Compositions And Methods For The Diagnosis And Treatment Of Tumor

Abstract

The present invention is directed to compositions of matter useful for the diagnosis and treatment of tumor in mammals and to methods of using those compositions of matter for the same.

Description

RELATED APPLICATIONS

[0001] The application is a continuation of and claims priority under 35 U.S.C. .sctn.120 to U.S. patent application Ser. No. 10/953,264, filed on Sep. 29, 2004, which is a continuation of and claims priority under 35 U.S.C. .sctn.120 to U.S. patent application Ser. No. 10/411,010, filed on Apr. 10, 2003, which claims the benefit under 35 U.S.C. .sctn.119 of U.S. Provisional Patent Application Ser. Nos. 60/404,809, filed Aug. 19, 2002, 60/378,885, filed May 8, 2002, and 60/373,160, filed Apr. 16, 2002, the entire disclosures of which are hereby incorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention is directed to compositions of matter useful for the diagnosis and treatment of tumor in mammals and to methods of using those compositions of matter for the same.

BACKGROUND OF THE INVENTION

[0003] Malignant tumors (cancers) are the second leading cause of death in the United States, after heart disease (Boring et al., CA Cancer J. Clin. 43:7 (1993)). Cancer is characterized by the increase in the number of abnormal, or neoplastic, cells derived from a normal tissue which proliferate to form a tumor mass, the invasion of adjacent tissues by these neoplastic tumor cells, and the generation of malignant cells which eventually spread via the blood or lymphatic system to regional lymph nodes and to distant sites via a process called metastasis. In a cancerous state, a cell proliferates under conditions in which normal cells would not grow. Cancer manifests itself in a wide variety of forms, characterized by different degrees of invasiveness and aggressiveness.

[0004] In attempts to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify transmembrane or otherwise membrane-associated polypeptides that are specifically expressed on the surface of one or more particular type(s) of cancer cell as compared to on one or more normal non-cancerous cell(s). Often, such membrane-associated polypeptides are more abundantly expressed on the surface of the cancer cells as compared to on the surface of the non-cancerous cells. The identification of such tumor-associated cell surface antigen polypeptides has given rise to the ability to specifically target cancer cells for destruction via antibody-based therapies. In this regard, it is noted that antibody-based therapy has proved very effective in the treatment of certain cancers. For example, HERCEPTIN.RTM. and RITUXAN.RTM. (both from Genentech Inc., South San Francisco, Calif.) are antibodies that have been used successfully to treat breast cancer and non-Hodgkin's lymphoma, respectively. More specifically, HERCEPTIN.RTM. is a recombinant DNA-derived humanized monoclonal antibody that selectively binds to the extracellular domain of the human epidermal growth factor receptor 2 (HER2) proto-oncogene. HER2 protein overexpression is observed in 25-30% of primary breast cancers. RITUXAN.RTM. is a genetically engineered chimeric murine/human monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes. Both these antibodies are recombinantly produced in CHO cells.

[0005] In other attempts to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify (1) non-membrane-associated polypeptides that are specifically produced by one or more particular type(s) of cancer cell(s) as compared to by one or more particular type(s) of non-cancerous normal cell(s), (2) polypeptides that are produced by cancer cells at an expression level that is significantly higher than that of one or more normal non-cancerous cell(s), or (3) polypeptides whose expression is specifically limited to only a single (or very limited number of different) tissue type(s) in both the cancerous and non-cancerous state (e.g., normal prostate and prostate tumor tissue). Such polypeptides may remain intracellularly located or may be secreted by the cancer cell. Moreover, such polypeptides may be expressed not by the cancer cell itself, but rather by cells which produce and/or secrete polypeptides having a potentiating or growth-enhancing effect on cancer cells. Such secreted polypeptides are often proteins that provide cancer cells with a growth advantage over normal cells and include such things as, for example, angiogenic factors, cellular adhesion factors, growth factors, and the like. Identification of antagonists of such non-membrane associated polypeptides would be expected to serve as effective therapeutic agents for the treatment of such cancers. Furthermore, identification of the expression pattern of such polypeptides would be useful for the diagnosis of particular cancers in mammals.

[0006] Despite the above identified advances in mammalian cancer therapy, there is a great need for additional diagnostic and therapeutic agents capable of detecting the presence of tumor in a mammal and for effectively inhibiting neoplastic cell growth, respectively. Accordingly, it is an objective of the present invention to identify: (1) cell membrane-associated polypeptides that are more abundantly expressed on one or more type(s) of cancer cell(s) as compared to on normal cells or on other different cancer cells, (2) non-membrane-associated polypeptides that are specifically produced by one or more particular type(s) of cancer cell(s) (or by other cells that produce polypeptides having a potentiating effect on the growth of cancer cells) as compared to by one or more particular type(s) of non-cancerous normal cell(s), (3) non-membrane-associated polypeptides that are produced by cancer cells at an expression level that is significantly higher than that of one or more normal non-cancerous cell(s), or (4) polypeptides whose expression is specifically limited to only a single (or very limited number of different) tissue type(s) in both a cancerous and non-cancerous state (e.g., normal prostate and prostate tumor tissue), and to use those polypeptides, and their encoding nucleic acids, to produce compositions of matter useful in the therapeutic treatment and diagnostic detection of cancer in mammals. It is also an objective of the present invention to identify cell membrane-associated, secreted or intracellular polypeptides whose expression is limited to a single or very limited number of tissues, and to use those polypeptides, and their encoding nucleic acids, to produce compositions of matter useful in the therapeutic treatment and diagnostic detection of cancer in mammals.

SUMMARY OF THE INVENTION

A. Embodiments

[0007] In the present specification, Applicants describe for the first time the identification of various cellular polypeptides (and their encoding nucleic acids or fragments thereof) which are expressed to a greater degree on the surface of or by one or more types of cancer cell(s) as compared to on the surface of or by one or more types of normal non-cancer cells. Alternatively, such polypeptides are expressed by cells which produce and/or secrete polypeptides having a potentiating or growth-enhancing effect on cancer cells. Again alternatively, such polypeptides may not be overexpressed by tumor cells as compared to normal cells of the same tissue type, but rather may be specifically expressed by both tumor cells and normal cells of only a single or very limited number of tissue types (preferably tissues which are not essential for life, e.g., prostate, etc.). All of the above polypeptides are herein referred to as Tumor-associated Antigenic Target polypeptides ("TAT" polypeptides) and are expected to serve as effective targets for cancer therapy and diagnosis in mammals.

[0008] Accordingly, in one embodiment of the present invention, the invention provides an isolated nucleic acid molecule having a nucleotide sequence that encodes a tumor-associated antigenic target polypeptide or fragment thereof (a "TAT" polypeptide).

[0009] In certain aspects, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity, to (a) a DNA molecule encoding a full-length TAT polypeptide having an amino acid sequence as disclosed herein, a TAT polypeptide amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane TAT polypeptide, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of a full-length TAT polypeptide amino acid sequence as disclosed herein, or (b) the complement of the DNA molecule of (a).

[0010] In other aspects, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity, to (a) a DNA molecule comprising the coding sequence of a full-length TAT polypeptide cDNA as disclosed herein, the coding sequence of a TAT polypeptide lacking the signal peptide as disclosed herein, the coding sequence of an extracellular domain of a transmembrane TAT polypeptide, with or without the signal peptide, as disclosed herein or the coding sequence of any other specifically defined fragment of the full-length TAT polypeptide amino acid sequence as disclosed herein, or (b) the complement of the DNA molecule of (a).

[0011] In further aspects, the invention concerns an isolated nucleic acid molecule comprising a nucleotide sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% nucleic acid sequence identity, to (a) a DNA molecule that encodes the same mature polypeptide encoded by the full-length coding region of any of the human protein cDNAs deposited with the ATCC as disclosed herein, or (b) the complement of the DNA molecule of (a).

[0012] Another aspect of the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a TAT polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated, or is complementary to such encoding nucleotide sequence, wherein the transmembrane domain(s) of such polypeptide(s) are disclosed herein. Therefore, soluble extracellular domains of the herein described TAT polypeptides are contemplated.

[0013] In other aspects, the present invention is directed to isolated nucleic acid molecules which hybridize to (a) a nucleotide sequence encoding a TAT polypeptide having a full-length amino acid sequence as disclosed herein, a TAT polypeptide amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane TAT polypeptide, with or without the signal peptide, as disclosed herein or any other specifically deemed fragment of a full-length TAT polypeptide amino acid sequence as disclosed herein, or (b) the complement of the nucleotide sequence of (a). In this regard, an embodiment of the present invention is directed to fragments of a full-length TAT polypeptide coding sequence, or the complement thereof, as disclosed herein, that may find use as, for example, hybridization probes useful as, for example, diagnostic probes, antisense oligonucleotide probes, or for encoding fragments of a full-length TAT polypeptide that may optionally encode a polypeptide comprising a binding site for an anti-TAT polypeptide antibody, a TAT binding oligopeptide or other small organic molecule that binds to a TAT polypeptide. Such nucleic acid fragments are usually at least about 5 nucleotides in length, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 nucleotides in length, wherein in this context the term "about" means the referenced nucleotide sequence length plus or minus 10% of that referenced length. It is noted that novel fragments of a TAT polypeptide-encoding nucleotide sequence may be determined in a routine manner by aligning the TAT polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of a number of well known sequence alignment programs and determining which TAT polypeptide-encoding nucleotide sequence fragment(s) are novel. All of such novel fragments of TAT polypeptide-encoding nucleotide sequences are contemplated herein. Also contemplated are the TAT polypeptide fragments encoded by these nucleotide molecule fragments, preferably those TAT polypeptide fragments that comprise a binding site for an anti-TAT antibody, a TAT binding oligopeptide or other small organic molecule that binds to a TAT polypeptide.

[0014] In another embodiment, the invention provides isolated TAT polypeptides encoded by any of the isolated nucleic acid sequences hereinabove identified.

[0015] In a certain aspect, the invention concerns an isolated TAT polypeptide, comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity, to a TAT polypeptide having a full-length amino acid sequence as disclosed herein, a TAT polypeptide amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane TAT polypeptide protein, with or without the signal peptide, as disclosed herein, an amino acid sequence encoded by any of the nucleic acid sequences disclosed herein or any other specifically defined fragment of a full-length TAT polypeptide amino acid sequence as disclosed herein.

[0016] In a further aspect, the invention concerns an isolated TAT polypeptide comprising an amino acid sequence having at least about 80% amino acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity, to an amino acid sequence encoded by any of the human protein cDNAs deposited with the ATCC as disclosed herein.

[0017] In a specific aspect, the invention provides an isolated TAT polypeptide without the N-terminal signal sequence and/or without the initiating methionine and is encoded by a nucleotide sequence that encodes such an amino acid sequence as hereinbefore described. Processes for producing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the TAT polypeptide and recovering the TAT polypeptide from the cell culture.

[0018] Another aspect of the invention provides an isolated TAT polypeptide which is either transmembrane domain-deleted or transmembrane domain-inactivated. Processes for producing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the TAT polypeptide and recovering the TAT polypeptide from the cell culture.

[0019] In other embodiments of the present invention, the invention provides vectors comprising DNA encoding any of the herein described polypeptides. Host cells comprising any such vector are also provided. By way of example, the host cells may be CHO cells, E. coli cells, or yeast cells. A process for producing any of the herein described polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture.

[0020] In other embodiments, the invention provides isolated chimeric polypeptides comprising any of the herein described TAT polypeptides fused to a heterologous (non-TAT) polypeptide. Example of such chimeric molecules comprise any of the herein described TAT polypeptides fused to a heterologous polypeptide such as, for example, an epitope tag sequence or a Fc region of an immunoglobulin.

[0021] In another embodiment, the invention provides an antibody which binds, preferably specifically, to any of the above or below described polypeptides. Optionally, the antibody is a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody, single-chain antibody or antibody that competitively inhibits the binding of an anti-TAT polypeptide antibody to its respective antigenic epitope. Antibodies of, the present invention may optionally be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The antibodies of the present invention may optionally be produced in CHO cells or bacterial cells and preferably induce death of a cell to which they bind. For diagnostic purposes, the antibodies of the present invention may be detectably labeled, attached to a solid support, or the like.

[0022] In other embodiments of the present invention, the invention provides vectors comprising DNA encoding any of the herein described antibodies. Host cell comprising any such vector are also provided. By way of example, the host cells may be CHO cells, E. coli cells, or yeast cells. A process for producing any of the herein described antibodies is further provided and comprises culturing host cells under conditions suitable for expression of the desired antibody and recovering the desired antibody from the cell culture.

[0023] In another embodiment, the invention provides oligopeptides ("TAT binding oligopeptides") which bind, preferably specifically, to any of the above or below described TAT polypeptides. Optionally, the TAT binding oligopeptides of the present invention may be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The TAT binding oligopeptides of the present invention may optionally be produced in CHO cells or bacterial cells and preferably induce death of a cell to which they bind. For diagnostic purposes, the TAT binding oligopeptides of the present invention may be detectably labeled, attached to a solid support, or the like.

[0024] In other embodiments of the present invention, the invention provides vectors comprising DNA encoding any of the herein described TAT binding oligopeptides. Host cell comprising any such vector are also provided. By way of example, the host cells may be CHO cells, E. coli cells, or yeast cells. A process for producing any of the herein described TAT binding oligopeptides is further provided and comprises culturing host cells under conditions suitable for expression of the desired oligopeptide and recovering the desired oligopeptide from the cell culture.

[0025] In another embodiment, the invention provides small organic molecules ("TAT binding organic molecules") which bind, preferably specifically, to any of the above or below described TAT polypeptides. Optionally, the TAT binding organic molecules of the present invention may be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The TAT binding organic molecules of the present invention preferably induce death of a cell to which they bind. For diagnostic purposes, the TAT binding organic molecules of the present invention may be detectably labeled, attached to a solid support, or the like.

[0026] In a still further embodiment, the invention concerns a composition of matter comprising a TAT polypeptide as described herein, a chimeric TAT polypeptide as described herein, an anti-TAT antibody as described herein, a TAT binding oligopeptide as described herein, or a TAT binding organic molecule as described herein, in combination with a carrier. Optionally, the carrier is a pharmaceutically acceptable carrier.

[0027] In yet another embodiment, the invention concerns an article of manufacture comprising a container and a composition of matter contained within the container, wherein the composition of matter may comprise a TAT polypeptide as described herein, a chimeric TAT polypeptide as described herein, an anti-TAT antibody as described herein, a TAT binding oligopeptide as described herein, or a TAT binding organic molecule as described herein. The article may further optionally comprise a label affixed to the container, or a package insert included with the container, that refers to the use of the composition of matter for the therapeutic treatment or diagnostic detection of a tumor.

[0028] Another embodiment of the present invention is directed to the use of a TAT polypeptide as described herein, a chimeric TAT polypeptide as described herein, an anti-TAT polypeptide antibody as described herein, a TAT binding oligopeptide as described herein, or a TAT binding organic molecule as described herein, for the preparation of a medicament useful in the treatment of a condition which is responsive to the TAT polypeptide, chimeric TAT polypeptide, anti-TAT polypeptide antibody, TAT binding oligopeptide, or TAT binding organic molecule.

B. Additional Embodiments

[0029] Another embodiment of the present invention is directed to a method for inhibiting the growth of a cell that expresses a TAT polypeptide, wherein the method comprises contacting the cell with an antibody, an oligopeptide or a small organic molecule that binds to the TAT polypeptide, and wherein the binding of the antibody, oligopeptide or organic molecule to the TAT polypeptide causes inhibition of the growth of the cell expressing the TAT polypeptide. In preferred embodiments, the cell is a cancer cell and binding of the antibody, oligopeptide or organic molecule to the TAT polypeptide causes death of the cell expressing the TAT polypeptide. Optionally, the antibody is a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody, or single-chain antibody. Antibodies, TAT binding oligopeptides and TAT binding organic molecules employed in the methods of the present invention may optionally be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The antibodies and TAT binding oligopeptides employed in the methods of the present invention may optionally be produced in CHO cells or bacterial cells.

[0030] Yet another embodiment of the present invention is directed to a method of therapeutically treating a mammal having a cancerous tumor comprising cells that express a TAT polypeptide, wherein the method comprises administering to the mammal a therapeutically effective amount of an antibody, an oligopeptide or a small organic molecule that binds to the TAT polypeptide, thereby resulting in the effective therapeutic treatment of the tumor. Optionally, the antibody is a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody, or single-chain antibody. Antibodies, TAT binding oligopeptides and TAT binding organic molecules employed in the methods of the present invention may optionally be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The antibodies and oligopeptides employed in the methods of the present invention may optionally be produced in CHO cells or bacterial cells.

[0031] Yet another embodiment of the present invention is directed to a method of determining the presence of a TAT polypeptide in a sample suspected of containing the TAT polypeptide, wherein the method comprises exposing the sample to an antibody, oligopeptide or small organic molecule that binds to the TAT polypeptide and determining binding of the antibody, oligopeptide or organic molecule to the TAT polypeptide in the sample, wherein the presence of such binding is indicative of the presence of the TAT polypeptide in the sample. Optionally, the sample may contain cells (which may be cancer cells) suspected of expressing the TAT polypeptide. The antibody, TAT binding oligopeptide or TAT binding organic molecule employed in the method may optionally be detectably labeled, attached to a solid support, or the like.

[0032] A further embodiment of the present invention is directed to a method of diagnosing the presence of a tumor in a mammal, wherein the method comprises detecting the level of expression of a gene encoding a TAT polypeptide (a) in a test sample of tissue cells obtained from said mammal, and (b) in a control sample of known normal non-cancerous cells of the same tissue origin or type, wherein a higher level of expression of the TAT polypeptide in the test sample, as compared to the control sample, is indicative of the presence of tumor in the mammal from which the test sample was obtained.

[0033] Another embodiment of the present invention is directed to a method of diagnosing the presence of a tumor in a mammal, wherein the method comprises (a) contacting a test sample comprising tissue cells obtained from the mammal with an antibody, oligopeptide or small organic molecule that binds to a TAT polypeptide and (b) detecting the formation of a complex between the antibody, oligopeptide or small organic molecule and the TAT polypeptide in the test sample, wherein the formation of a complex is indicative of the presence of a tumor in the mammal. Optionally, the antibody, TAT binding oligopeptide or TAT binding organic molecule employed is detectably labeled, attached to a solid support, or the like, and/or the test sample of tissue cells is obtained from an individual suspected of having a cancerous tumor.

[0034] Yet another embodiment of the present invention is directed to a method for treating or preventing a cell proliferative disorder associated with altered, preferably increased, expression or activity of a TAT polypeptide, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of a TAT polypeptide. Preferably, the cell proliferative disorder is cancer and the antagonist of the TAT polypeptide is an anti-TAT polypeptide antibody, TAT binding oligopeptide, TAT binding organic molecule or antisense oligonucleotide. Effective treatment or prevention of the cell proliferative disorder may be a result of direct killing or growth inhibition of cells that express a TAT polypeptide or by antagonizing the cell growth potentiating activity of a TAT polypeptide.

[0035] Yet another embodiment of the present invention is directed to a method of binding an antibody, oligopeptide or small organic molecule to a cell that expresses a TAT polypeptide, wherein the method comprises contacting a cell that expresses a TAT polypeptide with said antibody, oligopeptide or small organic molecule under conditions which are suitable for binding of the antibody, oligopeptide or small organic molecule to said TAT polypeptide and allowing binding therebetween.

[0036] Other embodiments of the present invention are directed to the use of (a) a TAT polypeptide, (b) a nucleic acid encoding a TAT polypeptide or a vector or host cell comprising that nucleic acid, (c) an anti-TAT polypeptide antibody, (d) a TAT-binding oligopeptide, or (e) a TAT-binding small organic molecule in the preparation of a medicament useful for (i) the therapeutic treatment or diagnostic detection of a cancer or tumor, or (ii) the therapeutic treatment or prevention of a cell proliferative disorder.

[0037] Another embodiment of the present invention is directed to a method for inhibiting the growth of a cancer cell, wherein the growth of said cancer cell is at least in part dependent upon the growth potentiating effect(s) of a TAT polypeptide (wherein the TAT polypeptide may be expressed either by the cancer cell itself or a cell that produces polypeptide(s) that have a growth potentiating effect on cancer cells), wherein the method comprises contacting the TAT polypeptide with an antibody, an oligopeptide or a small organic molecule that binds to the TAT polypeptide, thereby antagonizing the growth-potentiating activity of the TAT polypeptide and, in turn, inhibiting the growth of the cancer cell. Preferably the growth of the cancer cell is completely inhibited. Even more preferably, binding of the antibody, oligopeptide or small organic molecule to the TAT polypeptide induces the death of the cancer cell. Optionally, the antibody is a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody, or single-chain antibody. Antibodies, TAT binding oligopeptides and TAT binding organic molecules employed in the methods of the present invention may optionally be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The antibodies and TAT binding oligopeptides employed in the methods of the present invention may optionally be produced in CHO cells or bacterial cells.

[0038] Yet another embodiment of the present invention is directed to a method of therapeutically treating a tumor in a mammal, wherein the growth of said tumor is at least in part dependent upon the growth potentiating effect(s) of a TAT polypeptide, wherein the method comprises administering to the mammal a therapeutically effective amount of an antibody, an oligopeptide or a small organic molecule that binds to the TAT polypeptide, thereby antagonizing the growth potentiating activity of said TAT polypeptide and resulting in the effective therapeutic treatment of the tumor. Optionally, the antibody is a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody, or single-chain antibody. Antibodies, TAT binding oligopeptides and TAT binding organic molecules employed in the methods of the present invention may optionally be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The antibodies and oligopeptides employed in the methods of the present invention may optionally be produced in CHO cells or bacterial cells.

C. Further Additional Embodiments

[0039] In yet further embodiments, the invention is directed to the following set of potential claims for this application:

[0040] 1. Isolated nucleic acid having a nucleotide sequence that has at least 80% nucleic acid sequence identity to:

[0041] (a) a DNA molecule encoding the amino acid sequence shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32);

[0042] (b) a DNA molecule encoding the amino acid sequence shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0043] (c) a DNA molecule encoding an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), with its associated signal peptide;

[0044] (d) a DNA molecule encoding an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0045] (e) the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS: 1-16);

[0046] (f) the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or

[0047] (g) the complement of (a), (b), (c), (d), (e) or (f).

[0048] 2. Isolated nucleic acid having:

[0049] (a) a nucleotide sequence that encodes the amino acid sequence shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32);

[0050] (b) a nucleotide sequence that encodes the amino acid sequence shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0051] (c) a nucleotide sequence that encodes an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), with its associated signal peptide;

[0052] (d) a nucleotide sequence that encodes an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0053] (e) the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS: 1-16);

[0054] (f) the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or

[0055] (g) the complement of (a), (b), (c), (d), (e) or (f).

[0056] 3. Isolated nucleic acid that hybridizes to:

[0057] (a) a nucleic acid that encodes the amino acid sequence shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32);

[0058] (b) a nucleic acid that encodes the amino acid sequence shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0059] (c) a nucleic acid that encodes an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), with its associated signal peptide;

[0060] (d) a nucleic acid that encodes an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32), lacking its associated signal peptide;

[0061] (e) the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS: 1-16);

[0062] (f) the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or

[0063] (g) the complement of (a), (b), (c), (d), (e) or (f).

[0064] 4. The nucleic acid of claim 3, wherein the hybridization occurs under stringent conditions.

[0065] 5. The nucleic acid of claim 3 which is at least about 5 nucleotides in length.

[0066] 6. An expression vector comprising the nucleic acid of claim 1, 2 or 3.

[0067] 7. The expression vector of claim 6, wherein said nucleic acid is operably linked to control sequences recognized by a host cell transformed with the vector.

