTTAGCTT

ACTGCGACGA

CGCCCAGCGA

GCCGCCGCTC

GAGACAACGA

AGCTGCTGGG

TCATCAAAAA

CAGAACTGGA

CGGGAATGGT

ACTCCCGGCC

GCTGGGGGTT

GACCGCATAT

GGGAGGGCAT

GCGTCCCGCC

GCATTTGGCT

GACGTGGTGT

CTGGAATCGT

CCGCGGCCGC

TCTGGGCGGT

GCGGGGTCTC

TATTTGACAC

TCCAGCCGGC

GGGGGTGGGG

GTAGGAGAGC

CGGGGTTCAC

AACTTTOTGC

AAGGGCGCCA

GCTCCGGGCT

CGTCTCCGAG

CACCAAGACC

GGCGCGCACT

TCCCGCTTTG

CACCA ACAGG

GGAGGAGAAC

GGATATCTGG

CGGGCTCTGC

CGGCGGTGGC

AGGAGACATG

CATCATCATC

TCGGGGTAAA

TTTTAAGACT

GGTCGGACAT

GCTTTGCGGT

CGCCTGTGTG

TTAAATTTCG

CGCCTGTCCC

TTTTAAAAAG

TGGGTAGGCG

TGACTTGGAA

CCTCGGGTGT

TCCAGAACAG

TGGCGCAGTT

CCCCCTTGTA

GAGAGAAAGA

ACGGGGGCGG

GGCTAGGGCG

GCAGCCGCTA

CTTGGATTTT

GGGCCGATTT

CCCGGGGGAG

ATAGCAGGGG

ACCCAGCCGC

GCGCGCTGCG

TGTGCCCCGC

TAACTATGACC

TTCTACCAGC

P.AGAAATTCG

TCGCCCTCCT

GGGAGCTTCT

GTGAACCAGA

CAGGACTGTA

3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 WO 97/25860 WO 9725860PCT/US97/00582.

