	CHART 1


	Original Residue	Exemplary Substitutions

	Ala	Ser
	Arg	Lys
	Asn	Gln, His
	Asp	Glu
	Cys	Ser
	Gln	Asn
	Glu	Asp
	Gly	Pro
	His	Asn, Gln
	Ile	Leu, Val
	Leu	Ile, Val
	Lys	Arg, Gln, Glu
	Met	Leu, Ile
	Phe	Met, Leu, Tyr
	Ser	Thr
	Thr	Ser
	Trp	Tyr
	Tyr	Trp, Phe
	Val	Ile, Leu

Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those shown in Chart I. For example, substitutions may be made full length to more significantly affect one or more of the following: the structure of the polypeptide backbone in the area of the alteration (e.g., the alpha-helical or beta-sheet structure); the charge or hydrophobicity of the molecule at the target site; and the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the polypeptide's properties are those in which (a) a hydrophilic residue, e.g. seryl or threonyl is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g. glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g. phenylalanine, is substituted for (or by) one not having a side chain, e.g. glycine.[0139]

The variants typically exhibit the same qualitative biological activity and will elicit the same immune response as the naturally-occurring analogue, although variants also are selected to modify the characteristics of the CA proteins as needed. Alternatively, the variant may be designed such that the biological activity of the CA protein is altered. For example, glycosylation sites may be altered or removed, dominant negative mutations created, etc.[0140]

Covalent modifications of CA polypeptides are included within the scope of this invention, for example for use in screening. One type of covalent modification includes reacting targeted amino acid residues of a CA polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N-or C-terminal residues of a CA polypeptide. Derivatization with bifunctional agents is useful, for instance, for crosslinking CA polypeptides to a water-insoluble support matrix or surface for use in the method for purifying anti-CA antibodies or screening assays, as is more fully described below. Commonly used crosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-1,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.[0141]

Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl, threonyl or tyrosyl residues, methylation of the a-amino groups of lysine, arginine, and histidine side chains [T. E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.[0142]

Another type of covalent modification of the CA polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide. “Altering the native glycosylation pattern” is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence CA polypeptide, and/or adding one or more glycosylation sites that are not present in the native sequence CA polypeptide.[0143]

Addition of glycosylation sites to CA polypeptides may be accomplished by altering the amino acid sequence thereof. The alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence CA polypeptide (for O-linked glycosylation sites). The CA amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the CA polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.[0144]

Another means of increasing the number of carbohydrate moieties on the CA polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330 published 11 Sep. 1987, and in Aplin and Wriston, LA Crit. Rev. Biochem., pp. 259-306 (1981).[0145]

Removal of carbohydrate moieties present on the CA polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al., Meth. Enzymol., 138:350 (1987).[0146]

Another type of covalent modification of CA comprises linking the CA polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.[0147]

CA polypeptides of the present invention may also be modified in a way to form chimeric molecules comprising a CA polypeptide fused to another, heterologous polypeptide or amino acid sequence. In one embodiment, such a chimeric molecule comprises a fusion of a CA polypeptide with a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally placed at the amino-or carboxyl-terminus of the CA polypeptide, although internal fusions may also be tolerated in some instances. The presence of such epitope-tagged forms of a CA polypeptide can be detected using an antibody against the tag polypeptide. Also, provision of the epitope tag enables the CA polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. In an alternative embodiment, the chimeric molecule may comprise a fusion of a CA polypeptide with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule, such a fusion could be to the Fe region of an IgG molecule.[0148]

Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and Cellular Biology, 5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194 (1992)]; tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the[0149]T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)].

Also included with the definition of CA protein in one embodiment are other CA proteins of the CA family, and CA proteins from other organisms, which are cloned and expressed as outlined below. Thus, probe or degenerate polymerase chain reaction (PCR) primer sequences may be used to find other related CA proteins from humans or other organisms. As will be appreciated by those in the art, particularly useful probe and/or PCR primer sequences include the unique areas of the CA nucleic acid sequence. As is generally known in the art, preferred PCR primers are from about 15 to about 35 nucleotides in length, with from about 20 to about 30 being preferred, and may contain inosine as needed. The conditions for the PCR reaction are well known in the art.[0150]

In addition, as is outlined herein, CA proteins can be made that are longer than those encoded by the nucleic acids of the figures, for example, by the elucidation of additional sequences, the addition of epitope or purification tags, the addition of other fusion sequences, etc.[0151]

CA proteins may also be identified as being encoded by CA nucleic acids. Thus, CA proteins are encoded by nucleic acids that will hybridize to the sequences of the sequence listings, or their complements, as outlined herein.[0152]

CA Antigens and Antibodies Thereto[0153]

In one embodiment, the invention provides CA specific antibodies. In a preferred embodiment, when the CA protein is to be used to generate antibodies, for example for immunotherapy, the CA protein should share at least one epitope or determinant with the full-length protein. By “epitope” or “determinant” herein is meant a portion of a protein that will generate and/or bind an antibody or T-cell receptor in the context of MHC. Thus, in most instances, antibodies made to a smaller CA protein will be able to bind to the full-length protein. In a preferred embodiment, the epitope is unique; that is, antibodies generated to a unique epitope show little or no cross-reactivity.[0154]

Any polypeptide sequence encoded by the CA polynucleotide sequences may be analyzed to determine certain preferred regions of the polypeptide. Regions of high antigenicity are determined from data by DNASTAR analysis by choosing values that represent regions of the polypeptide that are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response. For example, the amino acid sequence of a polypeptide encoded by a CA polynucleotide sequence may be analyzed using the default parameters of the DNASTAR computer algorithm (DNASTAR, Inc., Madison, Wis.; http://www.dnastar.com/).[0155]

Polypeptide features that may be routinely obtained using the DNASTAR computer algorithm include, but are not limited to, Gamier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions (Gamier et al.[0156]J. Mol. Biol.,120: 97 (1978)); Chou-Fasman alpha-regions, beta-regions, and turn-regions (Adv. in Enzymol.,47:45-148 (1978)); Kyte-Doolittle hydrophi lic regions and hydrophobic regions (J. Mol. Biol.,157:105-132 (1982)); Eisenberg alpha- and beta-amphipathic regions; Karplus-Schulz flexible regions; Emini surface-forming regions (J. Virol.,55(3):836-839 (1985)); and Jameson-Wolf regions of high antigenic index (CABIOS,4(1):181-186 (1988)). Kyte-Doolittle hydrophilic regions and hydrophobic regions, Emini surface-forming regions, and Jameson-Wolf regions of high antigenic index (i.e., containing four or more contiguous amino acids having an antigenic index of greater than or equal to 1.5, as identified using the default parameters of the Jameson-Wolf program) can routinely be used to determine polypeptide regions that exhibit a high degree of potential for antigenicity. One approach for preparing antibodies to a protein is the selection and preparation of an amino acid sequence of all or part of the protein, chemically synthesizing the sequence and injecting it into an appropriate animal, typically a rabbit, hamster or a mouse. Oligopeptides can be selected as candidates for the production of an antibody to the CA protein based upon the oligopeptides lying in hydrophilic regions, which are thus likely to be exposed in the mature protein. Additional oligopeptides can be determined using, for example, the Antigenicity Index, Welling, G. W. et al.,FEBS Lett.188:215-218 (1985), incorporated herein by reference.

In one embodiment, the term “antibody” includes antibody fragments, as are known in the art, including Fab, Fab[0157]₂, single chain antibodies (Fv for example), chimeric antibodies, etc., either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies.

Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include a protein encoded by a nucleic acid of the figures or fragment thereof or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants that may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.[0158]

The antibodies may, alternatively, be monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein,[0159]Nature,256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro. The immunizing agent will typically include a polypeptide encoded by a nucleic acid of Tables 1-27, or fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes (“PBLs”) are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse mycloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.

Monoclonal antibody technology is used in implementing research, diagnosis and therapy. Monoclonal antibodies are used in radioimmunoassays, enzyme-linked immunosorbent assays, immunocytopathology, and flow cytometry for in vitro diagnosis, and in vivo for diagnosis and immunotherapy of human disease. Waldmann, T. A. (1991)[0160]Science252:1657-1662. In particular, monoclonal antibodies have been widely applied to the diagnosis and therapy of cancer, wherein it is desirable to target malignant lesions while avoiding normal tissue. See, e.g., U.S. Pat. No. 4,753,894 to Frankel, et al.; U.S. Pat. No. 4,938,948 to Ring et al.; and U.S. Pat. No. 4,956,453 to Bjorn et al.

In one embodiment, the antibodies are bispecific antibodies. Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. A number of “humanized” antibody molecules comprising an antigen-binding site derived from a non-human immunoglobulin have been described, including chimeric antibodies having rodent V regions and their associated CDRs fused to human constant domains (Winter et al. (1991)[0161]Nature349:293-299; Lobuglio et al. (1989)Proc. Nat. Acad. Sci. USA86:4220-4224; Shaw et al. (1987)J Immunol.138:4534-4538; and Brown et al. (1987)Cancer Res.47:3577-3583), rodent CDRs grafted into a human supporting FR prior to fusion with an appropriate human antibody constant domain (Riechmann et al. (1988)Nature332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536; and Jones et al. (1986)Nature321:522-525), and rodent CDRs supported by recombinantly veneered rodent FRs (European Patent Publication No. 519,596, published Dec. 23, 1992). These “humanized” molecules are designed to minimize unwanted immunological response toward rodent antihuman antibody molecules which limits the duration and effectiveness of therapeutic applications of those moieties in human recipients. In the present case, one of the binding specificities is for a protein encoded by a nucleic acid of Tables 1-27, or a fragment thereof, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit, preferably one that is tumor specific.

In a preferred embodiment, the antibodies to CA are capable of reducing or eliminating the biological function of CA, as is described below. That is, the addition of anti-CA antibodies (either polyclonal or preferably monoclonal) to CA (or cells containing CA) may reduce or eliminate the CA activity. Generally, at least a 25% decrease in activity is preferred, with at least about 50% being particularly preferred and about a 95-100% decrease being especially preferred.[0162]

In a preferred embodiment the antibodies to the CA proteins are humanized antibodies. “Humanized” antibodies refer to a molecule having an antigen binding site that is substantially derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule based upon the structure and/or sequence of a human immunoglobulin. The antigen binding site may comprise either complete variable domains fused onto constant domains or only the complementarity determining regions (CDRs) grafted onto appropriate framework regions in the variable domains. Antigen binding sites may be wild type or modified by one or more amino acid substitutions, e.g., modified to resemble human immunoglobulin more closely. Alternatively, a humanized antibody may be derived from a chimeric antibody that retains or substantially retains the antigen-binding properties of the parental, non-human, antibody but which exhibits diminished immunogenicity as compared to the parental antibody when administered to humans. The phrase “chimeric antibody,” as used herein, refers to an antibody containing sequence derived from two different antibodies (see, e.g., U.S. Pat. No. 4,816,567) that typically originate from different species. Typically, in these chimeric antibodies, the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals, while the constant portions are homologous to the sequences in antibodies derived from another. Most typically, chimeric antibodies comprise human and murine antibody fragments, generally human constant and mouse variable regions. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework residues (FR) regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al.,[0163]Nature,321:522-525 (1986); Riechmann et al.,Nature,332:323-329 (1988); and Presta,Curr. Op. Struct. Biol.,2:593-596 (1992)). One clear advantage to such chimeric forms is that, for example, the variable regions can conveniently be derived from presently known sources using readily available hybridomas or B cells from non human host organisms in combination with constant regions derived from, for example, human cell preparations. While the variable region has the advantage of ease of preparation, and the specificity is not affected by its source, the constant region being human, is less likely to elicit an immune response from a human subject when the antibodies are injected than would the constant region from a non-human source. However, the definition is not limited to this particular example.

Because humanized antibodies are far less immunogenic in humans than the parental mouse monoclonal antibodies, they can be used for the treatment of humans with far less risk of anaphylaxis. Thus, these antibodies may be preferred in therapeutic applications that involve in vivo administration to a human such as, e.g., use as radiation sensitizers for the treatment of neoplastic disease or use in methods to reduce the side effects of, e.g., cancer therapy. Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al.,[0164]Nature321:522-525 (1986); Riechmann et al.,Nature332:323-327 (1988); Verhoeyen et al.,Science239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies [Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boemer et al., J. Immunol., 147(1):86-95 (1991)]. Humanized antibodies may be achieved by a variety of methods including, for example: (1) grafting the non-human complementarity determining regions (CDRs) onto a human framework and constant region (a process referred to in the art as “humanizing”), or, alternatively, (2) transplanting the entire non-human variable domains, but “cloaking” them with a human-like surface by replacement of surface residues (a process referred to in the art as “veneering”). In the present invention, humanized antibodies will include both “humanized” and “veneered” antibodies. Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/[0165]Technology 10, 779-783 (1992); Lonberg et al.,Nature368 856-859 (1994); Morrison,Nature368, 812-13 (1994); Fishwild et al.,Nature Biotechnology14, 845-51 (1996); Neuberger,Nature Biotechnology14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol.13 65-93 (1995); Jones et al.,Nature321:522-525 (1986); Morrison et al.,Proc. Natl. Acad. Sci, U.S.A.,81:6851-6855 (1984); Morrison and Oi,Adv. Immunol.,44:65-92 (1988); Verhoeyer et al.,Science239:1534-1536 (1988); Padlan,Molec. Immun.28:489-498 (1991); Padlan,Molec. Immunol.31(3):169-217 (1994); and Kettleborough, C. A. et al.,Protein Eng.4(7):773-83 (1991) each of which is incorporated herein by reference.

The phrase “complementarity determining region” refers to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. See, e.g., Chothia et al.,[0166]J. Mol. Biol.196:901-917 (1987); Kabat et al., U.S. Dept. of Health and Human Services NIH Publication No. 91-3242 (1991). The phrase “constant region” refers to the portion of the antibody molecule that confers effector functions. In the present invention, mouse constant regions are substituted by human constant regions. The constant regions of the subject humanized antibodies are derived from human immunoglobulins. The heavy chain constant region can be selected from any of the five isotypes: alpha, delta, epsilon, gamma or mu. One method of humanizing antibodies comprises aligning the non-human heavy and light chain sequences to human heavy and light chain sequences, selecting and replacing the non-human framework with a human framework based on such alignment, molecular modeling to predict the conformation of the humanized sequence and comparing to the conformation of the parent antibody. This process is followed by repeated back mutation of residues in the CDR region that disturb the structure of the CDRs until the predicted conformation of the humanized sequence model closely approximates the conformation of the non-human CDRs of the parent non-human antibody. Such humanized antibodies may be further derivatized to facilitate uptake and clearance, e.g, via Ashwell receptors. See, e.g., U.S. Pat. Nos. 5,530,101 and 5,585,089 which are incorporated herein by reference.

Humanized antibodies to CA polypeptides can also be produced using transgenic animals that are engineered to contain human immunoglobulin loci. For example, WO 98/24893 discloses transgenic animals having a human Ig locus wherein the animals do not produce functional endogenous immunoglobulins due to the inactivation of endogenous heavy and light chain loci. WO 91/10741 also discloses transgenic non-primate mammalian hosts capable of mounting an immune response to an immunogen, wherein the antibodies have primate constant and/or variable regions, and wherein the endogenous immunoglobulin-encoding loci are substituted or inactivated. WO 96/30498 discloses the use of the Cre/Lox system to modify the immunoglobulin locus in a mammal, such as to replace all or a portion of the constant or variable region to form a modified antibody molecule. WO 94/02602 discloses non-human mammalian hosts having inactivated endogenous Ig loci and functional human Ig loci. U.S. Pat. No. 5,939,598 discloses methods of making transgenic mice in which the mice lack endogenous heavy chains, and express an exogenous immunoglobulin locus comprising one or more xenogeneic constant regions.[0167]

Using a transgenic animal described above, an immune response can be produced to a selected antigenic molecule, and antibody-producing cells can be removed from the animal and used to produce hybridomas that secrete human monoclonal antibodies. Immunization protocols, adjuvants, and the like are known in the art, and are used in immunization of, for example, a transgenic mouse as described in WO 96/33735. The monoclonal antibodies can be tested for the ability to inhibit or neutralize the biological activity or physiological effect of the corresponding protein.[0168]

In the present invention, CA polypeptides of the invention and variants thereof are used to immunize a transgenic animal as described above. Monoclonal antibodies are made using methods known in the art, and the specificity of the antibodies is tested using isolated CA polypeptides. Methods for preparation of the human or primate CA or an epitope thereof include, but are not limited to chemical synthesis, recombinant DNA techniques or isolation from biological samples. Chemical synthesis of a peptide can be performed, for example, by the classical Merrifeld method of solid phase peptide synthesis (Merrifeld,[0169]J. Am. Chem. Soc.85:2149, 1963 which is incorporated by reference) or the FMOC strategy on a Rapid Automated Multiple Peptide Synthesis system (E. I. du Pont de Nemours Company, Wilmington, Del.) (Caprino and Han,J. Org. Chem.37:3404, 1972 which is incorporated by reference).

Polyclonal antibodies can be prepared by immunizing rabbits or other animals by injecting antigen followed by subsequent boosts at appropriate intervals. The animals are bled and sera assayed against purified CA proteins usually by ELISA or by bioassay based upon the ability to block the action of CA proteins. When using avian species, e.g., chicken, turkey and the like, the antibody can be isolated from the yolk of the egg. Monoclonal antibodies can be prepared after the method of Milstein and Kohler by fusing splenocytes from immunized mice with continuously replicating tumor cells such as myeloma or lymphoma cells. (Milstein and Kohler,[0170]Nature256:495-497, 1975; Gulfre and Milstein,Methods in Enzymology: Immunochemical Techniques73:1-46, Langone and Banatis eds., Academic Press, 1981 which are incorporated by reference). The hybridoma cells so formed are then cloned by limiting dilution methods and supernates assayed for antibody production by ELISA, RIA or bioassay.

The unique ability of antibodies to recognize and specifically bind to target proteins provides an approach for treating an overexpression of the protein. Thus, another aspect of the present invention provides for a method for preventing or treating diseases involving overexpression of a CA polypeptide by treatment of a patient with specific antibodies to the CA protein.[0171]

Specific antibodies, either polyclonal or monoclonal, to the CA proteins can be produced by any suitable method known in the art as discussed above. For example, murine or human monoclonal antibodies can be produced by hybridoma technology or, alternatively, the CA proteins, or an immunologically active fragment thereof, or an anti-idiotypic antibody, or fragment thereof can be administered to an animal to elicit the production of antibodies capable of recognizing and binding to the CA proteins. Such antibodies can be from any class of antibodies including, but not limited to IgG, IgA, IgM, IgD, and IgE or in the case of avian species, IgY and from any subclass of antibodies.[0172]

By immunotherapy is meant treatment of a cancer with an antibody raised against a CA protein. As used herein, immunotherapy can be passive or active. Passive immunotherapy as defined herein is the passive transfer of antibody to a recipient (patient). Active immunization is the induction of antibody and/or T-cell responses in a recipient (patient). Induction of an immune response is the result of providing the recipient with an antigen to which antibodies are raised. As appreciated by one of ordinary skill in the art, the antigen may be provided by injecting a polypeptide against which antibodies are desired to be raised into a recipient, or contacting the recipient with a nucleic acid capable of expressing the antigen and under conditions for expression of the antigen.[0173]

In a preferred embodiment, oncogenes which encode secreted growth factors may be inhibited by raising antibodies against CA proteins that are secreted proteins as described above. Without being bound by theory, antibodies used for treatment, bind and prevent the secreted protein from binding to its receptor, thereby inactivating the secreted CA protein.[0174]

In another preferred embodiment, the CA protein to which antibodies are raised is a transmembrane protein. Without being bound by theory, antibodies used for treatment, bind the extracellular domain of the CA protein and prevent it from binding to other proteins, such as circulating ligands or cell-associated molecules. The antibody may cause down-regulation of the transmembrane CA protein. As will be appreciated by one of ordinary skill in the art, the antibody may be a competitive, non-competitive or uncompetitive inhibitor of protein binding to the extracellular domain of the CA protein. The antibody is also an antagonist of the CA protein. Further, the antibody prevents activation of the transmembrane CA protein. In one aspect, when the antibody prevents the binding of other molecules to the CA protein, the antibody prevents growth of the cell. The antibody may also sensitize the cell to cytotoxic agents, including, but not limited to TNF-α, TNF-β, IL-1, INF-γ and IL-2, or chemotherapeutic agents including 5FU, vinblastine, actinomycin D, cisplatin, methotrexate, and the like. In some instances the antibody belongs to a sub-type that activates serum complement when complexed with the transmembrane protein thereby mediating cytotoxicity. Thus, cancers may be treated by administering to a patient antibodies directed against the transmembrane CA protein.[0175]

In another preferred embodiment, the antibody is conjugated to a therapeutic moiety. In one aspect the therapeutic moiety is a small molecule that modulates the activity of the CA protein. In another aspect the therapeutic moiety modulates the activity of molecules associated with or in close proximity to the CA protein. The therapeutic moiety may inhibit enzymatic activity such as protease or protein kinase activity associated with cancer.[0176]

In a preferred embodiment, the therapeutic moiety may also be a cytotoxic agent. In this method, radioisotopes, natural toxins, chemotherapy agents, or other substances (such as biological response modifiers) are chemically linked or conjugated to a monoclonal antibody to form “immunoconjugates” and “immunotoxins” which target the cytotoxic agent to tumor tissue or cells resulting in a reduction in the number of afflicted cells, thereby reducing symptoms associated with cancers, including lymphoma. Cytotoxic agents are numerous and varied and include, but are not limited to, cytotoxic drugs or toxins or active fragments of such toxins. Suitable toxins and their corresponding fragments include diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin and the like. Cytotoxic agents also include radiochemicals made by conjugating radioisotopes to antibodies raised against CA proteins, or binding of a radionuclide to a chelating agent that has been covalently attached to the antibody. Targeting the therapeutic moiety to transmembrane CA proteins not only serves to increase the local concentration of therapeutic moiety in the cancer of interest, i.e., lymphoma, but also serves to reduce deleterious side effects that may be associated with the therapeutic moiety. A number of investigators have used monoclonal antibodies as carriers of cytotoxic substances in attempts to selectively direct those agents to malignant tissue. More particularly, a number of monoclonal antibodies have been conjugated to toxins such as ricin, abrin, diphtheria toxin and Pseudomonas exotoxin or to enzymatically active portions (A chains) thereof via heterobifunctional agents. See, e.g., U.S. Pat. No. 4,753,894 to Frankel et al.; Nevelle, et al. (1982)[0177]Immunol Rev62:75-91; Ross et al. (1980)Eur. J. Biochem104; Vitteta et al. (1982)Immunol Rev62:158-183; Raso et al. (1982)Cancer Res42:457-464, and Trowbridge et al. (1981)Nature294:171-173.

