WO1999050289A2 - Transformation progression-related genes and their use - Google Patents

Abstract

Compositions and methods for cancer diagnosis, monitoring and therapy are disclosed. The compounds provided are progression-related proteins, and portions and variants thereof, as well as polynucleotides that encode such polypeptides, antibodies that bind to such polypeptides and agents that modulate expression of such polypeptides. Vaccines and pharmaceutical compositions comprising such compounds are also provided and may be used, for example, for cancer prevention and treatment. The polypeptides may also be used as markers for monitoring cancer progression in a patient.

Description

PROGRESSION RELATED GENES AND METHODS OF USE THEREFOR

TECHNICAL FIELD

The present invention relates generally to methods for detecting and treating cancer. The invention is more particularly related to polypeptide and nucleotide sequences associated with tumor progression, antibodies that bind to such polypeptides and agents that modulate the expression of such polypeptides. The compounds provided herein are generally useful for the diagnosis and/or therapy of diseases such as cancer.

BACKGROUND OF THE INVENTION

The carcinogenic process involves a series of sequential changes in the phenotype of a cell resulting in the acquisition of new properties or a further elaboration of transformation-associated traits by the evolving tumor cell. Although extensively studied, the precise genetic mechanisms underlying tumor cell progression during the development of most human cancers remain enigmas. Possible factors contributing to tumor progression include: activation of cellular genes that promote the cancer cell phenotype (i.e., oncogenes); activation or modification of genes that regulate genomic stability (i.e., DNA repair genes); loss or inactivation of cellular genes that function as inhibitors of the cancer cell phenotype (i.e., tumor suppressor genes); and/or combinations of these genetic changes in the same tumor cell. However, to develop improved diagnostic and therapeutic methods for cancer, it is necessary to identify genetic mechanisms leading to tumor progression.

One model system for defining the genetic and biochemical changes mediating tumor progression is the type 5 adenovirus (Ad5) / early passage rat embryo (RE) cell culture system (see, e.g., P.B. Fisher, in T.J. Slaga, ed., Tumor Promotion and Cocarcinogenesis In vitro, Mechanisms of Tumor Promotion (CRC Press, Boca Raton, FL), pp. 57-123, 1984). Transformation of secondary RE cells by Ad5 is often a sequential process resulting in the acquisition of and further elaboration of specific phenotypes by the transformed cell (see Fisher et al., Cancer Res. 59:3051-3057, 1979; Fisher et al., Nature 257:591-594, 1979;

SUBSTITUTE SHEET (RULE 25) phenotypes by the transformed cell (see Fisher et al., Cancer Res. 59:3051-3057, 1979; Fisher et al., Nature 257:591-594, 1979; Fisher et al., Cell 75:695-705, 1979; Babiss et al., Science 225:1099-1101, 1985). Progression in the Ad5-transformation model is characterized by the development of enhanced anchorage-independence and tumorigenic potential (as indicated by a reduced latency time for tumor formation in nude mice) by progressed cells. The progression phenotype in Ad5 -transformed RE cells can be induced by selection for growth in agar or tumor formation in nude mice, referred to as spontaneous-progression, by transfection with oncogenes (see Reddy et al., K.W. Adolph, ed., in Chromosome and Genetic Analysis, Methods in Molecular Genetics (Academic, Orlando, FL), Vol. 1, pp. 68-102, 1993), such as Ha-ras, v-src, v-raf or the E6/E7 region of human papillomavirus type (HPV)-18. Such progression is referred to as oncogene- mediated progression. Alternatively, the progression phenotype in Ad5-transformed RE cells can be induced by transfection with specific signal transducing genes (Su et al., Oncogene 9:1123-1132, 1994), such as protein kinase C. Such progression is referred to as growth factor-related, gene-induced progression.

Progression, induced spontaneously or after gene transfer, is a stable cellular trait that remains undiminished in Ad5-transformed RE cells even after extensive passage (- 100) in monolayer culture. However, a single-treatment with the demethylating agent 5-azacytidine (AZA) results in a stable reversion in transformation progression in >95% of cellular clones. The progression phenotype is also suppressed in somatic cell hybrids formed between normal or unprogressed transformed cells and progressed cells (Duigou et al., Mol. Cell. Biol. 10:2027-2034, 1990; Duigou et al., Oncogene 6:1813-1824, 1991; Reddy et al., K.W. Adolph, ed., in Chromosome and Genetic Analysis, Methods in Molecular Genetics (Academic, Orlando, FL), Vol. 1, pp. 68-102, 1993). These findings suggest that progression may result from the activation of specific progression-promoting genes or the selective inhibition of progression- suppressing genes, or possibly a combination of both processes.

The final stage in tumor progression is acquisition by transformed cells of the ability to invade local tissue, survive in the circulation and recolonize in a new area of the body, i.e., metastasis (Fidler et. al.. Cancer Res. 50:6130-6138, 1990; Liotta et al., Cell 64:321-336, 1991 ; Fidler et al., J Nat/. Cancer Inst. 57:1588-1592, 1995). Transfection of a Ha-ras oncogene into cloned rat embryo fibroblast (CREF) cells (Fisher et al., Proc. Natl. Acad. Sci. USA 79: 3527-3531, 1982) results in morphological transformation, anchorageindependence and acquisition of tumorigenic and metastatic potential (Boylon et al., Anticancer Res. 10:1)1-124, 1990; Boylon et al., Mol. Carcinog. 3:309-318, 1992; Su et al., Oncogene 5:309-318, 1993). Ha-ras-transformed CREF cells exhibit major changes in the transcription and steady-state levels of genes involved in suppression and induction of oncogenesis (Su et al., Oncogene 5:309-318, 1993; Su et al., Intl. J. Oncology 7:1279-1284, 1995). Simultaneous overexpression of the Ha-røs suppressor gene Krev-1 in Ha-røs-transformed CREF cells results in morphological reversion, suppression of agar growth capacity and a delay in in vivo oncogenesis. Reversion of transformation in Ha-ras+Krev-1 transformed CREF cells correlates with a return in the transcriptional and steady-state mRΝA profile to that of untransformed CREF cells. Following long latency times, Ha-rαs+Krev-l transformed CREF cells form both tumors and metastases in athymic nude mice. The patterns of gene expression changes observed during progression, progression suppression and escape from progression suppression supports the concept of "transcriptional switching" as a major component of Ha-rar-induced transformation.

Notwithstanding the foregoing advances, considerable work remains to elucidate the genetic mechanisms associated with tumor progression, and to develop improved methods for diagnosing and treating cancer. The present invention fulfills this need, and further provides other related advantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides compositions and methods for cancer diagnosis and therapy. Within certain aspects, the present invention provides isolated polypeptides comprising at least portion of a progression-related protein, or a variant thereof, wherein: (a) the progression-related protein comprises a sequence encoded by a nucleotide sequence recited in any one of Figures 1-12 (SEQ IN NOs:l-24); and (b) the portion retains at least one immunological and/or biological activity characteristic of the progression-related protein.

Within related aspects, the present invention provides isolated polynucleotides encoding a polypeptide as described above, as well as antisense polynucleotides comprising a sequence complementary to such a polynucleotide, expression vectors comprising any of the above polynucleotides and host cells transformed or transfected with such an expression vector.

Within further aspects, the present invention provides monoclonal antibodies, and antigen-binding fragments thereof, that specifically bind to a polypeptide as described above.

The present invention further provides, within other aspects, pharmaceutical compositions comprising: (a) a polypeptide, polynucleotide or antibody as described above, or an agent that inhibits expression of a polypeptide as described above; and (b) a physiologically acceptable carrier. Vaccines are also provided, comprising: (a) a polypeptide as described above; and (b) an immune response enhancer.

Within further aspects, methods are provided for inhibiting the progression of a cancer in a patient, comprising administering to a patient an agent that inhibits expression of a polypeptide as described above.