65

TGTGGAGCGG

CTGCGCGCAA

CCAGCTTGTA

TCTTCCCCTA

GCGCCTTCTC

GCCCCGAGCC

AGCGAAGCCC

GCAGCTTATT

GGGAGCTCAT

ACTGCCCATG

GGTTTTTAAA

CATTTTAATT

GATCTCTGAG

GAACTATCTA

GGAGCCAGTG

TTTGAACACT

CCCCCGCCCT

AGTAAAATAG

TGAAGTGTTC

GGGTGTGTCC

GAAGTTGCAC

ACCTTCATGG

CTTTTTTAAG

CCAACCCCCT

TGCACACCAC

TGGCCAGGAT

GATTACAGGT

TCCAGAGACC

ATTATTTTAA

ACAAGAAGAT

AAGGTCAGAG

GGTCCTCAAG

CTTCTCGGCC

AGACAGCGGC

CCTGCAGGAT

CCCTCTCAAC

TCCGTCCTCG

CCTGGTGCTC

GCCCAGGCCT

TAACGGGCCA

CACCTCTGAA

TTTGCAGCCC

CCTTCTGGCT

GCCCTGGGGC

AGTGAATGAA

CAAAAATGAG

AACTGCCTCA

GAAAAGCAAA

CTTGGACTTT

GGAGTTGCTA

TTGGTAAAGT

AAAGCCTCAT

TTTTCTTGTC

TGAGAGGAGT

ACGGAGTCTC

GGTTCAAGAG

CATGCCAGGC

GGTCTCTCCT

GTGAGCCAGG

TTTCTAACGT

TGTAACCTTG

GAGGAAGAAA

TCTGGATCAC

AGGTGCCACG

GCCGCCAAGC

AGCCCGAACC

CTGAGCGCCG

GACAGCAGCT

GATTCTCTGC

CATGAGGAGA

GTCAAAAGTG

CTCTTATTAG

ACCTTGGGCT

CCCTCCCCGT

TATCTTACAA

GGGGTGGCAG

TTGCTTCCCT

GGGCTGTGTT

AGAGTGGGTG

TCCTTGCCAA

TGGCAAAACT

AAGTCATACC

CCCTCAAAAA

TAAGTCTTAG

CATGCCATAA

AAGGGTGGCT

ACTCTGTCAC

ATTCTCCTGC

TAATTTTTGT

GACCTCACGA

GCACCAGGCT

ATTCATGCCT

CTAAAGGAGT

TCGATGTTGT

CTTCTGCTGG

TCTCCACACA

TCGTCTCAGA

CCGCCCGCGG

CCGCCTCAGA

CGCCCAAGTC

TCTCCTCGAC

CACCGCCCAC

GGCGGCTGGA

GAAGGAGAGA

TTAGCGTTTC

TTGTCTCCCA

CTCAATCCAC

GGAGTGTATG

CTTAACTTCC

TAGAGGCTAG

GGCTGAGGAG

AGTTGGACTT

GCAATTTTTT

AAGCAATTTG

TAGGAGGTGC

GTAAGAATTG

CCCAGCTGTC

GGCTAGATTG

TAGGCTGGAG

CTCAGCCTCC

AATTTTAGTA

TCCGCCCACC

TAGATGTGGC

TGTATTTGTA

GATTTCTATT

TTCTGTGGAA

AGGCCACAGC

TCAGCACAAC

GAAGCTGGCC

CCACAGCGTC

GTGCATCGAC

CTGCGCCTCG

GGAGTCCTCC

CACCAGCAGC

TACCTTTCCC

TAGCAGATCT

CTCCCATCCC

CCCCTCAGGA

TTCTTCTTAC

AATGAGGATA

GAGAAGTGGT

GCAGGGCCTG

CTGGGATCTT

TTTTTTTTCT

TTTTTTTATT

CAGCTATCAT

TTGGGAATGT

GCATCAATGT

TTTCCCTTTA

GTTCTTTTTT

TGCAGTGGCG

CAAGTAGCTG

GAGATGGGGT

TCGGCCTCCC

TCTTTGGGGA

CAGCATTAAT

TCCTTTCTTA

AAGAGGCAGG

AAACCTCCTC

TACGCAGCGC

TCCTACCAGG

TGCTCCACCT

CCCTCGGTGG

CAAGACTCCA

CCGCAGGGCA

GACTCTGGTA

ATTTTCATTG

GGAGAGATTT

TTCCCCTTAG

ATTTCATTTA

CTCCCGTTAA

AGAGAGGATT

GGGATTTAAT

CCTGAGTGCG

CTCAGCCTAT

TTTATTCCTT

TTTCATTTCC

TTGCAACACC

GCTTTGCTTT

CCTATCCTGG

TGAGACTCTT

TTTTTTTTTC

CAATCAACCT

GGACTACAGG

TTCATCGTGT

AAAGTGCTGG

GATAATTTTG

CTGGTAATTG

AAGAGGAGGA

CTCCTGGCAA

ACAGCCCACT

CTCCCTCCAC

4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 WO 97/25860 WO 9725860PCTIUS97/00582 66 TCGGAAGGAC TATCCTGCTG CCAAGAGGGT CAAGTTGGAC AGTGTCAGAG TCCTGAGACA

GATCAGCAAC

GAGGCGAACA

TGCCCTGCGT

CCTTAAAAAA

TGAAGAGGAC

GAACTCTTGT

CAATCACCTA

GTCTTGAGAC!