In another preferred embodiment, the CA protein against which the antibodies are raised is an intracellular protein. In this case, the antibody may be conjugated to a protein that facilitates entry into the cell. In one case, the antibody enters the cell by endocytosis. In another embodiment, a nucleic acid encoding the antibody is administered to the individual or cell. Moreover, wherein the CA protein can be targeted within a cell, e.g., the nucleus, an antibody thereto contains a signal for that target localization, e.g., a nuclear localization signal.[0178]

The CA antibodies of the invention specifically bind to CA proteins. By “specifically bind” herein is meant that the antibodies bind to the protein with a binding constant in the range of 10[0179]⁻⁴-10⁻⁶M⁻¹, with a preferred range being 10⁻⁷-10⁻⁹M⁻¹.

In a preferred embodiment, the CA protein is purified or isolated after expression. CA proteins may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, and chromatofocusing. For example, the CA protein may be purified using a standard anti-CA antibody column. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. For general guidance in suitable purification techniques, see Scopes, R., Protein Purification, Springer-Verlag, NY (1982). The degree of purification necessary will vary depending on the use of the CA protein. In some instances no purification will be necessary.[0180]

Detection of Cancer Phenotype[0181]

Once expressed and purified if necessary, the CA proteins and nucleic acids are useful in a number of applications. In one aspect, the expression levels of genes are determined for different cellular states in the cancer phenotype; that is, the expression levels of genes in normal tissue and in cancer tissue (and in some cases, for varying severities of lymphoma that relate to prognosis, as outlined below) are evaluated to provide expression profiles. An expression profile of a particular cell state or point of development is essentially a “fingerprint” of the state; while two states may have any particular gene similarly expressed, the evaluation of a number of genes simultaneously allows the generation of a gene expression profile that is unique to the state of the cell. By comparing expression profiles of cells in different states, information regarding which genes are important (including both up- and down-regulation of genes) in each of these states is obtained. Then, diagnosis may be done or confirmed: does tissue from a particular patient have the gene expression profile of normal or cancer tissue.[0182]

“Differential expression,” or equivalents used herein, refers to both qualitative as well as quantitative differences in the temporal and/or cellular expression patterns of genes, within and among the cells. Thus, a differentially expressed gene can qualitatively have its expression altered, including an activation or inactivation, in, for example, normal versus cancer tissue. That is, genes may be turned on or turned off in a particular state, relative to another state. As is apparent to the skilled artisan, any comparison of two or more states can be made. Such a qualitatively regulated gene will exhibit an expression pattern within a state or cell type which is detectable by standard techniques in one such state or cell type, but is not detectable in both. Alternatively, the determination is quantitative in that expression is increased or decreased; that is, the expression of the gene is either up-regulated, resulting in an increased amount of transcript, or down-regulated, resulting in a decreased amount of transcript. The degree to which expression differs need only be large enough to quantify via standard characterization techniques as outlined below, such as by use of Affymetrix GeneChip® expression arrays, Lockhart, Nature Biotechnology, 14:1675-1680 (1996), hereby expressly incorporated by reference. Other techniques include, but are not limited to, quantitative reverse transcriptase PCR, Northern analysis and RNase protection. As outlined above, preferably the change in expression (i.e. upregulation or downregulation) is at least about 50%, more preferably at least about 100%, more preferably at least about 150%, more preferably, at least about 200%, with from 300 to at least 1000% being especially preferred.[0183]

As will be appreciated by those in the art, this may be done by evaluation at either the gene transcript, or the protein level; that is, the amount of gene expression may be monitored using nucleic acid probes to the DNA or RNA equivalent of the gene transcript, and the quantification of gene expression levels, or, alternatively, the final gene product itself (protein) can be monitored, for example through the use of antibodies to the CA protein and standard immunoassays (ELISAs, etc.) or other techniques, including mass spectroscopy assays, 2D gel electrophoresis assays, etc. Thus, the proteins corresponding to CA genes, i.e. those identified as being important in a particular cancer phenotype, i.e., lymphoma, can be evaluated in a diagnostic test specific for that cancer.[0184]

In a preferred embodiment, gene expression monitoring is done and a number of genes, i.e. an expression profile, is monitored simultaneously, although multiple protein expression monitoring can be done as well. Similarly, these assays may be done on an individual basis as well.[0185]

In this embodiment, the CA nucleic acid probes may be attached to biochips as outlined herein for the detection and quantification of CA sequences in a particular cell. The assays are done as is known in the art. As will be appreciated by those in the art, any number of different CA sequences may be used as probes, with single sequence assays being used in some cases, and a plurality of the sequences described herein being used in other embodiments. In addition, while solid-phase assays are described, any number of solution based assays may be done as well.[0186]

In a preferred embodiment, both solid and solution based assays may be used to detect CA sequences that are up-regulated or down-regulated in cancers as compared to normal tissue. In instances where the CA sequence has been altered but shows the same expression profile or an altered expression profile, the protein will be detected as outlined herein.[0187]

In a preferred embodiment nucleic acids encoding the CA protein are detected. Although DNA or RNA encoding the CA protein may be detected, of particular interest are methods wherein the mRNA encoding a CA protein is detected. The presence of mRNA in a sample is an indication that the CA gene has been transcribed to form the mRNA, and suggests that the protein is expressed. Probes to detect the mRNA can be any nucleotide/deoxynucleotide probe that is complementary to and base pairs with the mRNA and includes but is not limited to oligonucleotides, cDNA or RNA. Probes also should contain a detectable label, as defined herein. In one method the mRNA is detected after immobilizing the nucleic acid to be examined on a solid support such as nylon membranes and hybridizing the probe with the sample. Following washing to remove the non-specifically bound probe, the label is detected. In another method detection of the mRNA is performed in situ. In this method permeabilized cells or tissue samples are contacted with a detectably labeled nucleic acid probe for sufficient time to allow the probe to hybridize with the target mRNA. Following washing to remove the non-specifically bound probe, the label is detected. For example a digoxygenin labeled riboprobe (RNA probe) that is complementary to the mRNA encoding a CA protein is detected by binding the digoxygenin with an anti-digoxygenin secondary antibody and developed with nitro blue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate.[0188]

In a preferred embodiment, any of the three classes of proteins as described herein (secreted, transmembrane or intracellular proteins) are used in diagnostic assays. The CA proteins, antibodies, nucleic acids, modified proteins and cells containing CA sequences are used in diagnostic assays. This can be done on an individual gene or corresponding polypeptide level, or as sets of assays.[0189]

As described and defined herein, CA proteins find use as markers of cancers, including lymphomas such as, but not limited to, Hodgkin's and non-Hodgkin's lymphoma. Detection of these proteins in putative cancer tissue or patients allows for a determination or diagnosis of the type of cancer. Numerous methods known to those of ordinary skill in the art find use in detecting cancers. In one embodiment, antibodies are used to detect CA proteins. A preferred method separates proteins from a sample or patient by electrophoresis on a gel (typically a denaturing and reducing protein gel, but may be any other type of gel including isoelectric focusing gels and the like). Following separation of proteins, the CA protein is detected by immunoblotting with antibodies raised against the CA protein. Methods of immunoblotting are well known to those of ordinary skill in the art.[0190]

In another preferred method, antibodies to the CA protein find use in in situ imaging techniques. In this method cells are contacted with from one to many antibodies to the CA protein(s). Following washing to remove non-specific antibody binding, the presence of the antibody or antibodies is detected. In one embodiment the antibody is detected by incubating with a secondary antibody that contains a detectable label. In another method the primary antibody to the CA protein(s) contains a detectable label. In another preferred embodiment each one of multiple primary antibodies contains a distinct and detectable label. This method finds particular use in simultaneous screening for a plurality of CA proteins. As will be appreciated by one of ordinary skill in the art, numerous other histological imaging techniques are useful in the invention.[0191]

In a preferred embodiment the label is detected in a fluorometer that has the ability to detect and distinguish emissions of different wavelengths. In addition, a fluorescence activated cell sorter (FACS) can be used in the method.[0192]

In another preferred embodiment, antibodies find use in diagnosing cancers from blood samples. As previously described, certain CA proteins are secreted/circulating molecules. Blood samples, therefore, are useful as samples to be probed or tested for the presence of secreted CA proteins. Antibodies can be used to detect the CA proteins by any of the previously described immunoassay techniques including ELISA, immunoblotting (Western blotting), immunoprecipitation, BIACORE technology and the like, as will be appreciated by one of ordinary skill in the art.[0193]

In a preferred embodiment, in situ hybridization of labeled CA nucleic acid probes to tissue arrays is done. For example, arrays of tissue samples, including CA tissue and/or normal tissue, are made. In situ hybridization as is known in the art can then be done.[0194]

It is understood that when comparing the expression fingerprints between an individual and a standard, the skilled artisan can make a diagnosis as well as a prognosis. It is further understood that the genes that indicate diagnosis may differ from those that indicate prognosis.[0195]

In a preferred embodiment, the CA proteins, antibodies, nucleic acids, modified proteins and cells containing CA sequences are used in prognosis assays. As above, gene expression profiles can be generated that correlate to cancer, especially lymphoma, severity, in terms of long term prognosis. Again, this may be done on either a protein or gene level, with the use of genes being preferred. As above, the CA probes are attached to biochips for the detection and quantification of CA sequences in a tissue or patient. The assays proceed as outlined for diagnosis.[0196]

Screening for CA-Targeted Drugs[0197]

In one embodiment, any of the CA sequences as described herein are used in drug screening assays. The CA proteins, antibodies, nucleic acids, modified proteins and cells containing CA sequences are used in drug screening assays or by evaluating the effect of drug candidates on a “gene expression profile” or expression profile of polypeptides. In one embodiment, the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring for expression profile genes after treatment with a candidate agent, Zlokarnik, et al., Science 279, 84-8 (1998), Heid, et al., Genome Res., 6:986-994 (1996).[0198]

In another embodiment, the CA proteins, antibodies, nucleic acids, modified proteins and cells containing the native or modified CA proteins are used in screening assays. That is, the present invention provides novel methods for screening for compositions that modulate the cancer phenotype. As above, this can be done by screening for modulators of gene expression or for modulators of protein activity. Similarly, this may be done on an individual gene or protein level or by evaluating the effect of drug candidates on a “gene expression profile”. In a preferred embodiment, the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring for expression profile genes after treatment with a candidate agent, see Zlokarnik, supra.[0199]

Having identified the CA genes herein, a variety of assays to evaluate the effects of agents on gene expression may be executed. In a preferred embodiment, assays may be run on an individual gene or protein level. That is, having identified a particular gene as aberrantly regulated in cancer, candidate bioactive agents may be screened to modulate the gene's regulation. “Modulation” thus includes both an increase and a decrease in gene expression or activity. The preferred amount of modulation will depend on the original change of the gene expression in normal versus tumor tissue, with changes of at least 10%, preferably 50%, more preferably 100-300%, and in some embodiments 300-1000% or greater. Thus, if a gene exhibits a 4 fold increase in tumor compared to normal tissue, a decrease of about four fold is desired; a 10 fold decrease in tumor compared to normal tissue gives a 10 fold increase in expression for a candidate agent is desired, etc. Alternatively, where the CA sequence has been altered but shows the same expression profile or an altered expression profile, the protein will be detected as outlined herein.[0200]

As will be appreciated by those in the art, this may be done by evaluation at either the gene or the protein level; that is, the amount of gene expression may be monitored using nucleic acid probes and the quantification of gene expression levels, or, alternatively, the level of the gene product itself can be monitored, for example through the use of antibodies to the CA protein and standard immunoassays. Alternatively, binding and bioactivity assays with the protein may be done as outlined below.[0201]

In a preferred embodiment, gene expression monitoring is done and a number of genes, i.e. an expression profile, is monitored simultaneously, although multiple protein expression monitoring can be done as well.[0202]

In this embodiment, the CA nucleic acid probes are attached to biochips as outlined herein for the detection and quantification of CA sequences in a particular cell. The assays are further described below.[0203]

Generally, in a preferred embodiment, a candidate bioactive agent is added to the cells prior to analysis. Moreover, screens are provided to identify a candidate bioactive agent that modulates a particular type of cancer, modulates CA proteins, binds to a CA protein, or interferes between the binding of a CA protein and an antibody.[0204]

The term “candidate bioactive agent” or “drug candidate” or grammatical equivalents as used herein describes any molecule, e.g., protein, oligopeptide, small organic or inorganic molecule, polysaccharide, polynucleotide, etc., to be tested for bioactive agents that are capable of directly or indirectly altering either the cancer phenotype, binding to and/or modulating the bioactivity of a CA protein, or the expression of a CA sequence, including both nucleic acid sequences and protein sequences. In a particularly preferred embodiment, the candidate agent suppresses a CA phenotype, for example to a normal tissue fingerprint. Similarly, the candidate agent preferably suppresses a severe CA phenotype. Generally a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.[0205]

In one aspect, a candidate agent will neutralize the effect of a CA protein. By “neutralize” is meant that activity of a protein is either inhibited or counter acted against so as to have substantially no effect on a cell.[0206]

Candidate agents encompass numerous chemical classes, though typically they are organic or inorganic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 Daltons. Preferred small molecules are less than 2000, or less than 1500 or less than 1000 or less than 500 D. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides.[0207]

Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, or amidification to produce structural analogs.[0208]

In one embodiment, the candidate bioactive agents are proteins. By “protein” herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides. The protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures. Thus “amino acid”, or “peptide residue”, as used herein means both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline and norleucine are considered amino acids for the purposes of the invention. “Amino acid” also includes imino acid residues such as proline and hydroxyproline. The side chains may be in either the (R) or the (S) configuration. In the preferred embodiment, the amino acids are in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradations.[0209]

In a preferred embodiment, the candidate bioactive agents are naturally occurring proteins or fragments of naturally occurring proteins. Thus, for example, cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, may be used. In this way libraries of prokaryotic and eukaryotic proteins may be made for screening in the methods of the invention. Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred.[0210]

In another preferred embodiment, the candidate bioactive agents are peptides of from about 5 to about 30 amino acids, with from about 5 to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred. The peptides may be digests of naturally occurring proteins as is outlined above, random peptides, or “biased” random peptides. By “randomized” or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or amino acid at any position. The synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.[0211]

In one embodiment, the library is fully randomized, with no sequence preferences or constants at any position. In a preferred embodiment, the library is biased. That is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities. For example, in a preferred embodiment, the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of nucleic acid binding domains, the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.[0212]

In one embodiment, the candidate bioactive agents are nucleic acids. As described generally for proteins, nucleic acid candidate bioactive agents may be naturally occurring nucleic acids, random nucleic acids, or “biased” random nucleic acids. In another embodiment, the candidate bioactive agents are organic chemical moieties, a wide variety of which are available in the literature.[0213]

In assays for testing alteration of the expression profile of one or more CA genes, after the candidate agent has been added and the cells allowed to incubate for some period of time, a nucleic acid sample containing the target sequences to be analyzed is prepared. The target sequence is prepared using known techniques (e.g., converted from RNA to labeled cDNA, as described above) and added to a suitable microarray. For example, an in vitro reverse transcription with labels covalently attached to the nucleosides is performed. Generally, the nucleic acids are labeled with a label as defined herein, especially with biotin-FITC or PE, Cy3 and Cy5.[0214]

As will be appreciated by those in the art, these assays can be direct hybridization assays or can comprise “sandwich assays”, which include the use of multiple probes, as is generally outlined in U.S. Pat. Nos. 5,681,702, 5,597,909, 5,545,730, 5,594,117, 5,591,584, 5,571,670, 5,580,731, 5,571,670, 5,591,584, 5,624,802, 5,635,352, 5,594,118, 5,359,100, 5,124,246 and 5,681,697, all of which are hereby incorporated by reference. In this embodiment, in general, the target nucleic acid is prepared as outlined above, and then added to the biochip comprising a plurality of nucleic acid probes, under conditions that allow the formation of a hybridization complex.[0215]

A variety of hybridization conditions may be used in the present invention, including high, moderate and low stringency conditions as outlined above. The assays are generally run under stringency conditions that allow formation of the label probe hybridization complex only in the presence of target. Stringency can be controlled by altering a step parameter that is a thermodynamic variable, including, but not limited to, temperature, formamide concentration, salt concentration, chaotropic salt concentration, pH, organic solvent concentration, etc. These parameters may also be used to control non-specific binding, as is generally outlined in U.S. Pat. No. 5,681,697. Thus it may be desirable to perform certain steps at higher stringency conditions to reduce non-specific binding.[0216]

The reactions outlined herein may be accomplished in a variety of ways, as will be appreciated by those in the art. Components of the reaction may be added simultaneously, or sequentially, in any order, with preferred embodiments outlined below. In addition, the reaction may include a variety of other reagents in the assays. These include reagents like salts, buffers, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal hybridization and detection, and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used, depending on the sample preparation methods and purity of the target. In addition, either solid phase or solution based (i.e., kinetic PCR) assays may be used.[0217]

Once the assay is run, the data are analyzed to determine the expression levels, and changes in expression levels as between states, of individual genes, forming a gene expression profile.[0218]

In a preferred embodiment, as for the diagnosis and prognosis applications, having identified the differentially expressed gene(s) or mutated gene(s) important in any one state, screens can be run to test for alteration of the expression of the CA genes individually. That is, screening for modulation of regulation of expression of a single gene can be done. Thus, for example, in the case of target genes whose presence or absence is unique between two states, screening is done for modulators of the target gene expression.[0219]

In addition, screens can be done for novel genes that are induced in response to a candidate agent. After identifying a candidate agent based upon its ability to suppress a CA expression pattern leading to a normal expression pattern, or modulate a single CA gene expression profile so as to mimic the expression of the gene from normal tissue, a screen as described above can be performed to identify genes that are specifically modulated in response to the agent. Comparing expression profiles between normal tissue and agent treated CA tissue reveals genes that are not expressed in normal tissue or CA tissue, but are expressed in agent treated tissue. These agent specific sequences can be identified and used by any of the methods described herein for CA genes or proteins. In particular these sequences and the proteins they encode find use in marking or identifying agent-treated cells. In addition, antibodies can be raised against the agent-induced proteins and used to target novel therapeutics to the treated CA tissue sample.[0220]

Thus, in one embodiment, a candidate agent is administered to a population of CA cells, that thus has an associated CA expression profile. By “administration” or “contacting” herein is meant that the candidate agent is added to the cells in such a manner as to allow the agent to act upon the cell, whether by uptake and intracellular action, or by action at the cell surface. In some embodiments, nucleic acid encoding a proteinaceous candidate agent (i.e. a peptide) may be put into a viral construct such as a retroviral construct and added to the cell, such that expression of the peptide agent is accomplished; see PCT US97/01019, hereby expressly incorporated by reference.[0221]

Once the candidate agent has been administered to the cells, the cells can be washed if desired and are allowed to incubate under preferably physiological conditions for some period of time. The cells are then harvested and a new gene expression profile is generated, as outlined herein.[0222]

Thus, for example, CA tissue may be screened for agents that reduce or suppress the CA phenotype. A change in at least one gene of the expression profile indicates that the agent has an effect on CA activity. By defining such a signature for the CA phenotype, screens for new drugs that alter the phenotype can be devised. With this approach, the drug target need not be known and need not be represented in the original expression screening platform, nor does the level of transcript for the target protein need to change.[0223]

In a preferred embodiment, as outlined above, screens may be done on individual genes and gene products (proteins). That is, having identified a particular differentially expressed gene as important in a particular state, screening of modulators of either the expression of the gene or the gene product itself can be done. The gene products of differentially expressed genes are sometimes referred to herein as “CA proteins” or “CAP”. The CAP may be a fragment, or alternatively, be the full-length protein to the fragment encoded by the nucleic acids of Tables 1-27 (human genomic sequences of SEQ ID NOS: 4, 10, 16, 26, 32, 38, 50, 56, 66, 74, 77, 83, 93, 99, 105, 111, 117, 125, 133, 139, 145, 151, 163, 169, 179, 189, 195, and 201, and sequences of SEQ ID NOS: 5, 11, 17, 19, 21, 27, 33, 39, 51, 57, 59, 61, 67, 69, 75, 78, 84, 86, 88, 94, 100, 106, 112, 118, 120, 126, 134, 140, 146, 152, 154, 156, 158, 164, 170, 172, 174, 180, 182, 184, 190, 196, and 202 corresponding to the human mRNAs generated therefrom). In a preferred embodiment, the CAP is selected from the human protein sequences shown in Tables 1-27 (of SEQ ID NOS: 6, 12, 18, 20, 22, 28, 34, 40, 52, 58, 60, 62, 68, 70, 76, 79, 85, 87, 89, 95, 101, 107, 113, 119, 121, 127, 135, 141, 147, 153, 155, 157, 159, 165, 171, 173, 175, 181, 183, 185, 191, 197 and 203). In another embodiment, the sequences are sequence variants as further described herein.[0224]

Preferably, the CAP is a fragment approximately 14 to 24 amino acids in length. More preferably the fragment is a soluble fragment. Preferably, the fragment includes a non-transmembrane region. In a preferred embodiment, the fragment has an N-terminal Cys to aid in solubility. In one embodiment, the C-terminus of the fragment is kept as a free acid and the N-terminus is a free amine to aid in coupling, e.g., to a cysteine.[0225]

In one embodiment the CA proteins are conjugated to an immunogenic agent as discussed herein. In one embodiment the CA protein is conjugated to BSA.[0226]