The present invention further provides, within other aspects, methods for detecting expression of a progression-related gene in a cell sample, comprising steps of: (a) obtaining RNA from a cell sample; (b) contacting the RNA with a labeled probe capable of hybridizing a progression-related mRNA under conditions permitting specific hybridization of the probe and the RNA; and (c) determining the presence of RNA hybridized to the probe, and therefrom detecting the expression of a progression-related gene in the sample.

In another aspect, the present invention provides methods for determining whether cells are in progression, comprising the steps of: (a) measuring expression of a progression-related gene in a sample of cells; and (b) comparing the expression measured in step (a) with the expression of the progression-related gene in cells that are not in progression, and therefrom determining whether the cells are in progression. The present invention further provides methods for determining whether a cancer in a patient is in progression, comprising detecting, in a biological sample obtained from a patient, a polynucleotide or polypeptide as described above, and therefrom determining whether a cancer in the patient is in progression.

Within further aspects, the present invention provides methods for monitoring the progression of a cancer in a patient, comprising: (a) detecting, in a biological sample obtained from a patient, an amount of a polynucleotide or polypeptide as described above at a first point in time; (b) repeating step (a) at a subsequent point in time; and (c) comparing the amounts of polypeptide detected in steps (a) and (b), and therefrom monitoring the progression of a cancer in the patient.

The present invention further provides, within other aspects, diagnostic kits, comprising: (a) a monoclonal antibody or fragment thereof as described above; and (b) a second monoclonal antibody or fragment thereof that binds to (i) a monoclonal antibody recited in step (a); or (ii) a polypeptide as described above; wherein the second monoclonal antibody is conjugated to a reporter group.

Within still further aspects, the present invention provides methods for preparing a polypeptide as described above, comprising the steps of: (a) culturing a host cell as described above under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.

Methods are further provided for producing cells that are resistant to progression or for protecting cells from chemotherapeutic damage, comprising inhibiting or eliminating the expression of a progression-related gene in the cells.

Cells transformed or transfected with a reporter gene under the control of a human progression-related gene promoter, or regulatory element thereof are also provided.

Within other aspects, methods are provided for identifying an agent that modulates the expression of a progression-related gene, comprising the steps of: (a) contacting a candidate agent with a cell transformed or transfected with a reporter gene under the control of a progression-related gene promoter or a regulatory element thereof under conditions and for a time sufficient to allow the candidate agent to interact with the promoter or regulatory element thereof; and (b) determining the effect of the candidate agent on the level of reporter protein produced by the cell, and therefrom identifying an agent that modulates expression of a progression-related gene.

Within further aspects, the present invention provides methods for identifying an agent that modulates the ability of a progression-related gene to induce progression, comprising the steps of: (a) contacting a candidate agent with a polypeptide as described above, under conditions and for a time sufficient to allow the candidate agent and polypeptide to interact; and (b) determining the effect of the candidate agent on the ability of the polypeptide to induce progression, and therefrom identifying an agent that modulates the ability of the progression-related gene to induce progression.

These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 presents partial nucleotide sequences of a rat progression related gene referred to herein as ZNPE-1. 5' and 3' partial sequences are shown, as indicated (SEQ ID NOs: 1 and 2 respectively).

Figure 2 presents partial nucleotide sequences of a rat progression related gene referred to herein as ZNPE-119. 5' and 3' partial sequences are shown, as indicated (SEQ ID NOs: 3 and 4 respectively).

Figure 3 presents partial nucleotide sequences of a rat progression related gene referred to herein as ZNPE-12. 5' and 3' partial sequences are shown, as indicated (SEQ ID NOs: 5 and 6 respectively).

Figure 4 presents partial nucleotide sequences of a rat progression related gene referred to herein as ZNPE-120. 5' and 3' partial sequences are shown, as indicated (SEQ ID NOs: 7 and 8 respectively). Figure 5 presents partial nucleotide sequences of a rat progression related gene referred to herein as ZNPE-15. 5' and 3' partial sequences are shown, as indicated (SEQ ID NOs: 9 and 10 respectively).

Figure 6 presents partial nucleotide sequences of a rat progression related gene referred to herein as ZNPE-28. 5' and 3' partial sequences are shown, as indicated (SEQ ID NOs: 11 and 12 respectively).

Figure 7 presents partial nucleotide sequences of a rat progression related gene referred to herein as ZNPE-37. 5' and 3' partial sequences are shown, as indicated (SEQ ID NOs: 13 and 14 respectively).

Figure 8 presents partial nucleotide sequences of a rat progression related gene referred to herein as ZNPE-38. 5' and 3' partial sequences are shown, as indicated (SEQ ID NOs: 15 and 16 respectively).

Figure 9 presents partial nucleotide sequences of a rat progression related gene referred to herein as ZNPE-43. 5' and 3' partial sequences are shown, as indicated (SEQ ID NOs: 17 and 18 respectively).

Figure 10 presents partial nucleotide sequences of a rat progression related gene referred to herein as ZNPE-45. 5' and 3' partial sequences are shown, as indicated (SEQ ID NOs: 19 and 20 respectively).

Figure 11 presents partial nucleotide sequences of a rat progression related gene referred to herein as ZNPE-62-1. 5' and 3' partial sequences are shown, as indicated (SEQ ID NOs: 21 and 22 respectively).

Figure 12 presents partial nucleotide sequences of a rat progression related gene referred to herein as ZNPE-62-2. 5' and 3' partial sequences are shown, as indicated (SEQ ID NOs: 23 and 24 respectively).

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention is generally directed to compositions and methods for cancer diagnosis, monitoring and therapy. The compositions described herein may include one or more polypeptides, nucleic acid sequences and/or antibodies. Polypeptides of the present invention generally comprise at least a portion of a tumor progression-related protein, or a variant thereof. Nucleic acid sequences of the subject invention generally comprise a DNA or RNA sequence that encodes such a polypeptide, or that is complementary to such a coding sequence. Antibodies are generally immune system proteins, or antigen-binding fragments thereof, that are capable of binding to a portion of a polypeptide as described above.

The present invention is based, in part, on the identification of certain cDNA molecules that correspond to progression-related mRNA molecules. As used herein, a "progression-related" mRNA is a mRNA whose expression correlates with tumor cell progression (i.e., the level of RNA is at least 2-fold higher in progressing tumor cells). A progression-related cDNA molecule comprises the sequence of a progression-related mRNA (and/or a complementary sequence). Similarly, a progression- related protein or polypeptide comprises a sequence encoded by a progression-related mRNA, where the level of protein or polypeptide correlates with tumor cell progression (i.e., the level of protein is at least 2-fold higher in progressing tumor cells). Progression- related sequences described herein may also be referred to as progression related genes (PRGs).

PROGRESSION-RELATED POLYNUCLEOTIDES

Any polynucleotide that encodes a progression-related polypeptide, or a portion or variant thereof as described herein, is encompassed by the present invention. Such polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. Additional non- coding sequences may, but need not, be present within a polynucleotide of the present invention, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.

Progression-related polynucleotides may be prepared using any of a variety of techniques. For example, such a polynucleotide may be amplified from human genomic DNA, from tumor cDNA or from cDNA prepared from any of a variety of tumor-derived cell lines (typically cell lines characterized by a progression phenotype), via polymerase chain reaction (PCR). For this approach, sequence-specific primers may be designed based on the sequences provided herein, and may be purchased or synthesized. An amplified portion may then be used to isolate a full length gene from a human genomic DNA library or from a tumor cDNA library, using well known techniques, as described below. Alternatively, a full length gene can be constructed from multiple PCR fragments. cDNA molecules encoding a native progression-related protein, or a portion thereof, may also be prepared by screening a cDNA library prepared from mRNA of a cell that is in progression, such as El l-NMT or MCF-7 cells, as described herein. Such libraries may be commercially available, or may be prepared using standard techniques (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989, and references cited therein). A library may be a cDNA expression library and may, but need not, be subtracted using well known subtractive hybridization techniques.