AAAGAACTTT

TGTTTTTAAAS

AAATAACTTT

CCTAGTATTA

AAGTTGATTT

GCCAAATCTT

CCTGTTTGTT

CTCTGTTGAA

TTCCTGGAGA

GAGAAGGTGA

AAGAGGCATA

TTAACCA.GTG

AACCGAAAA~T

CACAACGTCT

GACCAGATCC

GCCACAGCAT

TTGTTGCGGA

GCGTAAGGAA

TGAACTTGTT

TGAAAGATTT

TTTATGCTTA

AAATTTTAAG

AATAAAACGT

TAGGTACTAT

TTTTCTATTG

AAGTTGTGAA

TTCATCAATT

ATGGGTCTGG

CTATGATAAC

GAGGTAGGCA

AGGACTGGGG

CCAGTCCCCT

GCACCAGCCC

TGGAGCGCCA

CGGAGTTGGA

AC!ATCCTGTC

AACGACGAGA

AAGTAAGGAA

TCAA.ATGCAT

AGCCATAATG

CCATCTTTTT

ATTTACACAA

TTATAGCAGT

AAACCCTAAT

TTTTTAGAA.A

TGTTTTGTTT

GCCCCTTCAG

GGGCCTTAAG

AGCCAGAGTT

AAGGAGATAC

AGTTGGGAGG

GCTCACTCCA

CAGGTCCTCG

GAGGAGGAAC

AAACAATGAA

CGTCCAAGCA

ACAGTTGAAA

AACGATTCCT

GATCAAATGC

TAA.ACTGCCT

TTTTTCTTTA

TGTTTCTCTG

TACACAGAAT

TTTTTTTATT

AAATAAAATA.

CGTTTCTTCC

AGGGCGGTCT

GTCTTTAAGT

GACAGTTAGA

AAGGTCAA

AAGGTGAGGA

P.ACCCAGGAA

GACACCGAGG

GAGCTAAAAC

AAGGCCCCCA

GAGGAGCAAA

CACAAACTTG

TCTAACAGAA

AACCTCACAA

CAAATTGGAC

ACAGATTTGT

TAAATATTGC

TTCAATCCTA

TAAGTACATT

ACTGGCAAAT

CCCTCCCAAC

TAAGAAAGGC

TCTTGGAGGT

AGGAATGGCA.

AGGTAGCAGT

AGAAACTCCT

TT

AGAATGTCAA

GGAGCTTTTT

AGGTAGTTAT

AGCTCATTTC

AACAGCTACG

ATGTCCTGAG

CCTTGGCTGA

TTTGGGCATA

ATTTAAGAAT

CATTAAATGT

GTATATAGTA

TTGCTTTTTA

ATATCATTGA

CACCACCATC

AAGAGTTTTC

TCTAAGATGC

GAAGGCAGGT

TAAGTACACA

GTTACTTTAG

6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8082 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 4480 base pairs TYPE: nucleic acid STRAN~DEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:12: AGGGTTACAC GTCTTAACTC AGAGTTGCAA CAGGCTTGAA CAAGCCCAGG TACCTAGGGC CGAGTCACCG TTAAAACTAA CAGACCATAA AAGGAAAGGA GACTAGGAGT ACCGGATCTG ACTCACAGGC CACCTGGCAG GAAGAGATAA CCGACATTCA GGACGTCCCA GCCCGCACGT ACTCTTACCA TGTTACAACC ATGATTCAAA CCTGCCAATG TGTGTAGCTA TACCTTATCA CCTCATCTTG AATCATATGT GAACATGTCT ATATGCTTCG TTTAAATCCA CCAATCCCCG