[0068] 8. A host cell comprising the expression vector of claim 7.

[0069] 9. The host cell of claim 8 which is a CHO cell, an E. coli cell or a yeast cell.

[0070] 10. A process for producing a polypeptide comprising culturing the host cell of claim 8 under conditions suitable for expression of said polypeptide and recovering said polypeptide from the cell culture.

[0071] 11. An isolated polypeptide having at least 80% amino acid sequence identity to:

[0072] (a) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32);

[0073] (b) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0074] (c) an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), with its associated signal peptide;

[0075] (d) an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0076] (e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or

[0077] (f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16).

[0078] 12. An isolated polypeptide having:

[0079] (a) the amino acid sequence shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32);

[0080] (b) the amino acid sequence shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide sequence;

[0081] (c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), with its associated signal peptide sequence;

[0082] (d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide sequence;

[0083] (e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or

[0084] (f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16).

[0085] 13. A chimeric polypeptide comprising the polypeptide of claim 11 or 12 fused to a heterologous polypeptide.

[0086] 14. The chimeric polypeptide of claim 13, wherein said heterologous polypeptide is an epitope tag sequence or an Fc region of an immunoglobulin.

[0087] 15. An isolated antibody that binds to a polypeptide having at least 80% amino acid sequence identity to:

[0088] (a) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32);

[0089] (b) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0090] (c) an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), with its associated signal peptide;

[0091] (d) an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32), lacking its associated signal peptide;

[0092] (e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or

[0093] (f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16).

[0094] 16. An isolated antibody that binds to a polypeptide having:

[0095] (a) the amino acid sequence shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32);

[0096] (b) the amino acid sequence shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide sequence;

[0097] (c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), with its associated signal peptide sequence;

[0098] (d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide sequence;

[0099] (e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or

[0100] (f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS: 1-16).

[0101] 17. The antibody of claim 15 or 16 which is a monoclonal antibody.

[0102] 18. The antibody of claim 15 or 16 which is an antibody fragment.

[0103] 19. The antibody of claim 15 or 16 which is a chimeric or a humanized antibody.

[0104] 20. The antibody of claim 15 or 16 which is conjugated to a growth inhibitory agent.

[0105] 21. The antibody of claim 15 or 16 which is conjugated to a cytotoxic agent.

[0106] 22. The antibody of claim 21, wherein the cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.

[0107] 23. The antibody of claim 21, wherein the cytotoxic agent is a toxin.

[0108] 24. The antibody of claim 23, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.

[0109] 25. The antibody of claim 23, wherein the toxin is a maytansinoid.

[0110] 26. The antibody of claim 15 or 16 which is produced in bacteria.

[0111] 27. The antibody of claim 15 or 16 which is produced in CHO cells.

[0112] 28. The antibody of claim 15 or 16 which induces death of a cell to which it binds.

[0113] 29. The antibody of claim 15 or 16 which is detectably labeled.

[0114] 30. An isolated nucleic acid having a nucleotide sequence that encodes the antibody of claim 15 or 16.

[0115] 31. An expression vector comprising the nucleic acid of claim 30 operably linked to control sequences recognized by a host cell transformed with the vector.

[0116] 32. A host cell comprising the expression vector of claim 31.

[0117] 33. The host cell of claim 32 which is a CHO cell, an E. coli cell or a yeast cell.

[0118] 34. A process for producing an antibody comprising culturing the host cell of claim 32 under conditions suitable for expression of said antibody and recovering said antibody from the cell culture.

[0119] 35. An isolated oligopeptide that binds to a polypeptide having at least 80% amino acid sequence identity to:

[0120] (a) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32);

[0121] (b) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0122] (c) an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), with its associated signal peptide;

[0123] (d) an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0124] (e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or

[0125] (f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS: 1-16).

[0126] 36. An isolated oligopeptide that binds to a polypeptide having:

[0127] (a) the amino acid sequence shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32);

[0128] (b) the amino acid sequence shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide sequence;

[0129] (c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), with its associated signal peptide sequence;

[0130] (d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide sequence;

[0131] (e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or

[0132] (f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16).

[0133] 37. The oligopeptide of claim 35 or 36 which is conjugated to a growth inhibitory agent.

[0134] 38. The oligopeptide of claim 35 or 36 which is conjugated to a cytotoxic agent.

[0135] 39. The oligopeptide of claim 38, wherein the cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.

[0136] 40. The oligopeptide of claim 38, wherein the cytotoxic agent is a toxin.

[0137] 41. The oligopeptide of claim 40, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.

[0138] 42. The oligopeptide of claim 40, wherein the toxin is a maytansinoid.

[0139] 43. The oligopeptide of claim 35 or 36 which induces death of a cell to which it binds.

[0140] 44. The oligopeptide of claim 35 or 36 which is detectably labeled.

[0141] 45. A TAT binding organic molecule that binds to a polypeptide having at least 80% amino acid sequence identity to:

[0142] (a) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32);

[0143] (b) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0144] (c) an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), with its associated signal peptide;

[0145] (d) an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0146] (e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or

[0147] (f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16).

[0148] 46. The organic molecule of claim 45 that binds to a polypeptide having:

[0149] (a) the amino acid sequence shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32);

[0150] (b) the amino acid sequence shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide sequence;

[0151] (c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), with its associated signal peptide sequence;

[0152] (d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide sequence;

[0153] (e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or

[0154] (f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS: 1-16).

[0155] 47. The organic molecule of claim 45 or 46 which is conjugated to a growth inhibitory agent.

[0156] 48. The organic molecule of claim 45 or 46 which is conjugated to a cytotoxic agent.

[0157] 49. The organic molecule of claim 48, wherein the cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.

[0158] 50. The organic molecule of claim 48, wherein the cytotoxic agent is a toxin.

[0159] 51. The organic molecule of claim 50, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.

[0160] 52. The organic molecule of claim 50, wherein the toxin is a maytansinoid.

[0161] 53. The organic molecule of claim 45 or 46 which induces death of a cell to which it binds.

[0162] 54. The organic molecule of claim 45 or 46 which is detectably labeled.

[0163] 55. A composition of matter comprising:

[0164] (a) the polypeptide of claim 11;

[0165] (b) the polypeptide of claim 12;

[0166] (c) the chimeric polypeptide of claim 13;

[0167] (d) the antibody of claim 15;

[0168] (e) the antibody of claim 16;

[0169] (f) the oligopeptide of claim 35;

[0170] (g) the oligopeptide of claim 36;

[0171] (h) the TAT binding organic molecule of claim 45; or

[0172] (i) the TAT binding organic molecule of claim 46; in combination with a carrier.

[0173] 56. The composition of matter of claim 55, wherein said carrier is a pharmaceutically acceptable carrier.

[0174] 57. An article of manufacture comprising:

[0175] (a) a container; and

[0176] (b) the composition of matter of claim 55 contained within said container.

[0177] 58. The article of manufacture of claim 57 further comprising a label affixed to said container, or a package insert included with said container, referring to the use of said composition of matter for the therapeutic treatment of or the diagnostic detection of a cancer.

[0178] 59. A method of inhibiting the growth of a cell that expresses a protein having at least 80% amino acid sequence identity to:

[0179] (a) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32);

[0180] (b) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0181] (c) an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), with its associated signal peptide;

[0182] (d) an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0183] (e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or

[0184] (f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS: 1-16), said method comprising contacting said cell with an antibody, oligopeptide or organic molecule that binds to said protein, the binding of said antibody, oligopeptide or organic molecule to said protein thereby causing an inhibition of growth of said cell.

[0185] 60. The method of claim 59, wherein said antibody is a monoclonal antibody.

[0186] 61. The method of claim 59, wherein said antibody is an antibody fragment.

[0187] 62. The method of claim 59, wherein said antibody is a chimeric or a humanized antibody.

[0188] 63. The method of claim 59, wherein said antibody, oligopeptide or organic molecule is conjugated to a growth inhibitory agent.

[0189] 64. The method of claim 59, wherein said antibody, oligopeptide or organic molecule is conjugated to a cytotoxic agent.

[0190] 65. The method of claim 64, wherein said cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.

[0191] 66. The method of claim 64, wherein the cytotoxic agent is a toxin.

[0192] 67. The method of claim 66, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.

[0193] 68. The method of claim 66, wherein the toxin is a maytansinoid.

[0194] 69. The method of claim 59, wherein said antibody is produced in bacteria.

[0195] 70. The method of claim 59, wherein said antibody is produced in CHO cells.

[0196] 71. The method of claim 59, wherein said cell is a cancer cell.

[0197] 72. The method of claim 71, wherein said cancer cell is further exposed to radiation treatment or a chemotherapeutic agent.

[0198] 73. The method of claim 71, wherein said cancer cell is selected from the group consisting of a breast cancer cell, a colorectal cancer cell, a lung cancer cell, an ovarian cancer cell, a central nervous system cancer cell, a liver cancer cell, a bladder cancer cell, a pancreatic cancer cell, a cervical cancer cell, a melanoma cell and a leukemia cell.

[0199] 74. The method of claim 71, wherein said protein is more abundantly expressed by said cancer cell as compared to a normal cell of the same tissue origin.

[0200] 75. The method of claim 59 which causes the death of said cell.

[0201] 76. The method of claim 59, wherein said protein has:

[0202] (a) the amino acid sequence shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32);

[0203] (b) the amino acid sequence shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide sequence;

[0204] (c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), with its associated signal peptide sequence;

[0205] (d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide sequence;

[0206] (e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or

[0207] (f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS: 1-16).

[0208] 77. A method of therapeutically treating a mammal having a cancerous tumor comprising cells that express a protein having at least 80% amino acid sequence identity to:

[0209] (a) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32);

[0210] (b) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0211] (c) an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), with its associated signal peptide;

[0212] (d) an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0213] (e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or

[0214] (f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS: 1-16), said method comprising administering to said mammal a therapeutically effective amount of an antibody, oligopeptide or organic molecule that binds to said protein, thereby effectively treating said mammal.

[0215] 78. The method of claim 77, wherein said antibody is a monoclonal antibody.

[0216] 79. The method of claim 77, wherein said antibody is an antibody fragment.

[0217] 80. The method of claim 77, wherein said antibody is a chimeric or a humanized antibody.

[0218] 81. The method of claim 77, wherein said antibody, oligopeptide or organic molecule is conjugated to a growth inhibitory agent.

[0219] 82. The method of claim 77, wherein said antibody, oligopeptide or organic molecule is conjugated to a cytotoxic agent.

[0220] 83. The method of claim 82, wherein said cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.

[0221] 84. The method of claim 82, wherein the cytotoxic agent is a toxin.

[0222] 85. The method of claim 84, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.

[0223] 86. The method of claim 84, wherein the toxin is a maytansinoid.

[0224] 87. The method of claim 77, wherein said antibody is produced in bacteria.

[0225] 88. The method of claim 77, wherein said antibody is produced in CHO cells.

[0226] 89. The method of claim 77, wherein said tumor is further exposed to radiation treatment or a chemotherapeutic agent.

[0227] 90. The method of claim 77, wherein said tumor is a breast tumor, a colorectal tumor, a lung tumor, an ovarian tumor, a central nervous system tumor, a liver tumor, a bladder tumor, a pancreatic tumor, or a cervical tumor.

[0228] 91. The method of claim 77, wherein said protein is more abundantly expressed by the cancerous cells of said tumor as compared to a normal cell of the same tissue origin.

[0229] 92. The method of claim 77, wherein said protein has:

[0230] (a) the amino acid sequence shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32);

[0231] (b) the amino acid sequence shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide sequence;

[0232] (c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), with its associated signal peptide sequence;

[0233] (d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide sequence;

[0234] (e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS: 1-16); or

[0235] (f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS: 1-16).

[0236] 93. A method of determining the presence of a protein in a sample suspected of containing said protein, wherein said protein has at least 80% amino acid sequence identity to:

[0237] (a) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32);

[0238] (b) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0239] (c) an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), with its associated signal peptide;

[0240] (d) an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0241] (e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or

[0242] (f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS: 1-16), said method comprising exposing said sample to an antibody, oligopeptide or organic molecule that binds to said protein and determining binding of said antibody, oligopeptide or organic molecule to said protein in said sample, wherein binding of the antibody, oligopeptide or organic molecule to said protein is indicative of the presence of said protein in said sample.

[0243] 94. The method of claim 93, wherein said sample comprises a cell suspected of expressing said protein.

[0244] 95. The method of claim 94, wherein said cell is a cancer cell.

[0245] 96. The method of claim 93, wherein said antibody, oligopeptide or organic molecule is detectably labeled.

[0246] 97. The method of claim 93, wherein said protein has:

[0247] (a) the amino acid sequence shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32);

[0248] (b) the amino acid sequence shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide sequence;

[0249] (c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), with its associated signal peptide sequence;

[0250] (d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide sequence;

[0251] (e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or

[0252] (f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS: 1-16).

[0253] 98. A method of diagnosing the presence of a tumor in a mammal, said method comprising determining the level of expression of a gene encoding a protein having at least 80% amino acid sequence identity to:

[0254] (a) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32);

[0255] (b) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0256] (c) an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), with its associated signal peptide;

[0257] (d) an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0258] (e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or

[0259] (f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS: 1-16), in a test sample of tissue cells obtained from said mammal and in a control sample of known normal cells of the same tissue origin, wherein a higher level of expression of said protein in the test sample, as compared to the control sample, is indicative of the presence of tumor in the mammal from which the test sample was obtained.

[0260] 99. The method of claim 98, wherein the step of determining the level of expression of a gene encoding said protein comprises employing an oligonucleotide in an in situ hybridization or RT-PCR analysis.

[0261] 100. The method of claim 98, wherein the step determining the level of expression of a gene encoding said protein comprises employing an antibody in an immunohistochemistry or Western blot analysis.

[0262] 101. The method of claim 98, wherein said protein has:

[0263] (a) the amino acid sequence shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32);

[0264] (b) the amino acid sequence shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide sequence;

[0265] (c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), with its associated signal peptide sequence;

[0266] (d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide sequence;

[0267] (e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or

[0268] (f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS: 1-16).

[0269] 102. A method of diagnosing the presence of a tumor in a mammal, said method comprising contacting a test sample of tissue cells obtained from said mammal with an antibody, oligopeptide or organic molecule that binds to a protein having at least 80% amino acid sequence identity to:

[0270] (a) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32);

[0271] (b) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0272] (c) an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), with its associated signal peptide;

[0273] (d) an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0274] (e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or

[0275] (f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16), and detecting the formation of a complex between said antibody, oligopeptide or organic molecule and said protein in the test sample, wherein the formation of a complex is indicative of the presence of a tumor in said mammal.

[0276] 103. The method of claim 102, wherein said antibody, oligopeptide or organic molecule is detectably labeled.

[0277] 104. The method of claim 102, wherein said test sample of tissue cells is obtained from an individual suspected of having a cancerous tumor.

[0278] 105. The method of claim 102, wherein said protein has:

[0279] (a) the amino acid sequence shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32);

[0280] (b) the amino acid sequence shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide sequence;

[0281] (c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), with its associated signal peptide sequence;

[0282] (d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide sequence;

[0283] (e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or

[0284] (f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16).

[0285] 106. A method for treating or preventing a cell proliferative disorder associated with increased expression or activity of a protein having at least 80% amino acid sequence identity to:

[0286] (a) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32);

[0287] (b) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0288] (c) an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), with its associated signal peptide;

[0289] (d) an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0290] (e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or

[0291] (f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16), said method comprising administering to a subject in need of such treatment an effective amount of an antagonist of said protein, thereby effectively treating or preventing said cell proliferative disorder.

[0292] 107. The method of claim 106, wherein said cell proliferative disorder is cancer.

[0293] 108. The method of claim 106, wherein said antagonist is an anti-TAT polypeptide antibody, TAT binding oligopeptide, TAT binding organic molecule or antisense oligonucleotide.

[0294] 109. A method of binding an antibody, oligopeptide or organic molecule to a cell that expresses a protein having at least 80% amino acid sequence identity to:

[0295] (a) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32);

[0296] (b) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0297] (c) an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), with its associated signal peptide;

[0298] (d) an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0299] (e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or

[0300] (f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS: 1-16), said method comprising contacting said cell with an antibody, oligopeptide or organic molecule that binds to said protein and allowing the binding of the antibody, oligopeptide or organic molecule to said protein to occur, thereby binding said antibody, oligopeptide or organic molecule to said cell.

[0301] 110. The method of claim 109, wherein said antibody is a monoclonal antibody.

[0302] 111. The method of claim 109, wherein said antibody is an antibody fragment.

[0303] 112. The method of claim 109, wherein said antibody is a chimeric or a humanized antibody.

[0304] 113. The method of claim 109, wherein said antibody, oligopeptide or organic molecule is conjugated to a growth inhibitory agent.

[0305] 114. The method of claim 109, wherein said antibody, oligopeptide or organic molecule is conjugated to a cytotoxic agent.

[0306] 115. The method of claim 114, wherein said cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.

[0307] 116. The method of claim 114, wherein the cytotoxic agent is a toxin.

[0308] 117. The method of claim 116, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.

[0309] 118. The method of claim 116, wherein the toxin is a maytansinoid.

[0310] 119. The method of claim 109, wherein said antibody is produced in bacteria.

[0311] 120. The method of claim 109, wherein said antibody is produced in CHO cells.

[0312] 121. The method of claim 109, wherein said cell is a cancer cell.

[0313] 122. The method of claim 121, wherein said cancer cell is further exposed to radiation treatment or a chemotherapeutic agent.

[0314] 123. The method of claim 121, wherein said cancer cell is selected from the group consisting of a breast cancer cell, a colorectal cancer cell, a lung cancer cell, an ovarian cancer cell, a central nervous system cancer cell, a liver cancer cell, a bladder cancer cell, a pancreatic cancer cell, a cervical cancer cell, a melanoma cell and a leukemia cell.

[0315] 124. The method of claim 123, wherein said protein is more abundantly expressed by said cancer cell as compared to a normal cell of the same tissue origin.

[0316] 125. The method of claim 109 which causes the death of said cell.

[0317] 126. Use of a nucleic acid as claimed in any of claims 1 to 5 or 30 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.

[0318] 127. Use of a nucleic acid as claimed in any of claims 1 to 5 or 30 in the preparation of a medicament for treating a tumor.

[0319] 128. Use of a nucleic acid as claimed in any of claims 1 to 5 or 30 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.

[0320] 129. Use of an expression vector as claimed in any of claims 6, 7 or 31 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.

[0321] 130. Use of an expression vector as claimed in any of claims 6, 7 or 31 in the preparation of medicament for treating a tumor.

[0322] 131. Use of an expression vector as claimed in any of claims 6, 7 or 31 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.

[0323] 132. Use of a host cell as claimed in any of claims 8, 9, 32, or 33 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.

[0324] 133. Use of a host cell as claimed in any of claims 8, 9, 32 or 33 in the preparation of a medicament for treating a tumor.

[0325] 134. Use of a host cell as claimed in any of claims 8, 9, 32 or 33 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.

[0326] 135. Use of a polypeptide as claimed in any of claims 11 to 14 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.

[0327] 136. Use of a polypeptide as claimed in any of claims 11 to 14 in the preparation of a medicament for treating a tumor.

[0328] 137. Use of a polypeptide as claimed in any of claims 11 to 14 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.

[0329] 138. Use of an antibody as claimed in any of claims 15 to 29 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.

[0330] 139. Use of an antibody as claimed in any of claims 15 to 29 in the preparation of a medicament for treating a tumor.

[0331] 140. Use of an antibody as claimed in any of claims 15 to 29 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.

[0332] 141. Use of an oligopeptide as claimed in any of claims 35 to 44 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.

[0333] 142. Use of an oligopeptide as claimed in any of claims 35 to 44 in the preparation of a medicament for treating a tumor.

[0334] 143. Use of an oligopeptide as claimed in any of claims 35 to 44 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.

[0335] 144. Use of a TAT binding organic molecule as claimed in any of claims 45 to 54 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.

[0336] 145. Use of a TAT binding organic molecule as claimed in any of claims 45 to 54 in the preparation of a medicament for treating a tumor.

[0337] 146. Use of a TAT binding organic molecule as claimed in any of claims 45 to 54 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.

[0338] 147. Use of a composition of matter as claimed in any of claims 55 or 56 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.

[0339] 148. Use of a composition of matter as claimed in any of claims 55 or 56 in the preparation of a medicament for treating a tumor.

[0340] 149. Use of a composition of matter as claimed in any of claims 55 or 56 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.

[0341] 150. Use of an article of manufacture as claimed in any of claims 57 or 58 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.

[0342] 151. Use of an article of manufacture as claimed in any of claims 57 or 58 in the preparation of a medicament for treating a tumor.

[0343] 152. Use of an article of manufacture as claimed in any of claims 57 or 58 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.

[0344] 153. A method for inhibiting the growth of a cell, wherein the growth of said cell is at least in part dependent upon a growth potentiating effect of a protein having at least 80% amino acid sequence identity to:

[0345] (a) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32);

[0346] (b) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0347] (c) an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), with its associated signal peptide;

[0348] (d) an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0349] (e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or

[0350] (f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16), said method comprising contacting said protein with an antibody, oligopeptide or organic molecule that binds to said protein, there by inhibiting the growth of said cell.

[0351] 154. The method of claim 153, wherein said cell is a cancer cell.

[0352] 155. The method of claim 153, wherein said protein is expressed by said cell.

[0353] 156. The method of claim 153, wherein the binding of said antibody, oligopeptide or organic molecule to said protein antagonizes a cell growth-potentiating activity of said protein.

[0354] 157. The method of claim 153, wherein the binding of said antibody, oligopeptide or organic molecule to said protein induces the death of said cell.

[0355] 158. The method of claim 153, wherein said antibody is a monoclonal antibody.

[0356] 159. The method of claim 153, wherein said antibody is an antibody fragment.

[0357] 160. The method of claim 153, wherein said antibody is a chimeric or a humanized antibody.

[0358] 161. The method of claim 153, wherein said antibody, oligopeptide or organic molecule is conjugated to a growth inhibitory agent.

[0359] 162. The method of claim 153, wherein said antibody, oligopeptide or organic molecule is conjugated to a cytotoxic agent.

[0360] 163. The method of claim 162, wherein said cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.

[0361] 164. The method of claim 162, wherein the cytotoxic agent is a toxin.

[0362] 165. The method of claim 164, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.

[0363] 166. The method of claim 164, wherein the toxin is a maytansinoid.

[0364] 167. The method of claim 153, wherein said antibody is produced in bacteria.

[0365] 168. The method of claim 153, wherein said antibody is produced in CHO cells.

[0366] 169. The method of claim 153, wherein said protein has:

[0367] (a) the amino acid sequence shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32);

[0368] (b) the amino acid sequence shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide sequence;

[0369] (c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), with its associated signal peptide sequence;

[0370] (d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide sequence;

[0371] (e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or

[0372] (f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS: 1-16).

[0373] 170. A method of therapeutically treating a tumor in a mammal, wherein the growth of said tumor is at least in part dependent upon a growth potentiating effect of a protein having at least 80% amino acid sequence identity to:

[0374] (a) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32);

[0375] (b) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0376] (c) an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), with its associated signal peptide;

[0377] (d) an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide;

[0378] (e) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or

[0379] (f) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16), said method comprising contacting said protein with an antibody, oligopeptide or organic molecule that binds to said protein, thereby effectively treating said tumor.

[0380] 171. The method of claim 170, wherein said protein is expressed by cells of said tumor.

[0381] 172. The method of claim 170, wherein the binding of said antibody, oligopeptide or organic molecule to said protein antagonizes a cell growth-potentiating activity of said protein.

[0382] 173. The method of claim 170, wherein said antibody is a monoclonal antibody.