There are many types of screens that may be employed, including any of a variety of standard hybridization methods. For initial screens, conventional subtractive hybridization techniques may be used.

A progression-related cDNA molecule may be sequenced using well known techniques employing such enzymes as Klenow fragment of DNA polymerase I, Sequenase® (US Biochemical Corp., Cleveland OH) Taq polymerase (Perkin Elmer, Foster City CA), thermostable T7 polymerase (Amersham, Chicago, IL) or combinations of recombinant polymerases and proofreading exonucleases such as the ELONGASE Amplification System (Gibco BRL, Gaithersburg, MD). An automated sequencing system may be used, using instruments available from commercial suppliers such as Perkin Elmer and Pharmacia.

The sequence of a partial cDNA may be used to identify a polynucleotide sequence that encodes a full length progression-related protein using any of a variety of standard techniques. Within such techniques, a library (cDNA or genomic) is screened using one or more polynucleotide probes or primers suitable for amplification. Preferably, a library is size-selected to include larger molecules. Random primed libraries may also be preferred for identifying 5' and upstream regions of genes. Genomic libraries are preferred for obtaining introns and extending 5' sequence.

For hybridization techniques, a partial sequence may be labeled (e.g., by nick-translation or end-labeling with ³²P) using well known techniques. A bacterial or bacteriophage library is then screened by hybridizing filters containing denatured bacterial colonies (or lawns containing phage plaques) with the labeled probe (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989). Hybridizing colonies or plaques are selected and expanded, and the DNA is isolated for further analysis. cDNA clones may be analyzed to determine the amount of additional sequence by, for example, PCR using a primer from the partial sequence and a primer from the vector. Restriction maps and partial sequences may be generated to identify one or more overlapping clones. The complete sequence may then be determined using standard techniques, which may involve generating a series of deletion clones. The resulting overlapping sequences are then assembled into a single contiguous sequence. A full length cDNA molecule can be generated by ligating suitable fragments, using well known techniques.

Alternatively, there are numerous amplification techniques for obtaining a full length coding sequence from a partial cDNA sequence. Within such techniques, amplification is generally performed via PCR. Any of a variety of commercially available kits may be used to perform the amplification step. Primers may be designed using, for example, software well known in the art. Primers are preferably 22-30 nucleotides in length, have a GC content of at least 50% and anneal to the target sequence at temperatures of about 68°C to 72°C. The amplified region may be sequenced as described above, and overlapping sequences assembled into a contiguous sequence.

One such amplification technique is inverse PCR (see Triglia et al., Nucl. Acids Res. 16:8186, 1988), which uses restriction enzymes to generate a fragment in the known region of the gene. The fragment is then circularized by intramolecular ligation and used as a template for PCR with divergent primers derived from the known region. Within an alternative approach, sequences adjacent to a partial sequence may be retrieved by amplification with a primer to a linker sequence and a primer specific to a known region. The amplified sequences are typically subjected to a second round of amplification with the same linker primer and a second primer specific to the known region. A variation on this procedure, which employs two primers that initiate extension in opposite directions from the known sequence, is described in WO 96/38591. Additional techniques include capture PCR (Lagerstrom et al., PCR Methods Applic. 7:111-19, 1991), walking PCR (Parker et al., Nucl. Acids. Res. 79:3055-60,1991) and rapid amplification of cDNA end (RACE) procedures (see Jiang et al., Oncogene 70:1855-1864, 1995; Jiang et al., Oncogene 77:2477-2486, 1995). Other methods employing amplification may also be employed to obtain a full length cDNA sequence.

In certain instances, it is possible to obtain a full length cDNA sequence by analysis of sequences provided in an expressed sequence tag (EST) database, such as that available from GenBank. Searches for overlapping ESTs may generally be performed using well known programs (e.g., NCBI BLAST searches), and such ESTs may be used to generate a contiguous full length sequence.

Nucleic acid sequences of cDNA molecules corresponding to certain rat progression-related genes are provided in Figures 1-12 and SEQ ID NOs: 1-24. These sequences were identified based upon their upregulation in El IT cells, as compared to El 1 cells. Rat progression-related genes display elevated expression in progressed Ad5- transformed cells (spontaneous, oncogene-induced and growth factor-related, gene- induced) versus unprogressed cells (parental Ad5 -transformed, AZA-suppressed, and suppressed hybrids) using subtraction hybridization (Jiang et al., Mol. Cell. Different. 7:285-299, 1993). The polynucleotides specifically recited herein, as well as full length polynucleotides comprising such sequences, other portions of full length polynucleotides, and sequences complementary to all or a portion of such full length molecules, are specifically encompassed by the present invention.

Human homologues of the progression-related sequences recited herein may be isolated based on hybridization to a rat sequence probe, and are also encompassed by the present invention. Human homologues may be identified using libraries prepared from cells that are in progression, including most human tumor cell lines (e.g., MCF-7). These sequences are detected at a lower level, if at all, in normal tissues examined by Northern blot analysis. Homologues from other species are also contemplated, and may generally be prepared as described herein for the human homolog.

Variants of the recited polynucleotide sequences are also provided herein. Polynucleotide variants may contain one or more substitutions, deletions, insertions and/or modifications such that the antigenic, immunogenic and/or biological properties of the encoded polypeptide are not diminished. The effect on the properties of the encoded polypeptide may generally be assessed as described herein. Preferred variants contain nucleotide substitutions, deletions, insertions and/or modifications at no more than 20%, preferably at no more than 10%, of the nucleotide positions. Certain variants are substantially homologous to a native gene, or a potion or complement thereof. Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence encoding a progression-related protein (or a complementary sequence). Suitable moderately stringent conditions include prewashing in a solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C-65°C, 5 X SSC, overnight; followed by washing twice at 65°C for 20 minutes with each of 2X, 0.5X and 0.2X SSC containing 0.1 % SDS). Such hybridizing DNA sequences are also within the scope of this invention.

It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention.

As noted above, antisense polynucleotides and portions of any of the above sequences are also contemplated by the present invention. Such polynucleotides may generally be prepared by any method known in the art, including chemical synthesis by, for example, solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro or in vivo transcription of DNA sequences encoding a progression-related protein, or a portion thereof, provided that the DNA is incorporated into a vector downstream of a suitable RNA polymerase promoter (such as T3, T7 or SP6). Large amounts of RNA probe may be produced by incubating labeled nucleotides with a linearized progression-related gene fragment downstream of such a promoter in the presence of the appropriate RNA polymerase. Certain portions of a polynucleotide may be used to prepare an encoded polypeptide, as described herein. In addition, or alternatively, a portion may function as a probe (e.g., for diagnostic purposes, such as to monitor or study the progression of cancer), and may be labeled by a variety of reporter groups, such as radionuclides, fluorescent dyes and enzymes. Such portions are preferably at least 10 nucleotides in length, more preferably at least 12 nucleotides in length and still more preferably at least 15 nucleotides in length. Within certain preferred embodiments, a portion for use as a probe comprises a sequence that is unique to a progression-related gene. A portion of a sequence complementary to a coding sequence (i.e., an antisense polynucleotide) may also be used as a probe or to modulate gene expression. cDNA constructs that can be transcribed into antisense RNA may also be introduced into cells of tissues to facilitate the production of antisense RNA.

Any polynucleotide may be further modified to increase stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queosine and wybutosine, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine and uridine.