CACGCCCAGA

ATACAGAACA

GCCCCAGCCC

TCATTCGAAT

TGAAATAACC

TAACTATGCA

120 180 240 300 360 WO 97/25860 WO 9725860PCTIUS97/00582 67

TCTGCTTCTG

GTTGGGCGCG

AACCCTCTTG

CCTCCTGAGG

GGAGATCCTC

TCGTGTCTTG

TTGGACTCAG

TCAGGACCCT

CTCCCCCCGC

AGCCCGTATC

CTCCATCTGA

TTTGTCTGAT

TGGGACAATC

ACCGAGCACG

TCGCGCCAGC

CAGAATACAA

AGCTGATCCA

AACAGGTAGT

AAGAGTATAG

TTGCCATCAA

GGGTGAAAGA

CTCACACTGT

TTGAAACATC

AGATTCGGCA

TGAGCTGCAA

CGTACGGACA

CTCCTCCTTG

TTTCCCCGAG

AGGAAGAATC

CCCTTATAGA

GAGAAGAGGA

CTGGAAGACT

TACGCCCGCT

CAAGTCCTCC

CTGATTGCAT

GAAAGGTCCT

CGCCCAGAGA

CCCTGTCTTG

ATCTGGGTTT

CAGCGCCTCC

AGCCCTGGAA

GGAGGGATAC

CTCTTTGTTT

CGTTGGATTT

GCTAACAACC

TGATCAGTCG

AAGCGGTGGG

GCTTGTGGTG

GAACCATTTT

CATTGATGGG

TGCCATGCGG

CAACACCAAG

TTCAGATGAT

TGAGTCTCGO

AGCCAAGACC

GCATAAACTG

GTGTGTGCTG

CCCTGACCAG

GGCAGAACCC

CCCACCTGAC

TCCCAAAGTC

TCTCCTGTTG

AGTGGAGCCG

TCGGGGACGA

TCTGCTTCCC

GAAGAGACTG

CCGAGTGGCC

CTCCGGAGGT

TCACCGACCA

TCTTGTCTTG

TGGTCGAAGG

GTTTGGGCGG

GACGTTCCAA

GTGGTTTTGG

TGGGTTTTAC

GTCTGTCTAA

CCCTTGAGTC

GTCGAGATCA

GCAGGAGCTC

GTGGGCGCTA

GTGGACGAGT

GAGACGTGTT

GACCAGTACA

TCCTTTGAAG

GTGCCAATGG

CAGGCCCAGG

CGACCAGGTG

CGGAAACTGA

TCCTGACACC

GTGGCCTACA

TTTGTGGACC

CCTTTGGTTG

CCTCCCCCGA

GAAGAACCTC

CCTGCTAGAC

CGCGAGGTGG

CAAACCCTAT

TGTGCCCGCA

TCGGCTCGGT

CTTTTCATTT

CCCACCGGGA

TCCTGTGCGC

AGAAGGCCCA

GTCAGAGAAG

GGGTGTCTGG

TTGGAGGAGA

GTCGAAGCCG

TCTGTGCCCT

TCACTCTAGA

AGAAAGGTCC

CTGGTTTGGC

GAGGCGTGGG

ATGATCCCAC

TACTGGACAT

TGCGCACAGG

ACATCCATCA

TGCTGGTGGG

ACCTTGCTCG

TGGAGGATGC

ACCCGCCTGA

AGGTTAAGGA

TTGTCACCTG

CGAATTGGCT

CTTCTTCCTC

AACCTGTCCT

CTCCGTACCC

CTCGACTCGA

CGCCAGACTC

AAAAGCCCCA TGCTAGAGCT

GGTACCTGTG

CATTGGGCGC

TGGGGGCTCG

GGTAAGCCGG

GTGTTCAGTT

GGGCTTCGGT

GAGCTGACGA

AGCCCGGTTC

GGGTCCAGGA

CGCGGCGCGT

AATTTTCTTT

CCATTGGAAG

TCTCCGGAGG

AGCCCCTGTA

AAAGAGTGCC

TATAGAGGAC

CTTAGACACA

GGAGGGCTTC

GTACAGGGAG

CAACAAGTGT

CAGCTATGGC

CTTCTACACA

TGAGAGTGGC

CCTGATTTTC

GGAGAGCTTG

TCCTGTTTCC

TCTCTATCCT

CCCAGAGGAC

TGTACCTACA

GGCGGCCCCT

CACCTCCCAG

TTTTCCAATA

TTGGGGGTCT

TCCGGGATCT

CCGGCATCTG

CGTCTCAGTT

TTCTCAGGGT

GCTCGGACTT

TTTGGGGCTC

CCCTCGGCAC

CTGTCTGTTA

GAAGCTACCA

GACGTCCGAG

TCGGGGACAG

GAAGCGATGA

CTGACCATCC

TCCTACCGGA

GCAGGTCAAG

CTCTGTGTAT

CAGATCAAGC

GACCTGGCCG

ATCCCCTACA

CTAGTACGTG

CCTGGCTGCA

CGCCAGAAGC

GCATTTAGCC

CCTAAACCTG

GCTCTAACTA

CCAAATTCCC

GCCCCGCCAA

TCCCCTGTGG

GCCTTTCCGC

420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 WO 97/25860 WO 9725860PCT/US97/00582 68 TTAGACAAGG GGCTGGCGGC CAGATACAAT ACTGGCCATT CTCAGCGGCC GACATATATA ACTGGAAACA ACACAACCCC AATCTGTCTT GCTTACCCAI