In a preferred embodiment, screening is done to alter the biological function of the expression product of the CA gene. Again, having identified the importance of a gene in a particular state, screening for agents that bind and/or modulate the biological activity of the gene product can be run as is more fully outlined below.[0227]

In a preferred embodiment, screens are designed to first find candidate agents that can bind to CA proteins, and then these agents may be used in assays that evaluate the ability of the candidate agent to modulate the CAP activity and the cancer phenotype. Thus, as will be appreciated by those in the art, there are a number of different assays that may be run; binding assays and activity assays.[0228]

In a preferred embodiment, binding assays are done. In general, purified or isolated gene product is used; that is, the gene products of one or more CA nucleic acids are made. In general, this is done as is known in the art. For example, antibodies are generated to the protein gene products, and standard immunoassays are run to determine the amount of protein present. Alternatively, cells comprising the CA proteins can be used in the assays.[0229]

Thus, in a preferred embodiment, the methods comprise combining a CA protein and a candidate bioactive agent, and determining the binding of the candidate agent to the CA protein. Preferred embodiments utilize the human or mouse CA protein, although other mammalian proteins may also be used, for example for the development of animal models of human disease. In some embodiments, as outlined herein, variant or derivative CA proteins may be used.[0230]

Generally, in a preferred embodiment of the methods herein, the CA protein or the candidate agent is non-diffusably bound to an insoluble support having isolated sample receiving areas (e.g. a microtiter plate, an array, etc.). The insoluble support may be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening. The surface of such supports may be solid or porous and of any convenient shape. Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads. These are typically made of glass, plastic (e.g., polystyrene), polysaccharides, nylon or nitrocellulose, Teflon®, etc. Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples.[0231]

The particular manner of binding of the composition is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the composition and is nondiffusable. Preferred methods of binding include the use of antibodies (which do not sterically block either the ligand binding site or activation sequence when the protein is bound to the support), direct binding to “sticky” or ionic supports, chemical crosslinking, the synthesis of the protein or agent on the surface, etc. Following binding of the protein or agent, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety.[0232]

In a preferred embodiment, the CA protein is bound to the support, and a candidate bioactive agent is added to the assay. Alternatively, the candidate agent is bound to the support and the CA protein is added. Novel binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc. Of particular interest are screening assays for agents that have a low toxicity for human cells. A wide variety of assays may be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.) and the like.[0233]

The determination of the binding of the candidate bioactive agent to the CA protein may be done in a number of ways. In a preferred embodiment, the candidate bioactive agent is labeled, and binding determined directly. For example, this may be done by attaching all or a portion of the CA protein to a solid support, adding a labeled candidate agent (for example a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support. Various blocking and washing steps may be utilized as is known in the art.[0234]

By “labeled” herein is meant that the compound is either directly or indirectly labeled with a label which provides a detectable signal, e.g. radioisotope, fluorescers, enzyme, antibodies, particles such as magnetic particles, chemiluminescers, or specific binding molecules, etc. Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc. For the specific binding members, the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known procedures, as outlined above. The label can directly or indirectly provide a detectable signal.[0235]

In some embodiments, only one of the components is labeled. For example, the proteins (or proteinaceous candidate agents) may be labeled at tyrosine positions using[0236]¹²⁵I, or with fluorophores. Alternatively, more than one component may be labeled with different labels; using¹²⁵I for the proteins, for example, and a fluorophore for the candidate agents.

In a preferred embodiment, the binding of the candidate bioactive agent is determined through the use of competitive binding assays. In this embodiment, the competitor is a binding moiety known to bind to the target molecule (i.e. CA protein), such as an antibody, peptide, binding partner, ligand, etc. Under certain circumstances, there may be competitive binding as between the bioactive agent and the binding moiety, with the binding moiety displacing the bioactive agent.[0237]

In one embodiment, the candidate bioactive agent is labeled. Either the candidate bioactive agent, or the competitor, or both, is added first to the protein for a time sufficient to allow binding, if present. Incubations may be performed at any temperature which facilitates optimal activity, typically between 4 and 40° C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high throughput screening. Typically between 0.1 and 1 hour will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding.[0238]

In a preferred embodiment, the competitor is added first, followed by the candidate bioactive agent. Displacement of the competitor is an indication that the candidate bioactive agent is binding to the CA protein and thus is capable of binding to, and potentially modulating, the activity of the CA protein. In this embodiment, either component can be labeled. Thus, for example, if the competitor is labeled, the presence of label in the wash solution indicates displacement by the agent. Alternatively, if the candidate bioactive agent is labeled, the presence of the label on the support indicates displacement.[0239]

In an alternative embodiment, the candidate bioactive agent is added first, with incubation and washing, followed by the competitor. The absence of binding by the competitor may indicate that the bioactive agent is bound to the CA protein with a higher affinity. Thus, if the candidate bioactive agent is labeled, the presence of the label on the support, coupled with a lack of competitor binding, may indicate that the candidate agent is capable of binding to the CA protein.[0240]

In a preferred embodiment, the methods comprise differential screening to identity bioactive agents that are capable of modulating the activity of the CA proteins. In this embodiment, the methods comprise combining a CA protein and a competitor in a first sample. A second sample comprises a candidate bioactive agent, a CA protein and a competitor. The binding of the competitor is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to the CA protein and potentially modulating its activity. That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is capable of binding to the CA protein.[0241]

Alternatively, a preferred embodiment utilizes differential screening to identify drug candidates that bind to the native CA protein, but cannot bind to modified CA proteins. The structure of the CA protein may be modeled, and used in rational drug design to synthesize agents that interact with that site. Drug candidates that affect CA bioactivity are also identified by screening drugs for the ability to either enhance or reduce the activity of the protein.[0242]

Positive controls and negative controls may be used in the assays. Preferably all control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples is for a time sufficient for the binding of the agent to the protein. Following incubation, all samples are washed free of non-specifically bound material and the amount of bound, generally labeled agent determined. For example, where a radiolabel is employed, the samples may be counted in a scintillation counter to determine the amount of bound compound.[0243]

A variety of other reagents may be included in the screening assays. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used. The mixture of components may be added in any order that provides for the requisite binding.[0244]

Screening for agents that modulate the activity of CA proteins may also be done. In a preferred embodiment, methods for screening for a bioactive agent capable of modulating the activity of CA proteins comprise the steps of adding a candidate bioactive agent to a sample of CA proteins, as above, and determining an alteration in the biological activity of CA proteins. “Modulating the activity of a CA protein” includes an increase in activity, a decrease in activity, or a change in the type or kind of activity present. Thus, in this embodiment, the candidate agent should both bind to CA proteins (although this may not be necessary), and alter its biological or biochemical activity as defined herein. The methods include both in vitro screening methods, as are generally outlined above, and in vivo screening of cells for alterations in the presence, distribution, activity or amount of CA proteins.[0245]

Thus, in this embodiment, the methods comprise combining a CA sample and a candidate bioactive agent, and evaluating the effect on CA activity. By “CA activity” or grammatical equivalents herein is meant one of the CA protein's biological activities, including, but not limited to, its role in tumorigenesis, including cell division, preferably in lymphatic tissue, cell proliferation, tumor growth and transformation of cells. In one embodiment, CA activity includes activation of or by a protein encoded by a nucleic acid of Tables 1-27. An inhibitor of CA activity is the inhibition of any one or more CA activities.[0246]

In a preferred embodiment, the activity of the CA protein is increased; in another preferred embodiment, the activity of the CA protein is decreased. Thus, bioactive agents that are antagonists are preferred in some embodiments, and bioactive agents that are agonists may be preferred in other embodiments.[0247]

In a preferred embodiment, the invention provides methods for screening for bioactive agents capable of modulating the activity of a CA protein. The methods comprise adding a candidate bioactive agent, as defined above, to a cell comprising CA proteins. Preferred cell types include almost any cell. The cells contain a recombinant nucleic acid that encodes a CA protein. In a preferred embodiment, a library of candidate agents is tested on a plurality of cells.[0248]

In one aspect, the assays are evaluated in the presence or absence or previous or subsequent exposure of physiological signals, for example hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcinogenics, or other cells (i.e. cell-cell contacts). In another example, the determinations are determined at different stages of the cell cycle process.[0249]

In this way, bioactive agents are identified. Compounds with pharmacological activity are able to enhance or interfere with the activity of the CA protein.[0250]

Applications of the Invention[0251]

In one embodiment, a method of inhibiting cancer cell division is provided. In another embodiment, a method of inhibiting tumor growth is provided. In a further embodiment, methods of treating cells or individuals with cancer are provided.[0252]

The method comprises administration of a cancer inhibitor. In particular embodiments, the cancer inhibitor is an antisense molecule, a pharmaceutical composition, a therapeutic agent or small molecule, or a monoclonal, polyclonal, chimeric or humanized antibody. In particular embodiments, a therapeutic agent is coupled with a an antibody, preferable a monoclonal antobody.[0253]

In other embodiments, methods for detection or diagnosis of cancer cells in an individual are provided. In particular embodiments, the diagnostic/detection agent is a small molecule that pereferentially binds to a CAP according to the invention. In one embodiment, the diagnostic/detection agent is an antibody, preferably a monoclonal antobody, preferably linked to a detectable agent.[0254]

In other embodiments of the invention, animal models and transgenic animals are provided, which find use in generating animal models of cancers, particularly lymphomas and carcinomas.[0255]

(a) Antisense Molecules[0256]

In one embodiment, the cancer inhibitor is an antisense molecule. Antisense molecules as used herein include antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences for cancer molecules. Antisense or sense oligonucleotides, according to the present invention, comprise a fragment generally at least about 14 nucleotides, preferably from about 14 to 30 nucleotides. The ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, for example, Stein and Cohen, Cancer Res. 48:2659, (1988) and van der Krol et al., BioTechniques 6:958, (1988).[0257]

Antisense molecules may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell. Alternatively, a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448. It is understood that the use of antisense molecules or knock out and knock in models may also be used in screening assays as discussed above, in addition to methods of treatment.[0258]

(b) Pharmaceutical Compositions[0259]

Pharmaceutical compositions encompassed by the present invention include as active agent, the polypeptides, polynucleotides, antisense oligonucleotides, or antibodies of the invention disclosed herein in a therapeutically effective amount. An “effective amount” is an amount sufficient to effect beneficial or desired results, including clinical results. An effective amount can be administered in one or more administrations. For purposes of this invention, an effective amount of an adenoviral vector is an amount that is sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of the disease state.[0260]

The compositions can be used to treat cancer as well as metastases of primary cancer. In addition, the pharmaceutical compositions can be used in conjunction with conventional methods of cancer treatment, e.g., to sensitize tumors to radiation or conventional chemotherapy. The terms “treatment”, “treating”, “treat” and the like are used herein to generally refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease. “Treatment” as used herein covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease or symptom from occurring in a subject which may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease symptom, i.e., arresting its development; or (c) relieving the disease symptom, i.e., causing regression of the disease or symptom.[0261]

Where the pharmaceutical composition comprises an antibody that specifically binds to a gene product encoded by a differentially expressed polynucleotide, the antibody can be coupled to a drug for delivery to a treatment site or coupled to a detectable label to facilitate imaging of a site comprising cancer cells, such as prostate cancer cells. Methods for coupling antibodies to drugs and detectable labels are well known in the art, as are methods for imaging using detectable labels.[0262]

A “patient” for the purposes of the present invention includes both humans and other animals, particularly mammals, and organisms. Thus the methods are applicable to both human therapy and veterinary applications. In the preferred embodiment the patient is a mammal, and in the most preferred embodiment the patient is human.[0263]

The term “therapeutically effective amount” as used herein refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect. The effect can be detected by, for example, chemical markers or antigen levels. Therapeutic effects also include reduction in physical symptoms, such as decreased body temperature. The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. The effective amount for a given situation is determined by routine experimentation and is within the judgment of the clinician. For purposes of the present invention, an effective dose will generally be from about 0.01 mg/kg to about 5 mg/kg, or about 0.01 mg/kg to about 50 mg/kg or about 0.05 mg/kg to about 10 mg/kg of the compositions of the present invention in the individual to which it is administered.[0264]

A pharmaceutical composition can also contain a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which can be administered without undue toxicity. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in therapeutic compositions can include liquids such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, can also be present in such vehicles. Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. Liposomes are included within the definition of a pharmaceutically acceptable carrier. Pharmaceutically acceptable salts can also be present in the pharmaceutical composition, e.g., mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in[0265]Remington: The Science and Practice of Pharmacy(1995) Alfonso Gennaro, Lippincott, Williams, & Wilkins.

The pharmaceutical compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like. Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically-active compounds. Diluents known to the art include aqueous media, vegetable and animal oils and fats. Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents.[0266]

The pharmaceutical compositions of the present invention comprise a CA protein in a form suitable for administration to a patient. In the preferred embodiment, the pharmaceutical compositions are in a water soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts. “Pharmaceutically acceptable acid addition salt” refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. “Pharmaceutically acceptable base addition salts” include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.[0267]

The pharmaceutical compositions may also include one or more of the following: carrier proteins such as serum albumin; buffers; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol. Additives are well known in the art, and are used in a variety of formulations.[0268]

The compounds having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host, as previously described. The agents may be administered in a variety of ways, orally, parenterally e.g., subcutaneously, intraperitoneally, intravascularly, etc. Depending upon the manner of introduction, the compounds may be formulated in a variety of ways. The concentration of therapeutically active compound in the formulation may vary from about 0.1-100% wgt/vol. Once formulated, the compositions contemplated by the invention can be (1) administered directly to the subject (e.g., as polynucleotide, polypeptides, small molecule agonists or antagonists, and the like); or (2) delivered ex vivo, to cells derived from the subject (e.g., as in ex vivo gene therapy). Direct delivery of the compositions will generally be accomplished by parenteral injection, e.g., subcutaneously, intraperitoneally, intravenously or intramuscularly, intratumoral or to the interstitial space of a tissue. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal applications, needles, and gene guns or hyposprays. Dosage treatment can be a single dose schedule or a multiple dose schedule.[0269]

Methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and described in e.g., International Publication No. WO 93/14778. Examples of cells useful in ex vivo applications include, for example, stem cells, particularly hematopoetic, lymph cells, macrophages, dendritic cells, or tumor cells. Generally, delivery of nucleic acids for both ex vivo and in vitro applications can be accomplished by, for example, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei, all well known in the art.[0270]

Once differential expression of a gene corresponding to a CA polynucleotide described herein has been found to correlate with a proliferative disorder, such as neoplasia, dysplasia, and hyperplasia, the disorder can be amenable to treatment by administration of a therapeutic agent based on the provided polynucleotide, corresponding polypeptide or other corresponding molecule (e.g., antisense, ribozyme, etc.). In other embodiments, the disorder can be amenable to treatment by administration of a small molecule drug that, for example, serves as an inhibitor (antagonist) of the function of the encoded gene product of a gene having increased expression in cancerous cells relative to normal cells or as an agonist for gene products that are decreased in expression in cancerous cells (e.g., to promote the activity of gene products that act as tumor suppressors).[0271]

The dose and the means of administration of the inventive pharmaceutical compositions are determined based on the specific qualities of the therapeutic composition, the condition, age, and weight of the patient, the progression of the disease, and other relevant factors. For example, administration of polynucleotide therapeutic compositions agents includes local or systemic administration, including injection, oral administration, particle gun or catheterized administration, and topical administration. Preferably, the therapeutic polynucleotide composition contains an expression construct comprising a promoter operably linked to a polynucleotide of at least 12, 22, 25, 30, or 35 contiguous nt of the polynucleotide disclosed herein. Various methods can be used to administer the therapeutic composition directly to a specific site in the body. For example, a small metastatic lesion is located and the therapeutic composition injected several times in several different locations within the body of tumor. Alternatively, arteries that serve a tumor are identified, and the therapeutic composition injected into such an artery, in order to deliver the composition directly into the tumor. A tumor that has a necrotic center is aspirated and the composition injected directly into the now empty center of the tumor. An antisense composition is directly administered to the surface of the tumor, for example, by topical application of the composition. X-ray imaging is used to assist in certain of the above delivery methods.[0272]

Targeted delivery of therapeutic compositions containing an antisense polynucleotide, subgenomic polynucleotides, or antibodies to specific tissues can also be used. Receptor-mediated DNA delivery techniques are described in, for example, Findeis et al.,[0273]Trends Biotechnol. (1993) 11:202; Chiou et al.,Gene Therapeutics: Methods And Applications Of Direct Gene Transfer(J. A. Wolff, ed.) (1994); Wu et al.,J. Biol. Chem. (1988) 263:621; Wu et al.,J. Biol. Chem. (1994) 269:542; Zenke et al.,Proc. Natl. Acad. Sci. (USA) (1990) 87:3655; Wu et al.,J. Biol. Chem. (1991) 266:338. Therapeutic compositions containing a polynucleotide are administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol. Concentration ranges of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA can also be used during a gene therapy protocol. Factors such as method of action (e.g., for enhancing or inhibiting levels of the encoded gene product) and efficacy of transformation and expression are considerations that will affect the dosage required for ultimate efficacy of the antisense subgenomic polynucleotides. Where greater expression is desired over a larger area of tissue, larger amounts of antisense subgenomic polynucleotides or the same amounts re-administered in a successive protocol of administrations, or several administrations to different adjacent or close tissue portions of, for example, a tumor site, may be required to effect a positive therapeutic outcome. In all cases, routine experimentation in clinical trials will determine specific ranges for optimal therapeutic effect.

The therapeutic polynuctleotides and polypeptides of the present invention can be delivered using gene delivery vehicles. The gene delivery vehicle can be of viral or non-viral origin (see generally, Jolly,[0274]Cancer Gene Therapy(1994) 1:51; Kimura,Human Gene Therapy(1994) 5:845; Connelly,Human Gene Therapy(1995) 1:185; and Kaplitt,Nature Genetics(1994) 6:148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence can be either constitutive or regulated.

Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art. Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Pat. No. 5,219,740; WO 93/11230; WO 93/10218; U.S. Pat. No. 4,777,127; GB Patent No. 2,200,651; EP 0 345 242; and WO 91/02805), alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and adeno-associated virus (AAV) vectors (see, e.g., WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655). Administration of DNA linked to killed adenovirus as described in Curiel,[0275]Hum. Gene Ther. (1992) 3:147 can also be employed.

Non-viral delivery vehicles and methods can also be employed, including, but not limited to, polycationic condensed DNA linked or unlinked to killed adenovirus alone (see, e.g., Curiel,[0276]Hum. Gene Ther. (1992) 3:147); ligand-linked DNA (see, e.g., Wu,J. Biol. Chem. (1989) 264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S. Pat. No. 5,814,482; WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nucleic charge neutralization or fusion with cell membranes. Naked DNA can also be employed. Exemplary naked DNA introduction methods are described in WO 90/11092 and U.S. Pat. No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in U.S. Pat. No. 5,422,120; WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additional approaches are described in Philip,Mol. Cell Biol. (1994) 14:2411, and in Woffendin,Proc. Natl. Acad. Sci. (1994) 91:1581.

Further non-viral delivery suitable for use includes mechanical delivery systems such as the approach described in Woffendin et al.,[0277]Proc. Natl. Acad. Sci. USA (1994) 91(24):11581. Moreover, the coding sequence and the product of expression of such can be delivered through deposition of photopolymerized hydrogel materials or use of ionizing radiation (see, e.g., U.S. Pat. No. 5,206,152 and WO 92/11033). Other conventional methods for gene delivery that can be used for delivery of the coding sequence include, for example, use of hand-held gene transfer particle gun (see, e.g., U.S. Pat. No. 5,149,655); use of ionizing radiation for activating transferred gene (see, e.g., U.S. Pat. No. 5,206,152 and WO 92/11033).