[0383] 174. The method of claim 170, wherein said antibody is an antibody fragment.

[0384] 175. The method of claim 170, wherein said antibody is a chimeric or a humanized antibody.

[0385] 176. The method of claim 170, wherein said antibody, oligopeptide or organic molecule is conjugated to a growth inhibitory agent.

[0386] 177. The method of claim 170, wherein said antibody, oligopeptide or organic molecule is conjugated to a cytotoxic agent.

[0387] 178. The method of claim 177, wherein said cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.

[0388] 179. The method of claim 177, wherein the cytotoxic agent is a toxin.

[0389] 180. The method of claim 179, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.

[0390] 181. The method of claim 179, wherein the toxin is a maytansinoid.

[0391] 182. The method of claim 170, wherein said antibody is produced in bacteria.

[0392] 183. The method of claim 170, wherein said antibody is produced in CHO cells.

[0393] 184. The method of claim 170, wherein said protein has:

[0394] (a) the amino acid sequence shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32);

[0395] (b) the amino acid sequence shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide sequence;

[0396] (c) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), with its associated signal peptide sequence;

[0397] (d) an amino acid sequence of an extracellular domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS: 17-32), lacking its associated signal peptide sequence;

[0398] (e) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS: 1-16); or

[0399] (f) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID NOS: 1-16).

[0400] Yet further embodiments of the present invention will be evident to the skilled artisan upon a reading of the present specification.

BRIEF DESCRIPTION OF THE DRAWINGS

[0401] FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a TAT293 cDNA, wherein SEQ ID NO:1 is a clone designated herein as "DNA293550".

[0402] FIG. 2 shows a nucleotide sequence (SEQ ID NO:2) of a TAT294 cDNA, wherein SEQ ID NO:2 is a clone designated herein as "DNA 150813".

[0403] FIG. 3 shows a nucleotide sequence (SEQ ID NO:3) of a TAT295 cDNA, wherein SEQ ID NO:3 is a clone designated herein as "DNA225785".

[0404] FIG. 4 shows a nucleotide sequence (SEQ ID NO:4) of a TAT296 cDNA, wherein SEQ ID NO:4 is a clone designated herein as "DNA226313".

[0405] FIG. 5 shows a nucleotide sequence (SEQ ID NO:5) of a TAT297 cDNA, wherein SEQ ID NO:5 is a clone designated herein as "DNA103365".

[0406] FIG. 6 shows a nucleotide sequence (SEQ ID NO:6) of a TAT298 cDNA, wherein SEQ ID NO:6 is a clone designated herein as "DNA88158".

[0407] FIG. 7 shows a nucleotide sequence (SEQ ID NO:7) of a TAT347 cDNA, wherein SEQ ID NO:7 is a clone designated herein as "DNA225800".

[0408] FIG. 8 shows a nucleotide sequence (SEQ ID NO:8) of a TAT299 cDNA, wherein SEQ ID NO:8 is a clone designated herein as "DNA103386".

[0409] FIG. 9 shows a nucleotide sequence (SEQ ID NO:9) of a TAT351 cDNA, wherein SEQ ID NO:9 is a clone designated herein as "DNA273438".

[0410] FIG. 10 shows a nucleotide sequence (SEQ ID NO:10) of a TAT300 cDNA, wherein SEQ ID NO:10 is a clone designated herein as "DNA225865".

[0411] FIG. 11 shows a nucleotide sequence (SEQ ID NO:11) of a TAT301 cDNA, wherein SEQ ID NO:11 is a clone designated herein as "DNA196665".

[0412] FIG. 12 shows a nucleotide sequence (SEQ ID NO:12) of a TAT352 cDNA, wherein SEQ ID NO:12 is a clone designated herein as "DNA287183".

[0413] FIGS. 13A-B show a nucleotide sequence (SEQ ID NO:13) of a TAT302 cDNA, wherein SEQ ID NO:13 is a clone designated herein as "DNA226403".

[0414] FIG. 14 shows a nucleotide sequence (SEQ ID NO:14) of a TAT303 cDNA, wherein SEQ ID NO:14 is a clone designated herein as "DNA73736".

[0415] FIG. 15 shows a nucleotide sequence (SEQ ID NO:15) of a TAT304 cDNA, wherein SEQ ID NO:15 is a clone designated herein as "DNA299884".

[0416] FIG. 16 shows a nucleotide sequence (SEQ ID NO:16) of a TAT305 cDNA, wherein SEQ ID NO:16 is a clone designated herein as "DNA299885".

[0417] FIG. 17 shows the amino acid sequence (SEQ ID NO:17) derived from the coding sequence of SEQ ID NO:1 shown in FIG. 1.

[0418] FIG. 18 shows the amino acid sequence (SEQ ID NO:18) derived from the coding sequence of SEQ ID NO:2 shown in FIG. 2.

[0419] FIG. 19 shows the amino acid sequence (SEQ ID NO:19) derived from the coding sequence of SEQ ID NO:3 shown in FIG. 3.

[0420] FIG. 20 shows the amino acid sequence (SEQ ID NO:20) derived from the coding sequence of SEQ ID NO:4 shown in FIG. 4.

[0421] FIG. 21 shows the amino acid sequence (SEQ ID NO:21) derived from the coding sequence of SEQ ID NO:5 shown in FIG. 5.

[0422] FIG. 22 shows the amino acid sequence (SEQ ID NO:22) derived from the coding sequence of SEQ ID NO:6 shown in FIG. 6.

[0423] FIG. 23 shows the amino acid sequence (SEQ ID NO:23) derived from the coding sequence of SEQ ID NO:7 shown in FIG. 7.

[0424] FIG. 24 shows the amino acid sequence (SEQ ID NO:24) derived from the coding sequence of SEQ ID NO:8 shown in FIG. 8.

[0425] FIG. 25 shows the amino acid sequence (SEQ ID NO:25) derived from the coding sequence of SEQ ID NO:9 shown in FIG. 9.

[0426] FIG. 26 shows the amino acid sequence (SEQ ID NO:26) derived from the coding sequence of SEQ ID NO:10 shown in FIG. 10.

[0427] FIG. 27 shows the amino acid sequence (SEQ ID NO:27) derived from the coding sequence of SEQ ID NO:11 shown in FIG. 11.

[0428] FIG. 28 shows the amino acid sequence (SEQ ID NO:28) derived from the coding sequence of SEQ ID NO:12 shown in FIG. 12.

[0429] FIG. 29 shows the amino acid sequence (SEQ ID NO:29) derived from the coding sequence of SEQ ID NO:13 shown in FIGS. 13A-B.

[0430] FIG. 30 shows the amino acid sequence (SEQ ID NO:30) derived from the coding sequence of SEQ ID NO:14 shown in FIG. 14.

[0431] FIG. 31 shows the amino acid sequence (SEQ ID NO :31) derived from the coding sequence of SEQ ID NO:15 shown in FIG. 15.

[0432] FIG. 32 shows the amino acid sequence (SEQ ID NO:32) derived from the coding sequence of SEQ ID NO:16 shown in FIG. 16.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions

[0433] The terms "TAT polypeptide" and "TAT" as used herein and when immediately followed by a numerical designation, refer to various polypeptides, wherein the complete designation (i.e., TAT/number) refers to specific polypeptide sequences as described herein. The terms "TAT/number polypeptide" and "TAT/number" wherein the term "number" is provided as an actual numerical designation as used herein encompass native sequence polypeptides, polypeptide variants and fragments of native sequence polypeptides and polypeptide variants (which are further defined herein). The TAT polypeptides described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. The term "TAT polypeptide" refers to each individual TAT/number polypeptide disclosed herein. All disclosures in this specification which refer to the "TAT polypeptide" refer to each of the polypeptides individually as well as jointly. For example, descriptions of the preparation of, purification of, derivation of, formation of antibodies to or against, formation of TAT binding oligopeptides to or against, formation of TAT binding organic molecules to or against, administration of, compositions containing, treatment of a disease with, etc., pertain to each polypeptide of the invention individually. The term "TAT polypeptide" also includes variants of the TAT/number polypeptides disclosed herein.

[0434] A "native sequence TAT polypeptide" comprises a polypeptide having the same amino acid sequence as the corresponding TAT polypeptide derived from nature. Such native sequence TAT polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. The term "native sequence TAT polypeptide" specifically encompasses naturally-occurring truncated or secreted forms of the specific TAT polypeptide (e.g., an extracellular domain sequence), naturally-occurring variant forms (e.g., alternatively spliced forms) and naturally-occurring allelic variants of the polypeptide. In certain embodiments of the invention, the native sequence TAT polypeptides disclosed herein are mature or full-length native sequence polypeptides comprising the full-length amino acids sequences shown in the accompanying figures. Start and stop codons (if indicated) are shown in bold font and underlined in the figures. Nucleic acid residues indicated as "N" in the accompanying figures are any nucleic acid residue. However, while the TAT polypeptides disclosed in the accompanying figures are shown to begin with methionine residues designated herein as amino acid position 1 in the figures, it is conceivable and possible that other methionine residues located either upstream or downstream from the amino acid position 1 in the figures may be employed as the starting amino acid residue for the TAT polypeptides.

[0435] The TAT polypeptide "extracellular domain" or "ECD" refers to a form of the TAT polypeptide which is essentially free of the transmembrane and cytoplasmic domains. Ordinarily, a TAT polypeptide ECD will have less than 1% of such transmembrane and/or cytoplasmic domains and preferably, will have less than 0.5% of such domains. It will be understood that any transmembrane domains identified for the TAT polypeptides of the present invention are identified pursuant to criteria routinely employed in the art for identifying that type of hydrophobic domain. The exact boundaries of a transmembrane domain may vary but most likely by no more than about 5 amino acids at either end of the domain as initially identified herein. Optionally, therefore, an extracellular domain of a TAT polypeptide may contain from about 5 or fewer amino acids on either side of the transmembrane domain/extracellular domain boundary as identified in the Examples or specification and such polypeptides, with or without the associated signal peptide, and nucleic acid encoding them, are contemplated by the present invention.

[0436] The approximate location of the "signal peptides" of the various TAT polypeptides disclosed herein may be shown in the present specification and/or the accompanying figures. It is noted, however, that the C-terminal boundary of a signal peptide may vary, but most likely by no more than about 5 amino acids on either side of the signal peptide C-terminal boundary as initially identified herein, wherein the C-terminal boundary of the signal peptide may be identified pursuant to criteria routinely employed in the art for identifying that type of amino acid sequence element (e.g., Nielsen et al., Prot. Eng. 10:1-6 (1997) and von Heinje et al., Nucl. Acids. Res. 14:4683-4690 (1986)). Moreover, it is also recognized that, in some cases, cleavage of a signal sequence from a secreted polypeptide is not entirely uniform, resulting in more than one secreted species. These mature polypeptides, where the signal peptide is cleaved within no more than about 5 amino acids on either side of the C-terminal boundary of the signal peptide as identified herein, and the polynucleotides encoding them, are contemplated by the present invention.

[0437] "TAT polypeptide variant" means a TAT polypeptide, preferably an active TAT polypeptide, as defined herein having at least about 80% amino acid sequence identity with a full-length native sequence TAT polypeptide sequence as disclosed herein, a TAT polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a TAT polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length TAT polypeptide sequence as disclosed herein (such as those encoded by a nucleic acid that represents only a portion of the complete coding sequence for a full-length TAT polypeptide). Such TAT polypeptide variants include, for instance, TAT polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the full-length native amino acid sequence. Ordinarily, a TAT polypeptide variant will have at least about 80% amino acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, to a full-length native sequence TAT polypeptide sequence as disclosed herein, a TAT polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a TAT polypeptide, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of a full-length TAT polypeptide sequence as disclosed herein. Ordinarily, TAT variant polypeptides are at least about 10 amino acids in length, alternatively at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600 amino acids in length, or more. Optionally, TAT variant polypeptides will have no more than one conservative amino acid substitution as compared to the native TAT polypeptide sequence, alternatively no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitution as compared to the native TAT polypeptide sequence.

[0438] "Percent (%) amino acid sequence identity" with respect to the TAT polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific TAT polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 below. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code shown in Table 1 below has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, Calif. or may be compiled from the source code provided in Table 1 below. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0439] In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. As examples of % amino acid sequence identity calculations using this method, Tables 2 and 3 demonstrate how to calculate the % amino acid sequence identity of the amino acid sequence designated "Comparison Protein" to the amino acid sequence designated "TAT", wherein "TAT" represents the amino acid sequence of a hypothetical TAT polypeptide of interest, "Comparison Protein" represents the amino acid sequence of a polypeptide against which the "TAT" polypeptide of interest is being compared, and "X, "Y" and "Z" each represent different hypothetical amino acid residues. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0440] "TAT variant polynucleotide" or "TAT variant nucleic acid sequence" means a nucleic acid molecule which encodes a TAT polypeptide, preferably an active TAT polypeptide, as defined herein and which has at least about 80% nucleic acid sequence identity with a nucleotide acid sequence encoding a full-length native sequence TAT polypeptide sequence as disclosed herein, a full-length native sequence TAT polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a TAT polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length TAT polypeptide sequence as disclosed herein (such as those encoded by a nucleic acid that represents only a portion of the complete coding sequence for a full-length TAT polypeptide). Ordinarily, a TAT variant polynucleotide will have at least about 80% nucleic acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity with a nucleic acid sequence encoding a full-length native sequence TAT polypeptide sequence as disclosed herein, a full-length native sequence TAT polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a TAT polypeptide, with or without the signal sequence, as disclosed herein or any other fragment of a full-length TAT polypeptide sequence as disclosed herein. Variants do not encompass the native nucleotide sequence.

[0441] Ordinarily, TAT variant polynucleotides are at least about 5 nucleotides in length, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 nucleotides in length, wherein in this context the term "about" means the referenced nucleotide sequence length plus or minus 10% of that referenced length.

[0442] "Percent (%) nucleic acid sequence identity" with respect to TAT-encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the TAT nucleic acid sequence of interest, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. For purposes herein, however, % nucleic acid sequence identity values are generated using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 1 below. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code shown in Table 1 below has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, Calif. or may be compiled from the source code provided in Table 1 below. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0443] In situations where ALIGN-2 is employed for nucleic acid sequence comparisons, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows: 100 times the fraction W/Z where W is the number of nucleotides scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C. As examples of % nucleic acid sequence identity calculations, Tables 4 and 5, demonstrate how to calculate the % nucleic acid sequence identity of the nucleic acid sequence designated "Comparison DNA" to the nucleic acid sequence designated "TAT-DNA", wherein "TAT-DNA" represents a hypothetical TAT-encoding nucleic acid sequence of interest, "Comparison DNA" represents the nucleotide sequence of a nucleic acid molecule against which the "TAT-DNA" nucleic acid molecule of interest is being compared, and "N", "L" and "V" each represent different hypothetical nucleotides. Unless specifically stated otherwise, all % nucleic acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0444] In other embodiments, TAT variant polynucleotides are nucleic acid molecules that encode a TAT polypeptide and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding a fall-length TAT polypeptide as disclosed herein. TAT variant polypeptides may be those that are encoded by a TAT variant polynucleotide.

[0445] The term "full-length coding region" when used in reference to a nucleic acid encoding a TAT polypeptide refers to the sequence of nucleotides which encode the full-length TAT polypeptide of the invention (which is often shown between start and stop codons, inclusive thereof, in the accompanying figures). The term "full-length coding region" when used in reference to an ATCC deposited nucleic acid refers to the TAT polypeptide-encoding portion of the cDNA that is inserted into the vector deposited with the ATCC (which is often shown between start and stop codons, inclusive thereof, in the accompanying figures).

[0446] "Isolated," when used to describe the various TAT polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the TAT polypeptide natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.

[0447] An "isolated" TAT polypeptide-encoding nucleic acid or other polypeptide-encoding nucleic acid is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the polypeptide-encoding nucleic acid. An isolated polypeptide-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated polypeptide-encoding nucleic acid molecules therefore are distinguished from the specific polypeptide-encoding nucleic acid molecule as it exists in natural cells. However, an isolated polypeptide-encoding nucleic acid molecule includes polypeptide-encoding nucleic acid molecules contained in cells that ordinarily express the polypeptide where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.

[0448] The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0449] Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0450] "Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

[0451] "Stringent conditions" or "high stringency conditions", as defined herein, may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50.degree. C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42.degree. C.; or (3) overnight hybridization in a solution that employs 50% formamide, 5.times.SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5.times.Denhardt's solution, sonicated salmon sperm DNA (50 .mu.g/ml), 0.1% SDS, and 10% dextran sulfate at 42.degree. C., with a 10 minute wash at 42.degree. C. in 0.2.times.SSC (sodium chloride/sodium citrate) followed by minute high-stringency wash consisting of 0.1.times.SSC containing EDTA at 55.degree. C.

[0452] "Moderately stringent conditions" may be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent that those described above. An example of moderately stringent conditions is overnight incubation at 37.degree. C. in a solution comprising: 20% formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5.times.Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1.times.SSC at about 37-50.degree. C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.

[0453] The term "epitope tagged" when used herein refers to a chimeric polypeptide comprising a TAT polypeptide or anti-TAT antibody fused to a "tag polypeptide". The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused. The tag polypeptide preferably also is fairly unique so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues).

[0454] "Active" or "activity" for the purposes herein refers to form(s) of a TAT polypeptide which retain a biological and/or an immunological activity of native or naturally-occurring TAT, wherein "biological" activity refers to a biological function (either inhibitory or stimulatory) caused by a native or naturally-occurring TAT other than the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring TAT and an "immunological" activity refers to the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring TAT.

[0455] The term "antagonist" is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native TAT polypeptide disclosed herein. In a similar manner, the term "agonist" is used in the broadest sense and includes any molecule that mimics a biological activity of a native TAT polypeptide disclosed herein. Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native TAT polypeptides, peptides, antisense oligonucleotides, small organic molecules, etc. Methods for identifying agonists or antagonists of a TAT polypeptide may comprise contacting a TAT polypeptide with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the TAT polypeptide.

[0456] "Treating" or "treatment" or "alleviation" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. A subject or mammal is successfully "treated" for a TAT polypeptide-expressing cancer if, after receiving a therapeutic amount of an anti-TAT antibody, TAT binding oligopeptide or TAT binding organic molecule according to the methods of the present invention, the patient shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of cancer cells or absence of the cancer cells; reduction in the tumor size; inhibition (i.e., slow to some extent and preferably stop) of cancer cell infiltration into peripheral organs including the spread of cancer into soft tissue and bone; inhibition (i.e., slow to some extent and preferably stop) of tumor metastasis; inhibition, to some extent, of tumor growth; and/or relief to some extent, one or more of the symptoms associated with the specific cancer; reduced morbidity and mortality, and improvement in quality of life issues. To the extent the anti-TAT antibody or TAT binding oligopeptide may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. Reduction of these signs or symptoms may also be felt by the patient.

[0457] The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician. For cancer therapy, efficacy can be measured, for example, by assessing the time to disease progression (TTP) and/or determining the response rate (RR). Metastasis can be determined by staging tests and by bone scan and tests for calcium level and other enzymes to determine spread to the bone. CT scans can also be done to look for spread to the pelvis and lymph nodes in the area. Chest X-rays and measurement of liver enzyme levels by known methods are used to look for metastasis to the lungs and liver, respectively. Other routine methods for monitoring the disease include transrectal ultrasonography (TRUS) and transrectal needle biopsy (TRNB).

[0458] For bladder cancer, which is a more localized cancer, methods to determine progress of disease include urinary cytologic evaluation by cystoscopy, monitoring for presence of blood in the urine, visualization of the urothelial tract by sonography or an intravenous pyelogram, computed tomography (CT) and magnetic resonance imaging (MRI). The presence of distant metastases can be assessed by CT of the abdomen, chest x-rays, or radionuclide imaging of the skeleton.

[0459] "Chronic" administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. "Intermittent" administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.

[0460] "Mammal" for purposes of the treatment of, alleviating the symptoms of or diagnosis of a cancer refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is human.

[0461] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0462] "Carriers" as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN.RTM., polyethylene glycol (PEG), and PLURONICS.RTM..

[0463] By "solid phase" or "solid support" is meant a non-aqueous matrix to which an antibody, TAT binding oligopeptide or TAT binding organic molecule of the present invention can adhere or attach. Examples of solid phases encompassed herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g., an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Pat. No. 4,275,149.

[0464] A "liposome" is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as a TAT polypeptide, an antibody thereto or a TAT binding oligopeptide) to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.

[0465] A "small" molecule or "small" organic molecule is defined herein to have a molecular weight below about 500 Daltons.

[0466] An "effective amount" of a polypeptide, antibody, TAT binding oligopeptide, TAT binding organic molecule or an agonist or antagonist thereof as disclosed herein is an amount sufficient to carry out a specifically stated purpose. An "effective amount" may be determined empirically and in a routine manner, in relation to the stated purpose.

[0467] The term "therapeutically effective amount" refers to an amount of an antibody, polypeptide, TAT binding oligopeptide, TAT binding organic molecule or other drug effective to "treat" a disease or disorder in a subject or mammal. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. See the definition herein of "treating". To the extent the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic.

[0468] A "growth inhibitory amount" of an anti-TAT antibody, TAT polypeptide, TAT binding oligopeptide or TAT binding organic molecule is an amount capable of inhibiting the growth of a cell, especially tumor, e.g., cancer cell, either in vitro or in vivo. A "growth inhibitory amount" of an anti-TAT antibody, TAT polypeptide, TAT binding oligopeptide or TAT binding organic molecule for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner.

[0469] A "cytotoxic amount" of an anti-TAT antibody, TAT polypeptide, TAT binding oligopeptide or TAT binding organic molecule is an amount capable of causing the destruction of a cell, especially tumor, e.g., cancer cell, either in vitro or in vivo. A "cytotoxic amount" of an anti-TAT antibody, TAT polypeptide, TAT binding oligopeptide or TAT binding organic molecule for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner.

[0470] The term "antibody" is used in the broadest sense and specifically covers, for example, single anti-TAT monoclonal antibodies (including agonist, antagonist, and neutralizing antibodies), anti-TAT antibody compositions with polyepitopic specificity, polyclonal antibodies, single chain anti-TAT antibodies, and fragments of anti-TAT antibodies (see below) as long as they exhibit the desired biological or immunological activity. The term "immunoglobulin" (Ig) is used interchangeable with antibody herein.

[0471] An "isolated antibody" is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0472] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains (an IgM antibody consists of 5 of the basic heterotetramer unit along with an additional polypeptide called J chain, and therefore contain 10 antigen binding sites, while secreted IgA antibodies can polymerize to form polyvalent assemblages comprising 2-5 of the basic 4-chain units along with J chain). In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to a H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (V.sub.H) followed by three constant domains (C.sub.H) for each of the .alpha. and .gamma. chains and four C.sub.H domains for .mu. and .epsilon. isotypes. Each L chain has at the N-terminus, a variable domain (V.sub.L) followed by a constant domain (C.sub.L) at its other end. The V.sub.L is aligned with the V.sub.H and the C.sub.L is aligned with the first constant domain of the heavy chain (C.sub.H1). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a V.sub.H and V.sub.L together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6.

[0473] The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (C.sub.H), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated .alpha., .delta., .epsilon., .gamma., and .mu., respectively. The .gamma. and .alpha. classes are further divided into subclasses on the basis of relatively minor differences in C.sub.H sequence and function, e.g., humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0474] The term "variable" refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and define specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable domains. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called "hypervariable regions" that are each 9-12 amino acids long. The variable domains of native heavy and light chains each comprise four FRs, largely adopting a .beta.-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the .beta.-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).