TCCTGACCTC

ACAATGGACG

CTGATTGGGA

TCCTAGCGGG

TGGTACAAGA

GCATGTATAC

TCATCTGGCA

GGTATACACT

AAGAAGAAAA

GAGAGTTGAG

TGGGAGTTCG

GACACTGGGC

CTCCCTCTTG

CAGGATAACT

GCACTCAGTC

AGCCACTGGC

TAAGGTGACC

CTTGCTTACC

GCCCCGCGGT

TGAACCAGAA

ACTGGCCTGG

CCAGTTAAAG

TTATCAGGGC

CCGGTCACCC

GCCAGTACAA

AAACCCTTAT

TCTGAAGGAT

TGAGTGGAAA

TGAAGAGAAG-

CCCCACCTTG

TTTCACCACC

TCTCCGAGGG

GGCTGCGGAG

CCCTTATGAT

GGCAGCACCA

GCAGGATTTA

GAAAGATAAA

TCGAATCTTG

AAAGGGGCCA

CAGAGATTGC

GCCCTAGATA

CTTGAAG-TTG

CTCACCCAGG

AGCAAGAGAT

CATTCCTTCT

AAATTAAAAG

ATTCCTCTTC

CAGGACAAGC

GCAGAGACTG

GCCACCGCGA

ATAAAGCCAC

TGGAATACGC

GATTTGAGAG

AACCTACTCA

GCTTTCTTCT

GACTCTGAAA

CCCTTTTCTA

CAGCCCACTT

CAGAGAGTGC

CTCCCGAAAG

GCTAAAGGTA

GCGGCACGAC

ACTCCCTCAG

CCAGATGATC

GATATCAGGG

CTTAAGGAGG

ATACGTAGAG

GCCGCCGTAG

AAGCTAGATA

CCTAAGAAA~C

AAGATTAGGG

GGGGGCAGCC

CCCCTGGACA

ACCGTTGGAC

TACATGTTCC

CTCAGATCCA

AGATTTTAAC

CAAAATCTCC

GCGGGATGGG

CTCCTGTCTC

ATATCAGGAG

CTCTGCTACC

AGGTCAACAA

GCACCCTGCC

GCCTCCGGCT

TGGGGCTTTC

*AGGATCCGGT

GGGATGATAT

TCTTAGAGGC

AGATCGATGA

GGAGACACCT

GCCCCACCAA

CCTTCCTAGA

CAGGACAAAT

CCAAGCTACA

CAGAAAGAAT

AAAAAGATGA

TTCAGGGTCA

AGGATCAATG

CCAGCGGCTC

AGGTCAGGGC

AGTCACCTTT

ACTCAGCGAC

TACAGATCGA

GGACTGCCCA

TTTTGAAGAA

CCTTCAGTTA

AGAAATAGAC

GTTGGCGCTC

CATTAAACAG

GCTCTTAGAC

TGTTAAGAAG

AAGAGTAGAA

TCCCACCCAT

GAGCCCAGAA

GGGACAGTTG

GGCTCTCACC

ACAGCAACTT

CAGGAAACAT

TGCATTCCCA

ACGCCTTTAT

TTTGGCTCAG

GAGACTTAAG

GACAAATGTC

GAGAATAGAA

TTATAACAAG

GAGAGACCGA

AGAGAAAAGG

TGCGTATTGC

CGAAGACCCC

CAGGAGCCCC

CTGGTGGACA

CGGACGGCCT

CGGGTTCAGC

TACCCTCTGC

GGAGGGACCC

GAAGATGAAT

TCTTGGGTCA

CAACAGCCTC

TACCCCATGT

CAAGGCATCC

CCCGGCACTG

GATATTCATC

ACTTGGTATA

AGCCAGCCCT

A~CTTGGACAA

AACCAGATAG

TTACAGGCCC

GTTTTGGGGG

CTTACAAGAC

CGCCAGTTGC

GTAAAACAAG

GAAGCTTATC

TCCATGTCCT

AATTTACAAG

AGAGAGACAC

AAAAGAAACA

GGAGAGAGGG

AAAGAAAGAG

GCCCACAGAC

CCCCTGAGCC

CAGGAGCCCA

GGGTACAAGG

TGGCTACTGG

TGGGCCGTGA

GAGTAACCGG