The administration of the CA proteins and modulators of the present invention can be done in a variety of ways as discussed above, including, but not limited to, orally, subcutaneously, intravenously, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly. In some instances, for example, in the treatment of wounds and inflammation, the CA proteins and modulators may be directly applied as a solution or spray.[0278]

In a preferred embodiment, CA proteins and modulators are administered as therapeutic agents, and can be formulated as outlined above. Similarly, CA genes (including both the full-length sequence, partial sequences, or regulatory sequences of the CA coding regions) can be administered in gene therapy applications, as is known in the art. These CA genes can include antisense applications, either as gene therapy (i.e. for incorporation into the genome) or as antisense compositions, as will be appreciated by those in the art.[0279]

Thus, in one embodiment, methods of modulating CA gene activity in cells or organisms are provided. In one embodiment, the methods comprise administering to a cell an anti-CA antibody that reduces or eliminates the biological activity of an endogenous CA protein. Alternatively, the methods comprise administering to a cell or organism a recombinant nucleic acid encoding a CA protein. As will be appreciated by those in the art, this may be accomplished in any number of ways. In a preferred embodiment, for example when the CA sequence is down-regulated in cancer, the activity of the CA gene product is increased by increasing the amount of CA expression in the cell, for example by overexpressing the endogenous CA gene or by administering a gene encoding the CA sequence, using known gene-therapy techniques. In a preferred embodiment, the gene therapy techniques include the incorporation of the exogenous gene using enhanced homologous recombination (EHR), for example as described in PCT/US93/03868, hereby incorporated by reference in its entirety. Alternatively, for example when the CA sequence is up-regulated in cancer, the activity of the endogenous CA gene is decreased, for example by the administration of a CA antisense nucleic acid.[0280]

(c) Vaccines[0281]

In a preferred embodiment, CA genes are administered as DNA vaccines, either single genes or combinations of CA genes. Naked DNA vaccines are generally known in the art. Brower, Nature Biotechnology, 16:1304-1305 (1998).[0282]

In one embodiment, CA genes of the present invention are used as DNA vaccines. Methods for the use of genes as DNA vaccines are well known to one of ordinary skill in the art, and include placing a CA gene or portion of a CA gene under the control of a promoter for expression in a patient with cancer. The CA gene used for DNA vaccines can encode full-length CA proteins, but more preferably encodes portions of the CA proteins including peptides derived from the CA protein. In a preferred embodiment a patient is immunized with a DNA vaccine comprising a plurality of nucleotide sequences derived from a CA gene. Similarly, it is possible to immunize a patient with a plurality of CA genes or portions thereof. Without being bound by theory, expression of the polypeptide encoded by the DNA vaccine, cytotoxic T-cells, helper T-cells and antibodies are induced that recognize and destroy or eliminate cells expressing CA proteins.[0283]

In a preferred embodiment, the DNA vaccines include a gene encoding an adjuvant molecule with the DNA vaccine. Such adjuvant molecules include cytokines that increase the immunogenic response to the CA polypeptide encoded by the DNA vaccine. Additional or alternative adjuvants are known to those of ordinary skill in the art and find use in the invention.[0284]

(d) Antibodies[0285]

In one embodiment, a cancer inhibitor is an antibody as discussed above. In one embodiment, the CA proteins of the present invention may be used to generate polyclonal and monoclonal antibodies to CA proteins, which are useful as described herein. Similarly, the CA proteins can be coupled, using standard technology, to affinity chromatography columns. These columns may then be used to purify CA antibodies. In a preferred embodiment, the antibodies are generated to epitopes unique to a CA protein; that is, the antibodies show little or no cross-reactivity to other proteins. These antibodies find use in a number of applications. For example, the CA antibodies may be coupled to standard affinity chromatography columns and used to purify CA proteins. The antibodies may also be used therapeutically as blocking polypeptides, as outlined above, since they will specifically bind to the CA protein.[0286]

The present invention further provides methods for detecting the presence of and/or measuring a level of a polypeptide in a biological sample, which CA polypeptide is encoded by a CA polynucleotide that is differentially expressed in a cancer cell, using an antibody specific for the encoded polypeptide. The methods generally comprise: a) contacting the sample with an antibody specific for a polypeptide encoded by a CA polynucleotide that is differentially expressed in a prostate cancer cell; and b) detecting binding between the antibody and molecules of the sample.[0287]

Detection of specific binding of the antibody specific for the encoded cancer-associated polypeptide, when compared to a suitable control is an indication that encoded polypeptide is present in the sample. Suitable controls include a sample known not to contain the encoded CA polypeptide or known not to contain elevated levels of the polypeptide; such as normal tissue, and a sample contacted with an antibody not specific for the encoded polypeptide, e.g., an anti-idiotype antibody. A variety of methods to detect specific antibody-antigen interactions are known in the art and can be used in the method, including, but not limited to, standard immunohistological methods, immunoprecipitation, an enzyme immunoassay, and a radioimmunoassay. In general, the specific antibody will be detectably labeled, either directly or indirectly. Direct labels include radioisotopes; enzymes whose products are detectable (e.g., luciferase, β-galactosidase, and the like); fluorescent labels (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, and the like); fluorescence emitting metals, e.g.,[0288]¹⁵²Eu, or others of the lanthanide series, attached to the antibody through metal chelating groups such as EDTA; chemiluminescent compounds, e.g., luminol, isoluminol, acridinium salts, and the like; bioluminescent compounds, e.g., luciferin, aequorin (green fluorescent protein), and the like. The antibody may be attached (coupled) to an insoluble support, such as a polystyrene plate or a bead. Indirect labels include second antibodies specific for antibodies specific for the encoded polypeptide (“first specific antibody”), wherein the second antibody is labeled as described above; and members of specific binding pairs, e.g., biotin-avidin, and the like. The biological sample may be brought into contact with and immobilized on a solid support or carrier, such as nitrocellulose, that is capable of immobilizing cells, cell particles, or soluble proteins. The support may then be washed with suitable buffers, followed by contacting with a detectably-labeled first specific antibody. Detection methods are known in the art and will be chosen as appropriate to the signal emitted by the detectable label. Detection is generally accomplished in comparison to suitable controls, and to appropriate standards.

In some embodiments, the methods are adapted for use in vivo, e.g., to locate or identify sites where cancer cells are present. In these embodiments, a detectably-labeled moiety, e.g., an antibody, which is specific for a cancer-associated polypeptide is administered to an individual (e.g., by injection), and labeled cells are located using standard imaging techniques, including, but not limited to, magnetic resonance imaging, computed tomography scanning, and the like. In this manner, cancer cells are differentially labeled.[0289]

(e) Detection and Diagnosis of Cancers[0290]

Without being bound by theory, it appears that the various CA sequences are important in cancers. Accordingly, disorders based on mutant or variant CA genes may be determined. In one embodiment, the invention provides methods for identifying cells containing variant CA genes comprising determining all or part of the sequence of at least one endogenous CA genes in a cell. As will be appreciated by those in the art, this may be done using any number of sequencing techniques. In a preferred embodiment, the invention provides methods of identifying the CA genotype of an individual comprising determining all or part of the sequence of at least one CA gene of the individual. This is generally done in at least one tissue of the individual, and may include the evaluation of a number of tissues or different samples of the same tissue. The method may include comparing the sequence of the sequenced CA gene to a known CA gene, i.e., a wild-type gene. As will be appreciated by those in the art, alterations in the sequence of some CA genes can be an indication of either the presence of the disease, or propensity to develop the disease, or prognosis evaluations.[0291]

The sequence of all or part of the CA gene can then be compared to the sequence of a known CA gene to determine if any differences exist. This can be done using any number of known homology programs, such as Bestfit, etc. In a preferred embodiment, the presence of a difference in the sequence between the CA gene of the patient and the known CA gene is indicative of a disease state or a propensity for a disease state, as outlined herein.[0292]

In a preferred embodiment, the CA genes are used as probes to determine the number of copies of the CA gene in the genome. For example, some cancers exhibit chromosomal deletions or insertions, resulting in an alteration in the copy number of a gene.[0293]

In another preferred embodiment CA genes are used as probes to determine the chromosomal location of the CA genes. Information such as chromosomal location finds use in providing a diagnosis or prognosis in particular when chromosomal abnormalities such as translocations, and the like are identified in CA gene loci.[0294]

The present invention provides methods of using the polynucleotides described herein for detecting cancer cells, facilitating diagnosis of cancer and the severity of a cancer (e.g., tumor grade, tumor burden, and the like) in a subject, facilitating a determination of the prognosis of a subject, and assessing the responsiveness of the subject to therapy (e.g., by providing a measure of therapeutic effect through, for example, assessing tumor burden during or following a chemotherapeutic regimen). Detection can be based on detection of a polynucleotide that is differentially expressed in a cancer cell, and/or detection of a polypeptide encoded by a polynucleotide that is differentially expressed in a cancer cell. The detection methods of the invention can be conducted in vitro or in vivo, on isolated cells, or in whole tissues or a bodily fluid e.g., blood, plasma, serum, urine, and the like).[0295]

In some embodiments, methods are provided for detecting a cancer cell by detecting expression in the cell of a transcript that is differentially expressed in a cancer cell. Any of a variety of known methods can be used for detection, including, but not limited to, detection of a transcript by hybridization with a polynucleotide that hybridizes to a polynucleotide that is differentially expressed in a prostate cancer cell; detection of a transcript by a polymerase chain reaction using specific oligonucleotide primers; in situ hybridization of a cell using as a probe a polynucleotide that hybridizes to a gene that is differentially expressed in a prostate cancer cell. The methods can be used to detect and/or measure mRNA levels of a gene that is differentially expressed in a cancer cell. In some embodiments, the methods comprise: a) contacting a sample with a polynucleotide that corresponds to a differentially expressed gene described herein under conditions that allow hybridization; and b) detecting hybridization, if any.[0296]

Detection of differential hybridization, when compared to a suitable control, is an indication of the presence in the sample of a polynucleotide that is differentially expressed in a cancer cell. Appropriate controls include, for example, a sample that is known not to contain a polynucleotide that is differentially expressed in a cancer cell, and use of a labeled polynucleotide of the same “sense” as the polynucleotide that is differentially expressed in the cancer cell. Conditions that allow hybridization are known in the art, and have been described in more detail above. Detection can also be accomplished by any known method, including, but not limited to, in situ hybridization, PCR (polymerase chain reaction), RT-PCR (reverse transcription-PCR), TMA, bDNA, and Nasbau and “Northern” or RNA blotting, or combinations of such techniques, using a suitably labeled polynucleotide. A variety of labels and labeling methods for polynucleotides are known in the art and can be used in the assay methods of the invention. Specificity of hybridization can be determined by comparison to appropriate controls.[0297]

Polynucleotides generally comprising at least 10 nt, at least 12 nt or at least 15 contiguous nucleotides of a polynucleotide provided herein, such as, for example, those having the sequence as depicted in Tables 1-27, are used for a variety of purposes, such as probes for detection of and/or measurement of, transcription levels of a polynucleotide that is differentially expressed in a prostate cancer cell. As will be readily appreciated by the ordinarily skilled artisan, the probe can be detectably labeled and contacted with, for example, an array comprising immobilized polynucleotides obtained from a test sample (e.g., mRNA). Alternatively, the probe can be immobilized on an array and the test sample detectably labeled. These and other variations of the methods of the invention are well within the skill in the art and are within the scope of the invention.[0298]

Nucleotide probes are used to detect expression of a gene corresponding to the provided polynucleotide. In Northern blots, mRNA is separated electrophoretically and contacted with a probe. A probe is detected as hybridizing to an mRNA species of a particular size. The amount of hybridization can be quantitated to determine relative amounts of expression, for example under a particular condition. Probes are used for in situ hybridization to cells to detect expression. Probes can also be used in vivo for diagnostic detection of hybridizing sequences. Probes are typically labeled with a radioactive isotope. Other types of detectable labels can be used such as chromophores, fluorophores, and enzymes. Other examples of nucleotide hybridization assays are described in WO92/02526 and U.S. Pat. No. 5,124,246.[0299]

PCR is another means for detecting small amounts of target nucleic acids (see, e.g., Mullis et al.,[0300]Meth. Enzymol. (1987) 155:335; U.S. Pat. No. 4,683,195; and U.S. Pat. No. 4,683,202). Two primer oligonucleotides that hybridize with the target nucleic acids are used to prime the reaction. The primers can be composed of sequence within or 3′ and 5′ to the CA polynucleotides disclosed herein. Alternatively, if the primers are 3′ and 5′ to these polynucleotides, they need not hybridize to them or the complements. After amplification of the target with a thermostable polymerase, the amplified target nucleic acids can be detected by methods known in the art, e.g., Southern blot. mRNA or cDNA can also be detected by traditional blotting techniques (e.g., Southern blot, Northern blot, etc.) described in Sambrook et al., “Molecular Cloning: A Laboratory Manual” (New York, Cold Spring Harbor Laboratory, 1989) (e.g., without PCR amplification). In general, mRNA or cDNA generated from mRNA using a polymerase enzyme can be purified and separated using gel electrophoresis, and transferred to a solid support, such as nitrocellulose. The solid support is exposed to a labeled probe, washed to remove any unhybridized probe, and duplexes containing the labeled probe are detected.

Methods using PCR amplification can be performed on the DNA from a single cell, although it is convenient to use at least about 10[0301]⁵cells. The use of the polymerase chain reaction is described in Saiki et al. (1985)Science239:487, and a review of current techniques may be found in Sambrook, et al.Molecular Cloning: A Laboratory Manual, CSH Press 1989, pp.14.2-14.33. A detectable label may be included in the amplification reaction. Suitable detectable labels include fluorochromes, (e.g. fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM), 2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein, 6-carboxy-X-rhodamine (ROX), 6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein (5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA)), radioactive labels, (e.g.³²P,³⁵S,³H, etc.), and the like. The label may be a two stage system, where the polynucleotides is conjugated to biotin, haptens, etc. having a high affinity binding partner, e.g. avidin, specific antibodies, etc., where the binding partner is conjugated to a detectable label. The label may be conjugated to one or both of the primers. Alternatively, the pool of nucleotides used in the amplification is labeled, so as to incorporate the label into the amplification product.

The detection methods can be provided as part of a kit. Thus, the invention further provides kits for detecting the presence and/or a level of a polynucleotide that is differentially expressed in a cancer cell (e.g., by detection of an mRNA encoded by the differentially expressed gene of interest), and/or a polypeptide encoded thereby, in a biological sample. Procedures using these kits can be performed by clinical laboratories, experimental laboratories, medical practitioners, or private individuals. The kits of the invention for detecting a polypeptide encoded by a polynucleotide that is differentially expressed in a cancer cell may comprise a moiety that specifically binds the polypeptide, which may be an antibody that binds the polypeptide or fragment thereof. The kits of the invention used for detecting a polynucleotide that is differentially expressed in a prostate cancer cell may comprise a moiety that specifically hybridizes to such a polynucleotide. The kit may optionally provide additional components that are useful in the procedure, including, but not limited to, buffers, developing reagents, labels, reacting surfaces, means for detection, control samples, standards, instructions, and interpretive information. Accordingly, the present invention provides kits for detecting prostate cancer comprising at least one of polynucleotides having the sequence as shown in Tables 1-27 or fragments thereof.[0302]

The present invention further relates to methods of detecting/diagnosing a neoplastic or preneoplastic condition in a mammal (for example, a human). “Diagnosis” as used herein generally includes determination of a subject's susceptibility to a disease or disorder, determination as to whether a subject is presently affected by a disease or disorder, prognosis of a subject affected by a disease or disorder (e.g., identification of pre-metastatic or metastatic cancerous states, stages of cancer, or responsiveness of cancer to therapy), and therametrics (e.g., monitoring a subject's condition to provide information as to the effect or efficacy of therapy).[0303]

The terms “treatment”, “treating”, “treat” and the like are used herein to generally refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease. “Treatment” as used herein covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease or symptom from occurring in a subject which may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease symptom, i.e., arresting its development; or (c) relieving the disease symptom, i.e., causing regression of the disease or symptom.[0304]

An “effective amount” is an amount sufficient to effect beneficial or desired results, including clinical results. An effective amount can be administered in one or more administrations.[0305]

A “cell sample” encompasses a variety of sample types obtained from an individual and can be used in a diagnostic or monitoring assay. The definition encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom, and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as proteins or polynucleotides. The term “cell sample” encompasses a clinical sample, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluid, and tissue samples.[0306]

As used herein, the terms “neoplastic cells”, “neoplasia”, “tumor”, “tumor cells”, “cancer” and “cancer cells”, (used interchangeably) refer to cells which exhibit relatively autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation (i.e., de-regulated cell division). Neoplastic cells can be malignant or benign.[0307]

The terms “individual,” “subject,” “host,” and “patient,” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and so on. Examples of conditions that can be detected/diagnosed in accordance with these methods include cancers. Polynucleotides corresponding to genes that exhibit the appropriate expression pattern can be used to detect cancer in a subject. For a review of markers of cancer, see, e.g., Hanahan et al. Cell 100:57-70 (2000).[0308]

One detection/diagnostic method comprises: (a) obtaining from a mammal (e.g., a human) a biological sample, (b) detecting the presence in the sample of a CA protein and (c) comparing the amount of product present with that in a control sample. In accordance with this method, the presence in the sample of elevated levels of a CA gene product indicates that the subject has a neoplastic or preneoplastic condition.[0309]

Biological samples suitable for use in this method include biological fluids such as serum, plasma, pleural effusions, urine and cerebro-spinal fluid, CSF, tissue samples (e.g., mammary tumor or prostate tissue slices) can also be used in the method of the invention, including samples derived from biopsies. Cell cultures or cell extracts derived, for example, from tissue biopsies can also be used.[0310]

The compound is preferably a binding protein, e.g., an antibody, polyclonal or monoclonal, or antigen binding fragment thereof, which can be labeled with a detectable marker (e.g., fluorophore, chromophore or isotope, etc). Where appropriate, the compound can be attached to a solid support such as a bead, plate, filter, resin, etc. Determination of formation of the complex can be effected by contacting the complex with a further compound (e.g., an antibody) that specifically binds to the first compound (or complex). Like the first compound, the further compound can be attached to a solid support and/or can be labeled with a detectable marker.[0311]

The identification of elevated levels of CA protein in accordance with the present invention makes possible the identification of subjects (patients) that are likely to benefit from adjuvant therapy. For example, a biological sample from a post primary therapy subject (e.g., subject having undergone surgery) can be screened for the presence of circulating CA protein, the presence of elevated levels of the protein, determined by studies of normal populations, being indicative of residual tumor tissue. Similarly, tissue from the cut site of a surgically removed tumor can be examined (e.g., by immunofluorescence), the presence of elevated levels of product (relative to the surrounding tissue) being indicative of incomplete removal of the tumor. The ability to identify such subjects makes it possible to tailor therapy to the needs of the particular subject. Subjects undergoing non-surgical therapy, e.g., chemotherapy or radiation therapy, can also be monitored, the presence in samples from such subjects of elevated levels of CA protein being indicative of the need for continued treatment. Staging of the disease (for example, for purposes of optimizing treatment regimens) can also be effected, for example, by biopsy e.g., with antibody specific for a CA protein.[0312]

(f) Animal Models and Transgenics[0313]

In another preferred embodiment CA genes find use in generating animal models of cancers, particularly lymphomas and carcinomas. As is appreciated by one of ordinary skill in the art, when the CA gene identified is repressed or diminished in CA tissue, gene therapy technology wherein antisense RNA directed to the CA gene will also diminish or repress expression of the gene. An animal generated as such serves as an animal model of CA that finds use in screening bioactive drug candidates. Similarly, gene knockout technology, for example as a result of homologous recombination with an appropriate gene targeting vector, will result in the absence of the CA protein. When desired, tissue-specific expression or knockout of the CA protein may be necessary.[0314]

It is also possible that the CA protein is overexpressed in cancer. As such, transgenic animals can be generated that overexpress the CA protein. Depending on the desired expression level, promoters of various strengths can be employed to express the transgene. Also, the number of copies of the integrated transgene can be determined and compared for a determination of the expression level of the transgene. Animals generated by such methods find use as animal models of CA and are additionally useful in screening for bioactive molecules to treat cancer.[0315]

Characterization of CA sequences[0316]

The CA nucleic acid sequences of the invention are depicted in Tables 1-27. The sequences in each Table include genomic DNA sequence (mouse genomic sequences mDxx-yyy; human genomic sequences hDxx-yyy), sequence corresponding to the mRNA(s) generated therefrom (mRxx-yyy; hRxx-yyy) and amino acid sequences of the proteins (mPxx-yyy; hPxx-yyy encoded by the mRNA for both mouse and human genes. N/A indicates a gene that has been identified, but for which there has not been a name ascribed.[0317]

The mouse and human genomic DNA sequence, sequence corresponding to the mRNA(s) generated therefrom and amino acid sequences of the proteins as shown in Tables 1-27 are described according to SEQ ID NOS as follows in Table 28.[0318]