[0475] The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a "complementarity determining region"or "CDR" (e.g. around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the V.sub.L, and around about 1-35 (H1), 50-65 (H2) and 95-102 (H3) in the V.sub.H; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a "hypervariable loop" (e.g. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the V.sub.L, and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the V.sub.H; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0476] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.

[0477] The monoclonal antibodies herein include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g. Old World Monkey, Ape etc), and human constant region sequences.

[0478] An "intact" antibody is one which comprises an antigen-binding site as well as a C.sub.L and at least heavy chain constant domains, C.sub.H1, C.sub.H2 and C.sub.H3. The constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.

[0479] "Antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab').sub.2, and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0480] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (V.sub.H), and the first constant domain of one heavy chain (C.sub.H1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab').sub.2 fragment which roughly corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the C.sub.H1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0481] The Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.

[0482] "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0483] "Single-chain Fv" also abbreviated as "sFv" or "scFv" are antibody fragments that comprise the V.sub.H and V.sub.L antibody domains connected into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the V.sub.H and V.sub.L domains which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra.

[0484] The term "diabodies" refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short linkers (about 5-10 residues) between the V.sub.H and V.sub.L domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments in which the V.sub.H and V.sub.L domains of the two antibodies are present on different polypeptide chains. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0485] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. 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 hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0486] A "species-dependent antibody," e.g., a mammalian anti-human IgE antibody, is an antibody which has a stronger binding affinity for an antigen from a first mammalian species than it has for a homologue of that antigen from a second mammalian species. Normally, the species-dependent antibody "bind specifically" to a human antigen (i.e., has a binding affinity (Kd) value of no more than about 1.times.10.sup.-7 M, preferably no more than about 1.times.10.sup.-8 and most preferably no more than about 1.times.10.sup.-9 M) but has a binding affinity for a homologue of the antigen from a second non-human mammalian species which is at least about 50 fold, or at least about 500 fold, or at least about 1000 fold, weaker than its binding affinity for the human antigen. The species-dependent antibody can be of any of the various types of antibodies as defined above, but preferably is a humanized or human antibody.

[0487] A "TAT binding oligopeptide" is an oligopeptide that binds, preferably specifically, to a TAT polypeptide as described herein. TAT binding oligopeptides may be chemically synthesized using known oligopeptide synthesis methodology or may be prepared and purified using recombinant technology. TAT binding oligopeptides are usually at least about 5 amino acids in length, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids in length or more, wherein such oligopeptides that are capable of binding, preferably specifically, to a TAT polypeptide as described herein. TAT binding oligopeptides may be identified without undue experimentation using well known techniques. In this regard, it is noted that techniques for screening oligopeptide libraries for oligopeptides that are capable of specifically binding to a polypeptide target are well known in the art (see, e.g., U.S. Pat. Nos. 5,556,762, 5,750,373, 4,708,871, 4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCT Publication Nos. WO 84/03506 and W084/03564; Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 81:3998-4002(1984); Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 82:178-182(1985); Geysen et al., in Synthetic Peptides as Antigens, 130-149(1986); Geysen et al., J. Immunol. Meth., 102:259-274 (1987); Schoofs et al., J. Immunol., 140:611-616 (1988), Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6378; Lowman, H. B. et al. (1991) Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8363, and Smith, G. P. (1991) Current Opin. Biotechnol., 2:668).

[0488] A "TAT binding organic molecule" is an organic molecule other than an oligopeptide or antibody as defined herein that binds, preferably specifically, to a TAT polypeptide as described herein. TAT binding organic molecules may be identified and chemically synthesized using known methodology (see, e.g., PCT Publication Nos. WO00/00823 and WO00/39585). TAT binding organic molecules are usually less than about 2000 daltons in size, alternatively less than about 1500, 750, 500, 250 or 200 daltons in size, wherein such organic molecules that are capable of binding, preferably specifically, to a TAT polypeptide as described herein may be identified without undue experimentation using well known techniques. In this regard, it is noted that techniques for screening organic molecule libraries for molecules that are capable of binding to a polypeptide target are well known in the art (see, e.g., PCT Publication Nos. WO00/00823 and WO00/39585).

[0489] An antibody, oligopeptide or other organic molecule "which binds" an antigen of interest, e.g. a tumor-associated polypeptide antigen target, is one that binds the antigen with sufficient affinity such that the antibody, oligopeptide or other organic molecule is useful as a diagnostic and/or therapeutic agent in targeting a cell or tissue expressing the antigen, and does not significantly cross-react with other proteins. In such embodiments, the extent of binding of the antibody, oligopeptide or other organic molecule to a "non-target" protein will be less than about 10% of the binding of the antibody, oligopeptide or other organic molecule to its particular target protein as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA). With regard to the binding of an antibody, oligopeptide or other organic molecule to a target molecule, the term "specific binding" or "specifically binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide target means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target. The term "specific binding" or "specifically binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a Kd for the target of at least about 10.sup.-4 M, alternatively at least about 10.sup.-5 M, alternatively at least about 10.sup.-6 M, alternatively at least about 10.sup.-7 M, alternatively at least about 10.sup.-8 M, alternatively at least about 10.sup.-9 M, alternatively at least about 10.sup.-10 M, alternatively at least about 10.sup.11 M, alternatively at least about 10.sup.-12 M, or greater. In one embodiment, the term "specific binding" refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

[0490] An antibody, oligopeptide or other organic molecule that "inhibits the growth of tumor cells expressing a TAT polypeptide" or a "growth inhibitory" antibody, oligopeptide or other organic molecule is one which results in measurable growth inhibition of cancer cells expressing or overexpressing the appropriate TAT polypeptide. The TAT polypeptide may be a transmembrane polypeptide expressed on the surface of a cancer cell or may be a polypeptide that is produced and secreted by a cancer cell. Preferred growth inhibitory anti-TAT antibodies, oligopeptides or organic molecules inhibit growth of TAT-expressing tumor cells by greater than 20%, preferably from about 20% to about 50%, and even more preferably, by greater than 50% (e.g., from about 50% to about 100%) as compared to the appropriate control, the control typically being tumor cells not treated with the antibody, oligopeptide or other organic molecule being tested. In one embodiment, growth inhibition can be measured at an antibody concentration of about 0.1 to 30 .mu.g/ml or about 0.5 nM to 200 nM in cell culture, where the growth inhibition is determined 1-10 days after exposure of the tumor cells to the antibody. Growth inhibition of tumor cells in vivo can be determined in various ways such as is described in the Experimental Examples section below. The antibody is growth inhibitory in vivo if administration of the anti-TAT antibody at about 1 .mu.g/kg to about 100 mg/kg body weight results in reduction in tumor size or tumor cell proliferation within about 5 days to 3 months from the first administration of the antibody, preferably within about 5 to 30 days.

[0491] An antibody, oligopeptide or other organic molecule which "induces apoptosis" is one which induces programmed cell death as determined by binding of annexin V, fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies). The cell is usually one which overexpresses a TAT polypeptide. Preferably the cell is a tumor cell, e.g., a prostate, breast, ovarian, stomach, endometrial, lung, kidney, colon, bladder cell. Various methods are available for evaluating the cellular events associated with apoptosis. For example, phosphatidyl serine (PS) translocation can be measured by annexin binding; DNA fragmentation can be evaluated through DNA laddering; and nuclear/chromatin condensation along with DNA fragmentation can be evaluated by any increase in hypodiploid cells. Preferably, the antibody, oligopeptide or other organic molecule which induces apoptosis is one which results in about 2 to 50 fold, preferably about 5 to 50 fold, and most preferably about 10 to 50 fold, induction of annexin binding relative to untreated cell in an annexin binding assay.

[0492] Antibody "effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0493] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies "arm" the cytotoxic cells and are absolutely required for such killing. The primary cells for mediating ADCC, NK cells, express Fc.gamma.RIII only, whereas monocytes express Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al. (USA) 95:652-656 (1998).

[0494] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. The preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII subclasses, including allelic variants and alternatively spliced forms of these receptors. Fc.gamma.RII receptors include Fc.gamma.RIIA (an "activating receptor") and Fc.gamma.RIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor Fc.gamma.RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (see review M. in Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

[0495] "Human effector cells" are leukocytes which express one or more FcRs and perform effector functions. Preferably, the cells express at least Fc.gamma.RIII and perform ADCC effector function. Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferred. The effector cells may be isolated from a native source, e.g., from blood.

[0496] "Complement dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) which are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed.

[0497] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, multiple myeloma and B-cell lymphoma, brain, as well as head and neck cancer, and associated metastases.

[0498] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders that are associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

[0499] "Tumor", as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0500] An antibody, oligopeptide or other organic molecule which "induces cell death" is one which causes a viable cell to become nonviable. The cell is one which expresses a TAT polypeptide, preferably a cell that overexpresses a TAT polypeptide as compared to a normal cell of the same tissue type. The TAT polypeptide may be a transmembrane polypeptide expressed on the surface of a cancer cell or may be a polypeptide that is produced and secreted by a cancer cell. Preferably, the cell is a cancer cell, e.g., a breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic or bladder cell. Cell death in vitro may be determined in the absence of complement and immune effector cells to distinguish cell death induced by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). Thus, the assay for cell death may be performed using heat inactivated serum (i.e., in the absence of complement) and in the absence of immune effector cells. To determine whether the antibody, oligopeptide or other organic molecule is able to induce cell death, loss of membrane integrity as evaluated by uptake of propidium iodide (PI), trypan blue (see Moore et al. Cytotechnology 17:1-11 (1995)) or 7AAD can be assessed relative to untreated cells. Preferred cell death-inducing antibodies, oligopeptides or other organic molecules are those which induce PI uptake in the PI uptake assay in BT474 cells.

[0501] A "TAT-expressing cell" is a cell which expresses an endogenous or transfected TAT polypeptide either on the cell surface or in a secreted form. A "TAT-expressing cancer" is a cancer comprising cells that have a TAT polypeptide present on the cell surface or that produce and secrete a TAT polypeptide. A "TAT-expressing cancer" optionally produces sufficient levels of TAT polypeptide on the surface of cells thereof, such that an anti-TAT antibody, oligopeptide ot other organic molecule can bind thereto and have a therapeutic effect with respect to the cancer. In another embodiment, a "TAT-expressing cancer" optionally produces and secretes sufficient levels of TAT polypeptide, such that an anti-TAT antibody, oligopeptide ot other organic molecule antagonist can bind thereto and have a therapeutic effect with respect to the cancer. With regard to the latter, the antagonist may be an antisense oligonucleotide which reduces, inhibits or prevents production and secretion of the secreted TAT polypeptide by tumor cells. A cancer which "overexpresses" a TAT polypeptide is one which has significantly higher levels of TAT polypeptide at the cell surface thereof, or produces and secretes, compared to a noncancerous cell of the same tissue type. Such overexpression may be caused by gene amplification or by increased transcription or translation. TAT polypeptide overexpression may be determined in a diagnostic or prognostic assay by evaluating increased levels of the TAT protein present on the surface of a cell, or secreted by the cell (e.g., via an immunohistochemistry assay using anti-TAT antibodies prepared against an isolated TAT polypeptide which may be prepared using recombinant DNA technology from an isolated nucleic acid encoding the TAT polypeptide; FACS analysis, etc.). Alternatively, or additionally, one may measure levels of TAT polypeptide-encoding nucleic acid or mRNA in the cell, e.g., via fluorescent in situ hybridization using a nucleic acid based probe corresponding to a TAT-encoding nucleic acid or the complement thereof; (FISH; see WO98/45479 published October, 1998), Southern blotting, Northern blotting, or polymerase chain reaction (PCR) techniques, such as real time quantitative PCR (RT-PCR). One may also study TAT polypeptide overexpression by measuring shed antigen in a biological fluid such as serum, e.g., using antibody-based assays (see also, e.g., U.S. Pat. No. 4,933,294 issued Jun. 12, 1990; WO91/05264 published Apr. 18, 1991; U.S. Pat. No. 5,401,638 issued Mar. 28, 1995; and Sias et al., J. Immunol. Methods 132:73-80 (1990)). Aside from the above assays, various in vivo assays are available to the skilled practitioner. For example, one may expose cells within the body of the patient to an antibody which is optionally labeled with a detectable label, e.g., a radioactive isotope, and binding of the antibody to cells in the patient can be evaluated, e.g., by external scanning for radioactivity or by analyzing a biopsy taken from a patient previously exposed to the antibody.

[0502] As used herein, the term "immunoadhesin" designates antibody-like molecules which combine the binding specificity of a heterologous protein (an "adhesin") with the effector functions of immunoglobulin constant domains. Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (i.e., is "heterologous"), and an immunoglobulin constant domain sequence. The adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand. The immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM.

[0503] The word "label" when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody, oligopeptide or other organic molecule so as to generate a "labeled" antibody, oligopeptide or other organic molecule. The label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.

[0504] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., At.sup.211, I.sup.131, I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32 and radioactive isotopes of Lu), chemotherapeutic agents e.g. methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, and the various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below. A tumoricidal agent causes destruction of tumor cells.

[0505] A "growth inhibitory agent" when used herein refers to a compound or composition which inhibits growth of a cell, especially a TAT-expressing cancer cell, either in vitro or in vivo. Thus, the growth inhibitory agent may be one which significantly reduces the percentage of TAT-expressing cells in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce G1 arrest and M-phase arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest G1 also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel and docetaxel) are anticancer drugs both derived from the yew tree. Docetaxel (TAXOTERE.RTM., Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL.RTM., Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.

[0506] "Doxorubicin" is an anthracycline antibiotic. The full chemical name of doxorubicin is (8S-cis)-10-[(3-amino-2,3,6-trideoxy-.alpha.-L-lyxo-hexapyranosyl)oxy]-7,- 8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-napht- hacenedione.

[0507] The term "cytokine" is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-.alpha. and -.beta.; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-.beta.; platelet-growth factor; transforming growth factors (TGFs) such as TGF-.alpha. and TGF-.beta.; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon -.alpha., -.beta., and -.gamma.; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such as TNF-.alpha. or TNF-.beta.; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

[0508] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products. TABLE-US-00001 TABLE 2 TAT XXXXXXXXXXXXXXX (Length = 15 amino acids) Comparison XXXXXYYYYYYY (Length = 12 amino acids) Protein % amino acid sequence identity = (the number of identically matching amino acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of amino acid residues of the TAT polypeptide) = 5 divided by 15 = 33.3%

[0509] TABLE-US-00002 TABLE 3 TAT XXXXXXXXXX (Length = 10 amino acids) Comparison XXXXXYYYYYYZZYZ (Length = 15 amino acids) Protein % amino acid sequence identity = (the number of identically matching amino acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of amino acid residues of the TAT polypeptide) = 5 divided by 10 = 50%

[0510] TABLE-US-00003 TABLE 4 TAT-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides) Comparison NNNNNNLLLLLLLLLL (Length = 16 nucleotides) DNA % nucleic acid sequence identity = (the number of identically matching nucleotides between the two nucleic acid sequences as determined by ALIGN-2) divided by (the total number of nucleotides of the TAT-DNA nucleic acid sequence) = 6 divided by 14 = 42.9%

[0511] TABLE-US-00004 TABLE 5 TAT-DNA NNNNNNNNNNNN (Length = 12 nucleotides) Comparison DNA NNNNLLLVV (Length = 9 nucleotides) % nucleic acid sequence identity = (the number of identically matching nucleotides between the two nucleic acid sequences as determined by ALIGN-2) divided by (the total number of nucleotides of the TAT-DNA nucleic acid sequence) = 4 divided by 12 = 33.3%

II. Compositions and Methods of the Invention

[0512] A. Anti-TAT Antibodies

[0513] In one embodiment, the present invention provides anti-TAT antibodies which may find use herein as therapeutic and/or diagnostic agents. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies. [0514] 1. Polyclonal Antibodies

[0515] Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen (especially when synthetic peptides are used) to a protein that is immunogenic in the species to be immunized. For example, the antigen can be conjugated to keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctional or derivatizing agent, e.g., maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR, where R and R.sup.1 are different alkyl groups.

[0516] Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 .mu.g or 5 .mu.g of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later, the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response. [0517] 2. Monoclonal Antibodies

[0518] Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

[0519] In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. After immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)).

[0520] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium which medium preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the selective culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

[0521] Preferred fusion partner myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a selective medium that selects against the unfused parental cells. Preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 and derivatives e.g., X63-Ag8-653 cells available from the American Type Culture Collection, Manassas, Va., USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0522] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0523] The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson et al., Anal. Biochem., 107:220 (1980).

[0524] Once hybridoma cells that produce antibodies of the desired specificity, affinity, and/or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal e.g., by i.p. injection of the cells into mice.

[0525] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography (e.g., using protein A or protein G-Sepharose) or ion-exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.

[0526] DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Revs. 130:151-188 (1992).

[0527] In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res. 21:2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.

[0528] The DNA that encodes the antibody may be modified to produce chimeric or fusion antibody polypeptides, for example, by substituting human heavy chain and light chain constant domain (C.sub.H and C.sub.L) sequences for the homologous murine sequences (U.S. Pat. No. 4,816,567; and Morrison, et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by fusing the immunoglobulin coding sequence with all or part of the coding sequence for a non-immunoglobulin polypeptide (heterologous polypeptide). The non-immunoglobulin polypeptide sequences can substitute for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen. [0529] 3. Human and Humanized Antibodies

[0530] The anti-TAT antibodies of the invention may further comprise humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from 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 which 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 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., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].

[0531] 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 which 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., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (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.

[0532] The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity and HAMA response (human anti-mouse antibody) when the antibody is intended for human therapeutic use. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human V domain sequence which is closest to that of the rodent is identified and the human framework region (FR) within it accepted for the humanized antibody (Sims et al., J. Immunol. 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993)).

[0533] It is further important that antibodies be humanized with retention of high binding affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0534] Various forms of a humanized anti-TAT antibody are contemplated. For example, the humanized antibody may be an antibody fragment, such as a Fab, which is optionally conjugated with one or more cytotoxic agent(s) in order to generate an immunoconjugate. Alternatively, the humanized antibody may be an intact antibody, such as an intact IgG1 antibody.

[0535] As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (J.sub.H) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno. 7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669 (all of GenPharm); U.S. Pat. No. 5,545,807; and WO 97/17852.

[0536] Alternatively, phage display technology (McCafferty et al., Nature 348:552-553 [1990]) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B-cell. Phage display can be performed in a variety of formats, reviewed in, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V-gene segments can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905.

[0537] As discussed above, human antibodies may also be generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275). [0538] 4. Antibody Fragments

[0539] In certain circumstances there are advantages of using antibody fragments, rather than whole antibodies. The smaller size of the fragments allows for rapid clearance, and may lead to improved access to solid tumors.

[0540] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab').sub.2 fragments (Carter et al., Bio/Technology 10: 163-167 (1992)). According to another approach, F(ab').sub.2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab').sub.2 fragment with increased in vivo half-life comprising a salvage receptor binding epitope residues are described in U.S. Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458. Fv and sFv are the only species with intact combining sites that are devoid of constant regions; thus, they are suitable for reduced nonspecific binding during in vivo use. sFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an sFv. See Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment may also be a "linear antibody", e.g., as described in U.S. Pat. 5,641,870 for example. Such linear antibody fragments may be monospecific or bispecific.

[0541] 5. Bispecific Antibodies

[0542] Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of a TAT protein as described herein. Other such antibodies may combine a TAT binding site with a binding site for another protein. Alternatively, an anti-TAT arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD3), or Fc receptors for IgG (Fc.gamma.R), such as Fc.gamma.RI (CD64), Fc.gamma.RII (CD32) and Fc.gamma.RIII (CD16), so as to focus and localize cellular defense mechanisms to the TAT-expressing cell. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express TAT. These antibodies possess a TAT-binding arm and an arm which binds the cytotoxic agent (e.g., saporin, anti-interferon-.alpha., vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab').sub.2 bispecific antibodies).

[0543] WO 96/16673 describes a bispecific anti-ErbB2/anti-Fc.gamma.RIII antibody and U.S. Pat. No. 5,837,234 discloses a bispecific anti-ErbB2/anti-Fc.gamma.RI antibody. A bispecific anti-ErbB2/Fc.alpha. antibody is shown in WO98/02463. U.S. Pat. No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3 antibody.

[0544] Methods for making bispecific antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al., EMBO J. 10:3655-3659 (1991).

[0545] According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. Preferably, the fusion is with an Ig heavy chain constant domain, comprising at least part of the hinge, C.sub.H2, and C.sub.H3 regions. It is preferred to have the first heavy-chain constant region (C.sub.H1) containing the site necessary for light chain bonding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host cell. This provides for greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yield of the desired bispecific antibody. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios have no significant affect on the yield of the desired chain combination.

[0546] In a preferred embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology 121:210 (1986).

[0547] According to another approach described in U.S. Patent No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the C.sub.H3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

[0548] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.

[0549] Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab').sub.2 fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

[0550] Recent progress has facilitated the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab').sub.2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

[0551] Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5): 1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a V.sub.H connected to a V.sub.L by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the V.sub.H and V.sub.L domains of one fragment are forced to pair with the complementary V.sub.L and V.sub.H domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).

[0552] Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991). [0553] 6. Heteroconjugate Antibodies

[0554] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U.S. Pat. No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat. No. 4,676,980. [0555] 7. Multivalent Antibodies

[0556] A multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind. The antibodies of the present invention can be multivalent antibodies (which are other than of the IgM class) with three or more antigen binding sites (e.g. tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody. The multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. The preferred dimerization domain comprises (or consists of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region. The preferred multivalent antibody herein comprises (or consists of) three to about eight, but preferably four, antigen binding sites. The multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable domains. For instance, the polypeptide chain(s) may comprise VD1-(X1).sub.n-VD2-(X2).sub.n-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent an amino acid or polypeptide, and n is 0 or 1. For instance, the polypeptide chain(s) may comprise: VH--CH1-flexible linker-VH--CH1-Fc region chain; or VH--CH1--VH--CH1-Fc region chain. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable domain polypeptides. The multivalent antibody herein may, for instance, comprise from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated here comprise a light chain variable domain and, optionally, further comprise a CL domain. [0557] 8. Effector Function Engineering

[0558] It may be desirable to modify the antibody of the invention with respect to effector function, e.g., so as to enhance antigen-dependent cell-mediated cyotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) of the antibody. This may be achieved by introducing one or more amino acid substitutions in an Fc region of the antibody. Alternatively or additionally, cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989).

[0559] To increase the serum half life of the antibody, one may incorporate a salvage receptor binding epitope into the antibody (especially an antibody fragment) as described in U.S. Pat. No. 5,739,277, for example. As used herein, the term "salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, or IgG.sub.4) that is responsible for increasing the in vivo serum half-life of the IgG molecule. [0560] 9. Immunoconjugates

[0561] The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).

[0562] Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include .sup.212Bi, .sup.131I, .sup.131In, .sup.90Y, and .sup.186Re. Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science,238: 1098(1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.

[0563] Conjugates of an antibody and one or more small molecule toxins, such as a calicheamicin, maytansinoids, a trichothene, and CC 1065, and the derivatives of these toxins that have toxin activity, are also contemplated herein.

Maytansine and Maytansinoids

[0564] In one preferred embodiment, an anti-TAT antibody (full length or fragments) of the invention is conjugated to one or more maytansinoid molecules.

[0565] Maytansinoids are mitototic inhibitors which act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and derivatives and analogues thereof are disclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533, the disclosures of which are hereby expressly incorporated by reference.