Nucleotide sequences as described herein may be joined to a variety of other nucleotide sequences using established recombinant DNA techniques. For example, a polynucleotide may be cloned into any of a variety of cloning vectors, including plasmids, phagemids, lambda phage derivatives and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors. In general, a vector will contain an origin of replication functional in at least one organism, convenient restriction endonuclease sites and one or more selectable markers. Additional initial, terminal and/or intermediate DNA sequences that, for example, facilitate construction of readily expressed vectors may also be present. For example, regulatory elements required for expression include promoter sequences to bind RNA polymerase and transcription initiation sequences for ribosome binding. A bacterial expression vector may include a promoter such as the lac promoter and for transcription initiation the ShineDalgarno sequence and the start codon AUG. Similarly, a eukaryotic expression vector may include a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome. Such vectors may be obtained commercially or assembled from the sequences described by methods well-known in the art, for example, the methods described above for constructing vectors in general. Other elements that may be present in a vector will depend upon the desired use, and will be apparent to those of ordinary skill in the art.

For example, insert and vector DNA can both be exposed to a restriction enzyme to create complementary ends on both molecules which base pair with each other and are then ligated together with DNA ligase. Alternatively, linkers can be ligated to the insert DNA which correspond to a restriction site in the vector DNA, which is then digested with the restriction enzyme which cuts at that site. Other means are also available and known to an ordinary skilled practitioner.

Vectors as described herein may generally be transfected into a suitable host cell, such as a mammalian cell, by methods well-known in the art Such methods include calcium phosphate precipitation, electroporation and microinjection.

PROGRESSION-RELATED POLYPEPTIDES

Polypeptides within the scope of the present invention comprise at least a portion of a progression-related protein or variant thereof, where the portion is immunologically and/or biologically active. A polypeptide may further comprise additional sequences, which may or may not be derived from a native progression-related protein. Such sequences may (but need not) possess immunogenic or antigenic properties and/or a biological activity.

A polypeptide is "immunologically active," within the context of the present invention if it is recognized (i.e., specifically bound) by a B-cell and/or T-cell surface antigen receptor. Immunological activity may generally be assessed using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Such techniques include screening polypeptides derived from the native polypeptide for the ability to react with antigen-specific antisera and/or T-cell lines or clones, which may be prepared using well known techniques. An immunologically active portion of a progression-related protein reacts with such antisera and/or T-cells at a level that is not substantially lower than the reactivity of the full length polypeptide (e.g., in an ELISA and/or T-cell reactivity assay). Such screens may generally be performed using methods well known to those of ordinary skill in the art, such as those described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. B-cell and T-cell epitopes may also be predicted via computer analysis.

Similarly, a polypeptide is "biologically active" if it possesses one or more structural, regulatory and/or biochemical functions of the native progression-related protein. For example, a polypeptide may induce progression in cells at levels comparable to the level of native protein. Appropriate assays designed to evaluate the activity may then be designed based on existing assays known in the art, and on the assays provided herein.

As noted above, polypeptides may comprise one or more portions of a variant of an endogenous protein, where the portion is immunologically and/or biologically active (i.e., the portion exhibits one or more antigenic, immunogenic and/or biological properties characteristic of the full length protein). Preferably, such a portion is at least as active as the full length protein within one or more assays to detect such properties. A polypeptide "variant," as used herein, is a polypeptide that differs from a native protein in substitutions, insertions, deletions and/or amino acid modifications, such that the antigenic, immunogenic and/or biological properties of the native protein are not substantially diminished. A variant preferably retains at least 80% sequence identity to a native sequence, more preferably at least 90% identity, and even more preferably at least 95% identity. Guidance in determining which and how many amino acid residues may be substituted, inserted, deleted and/or modified without diminishing immunological and/or biological activity may be found using any of a variety of computer programs known in the art, such as DNAStar software. Properties of a variant may generally be evaluated by assaying the reactivity of the variant with antisera and/or T-cells as described above and/or evaluating a biological property characteristic of the native protein.

Preferably, a variant contains conservative substitutions. A "conservative substitution" is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Amino acid substitutions may generally be made on the basis of similarity on polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also, or alternatively, contain nonconservative changes.

Variants within the scope of this invention also include polypeptides in which the primary amino acid structure of a native protein is modified by forming covalent or aggregative conjugates with other polypeptides or chemical moieties such as glycosyl groups, lipids, phosphate, acetyl groups and the like. Covalent derivatives may be prepared, for example, by linking particular functional groups to amino acid side chains or at the N- or C-termini.

The present invention also includes polypeptides with or without associated native-pattern glycosylation. Polypeptides expressed in yeast or mammalian expression systems may be similar to or slightly different in molecular weight and glycosylation pattern than the native molecules, depending upon the expression system. Expression of DNA in bacteria such as E. coli provides non-glycosylated molecules. N- glycosylation sites of eukaryotic proteins are characterized by the amino acid triplet Asn- Aj-Z, where A_] is any amino acid except Pro, and Z is Ser or Thr. Variants having inactivated N-glycosylation sites can be produced by techniques known to those of ordinary skill in the art, such as oligonucleotide synthesis and ligation or site-specific mutagenesis techniques, and are within the scope of this invention. Alternatively, N- linked glycosylation sites can be added to a polypeptide.

As noted above, polypeptides may further comprise sequences that are not related to an endogenous progression-related protein. For example, an N-terminal signal (or leader) sequence may be present, which co-translationally or post-translationally directs transfer of the polypeptide from its site of synthesis to a site inside or outside of the cell membrane or wall (e.g., the yeast α-factor leader). The polypeptide may also comprise a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e g., poly-His or hemagglutinin), or to enhance binding of the polypeptide to a solid support. Fusion proteins capped with such peptides may also be resistant to intracellular degradation in E. coli. Protein fusions encompassed by this invention further include, for example, polypeptides conjugated to an immunoglobulin Fc region or a leucine zipper domain as described, for example, in published PCT Application WO 94/10308. Polypeptides comprising leucine zippers may, for example, be oligomeric, dimeric or trimeric. All of the above protein fusions may be prepared by chemical linkage or as fusion proteins, as described below.

Also included within the present invention are alleles of a progression- related protein. Alleles are alternative forms of a native protein resulting from one or more genetic mutations (which may be amino acid deletions, additions and/or substitutions), resulting in an altered mRNA. Allelic proteins may differ in sequence, but overall structure and function are substantially similar.

Progression-related polypeptides, variants and portions thereof may generally be prepared from nucleic acid encoding the desired polypeptide using well known techniques. To prepare an endogenous protein, an isolated cDNA may be used. To prepare a variant polypeptide, standard mutagenesis techniques, such as oligonucleotide-directed site-specific mutagenesis may be used, and sections of the DNA sequence may be removed to permit preparation of truncated polypeptides. Briefly, host cells of a vector system containing a progression-related sequence under suitable conditions permitting production of the polypeptide may be grown, and the polypeptide so produced may then be recovered.

In general, any of a variety of expression vectors known to those of ordinary skill in the art may be employed to express recombinant polypeptides of this invention. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA sequence that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast, insect cells and animal cells. Preferably, the host cells employed are E. coli, yeast, primary mammalian cells or a mammalian cell line such as COS, Vero, HeLa, fibroblast NIH3T3, CHO, Ltk^" or CV1. Following expression, supernatants from host/vector systems which secrete recombinant protein or polypeptide into culture media may be first concentrated using a commercially available filter. Following concentration, the concentrate may be applied to a suitable purification matrix such as an affinity matrix or an ion exchange resin. One or more reverse phase HPLC steps can be employed to further purify a recombinant polypeptide.

Portions and other variants having fewer than about 100 amino acids, and generally fewer than about 50 amino acids, may also be generated by synthetic means, using techniques well known to those of ordinary skill in the art. For example, such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J Am. Chem. Soc. 55:2149-2146, 1963. Various modified solid phase techniques are also available (e.g., the method of Roberge et al., Science 269:202-204, 1995). Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Applied BioSystems, Inc. (Foster City, CA), and may be operated according to the manufacturer's instructions.

In general, polypeptides and polynucleotides as described herein are isolated. An "isolated" polypeptide or polynucleotide is one that is removed from its original environment. For example, a naturally-occurring protein is isolated if it is separated from some or all of the coexisting materials in the natural system. A polynucleotide is considered to be isolated if, for example, it is cloned into a vector that is not a part of the natural environment.