TABLE 28


DESIGNATION	SEQ ID NO	TYPE OF SEQUENCE

mD07-23a	SEQ ID NO: 1	mouse genomic sequence

mR07-23a	SEQ ID NO: 2	mouse mRNA sequence

mP07-23a	SEQ ID NO: 3	mouse protein sequence

hD07-23a	SEQ ID NO: 4	human genomic sequence

hR07-23a	SEQ ID NO: 5	human mRNA sequence

hP07-23a	SEQ ID NO: 6	human protein sequence

mD07-24a	SEQ ID NO: 7	mouse genomic sequence

mR07-24a	SEQ ID NO: 8	mouse mRNA sequence

mP07-24a	SEQ ID NO: 9	mouse protein sequence

hD07-24a	SEQ ID NO: 10	human genomic sequence

hR07-24a	SEQ ID NO: 11	human mRNA sequence

hP07-24a	SEQ ID NO: 12	human protein sequence

mD07-24a	SEQ ID NO: 13	mouse genomic sequence

mR07-24a	SEQ ID NO: 14	mouse mRNA sequence

mP07-24a	SEQ ID NO: 15	mouse protein sequence

hD07-24a	SEQ ID NO: 16	human genomic sequence

hR07-24a.1	SEQ ID NO: 17	human mRNA sequence

hP07-24a.1	SEQ ID NO: 18	human protein sequence

hR07-24a.2	SEQ ID NO: 19	human mRNA sequence

hP07-24a.2	SEQ ID NO: 20	human protein sequence

hR07-24a.3	SEQ ID NO: 21	human mRNA sequence

hP07-24a.3	SEQ ID NO: 22	human protein sequence

mD07-125a	SEQ ID NO: 23	mouse genomic sequence

mR07-125a	SEQ ID NO: 24	mouse mRNA sequence

mP07-125a	SEQ ID NO: 25	mouse protein sequence

hD07-125a	SEQ ID NO: 26	human genomic sequence

hR07-125a	SEQ ID NO: 27	human mRNA sequence

hP07-125a	SEQ ID NO: 28	human protein sequence

mD07-153a	SEQ ID NO: 29	mouse genomic sequence

mR07-153a	SEQ ID NO: 30	mouse mRNA sequence

mP07-153a	SEQ ID NO: 31	mouse protein sequence

hD07-153a	SEQ ID NO: 32	human genomic sequence

hR07-153a	SEQ ID NO: 33	human mRNA sequence

hP07-153a	SEQ ID NO: 34	human protein sequence

mD07-204a	SEQ ID NO: 35	mouse genomic sequence

mR07-204a	SEQ ID NO: 36	mouse mRNA sequence

mP07-204a	SEQ ID NO: 37	mouse protein sequence

hD07-204a	SEQ ID NO: 38	human genomic sequence

hR07-204a	SEQ ID NO: 39	human mRNA sequence

hP07-204a	SEQ ID NO: 40	human protein sequence

mD07-205a	SEQ ID NO: 41	mouse genomic sequence

mR07-205a.1	SEQ ID NO: 42	mouse mRNA sequence

mP07-205a.1	SEQ ID NO: 43	mouse protein sequence

mR07-205a.2	SEQ ID NO: 44	mouse mRNA sequence

mP07-205a.2	SEQ ID NO: 45	mouse protein sequence

mR07-205a.3	SEQ ID NO: 46	mouse mRNA sequence

mP07-205a.3	SEQ ID NO: 47	mouse protein sequence

mR07-205a.4	SEQ ID NO: 48	mouse mRNA sequence

mP07-205a.4	SEQ ID NO: 49	mouse protein sequence

hD07-205a	SEQ ID NO: 50	human genomic sequence

hR07-205a	SEQ ID NO: 51	human mRNA sequence

hP07-205a	SEQ ID NO: 52	human protein sequence

mD07-210a	SEQ ID NO: 53	mouse genomic sequence

mR07-210a	SEQ ID NO: 54	mouse mRNA sequence

mP07-210a	SEQ ID NO: 55	mouse protein sequence

hD07-210a	SEQ ID NO: 56	human genomic sequence

hR07-210a.1	SEQ ID NO: 57	human mRNA sequence

hP07-210a.1	SEQ ID NO: 58	human protein sequence

hR07-210a.2	SEQ ID NO: 59	human mRNA sequence

hP07-210a.2	SEQ ID NO: 60	human protein sequence

hP07-210a.3	SEQ ID NO: 61	human mRNA sequence

hP07-210a.3	SEQ ID NO: 62	human protein sequence

mD07-211a	SEQ ID NO: 63	mouse genomic sequence

mR07-211a	SEQ ID NO: 64	mouse mRNA sequence

mP07-211a	SEQ ID NO: 65	mouse protein sequence

hD07-211a	SEQ ID NO: 66	human genomic sequence

hR07-211a.1	SEQ ID NO: 67	human mRNA sequence

hP07-211a.1	SEQ ID NO: 68	human protein sequence

hR07-211a.2	SEQ ID NO: 69	human mRNA sequence

hP07-211a.2	SEQ ID NO: 70	human protein sequence

mD07-220a	SEQ ID NO: 71	mouse genomic sequence

mR07-220a	SEQ ID NO: 72	mouse mRNA sequence

mP07-220a	SEQ ID NO: 73	mouse protein sequence

hD07-220a.1	SEQ ID NO: 74	human genomic sequence

hR07-220a.1	SEQ ID NO: 75	human mRNA sequence

hP07-220a.1	SEQ ID NO: 76	human protein sequence

hD07-220a.2	SEQ ID NO: 77	human genomic sequence

hR07-220a.2	SEQ ID NO: 78	human mRNA sequence

hP07-220a.2	SEQ ID NO: 79	human protein sequence

mD07-221a	SEQ ID NO: 80	mouse genomic sequence

mR07-221a	SEQ ID NO: 81	mouse mRNA sequence

mP07-221a	SEQ ID NO: 82	mouse protein sequence

hD07-221a	SEQ ID NO: 83	human genomic sequence

hR07-221a.1	SEQ ID NO: 84	human mRNA sequence

hP07-221a.1	SEQ ID NO: 85	human protein sequence

hR07-221a.2	SEQ ID NO: 86	human mRNA sequence

hP07-221a.2	SEQ ID NO: 87	human protein sequence

hR07-221a.3	SEQ ID NO: 88	human mRNA sequence

hP07-221a.3	SEQ ID NO: 89	human protein sequence

mD07-239a	SEQ ID NO: 90	mouse genomic sequence

mR07-239a	SEQ ID NO: 91	mouse mRNA sequence

mP07-239a	SEQ ID NO: 92	mouse protein sequence

hD07-239a	SEQ ID NO: 93	human genomic sequence

hR07-239a	SEQ ID NO: 94	human mRNA sequence

hP07-239a	SEQ ID NO: 95	human protein sequence

mD12-017	SEQ ID NO: 96	mouse genomic sequence

mR12-017	SEQ ID NO: 97	mouse mRNA sequence

mP12-017	SEQ ID NO: 98	mouse protein sequence

hD12-017	SEQ ID NO: 99	human genomic sequence

hR12-017	SEQ ID NO: 100	human mRNA sequence

hP12-017	SEQ ID NO: 101	human protein sequence

mD12-027	SEQ ID NO: 102	mouse genomic sequence

mR12-027	SEQ ID NO: 103	mouse mRNA sequence

mP12-027	SEQ ID NO: 104	mouse protein sequence

hD12-027	SEQ ID NO: 105	human genomic sequence

hR12-027	SEQ ID NO: 106	human mRNA sequence

hP12-027	SEQ ID NO: 107	human protein sequence

mD13-010	SEQ ID NO: 108	mouse genomic sequence

mR13-010	SEQ ID NO: 109	mouse mRNA sequence

mP13-010	SEQ ID NO: 110	mouse protein sequence

hD13-010	SEQ ID NO: 111	human genomic sequence

hR13-010	SEQ ID NO: 112	human mRNA sequence

hP13-010	SEQ ID NO: 113	human protein sequence

mD13-011	SEQ ID NO: 114	mouse genomic sequence

mR13-011	SEQ ID NO: 115	mouse mRNA sequence

mP13-011	SEQ ID NO: 116	mouse protein sequence

hD13-011	SEQ ID NO: 117	human genomic sequence

hR13-011.1	SEQ ID NO: 118	human mRNA sequence

hP13-011.1	SEQ ID NO: 119	human protein sequence

hR13-011.2	SEQ ID NO: 120	human mRNA sequence

hP13-011.2	SEQ ID NO: 121	human protein sequence

mD13-017	SEQ ID NO: 122	mouse genomic sequence

mR13-017	SEQ ID NO: 123	mouse mRNA sequence

mP13-017	SEQ ID NO: 124	mouse protein sequence

hD13-017	SEQ ID NO: 125	human genomic sequence

hR13-017	SEQ ID NO: 126	human mRNA sequence

hP13-017	SEQ ID NO: 127	human protein sequence

mD13-019	SEQ ID NO: 128	mouse genomic sequence

mR13-019.1	SEQ ID NO: 129	mouse mRNA sequence

mP13-019.1	SEQ ID NO: 130	mouse protein sequence

mR13-019.2	SEQ ID NO: 131	mouse mRNA sequence

mP13-019.2	SEQ ID NO: 132	mouse protein sequence

hD13-019	SEQ ID NO: 133	human genomic sequence

hR13-019	SEQ ID NO: 134	human mRNA sequence

hP13-019	SEQ ID NO: 135	human protein sequence

mD13-023	SEQ ID NO: 136	mouse genomic sequence

mR13-023	SEQ ID NO: 137	mouse mRNA sequence

mP13-023	SEQ ID NO: 138	mouse protein sequence

hD13-023	SEQ ID NO: 139	human genomic sequence

hR13-023	SEQ ID NO: 140	human mRNA sequence

hP13-023	SEQ ID NO: 141	human protein sequence

mD13-026	SEQ ID NO: 142	mouse genomic sequence

mR13-026	SEQ ID NO: 143	mouse mRNA sequence

mP13-026	SEQ ID NO: 144	mouse protein sequence

mD13-026	SEQ ID NO: 145	human genomic sequence

hR13-026	SEQ ID NO: 146	human mRNA sequence

hP13-026	SEQ ID NO: 147	human protein sequence

mD13-028	SEQ ID NO: 148	mouse genomic sequence

mR13-028	SEQ ID NO: 149	mouse mRNA sequence

mP13-028	SEQ ID NO: 150	mouse protein sequence

hD13-028	SEQ ID NO: 151	human genomic sequence

hR13-028.1	SEQ ID NO: 152	human mRNA sequence

hP13-028.1	SEQ ID NO: 153	human protein sequence

hR13-028.2	SEQ ID NO: 154	human mRNA sequence

hP13-028.2	SEQ ID NO: 155	human protein sequence

hR13-028.3	SEQ ID NO: 156	human mRNA sequence

hP13-028.3	SEQ ID NO: 157	human protein sequence

hR13-028.4	SEQ ID NO: 158	human mRNA sequence

hP13-028.4	SEQ ID NO: 159	human protein sequence

mD13-036	SEQ ID NO: 160	mouse genomic sequence

mR13-036	SEQ ID NO: 161	mouse mRNA sequence

mP13-036	SEQ ID NO: 162	mouse protein sequence

mD13-036	SEQ ID NO: 163	human genomic sequence

hR13-036	SEQ ID NO: 164	human mRNA sequence

hP13-036	SEQ ID NO: 165	human protein sequence

mD13-060	SEQ ID NO: 166	mouse genomic sequence

mR13-060	SEQ ID NO: 167	mouse mRNA sequence

mP13-060	SEQ ID NO: 168	mouse protein sequence

hD13-060	SEQ ID NO: 169	human genomic sequence

hR13-060.1	SEQ ID NO: 170	human mRNA sequence

hP13-060.1	SEQ ID NO: 171	human protein sequence

hR13-060.2	SEQ ID NO: 172	human mRNA sequence

hP13-060.2	SEQ ID NO: 173	human protein sequence

hR13-060.3	SEQ ID NO: 174	human mRNA sequence

hP13-060.3	SEQ ID NO: 175	human protein sequence

mD13-065	SEQ ID NO: 176	mouse genomic sequence

mR13-065	SEQ ID NO: 177	mouse mRNA sequence

mP13-065	SEQ ID NO: 178	mouse protein sequence

hD13-065	SEQ ID NO: 179	human genomic sequence

hR13-065.1	SEQ ID NO: 180	human mRNA sequence

hP13-065.1	SEQ ID NO: 181	human protein sequence

hR13-065.2	SEQ ID NO: 182	human mRNA sequence

hP13-065.2	SEQ ID NO: 183	human protein sequence

hR13-065.3	SEQ ID NO: 184	human mRNA sequence

hP13-065.3	SEQ ID NO: 185	human protein sequence

mD14-032	SEQ ID NO: 186	mouse genomic sequence

mR14-032	SEQ ID NO: 187	mouse mRNA sequence

mP14-032	SEQ ID NO: 188	mouse protein sequence

hD14-032	SEQ ID NO: 189	human genomic sequence

hR14-032	SEQ ID NO: 190	human mRNA sequence

hP14-032	SEQ ID NO: 191	human protein sequence

mD14-033	SEQ ID NO: 192	mouse genomic sequence

mR14-033	SEQ ID NO: 193	mouse mRNA sequence

mP14-033	SEQ ID NO: 194	mouse protein sequence

hD14-033	SEQ ID NO: 195	human genomic sequence

hR14-033	SEQ ID NO: 196	human mRNA sequence

hP14-033	SEQ ID NO: 197	human protein sequence

mD14-034	SEQ ID NO: 198	mouse genomic sequence

mR14-034	SEQ ID NO: 199	mouse mRNA sequence

mP14-034	SEQ ID NO: 200	mouse protein sequence

hD14-034	SEQ ID NO: 201	human genomic sequence

hR14-034	SEQ ID NO: 202	human mRNA sequence

hP14-034	SEQ ID NO: 203	human protein sequence

The CA sequences were analyzed by Panther™ (Molecular Diagnostics, Palo Alto, Calif.) software designed to detect homologs and enable prediction of molecular function through a system for protein functional classification. Human Gene Ontlogy annotations were prepared in accordance with the Gene Ontology Consortium (Gene Ontology: tool for the unification of biology. The Gene Ontology Consortium[0319]Nature Genet.25: 25-29 (2000)). Similar analysis was carried out by determining IPR information regarding the CA polypeptides from InterPro, which is an integrated documentation resource for protein families, domains and functional sites (Apweiler at al. Bioinformatics 16(12): 1145-1150 (2000)).

The CA sequences may be classified according to the following predicted general classifications of function by Panther™ analysis, human gene ontology and IPR domain information for polypeptides SEQ ID NOS: 6, 12, 18, 20, 22, 28, 34, 40, 52, 58, 60, 62, 68, 70, 76, 79, 85, 87, 89, 95, 101, 107, 113, 119, 121, 127, 135, 141, 147, 153, 155, 157, 159, 165, 171, 173, 175, 181, 183, 185, 191, 197 and 203 shown in Tables 1-27.[0320]

TABLE 29


HUMAN
PROTEIN	CLASSIFICATION

hP07-023a	HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 6)	No Panther Hit

HUMAN GENE ONTOLOGY

PROCESS

	stress response > defence response

	developmental processes > fertilization

FUNCTION

	molecular_function unknown > lymphocyte antigen

	ligand binding or carrier > calcium binding

LOCATION

	cell > plasma membrane

	cell > membrane fraction

	lysosome > lysosomal membrane

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR000082 (SEA)

	NULL (THR RICH)

	IPR000561 (EGF 2)

hP07-24a

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 12)

FAMILY (SUBFAMILY)

CELL SURFACE GLYCOPROTEIN MUC18-

RELATED HUMAN GENE ONTOLOGY PROCESS

	cell communication > cell adhesion

	defence response > immune response

	neurogenesis > central nervous system development

	transcription, DNA-dependent > transcription

	regulation

	microtubule-based process

	nuclear congression

	peptidoglycan catabolism > microtubule-based

	movement

FUNCTION

	B cell receptor

	defense/immunity protein > immunoglobulin

	enzyme > nitric oxide synthase

	GO molecular function > cell adhesion

	GO molecular function > cell adhesion

	nucleic acid binding >DNA binding

LOCATION

	cell > membrane fraction

	cell > plasma membrane

	plasma membrane > integral plasma membrane

	protein

mitochondrial membrane > mitochondrial inner

	membrane

adherens junction > cell—cell adherens junction

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR003599 (IG)

	IPR003006 (ig)

hP07-053a.1

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 18)

FAMILY (SUBFAMILY)

TUMOR NECROSIS FACTOR-RELATED

(TNF-RELATED APOPTOSIS INDUCING LIGAND)

MOLECULAR FUNCTIONS

	Signaling molecule > Cytokine > Other cytokine

BIOLOGICAL PROCESS

Signal transduction > Intracellular signaling

	cascade > NF-kappaB cascade

Signal transduction > Cell communication >

	Ligand-mediated signaling

Signal transduction > Cell surface receptor

	mediated signal transduction > Cytokine and chemokine
	mediated signaling pathway

	Apoptosis > Induction of apoptosis

HUMAN GENE ONTOLOGY

PROCESS

	cell death > apoptosis

	defence response > immune response

	apoptosis > induction of apoptosis

	cell communication > signal transduction

	cell communication > cell—cell signaling

FUNCTION

	molecular_function unknown > lymphocyte antigen

LOCATION

	cell > membrane fraction

	cell > soluble fraction

	plasma membrane > integral plasma membrane

	protein

	HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)
	IPR003263 (sp P50591 TRAI HUMAN)

hP07-053a.2	HUMAN PANTHER CLASSIFICATIONS
(SEQ ID NO: 20)	FAMILY (SUBFAMILY)
	TUMOR NECROSIS FACTOR-RELATED
	(TNF-RELATED APOPTOSIS INDUCING LIGAND)
	MOLECULAR FUNCTIONS

	Signaling molecule > Cytokine > Other cytokine

BIOLOGICAL PROCESS

Signal transduction > Intracellular signaling

	cascade > NF-kappaB cascade

Signal transduction > Cell communication >

	Ligand-mediated signaling

Signal transduction > Cell surface receptor

	Apoptosis > Induction of apoptosis

HUMAN GENE ONTOLOGY

PROCESS

FUNCTION

	molecular_function unknown > lymphocyte antigen

LOCATION

	protein

	HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

IPR003263 (sp P50591 TRAI HUMAN)

hP07-053a.3

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 22)

FAMILY (SUBFAMILY)

TUMOR NECROSIS FACTOR-RELATED

	(TNF-RELATED APOPTOSIS INDUCING LIGAND)

MOLECULAR FUNCTIONS

	Signaling molecule > Cytokine > Other cytokine

BIOLOGICAL PROCESS

Signal transduction > Intracellular signaling

	cascade > NF-kappaB cascade

Signal transduction > Cell communication >

	Ligand-mediated signaling

Signal transduction > Cell surface receptor

	mediated signal transduction > Cytokine and chemokine
	mediated signaling pathway
	Apoptosis > Induction of apoptosis

	HUMAN GENE ONTOLOGY

PROCESS

	defence response > immune response

	cell death > apoptosis

	apoptosis > induction of apoptosis

	cell communication > signal transduction

	cell communication > cell—cell signaling

FUNCTION

	molecular_function unknown > lymphocyte antigen

	enzyme > nitric oxide synthase

	GO molecular function > cell cycle regulator

	enzyme > protein kinase

	nucleotide binding > ATP binding

LOCATION

	cell > membrane fraction

	plasma membrane > integral plasma membrane

	protein

	cell > soluble fraction

	GO cellular component > extracellular

	mitochondrial membrane > mitochondrial inner

	membrane

	HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR000478 (TNF)

	IPR000478 (TNF)

	IPR000478 (TNF 2)

	IPR000478 (TNF 1)

	IPR003263 (sp P50591 TRAI HUMAN)

	IPR003636 (sp P41047 FASL MOUSE)

hP07-125a

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 28)

FAMILY (SUBFAMILY)

	CD40L RECEPTOR-RELATED (CD27L RECEPTOR)

MOLECULAR FUNCTIONS

	Molecular function unclassified

BIOLOGICAL PROCESS

Immunity and defense > T-cell mediated

	immunity

	HUMAN GENE ONTOLOGY

PROCESS

induction of apoptosis by extracellular signals >

	induction of apoptosis via death domain receptors

	cell death > apoptosis

	apoptosis > anti-apoptosis

	apoptosis > induction of apoptosis

	cell communication > signal transduction

FUNCTION

	glycosaminoglycan binding > hyaluronic acid binding

	GO molecular function > apoptosis inhibitor

	O-glucosyl hydrolase

	antimicrobial response protein > lysozyme

	molecular_function unknown > lymphocyte antigen

	electron carrier > iron-sulfur electron transfer

	carrier

LOCATION

	protein

integral plasma membrane protein > integral

	plasma membrane proteoglycan

	cell > soluble fraction

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR001368 (TNFR)

	IPR001368 (TNFRc6)

	IPR001368 (TNFR NGFR 2)

	NULL (CYS RICH)

	IPR000561 (EGF 2)

	IPR001368 (TNFR NGFR 1)

	IPR001368 (sp P26842 CD27 HUMAN)

hP14-034

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 203)

FAMILY (SUBFAMILY)

INTERFERON INDUCIBLE

	TRANSMEMBRANE PROTEIN (INTERFERON INDUCIBLE
	TRANSMEMBRANE PROTEIN)

MOLECULAR FUNCTIONS

	Molecular function unknown

BIOLOGICAL PROCESS

	Cell proliferation and differentiation

HUMAN GENE ONTOLOGY

PROCESS

	defence response > immune response

	cell cycle > cell cycle control

	cell proliferation > negative control of cell

	proliferation

FUNCTION

defense/immunity protein > antiviral response

	protein

LOCATION

	cell > membrane fraction

	cell > plasma membrane

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	No Domain Hit

hP07-153a

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 34)

FAMILY (SUBFAMILY)

	LAMININ-RELATED HUMAN GENE ONTOLOGY

PROCESS

	cell growth and maintenance > cell proliferation

	protein metabolism and modification

	macromolecule catabolism > proteolysis and

	peptidolysis

	developmental processes > sex differentiation

	cell communication > signal transduction

	apoptotic program > caspase activation

FUNCTION

	ligand binding or carrier > calcium binding

	serine-type endopeptidase > trypsin

	enzyme inhibitor > proteinase inhibitor

	serine-type endopeptidase > trypsin

	blood coagulation factor > protein C (activated)

	serine-type endopeptidase > trypsin

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR002383 (GLABLOOD)

	IPR000294 (GLA)

	IPR001791 (LamG)

	IPR000294 (gla)

	IPR001791 (laminin G)

	IPR001791 (LAM G DOMAIN 2)

	IPR000294 (GLU CARBOXYLATION)

hP07-204a

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 40)

FAMILY (SUBFAMILY)

	WNT PROTEIN (WNT)

MOLECULAR FUNCTIONS

	Signaling molecule > Other signaling molecule

BIOLOGICAL PROCESS

	Developmental processes

HUMAN GENE ONTOLOGY

PROCESS

cell surface receptor linked signal transduction >

	fz2 receptor signaling pathway

	GO biological process > developmental processes

	developmental processes > embryogenesis and

	morphogenesis

	cell communication > signal transduction

	cell communication > cell—cell signaling

FUNCTION

	GO molecular function > cell cycle regulator

LOCATION

	extracellular > extracellular space

	extracellular > extracellular matrix

	cell > soluble fraction

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR000970 (WNTPROTEIN)

	IPR000970 (WNT1)

	IPR000970 (wnt)

	NULL (CYS RICH)

	IPR000970 (WNT1)

hP07-205a

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 52)

FAMILY (SUBFAMILY)

PLATELET ENDOTHELIAL CELL

	ADHESION MOLECULE (PECAM-1)(PLATELET
	ENDOTHELIAL CELL ADHESION MOLECULE
	PRECURSOR (PECAM-1) (CD31 ANTIGEN))

MOLECULAR FUNCTIONS

	Receptor

	Cell adhesion molecule > Other cell adhesion

	molecule

Defense/immunity protein > Immunoglobulin

	receptor family member

BIOLOGICAL PROCESS

	Cell adhesion

HUMAN GENE ONTOLOGY

PROCESS

	cell growth and maintenance > cell motility

	cell communication > cell recognition

	cell communication > signal transduction

	cell communication > cell adhesion

FUNCTION

	B cell receptor

	defense/immunity protein > immunoglobulin

	GO molecular function > cell adhesion

LOCATION

	plasma membrane > intercellular junction

	cell > plasma membrane

	integral plasma membrane protein > integral

	plasma membrane proteoglycan

	cell > membrane fraction

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR003599 (IG)

	IPR003006 (ig)

hP07-210a.1

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 58))

FAMILY (SUBFAMILY)

OLIGOPEPTIDE TRANSPORTER-RELATED

	(gb def: (ab000280) peptide/histidine transporter [rattus
	norvegicus])

MOLECULAR FUNCTIONS

	Molecular function unclassified

BIOLOGICAL PROCESS

	Biological process unclassified

HUMAN GENE ONTOLOGY

PROCESS

	peptide transport > oligopeptide transport

FUNCTION

	serine carboxypeptidase > carboxypeptidase D

LOCATION

	cell > membrane fraction

	plasma membrane > integral plasma membrane

	protein

	HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR000109 (PTR2)

	IPR000109 (PTR2 2)