Maytansinoid-Antibody Conjugates

[0566] In an attempt to improve their therapeutic index, maytansine and maytansinoids have been conjugated to antibodies specifically binding to tumor cell antigens. Immunoconjugates containing maytansinoids and their therapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1, the disclosures of which are hereby expressly incorporated by reference. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugates comprising a maytansinoid designated DM1 linked to the monoclonal antibody C242 directed against human colorectal cancer. The conjugate was found to be highly cytotoxic towards cultured colon cancer cells, and showed antitumor activity in an in vivo tumor growth assay. Chari et al., Cancer Research 52:127-131 (1992) describe immunoconjugates in which a maytansinoid was conjugated via a disulfide linker to the murine antibody A7 binding to an antigen on human colon cancer cell lines, or to another murine monoclonal antibody TA. 1 that binds the HER-2/neu oncogene. The cytotoxicity of the TA. 1-maytansonoid conjugate was tested in vitro on the human breast cancer cell line SK-BR-3, which expresses 3.times.10.sup.5 HER-2 surface antigens per cell. The drug conjugate achieved a degree of cytotoxicity similar to the free maytansonid drug, which could be increased by increasing the number of maytansinoid molecules per antibody molecule. The A7-maytansinoid conjugate showed low systemic cytotoxicity in mice.

Anti-TAT Polypeptide Antibody-Maytansinoid Conjugates (Immunoconjugates)

[0567] Anti-TAT antibody-maytansinoid conjugates are prepared by chemically linking an anti-TAT antibody to a maytansinoid molecule without significantly diminishing the biological activity of either the antibody or the maytansinoid molecule. An average of 3-4 maytansinoid molecules conjugated per antibody molecule has shown efficacy in enhancing cytotoxicity of target cells without negatively affecting the function or solubility of the antibody, although even one molecule of toxin/antibody would be expected to enhance cytotoxicity over the use of naked antibody. Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources. Suitable maytansinoids are disclosed, for example, in U.S. Pat. No. 5,208,020 and in the other patents and nonpatent publications referred to hereinabove. Preferred maytansinoids are maytansinol and maytansinol analogues modified in the aromatic ring or at other positions of the maytansinol molecule, such as various maytansinol esters.

[0568] There are many linking groups known in the art for making antibody-maytansinoid conjugates, including, for example, those disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, and Chari et al., Cancer Research 52:127-131 (1992). The linking groups include disufide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, or esterase labile groups, as disclosed in the above-identified patents, disulfide and thioether groups being preferred.

[0569] Conjugates of the antibody and maytansinoid may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succininidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agents include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlssonet al., Biochem. J. 173:723-737 [1978]) and N-succininidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a disulfide linkage.

[0570] The linker may be attached to the maytansinoid molecule at various positions, depending on the type of the link. For example, an ester linkage may be formed by reaction with a hydroxyl group using conventional coupling techniques. The reaction may occur at the C-3 position having a hydroxyl group, the C-14 position modified with hyrdoxymethyl, the C-15 position modified with a hydroxyl group, and the C-20 position having a hydroxyl group. In a preferred embodiment, the linkage is formed at the C-3 position of maytansinol or a maytansinol analogue.

Calicheamicin

[0571] Another immunoconjugate of interest comprises an anti-TAT antibody conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of conjugates of the calicheamicin family, see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296 (all to American Cyanamid Company). Structural analogues of calicheamicin which may be used include, but are not limited to, .gamma..sub.1.sup.I, .alpha..sub.2.sup.I, .alpha..sub.3.sup.I, N-acetyl-.gamma..sub.1.sup.I, PSAG and .theta..sup.I.sub.1 (Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998) and the aforementioned U.S. patents to American Cyanamid). Another anti-tumor drug that the antibody can be conjugated is QFA which is an antifolate. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Therefore, cellular uptake of these agents through antibody mediated internalization greatly enhances their cytotoxic effects.

Other Cytotoxic Agents

[0572] Other antitumor agents that can be conjugated to the anti-TAT antibodies of the invention include BCNU, streptozoicin, vincristine and 5-fluorouracil, the family of agents known collectively LL-E33288 complex described in U.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No. 5,877,296).

[0573] Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, WO 93/21232 published Oct. 28, 1993.

[0574] The present invention further contemplates an immunoconjugate formed between an antibody and a compound with nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

[0575] For selective destruction of the tumor, the antibody may comprise a highly radioactive atom. A variety of radioactive isotopes are available for the production of radioconjugated anti-TAT antibodies. Examples include At.sup.211, I.sup.131, I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32, Pb.sup.212 and radioactive isotopes of Lu. When the conjugate is used for diagnosis, it may comprise a radioactive atom for scintigraphic studies, for example tc.sup.99m or I.sup.123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

[0576] The radio- or other labels may be incorporated in the conjugate in known ways. For example, the peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen. Labels such as tc.sup.99m or I.sup.123 , Re.sup.186, Re.sup.188 and In.sup.111 can be attached via a cysteine residue in the peptide. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123. "Monoclonal Antibodies in Immunoscintigraphy" (Chatal,CRC Press 1989) describes other methods in detail.

[0577] Conjugates of the antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026. The linker may be a "cleavable linker" facilitating release of the cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat. No. 5,208,020) may be used.

[0578] Alternatively, a fusion protein comprising the anti-TAT antibody and cytotoxic agent may be made, e.g., by recombinant techniques or peptide synthesis. The length of DNA may comprise respective regions encoding the two portions of the conjugate either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate.

[0579] In yet another embodiment, the antibody may be conjugated to a "receptor" (such streptavidin) for utilization in tumor pre-targeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e.g., avidin) which is conjugated to a cytotoxic agent (e.g., a radionucleotide). [0580] 10. Immunoliposomes

[0581] The anti-TAT antibodies disclosed herein may also be formulated as immunoliposomes. A "liposome" is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; and WO97/38731 published Oct. 23, 1997. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.

[0582] Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem. 257:286-288 (1982) via a disulfide interchange reaction. A chemotherapeutic agent is optionally contained within the liposome. See Gabizon et al., J. National Cancer Inst. 81(19): 1484 (1989).

[0583] B. TAT Binding Oligopeptides

[0584] TAT binding oligopeptides of the present invention are oligopeptides that bind, preferably specifically, to a TAT polypeptide as described herein. TAT binding oligopeptides may be chemically synthesized using known oligopeptide synthesis methodology or may be prepared and purified using recombinant technology. TAT binding oligopeptides are usually at least about 5 amino acids in length, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids in length or more, wherein such oligopeptides that are capable of binding, preferably specifically, to a TAT polypeptide as described herein. TAT binding oligopeptides may be identified without undue experimentation using well known techniques. In this regard, it is noted that techniques for screening oligopeptide libraries for oligopeptides that are capable of specifically binding to a polypeptide target are well known in the art (see, e.g., U.S. Pat. Nos. 5,556,762, 5,750,373, 4,708,871, 4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCT Publication Nos. WO 84/03506 and W084/03564; Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 81:3998-4002 (1984); Geysen et al., Proc. Natl. Acad. Sci. U.S.A., 82:178-182 (1985); Geysen et al., in Synthetic Peptides as Antigens, 130-149 (1986); Geysen et al., J. Immunol. Meth., 102:259-274 (1987); Schoofs et al., J. Immunol., 140:611-616 (1988), Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6378; Lowman, H. B. et al. (1991) Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8363, and Smith, G. P. (1991) Current Opin. Biotechnol., 2:668).

[0585] In this regard, bacteriophage (phage) display is one well known technique which allows one to screen large oligopeptide libraries to identify member(s) of those libraries which are capable of specifically binding to a polypeptide target. Phage display is a technique by which variant polypeptides are displayed as fusion proteins to the coat protein on the surface of bacteriophage particles (Scott, J. K. and Smith, G. P. (1990) Science 249: 386). The utility of phage display lies in the fact that large libraries of selectively randomized protein variants (or randomly cloned cDNAs) can be rapidly and efficiently sorted for those sequences that bind to a target molecule with high affinity. Display of peptide (Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6378) or protein (Lowman, H. B. et al. (1991) Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8363) libraries on phage have been used for screening millions of polypeptides or oligopeptides for ones with specific binding properties (Smith, G. P. (1991) Current Opin. Biotechnol., 2:668). Sorting phage libraries of random mutants requires a strategy for constructing and propagating a large number of variants, a procedure for affinity purification using the target receptor, and a means of evaluating the results of binding enrichments. U.S. Pat. Nos. 5,223,409, 5,403,484, 5,571,689, and 5,663,143.

[0586] Although most phage display methods have used filamentous phage, lambdoid phage display systems (WO 95/34683; U.S. Pat. No. 5,627,024), T4 phage display systems (Ren et al., Gene, 215: 439 (1998); Zhu et al., Cancer Research, 58(15): 3209-3214 (1998); Jiang et al., Infection & Immunity, 65(11): 4770-4777(1997); Ren et al., Gene, 195(2):303-311 (1997); Ren, Protein Sci., 5: 1833 (1996); Efimov et al., Virus Genes, 10: 173 (1995)) and T7 phage display systems (Smith and Scott, Methods in Enzymology, 217: 228-257 (1993); U.S. Pat. No. 5,766,905) are also known.

[0587] Many other improvements and variations of the basic phage display concept have now been developed. These improvements enhance the ability of display systems to screen peptide libraries for binding to selected target molecules and to display functional proteins with the potential of screening these proteins for desired properties. Combinatorial reaction devices for phage display reactions have been developed (WO 98/14277) and phage display libraries have been used to analyze and control bimolecular interactions (WO 98/20169; WO 98/20159) and properties of constrained helical peptides (WO 98/20036). WO 97/35196 describes a method of isolating an affinity ligand in which a phage display library is contacted with one solution in which the ligand will bind to a target molecule and a second solution in which the affinity ligand will not bind to the target molecule, to selectively isolate binding ligands. WO 97/46251 describes a method of biopanning a random phage display library with an affinity purified antibody and then isolating binding phage, followed by a micropanning process using microplate wells to isolate high affinity binding phage. The use of Staphlylococcus aureus protein A as an affinity tag has also been reported (Li et al. (1998) Mol Biotech., 9:187). WO 97/47314 describes the use of substrate subtraction libraries to distinguish enzyme specificities using a combinatorial library which may be a phage display library. A method for selecting enzymes suitable for use in detergents using phage display is described in WO 97/09446. Additional methods of selecting specific binding proteins are described in U.S. Pat. Nos. 5,498,538, 5,432,018, and WO 98/15833.

[0588] Methods of generating peptide libraries and screening these libraries are also disclosed in U.S. Pat. Nos. 5,723,286, 5,432,018, 5,580,717, 5,427,908, 5,498,530, 5,770,434, 5,734,018, 5,698,426, 5,763,192, and 5,723,323.

[0589] C. TAT Binding Organic Molecules

[0590] TAT binding organic molecules are organic molecules other than oligopeptides or antibodies as defined herein that bind, preferably specifically, to a TAT polypeptide as described herein. TAT binding organic molecules may be identified and chemically synthesized using known methodology (see, e.g., PCT Publication Nos. WO00/00823 and WO00/39585). TAT binding organic molecules are usually less than about 2000 daltons in size, alternatively less than about 1500, 750, 500, 250 or 200 daltons in size, wherein such organic molecules that are capable of binding, preferably specifically, to a TAT polypeptide as described herein may be identified without undue experimentation using well known techniques. In this regard, it is noted that techniques for screening organic molecule libraries for molecules that are capable of binding to a polypeptide target are well known in the art (see, e.g., PCT Publication Nos. WO00/00823 and WO00/39585). TAT binding organic molecules may be, for example, aldehydes, ketones, oximes, hydrazones, semicarbazones, carbazides, primary amines, secondary amines, tertiary amines, N-substituted hydrazines, hydrazides, alcohols, ethers, thiols, thioethers, disulfides, carboxylic acids, esters, amides, ureas, carbamates, carbonates, ketals, thioketals, acetals, thioacetals, aryl halides, aryl sulfonates, alkyl halides, alkyl sulfonates, aromatic compounds, heterocyclic compounds, anilines, alkenes, alkynes, diols, amino alcohols, oxazolidines, oxazolines, thiazolidines, thiazolines, enamines, sulfonamides, epoxides, aziridines, isocyanates, sulfonyl chlorides, diazo compounds, acid chlorides, or the like.

[0591] D. Screening for Anti-TAT Antibodies, TAT Binding Oligopeptides and TAT Binding Organic Molecules With the Desired Properties

[0592] Techniques for generating antibodies, oligopeptides and organic molecules that bind to TAT polypeptides have been described above. One may further select antibodies, oligopeptides or other organic molecules with certain biological characteristics, as desired.

[0593] The growth inhibitory effects of an anti-TAT antibody, oligopeptide or other organic molecule of the invention may be assessed by methods known in the art, e.g., using cells which express a TAT polypeptide either endogenously or following transfection with the TAT gene. For example, appropriate tumor cell lines and TAT-transfected cells may treated with an anti-TAT monoclonal antibody, oligopeptide or other organic molecule of the invention at various concentrations for a few days (e.g., 2-7) days and stained with crystal violet or MTT or analyzed by some other colorimetric assay. Another method of measuring proliferation would be by comparing .sup.3H-thymidine uptake by the cells treated in the presence or absence an anti-TAT antibody, TAT binding oligopeptide or TAT binding organic molecule of the invention. After treatment, the cells are harvested and the amount of radioactivity incorporated into the DNA quantitated in a scintillation counter. Appropriate positive controls include treatment of a selected cell line with a growth inhibitory antibody known to inhibit growth of that cell line. Growth inhibition of tumor cells in vivo can be determined in various ways known in the art. Preferably, the tumor cell is one that overexpresses a TAT polypeptide. Preferably, the anti-TAT antibody, TAT binding oligopeptide or TAT binding organic molecule will inhibit cell proliferation of a TAT-expressing tumor cell in vitro or in vivo by about 25-100% compared to the untreated tumor cell, more preferably, by about 30-100%, and even more preferably by about 50-100% or 70-100%, in one embodiment, at an antibody concentration of about 0.5 to 30 .mu.g/ml. Growth inhibition can be measured at an antibody concentration of about 0.5 to 30 .mu.g/ml or about 0.5 nM to 200 nM in cell culture, where the growth inhibition is determined 1-10 days after exposure of the tumor cells to the antibody. The antibody is growth inhibitory in vivo if administration of the anti-TAT antibody at about 1 .mu.g/kg to about 100 mg/kg body weight results in reduction in tumor size or reduction of tumor cell proliferation within about 5 days to 3 months from the first administration of the antibody, preferably within about 5 to 30 days.

[0594] To select for an anti-TAT antibody, TAT binding oligopeptide or TAT binding organic molecule which induces cell death, loss of membrane integrity as indicated by, e.g., propidium iodide (PI), trypan blue or 7AAD uptake may be assessed relative to control. A PI uptake assay can be performed in the absence of complement and immune effector cells. TAT polypeptide-expressing tumor cells are incubated with medium alone or medium containing the appropriate anti-TAT antibody (e.g., at about 10 .mu.g/ml), TAT binding oligopeptide or TAT binding organic molecule. The cells are incubated for a 3 day time period. Following each treatment, cells are washed and aliquoted into 35 mm strainer-capped 12.times.75 tubes (1 ml per tube, 3 tubes per treatment group) for removal of cell clumps. Tubes then receive PI (10 .mu.g/ml). Samples may be analyzed using a FACSCAN.RTM. flow cytometer and FACSCONVERT.RTM. CellQuest software (Becton Dickinson). Those anti-TAT antibodies, TAT binding oligopeptides or TAT binding organic molecules that induce statistically significant levels of cell death as determined by PI uptake may be selected as cell death-inducing anti-TAT antibodies, TAT binding oligopeptides or TAT binding organic molecules.

[0595] To screen for antibodies, oligopeptides or other organic molecules which bind to an epitope on a TAT polypeptide bound by an antibody of interest, a routine cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. This assay can be used to determine if a test antibody, oligopeptide or other organic molecule binds the same site or epitope as a known anti-TAT antibody. Alternatively, or additionally, epitope mapping can be performed by methods known in the art. For example, the antibody sequence can be mutagenized such as by alanine scanning, to identify contact residues. The mutant antibody is initailly tested for binding with polyclonal antibody to ensure proper folding. In a different method, peptides corresponding to different regions of a TAT polypeptide can be used in competition assays with the test antibodies or with a test antibody and an antibody with a characterized or known epitope.

[0596] E. Antibody Dependent Enzyme Mediated Prodrug Therapy (ADEPT) The antibodies of the present invention may also be used in ADEPT by conjugating the antibody to a prodrug-activating enzyme which converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see WO81/01145) to an active anti-cancer drug. See, for example, WO 88/07378 and U.S. Pat. No. 4,975,278.

[0597] The enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such a way so as to covert it into its more active, cytotoxic form.

[0598] Enzymes that are useful in the method of this invention include, but are not limited to, alkaline phosphatase useful for converting phosphate-containing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminase useful for converting non-toxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil; proteases, such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins (such as cathepsins B and L), that are useful for converting peptide-containing prodrugs into free drugs; D-alanylcarboxypeptidases, useful for converting prodrugs that contain D-amino acid substituents; carbohydrate-cleaving enzymes such as .beta.-galactosidase and neuraminidase useful for converting glycosylated prodrugs into free drugs; .beta.-lactamase useful for converting drugs derivatized with .beta.-lactams into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, useful for converting drugs derivatized at their amine nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, into free drugs. Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes", can be used to convert the prodrugs of the invention into free active drugs (see, e.g., Massey, Nature 328:457-458 (1987)). Antibody-abzyme conjugates can be prepared as described herein for delivery of the abzyme to a tumor cell population.

[0599] The enzymes of this invention can be covalently bound to the anti-TAT antibodies by techniques well known in the art such as the use of the heterobifunctional crosslinking reagents discussed above. Alternatively, fusion proteins comprising at least the antigen binding region of an antibody of the invention linked to at least a functionally active portion of an enzyme of the invention can be constructed using recombinant DNA techniques well known in the art (see, e.g., Neuberger et al., Nature 312:604-608 (1984).

[0600] F. Full-Length TAT Polypeptides

[0601] The present invention also provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as TAT polypeptides. In particular, cDNAs (partial and full-length) encoding various TAT polypeptides have been identified and isolated, as disclosed in further detail in the Examples below.

[0602] As disclosed in the Examples below, various cDNA clones have been deposited with the ATCC. The actual nucleotide sequences of those clones can readily be determined by the skilled artisan by sequencing of the deposited clone using routine methods in the art. The predicted amino acid sequence can be determined from the nucleotide sequence using routine skill. For the TAT polypeptides and encoding nucleic acids described herein, in some cases, Applicants have identified what is believed to be the reading frame best identifiable with the sequence information available at the time.

[0603] G. Anti-TAT Antibody and TAT Polypeptide Variants

[0604] In addition to the anti-TAT antibodies and full-length native sequence TAT polypeptides described herein, it is contemplated that anti-TAT antibody and TAT polypeptide variants can be prepared. Anti-TAT antibody and TAT polypeptide variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA, and/or by synthesis of the desired antibody or polypeptide. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the anti-TAT antibody or TAT polypeptide, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.

[0605] Variations in the anti-TAT antibodies and TAT polypeptides described herein, can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S. Pat. No. 5,364,934. Variations may be a substitution, deletion or insertion of one or more codons encoding the antibody or polypeptide that results in a change in the amino acid sequence as compared with the native sequence antibody or polypeptide. Optionally the variation is by substitution of at least one amino acid with any other amino acid in one or more of the domains of the anti-TAT antibody or TAT polypeptide. Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the anti-TAT antibody or TAT polypeptide with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.

[0606] Anti-TAT antibody and TAT polypeptide fragments are provided herein. Such fragments may be truncated at the N-terminus or C-terminus, or may lack internal residues, for example, when compared with a full length native antibody or protein. Certain fragments lack amino acid residues that are not essential for a desired biological activity of the anti-TAT antibody or TAT polypeptide.

[0607] Anti-TAT antibody and TAT polypeptide fragments may be prepared by any of a number of conventional techniques. Desired peptide fragments may be chemically synthesized. An alternative approach involves generating antibody or polypeptide fragments by enzymatic digestion, e.g., by treating the protein with an enzyme known to cleave proteins at sites defined by particular amino acid residues, or by digesting the DNA with suitable restriction enzymes and isolating the desired fragment. Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired antibody or polypeptide fragment, by polymerase chain reaction (PCR). Oligonucleotides that define the desired termini of the DNA fragment are employed at the 5' and 3' primers in the PCR. Preferably, anti-TAT antibody and TAT polypeptide fragments share at least one biological and/or immunological activity with the native anti-TAT antibody or TAT polypeptide disclosed herein.

[0608] In particular embodiments, conservative substitutions of interest are shown in Table 6 under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 6, or as further described below in reference to amino acid classes, are introduced and the products screened. TABLE-US-00005 TABLE 6 Original Exemplary Preferred Residue Substitutions Substitutions Ala (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe; leu norleucine Leu (L) norleucine; ile; val; ile met; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; leu ala; norleucine

[0609] Substantial modifications in function or immunological identity of the anti-TAT antibody or TAT polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties: [0610] (1) hydrophobic: norleucine, met, ala, val, leu, ile; [0611] (2) neutral hydrophilic: cys, ser, thr; [0612] (3) acidic: asp, glu; [0613] (4) basic: asn, gin, his, lys, arg; [0614] (5) residues that influence chain orientation: gly, pro; and [0615] (6) aromatic: trp, tyr, phe.

[0616] Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.

[0617] The variations can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)], restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or other known techniques can be performed on the cloned DNA to produce the anti-TAT antibody or TAT polypeptide variant DNA.

[0618] Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant [Cunningham and Wells, Science, 244:1081-1085 (1989)]. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions [Creighton, The Proteins, (W. H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. If alanine substitution does not yield adequate amounts of variant, an isoteric amino acid can be used.

[0619] Any cysteine residue not involved in maintaining the proper conformation of the anti-TAT antibody or TAT polypeptide also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the anti-TAT antibody or TAT polypeptide to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).

[0620] A particularly preferred type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as herein disclosed. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and human TAT polypeptide. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.

[0621] Nucleic acid molecules encoding amino acid sequence variants of the anti-TAT antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the anti-TAT antibody.

[0622] H. Modifications of Anti-TAT Antibodies and TAT Polypeptides

[0623] Covalent modifications of anti-TAT antibodies and TAT polypeptides are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of an anti-TAT antibody or TAT polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N-- or C-terminal residues of the anti-TAT antibody or TAT polypeptide. Derivatization with bifunctional agents is useful, for instance, for crosslinking anti-TAT antibody or TAT polypeptide to a water-insoluble support matrix or surface for use in the method for purifying anti-TAT antibodies, and vice-versa. 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.

[0624] 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 or threonyl 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.

[0625] Another type of covalent modification of the anti-TAT antibody or TAT polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the antibody or polypeptide. "Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence anti-TAT antibody or TAT polypeptide (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence anti-TAT antibody or TAT polypeptide. In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.

[0626] Glycosylation of antibodies and other polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0627] Addition of glycosylation sites to the anti-TAT antibody or TAT polypeptide is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original anti-TAT antibody or TAT polypeptide (for O-linked glycosylation sites). The anti-TAT antibody or TAT polypeptide amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the anti-TAT antibody or TAT polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.

[0628] Another means of increasing the number of carbohydrate moieties on the anti-TAT antibody or TAT 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, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0629] Removal of carbohydrate moieties present on the anti-TAT antibody or TAT 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).

[0630] Another type of covalent modification of anti-TAT antibody or TAT polypeptide comprises linking the antibody or polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), 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. The antibody or polypeptide also may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).

[0631] The anti-TAT antibody or TAT polypeptide of the present invention may also be modified in a way to form chimeric molecules comprising an anti-TAT antibody or TAT polypeptide fused to another, heterologous polypeptide or amino acid sequence.