ANTIBODIES AND FRAGMENTS THEREOF

The present invention further provides antibodies, and antigen-binding fragments thereof, that specifically bind to a progression-related protein. As used herein, an antibody, or antigen-binding fragment, is said to "specifically bind" to a progression- related protein if it reacts at a detectable level (within, for example, an ELISA) with a progression-related protein or a portion or variant thereof, and does not react detectably with unrelated proteins. For certain embodiments, antibodies that inhibit progression induced by a progression-related sequence are preferred.

Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies as described herein, or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies. Alternatively, monoclonal antibodies may be produced by in vitro techniques known to a person of ordinary skill in the art.

Polypeptides comprising specific portions of a progression-related protein may be selected for the generation of antibodies using methods well known in the art. In general, hydrophilic regions are more immunogenic than the hydrophobic regions. Such hydrophilic portions may be preferred for the generation of antibodies.

In one such technique, an immunogen comprising the polypeptide is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep or goats). In this step, the polypeptides of this invention may serve as the immunogen without modification. Alternatively, particularly for relatively short polypeptides, a superior immune response may be elicited if the polypeptide is joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin. The immunogen is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically. Polyclonal antibodies specific for the polypeptide may then be purified from such antisera by, for example, affinity chromatography using the polypeptide coupled to a suitable solid support.

Monoclonal antibodies specific for the antigenic polypeptide of interest may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. :51 1-519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the polypeptide of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. A variety of fusion techniques may be employed. For example, the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells, but not myeloma cells. A preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and their culture supernatants tested for binding activity against the polypeptide. Hybridomas having high reactivity and specificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies. In addition, various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. The antibodies of this invention may be used in the purification process in, for example, an affinity chromatography step. Antibodies with a high degree of specificity for a progression-related protein may then be selected. Such antibodies may be used, for example, to detect the expression of a progression-related protein in living animals, in humans, or in biological tissues or fluids isolated from animals or humans.

Within certain embodiments, the use of antigen-binding fragments of antibodies may be preferred. Such fragments include Fab fragments, which may be prepared using standard techniques. Briefly, immunoglobulins may be purified from rabbit serum by affinity chromatography on Protein A bead columns (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988) and digested by papain to yield Fab and Fc fragments. The Fab and Fc fragments may be separated by affinity chromatography on protein A bead columns.

METHODS FOR IDENTIFYING BINDING AGENTS AND MODULATING AGENTS

The present invention further provides methods for identifying compounds that bind to and/or modulate the activity of a progression-related protein. Such agents may generally be identified by contacting a polypeptide as provided herein with a candidate compound or agent under conditions and for a time sufficient to allow interaction with the polypeptide. Any of a variety of well known binding assays may then be performed to assess the ability of the candidate compound to bind to the polypeptide, and assays for a biological activity of the polypeptide may be performed to identify agents that modulate (i.e., enhance or inhibit) the biological activity of the protein. Depending on the design of the assay, a polypeptide may be free in solution, affixed to a solid support, present on a cell surface or located intracellularly. Large scale screens may be performed using automation.

Alternatively, compounds may be screened for the ability to modulate expression (e.g., transcription) of a progression-related protein. For such assays a promoter for a progression-related protein may be isolated using standard techniques. The present invention provides nucleic acid molecules comprising such a promoter or a cis- or trans-acting regulatory element thereof. Such regulatory elements may activate or suppress expression of a progression-related protein.

One method for identifying a promoter region uses a PCR-based method to clone unknown genomic DNA sequences adjacent to a known cDNA sequence (e.g., a human PromoterFinder™DNA Walking Kit, available from Clontech). This approach may generate a 5' flanking region, which may be subcloned and sequenced using standard methods. Primer extension and/or RNase protection analyses may be used to verify the transcriptional start site deduced from the cDNA.

To define the boundary of the promoter region, putative promoter inserts of varying sizes may be subcloned into a heterologous expression system containing a suitable reporter gene without a promoter or enhancer may be employed. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase or the Green Fluorescent Protein gene, and may be generated using well known techniques Internal deletion constructs may be generated using unique internal restriction sites or by partial digestion of non-unique restriction sites. Constructs may then be transfected into cells that display high levels of a progression-related protein (e.g., El 1-NMT). In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the progression-related gene promoter.

Once a functional promoter is identified, cis- and trans-acting elements may be located. Cis-acting sequences may generally be identified based on homology to previously characterized transcriptional motifs. Point mutations may then be generated within the identified sequences to evaluate the regulatory role of such sequences. Such mutations may be generated using site-specific mutagenesis techniques or a PCR-based strategy. The altered promoter is then cloned into a reporter gene expression vector, as described above, and the effect of the mutation on reporter gene expression is evaluated. Trans-acting factors that bind to cis-acting sequences may be identified using assays such as gel shift assays. Proteins displaying binding activity within such assays may be partially digested, and the resulting peptides separated and sequenced. Peptide sequences may be used to design degenerate primers for use within RT-PCR to identify cDNAs encoding the trans-acting factors.

To evaluate the effect of a candidate agent on expression of a progression- related protein, a promoter or regulatory element thereof may be operatively linked to a reporter gene as described above. Such a construct may be transfected into a suitable host cell, such as El 1-NMT or transfected forms of CREF Trans 6, including CREF-Trans 6:4NMT (expressing PTI-1), T24 (expressing ras), CREF-src (expressing src) and CREF- HPV (expressing HPV). Clones that constitutively express high levels of reporter protein may be selected and used within a variety of screens. Such clones are encompassed by the present invention.

Within one preferred screen, cells may be used to screen a combinatorial small molecule library. Briefly, cells are incubated with the library (e.g., overnight). Cells are then lysed and the supernatant is analyzed for reporter gene activity according to standard protocols. Compounds that result in a decrease in reporter gene activity are inhibitors of progression-related gene transcription, and may be used to inhibit DNA damage and repair pathways, cancer progression and/or oncogene mediated transformation.

This invention further provides methods for identifying agents capable of inducing DNA damage and repair pathways, cancer progression and/or oncogene mediated transformation. Briefly, candidate compounds may be tested as described above, except that the cells employed (which comprise a progression-related gene promoter or regulatory element thereof operatively linked to a reporter gene) are not in progression. For example, CREF-Trans 6 cells may be employed. Within such assays, an increase in expression of the reporter gene after the contact indicates that the compound is capable of inducing DNA damage and repair pathways, cancer progression or oncogene mediated transformation.

Within other embodiments, cells may comprise one or more exogenous suicidal genes under the control of a promoter or regulatory element of a progression- related protein. Such suicidal genes disrupt the normal progress of the cell following transcription from the promoter. Preferably, the switching on of the suicidal gene will lead to cell death or halt in cell growth. Example of such genes are genes which lead to apoptosis. PHARMACEUTICAL COMPOSITIONS AND VACCINES

Within certain aspects, compounds such as polypeptides, antibodies, nucleic acid molecules and/or other agents that modulate expression or activity of a progression-related protein may be incorporated into pharmaceutical compositions or vaccines. Pharmaceutical compositions comprise one or more such compounds and a physiologically acceptable carrier. Certain vaccines may comprise one or more polypeptides and an immune response enhancer, such as an adjuvant or a liposome (into which the compound is incorporated). Pharmaceutical compositions and vaccines may additionally contain a delivery system, such as biodegradable microspheres which are disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075,109. Pharmaceutical compositions and vaccines within the scope of the present invention may also contain other compounds, which may be biologically active or inactive.

A pharmaceutical composition or vaccine may contain DNA encoding an antisense polynucleotide or a polypeptide as described above, such that the polynucleotide or polypeptide is generated in situ. The DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacteria and viral expression systems. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminating signal). Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette-Guerriή) that expresses an immunogenic portion of the polypeptide on its cell surface. In a preferred embodiment, the DNA may be introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be "naked," as described, for example, in Ulmer et al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells. While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration. Such carriers may be aqueous or non- aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, saline and buffered media.