	IPR001117 (MULTICOPPER OXIDASE1)

hP07-210a.2

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 60)

FAMILY (SUBFAMILY)

OLIGOPEPTIDE TRANSPORTER-RELATED

	(gb def: (ab000280) peptide/histidine transporter [rattus
	norvegidus])

MOLECULAR FUNCTIONS

	Molecular function unclassified

BIOLOGICAL PROCESS

	Biological process unclassified

HUMAN GENE ONTOLOGY

PROCESS

	peptide transport > oligopeptide transport

FUNCTION

	serine carboxypeptidase > carboxypeptidase D

LOCATION

	cell > membrane fraction

	plasma membrane > integral plasma membrane

	protein

	HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR000109 (PTR2)

	IPR000109 (PTR2 2)

	IPR001117 (MULTICOPPER OXIDASE 1)

hP07-210a.3

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 62)

FAMILY (SUBFAMILY)

OLIGOPEPTIDE TRANSPORTER-RELATED

	(gb def: (ab000280) peptide/histidine transporter
	[rattus norvegicus])

MOLECULAR FUNCTIONS

	Molecular function unclassified

BIOLOGICAL PROCESS

	Biological process unclassified

HUMAN GENE ONTOLOGY

PROCESS

	peptide transport > oligopeptide transport

	neurogenesis > central nervous system

	development

transcription, DNA-dependent > transcription

	regulation

	cell death > apoptosis

	microtubule-based process

	nuclear congression

	peptidoglycan catabolism > microtubule-based

	movement

FUNCTION

	serine carboxypeptidase > carboxypeptidase D

	enzyme > nitric oxide synthase

	GO molecular function > cell cycle regulator

	ligand binding or carrier > electron transfer

	enzyme > sarcosine dehydrogenase

LOCATION

	cell > membrane fraction

	plasma membrane > integral plasma membrane

	protein

	GO cellular component > extracellular

	mitochondrial membrane > mitochondrial inner

	membrane

	extracellular > extracellular space

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR000109 (PTR2)

	NULL (ALA RICH)

	IPR000109 (PTR2 2)

	IPR001117 (MULTICOPPER OXIDASE1)

hP07-211a.1

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 68)	No Panther Hit

	HUMAN GENE ONTOLOGY
	No Gene Ontology

	HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)
	No Domain Hit

hP07-211a.2

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 70)

No Panther Hit

	HUMAN GENE ONTOLOGY
	No Gene Ontology

	HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR000282 (CR2A)

hP07-220a.1

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 76)

FAMILY (SUBFAMILY)

INTERLEUKIN-1 RECEPTOR-RELATED

	(INTERLEUKIN-1 RECEPTOR-RELATED)

MOLECULAR FUNCTIONS

Receptor > Cytokine receptor > Interleukin

	receptor

BIOLOGICAL PROCESS

Signal transduction > Cell surface receptor

	mediated signal transduction > Cytokine and chemokine
	mediated signaling pathway

	HUMAN GENE ONTOLOGY

FUNCTION

	molecular_function unknown > lymphocyte antigen

	B cell receptor

	defense/immunity protein > immunoglobulin

	GO molecular function > cell adhesion

LOCATION

plasma membrane > integral plasma membrane

	protein

	cell > plasma membrane

	cell > membrane fraction

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR000157 (TIR)

	IPR000157 (TIR)

	IPR003006 (ig)

	IPR000157 (TOLL)

hP07-220a.2

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 79)

FAMILY (SUBFAMILY)

INTERLEUKIN-1 RECEPTOR-RELATED

	(INTERLEUKIN-1 RECEPTOR-RELATED)

MOLECULAR FUNCTIONS

Receptor > Cytokine receptor > Interleukin

	receptor

BIOLOGICAL PROCESS

Signal transduction > Cell surface receptor

FUNCTION

	molecular_function unknown > lymphocyte antigen

	B cell receptor

	defense/immunity protein > immunoglobulin

LOCATION

plasma membrane > integral plasma membrane

	protein

	cell > membrane fraction

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR003599 (IG)

hP07-221a.1

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 85)

FAMILY (SUBFAMILY)

COLLAGEN ALPHA CHAIN (COLLAGEN

	ALPHA 5(IV) CHAIN)

MOLECULAR FUNCTIONS

Extracellular matrix > Extracellular matrix

	structural protein

BIOLOGICAL PROCESS

	Biological process unclassified

HUMAN GENE ONTOLOGY

PROCESS

	cell communication > cell adhesion

	ectoderm development > epidermal differentiation

	mesoderm development > skeletal development

	complement activation > complement activation,

	classical pathway

	sensory perception > hearing

FUNCTION

	GO molecular function > cell adhesion

	blood coagulation factor > protein C (activated)

	serine-type endopeptidase

	protein binding > collagen binding

	defense/immunity protein > opsonin

	proteinase inhibitor > serine protease inhibitor

LOCATION

	fibrillar collagen > collagen type IV

	extracellular matrix > basement membrane

	extracellular matrix > collagen

	fibrillar collagen > collagen type III

	fibrillar collagen > collagen type I

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR000087 (Collagen)

	NULL (GLY RICH)

	IPR000694 (PRO RICH)

	IPR000087 (COLLAGEN REP)

hP07-221a.2

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 87)

FAMILY (SUBFAMILY)

COLLAGEN ALPHA CHAIN (COLLAGEN

	ALPHA 5(IV) CHAIN)

MOLECULAR FUNCTIONS

Extracellular matrix > Extracellular matrix

	structural protein

BIOLOGICAL PROCESS

	Biological process unclassified

HUMAN GENE ONTOLOGY

PROCESS

	classical pathway

	sensory perception > hearing

FUNCTION

LOCATION

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

hP07-221a.3

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 89)

FAMILY (SUBFAMILY)

COLLAGEN ALPHA CHAIN(COLLAGEN

	ALPHA 5(IV) CHAIN)

MOLECULAR FUNCTIONS

Extracellular matrix > Extracellular matrix

	structural protein

BIOLOGICAL PROCESS

	Biological process unclassified

HUMAN GENE ONTOLOGY

PROCESS

	cell communication > cell adhesion

	ectoderm development > epidermal

	differentiation

	mesoderm development > skeletal development

	complement activation > complement activation,

	classical pathway

	sensory perception > hearing

FUNCTION

LOCATION

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR001442 (C4)

	IPR000087 (Collagen)

	IPR001442 (C4)

	NULL (GLY RICH)

	IPR000694 (PRO RICH)

	IPR000087 (COLLAGEN REP)

	IPR001442 (sp P29400 CA54 HUMAN)

hP07-239a

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 95)

FAMILY (SUBFAMILY)

	SEMAPHORIN(SEMAPHORIN 6B)

MOLECULAR FUNCTIONS

Signaling molecule > Membrane-bound

	signaling molecule

BIOLOGICAL PROCESS

	Signal transduction > Cell communication

	Developmental processes > Ectoderm

	development > Neurogenesis

	HUMAN GENE ONTOLOGY

PROCESS

	ectoderm development > neurogenesis

	peptidoglycan catabolism > axon guidance

	axonogenesis

	defence response > immune response

	xenobiotic metabolism > drug resistance

	cell adhesion

FUNCTION

	GO molecular function > cell adhesion

	B cell receptor

	defense/immunity protein > immunoglobulin

	cell adhesion

	transmembrane receptor > cell adhesion receptor

	glucosidase > mannosyl-oligosaccharide

	glucosidase (processing A-glucosidase I)

LOCATION

	cell > membrane fraction

	GO cellular component > extracellular

	extracellular > extracellular space

	integral plasma membrane protein > integrin

	cytoplasm > endoplasmic reticulum

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR001627 (Sema)

hP12-017

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 101)

FAMILY (SUBFAMILY)

	CELL ADHESION MOLECULE-RELATED
	(INTEGRAL MEMBRANE GLYCOPROTEIN)

MOLECULAR FUNCTIONS

	MOLECULAR FUNCTION UNCLASSIFIED

BIOLOGICAL PROCESS

	BIOLOGICAL PROCESS UNCLASSIFIED

HUMAN GENE ONTOLOGY

PROCESS

	cell communication > cell adhesion

	protein modification > protein dephosphorylation

	protein modification > protein phosphorylation

	protein kinase cascade > MAPKKK cascade

	embryogenesis and morphogenesis > histogenesis

	and organogenesis

FUNCTION

	GO molecular function > cell adhesion

	B cell receptor

	defense/immunity protein > immunoglobulin

	protein kinase > protein tyrosine kinase

	nucleotide binding > ATP binding

	enzyme > protein kinase

LOCATION

plasma membrane > integral plasma membrane

	protein

	cell > plasma membrane

	cell > membrane fraction

	extracellular > extracellular matrix

	extracellular > extracellular space

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR001611 (LEURICHRPT)

	IPR000372 (LRRNT)

	IPR000483 (LRRCT)

	IPR003006 (ig)

	IPR000483 (LRRCT)

	IPR003885 (LRR SD22)

	NULL (LRR PS)

	IPR003598 (IGc2)

	IPR000372 (LRRNT)

	IPR003599 (IG)

	IPR003591 (LRR TYP)

	IPR001611 (LRR)

hP12-027

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 107)

FAMILY (SUBFAMILY)

NOT ANNOTATED HUMAN GENE

	ONTOLOGY

PROCESS

	cell death > apoptosis

	defence response > humoral defense mechanism

	GO biological process > developmental processes

	ectoderm development > epidermal differentiation

	cell communication > cell adhesion

FUNCTION

	B cell receptor

	defense/immunity protein > immunoglobulin

	signaling (initiator) caspase > caspase-2

	enzyme > sterol esterase

	GO molecular function > enzyme

	protein binding > profilin binding

LOCATION

	cell > membrane fraction

	extracellular > extracellular space

	cell > nucleus

	cytoplasm > cytoskeleton

	cell > plasma membrane

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR003596 (IGv)

	IPR003599 (IG)

	IPR003006 (ig)

hP13-010

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 113)

FAMILY (SUBFAMILY)

LEU RICH

	GLYCOPROTEIN (FIBROMODULIN)

MOLECULAR FUNCTIONS

	Extracellular matrix > Other extracellular matrix

BIOLOGICAL PROCESS

	Cell adhesion

HUMAN GENE ONTOLOGY

PROCESS

	skeletal development > cartilage condensation

	embryogenesis and morphogenesis > histogenesis and

	organogenesis

	mesoderm development > skeletal development

	cell communication > cell adhesion

	cell communication > signal transduction

FUNCTION

glycosaminoglycan binding > hyaluronic acid

	binding

	ligand binding or carrier > protein binding

	GO molecular function > cell adhesion

	ligand binding or carrier > glycosaminoglycan

	binding

	calcium binding > calcium sensing

LOCATION

	extracellular > extracellular matrix

	cell > plasma membrane

	extracellular > extracellular space

	cell > membrane fraction

	plasma membrane > integral plasma membrane protein

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR001611 (LEURICHRPT)

	NULL (LRR BAC)

	NULL (LRR PS)

	IPR000372 (LRRNT)

	IPR003591 (LRR TYP)

	IPR001611 (LRR)

	IPR000372 (LRRNT)

hP13-011.1

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 119)

FAMILY (SUBFAMILY)

PROTEIN-TYROSINE PHOSPHATASE

	(PROTEIN-TYROSINE PHOSPHATASE-CD45)

MOLECULAR FUNCTIONS

	Receptor > Other receptor

	Phosphatase > Protein phosphatase

BIOLOGICAL PROCESS

Protein metabolism and modification > Protein

	modification > Protein phosphorylation

	HUMAN GENE ONTOLOGY

PROCESS

	protein modification > protein dephosphorylation

	enzyme linked receptor protein signaling pathway

	> transmembrane receptor protein tyrosine phosphatase
	signaling pathway

isoprenoid catabolism > one-carbon compound

	metabolism

defasciculation of neuron > defasciculation of

	motor neuron

FUNCTION

protein tyrosine phosphatase > prenylated protein

	tyrosine phosphatase

protein phosphatase > protein tyrosine

	phosphatase

	enzyme > protein phosphatase
	transmembrane receptor

	protein tyrosine phosphatase > prenylated protein

	tyrosine phosphatase

protein tyrosine phosphatase > prenylated protein

	tyrosine phosphatase

LOCATION

plasma membrane > integral plasma membrane

	protein

	cell > plasma membrane

	cell > membrane fraction

	cell > cytoplasm

	cytoplasm > cytoskeleton

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR000242 (PRTYPHPHTASE)

	IPR000242 (PTPc)

	IPR003595 (PTPc motif)

	IPR000242 (Y phosphatase)

	IPR000387 (TYR PHOSPHATASE 2 2)

	IPR000242 (TYR PHOSPHATASE PTP 2)

	IPR000387 (TYR PHOSPHATASE 1)

hP13-011.2

HUMAN PANTHER CLASS LOCATIONS

(SEQ ID NO: 121)

FAMILY (SUBFAMILY)

PROTEIN-TYROSINE

	PHOSPHATASE(PROTEIN-TYROSINE PHOSPHATASE-CD45)

MOLECULAR FUNCTIONS

	Receptor > Other receptor

	Phosphatase > Protein phosphatase

BIOLOGICAL PROCESS

Protein metabolism and modification > Protein

	modification > Protein phosphorylation

	HUMAN GENE ONTOLOGY

PROCESS

	protein modification > protein dephosphorylation

	enzyme linked receptor protein signaling pathway >

	transmembrane receptor protein tyrosine phosphatase
	signaling pathway

isoprenoid catabolism > one-carbon compound

	metabolism

defasciculation of neuron > defasciculation of

	motor neuron

FUNCTION

protein tyrosine phosphatase > prenylated protein

	tyrosine phosphatase

protein phosphatase > protein tyrosine

	phosphatase

	tyrosine phosphatase

protein tyrosine phosphatase > prenylated protein

	tyrosine phosphatase

LOCATION

plasma membrane > integral plasma membrane

	protein

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR000242 (PRTYPHPHTASE)

	IPR000387 (TYR PHOSPHATASE 1)

	IPR000242 (PTPc)

	IPR001777 (FN3)

	IPR003595 (PTPc motif)

	IPR001777 (fn3)

	IPR000242 (Y phosphatase)

	IPR000387 (TYR PHOSPHATASE 2 2)

	NULL (THR RICH)

	IPR000242 (TYR PHOSPHATASE PTP 2)

hP13-017

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 127)

FAMILY (SUBFAMILY)

INOSITOL 1,4,5-TRISPHOSPHATE

	RECEPTOR (INOSITOL 1,4,5-TRISPHOSPHATE RECEPTOR
	TYPE 2)

MOLECULAR FUNCTIONS

	Receptor

	Ion channel > Ligand-gated ion channel > Other

	ligand-gated ion channel

BIOLOGICAL PROCESS

Signal transduction > Cell surface receptor

	mediated signal transduction > G-protein mediated signaling

	Transport > Ion transport > Cation transport

HUMAN GENE ONTOLOGY

PROCESS

di-, tri-valent inorganic cation transport > calcium

	ion transport

	ion transport > cation transport

	cell communication > signal transduction

	transport > ion transport

	chemosensory perception > olfaction

FUNCTION

	enzyme > 1D-myo-inositol-trisphosphate 3-kinase

	ligand binding or earner > calcium binding

LOCATION

	cytoplasm > endoplasmic reticulum

	cell > membrane fraction

	cell > plasma membrane

	plasma membrane > brush border

	plasma membrane > integral plasma membrane

	protein

	HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR000493 (INSP3RECEPTR)

	IPR003608 (MIR)

	IPR000699 (RYDR ITPR)

	IPR001682 (CHANNEL PORE CA NA)

hP13-019

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 135)

FAMILY (SUBFAMILY)

NKG2 TYPE II INTEGRAL MEMBRANE

	PROTEIN (NATURAL KILLER CELL SURFACE PROTEIN)

MOLECULAR FUNCTIONS

	Receptor > Other receptor

	Defense/immunity protein > Other defense and

	immunity protein

BIOLOGICAL PROCESS

Immunity and defense > Natural killer cell

	mediated immunity

	HUMAN GENE ONTOLOGY

PROCESS

	stress response > defence response

	humoral defense mechanism > antimicrobial

	response

	cell communication > cell adhesion

	defence response > cellular defense response

FUNCTION

	molecular_function unknown > lymphocyte antigen

	sugar binding > lectin

	protein binding > lipoprotein binding

	GO molecular function > ligand binding or carrier

	defense/immunity protein > major histocompatibility

	complex antigen

LOCATION

	cell > plasma membrane

	cell > membrane fraction

	plasma membrane > integral plasma membrane

	protein

	HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR001304 (CLECT)

	IPR001304 (lectin c)

	IPR001304 (C TYPE LECTIN 2)

hP13-023

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 141)

FAMILY (SUBFAMILY)

NEUROTENSIN RECEPTOR-RELATED (G

	PROTEIN-COUPLED RECEPTOR)

MOLECULAR FUNCTIONS

	Receptor > G-protein coupled receptor

BIOLOGICAL PROCESS

Signal transduction > Cell surface receptor

	mediated signal transduction > G-protein mediated signaling

	HUMAN GENE ONTOLOGY

PROCESS

cell surface receptor linked signal transduction >

	G protein linked receptor protein signaling pathway

	behavior > feeding behavior

	G protein signaling, linked to cAMP nucleotide

	second messenger > G protein signaling, adenylate cyclase
	inhibiting pathway

G protein linked receptor protein signaling

	pathway > tachykinin signaling pathway

G protein linked receptor protein signaling

	pathway > G protein signaling, linked to cyclic nucleotide
	second messenger

FUNCTION

enzyme > 2-acetyl-1-alkylglycerophosphocholine

	esterase

1-phosphatidylinositol 3-kinase > 1-

	phosphatidylinositol 3-kinase regulator

transcription factor > RNA polymerase II

	transcription factor

	enzyme inhibitor > protein kinase inhibitor

	protein binding > lipoprotein binding

LOCATION

	cell > membrane fraction

	plasma membrane > integral plasma membrane

	protein

	cell > plasma membrane

	cytoplasm > endoplasmic reticulum

	cytoplasm > Golgi apparatus

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR000276 (GPCRRHODOPSN)

	IPR000276 (7tm 1)

	IPR000276 (G PROTEIN RECEP F1 2)

	IPR000276 (G PROTEIN RECEP F1 1)

hP13-026

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 147)

FAMILY (SUBFAMILY)

TRANSFORMING GROWTH FACTOR

	SUPERFAMILY MEMBER
	HUMAN GENE ONTOLOGY

PROCESS

	oocyte construction > axis determination

	transmembrane receptor protein serine/threonine

	kinase signaling pathway > TGFbeta receptor signaling pathway

	cell communication > cell—cell signaling

	GO biological process > developmental processes

	skeletal development > ossification

FUNCTION

	ligand binding or carrier > protein binding

LOCATION

	GO cellular component > extracellular

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR003942 (TGFBETA4)

	IPR001839 (TGFB)

	IPR001111 (TGFb propeptide)

	IPR001839 (TGF-beta)

	IPR001839 (TGF BETA 2)

	IPR001839 (TGF BETA)

	IPR001839 (sp 000292 TGF4 HUMAN)

hP13-028.1

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 153)

FAMILY (SUBFAMILY)

HEMATOPOIETIC PROGENITOR CELL

	ANTIGEN CD34 HUMAN GENE ONTOLOGY

PROCESS

	defence response > humoral defense mechanism

FUNCTION

	molecular_function unknown > lymphocyte antigen

LOCATION

	cell > plasma membrane

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

NULL (THR RICH)

hP13-028.2

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 155)

FAMILY (SUBFAMILY)

HEMATOPOIETIC PROGENITOR CELL

	ANTIGEN CD34 HUMAN GENE ONTOLOGY

PROCESS

	defence response > humoral defense mechanism

FUNCTION

	molecular_function unknown > lymphocyte antigen

LOCATION

	cell > plasma membrane

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	NULL (THR RICH)

hP13-028.3

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 157)

FAMILY (SUBFAMILY)

HEMATOPOIETIC PROGENITOR CELL

	ANTIGEN CD34 HUMAN GENE ONTOLOGY

PROCESS

	defence response > humoral defense mechanism

FUNCTION

	molecular_function unknown > lymphocyte antigen

LOCATION

	cell > plasma membrane

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	NULL (THR RICH)

	IPR001472 (NLS BP)

hP13-028.4

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 159)

FAMILY (SUBFAMILY)

HEMATOPOIETIC PROGENITOR CELL

	ANTIGEN CD34HUMAN GENE ONTOLOGY

PROCESS

	defence response > humoral defense mechanism

FUNCTION

	molecular function unknown > lymphocyte antigen

LOCATION

	cell > plasma membrane

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	NULL (THR RICH)

hP13-036

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 165)

FAMILY (SUBFAMILY)

CELL ADHESION MOLECULE-RELATED

	HUMAN GENE ONTOLOGY

PROCESS

	cell communication > cell adhesion

	ectoderm development > neurogenesis

	protein modification > protein dephosphorylation

	protein modification > protein phosphorylation

FUNCTION

	ligand binding or carrier > calcium binding

	GO molecular function > cell adhesion

	B cell receptor

	defense/immunity protein > immunoglobulin

	enzyme > protein kinase

	protein kinase > protein tyrosine kinase

LOCATION

plasma membrane > integral plasma membrane

	protein

	cell > plasma membrane

	cell > membrane fraction

	extracellular > extracellular matrix

	extracellular matrix > basement membrane

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR003596 (IGv)

	IPR000561 (EGF 2)

	IPR001881 (EGF CA)

	IPR001261 (ARGE DAPE CPG2 1)

	IPR001687 (ATP GTP A)

	IPR003598 (IGc2)

	IPR000561 (EGF)

	IPR003599 (IG)

	IPR000561 (EGF)

	IPR003006 (ig)

	IPR001881 (EGFCA 2 6)

	IPR000152 (ASX HYDROXYL)

hP13-060.1

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 171)

FAMILY (SUBFAMILY)

TRANSFORMING GROWTH FACTOR BETA-

	RELATED (BONE MORPHOGENETIC PROTEIN 7)