ATAGATTATA

CGAAATTCCC

CCCTAATTAT

CATGGGAAGC

TAGTCCCTTG

GAGACTATAG

CAACTGTCCC

CGGTCTTAGA

rATTTGCTTT

-GTTACCACA

2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 GGGTTTCAAA AACAGCCCAA CGCTCTTTGA TGAGGCCTTA CACCGGGACT TGGCTGACTT WO 97/25860 WO 9 725860PCTIUS97/00581 69 TCGAGTCCAG CATCCCACTC TTATACTTCT TCAGTTTGTT GATGACCTTC TTCTAGGGGC CACTTCTGAG ACAGCATGCC ACCAGGGAAC AGAATCCCTC TTGCAGACTT TGGGGCGATT GGGCTATCGA GCTTCTGCCA GAAAGGCTCA AATTTGCCAG ACCCAGGTTA CTTATTTAGG CTATCAACTA AGGGATGGAC AGCGATGGCT GACTCCGGCT AGGAAACAGA CCGTGGCCA.A CATCCCAGCC CCAAGAAATG GCCGACAGCT ACGGGAATTC INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 565 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GCTGAGTAGT GCGCGAGCAA AATTTAAGCT ACAACAAGGC AAGGCTTGGC CGACAATTGC 4260 4320 4380 4440 4480 120 180 240 300 360 420 480 540

ATGAAGAATC

ACGCGTATCT

GTTAGGAGTC

CTTATGCAAT

CTTACAAGGA

TGCCTTATTA

CATTGCAGAG

CACCACATTG

CACCTGCATG

TGCTTAGGGT TAGGCGTTTT GAGGGGACTA, GGGTGTGTTT CCCTCAGGAT ATAGTAGTTT ACTCTTGTAG TCTTGCAACA GAGAAAAAGC ACCGTGCATG GGAAGGCAAC AGACGGGTCT ATATTGTATT TAAGTGCCTA GTGTGCACCT GGGTTGATGG AAGCAGAAGG CTTCA

GCGCTGCTTC

AGGCGAAAAG

CGCTTTTGCA.

TGCTTATGTA

CCGATTGGTG

GACATGGATT

GCTCGATACA

CCGGACCGTT

GCGATGTACG

CGGGGCTTCG

TAGGGAAGGG

ACGATGAGTT

GAAGTAAGGT

GGACGAACCA

ATAAACGCCA

GATTCCCTGA

GGCCAGATAT

GTTGTACGCG

GAAATGTAGT

AGCAACATGC

GGTACGATCG

CCGAATTCCG

TTTGACCATT

CGACTACGAG

INFORMATION FOR SEQ ID NO:14: Wi SEQUENCE CHARACTERISTICS: LENGTH: 1804 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GGATCCTCAG GGGTAACACC TTTTGG.AGGT GGGCATCTTC CTCATTCTCA GTTCATATCC TGCTGGCTTA ACACGTGGTG TTACTATATT TGTGGCCTTA AAGCTAGAAC TACAGAAGAC CTTTCATTTA AGAAGGGTGA AAAATTTCAA ATACAGAAGG AGACTGGTGG GAAGCAAGAT CAATCACTAC AGGAAAGAAT TGAGCAGTTA TGTAGCGCCT GCAGATTCCA TTCAGGCAGA AGAATGGT.LATJ TGGGGAGAAA AGATGCTGAA AGATTACTTC TGAATCCTGG AAATTAATGA TAGGAAGAGA GAGTGAAATG GCTGGGTGCA GTGGCTCATG CCTGTAATCC

GTGGTGCCAA

TATGATTATG

ATAATTAACA

GGTTATATCC

,1TTGGCAAAA.