Compositions of the present invention may be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous or intramuscular administration. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, a fixed oil, a fat, a wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactate polyglycolate) may also be employed as carriers for the pharmaceutical compositions of this invention. For certain topical applications, formulation as a cream or lotion, using well known components, is preferred.

Such compositions may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and/or preservatives. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like. Compositions of the present invention may also be formulated as a lyophilizate. Compounds may also be encapsulated within liposomes using well known technology.

Any of a variety of adjuvants may be employed in the vaccines of this invention to nonspecifically enhance the immune response. Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins. Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI), Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ), alum, biodegradable microspheres, monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.

The compositions described herein may be administered as part of a sustained release formulation (i.e., a formulation such as a capsule or sponge that effects a slow release of compound following administration). Such formulations may generally be prepared using well known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Sustained- release formulations may contain a polypeptide, polynucleotide or antibody dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane. Carriers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of cyclic peptide release. The amount of active compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.

CANCER THERAPY

In further aspects of the present invention, the compounds described herein may be used for therapy of cancer. Within such aspects, the compounds (which may be polypeptides, antibodies, nucleic acid molecules or other modulating agents) are preferably incorporated into pharmaceutical compositions or vaccines, as described above. Suitable patients for therapy may be any warm-blooded animal, preferably a human. A patient may or may not be afflicted with cancer, as determined by standard diagnostic methods. Accordingly, the above pharmaceutical compositions and vaccines may be used to prevent the development of cancer or to treat a patient afflicted with cancer. Compositions and vaccines may also be used to inhibit angiogenesis, and thereby inhibit tumor progression. Suitable agents for use in such compositions include antisense polynucleotides, ribozymes and small molecule inhibitors, which may generally be identified as described herein.

Within certain aspects, cells may be protected from therapeutic damage (e.g., due to chemotherapy or a physical agent such as gamma-irradiation) and/or rendered resistant to progression by inhibiting or eliminating the expression and/or activity of a progression-related protein in the cells. One preferred method for inhibiting the expression of a progression-related protein comprises providing an effective amount of antisense RNA in the cell. Such antisense technology can generally be used to control gene expression through triple-helix formation, which compromises the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors or regulatory molecules (see Gee et al., In Huber and Carr, Molecular and Immunologic Approaches, Futura Publishing Co. (Mt. Kisco, NY; 1994). Alternatively, an antisense molecule may be designed to hybridize with a control region of a gene (e.g. promoter, enhancer or transcription initiation site), and block transcription of the gene; or to block translation by inhibiting binding of a transcript to ribosomes. In general, the expression of a progression-related protein may be eliminated by deleting the gene or introducing mutation(s) into the gene.

Pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). The route, duration and frequency of administration will be determined by such factors as the condition of the patient, the type and severity of the patient's disease and the method of administration. Routes and frequency of administration may vary from individual to individual, and may be readily established using standard techniques. In general, the pharmaceutical compositions and vaccines may be administered by injection (e.g., intracutaneous, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally. Preferably, between 1 and 10 doses may be administered over a 52 week period. Preferably, 6 doses are administered, at intervals of 1 month, and booster vaccinations may be given periodically thereafter. Alternate protocols may be appropriate for individual patients.

In general, an appropriate dosage and treatment regimen provides the active compound(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit (e.g., an amount capable of inhibiting tumor cell angiogenesis). Such a benefit should results in an improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated patients as compared to non-treated patients.

Appropriate dosages of polypeptides, polynucleotides, antibodies and modulating agents may generally be determined using experimental models and/or clinical trials. In general, the use of the minimum dosage that is sufficient to provide effective therapy is preferred. Patients may generally be monitored for therapeutic effectiveness using assays suitable for the condition being treated or prevented, which will be familiar to those of ordinary skill in the art. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 mL to about 5 mL.

CANCER DETECTION. DIAGNOSIS AND MONITORING

Polypeptides, polynucleotides and antibodies, as described herein, may be used within a variety of methods for detecting a cancer, determining whether a cancer is in progression, and monitoring the progression and/or treatment of a cancer in a patient. Within such methods, any of a variety of methods may be used to detect the activity or the level of a progression-related protein or mRNA in a sample. Suitable biological samples include tumor or normal tissue biopsy, mastectomy, blood, lymph node, serum or urine samples, or other tissue, homogenate or extract thereof obtained from a patient.

Methods involving the use of an antibody may detect the presence or absence of a progression-related protein in any suitable biological sample. There are a variety of assay formats known to those of ordinary skill in the art for using an antibody to detect polypeptide markers in a sample. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. For example, the assay may be performed in a Western blot format, wherein a protein preparation from the biological sample is submitted to gel electrophoresis, transferred to a suitable membrane and allowed to react with the antibody. The presence of the antibody on the membrane may then be detected using a suitable detection reagent, as described below.

In another embodiment, the assay involves the use of antibody immobilized on a solid support to bind to the polypeptide and remove it from the remainder of the sample. The bound polypeptide may then be detected using a second antibody or reagent that contains a reporter group. Alternatively, a competitive assay may be utilized, in which a polypeptide is labeled with a reporter group and allowed to bind to the immobilized antibody after incubation of the antibody with the sample. The extent to which components of the sample inhibit the binding of the labeled polypeptide to the antibody is indicative of the reactivity of the sample with the immobilized antibody, and as a result, indicative of the concentration of polypeptide in the sample.

The solid support may be any material known to those of ordinary skill in the art to which the antibody may be attached. For example, the solid support may be a test well in a microtiter plate or a nitrocellulose filter or other suitable membrane. Alternatively, the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Patent No. 5,359,681.

The antibody may be immobilized on the solid support using a variety of techniques known to those in the art, which are amply described in the patent and scientific literature. In the context of the present invention, the term "immobilization" refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the antigen and functional groups on the support or may be a linkage by way of a cross-linking agent). Immobilization by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption may be achieved by contacting the antibody, in a suitable buffer, with the solid support for a suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and 1 day. In general, contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of antibody ranging from about 10 ng to about 1 μg, and preferably about 100-200 ng, is sufficient to immobilize an adequate amount of polypeptide.

Covalent attachment of antibody to a solid support may also generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the antibody. For example, the antibody may be covalently attached to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner using well known techniques.

In certain embodiments, the assay for detection of polypeptide in a sample is a two-antibody sandwich assay. This assay may be performed by first contacting an antibody that has been immobilized on a solid support, commonly the well of a microtiter plate, with the biological sample, such that the polypeptide within the sample are allowed to bind to the immobilized antibody. Unbound sample is then removed from the immobilized polypeptide-antibody complexes and a second antibody (containing a reporter group) capable of binding to a different site on the polypeptide is added. The amount of second antibody that remains bound to the solid support is then determined using a method appropriate for the specific reporter group.

More specifically, once the antibody is immobilized on the support as described above, the remaining protein binding sites on the support are typically blocked. Any suitable blocking agent known to those of ordinary skill in the art, such as bovine serum albumin or Tween 20™ (Sigma Chemical Co., St. Louis, MO). The immobilized antibody is then incubated with the sample, and polypeptide is allowed to bind to the antibody. The sample may be diluted with a suitable diluent, such as phosphate-buffered saline (PBS) prior to incubation. In general, an appropriate contact time (i.e., incubation time) is that period of time that is sufficient to detect the presence of polypeptide within a sample obtained from an individual with cancer. Preferably, the contact time is sufficient to achieve a level of binding that is at least 95% of that achieved at equilibrium between bound and unbound polypeptide. Those of ordinary skill in the art will recognize that the time necessary to achieve equilibrium may be readily determined by assaying the level of binding that occurs over a period of time. At room temperature, an incubation time of about 30 minutes is generally sufficient.