MOLECULAR FUNCTIONS

	Signaling molecule > Cytokine > Other cytokine

BIOLOGICAL PROCESS

Signal transduction > Cell surface receptor

	mediated signal transduction > Receptor protein
	serine/threonine kinase signaling pathway

	HUMAN GENE ONTOLOGY

PROCESS

	skeletal development > ossification

	mesoderm development > skeletal development

	transmembrane receptor protein serine/threonine kinase

	signaling pathway > TGFbeta receptor signaling pathway

	cell communication > cell—cell signaling

	gametogenesis > spermatogenesis

FUNCTION

	ligand binding or earner > protein binding

	GO molecular function > cell cycle regulator

	metalloendopeptidase > astacin

	enzyme > arginine decarboxylase

LOCATION

	GO cellular component > extracellular

	extracellular > extracellular space

	cell > membrane fraction

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR001839 (TGFB)

	IPR001111 (TGFb propeptide)

	IPR001839 (TGF-beta)

	IPR001839 (TGF BETA 2)

	IPR001839 (TGF BETA)

	IPR001839 (sp P18075 BMP7 HUMAN)

hP13-060.2

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 173)

FAMILY (SUBFAMILY)

TRANSFORMING GROWTH FACTOR BETA-RELATED

	(BONE MORPHOGENETIC PROTEIN 7)

MOLECULAR FUNCTIONS

	Signaling molecule > Cytokine > Other cytokine

BIOLOGICAL PROCESS

Signal transduction > Cell surface receptor

PROCESS

	skeletal development > ossification

	mesoderm development > skeletal development

	transmembrane receptor protein serine/threonine

	kinase signaling pathway > TGFbeta receptor signaling pathway

	cell communication > cell—cell signaling

	gametogenesis > spermatogenesis

FUNCTION

	ligand binding or carrier > protein binding

	GO molecular function > cell cycle regulator

	metalloendopeptidase > astacin

LOCATION

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR001839 (TGFB)

	IPR001111 (TGFb propeptide)

	IPR001839 (TGF-beta)

	IPR001839 (TGF BETA 2)

	IPR001839 (TGF BETA)

	IPR001839 (sp P23359 BMP7 MOUSE)

hP13-060.3

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 175)

FAMILY (SUBFAMILY)

TRANSFORMING GROWTH FACTOR BETA-RELATED

	(BONE MORPHOGENETIC PROTEIN 7)

MOLECULAR FUNCTIONS

	Signaling molecule > Cytokine > Other cytokine

BIOLOGICAL PROCESS

Signal transduction > Cell surface receptor

PROCESS

	signaling pathway > TGFbeta receptor signaling pathway

	cell communication > cell—cell signaling

	gametogenesis > spermatogenesis

FUNCTION

	ligand binding or carrier > protein binding

	GO molecular function > cell cycle regulator

	metalloendopeptidase > astacin

	enzyme > arginine decarboxylase

LOCATION

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

hP13-065.1

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 181)

FAMILY (SUBFAMILY)

GLUTAMATE RECEPTOR-RELATED

	(GLUTAMATE RECEPTOR 1)

MOLECULAR FUNCTIONS

	Receptor

	Ion channel > Ligand-gated ion channel >

	Glutamate receptor

BIOLOGICAL PROCESS

Signal transduction > Intracellular signaling

	cascade > Calcium mediated signaling

	Transport > Ion transport > Cation transport

	Neuronal activities > Synaptic transmission >

	Nerve—nerve synaptic transmission

HUMAN GENE ONTOLOGY

PROCESS

G protein linked receptor protein signaling

	pathway > glutamate signaling pathway

	cell—cell signaling > synaptic transmission

	cell growth and maintenance > transport

	cytoplasm organization and biogenesis > ribosome

	biogenesis

	transport > ion transport

FUNCTION

	GO molecular function > enzyme

LOCATION

	protein

	cytoplasm > synaptic vesicle

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR001508 (NMDARECEPTOR)

	IPR001320 (PBPe)

	IPR001828 (ANF receptor)

	IPR001320 (lig chan)

	IPR001311 (SBP GLUR)

	IPR001622 (CHANNEL PORE K)

hP13-065.2

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 183)

FAMILY (SUBFAMILY)

GLUTAMATE RECEPTOR-RELATED

	(GLUTAMATE RECEPTOR 1)

MOLECULAR FUNCTIONS

	Receptor

	Ion channel > Ligand-gated ion channel > Glutamate

	receptor

BIOLOGICAL PROCESS

Signal transduction > Intracellular signaling

	cascade > Calcium mediated signaling

	Transport > Ion transport > Cation transport

	Neuronal activities > Synaptic transmission >

	Nerve—nerve synaptic transmission

	HUMAN GENE ONTOLOGY

PROCESS

G protein linked receptor protein signaling

	pathway > glutamate signaling pathway

	biogenesis

	transport > ion transport

FUNCTION

	GO molecular function > enzyme

LOCATION

	protein

	cytoplasm > synaptic vesicle

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

hP13-065.3

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 185)

FAMILY (SUBFAMILY)

GLUTAMATE RECEPTOR-RELATED

	(GLUTAMATE RECEPTOR 1)

MOLECULAR FUNCTIONS

	Receptor

	Ion channel > Ligand-gated ion channel > Glutamate

	receptor

BIOLOGICAL PROCESS

Signal transduction > Intracellular signaling

	cascade > Calcium mediated signaling

	Nerve—nerve synaptic transmission

	HUMAN GENE ONTOLOGY

PROCESS

G protein linked receptor protein signaling

	pathway > glutamate signaling pathway

	biogenesis

	transport > ion transport

FUNCTION

	GO molecular function > enzyme

LOCATION

	protein

	cytoplasm > synaptic vesicle

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

hP14-032

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 191)

FAMILY (SUBFAMILY)

CARCINOEMBRYONIC ANTIGEN

	(CARCINOEMBRYONIC ANTIGEN)

MOLECULAR FUNCTIONS

Cell adhesion molecule > CAM family adhesion

	molecule

BIOLOGICAL PROCESS

	Biological process unclassified

HUMAN GENE ONTOLOGY

PROCESS

	defence response > immune response

	protein-membrane targeting > post-translational

	membrane targeting

	cell communication > cell adhesion

	cell communication > signal transduction

	cell communication > cell—cell signaling

FUNCTION

	molecular_function unknown > tumor antigen

	B cell receptor

	defense/immunity protein > immunoglobulin

	GO molecular function > cell adhesion

	protein kinase > protein tyrosine kinase

	calmodulin regulated protein kinase > myosin

	light chain kinase

LOCATION

	extracellular > extracellular space

	plasma membrane > integral plasma membrane

	protein

	cell > membrane fraction

	cell > plasma membrane

	plasma membrane > peripheral plasma membrane

	protein

	HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR003598 (IGc2)

	IPR003599 (IG)

	IPR003006 (ig)

hP14-033

HUMAN PANTHER CLASSIFICATIONS

(SEQ ID NO: 197)

FAMILY (SUBFAMILY)

BETA-AMYLOID PRECURSOR PROTEIN-RELATED

	(ALZHEIMER' S DISEASE AMYLOID A4
	PROTEIN-RELATED)

MOLECULAR FUNCTIONS

	Receptor > Other receptor

BIOLOGICAL PROCESS

Signal transduction > Cell communication >

	Ligand-mediated signaling

Signal transduction > Cell surface receptor

	mediated signal transduction > G-protein mediated signaling

Neuronal activities > Synaptic transmission >

	Neurotransmitter release

	Apoptosis > Induction of apoptosis

	Cell structure and motility > Cell structure

HUMAN GENE ONTOLOGY

PROCESS

metal ion homeostasis > copper homeostasis

	cell death > apoptosis

	cell communication > signal transduction

	mating (sensu Saccharomyces) > pheromone
	response

	cell growth and maintenance > cell death

FUNCTION

	proteinase inhibitor > serine protease inhibitor

	nucleic acid binding > DNA binding

	enzyme inhibitor > proteinase inhibitor

	GO molecular function > cell adhesion

	GO molecular function > ligand binding or carrier

LOCATION

	cell > membrane fraction

	cytoplasm > Golgi apparatus

	extracellular > extracellular space

	cytoplasm > endoplasmic reticulum

	GO cellular component > extracellular

HUMAN PROTEIN DOMAINS (INTERPRO SIGNATURES)

	IPR002223 (BASIC PTASE)

	IPR001868 (A4 INTRA)

	IPR001868 (A4 EXTRA)

	IPR002223 (BPTI KUNITZ 1)

	IPR001868 (AMYLOIDA4)

	IPR001255 (BETAAMYLOID)

	IPR002223 (KU)

	IPR001868 (A4 EXTRA)

	IPR001868 (A4 EXTRA)

	IPR002223 (Kunitz BPTI)

	NULL (GLU RICH)

	IPR002223 (BPTI KUNITZ 2)

A CA protein (CAP) is a calcium binding protein wherein the CAP sequence is SEQ ID NOS: 6, 34, 113 and 165.[0321]

A CA protein (CAP) is a G-protein coupled receptor antagonist wherein the CAP sequence is SEQ ID NOS: 141, 181, 197.[0322]

A CA protein (CAP) is a tyrosine phosphatase wherein the CAP sequence is selected from the group consisting of SEQ ID NOS: 119 and 121.[0323]

A CA protein (CAP) is an amino acid transport protein wherein the CAP sequence is selected from the group consisting of SEQ ID NOS: 58, 60 and 62.[0324]

A CA protein (CAP) is an apoptosis-related protein wherein the CAP sequence is selected from the group consisting of SEQ ID NOS: 18, 20, 22 and 28.[0325]

A CA protein (CAP) is involved in signalling wherein the CAP sequence is selected from the group consisting of SEQ ID NOS: 34, 40, 79, 119, 121, 127, 141, 147, 171, 173, 175, 181, 183, 185, 191 and 197.[0326]

A CA protein (CAP) is a cell adhesion molecule wherein the CAP sequence is selected from the group consisting of SEQ ID NOS:12, 52, 76, 85, 87, 89, 95, 101, 107, 113, 135 and 165.[0327]

A CA protein (CAP) is a tyrosine kinase wherein the CAP sequence is selected from the group consisting of SEQ ID NOS: 165 and 191.[0328]

A CA protein (CAP) is expressed on a cell surface, wherein the CA protein is selected from the group consisting of SEQ ID NOS: 6, 12, 18, 20, 22, 28, 34, 40, 52, 58, 60, 62, 68, 70, 76, 79, 85, 87, 89, 95, 101, 107, 113, 119, 121, 127, 135, 141, 147, 153, 155, 157, 159, 165, 171, 173, 175, 181, 183, 185, 191, 197 and 203.[0329]

Certain aspects of the present invention are described in greater detail in the non-limiting examples that follow.[0330]

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all and only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.[0331]

Example 1Insertion Site Analysis Following Tumor Induction in Mice

Tumors are induced in mice using either mouse mammary tumor virus (MMTV) or murine leukemia virus (MLV). MMTV causes mammary adenocarcinomas and MLV causes a variety of different hematopoetic malignancies (primarily T- or B-cell lymphomas). Three routes of infection are used: (1) injection of neonates with purified virus preparations, (2) infection by milk-borne virus during nursing, and (3) genetic transmission of pathogenic proviruses via the germ-line (Akvr1 and/or Mtv2). The type of malignancy present in each affected mouse is determined by histological analysis of H&E-stained thin sections of formalin-fixed, paraffin-embedded biopsy samples. Host DNA sequences flanking all clonally-integrated proviruses in each tumor are recovered by nested anchored-PCR using two virus-specific primers and two primers specific for a 40 bp double stranded DNA anchor ligated to restriction enzyme digested tumor DNA. Amplified bands representing host/virus junction fragments are cloned and sequenced. Then the host sequences (called “tags”) are used to BLAST analyze the Celera mouse genomic sequence. For each individual tag, three parameters are recorded: (1) the mouse chromosome assignment, (2) base pair coordinates at which the integration occurred, and (3) provirus orientation. Using this information, all available tags from all analyzed tumors are mapped to the mouse genome. To identify the protooncogene targets of provirus insertion mutation, the provirus integration pattern at each cluster of integrants is analyzed relative to the locations of all known genes in the transcriptome. The presence of provirus at the same locus in two or more independent tumors is prima facie evidence that a protooncogene is present at or very near the proviral integration sites. This is because the genome is too large for random integrations to result in observable clustering. Any clustering that is detected is unequivocal evidence for biological selection during tumorigenesis. In order to identify the human orthologs of the protooncogene targets of provirus insertion mutation, a comparative analysis of syntenic regions of the mouse and human genomes is performed.[0332]

An example of PCR amplification of host/virus junction fragments is presented in FIG. 1. Lane 1 contains the amplification products from normal control DNA and[0333]lane 2 contains the amplification products from tumor DNA. The bands result from 5′ host/virus junction fragments present in the DNA samples.Lane 1 has bands from the env/3′ LTR junctions from all proviruses (upper) and the host/5′ LTR from the pathogenic endogenous Mtv2 provirus present in this particular mouse strain. This endogenous provirus is detected because its sequence is identical to the new clonally integrated proviruses in the tumor. All four new clonally integrated proviruses known to be in this tumor are readily detected.

Example 2Analysis of Quantitative RT-PCR: Comparative C_TMethod

The expression level of target genes is quantified using the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, California). The method is based on the quantitation of the initial copy number of target template in comparison to that of a reference (normalizer) housekeeper gene (Pre-Developed TaqMan® Assay Reagents Gene Expression Quantification Protocol, Applied Biosystems, 2001). Accumulation of DNA product with each PCR cycle is related to amplicon efficiency and the initial template concentration. Therefore the amplification efficiency of both the target and the normalizer must be approximately equal. The threshold cycle (C[0334]_T), which is dependent on the starting template copy number and the DNA amplification efficiency, is a PCR cycle during which PCR product growth is exponential. With a similar dynamic range for the target and normalizer, the comparative C_Tmethod is applicable.

An example of the comparative C[0335]_Tmethod of gene expression for quantitative RT-PCR is shown in FIG. 2. In the first step, assays are performed in quadruplicate on a normal tissue and several sample tissues. In these tissues, the means and standard deviations of C_Tvalues are determined for housekeeper genes (chosen as controls if shown to be biologically stable among various samples, irrespective of disease state) and for the target gene. FIG. 2 shows an example of average C_Tvalues for a housekeeper gene and target gene. These values can fall within a range from upper teens to 40 depending on the intrinsic expression level of the gene in the particular tissue. The coefficient of variance of all replicate sets cannot exceed 1.5%.

An assessment of how the ΔC[0336]_Tchanges with template dilution verifies that the efficiencies of the target and housekeeper amplicons are approximately equal if the log input amount of template RNA versus ΔC_Tplot has a slope <0.10. With the relative efficiencies verified for target and housekeeper, the ΔΔC_Tcomparative calculation becomes valid, as mentioned above. An example of the calculated difference between the C_Tvalues of target and housekeeper genes (ΔC_T) for various samples is shown in FIG. 3. The ΔΔC_Tis calculated for each sample by subtracting its ΔC_Tvalue from the ΔC_Tvalue of the baseline (calibrator) sample. If the expression is increased in some samples and decreased in others, ΔΔC_Twill be a mixture of negative and positive values. The final step in the calculation is to transform these values to absolute values. The formula for this is:

Comparative expression level=2^−ΔΔCT

The final value for the calibrator should always be one. FIG. 4 shows the ΔΔC[0337]_Tand comparative expression level for each sample from FIG. 3.

Example 3Detection of Elevated Levels of cDNA Associated with Cancer Using Arrays

cDNA sequences representing a variety of candidate CA genes to be screened for differential expression in cancer are assayed by hybridization on polynucleotide arrays. The cDNA sequences include cDNA clones isolated from cell lines or tissues of interest. The cDNA sequences analyzed also include polynucleotides comprising sequence overlap with sequences in the Unigene database, and which encode a variety of gene products of various origins, functionality, and levels of characterization. cDNAs are spotted onto reflective slides (Amersham) according to methods well known in the art at a density of 9,216 spots per slide representing 4,068 sequences (including controls) spotted in duplicate, with approximately 0.8 μl of an approximately 200 ng/μl solution of cDNA.[0338]

PCR products of selected cDNA clones corresponding to the gene products of interest are prepared in a 50% DMSO solution. These PCR products are spotted onto Amersham aluminum microarray slides at a density of 9216 clones per array using a Molecular Dynamics Generation III spotting robot. Clones are spotted in duplicate, for a total of 4608 different sequences per chip.[0339]

cDNA probes are prepared from total RNA obtained by laser capture microdissection (LCM, Arcturus Enginering Inc., Mountain View, Calif.) of tumor tissue samples and normal tissue samples isolated from patients.[0340]

Total RNA is first reverse transcribed into cDNA using a primer containing a T7 RNA polymerase promoter, followed by second strand DNA synthesis. cDNA is then transcribed in vitro to produce antisense RNA using the T7 promoter-mediated expression (see, e.g., Luo et al. (1999)[0341]Nature Med5:117-122), and the antisense RNA is then converted into cDNA. The second set of cDNAs are again transcribed in vitro, using the T7 promoter, to provide antisense RNA. This antisense RNA is then fluorescently labeled, or the RNA is again converted into cDNA, allowing for a third round of T7-mediated amplification to produce more antisense RNA. Thus the procedure provides for two or three rounds of in vitro transcription to produce the final RNA used for fluorescent labeling. Probes are labeled by making fluorescently labeled cDNA from the RNA starting material. Fluorescently labeled cDNAs prepared from the tumor RNA sample are compared to fluorescently labeled cDNAs prepared from normal cell RNA sample. For example, the cDNA probes from the normal cells are labeled with Cy3 fluorescent dye (green) and the cDNA probes prepared from suspected cancer cells are labeled with Cy5 fluorescent dye (red).

The differential expression assay is performed by mixing equal amounts of probes from tumor cells and normal cells of the same patient. The arrays are prehybridized by incubation for about 2 hrs at 60° C. in 5×SSC, 0.2% SDS, 1 mM EDTA, and then washing three times in water and twice in isopropanol. Following prehybridization of the array, the probe mixture is then hybridized to the array under conditions of high stringency (overnight at 42° C. in 50% formamide, 5×SSC, and 0.2% SDS. After hybridization, the array is washed at 55° C. three times as follows: 1) first wash in 1×SSC/0.2% SDS; 2) second wash in 0.1×SSC/0.2% SDS; and 3) third wash in 0.1×SSC.[0342]

The arrays are then scanned for green and red fluorescence using a Molecular Dynamics Generation III dual color laser-scanner/detector. The images are processed using BioDiscovery Autogene software, and the data from each scan set normalized. The experiment is repeated, this time labeling the two probes with the opposite color in order to perform the assay in both “color directions.” Each experiment is sometimes repeated with two more slides (one in each color direction). The data from each scan is normalized, and the level of fluorescence for each sequence on the array expressed as a ratio of the geometric mean of 8 replicate spots/genes from the four arrays or 4 replicate spots/gene from 2 arrays or some other permutation.[0343]

Normalization: The objective of normalization is to generate a cDNA library in which all transcripts expressed in a particular cell type or tissue are equally represented (S. M. Weissman, Mol. Biol. Med. 4(3):133-143 (1987); Patanjali, et al., Proc. Natl. Acad. Sci. USA 88(5):1943-1947 (1991)), and therefore isolation of as few as 30,000 recombinant clones in an optimally normalized library may represent the entire gene expression repertoire of a cell, estimated to number 10,000 per cell.[0344]

Total RNA is extracted from harvested cells using RNeasy™ Protect Kit (Qiagen, Valencia, Calif.), following manufacturer's recommended procedures. RNA is quantified using RiboGreen™ RNA quantification kit (Molecular Probes, Inc. Eugene, Oreg.). One μg of total RNA is reverse transcribed and PCR amplified using SMART™ PCR cDNA synthesis kit (ClonTech, Palo Alto, Calif.). The cDNA products are size-selected by agarose gel electrophoresis using standard procedures (Sambrook, J. T., et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, NY). The cDNA is extracted using Bio 101 Geneclean® II kit (Qbiogene, Carlsbad, Calif.). Normalization of the cDNA is carried out using kinetics of hybridization principles: 1.0 μg of cDNA is denatured by heat at 100° C. for 10 minutes, then incubated at 42° C. for 42 hours in the presence of 120 mM NaCl, 10 mM Tris.HCl (pH=8.0), 5 mM EDTA.Na+ and 50% formamide. Single-stranded cDNA (“normalized”) is purified by hydroxyapatite chromatography (#130-0520, BioRad, Hercules, Calif.) following the manufacturer's recommended procedures, amplified and converted to double-stranded cDNA by three cycles of PCR amplification, and cloned into plasmid vectors using standard procedures (Sambrook, J. T., et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, NY). All primers/adaptors used in the normalization and cloning process are provided by the manufacturer in the SMART™ PCR cDNA synthesis kit (ClonTech, Palo Alto, Calif.). Supercompetent cells (XL-2 Blue Ultracompetent Cells, Stratagene, Calif.) are transfected with the normalized cDNA libraries, plated on solid media and grown overnight at 36° C.[0345]

The sequences of 10,000 recombinants per normalized library are analyzed by capillary sequencing using the ABI PRISM 3700 DNA Analyzer (Applied Biosystems, California). To determine the representation of transcripts in a library, BLAST analysis is performed on the clone sequences to assign transcript identity to each isolated clone, i.e., the sequences of the isolated polynucleotides are first masked to eliminate low complexity sequences using the XBLAST masking program (Clayerie “Effective Large-Scale Sequence Similarity Searches,”[0346]Computer Methods for Macromolecular Sequence Analysis, Doolittle, ed.,Meth. Enzymol.266:212-227 Academic Press, NY, N.Y. (1996); see particularly Clayerie, in “Automated DNA Sequencing and Analysis Techniques” Adams et al., eds., Chap. 36, p. 267 Academic Press, San Diego, 1994 and Clayerie et al.Comput. Chem. (1993) 17:191). Generally, masking does not influence the final search results, except to eliminate sequences of relative little interest due to their low complexity, and to eliminate multiple “hits” based on similarity to repetitive regions common to multiple sequences, e.g., Alu repeats. The remaining sequences are then used in a BLASTN vs. GenBank search. The sequences are also used as query sequence in a BLASTX vs. NRP (non-redundant proteins) database search.