[0632] In one embodiment, such a chimeric molecule comprises a fusion of the anti-TAT antibody or TAT polypeptide with a tag polypeptide which 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 anti-TAT antibody or TAT polypeptide. The presence of such epitope-tagged forms of the anti-TAT antibody or TAT polypeptide can be detected using an antibody against the tag polypeptide. Also, provision of the epitope tag enables the anti-TAT antibody or TAT 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. 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)]; an .alpha.-tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)].

[0633] In an alternative embodiment, the chimeric molecule may comprise a fusion of the anti-TAT antibody or TAT polypeptide with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as an "immunoadhesin"), such a fusion could be to the Fc region of an IgG molecule. The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of an anti-TAT antibody or TAT polypeptide in place of at least one variable region within an Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusion includes the hinge, CH.sub.2 and CH.sub.3, or the hinge, CH.sub.1, CH.sub.2 and CH.sub.3 regions of an IgG1 molecule. For the production of immunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.

[0634] I. Preparation of Anti-TAT Antibodies and TAT Polypeptides

[0635] The description below relates primarily to production of anti-TAT antibodies and TAT polypeptides by culturing cells transformed or transfected with a vector containing anti-TAT antibody- and TAT polypeptide-encoding nucleic acid. It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare anti-TAT antibodies and TAT polypeptides. For instance, the appropriate amino acid sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques [see, e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)]. In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, Calif.) using manufacturer's instructions. Various portions of the anti-TAT antibody or TAT polypeptide may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired anti-TAT antibody or TAT polypeptide. [0636] 1. Isolation of DNA Encoding Anti-TAT Antibody or TAT Polypeptide

[0637] DNA encoding anti-TAT antibody or TAT polypeptide may be obtained from a cDNA library prepared from tissue believed to possess the anti-TAT antibody or TAT polypeptide mRNA and to express it at a detectable level. Accordingly, human anti-TAT antibody or TAT polypeptide DNA can be conveniently obtained from a cDNA library prepared from human tissue. The anti-TAT antibody- or TAT polypeptide-encoding gene may also be obtained from a genomic library or by known synthetic procedures (e.g., automated nucleic acid synthesis).

[0638] Libraries can be screened with probes (such as oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative means to isolate the gene encoding anti-TAT antibody or TAT polypeptide is to use PCR methodology [Sambrook et al., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].

[0639] Techniques for screening a cDNA library are well known in the art. The oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized. The oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened. Methods of labeling are well known in the art, and include the use of radiolabels like .sup.32P-labeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al., supra.

[0640] Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases. Sequence identity (at either the amino acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined using methods known in the art and as described herein.

[0641] Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al., supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA. [0642] 2. Selection and Transformation of Host Cells

[0643] Host cells are transfected or transformed with expression or cloning vectors described herein for anti-TAT antibody or TAT polypeptide production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. The culture conditions, such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.

[0644] Methods of eukaryotic cell transfection and prokaryotic cell transformation are known to the ordinarily skilled artisan, for example, CaCl.sub.2, CaPO.sub.4, liposome-mediated and electroporation. Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described in Sambrook et al., supra, or electroporation is generally used for prokaryotes. Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al., Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52:456-457 (1978) can be employed. General aspects of mammalian cell host system transfections have been described in U.S. Pat. No. 4,399,216. Transformations into yeast are typically carried out according to the method of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene, polyornithine, may also be used. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzymology, 185:527-537 (1990) and Mansour et al., Nature, 336:348-352 (1988).

[0645] Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes include but are not limited to eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as E. coli. Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. These examples are illustrative rather than limiting. Strain W3110 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example, strain W3110 may be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host, with examples of such hosts including E. coli W31 10 strain 1A2, which has the complete genotype tonA; E. coli W3110 strain 9E4, which has the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT kan.sup.r; E. coli W3110 strain 37D6, which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kan.sup.r; E. coli W3110 strain 40B4, which is strain 37D6 with a non-kanamycin resistant degP deletion mutation; and an E. coli strain having mutant periplasmic protease disclosed in U. S. Pat. No. 4,946,783 issued 7 Aug. 1990. Alternatively, in vitro methods of cloning, e.g., PCR or other nucleic acid polymerase reactions, are suitable.

[0646] Full length antibody, antibody fragments, and antibody fusion proteins can be produced in bacteria, in particular when glycosylation and Fc effector function are not needed, such as when the therapeutic antibody is conjugated to a cytotoxic agent (e.g., a toxin) and the immunoconjugate by itself shows effectiveness in tumor cell destruction. Full length antibodies have greater half life in circulation. Production in E. coli is faster and more cost efficient. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. No. 5,648,237 (Carter et. al.), U.S. Pat. No. 5,789,199 (Joly et al.), and U.S. Pat. No. 5,840,523 (Simmons et al.) which describes translation initiation regio (TIR) and signal sequences for optimizing expression and secretion, these patents incorporated herein by reference. After expression, the antibody is isolated from the E. coli cell paste in a soluble fraction and can be purified through, e.g., a protein A or G column depending on the isotype. Final purification can be carried out similar to the process for purifying antibody expressed e.g., in CHO cells.

[0647] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-TAT antibody- or TAT polypeptide-encoding vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 [1981]; EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al., Bio/Technology, 9:968-975 (1991)) such as, e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742 [1983]), K. fragilis (ATCC 12,424), K. drosophilarum (EP (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al., Bio/Technology, 8:135 (1990)), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278 [1988]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]); Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published 31 Oct. 1990); and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10 Jan. 1991), and Aspergillus hosts such as A. nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et al., Gene, 26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81: 1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479 [1985]). Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specific species that are exemplary of this class of yeasts may be found in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).

[0648] Suitable host cells for the expression of glycosylated anti-TAT antibody or TAT polypeptide are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells, such as cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.

[0649] However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51), TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[0650] Host cells are transformed with the above-described expression or cloning vectors for anti-TAT antibody or TAT polypeptide production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. [0651] 3. Selection and Use of a Replicable Vector

[0652] The nucleic acid (e.g., cDNA or genomic DNA) encoding anti-TAT antibody or TAT polypeptide may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression. Various vectors are publicly available. The vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan.

[0653] The TAT may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or it may be a part of the anti-TAT antibody- or TAT polypeptide-encoding DNA that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, 1 pp, or heat-stable enterotoxin II leaders. For yeast secretion the signal sequence may be, e.g., the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyveromyces .alpha.-factor leaders, the latter described in U.S. Pat. No. 5,010,182), or acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179 published 4 Apr. 1990), or the signal described in WO 90/13646 published 15 Nov. 1990. In mammalian cell expression, mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders.

[0654] Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2.mu. plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.

[0655] Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.

[0656] An example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the anti-TAT antibody- or TAT polypeptide-encoding nucleic acid, such as DHFR or thymidine kinase. An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitable selection gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1 gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].

[0657] Expression and cloning vectors usually contain a promoter operably linked to the anti-TAT antibody- or TAT polypeptide-encoding nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the .beta.-lactamase and lactose promoter systems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)]. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S. D.) sequence operably linked to the DNA encoding anti-TAT antibody or TAT polypeptide.

[0658] Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900 (1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

[0659] Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.

[0660] Anti-TAT antibody or TAT polypeptide transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an inmunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems.

[0661] Transcription of a DNA encoding the anti-TAT antibody or TAT polypeptide by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, .alpha.-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the vector at a position 5' or 3' to the anti-TAT antibody or TAT polypeptide coding sequence, but is preferably located at a site 5' from the promoter.

[0662] Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding anti-TAT antibody or TAT polypeptide.

[0663] Still other methods, vectors, and host cells suitable for adaptation to the synthesis of anti-TAT antibody or TAT polypeptide in recombinant vertebrate cell culture are described in Gething et al., Nature, 293:620-625 (1981); Mantei et al., Nature, 281:40-46 (1979); EP 117,060; and EP 117,058. [0664] 4. Culturing the Host Cells

[0665] The host cells used to produce the anti-TAT antibody or TAT polypeptide of this invention may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem.102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN.TM. drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan. [0666] 5. Detecting Gene Amplification/Expression

[0667] Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.

[0668] Gene expression, alternatively, may be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product. Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native sequence TAT polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to TAT DNA and encoding a specific antibody epitope. [0669] 6. Purification of Anti-TAT Antibody and TAT Polypeptide

[0670] Forms of anti-TAT antibody and TAT polypeptide may be recovered from culture medium or from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g. Triton-X 100) or by enzymatic cleavage. Cells employed in expression of anti-TAT antibody and TAT polypeptide can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents.

[0671] It may be desired to purify anti-TAT antibody and TAT polypeptide from recombinant cell proteins or polypeptides. The following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgG; and metal chelating columns to bind epitope-tagged forms of the anti-TAT antibody and TAT polypeptide. Various methods of protein purification may be employed and such methods are known in the art and described for example in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). The purification step(s) selected will depend, for example, on the nature of the production process used and the particular anti-TAT antibody or TAT polypeptide produced.

[0672] When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10: 163-167 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.

[0673] The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human .gamma.1, .gamma.2 or .gamma.4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human .gamma.3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a C.sub.H3 domain, the Bakerbond ABX.TM.resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE.TM. chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

[0674] Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt).

[0675] J. Pharmaceutical Formulations

[0676] Therapeutic formulations of the anti-TAT antibodies, TAT binding oligopeptides, TAT binding organic molecules and/or TAT polypeptides used in accordance with the present invention are prepared for storage by mixing the antibody, polypeptide, oligopeptide or organic molecule having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as acetate, Tris, phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; tonicifiers such as trehalose and sodium chloride; sugars such as sucrose, mannitol, trehalose or sorbitol; surfactant such as polysorbate; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN.RTM., PLURONICS.RTM. or polyethylene glycol (PEG). The antibody preferably comprises the antibody at a concentration of between 5-200 mg/ml, preferably between 10-100 mg/ml.

[0677] The formulations herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, in addition to an anti-TAT antibody, TAT binding oligopeptide, or TAT binding organic molecule, it may be desirable to include in the one formulation, an additional antibody, e.g., a second anti-TAT antibody which binds a different epitope on the TAT polypeptide, or an antibody to some other target such as a growth factor that affects the growth of the particular cancer. Alternatively, or additionally, the composition may further comprise a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal agent, and/or cardioprotectant. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

[0678] The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).

[0679] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT.RTM. (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.

[0680] The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0681] K. Diagnosis and Treatment with Anti-TAT Antibodies, TAT Binding Oligopeptides and TAT Binding Organic Molecules

[0682] To determine TAT expression in the cancer, various diagnostic assays are available. In one embodiment, TAT polypeptide overexpression may be analyzed by immunohistochemistry (IHC). Parrafin embedded tissue sections from a tumor biopsy may be subjected to the IHC assay and accorded a TAT protein staining intensity criteria as follows:

[0683] Score 0-no staining is observed or membrane staining is observed in less than 10% of tumor cells.

[0684] Score 1+-a faint/barely perceptible membrane staining is detected in more than 10% of the tumor cells. The cells are only stained in part of their membrane.

[0685] Score 2+-a weak to moderate complete membrane staining is observed in more than 10% of the tumor cells.

[0686] Score 3+-a moderate to strong complete membrane staining is observed in more than 10% of the tumor cells.

[0687] Those tumors with 0 or 1+ scores for TAT polypeptide expression may be characterized as not overexpressing TAT, whereas those tumors with 2+ or 3+ scores may be characterized as overexpressing TAT.

[0688] Alternatively, or additionally, FISH assays such as the INFORM.RTM. (sold by Ventana, Ariz.) or PATHVISION.RTM. (Vysis, Ill.) may be carried out on formalin-fixed, paraffin-embedded tumor tissue to determine the extent (if any) of TAT overexpression in the tumor.

[0689] TAT overexpression or amplification may be evaluated using an in vivo diagnostic assay, e.g., by administering a molecule (such as an antibody, oligopeptide or organic molecule) which binds the molecule to be detected and is tagged with a detectable label (e.g., a radioactive isotope or a fluorescent label) and externally scanning the patient for localization of the label.

[0690] As described above, the anti-TAT antibodies, oligopeptides and organic molecules of the invention have various non-therapeutic applications. The anti-TAT antibodies, oligopeptides and organic molecules of the present invention can be useful for diagnosis and staging of TAT polypeptide-expressing cancers (e.g., in radioimaging). The antibodies, oligopeptides and organic molecules are also useful for purification or immunoprecipitation of TAT polypeptide from cells, for detection and quantitation of TAT polypeptide in vitro, e.g., in an ELISA or a Western blot, to kill and eliminate TAT-expressing cells from a population of mixed cells as a step in the purification of other cells.

[0691] Currently, depending on the stage of the cancer, cancer treatment involves one or a combination of the following therapies: surgery to remove the cancerous tissue, radiation therapy, and chemotherapy. Anti-TAT antibody, oligopeptide or organic molecule therapy may be especially desirable in elderly patients who do not tolerate the toxicity and side effects of chemotherapy well and in metastatic disease where radiation therapy has limited usefulness. The tumor targeting anti-TAT antibodies, oligopeptides and organic molecules of the invention are useful to alleviate TAT-expressing cancers upon initial diagnosis of the disease or during relapse. For therapeutic applications, the anti-TAT antibody, oligopeptide or organic molecule can be used alone, or in combination therapy with, e.g., hormones, antiangiogens, or radiolabelled compounds, or with surgery, cryotherapy, and/or radiotherapy. Anti-TAT antibody, oligopeptide or organic molecule treatment can be administered in conjunction with other forms of conventional therapy, either consecutively with, pre- or post-conventional therapy. Chemotherapeutic drugs such as TAXOTERE.RTM. (docetaxel), TAXOL.RTM. (palictaxel), estramustine and mitoxantrone are used in treating cancer, in particular, in good risk patients. In the present method of the invention for treating or alleviating cancer, the cancer patient can be administered anti-TAT antibody, oligopeptide or organic molecule in conjuction with treatment with the one or more of the preceding chemotherapeutic agents. In particular, combination therapy with palictaxel and modified derivatives (see, e.g., EP0600517) is contemplated. The anti-TAT antibody, oligopeptide or organic molecule will be administered with a therapeutically effective dose of the chemotherapeutic agent. In another embodiment, the anti-TAT antibody, oligopeptide or organic molecule is administered in conjunction with chemotherapy to enhance the activity and efficacy of the chemotherapeutic agent, e.g., paclitaxel. The Physicians' Desk Reference (PDR) discloses dosages of these agents that have been used in treatment of various cancers. The dosing regimen and dosages of these aforementioned chemotherapeutic drugs that are therapeutically effective will depend on the particular cancer being treated, the extent of the disease and other factors familiar to the physician of skill in the art and can be determined by the physician.

[0692] In one particular embodiment, a conjugate comprising an anti-TAT antibody, oligopeptide or organic molecule conjugated with a cytotoxic agent is administered to the patient. Preferably, the immunoconjugate bound to the TAT protein is internalized by the cell, resulting in increased therapeutic efficacy of the immunoconjugate in killing the cancer cell to which it binds. In a preferred embodiment, the cytotoxic agent targets or interferes with the nucleic acid in the cancer cell. Examples of such cytotoxic agents are described above and include maytansinoids, calicheamicins, ribonucleases and DNA endonucleases.

[0693] The anti-TAT antibodies, oligopeptides, organic molecules or toxin conjugates thereof are administered to a human patient, in accord with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Intravenous or subcutaneous administration of the antibody, oligopeptide or organic molecule is preferred.

[0694] Other therapeutic regimens may be combined with the administration of the anti-TAT antibody, oligopeptide or organic molecule. The combined administration includes co-administration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities. Preferably such combined therapy results in a synergistic therapeutic effect.

[0695] It may also be desirable to combine administration of the anti-TAT antibody or antibodies, oligopeptides or organic molecules, with administration of an antibody directed against another tumor antigen associated with the particular cancer.

[0696] In another embodiment, the therapeutic treatment methods of the present invention involves the combined administration of an anti-TAT antibody (or antibodies), oligopeptides or organic molecules and one or more chemotherapeutic agents or growth inhibitory agents, including co-administration of cocktails of different chemotherapeutic agents. Chemotherapeutic agents include estramustine phosphate, prednimustine, cisplatin, 5-fluorouracil, melphalan, cyclophosphamide, hydroxyurea and hydroxyureataxanes (such as paclitaxel and doxetaxel) and/or anthracycline antibiotics. Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service Ed., M.C. Perry, Williams & Wilkins, Baltimore, Md. (1992).

[0697] The antibody, oligopeptide or organic molecule may be combined with an anti-hormonal compound; e.g., an anti-estrogen compound such as tamoxifen; an anti-progesterone such as onapristone (see, EP 616 812); or an anti-androgen such as flutamide, in dosages known for such molecules. Where the cancer to be treated is androgen independent cancer, the patient may previously have been subjected to anti-androgen therapy and, after the cancer becomes androgen independent, the anti-TAT antibody, oligopeptide or organic molecule (and optionally other agents as described herein) may be administered to the patient.

[0698] Sometimes, it may be beneficial to also co-administer a cardioprotectant (to prevent or reduce myocardial dysfunction associated with the therapy) or one or more cytokines to the patient. In addition to the above therapeutic regimes, the patient may be subjected to surgical removal of cancer cells and/or radiation therapy, before, simultaneously with, or post antibody, oligopeptide or organic molecule therapy. Suitable dosages for any of the above co-administered agents are those presently used and may be lowered due to the combined action (synergy) of the agent and anti-TAT antibody, oligopeptide or organic molecule.

[0699] For the prevention or treatment of disease, the dosage and mode of administration will be chosen by the physician according to known criteria. The appropriate dosage of antibody, oligopeptide or organic molecule will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody, oligopeptide or organic molecule is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, oligopeptide or organic molecule, and the discretion of the attending physician. The antibody, oligopeptide or organic molecule is suitably administered to the patient at one time or over a series of treatments. Preferably, the antibody, oligopeptide or organic molecule is administered by intravenous infusion or by subcutaneous injections. Depending on the type and severity of the disease, about 1 .mu.g/kg to about 50 mg/kg body weight (e.g., about 0.1-15 mg/kg/dose) of antibody can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A dosing regimen can comprise administering an initial loading dose of about 4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of the anti-TAT antibody. However, other dosage regimens may be useful. A typical daily dosage might range from about 1 .mu.g/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. The progress of this therapy can be readily monitored by conventional methods and assays and based on criteria known to the physician or other persons of skill in the art.

[0700] Aside from administration of the antibody protein to the patient, the present application contemplates administration of the antibody by gene therapy. Such administration of nucleic acid encoding the antibody is encompassed by the expression "administering a therapeutically effective amount of an antibody". See, for example, WO96/07321 published Mar. 14, 1996 concerning the use of gene therapy to generate intracellular antibodies.

[0701] There are two major approaches to getting the nucleic acid (optionally contained in a vector) into the patient's cells; in vivo and ex vivo. For in vivo delivery the nucleic acid is injected directly into the patient, usually at the site where the antibody is required. For ex vivo treatment, the patient's cells are removed, the nucleic acid is introduced into these isolated cells and the modified cells are administered to the patient either directly or, for example, encapsulated within porous membranes which are implanted into the patient (see, e.g., U.S. Pat. Nos. 4,892,538 and 5,283,187). There are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc. A commonly used vector for ex vivo delivery of the gene is a retroviral vector.

[0702] The currently preferred in vivo nucleic acid transfer techniques include transfection with viral vectors (such as adenovirus, Herpes simplex I virus, or adeno-associated virus) and lipid-based systems (useful lipids for lipid-mediated transfer of the gene are DOTMA, DOPE and DC-Chol, for example). For review of the currently known gene marking and gene therapy protocols see Anderson et al., Science 256:808-813 (1992). See also WO 93/25673 and the references cited therein.

[0703] The anti-TAT antibodies of the invention can be in the different forms encompassed by the definition of "antibody" herein. Thus, the antibodies include full length or intact antibody, antibody fragments, native sequence antibody or amino acid variants, humanized, chimeric or fusion antibodies, immunoconjugates, and functional fragments thereof. In fusion antibodies an antibody sequence is fused to a heterologous polypeptide sequence. The antibodies can be modified in the Fc region to provide desired effector functions. As discussed in more detail in the sections herein, with the appropriate Fc regions, the naked antibody bound on the cell surface can induce cytotoxicity, e.g., via antibody-dependent cellular cytotoxicity (ADCC) or by recruiting complement in complement dependent cytotoxicity, or some other mechanism. Alternatively, where it is desirable to eliminate or reduce effector function, so as to minimize side effects or therapeutic complications, certain other Fc regions may be used.

[0704] In one embodiment, the antibody competes for binding or bind substantially to, the same epitope as the antibodies of the invention. Antibodies having the biological characteristics of the present anti-TAT antibodies of the invention are also contemplated, specifically including the in vivo tumor targeting and any cell proliferation inhibition or cytotoxic characteristics.

[0705] Methods of producing the above antibodies are described in detail herein.

[0706] The present anti-TAT antibodies, oligopeptides and organic molecules are useful for treating a TAT-expressing cancer or alleviating one or more symptoms of the cancer in a mammal. Such a cancer includes prostate cancer, cancer of the urinary tract, lung cancer, breast cancer, colon cancer and ovarian cancer, more specifically, prostate adenocarcinoma, renal cell carcinomas, colorectal adenocarcinomas, lung adenocarcinomas, lung squamous cell carcinomas, and pleural mesothelioma. The cancers encompass metastatic cancers of any of the preceding. The antibody, oligopeptide or organic molecule is able to bind to at least a portion of the cancer cells that express TAT polypeptide in the mammal. In a preferred embodiment, the antibody, oligopeptide or organic molecule is effective to destroy or kill TAT-expressing tumor cells or inhibit the growth of such tumor cells, in vitro or in vivo, upon binding to TAT polypeptide on the cell. Such an antibody includes a naked anti-TAT antibody (not conjugated to any agent). Naked antibodies that have cytotoxic or cell growth inhibition properties can be further harnessed with a cytotoxic agent to render them even more potent in tumor cell destruction. Cytotoxic properties can be conferred to an anti-TAT antibody by, e.g., conjugating the antibody with a cytotoxic agent, to form an immunoconjugate as described herein. The cytotoxic agent or a growth inhibitory agent is preferably a small molecule. Toxins such as calicheamicin or a maytansinoid and analogs or derivatives thereof, are preferable.

[0707] The invention provides a composition comprising an anti-TAT antibody, oligopeptide or organic molecule of the invention, and a carrier. For the purposes of treating cancer, compositions can be administered to the patient in need of such treatment, wherein the composition can comprise one or more anti-TAT antibodies present as an immunoconjugate or as the naked antibody. In a further embodiment, the compositions can comprise these antibodies, oligopeptides or organic molecules in combination with other therapeutic agents such as cytotoxic or growth inhibitory agents, including chemotherapeutic agents. The invention also provides formulations comprising an anti-TAT antibody, oligopeptide or organic molecule of the invention, and a carrier. In one embodiment, the formulation is a therapeutic formulation comprising a pharmaceutically acceptable carrier.

[0708] Another aspect of the invention is isolated nucleic acids encoding the anti-TAT antibodies. Nucleic acids encoding both the H and L chains and especially the hypervariable region residues, chains which encode the native sequence antibody as well as variants, modifications and humanized versions of the antibody, are encompassed.

[0709] The invention also provides methods useful for treating a TAT polypeptide-expressing cancer or alleviating one or more symptoms of the cancer in a mammal, comprising administering a therapeutically effective amount of an anti-TAT antibody, oligopeptide or organic molecule to the mammal. The antibody, oligopeptide or organic molecule therapeutic compositions can be administered short term (acute) or chronic, or intermittent as directed by physician. Also provided are methods of inhibiting the growth of, and killing a TAT polypeptide-expressing cell.