GGTATTTTCT

CAGCACTTTG

120 180 240 300 360 420 WO 97/25860 WO 9725860PCTIUS97/00582 70

GGAGGCCGAG

GTGAAACCCC

CCTGTAATCC

AGGTTGCAGT

TCCGTCTAAA

TACTAAAGGT

GAAACACCAC

ACTTGATACT

CAAGTTAACA

TTGGGAAATC

CAAAGTGTGG

AGGTACAATG

TGGTAAACTT

ATTGATGTCA

GCTTCCACAA

AATGAACTAT

GTGCAAAATA

ACAAGGTGCA

TACAATAAAG

CAGAGTGCCA

CAGGATGCCT

GAAGAAGGAC

CTTCACTGCT

TTTA

TTGGGCGGAT

ATCTCTACTA

CAGCTACTCA

GAGCTGAGAT

AAACAAATAA

GCTTATTCCC

AAAATTAGGA

CTGCAGAAA~T

ACTGTGTGTC

CCTTGATAAT

ATGGGAATAT

ATGCCAGAAG

GTTCCACTAT

AAAGGAAGCT

ATGGTTGATA

ATTCACCGAG

GCAGATTTTG

GAATTTCCAA

TCTGGTGTCT

TATCCAGGTA

TGCCCTCAGG

CCTGATGAAA

ACAGAGCCAT

CACCTGAGGT

AAAAAAAAG

GGAGGCTGAG

CGCGCCACTG

GCAAACAGAA

TCTCTATTCG

AACTTGACAA

TGGCAAAACA

CAACTGTGAA

CTTTGCGACT

GGAATGGAAC

CTTTTCTTCA

ATGCTGTTGT

TATTCAATTT

TGCCTGCTCA

ATCTCTGGGC

GTTTAGCAAG

TCAAATGGAC

GCTCATTTGG

TGGTGAACCA

GCTGTCCAGA

GACCAACATT

AGTACCAGCC

CAGGAGTTCG

TACAAAATTA

GCAGCAGAAT

CACTCCAGCC

CAAAACAAAA

TGATTGGGAT

TGGTAGATAC

CTACACAGAA

ACCTCAGATT

AGAGGTTAAA

CACAAAAGTA

AGAAGCTCAG

TTCTGAAGAG

CCTTAAGGAA

GATTGCTGAT

TGCTAATATT

GTTAATTGAA

AGCTCCTGAA

AATTCTACAG

TGAAATACTG

ATCCCTCCAT

TGAATATGTT

AGGAGAAAAC

AGACTAGCCT

GCTGGACGTG

CACTTGAACC

TCGGCGACAA

CAAAAACGAG

GAGGTAAGGG

TATATCACAA

CATGCTGATG

CAAGGTCTAG

CTAGGACAAG

GCAATCAAAA

GTAATGAAAA

CCAATTTACA

GGAGATGGAA

GGTATGGCAT

CTTGTAGGAG

GACAATGAAT

GTTGCACTGT

ACAGAACTGG

GAACAGGTGG

GAATTGATGA

CAGTCCTTCT

TTCTAATTCA

GGCCAACATG

GTGGTGAGTG

TGGGAGGCGG

GAGCAAAAAC

AGAGCGAAAC

GTGACAATGT

CCAGAGAACA

GTTTATGCCA

CAAAAGATGC

GATGTTTTGG

CACTAAAACC

AAATAAGACA

TTGTCACTGA

AGTATTTGAA

ATATTAAAAG

AAAATCTTCT

ACACATCAAG

ATGGTGGGTT

TAACAAAGGG

AGCGAGGATA

ATCTGTGTTG

TGGGAGACTA

AGTAGCCTAT

480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1804

Claims (28)

Claims

1. A cellular immunogen for immunizing a host against the effects of the product of a target proto-oncogene, the overexpression of which target proto-oncogene is associated with a cancer, which cellular immunogen comprises host cells which have been transfected with at least one transgene construct comprising at least one transgene cognate to the target proto-oncogene and a strong promoter to drive the expression of the transgene in the transfected cells, the transgene encoding a gene product which induces host immunoreactivity to host self-determinants of the product of the target proto- oncogene gene

2. An immunogen according to claim 1 wherein the transgene comprises wild-type or mutant retroviral oncogene DNA; or wild-type or mutant proto-oncogene DNA of a species different from the host species.

3. An immunogen according to claim 2 wherein the transfected cells are non-dividing.

4. An immunogen according to claim 2 wherein the transgene comprises mutant retroviral oncogene DNA or mutant proto-oncogene DNA.

5. An immunogen according to claim 4 wherein the mutant DNA is nontransforming.

6. An immunogen according to claim 5 wherein the mutant DNA comprises a deletion mutation in a region of said DNA which is essential for transformation.