Unbound sample may then be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% Tween 20™. The second antibody, which contains a reporter group, may then be added to the solid support. Preferred reporter groups include enzymes (such as horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin. The conjugation of antibody to reporter group may be achieved using standard methods known to those of ordinary skill in the art.

The second antibody is then incubated with the immobilized antibody- polypeptide complex for an amount of time sufficient to detect the bound polypeptide. An appropriate amount of time may generally be determined by assaying the level of binding that occurs over a period of time. Unbound second antibody is then removed and bound second antibody is detected using the reporter group. The method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups. Biotin may be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups may generally be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products.

To determine whether cells are in progression, expression of a progression- related protein in the cells is evaluated and compared with the level of expression in cells that are not in progression. Briefly, the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal that corresponds to a predetermined cut-off value established from cells that are not in progression. In one preferred embodiment, the cut-off value is the average mean signal obtained when the immobilized antibody is incubated with samples from cells that are not in progression. In general, a sample generating a signal that is three standard deviations above the predetermined cut-off value may be considered positive for progression. In an alternate preferred embodiment, the cut-off value is determined using a Receiver Operator Curve, according to the method of Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, p. 106-7 (Little Brown and Co., 1985). Briefly, in this embodiment, the cut-off value may be determined from a plot of pairs of true positive rates (i.e., sensitivity) and false positive rates (100%-specificity) that correspond to each possible cut-off value for the diagnostic test result. The cut-off value on the plot that is the closest to the upper left-hand corner (i.e., the value that encloses the largest area) is the most accurate cut-off value, and a sample generating a signal that is higher than the cut-off value determined by this method may be considered positive. Alternatively, the cut-off value may be shifted to the left along the plot, to minimize the false positive rate, or to the right, to minimize the false negative rate. In general, a sample generating a signal that is higher than the cut-off value determined by this method is considered positive for progression.

In a related embodiment, the assay is performed in a flow- through or strip test format, wherein the antibody is immobilized on a membrane, such as nitrocellulose. In the flow-through test, the polypeptide within the sample bind to the immobilized antibody as the sample passes through the membrane. A second, labeled antibody then binds to the antibody-polypeptide complex as a solution containing the second antibody flows through the membrane. The detection of bound second antibody may then be performed as described above. In the strip test format, one end of the membrane to which antibody is bound is immersed in a solution containing the sample. The sample migrates along the membrane through a region containing second antibody and to the area of immobilized antibody. Concentration of second antibody at the area of immobilized antibody indicates the presence of cells in progression. Typically, the concentration of second antibody at that site generates a pattern, such as a line, that can be read visually. The absence of such a pattern indicates a negative result. In general, the amount of antibody immobilized on the membrane is selected to generate a visually discernible pattern when the biological sample contains a level of polypeptide that would be sufficient to generate a positive signal in the two-antibody sandwich assay, in the format discussed above. Preferably, the amount of antibody immobilized on the membrane ranges from about 25 ng to about 1 μg, and more preferably from about 50 ng to about 1 μg. Such tests can typically be performed with a very small amount of biological sample.

The presence or absence of cells in progression in a patient may also be determined by evaluating the level of mRNA encoding a progression-related protein within the biological sample (e.g., a biopsy, mastectomy and/or blood sample from a patient) relative to a predetermined cut-off value. Such an evaluation may be achieved using any of a variety of methods known to those of ordinary skill in the art such as, for example, in situ hybridization and amplification by polymerase chain reaction. Probes and primers for use within such assays may generally be designed based on the sequences provided herein, or on similar sequences identified in other individuals. Probes may be used within well known hybridization techniques, and may be labeled with a detection reagent to facilitate detection of the probe. Such reagents include, but are not limited to, radionuclides, fluorescent dyes and enzymes capable of catalyzing the formation of a detectable product.

Primers may generally be used within detection methods involving polymerase chain reaction (PCR), such as RT-PCR, in which PCR is applied in conjunction with reverse transcription. Typically, RNA is extracted from a sample tissue and is reverse transcribed to produce cDNA molecules. PCR amplification using specific primers generates a progression-related cDNA molecule, which may be separated and visualized using, for example, gel electrophoresis. Amplification is typically performed on samples obtained from matched pairs of tissue (tumor and non-tumor tissue from the same individual) or from unmatched pairs of tissue (tumor and non-tumor tissue from different individuals). The amplification reaction may be performed on several dilutions of cDNA spanning two orders of magnitude. A two-fold or greater increase in expression in several dilutions of the tumor sample as compared to the same dilutions of the non- tumor sample is typically considered positive.

Within certain specific embodiments, expression of a progression-related gene may be detected in a sample that contains cells by: (a) obtaining RNA from the cells; (b) contacting the RNA so obtained with a labeled (e.g., radioactively) probe specific for a progression-related gene under hybridizing conditions permitting specific hybridization of the probe and the RNA; and (c) determining the presence of RNA hybridized to the probe. As noted above, mRNA may be isolated and hybridized using any of a variety of procedures well-known to a person of ordinary skill in the art. The presence of mRNA hybridized to the probe may be determined by gel electrophoresis or other methods known in the art. By measuring the amount of the hybrid formed, the expression of the a progression-related protein by the cell can be determined. Alternatively, RNA obtained from the cells may be amplified by polymerase chain reaction (PCR) with appropriate primers derived from a known progression-related sequence. The presence of specific amplified DNA following PCR is an indicative of progression-related protein expression in the cells.

Certain in vivo diagnostic assays may be performed directly on the tumor. One such assay involves contacting tumor cells with an antibody or fragment thereof that binds to a progression-related protein. The bound antibody or fragment may then be detected directly or indirectly via a reporter group. Such antibodies may also be used in histological applications.

Within related aspects, the present invention provides methods for diagnosing the aggressiveness of cancer cells. Such methods are performed as described above, wherein an increase in the amount of the expression indicates that a cancer cell is more aggressive.

In other aspects of the present invention, the progression and/or response to treatment of a cancer may be monitored by performing any of the above assays over a period of time, and evaluating the change in the level of the response (i.e., the amount of polypeptide or mRNA detected). For example, the assays may be performed every month to every other month for a period of 1 to 2 years. In general, a cancer is progressing in those patients in whom the level of the response increases over time. In contrast, a cancer is not progressing when the signal detected either remains constant or decreases with time.

The present invention further provides kits for use within any of the above diagnostic methods. Such kits typically comprise two or more components necessary for performing the assay. Such components may be compounds, reagents and/or containers or equipment. For example, one container within a kit may contain a monoclonal antibody or fragment thereof that specifically binds to a progression-related polypeptide. Such antibodies or fragments may be provided attached to a support material, as described above. One or more additional containers may enclose elements, such as reagents or buffers, to be used in the assay. Such kits may also contain a detection reagent (e.g., an antibody) that contains a reporter group suitable for direct or indirect detection of antibody binding.

TRANSGENIC ORGANISMS

The present invention also provides transgenic nonhuman living organism expressing a progression-related protein. In an embodiment, the living organism is animal.

One means available for producing a transgenic animal, with a mouse as an example, is as follows: Female mice are mated, and the resulting fertilized eggs are dissected out of their oviducts. The eggs are stored in an appropriate medium. Progression-related DNA or cDNA is purified from a vector by methods well-known in the art. Inducible promoters may be fused with the coding region of the DNA to provide an experimental means to regulate expression of the trans-gene. Alternatively or in addition, tissue specific regulatory elements may be fused with the coding region to permit tissue-specific expression of the trans-gene. The DNA, in an appropriately buffered solution, is put into a microinjection needle (which may be made from capillary tubing using a pipes puller) and the egg to be injected is put in a depression slide. The needle is inserted into the pronucleus of the egg, and the DNA solution is injected. The injected egg is then transferred into the oviduct of a pseudopregnant mouse (a mouse stimulated by the appropriate hormones to maintain pregnancy but which is not actually pregnant), where it proceeds to the uterus, implants, and develops to term. As noted above, microinjection is not the only method for inserting DNA into the egg cell, and is used here only for exemplary purposes.