[0710] The invention also provides kits and articles of manufacture comprising at least one anti-TAT antibody, oligopeptide or organic molecule. Kits containing anti-TAT antibodies, oligopeptides or organic molecules find use, e.g., for TAT cell killing assays, for purification or immunoprecipitation of TAT polypeptide from cells. For example, for isolation and purification of TAT, the kit can contain an anti-TAT antibody, oligopeptide or organic molecule coupled to beads (e.g., sepharose beads). Kits can be provided which contain the antibodies, oligopeptides or organic molecules for detection and quantitation of TAT in vitro, e.g., in an ELISA or a Western blot. Such antibody, oligopeptide or organic molecule useful for detection may be provided with a label such as a fluorescent or radiolabel.

[0711] L. Articles of Manufacture and Kits

[0712] Another embodiment of the invention is an article of manufacture containing materials useful for the treatment of anti-TAT expressing cancer. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for treating the cancer condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an anti-TAT antibody, oligopeptide or organic molecule of the invention. The label or package insert indicates that the composition is used for treating cancer. The label or package insert will further comprise instructions for administering the antibody, oligopeptide or organic molecule composition to the cancer patient. Additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0713] Kits are also provided that are useful for various purposes, e.g., for TAT-expressing cell killing assays, for purification or immunoprecipitation of TAT polypeptide from cells. For isolation and purification of TAT polypeptide, the kit can contain an anti-TAT antibody, oligopeptide or organic molecule coupled to beads (e.g., sepharose beads). Kits can be provided which contain the antibodies, oligopeptides or organic molecules for detection and quantitation of TAT polypeptide in vitro, e.g., in an ELISA or a Western blot. As with the article of manufacture, the kit comprises a container and a label or package insert on or associated with the container. The container holds a composition comprising at least one anti-TAT antibody, oligopeptide or organic molecule of the invention. Additional containers may be included that contain, e.g., diluents and buffers, control antibodies. The label or package insert may provide a description of the composition as well as instructions for the intended in vitro or diagnostic use.

[0714] M. Uses for TAT Polypeptides and TAT-Polypeptide Encoding Nucleic Acids

[0715] Nucleotide sequences (or their complement) encoding TAT polypeptides have various applications in the art of molecular biology, including uses as hybridization probes, in chromosome and gene mapping and in the generation of anti-sense RNA and DNA probes. TAT-encoding nucleic acid will also be useful for the preparation of TAT polypeptides by the recombinant techniques described herein, wherein those TAT polypeptides may find use, for example, in the preparation of anti-TAT antibodies as described herein.

[0716] The full-length native sequence TAT gene, or portions thereof, may be used as hybridization probes for a cDNA library to isolate the full-length TAT cDNA or to isolate still other cDNAs (for instance, those encoding naturally-occurring variants of TAT or TAT from other species) which have a desired sequence identity to the native TAT sequence disclosed herein. Optionally, the length of the probes will be about 20 to about 50 bases. The hybridization probes may be derived from at least partially novel regions of the full length native nucleotide sequence wherein those regions may be determined without undue experimentation or from genomic sequences including promoters, enhancer elements and introns of native sequence TAT. By way of example, a screening method will comprise isolating the coding region of the TAT gene using the known DNA sequence to synthesize a selected probe of about 40 bases. Hybridization probes may be labeled by a variety of labels, including radionucleotides such as 32P or 35S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems. Labeled probes having a sequence complementary to that of the TAT gene of the present invention can be used to screen libraries of human cDNA, genomic DNA or mRNA to determine which members of such libraries the probe hybridizes to. Hybridization techniques are described in further detail in the Examples below. Any EST sequences disclosed in the present application may similarly be employed as probes, using the methods disclosed herein.

[0717] Other useful fragments of the TAT-encoding nucleic acids include antisense or sense oligonucleotides comprising a singe-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target TAT mRNA (sense) or TAT DNA (antisense) sequences. Antisense or sense oligonucleotides, according to the present invention, comprise a fragment of the coding region of TAT DNA. Such a fragment generally comprises 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).

[0718] Binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block transcription or translation of the target sequence by one of several means, including enhanced degradation of the duplexes, premature termination of transcription or translation, or by other means. Such methods are encompassed by the present invention. The antisense oligonucleotides thus may be used to block expression of TAT proteins, wherein those TAT proteins may play a role in the induction of cancer in mammals. Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar-phosphodiester backbones (or other sugar linkages, such as those described in WO 91/06629) and wherein such sugar linkages are resistant to endogenous nucleases. Such oligonucleotides with resistant sugar linkages are stable in vivo (i.e., capable of resisting enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide sequences.

[0719] Preferred intragenic sites for antisense binding include the region incorporating the translation initiation/start codon (5'-AUG/5'-ATG) or termination/stop codon (5'-UAA, 5'-UAG and 5-UGA/5'-TAA, 5'-TAG and 5'-TGA) of the open reading frame (ORF) of the gene. These regions refer to a portion of the mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation initiation or termination codon. Other preferred regions for antisense binding include: introns; exons; intron-exon junctions; the open reading frame (ORF) or "coding region," which is the region between the translation initiation codon and the translation termination codon; the 5' cap of an mRNA which comprises an N7-methylated guanosine residue joined to the 5'-most residue of the mRNA via a 5'-5' triphosphate linkage and includes 5' cap structure itself as well as the first 50 nucleotides adjacent to the cap; the 5' untranslated region (5'UTR), the portion of an mRNA in the 5' direction from the translation initiation codon, and thus including nucleotides between the 5' cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene; and the 3' untranslated region (3'UTR), the portion of an mRNA in the 3' direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3' end of an mRNA or corresponding nucleotides on the gene.

[0720] Specific examples of preferred antisense compounds useful for inhibiting expression of TAT proteins include oligonucleotides containing modified backbones or non-natural internucleoside linkages. Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotri-esters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and borano-phosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Preferred oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included. Representative United States patents that teach the preparation of phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos.: 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, each of which is herein incorporated by reference.

[0721] Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH.sub.2 component parts. Representative United States patents that teach the preparation of such oligonucleosides include, but are not limited to,. U.S. Pat. Nos.: 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, each of which is herein incorporated by reference.

[0722] In other preferred antisense oligonucleotides, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos.: 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0723] Preferred antisense oligonucleotides incorporate phosphorothioate backbones and/or heteroatom backbones, and in particular --CH.sub.2--NH--O--CH.sub.2--, --CH.sub.2--N(CH.sub.3)--O--CH.sub.2-- [known as a methylene (methylimino) or MMI backbone], --CH.sub.2--O--N(CH.sub.3)--CH.sub.2--, --CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2-- and --O--N(CH.sub.3)--CH.sub.2--CH.sub.2-- [wherein the native phosphodiester backbone is represented as --O--P--O--CH.sub.2--] described in the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are antisense oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

[0724] Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2' position: OH; F; 0-alkyl, S-alkyl, or N-alkyl; O-alkenyl, S-alkeynyl, or N-alkenyl; O-alkynyl, S-alkynyl or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C.sub.1 to C.sub.10 alkyl or C.sub.2 to C.sub.10 alkenyl and alkynyl. Particularly preferred are O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2).sub.nOCH.sub.3, O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nONH.sub.2, and O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3)].sub.2, where n and m are from 1 to about 10. Other preferred antisense oligonucleotides comprise one of the following at the 2' position: C.sub.1 to C.sub.10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3, SOCH.sub.3, SO.sub.2 CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2'-methoxyethoxy (2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2'-dimethylaminooxyethoxy, i.e., a O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE, as described in examples hereinbelow, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.2)--.

[0725] A further prefered modification includes Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. The linkage is preferably a methelyne (--CH.sub.2--).sub.n group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.

[0726] Other preferred modifications include 2'-methoxy (2'-O--CH.sub.3), 2'-aminopropoxy (2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2), 2'-allyl (2'-CH.sub.2--CH.dbd.CH.sub.2), 2'-O-allyl (2'-O--CH.sub.2--CH.dbd.CH.sub.2) and 2'-fluoro (2'-F). The 2'-modification may be in the arabino (up) position or ribo (down) position. A preferred 2'-arabino modification is 2'-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos.: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, each of which is herein incorporated by reference in its entirety.

[0727] Oligonucleotides may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C =C-CH.sub.3 or -CH.sub.2-C=CH) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido [5,4-b][1,4]benzoxazin-2(3 H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]-2one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2.degree. C. (Sanghvi et al, Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are preferred base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications. Representative United States patents that teach the preparation of modified nucleobases include, but are not limited to: U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; 5,681,941 and 5,750,692, each of which is herein incorporated by reference.

[0728] Another modification of antisense oligonucleotides chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. The compounds of the invention can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugates groups include cholesterols, lipids, cation lipids, phospholipids, cationic phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) and U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporated by reference.

[0729] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide. The present invention also includes antisense compounds which are chimeric compounds. "Chimeric" antisense compounds or "chimeras," in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA: DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Preferred chimeric antisense oligonucleotides incorporate at least one 2' modified sugar (preferably 2'-O--(CH.sub.2).sub.2--O--CH.sub.3) at the 3' terminal to confer nuclease resistance and a region with at least 4 contiguous 2-H sugars to confer RNase H activity. Such compounds have also been referred to in the art as hybrids or gapmers. Preferred gapmers have a region of 2'modified sugars (preferably 2'-O--(CH.sub.2).sub.2--O--CH.sub.3) at the 3'-terminal and at the 5' terminal separated by at least one region having at least 4 contiguous 2'-H sugars and preferably incorporate phosphorothioate backbone linkages. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference in its entirety.

[0730] The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives. The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption assisting formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.

[0731] Other examples of sense or antisense oligonucleotides include those oligonucleotides which are covalently linked to organic moieties, such as those described in WO 90/10048, and other moieties that increases affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(L-lysine). Further still, intercalating agents, such as ellipticine, and alkylating agents or metal complexes may be attached to sense or antisense oligonucleotides to modify binding specificities of the antisense or sense oligonucleotide for the target nucleotide sequence.

[0732] Antisense or sense oligonucleotides may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, CaPO.sub.4-mediated DNA transfection, electroporation, or by using gene transfer vectors such as Epstein-Barr virus. In a preferred procedure, an antisense or sense oligonucleotide is inserted into a suitable retroviral vector. A cell containing the target nucleic acid sequence is contacted with the recombinant retroviral vector, either in vivo or ex vivo. Suitable retroviral vectors include, but are not limited to, those derived from the murine retrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the double copy vectors designated DCT5A, DCT5B and DCT5C (see WO 90/13641).

[0733] Sense or antisense oligonucleotides also 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.

[0734] 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. The sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase.

[0735] Antisense or sense RNA or DNA molecules are generally at least about 5 nucleotides in length, alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 nucleotides in length, wherein in this context the term "about" means the referenced nucleotide sequence length plus or minus 10% of that referenced length.

[0736] The probes may also be employed in PCR techniques to generate a pool of sequences for identification of closely related TAT coding sequences.

[0737] Nucleotide sequences encoding a TAT can also be used to construct hybridization probes for mapping the gene which encodes that TAT and for the genetic analysis of individuals with genetic disorders. The nucleotide sequences provided herein may be mapped to a chromosome and specific regions of a chromosome using known techniques, such as in situ hybridization, linkage analysis against known chromosomal markers, and hybridization screening with libraries.

[0738] When the coding sequences for TAT encode a protein which binds to another protein (example, where the TAT is a receptor), the TAT can be used in assays to identify the other proteins or molecules involved in the binding interaction. By such methods, inhibitors of the receptor/ligand binding interaction can be identified. Proteins involved in such binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction. Also, the receptor TAT can be used to isolate correlative ligand(s). Screening assays can be designed to find lead compounds that mimic the biological activity of a native TAT or a receptor for TAT. Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates. Small molecules contemplated include synthetic organic or inorganic compounds. The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell based assays, which are well characterized in the art.

[0739] Nucleic acids which encode TAT or its modified forms can also be used to generate either transgenic animals or "knock out" animals which, in turn, are useful in the development and screening of therapeutically useful reagents. A transgenic animal (e.g., a mouse or rat) is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage. A transgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops. In one embodiment, cDNA encoding TAT can be used to clone genomic DNA encoding TAT in accordance with established techniques and the genomic sequences used to generate transgenic animals that contain cells which express DNA encoding TAT. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009. Typically, particular cells would be targeted for TAT transgene incorporation with tissue-specific enhancers. Transgenic animals that include a copy of a transgene encoding TAT introduced into the germ line of the animal at an embryonic stage can be used to examine the effect of increased expression of DNA encoding TAT. Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression. In accordance with this facet of the invention, an animal is treated with the reagent and a reduced incidence of the pathological condition, compared to untreated animals bearing the transgene, would indicate a potential therapeutic intervention for the pathological condition.

[0740] Alternatively, non-human homologues of TAT can be used to construct a TAT "knock out" animal which has a defective or altered gene encoding TAT as a result of homologous recombination between the endogenous gene encoding TAT and altered genomic DNA encoding TAT introduced into an embryonic stem cell of the animal. For example, cDNA encoding TAT can be used to clone genomic DNA encoding TAT in accordance with established techniques. A portion of the genomic DNA encoding TAT can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector [see e.g., Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologous recombination vectors]. The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected [see e.g., Li et al., Cell, 69:915 (1992)]. The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock out" animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knockout animals can be characterized for instance, for their ability to defend against certain pathological conditions and for their development of pathological conditions due to absence of the TAT polypeptide.

[0741] Nucleic acid encoding the TAT polypeptides may also be used in gene therapy. In gene therapy applications, genes are introduced into cells in order to achieve in vivo synthesis of a therapeutically effective genetic product, for example for replacement of a defective gene. "Gene therapy" includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA. Antisense RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes in vivo. It has already been shown that short antisense oligonucleotides can be imported into cells where they act as inhibitors, despite their low intracellular concentrations caused by their restricted uptake by the cell membrane. (Zamecnik et al., Proc. Natl. Acad. Sci. USA 83:4143-4146 [1986]). The oligonucleotides can be modified to enhance their uptake, e.g. by substituting their negatively charged phosphodiester groups by uncharged groups.

[0742] There are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc. The currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection (Dzau et al., Trends in Biotechnology 11, 205-210 [1993]). In some situations it is desirable to provide the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc. Where liposomes are employed, proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g. capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990). For review of gene marking and gene therapy protocols see Anderson et al., Science 256, 808-813 (1992).

[0743] The nucleic acid molecules encoding the TAT polypeptides or fragments thereof described herein are useful for chromosome identification. In this regard, there exists an ongoing need to identify new chromosome markers, since relatively few chromosome marking reagents, based upon actual sequence data are presently available. Each TAT nucleic acid molecule of the present invention can be used as a chromosome marker.

[0744] The TAT polypeptides and nucleic acid molecules of the present invention may also be used diagnostically for tissue typing, wherein the TAT polypeptides of the present invention may be differentially expressed in one tissue as compared to another, preferably in a diseased tissue as compared to a normal tissue of the same tissue type. TAT nucleic acid molecules will find use for generating probes for PCR, Northern analysis, Southern analysis and Western analysis.

[0745] This invention encompasses methods of screening compounds to identify those that mimic the TAT polypeptide (agonists) or prevent the effect of the TAT polypeptide (antagonists). Screening assays for antagonist drug candidates are designed to identify compounds that bind or complex with the TAT polypeptides encoded by the genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other cellular proteins, including e.g., inhibiting the expression of TAT polypeptide from cells. Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates.

[0746] The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays, which are well characterized in the art.

[0747] All assays for antagonists are common in that they call for contacting the drug candidate with a TAT polypeptide encoded by a nucleic acid identified herein under conditions and for a time sufficient to allow these two components to interact.

[0748] In binding assays, the interaction is binding and the complex formed can be isolated or detected in the reaction mixture. In a particular embodiment, the TAT polypeptide encoded by the gene identified herein or the drug candidate is immobilized on a solid phase, e.g., on a microtiter plate, by covalent or non-covalent attachments. Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the TAT polypeptide and drying. Alternatively, an immobilized antibody, e.g., a monoclonal antibody, specific for the TAT polypeptide to be immobilized can be used to anchor it to a solid surface. The assay is performed by adding the non-immobilized component, which may be labeled by a detectable label, to the immobilized component, e.g., the coated surface containing the anchored component. When the reaction is complete, the non-reacted components are removed, e.g., by washing, and complexes anchored on the solid surface are detected. When the originally non-immobilized component carries a detectable label, the detection of label immobilized on the surface indicates that complexing occurred. Where the originally non-immobilized component does not carry a label, complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex.

[0749] If the candidate compound interacts with but does not bind to a particular TAT polypeptide encoded by a gene identified herein, its interaction with that polypeptide can be assayed by methods well known for detecting protein-protein interactions. Such assays include traditional approaches, such as, e.g., cross-linking, co-immunoprecipitation, and co-purification through gradients or chromatographic columns. In addition, protein-protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers (Fields and Song, Nature (London), 340:245-246 (1989); Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)) as disclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89: 5789-5793 (1991). Many transcriptional activators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA-binding domain, the other one functioning as the transcription-activation domain. The yeast expression system described in the foregoing publications (generally referred to as the "two-hybrid system") takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA-binding domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain. The expression of a GAL1-lacZ reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for .beta.-galactosidase. A complete kit (MATCHMAKER.TM.) for identifying protein-protein interactions between two specific proteins using the two-hybrid technique is commercially available from Clontech. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions.

[0750] Compounds that interfere with the interaction of a gene encoding a TAT polypeptide identified herein and other intra- or extracellular components can be tested as follows: usually a reaction mixture is prepared containing the product of the gene and the intra- or extracellular component under conditions and for a time allowing for the interaction and binding of the two products. To test the ability of a candidate compound to inhibit binding, the reaction is run in the absence and in the presence of the test compound. In addition, a placebo may be added to a third reaction mixture, to serve as positive control. The binding (complex formation) between the test compound and the intra- or extracellular component present in the mixture is monitored as described hereinabove. The formation of a complex in the control reaction(s) but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of the test compound and its reaction partner.

[0751] To assay for antagonists, the TAT polypeptide may be added to a cell along with the compound to be screened for a particular activity and the ability of the compound to inhibit the activity of interest in the presence of the TAT polypeptide indicates that the compound is an antagonist to the TAT polypeptide. Alternatively, antagonists may be detected by combining the TAT polypeptide and a potential antagonist with membrane-bound TAT polypeptide receptors or recombinant receptors under appropriate conditions for a competitive inhibition assay. The TAT polypeptide can be labeled, such as by radioactivity, such that the number of TAT polypeptide molecules bound to the receptor can be used to determine the effectiveness of the potential antagonist. The gene encoding the receptor can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting. Coligan et al., Current Protocols in Immun., 1(2): Chapter 5 (1991). Preferably, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the TAT polypeptide and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the TAT polypeptide. Transfected cells that are grown on glass slides are exposed to labeled TAT polypeptide. The TAT polypeptide can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase. Following fixation and incubation, the slides are subjected to autoradiographic analysis. Positive pools are identified and sub-pools are prepared and re-transfected using an interactive sub-pooling and re-screening process, eventually yielding a single clone that encodes the putative receptor.

[0752] As an alternative approach for receptor identification, labeled TAT polypeptide can be photoaffinity-linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE and exposed to X-ray film. The labeled complex containing the receptor can be excised, resolved into peptide fragments, and subjected to protein micro-sequencing. The amino acid sequence obtained from micro-sequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor.

[0753] In another assay for antagonists, mammalian cells or a membrane preparation expressing the receptor would be incubated with labeled TAT polypeptide in the presence of the candidate compound. The ability of the compound to enhance or block this interaction could then be measured.

[0754] More specific examples of potential antagonists include an oligonucleotide that binds to the fusions of immunoglobulin with TAT polypeptide, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments. Alternatively, a potential antagonist may be a closely related protein, for example, a mutated form of the TAT polypeptide that recognizes the receptor but imparts no effect, thereby competitively inhibiting the action of the TAT polypeptide.

[0755] Another potential TAT polypeptide antagonist is an antisense RNA or DNA construct prepared using antisense technology, where, e.g., an antisense RNA or DNA molecule acts to block directly the translation of mRNA by hybridizing to targeted mRNA and preventing protein translation. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which encodes the mature TAT polypeptides herein, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix--see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan et al., Science, 251:1360 (1991)), thereby preventing transcription and the production of the TAT polypeptide. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the TAT polypeptide (antisense--Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRC Press: Boca Raton, Fla., 1988). The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the TAT polypeptide. When antisense DNA is used, oligodeoxyribonucleotides derived from the translation-initiation site, e.g., between about -10 and +10 positions of the target gene nucleotide sequence, are preferred.

[0756] Potential antagonists include small molecules that bind to the active site, the receptor binding site, or growth factor or other relevant binding site of the TAT polypeptide, thereby blocking the normal biological activity of the TAT polypeptide. Examples of small molecules include, but are not limited to, small peptides or peptide-like molecules, preferably soluble peptides, and synthetic non-peptidyl organic or inorganic compounds.

[0757] Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.

[0758] Ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage. Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques. For further details see, e.g., Rossi, Current Biology, 4:469-471 (1994), and PCT publication No. WO 97/33551 (published Sep. 18, 1997).

[0759] Nucleic acid molecules in triple-helix formation used to inhibit transcription should be single-stranded and composed of deoxynucleotides. The base composition of these oligonucleotides is designed such that it promotes triple-helix formation via Hoogsteen base-pairing rules, which generally require sizeable stretches of purines or pyrimidines on one strand of a duplex. For further details see, e.g., PCT publication No. WO 97/33551, supra.

[0760] These small molecules can be identified by any one or more of the screening assays discussed hereinabove and/or by any other screening techniques well known for those skilled in the art.

[0761] Isolated TAT polypeptide-encoding nucleic acid can be used herein for recombinantly producing TAT polypeptide using techniques well known in the art and as described herein. In turn, the produced TAT polypeptides can be employed for generating anti-TAT antibodies using techniques well known in the art and as described herein.

[0762] Antibodies specifically binding a TAT polypeptide identified herein, as well as other molecules identified by the screening assays disclosed hereinbefore, can be administered for the treatment of various disorders, including cancer, in the form of pharmaceutical compositions.

[0763] If the TAT polypeptide is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred. However, lipofections or liposomes can also be used to deliver the antibody, or an antibody fragment, into cells. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993).

[0764] The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

[0765] The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

[0766] All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

EXAMPLES

[0767] Commercially available reagents referred to in the examples were used according to manufacturer's instructions unless otherwise indicated. The source of those cells identified in the following examples, and throughout the specification, by ATCC accession numbers is the American Type Culture Collection, Manassas, Va.

EXAMPLE 1

Tissue Expression Profiling Using GeneExpress.RTM.