7. A cellular immunogen according to claim 6 wherein the host cells have been transfected with a plurality of transgene constructs, each construct encoding a different deletion mutation.

8. An immunogen according to claim 1 wherein the host cells have been transfected with a transgene cognate to a target proto-oncogene selected from the group of proto-oncogenes consisting of AKT-2, c-erbB-2, MDM-2, c-myc, c-myb, c-ras, c-src and c-yes.

9. An immunogen according to claim 1 wherein the cells comprise fibroblasts.

10. A method for preparing a cellular immunogen for immunizing a host against the effects of the product of a target proto-oncogene, the overexpression of which target proto-oncogene is associated with a cancer, the method comprising:

(a) excising cells from the host;

(b) transfecting the excised cells with at least one transgene construct comprising at least one transgene cognate to the target proto-oncogene and a strong promoter to drive the expression of the transgene in the transfected cells, the transgene encoding a gene product which induces host immunoreactivity to host self-determinants of the product of the target proto-oncogene gene.

11. A method according to claim 11 wherein the transgene comprises wild-type or mutant retroviral oncogene DNA; or wild-type or mutant proto-oncogene DNA of a species different from the host species.

12. A method according to claim 11 wherein the transfected cells are non-dividing.

13. A method according to claim 11 wherein the transgene comprises mutant retroviral oncogene DNA or mutant proto-oncogene DNA.

14. A method according to claim 13 wherein the mutant DNA is nontransforming.

15. A method according to claim 14 wherein the mutant DNA comprises a deletion mutation in a region of said DNA which is essential for transformation.

16. A method according to claim 15 wherein the host cells are transfected with a plurality of transgene constructs, each construct encoding a different deletion mutation.

17. A method according to claim 11 wherein the transgene is cognate to a target proto-oncogene selected from the group of proto-oncogenes consisting of AKT-2, c-erbB-2, MDM-2, c-myc, c-myb, c-ras, c-src and c-yes.

18. A method according to claim 1 wherein the excised cells comprise fibroblasts.

19. A method of vaccinating a host against disease associated with the overexpression of a target proto-oncogene comprising

(a) excising cells from the host;

(b) transfecting the excised cells with at least one transgene construct comprising at least one transgene cognate to the target proto-oncogene and a strong promoter to drive the expression of the transgene in the transfected cells, the transgene encoding a gene product which induces host immunoreactivity to host self-determinants of the product of the target proto-oncogene gene;

(c) returning the excised cells transfected with the transgene construct to the body of the host to obtain expression of the transgene in the host.

20. A method according to claim 19 wherein the transgene comprises wild-type or mutant retroviral oncogene DNA; or wild-type or mutant proto-oncogene DNA of a species different from the host species.

21. A method according to claim 20 wherein the transfected cells are rendered non-dividing prior to return to the body of the host.

22. A method according to claim 20 wherein the transgene comprises mutant retroviral oncogene DNA or mutant proto-oncogene DNA.

23. A method according to claim 22 wherein the mutant DNA is nontransforming.

24. A method according to claim 23 wherein the mutant DNA comprises a deletion mutation in a region of said DNA which is essential for transformation.

25. A method according to claim 24 wherein the host cells are transfected with a plurality of transgene constructs, each construct encoding a different deletion mutation.

26. A method according to claim 19 wherein the transgene is cognate to a target proto-oncogene selected from the group of proto-oncogenes consisting of AKT-2, c-erbB-2, MDM-2, c-myc, c-myb, c-ras, c-src and c-yes.

27. A method according to claim 19 wherein the excised host cells comprise fibroblasts.

28. A method of vaccinating a host against disease associated with the overexpression of a targeted proto-oncogene comprising

(a) excising cells from the host;

(b) transfecting the excised cells with at least one transgene construct comprising at least transgene and a strong promoter to drive the expression of the transgene in the transfected cells, wherein the transgene comprises

(1) wild-type or mutant cognate retroviral oncogene DNA; or

(2) wild-type or mutant cognate proto- oncogene DNA of a species different from the host species;

(c) returning the excised cells transfected with the transgene construct to the body of the host to obtain expression of the transgene in the host.