The following Example is offered by way of illustration and not by way of limitation. EXAMPLE Cloning and Sequencing of Rat Progression Related cDNAs This Example illustrates the isolation and initial characterization of progression-related genes.

Progression-related genes were cloned from a progressed mutant Ad5 (H5tsl25)-transformed rat embryo clone El 1-NMT. The isolation, properties and growth conditions of the El 1-NMT cells, as well as the parental unprogressed cells El l, have been described previously (see, e.g., Babiss et al., Science 225:1099-1101, 1985). Clones were isolated using subtraction hybridization as described (Jiang & Fisher, Mol. Cell. Different. 7:285-299, 1993). Briefly, an El 1-NMT cDNA library was subtracted with El 1 cDNA and probed with El 1 and El 1-NMT RNA. Clones that were upregulated in El 1-NMT cells were sequenced by the dideoxy-chain termination (Sanger) method (Su et al., Cancer Res. 55:1929-1938, 1993). Previously unknown sequences identified in this manner (3' and 5') are provided in Figures 1-12 and SEQ ID NOs:l-24.

From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

1. An isolated polypeptide comprising at least a portion of a progression-related protein, or a variant thereof, wherein:

(a) the progression-related protein comprises a sequence encoded by a nucleotide sequence recited in any one of SEQ ID NOs: 1-24; and

(b) the portion retains at least one immunological and/or biological activity characteristic of the progression-related protein.

2. A polypeptide according to claim 1 wherein the portion is immunologically active.

3. An isolated polynucleotide encoding a polypeptide according to claim 1.

4. An antisense polynucleotide comprising a sequence complementary to a polynucleotide encoding a polypeptide according to claim 1.

5. An expression vector comprising a polynucleotide according to claim 3.

6. A host cell transformed or transfected with an expression vector according to claim 5.

7. A pharmaceutical composition, comprising:

(a) a polypeptide according to claim 1 ; and

(b) a physiologically acceptable carrier.

8. A vaccine, comprising:

(a) a polypeptide according to claim 1 ; and (b) an immune response enhancer.

9. A pharmaceutical composition, comprising:

(a) an antisense polynucleotide according to claim 4; and

(b) a physiologically acceptable carrier.

10. A monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to a polypeptide according to claim 1.

11. A pharmaceutical composition, comprising:

(a) an antibody according to claim 10; and

(b) a physiologically acceptable carrier.

12. A method for inhibiting the progression of a cancer in a patient, comprising administering to a patient an agent that inhibits expression of a polypeptide according to claim 1

13. A method for detecting expression of a progression-related gene in a cell sample, comprising steps of:

(a) obtaining RNA from a cell sample;

(b) contacting the RNA with a labeled probe capable of hybridizing to a progression-related mRNA comprising a sequence recited in any one of SEQ ID NOs:l- 24 or a variant of any of the foregoing sequences under conditions permitting specific hybridization of the probe and the RNA; and

(c) determining the presence of RNA hybridized to the probe, and therefrom detecting the expression of the progression-related gene in the sample.

14. A method for determining whether cells are in progression, comprising the steps of: (a) measuring expression of a progression-related gene comprising a sequence recited in any one of SEQ ID NOs: 1-24 or a variant of any of the foregoing sequences in a sample of cells; and

(b) comparing the expression measured in step (a) with the expression of the progression-related gene in cells that are not in progression, and therefrom determining whether the cells are in progression.

15. A method for determining whether a cancer in a patient is in progression, comprising detecting, in a biological sample obtained from a patient, a polypeptide according to claim 1, and therefrom determining whether a cancer in the patient is in progression.

16. A method according to claim 15 wherein the biological sample is a portion of a tumor.

17. A method according to claim 15 wherein the step of detecting comprises contacting the biological sample with a monoclonal antibody that specifically recognizes a polypeptide according to claim 1.

18. A method for determining whether a cancer in a patient is in progression, comprising detecting, in a biological sample obtained from a patient, a polynucleotide encoding a polypeptide according to claim 1, or a portion thereof, and therefrom determining whether a cancer in the patient is in progression.

19. A method according to claim 18 wherein the biological sample is a portion of a tumor.

20. A method according to claim 18 wherein the step of detecting comprises: (a) preparing cDNA from RNA molecules in the biological sample; and

(b) specifically amplifying cDNA molecules that are capable of encoding at least a portion of a polypeptide according to claim 1.

21. A method for monitoring the progression of a cancer in a patient, comprising:

(a) detecting, in a biological sample obtained from a patient, an amount of a polypeptide according to claim 1 at a first point in time;

(b) repeating step (a) at a subsequent point in time; and

(c) comparing the amounts of polypeptide detected in steps (a) and (b), and therefrom monitoring the progression of a cancer in the patient.

22. A method according to claim 21 wherein the biological sample is a portion of a tumor.

23. A method according to claim 21 wherein the step of detecting comprises contacting a portion of the biological sample with a monoclonal antibody that specifically recognizes a polypeptide according to claim 1.

24. A method for monitoring the progression of a cancer in a patient, comprising:

(a) detecting, in a biological sample obtained from a patient, an amount of an RNA molecule encoding a polypeptide according to claim 1 at a first point in time;

(b) repeating step (a) at a subsequent point in time; and

(c) comparing the amounts of RNA molecules detected in steps (a) and (b), and therefrom monitoring the progression of a cancer in the patient.

25. A method according to claim 24 wherein the step of detecting comprises:

(a) preparing cDNA from RNA molecules in the biological sample; and

(b) specifically amplifying cDNA molecules that are capable of encoding at least a portion of a polypeptide according to claim 1.

26. A diagnostic kit, comprising:

(a) a monoclonal antibody or fragment thereof according to claim 10; and

(b) a second monoclonal antibody or fragment thereof that binds to (i) a monoclonal antibody recited in step (a); or

(ii) a polypeptide according to claim 1 ; wherein the second monoclonal antibody is conjugated to a reporter group.

27. A method for preparing a polypeptide according to claim 1, comprising the steps of:

(a) culturing a host cell according to claim 6 under conditions suitable for the expression of the polypeptide; and

(b) recovering the polypeptide from the host cell culture.

28. A method for producing cells that are resistant to progression, comprising inhibiting or eliminating the expression of a progression-related gene comprising a sequence recited in any one of SEQ ID NOs: 1-24 or a variant of any of the foregoing sequences in the cells.

29. A method for protecting cells from chemotherapeutic damage, comprising inhibiting or eliminating the expression of a progression-related gene comprising a sequence recited in any one of SEQ ID NOs: 1-24 or a variant of any of the foregoing sequences in the cells.

30. A cell transformed or transfected with a reporter gene under the control of a human progression-related gene promoter, or regulatory element thereof, wherein the progression-related gene comprises a sequence recited in any one of SEQ ID NOs: 1-24 or a variant of any of the foregoing sequences.

31. A method for identifying an agent that modulates the expression of a progression-related gene, comprising the steps of:

(a) contacting a candidate agent with a cell transformed or transfected with a reporter gene under the control of a progression-related gene promoter or a regulatory element thereof under conditions and for a time sufficient to allow the candidate agent to interact with the promoter or regulatory element thereof, wherein the progression-related gene comprises a sequence recited in any one of SEQ ID NOs: 1-24 or a variant of any of the foregoing sequences; and

(b) determining the effect of the candidate agent on the level of reporter protein produced by the cell, and therefrom identifying an agent that modulates expression of a progression-related gene.

32. A method for identifying an agent that modulates the ability of a progression-related protein to induce progression, comprising the steps of:

(a) contacting a candidate agent with a polypeptide according to claim 1, under conditions and for a time sufficient to allow the candidate agent and polypeptide to interact; and

(b) determining the effect of the candidate agent on the ability of the polypeptide to induce progression, and therefrom identifying an agent that modulates the ability of a progression-related protein to induce progression.