Automated sequencing reactions are performed using a Perkin-Elmer PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit containing AmpliTaq DNA Polymerase, FS, according to the manufacturer's directions. The reactions are cycled on a GeneAmp PCR System 9600 as per manufacturer's instructions, except that they are annealed at 20° C. or 30° C. for one minute. Sequencing reactions are ethanol precipitated, pellets are resuspended in 8 microliters of loading buffer, 1.5 microliters is loaded on a sequencing gel, and the data is collected by an ABI PRISM 3700 DNA Sequencer. (Applied Biosystems, Foster City, Calif.).[0347]

The number of times a sequence is represented in a library is determined by performing sequence identity analysis on the cloned cDNA sequences and assigning transcript identity to each isolated clone. First, each sequence is checked to determine if it is a bacterial, ribosomal, or mitochondrial contaminant. Such sequences are excluded from the subsequent analysis. Second, sequence artifacts, such as vector and repetitive elements, are masked and/or removed from each sequence.[0348]

The remaining sequences are compared via BLAST (Altschul et. al, J. Mol. Biol., 215:40, 1990) to GenBank and EST databases for gene identification and are compared with each other via FastA (Pearson & Lipman, PNAS, 85:2444, 1988) to calculate the frequency of cDNA appearance in the normalized cDNA library. The sequences are also searched against the GenBank and GeneSeq nucleotide databases using the BLASTN program (BLASTN 1.3 MP: Altschul et al., J. Mol. Bio. 215:403, 1990). Fourth, the sequences are analyzed against a non-redundant protein (NRP) database with the BLASTX program (BLASTX 1.3 MP: Altschul et al., supra). This protein database is a combination of the Swiss-Prot, PIR, and NCBI GenPept protein databases. The BLASTX program is run using the default BLOSUM-62 substitution matrix with the filter parameter: “xnu+seg”. The score cutoff utilized is 75. Assembly of overlapping clones into contigs is done using the program Sequencher (Gene Codes Corp.; Ann Arbor, Mich.). The assembled contigs are analyzed using the programs in the GCG package (Genetic Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711) Suite Version 10.1.[0349]

Example 4Detection of CA-Sequences in Human Cancer Cells and Tissues

DNA from prostate and breast cancer tissues and other human cancer tissues, human colon, normal human tissues including non-cancerous prostate, and from other human cell lines are extracted following the procedure of Delli Bovi et al. (1986, Cancer Res. 46:6333-6338). The DNA is resuspended in a solution containing 0.05 M Tris HCl buffer, pH 7.8, and 0.1 mM EDTA, and the amount of DNA recovered is determined by microfluorometry using Hoechst 33258 dye. Cesarone, C. et al., Anal Biochem 100:188-197 (1979).[0350]

Polymerase chain reaction (PCR) is performed using Taq polymerase following the conditions recommended by the manufacturer (Perkin Elmer Cetus) with regard to buffer, Mg[0351]²⁺, and nucleotide concentrations. Thermocycling is performed in a DNA cycler by denaturation at 94° C. for 3 min. followed by either 35 or 50 cycles of 94° C. for 1.5 min., 50° C. for 2 min. and 72° C. for 3 min. The ability of the PCR to amplify the selected regions of the CA gene is tested by using a cloned CA polynucleotide(s) as a positive template(s). Optimal Mg²⁺, primer concentrations and requirements for the different cycling temperatures are determined with these templates. The master mix recommended by the manufacturer is used. To detect possible contamination of the master mix components, reactions without template are routinely tested.

Southern blotting and hybridization are performed as described by Southern, E. M., (J. Mol. Biol. 98:503-517, 1975), using the cloned sequences labeled by the random primer procedure (Feinberg, A. P., et al., 1983, Anal. Biochem. 132:6-13). Prehybridization and hybridization are performed in a solution containing 6×SSPE, 5% Denhardt's, 0.5% SDS, 50% formamide, 100 μg/ml denaturated salmon testis DNA, incubated for 18 hrs at 42° C., followed by washings with 2×SSC and 0.5% SDS at room temperature and at 37° C. and finally in 0.1×SSC with 0.5% SDS at 68° C. for 30 min (Sambrook et al., 1989, in “Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor Lab. Press). For paraffin-embedded tissue sections the conditions described by Wright and Manos (1990, in “PCR Protocols”, Innis et al., eds., Academic Press, pp. 153-158) are followed using primers designed to detect a 250 bp sequence.[0352]

Example 5Detection of CA Sequences in Human Cancer Cells and Tissues

DNA from human cancer tissues, normal human tissues and from other human cell lines is extracted following the procedure of Delli Bovi et al. (1986, Cancer Res. 46:6333-6338). The DNA is resuspended in a solution containing 0.05 M Tris HCl buffer, pH 7.8, and 0.1 mM EDTA, and the amount of DNA recovered is determined by microfluorometry using Hoechst 33258 dye. Cesarone, C. et al., Anal Biochem 100:188-197 (1979).[0353]

Polymerase chain reaction (PCR) is performed using Taq polymerase following the conditions recommended by the manufacturer (Perkin Elmer Cetus) with regard to buffer, Mg[0354]²⁺, and nucleotide concentrations. Thermocycling is performed in a DNA cycler by denaturation at 94° C. for 3 min. followed by either 35 or 50 cycles of 94° C. for 1.5 min., 50° C. for 2 min. and 72° C. for 3 min. The ability of the PCR to amplify the selected regions of CA genes is tested by using a cloned CA polynucleotide(s) as a positive template(s). Optimal Mg²⁺, primer concentrations and requirements for the different cycling temperatures are determined with these templates. The master mix recommended by the manufacturer is used. To detect possible contamination of the master mix components, reactions without template are routinely tested.

Southern blotting and hybridization are performed as described by Southern, E. M., (J. Mol. Biol. 98:503-517, 1975), using the cloned sequences labeled by the random primer procedure (Feinberg, A. P., et al., 1983, Anal. Biochem. 132:6-13). Prehybridization and hybridization are performed in a solution containing 6×SSPE, 5% Denhardt's, 0.5% SDS, 50% formamide, 100 μg/ml denaturated salmon testis DNA, incubated for 18 hrs at 42° C., followed by washings with 2×SSC and 0.5% SDS at room temperature and at 37° C. and finally in 0.1×SSC with 0.5% SDS at 68° C. for 30 min (Sambrook et al., 1989, in “Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor Lab. Press). For paraffin-embedded tissue sections the conditions described by Wright and Manos (1990, in “PCR Protocols”, Innis et al., eds., Academic Press, pp. 153-158) are followed using primers designed to detect a 250 bp sequence.[0355]

Example 6Expression of Cloned Polynucleotides in Host Cells

To study the protein products of CA genes, restriction fragments from CA DNA are cloned into the expression vector pMT2 (Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press pp 16.17-16.22 (1989)) and transfected into COS cells grown in DMEM supplemented with 10% FCS. Transfections are performed employing calcium phosphate techniques (Sambrook, et al (1989) pp. 16.32-16.40, supra) and cell lysates are prepared forty-eight hours after transfection from both transfected and untransfected COS cells. Lysates are subjected to analysis by immunoblotting using anti-peptide antibody.[0356]

In immunoblotting experiments, preparation of cell lysates and electrophoresis are performed according to standard procedures. Protein concentration is determined using BioRad protein assay solutions. After semi-dry electrophoretic transfer to nitrocellulose, the membranes are blocked in 500 mM NaCl, 20 mM Tris, pH 7.5, 0.05% Tween-20 (TTBS) with 5% dry milk. After washing in TTBS and incubation with secondary antibodies (Amersham), enhanced chemiluminescence (ECL) protocols (Amersham) are performed as described by the manufacturer to facilitate detection.[0357]

Example 7Generation of Antibodies Against Polypeptides

Polypeptides, unique to CA genes are synthesized or isolated from bacterial or other (e.g., yeast, baculovirus) expression systems and conjugated to rabbit serum albumin (RSA) with m-maleimido benzoic acid N-hydroxysuccinimide ester (MBS) (Pierce, Rockford, Ill.). Immunization protocols with these peptides are performed according to standard methods. Initially, a pre-bleed of the rabbits is performed prior to immunization. The first immunization includes Freund's complete adjuvant and 500 μg conjugated peptide or 100 μg purified peptide. All subsequent immunizations, performed four weeks after the previous injection, include Freund's incomplete adjuvant with the same amount of protein. Bleeds are conducted seven to ten days after the immunizations.[0358]

For affinity purification of the antibodies, the corresponding CA polypeptide is conjugated to RSA with MBS, and coupled to CNBr-activated Sepharose (Pharmacia, Uppsala, Sweden). Antiserum is diluted 10-fold in 10 mM Tris-HCl, pH 7.5, and incubated overnight with the affinity matrix. After washing, bound antibodies are eluted from the resin with 100 mM glycine, pH 2.5.[0359]

Example 8Generation of Monoclonal Antibodies Against a CA Polypeptide

A non-denaturing adjuvant (Ribi, R730, Corixa, Hamilton Mont.) is rehydrated to 4 ml in phosphate buffered saline. 100 μl of this rehydrated adjuvant is then diluted with 400 μl of Hank's Balanced Salt Solution and this is then gently mixed with the cell pellet used for immunization. Approximately 500 μg conjugated peptide or 100 μg purified peptide and Freund's complete are injected into Balb/c mice via foot-pad, once a week. After 6 weeks of weekly injection, a drop of blood is drawn from the tail of each immunized animal to test the titer of antibodies against CA polypeptides using FACS analysis. When the titer reaches at least 1:2000, the mice are sacrificed in a CO[0360]₂chamber followed by cervical dislocation. Lymph nodes are harvested for hybridoma preparation. Lymphocytes from mice with the highest titer are fused with the mouse myeloma line X63-Ag8.653 using 35% polyethylene glycol 4000. Onday 10 following the fusion, the hybridoma supernatants are screened for the presence of CAP-specific monoclonal antibodies by fluorescence activated cell sorting (FACS). Conditioned medium from each hybridoma is incubated for 30 minutes with a combined aliquot of PC3, Colo-205, LnCap, or Panc-1 cells. After incubation, the cell samples are washed, resuspended in 0.1 ml diluent and incubated with 1 μg/ml of FITC conjugated F(ab′)₂fragment of goat anti-mouse IgG for 30 min at 4° C. The cells are washed, resuspended in 0.5 ml FACS diluent and analyzed using a FACScan cell analyzer (Becton Dickinson; San Jose, Calif.). Hybridoma clones are selected for further expansion, cloning, and characterization based on their binding to the surface of one or more of cell lines which express the CA polypeptide as assessed by FACS. A hybridoma making a monoclonal antibody designated mAbCA which binds an antigen designated Ag-CA.x and an epitope on that antigen designated Ag-CA.x. 1 is selected.

Example 9ELISA Assay for Detecting CA Related Antigens

To test blood samples for antibodies that bind specifically to recombinantly produced CA antigens, the following procedure is employed. After a recombinant CA related protein is purified, the recombinant protein is diluted in PBS to a concentration of 5 μg/ml (500 ng/100 μl). 100 microliters of the diluted antigen solution is added to each well of a 96-[0361]well Immulon 1 plate (Dynatech Laboratories, Chantilly, Va.), and the plate is then incubated for 1 hour at room temperature, or overnight at 4° C., and washed 3 times with 0.05% Tween 20 in PBS. Blocking to reduce nonspecific binding of antibodies is accomplished by adding to each well 200 μl of a 1% solution of bovine serum albumin in PBS/Tween 20 and incubation for 1 hour. After aspiration of the blocking solution, 100 μl of the primary antibody solution (anticoagulated whole blood, plasma, or serum), diluted in the range of {fraction (1/16)} to {fraction (1/2048)} in blocking solution, is added and incubated for 1 hour at room temperature or overnight at 4° C. The wells are then washed 3 times, and 100 μl of goat anti-human IgG antibody conjugated to horseradish peroxidase (Organon Teknika, Durham, N.C.), diluted{fraction (1/500)} or {fraction (1/1000)} in PBS/Tween 20, 100 μl of o-phenylenediamine dihydrochloride (OPD, Sigma) solution is added to each well and incubated for 5-15 minutes. The OPD solution is prepared by dissolving a 5 mg OPD tablet in 50ml 1% methanol in H₂O and adding 50 μl 30% H₂O₂immediately before use. The reaction is stopped by adding 25 l of 4M H₂SO₄. Absorbances are read at 490 nm in a microplate reader (Bio-Rad).

Example 10Identification and Characterization of CA Antigen on Cancer Cell Surface

A cell pellet of proximately 25 ul packed cell volume of a cancer cell preparation is lysed by first diluting the cells to 0.5 ml in water followed by freezing and thawing three times. The solution is centrifuged at 14,000 rpm. The resulting pellet, containing the cell membrane fragments, is resuspended in 50 μl of SDS sample buffer (Invitrogen, Carlsbad, Calif.). The sample is heated at 80° C. for 5 minutes and then centrifuged for 2 minutes at 14,000 rpm to remove any insoluble materials.[0362]

The samples are analyzed by Western blot using a 4 to 20% polyacrylamide gradient gel in Tris-Glycine SDS (Invitrogen; Carlsbad Calif.) following the manufacturer's directions. Ten microliters of membrane sample are applied to one lane on the polyacrylamide gel. A separate 10 μL sample is reduced first by the addition of 2 μL of dithiothreitol (100 mM) with heating at 80° C. for 2 minutes and then loaded into another lane. Pre-stained molecular weight markers See Blue Plus2 (Invitrogen; Carlsbad, Calif.) are used to assess molecular weight on the gel. The gel proteins are transferred to a nitrocellulose membrane using a transfer buffer of 14.4 g/l glycine, 3 g/l of Tris Base, 10% methanol, and 0.05% SDS. The membranes are blocked, probed with a CAP-specific monoclonal antibody (at a concentration of 0.5 ug/ml), and developed using the Invitrogen WesternBreeze Chromogenic Kit-AntiMouse according to the manufacturer's directions. In the reduced sample of the tumor cell membrane samples, a prominent band is observed migrating at a molecular weight within about 10% of the predicted molecular weight of the corresponding CA protein.[0363]

Example 11Preparation of Vaccines

The present invention also relates to a method of stimulating an immune response against cells that express CA polypeptides in a patient using CA polypeptides of the invention that act as an antigen produced by or associated with a malignant cell. This aspect of the invention provides a method of stimulating an immune response in a human against cancer cells or cells that express CA polynucleotides and polypeptides. The method comprises the step of administering to a human an immunogenic amount of a polypeptide comprising: (a) the amino acid sequence of a huma CA protein or (b) a mutein or variant of a polypeptide comprising the amino acid sequence of a human endogenous retrovirus CA protein.[0364]

Example 12Generation of Transgenic Animals Expressing Polypeptides as a Means for Testing Therapeutics

CA nucleic acids are used to generate genetically modified non-human animals, or site specific gene modifications thereof, in cell lines, for the study of function or regulation of prostate tumor-related genes, or to create animal models of diseases, including prostate cancer. The term “transgenic” is intended to encompass genetically modified animals having an exogenous CA gene(s) that is stably transmitted in the host cells where the gene(s) may be altered in sequence to produce a modified protein, or having an exogenous CA LTR promoter operably linked to a reporter gene. Transgenic animals may be made through a nucleic acid construct randomly integrated into the genome. Vectors for stable integration include plasmids, retroviruses and other animal viruses, YACs, and the like. Of interest are transgenic mammals, e.g. cows, pigs, goats, horses, etc., and particularly rodents, e.g. rats, mice, etc.[0365]

The modified cells or animals are useful in the study of CA gene function and regulation. For example, a series of small deletions and/or substitutions may be made in the CA genes to determine the role of different genes in tumorigenesis. Specific constructs of interest include, but are not limited to, antisense constructs to block CA gene expression, expression of dominant negative CA gene mutations, and over-expression of a CA gene. Expression of a CA gene or variants thereof in cells or tissues where it is not normally expressed or at abnormal times of development is provided. In addition, by providing expression of proteins derived from CA in cells in which it is otherwise not normally produced, changes in cellular behavior can be induced.[0366]

DNA constructs for random integration need not include regions of homology to mediate recombination. Conveniently, markers for positive and negative selection are included. For various techniques for transfecting mammalian cells, see Keown et al., Methods in Enzymology 185:527-537 (1990).[0367]

For embryonic stem (ES) cells, an ES cell line is employed, or embryonic cells are obtained freshly from a host, e.g. mouse, rat, guinea pig, etc. Such cells are grown on an appropriate fibroblast-feeder layer or grown in the presence of appropriate growth factors, such as leukemia inhibiting factor (LIF). When ES cells are transformed, they may be used to produce transgenic animals. After transformation, the cells are plated onto a feeder layer in an appropriate medium. Cells containing the construct may be detected by employing a selective medium. After sufficient time for colonies to grow, they are picked and analyzed for the occurrence of integration of the construct. Those colonies that are positive may then be used for embryo manipulation and blastocyst injection. Blastocysts are obtained from 4 to 6-week old superovulated females. The ES cells are trypsinized, and the modified cells are injected into the blastocoel of the blastocyst. After injection, the blastocysts are returned to each uterine horn of pseudopregnant females. Females are then allowed to go to term and the resulting chimeric animals screened for cells bearing the construct. By providing for a different phenotype of the blastocyst and the ES cells, chimeric progeny can be readily detected.[0368]

The chimeric animals are screened for the presence of the modified gene and males and females having the modification are mated to produce homozygous progeny. If the gene alterations cause lethality at some point in development, tissues or organs are maintained as allogeneic or congenic grafts or transplants, or in in vitro culture. The transgenic animals may be any non-human mammal, such as laboratory animals, domestic animals, etc. The transgenic animals are used in functional studies, drug screening, etc., e.g. to determine the effect of a candidate drug on prostate cancer, to test potential therapeutics or treatment regimens, etc.[0369]

Example 13Diagnostic Imaging Using CA Specific Antibodies

The present invention encompasses the use of antibodies to CA polypeptides to accurately stage cancer patients at initial presentation and for early detection of metastatic spread of cancer. Radioimmunoscintigraphy using monoclonal antibodies specific for CA polypeptides can provide an additional cancer-specific diagnostic test. The monoclonal antibodies of the instant invention are used for histopathological diagnosis of carcinomas.[0370]

Subcutaneous human xenografts of cancer cells in nude mice is used to test whether a technetium-99m ([0371]^99mTc)-labeled monoclonal antibody of the invention can successfully image the xenografted cancer by external gamma scintography as described for seminoma cells by Marks, et al., Brit. J. Urol. 75:225 (1995). Each monoclonal antibody specific for a CA polypeptide is purified from ascitic fluid of BALB/c mice bearing hybridoma tumors by affinity chromatography on protein A-Sepharose. Purified antibodies, including control monoclonal antibodies such as an avidin-specific monoclonal antibody (Skea, et al., J. Immunol. 151:3557 (1993)) are labeled with^99mTc following reduction, using the methods of Mather, et al., J. Nucl. Med. 31:692 (1990) and Zhang et al., Nucl. Med. Biol. 19:607 (1992). Nude mice bearing human cancer cells are injected intraperitoneally with 200-500 μCi of^99mTc-labeled antibody. Twenty-four hours after injection, images of the mice are obtained using a Siemens ZLC3700 gamma camera equipped with a 6 mm pinhole collimator set approximately 8 cm from the animal. To determine monoclonal antibody biodistribution following imaging, the normal organs and tumors are removed, weighed, and the radioactivity of the tissues and a sample of the injectate are measured. Additionally, CA-specific antibodies conjugated to antitumor compounds are used for cancer-specific chemotherapy.

Example 14Immunohistochemical Methods

Frozen tissue samples from cancer patients are embedded in an optimum cutting temperature (OCT) compound and quick-frozen in isopentane with dry ice. Cryosections are cut with a Leica 3050 CM mictrotome at thickness of 5 μm and thaw-mounted on vectabound-coated slides. The sections are fixed with ethanol at −20° C. and allowed to air dry overnight at room temperature. The fixed sections are stored at −80° C. until use. For immunohistochemistry, the tissue sections are retrieved and first incubated in blocking buffer (PBS, 5% normal goat serum, 0.1% Tween 20) for 30 minutes at room temperature, and then incubated with the CA protein-specific monoclonal antibody and control monoclonal antibodies diluted in blocking buffer (1 μg/ml) for 120 minutes. The sections are then washed three times with the blocking buffer. The bound monoclonal antibodies are detected with a goat anti-mouse IgG+IgM (H+L) F(ab′)[0372]²-peroxidase conjugates and the peroxidase substrate diaminobenzidine (1 mg/ml, Sigma Catalog No. D 5637) in 0.1 M sodium acetate buffer pH 5.05 and 0.003% hydrogen peroxide (Sigma cat. No. H1009). The stained slides are counter-stained with hematoxylin and examined under Nikon microscope.

Monoclonal antibody against a CA protein (antigen) is used to test reactivity with various cell lines from different types of tissues. Cells from different established cell lines are removed from the growth surface without using proteases, packed and embedded in OCT compound. The cells are frozen and sectioned, then stained using a standard IHC protocol. The CellArray™ technology is described in WO 01/43869. Normal tissue (human) obtained by surgical resection are frozen and mounted. Cryosections are cut with a Leica 3050 CM mictrotome at thickness of 5 μm and thaw-mounted on vectabound-coated slides. The sections are fixed with ethanol at −20° C. and allowed to air dry overnight at room temperature. PolyMICA™ Detection kit is used to determine binding of a CA-specific monoclonal antibody to normal tissue. Primary monoclonal antibody is used at a final concentration of 1 μg/ml.[0373]

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.[0374]

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.[0375]

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SEQUENCE LISTING

The patent application contains a lengthy “Sequence Listing” section. A copy of the “Sequence Listing” is available in electronic form from the USPTO

web site (http://seqdata.uspto.gov/sequence.html?DocID=20040170982). An electronic copy of the “Sequence Listing” will also be available from the

USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).