[0768] A proprietary database containing gene expression information (GeneExpress.RTM., Gene Logic Inc., Gaithersburg, Md.) was analyzed in an attempt to identify polypeptides (and their encoding nucleic acids) whose expression is significantly upregulated in a particular tumor tissue(s) of interest as compared to other tumor(s) and/or normal tissues. Specifically, analysis of the GeneExpress.RTM. database was conducted using either software available through Gene Logic Inc., Gaithersburg, Md., for use with the GeneExpress.RTM. database or with proprietary software written and developed at Genentech, Inc. for use with the GeneExpress.RTM. database. The rating of positive hits in the analysis is based upon several criteria including, for example, tissue specificity, tumor specificity and expression level in normal essential and/or normal proliferating tissues. The following is a list of molecules whose tissue expression profile as determined from an analysis of the GeneExpress.RTM. database evidences high tissue expression and significant upregulation of expression in a specific tumor or tumors as compared to other tumor(s) and/or normal tissues and optionally relatively low expression in normal essential and/or normal proliferating tissues. As such, the molecules listed below are excellent polypeptide targets for the diagnosis and therapy of cancer in mammals. TABLE-US-00006 upregulation of Molecule expression in: as compared to: DNA225785 (TAT295) breast tumor normal breast tissue DNA225785 (TAT295) colon tumor normal colon tissue DNA225785 (TAT295) lymphoid tumor normal lymphoid tissue DNA88158 (TAT298) ovarian tumor normal ovarian tissue DNA88158 (TAT298) uterine tumor normal uterine tissue DNA88158 (TAT298) breast tumor normal breast tissue DNA88158 (TAT298) kidney tumor normal kidney tissue DNA225800 (TAT347) ovarian tumor normal ovarian tissue DNA225800 (TAT347) uterine tumor normal uterine tissue DNA225800 (TAT347) breast tumor normal breast tissue DNA225800 (TAT347) kidney tumor normal kidney tissue DNA299884 (TAT304) lung tumor normal lung tissue DNA299884 (TAT304) colon tumor normal colon tissue DNA299884 (TAT304) rectum tumor normal rectum tissue DNA299884 (TAT304) ovarian tumor normal ovarian tissue DNA299884 (TAT304) breast tumor normal breast tissue DNA299884 (TAT304) uterine tumor normal uterine tissue DNA293550 (TAT293) breast tumor normal breast tissue DNA293550 (TAT293) colon tumor normal colon tissue DNA293550 (TAT293) rectum tumor normal rectum tissue DNA293550 (TAT293) uterine tumor normal uterine tissue DNA293550 (TAT293) lung tumor normal lung tissue DNA293550 (TAT293) ovarian tumor normal ovarian tissue DNA293550 (TAT293) pancreatic tumor normal pancreatic tissue DNA293550 (TAT293) prostate tumor normal prostate tissue DNA226313 (TAT296) skin tumor normal skin tissue DNA226313 (TAT296) melanoma tumor normal melanocyte tissue DNA225865 (TAT300) breast tumor normal breast tissue DNA225865 (TAT300) colon tumor normal colon tissue DNA225865 (TAT300) rectum tumor normal rectum tissue DNA225865 (TAT300) lung tumor normal lung tissue DNA225865 (TAT300) ovarian tumor normal ovarian tissue DNA225865 (TAT300) pancreatic tumor normal pancreatic tissue DNA225865 (TAT300) skin tumor normal skin tissue DNA225865 (TAT300) soft tissue tumor normal soft tissue DNA226403 (TAT302) breast tumor normal breast tissue DNA226403 (TAT302) prostate tumor normal prostate tissue DNA73736 (TAT303) uterine tumor normal uterine tissue DNA73736 (TAT303) lung tumor normal lung tissue DNA73736 (TAT303) ovarian tumor normal ovarian tissue DNA73736 (TAT303) testicular tumor normal testicular tissue DNA299885 (TAT305) skin tumor normal skin tissue

EXAMPLE 2

Quantitative Analysis of TAT mRNA Expression

[0769] In this assay, a 5' nuclease assay (for example, TaqMan.RTM.) and real-time quantitative PCR (for example, ABI Prizm 7700 Sequence Detection System.RTM. (Perkin Elmer, Applied Biosystems Division, Foster City, Calif.)), were used to find genes that are significantly overexpressed in a cancerous tumor or tumors as compared to other cancerous tumors or normal non-cancerous tissue. The 5' nuclease assay reaction is a fluorescent PCR-based technique which makes use of the 5' exonuclease activity of Taq DNA polymerase enzyme to monitor gene expression in real time. Two oligonucleotide primers (whose sequences are based upon the gene or EST sequence of interest) are used to generate an amplicon typical of a PCR reaction. A third oligonucleotide, or probe, is designed to detect nucleotide sequence located between the two PCR primers. The probe is non-extendible by Taq DNA polymerase enzyme, and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are on the probe. During the PCR amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner. The resultant probe fragments disassociate in solution, and signal from the released reporter dye is free from the quenching effect of the second fluorophore. One molecule of reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.

[0770] The 5' nuclease procedure is run on a real-time quantitative PCR device such as the ABI Prism 7700.TM. Sequence Detection. The system consists of a thermocycler, laser, charge-coupled device (CCD) camera and computer. The system amplifies samples in a 96-well format on a thermocycler. During amplification, laser-induced fluorescent signal is collected in real-time through fiber optics cables for all 96 wells, and detected at the CCD. The system includes software for running the instrument and for analyzing the data.

[0771] The starting material for the screen was mRNA isolated from a variety of different cancerous tissues. The mRNA is quantitated precisely, e.g., fluorometrically. As a negative control, RNA was isolated from various normal tissues of the same tissue type as the cancerous tissues being tested.

[0772] 5' nuclease assay data are initially expressed as Ct, or the threshold cycle. This is defined as the cycle at which the reporter signal accumulates above the background level of fluorescence. The .DELTA.Ct values are used as quantitative measurement of the relative number of starting copies of a particular target sequence in a nucleic acid sample when comparing cancer mRNA results to normal human mRNA results. As one Ct unit corresponds to 1 PCR cycle or approximately a 2-fold relative increase relative to normal, two units corresponds to a 4-fold relative increase, 3 units corresponds to an 8-fold relative increase and so on, one can quantitatively measure the relative fold increase in mRNA expression between two or more different tissues. Using this technique, the molecules listed below have been identified as being significantly overexpressed (i.e., at least 2-fold) in a particular tumor(s) as compared to their normal non-cancerous counterpart tissue(s) (from both the same and different tissue donors) and thus, represent excellent polypeptide targets for the diagnosis and therapy of cancer in mammals. TABLE-US-00007 upregulation of Molecule expression in: as compared to: DNA88158 (TAT298) ovarian tumor normal ovarian tissue DNA225800 (TAT347) ovarian tumor normal ovarian tissue DNA293550 (TAT293) breast tumor normal breast tissue DNA103386 (TAT299) lung tumor normal lung tissue DNA273438 (TAT351) lung tumor normal lung tissue DNA225865 (TAT300) colon tumor normal colon tissue DNA226403 (TAT302) breast tumor normal breast tissue DNA73736 (TAT303) ovarian tumor normal ovarian tissue

EXAMPLE 3

In situ Hybridization

[0773] In situ hybridization is a powerful and versatile technique for the detection and localization of nucleic acid sequences within cell or tissue preparations. It may be useful, for example, to identify sites of gene expression, analyze the tissue distribution of transcription, identify and localize viral infection, follow changes in specific mRNA synthesis and aid in chromosome mapping.

[0774] In situ hybridization was performed following an optimized version of the protocol by Lu and Gillett, Cell Vision 1: 169-176 (1994), using PCR-generated .sup.33P-labeled riboprobes. Briefly, formalin-fixed, paraffin-embedded human tissues were sectioned, deparaffinized, deproteinated in proteinase K (20 g/ml) for 15 minutes at 37.degree. C., and further processed for in situ hybridization as described by Lu and Gillett, supra. A [.sup.33-P] UTP-labeled antisense riboprobe was generated from a PCR product and hybridized at 55.degree. C. overnight. The slides were dipped in Kodak NTB2 nuclear track emulsion and exposed for 4 weeks.

.sup.33P-Riboprobe Synthesis

[0775] 6.0 .mu.l (125 mCi) of .sup.33P-UTP (Amersham BF 1002, SA<2000 Ci/mmol) were speed vac dried. To each tube containing dried .sup.33P-UTP, the following ingredients were added:

[0776] 2.0 .mu.l 5.times. transcription buffer

[0777] 1.0 .mu.l DTT (100 mM)

[0778] 2.0 .mu.l NTP mix (2.5 mM: 10.mu.; each of 10 mM GTP, CTP & ATP+10 .mu.H.sub.2O)

[0779] 1.0 .mu.l UTP (50 .mu.M)

[0780] 1.0 .mu.l Rnasin

[0781] 1.0 .mu.l DNA template (1 .mu.g)

[0782] 1.0 .mu.l H.sub.2O

[0783] 1.0 .mu.l RNA polymerase (for PCR products T3=AS, T7=S, usually)

[0784] The tubes were incubated at 37.degree. C. for one hour. 1.0 .mu.l RQ1 DNase were added, followed by incubation at 37.degree. C. for 15 minutes. 90 .mu.l TE (10 mM Tris pH 7.6/1 mM EDTA pH 8.0) were added, and the mixture was pipetted onto DE81 paper. The remaining solution was loaded in a Microcon-50 ultrafiltration unit, and spun using program 10 (6 minutes). The filtration unit was inverted over a second tube and spun using program 2 (3 minutes). After the final recovery spin, 100 .mu.l TE were added. 1 .mu.l of the final product was pipetted on DE81 paper and counted in 6 ml of Biofluor II.

[0785] The probe was run on a TBE/urea gel. 1-3 .mu.l of the probe or 5 .mu.l of RNA Mrk III were added to 3 .mu.l of loading buffer. After heating on a 95.degree. C. heat block for three minutes, the probe was immediately placed on ice. The wells of gel were flushed, the sample loaded, and run at 180-250 volts for 45 minutes. The gel was wrapped in saran wrap and exposed to XAR film with an intensifying screen in -70.degree. C. freezer one hour to overnight.

.sup.33P-Hybridization

[0786] A. Pretreatment of frozen sections

[0787] The slides were removed from the freezer, placed on aluminium trays and thawed at room temperature for 5 minutes. The trays were placed in 55.degree. C. incubator for five minutes to reduce condensation. The slides were fixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, and washed in 0.5.times.SSC for 5 minutes, at room temperature (25 ml 20.times.SSC+975 ml SQ H.sub.2O). After deproteination in 0.5 .mu.g/ml proteinase K for 10 minutes at 37.degree. C. (12.5 .mu.l of 10 mg/ml stock in 250 ml prewarmed RNase-free RNAse buffer), the sections were washed in 0.5.times.SSC for 10 minutes at room temperature. The sections were dehydrated in 70%, 95%, 100% ethanol, 2 minutes each.

[0788] B. Pretreatment of paraffin-embedded sections

[0789] The slides were deparaffinized, placed in SQ H.sub.2O, and rinsed twice in 2.times.SSC at room temperature, for 5 minutes each time. The sections were deproteinated in 20 .mu.g/ml proteinase K (500 .mu.l of 10 mg/ml in 250 ml RNase-free RNase buffer; 37.degree. C., 15 minutes)--human embryo, or 8.times.proteinase K (100 .mu.l in 250 ml Rnase buffer, 37.degree. C., 30 minutes)--formalin tissues. Subsequent rinsing in 0.5 x SSC and dehydration were performed as described above.

[0790] C. Prehybridization The slides were laid out in a plastic box lined with Box buffer (4.times.SSC, 50% formamide)--saturated filter paper.

[0791] D. Hybridization 1.0.times.10.sup.6 cpm probe and 1.0 .mu.l tRNA (50 mg/ml stock) per slide were heated at 95.degree. C. for 3 minutes. The slides were cooled on ice, and 48 .mu.l hybridization buffer were added per slide. After vortexing, 50 .mu.l .sup.33P mix were added to 50 .mu.l prehybridization on slide. The slides were incubated overnight at 55.degree. C.

[0792] E. Washes

[0793] Washing was done 2.times.10 minutes with 2.times.SSC, EDTA at room temperature (400 ml 20.times.SSC+16 ml 0.25M EDTA, V.sub.f=4 L), followed by RNaseA treatment at 37.degree. C. for 30 minutes (500 .mu.l of 10 mg/ml in 250 ml Rnase buffer=20 .mu.g/ml), The slides were washed 2.times.10 minutes with 2.times.SSC, EDTA at room temperature. The stringency wash conditions were as follows: 2 hours at 55.degree. C., 0.1.times.SSC, EDTA (20 ml 20.times.SSC+16 ml EDTA, V.sub.f=4 L).

[0794] F. Oligonucleotides

[0795] In situ analysis was performed on a variety of DNA sequences disclosed herein. The oligonucleotides employed for these analyses were obtained so as to be complementary to the nucleic acids (or the complements thereof) as shown in the accompanying figures.

[0796] G. Results

[0797] In situ analysis was performed on a variety of DNA sequences disclosed herein. The results from these analyses are as follows.

(1) DNA226313 (TAT296)

[0798] All normal tissues tested are negative for expression. 11/21 malignant melanoma tissue samples are positive for expression and of the positive cases 6 display an extremely strong signal.

(2) DNA103386 (TAT299)

[0799] A strong signal is observed in the malignant cells of a testicular neoplasm (seminoma) while normal testicular tissue is neagtive for expression.

[0800] (3) DNA273438 (TAT351)

[0801] A strong signal is observed in the malignant cells of a testicular neoplasm (seminoma) while normal testicular tissue is neagtive for expression.

(4) DNA73736 (TAT303)

[0802] Normal ovarian and endometrial tissues are negative for expression. On the other hand, 6 of 11 ovarian and 2 of 8 endometrial adenocarcinomas are positive for expression. Additionally, in another expreiment, 5 of 11 clear cell carcinomas, 18 of 26 serous cystadenocarcinomas and 15 of 21 endometrioid adenocarcinomas are positive for expression.

(5) DNA299885 (TAT305)

[0803] Normal skin tissue is negative for expression while 5 of 5 malignant melanoma samples tested are positive for expression.

EXAMPLE 4

Verification and Analysis of Differential TAT Polypeptide Expression by GEPIS

[0804] TAT polypeptides which may have been identified as a tumor antigen as described in one or more of the above Examples were analyzed and verified as follows. An expressed sequence tag (EST) DNA database (LIFESEQ.RTM., Incyte Pharmaceuticals, Palo Alto, Calif.) was searched and interesting EST sequences were identified by GEPIS. Gene expression profiling in silico (GEPIS) is a bioinformatics tool developed at Genentech, Inc. that characterizes genes of interest for new cancer therapeutic targets. GEPIS takes advantage of large amounts of EST sequence and library information to determine gene expression profiles. GEPIS is capable of determining the expression profile of a gene based upon its proportional correlation with the number of its occurrences in EST databases, and it works by integrating the LIFESEQ.RTM. EST relational database and Genentech proprietary information in a stringent and statistically meaningful way. In this example, GEPIS is used to identify and cross-validate novel tumor antigens, although GEPIS can be configured to perform either very specific analyses or broad screening tasks. For the initial screen, GEPIS is used to identify EST sequences from the LIFESEQ.RTM. database that correlate to expression in a particular tissue or tissues of interest (often a tumor tissue of interest). The EST sequences identified in this initial screen (or consensus sequences obtained from aligning multiple related and overlapping EST sequences obtained from the initial screen) were then subjected to a screen intended to identify the presence of at least one transmembrane domain in the encoded protein. Finally, GEPIS was employed to generate a complete tissue expression profile for the various sequences of interest. Using this type of screening bioinformatics, various TAT polypeptides (and their encoding nucleic acid molecules) were identified as being significantly overexpressed in a particular type of cancer or certain cancers as compared to other cancers and/or normal non-cancerous tissues. The rating of GEPIS hits is based upon several criteria including, for example, tissue specificity, tumor specificity and expression level in normal essential and/or normal proliferating tissues. The following is a list of molecules whose tissue expression profile as determined by GEPIS evidences high tissue expression and significant upregulation of expression in a specific tumor or tumors as compared to other tumor(s) and/or normal tissues and optionally relatively low expression in normal essential and/or normal proliferating tissues. As such, the molecules listed below are excellent polypeptide targets for the diagnosis and therapy of cancer in mammals. TABLE-US-00008 upregulation of Molecule expression in: as compared to: DNA225785 (TAT295) colon tumor normal colon tissue DNA88158 (TAT298) uterine tumor normal uterine tissue DNA225800 (TAT347) uterine tumor normal uterine tissue DNA293550 (TAT293) prostate tumor normal prostate tissue DNA293550 (TAT293) breast tumor normal breast tissue DNA293550 (TAT293) colon tumor normal colon tissue DNA293550 (TAT293) uterine tumor normal uterine tissue DNA293550 (TAT293) pancreatic tumor normal pancreatic tissue DNA293550 (TAT293) ovarian tumor normal ovarian tissue DNA293550 (TAT293) bladder tumor normal bladder tissue DNA293550 (TAT293) brain tumor normal brain tissue DNA103386 (TAT299) testicular tumor normal testicular tissue DNA273438 (TAT351) testicular tumor normal testicular tissue DNA225865 (TAT300) uterine tumor normal uterine tissue DNA225865 (TAT300) kidney tumor normal kidney tissue DNA225865 (TAT300) bone tumor normal bone tissue DNA225865 (TAT300) esophagus tumor normal esophagus tissue DNA225865 (TAT300) bladder tumor normal bladder tissue DNA226403 (TAT302) prostate tumor normal prostate tissue DNA73736 (TAT303) testicular tumor normal testicular tissue DNA73736 (TAT303) ovarian tumor normal ovarian tissue DNA73736 (TAT303) prostate tumor normal prostate tissue

EXAMPLE 5

Identification of TAT Polypeptides that are Specifically Expressed by or Overexpressed in Lymphoid Tissue(s)

[0805] Using the GeneExpress.RTM. and GEPIS-associated tissue expression analyses described in certain Examples above, various TAT polypeptides have been identified as being either specifically expressed on the surface of both normal and malignant B cells or overexpressed in cancerous lymphoid tissue(s) as compared to its normal tissue counterpart. By "specifically expressed" on a certain tissue or cell type, it is meant that the TAT polypeptide is expressed by that certain tissue or cell type and is not significantly expressed by any other tissue or cell type. TAT polypeptides that are specifically expressed on the surface of both normal and malignant B cells or overexpressed in cancerous lymphoid tissue(s) as compared to its normal tissue counterpart are excellent polypeptide targets for the treatment of B cell and other lymphoid-associated cancers including, for example, high, intermediate and low grade lymphomas (including B cell lymphomas such as, for example, mucosa-associated-lymphoid tissue B cell lymphoma and non-Hodgkin's lymphoma, mantle cell lymphoma, Burkitt's lymphoma, marginal zone lymphoma, diffuse large cell lymphoma, follicular lymphoma, and Hodgkin's lymphoma) and leukemias (including chronic lymphocytic leukemia), multiple myeloma, and other hematological and/or B cell-associated cancers. In this regard, we note that RITUXAN.RTM. (Genentech Inc., South San Francisco, Calif.) is an antibody that binds to the CD20 antigen that is specifically expressed on the surface of both normal and malignant B cells and which has been used successfully to treat non-Hodgkin's lymphoma in humans. The following are TAT polypeptides that have been herein identified as being specifically expressed on the surface of both normal and malignant B-cells or overexpressed in cancerous lymphoid tissue(s) as compared to their normal tissue counterpart and, therefore, will serve as excellent polypeptide targets for the treatment of B-cell and other lyumphoid-associated cancers: TAT294 (DNA150813), TAT297 (DNA103365), TAT301 (DNA 196665) and TAT352 (DNA287183).

EXAMPLE 6

Use of TAT as a Hybridization Probe

[0806] The following method describes use of a nucleotide sequence encoding TAT as a hybridization probe for, i.e., diagnosis of the presence of a tumor in a mammal.

[0807] DNA comprising the coding sequence of full-length or mature TAT as disclosed herein can also be employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring variants of TAT) in human tissue cDNA libraries or human tissue genomic libraries.

[0808] Hybridization and washing of filters containing either library DNAs is performed under the following high stringency conditions. Hybridization of radiolabeled TAT-derived probe to the filters is performed in a solution of 50% formamide, 5.times.SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2.times.Denhardt's solution, and 10% dextran sulfate at 42.degree. C. for 20 hours. Washing of the filters is performed in an aqueous solution of 0.1.times.SSC and 0.1% SDS at 42.degree. C.

[0809] DNAs having a desired sequence identity with the DNA encoding full-length native sequence TAT can then be identified using standard techniques known in the art.

EXAMPLE 7

Expression of TAT in E. coli

[0810] This example illustrates preparation of an unglycosylated form of TAT by recombinant expression in E. coli.

[0811] The DNA sequence encoding TAT is initially amplified using selected PCR primers. The primers should contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector. A variety of expression vectors may be employed. An example of a suitable vector is pBR322 (derived from E. coli; see Bolivar et al., Gene, 2:95 (1977)) which contains genes for ampicillin and tetracycline resistance. The vector is digested with restriction enzyme and dephosphorylated. The PCR amplified sequences are then ligated into the vector. The vector will preferably include sequences which encode for an antibiotic resistance gene, a trp promoter, a polyhis leader (including the first six STII codons, polyhis sequence, and enterokinase cleavage site), the TAT coding region, lambda transcriptional terminator, and an argU gene.

[0812] The ligation mixture is then used to transform a selected E. coli strain using the methods described in Sambrook et al., supra. Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing.

[0813] Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics. The overnight culture may subsequently be used to inoculate a larger scale culture. The cells are then grown to a desired optical density, during which the expression promoter is turned on.

[0814] After culturing the cells for several more hours, the cells can be harvested by centrifugation. The cell pellet obtained by the centrifugation can be solubilized using various agents known in the art, and the solubilized TAT protein can then be purified using a metal chelating column under conditions that allow tight binding of the protein.

[0815] TAT may be expressed in E. coli in a poly-His tagged form, using the following procedure. The DNA encoding TAT is initially amplified using selected PCR primers. The primers will contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector, and other useful sequences providing for efficient and reliable translation initiation, rapid purification on a metal chelation column, and proteolytic removal with enterokinase. The PCR-amplified, poly-His tagged sequences are then ligated into an expression vector, which is used to transform an E. coli host based on strain 52 (W3110 fuhA(tonA) Ion galE rpoHts(htpRts) clpP(lacIq). Transformants are first grown in LB containing 50 mg/ml carbenicillin at 30.degree. C. with shaking until an O.D.600 of 3-5 is reached. Cultures are then diluted 50-100 fold into CRAP media (prepared by mixing 3.57 g (NH.sub.4).sub.2SO.sub.4, 0.71 g sodium citrate.2H.sub.2O, 1.07 g KCl, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 mL water, as well as 110 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSO.sub.4) and grown for approximately 20-30 hours at 30.degree. C. with shaking. Samples are removed to verify expression by SDS-PAGE analysis, and the bulk culture is centrifuged to pellet the cells. Cell pellets are frozen until purification and refolding.

[0816] E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) is resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8 buffer. Solid sodium sulfite and sodium tetrathionate is added to make final concentrations of 0.1M and 0.02 M, respectively, and the solution is stirred overnight at 4.degree. C. This step results in a denatured protein with all cysteine residues blocked by sulfitolization. The solution is centrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. The supernatant is diluted with 3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micron filters to clarify. The clarified extract is loaded onto a 5 ml Qiagen Ni--NTA metal chelate column equilibrated in the metal chelate column buffer. The column is washed with additional buffer containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted with buffer containing 250 mM imidazole. Fractions containing the desired protein are pooled and stored at 4.degree. C. Protein concentration is estimated by its absorbance at 280 nm using the calculated extinction coefficient based on its amino acid sequence.

[0817] The proteins are refolded by diluting the sample slowly into freshly prepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Refolding volumes are chosen so that the final protein concentration is between 50 to 100 micrograms/ml. The refolding solution is stirred gently at 4.degree. C. for 12-36 hours. The refolding reaction is quenched by the addition of TFA to a final concentration of 0.4% (pH of approximately 3). Before further purification of the protein, the solution is filtered through a 0.22 micron filter and acetonitrile is added to 2-10% final concentration. The refolded protein is chromatographed on a Poros R1/H reversed phase column using a mobile buffer of 0.1% TFA with elution with a gradient of acetonitrile from 10 to 80%. Aliquots of fractions with A280 absorbance are analyzed on SDS polyacrylamide gels and fractions containing homogeneous refolded protein are pooled. Generally, the properly refolded species of most proteins are eluted at the lowest concentrations of acetonitrile since those species are the most compact with their hydrophobic interiors shielded from interaction with the reversed phase resin. Aggregated species are usually eluted at higher acetonitrile concentrations. In addition to resolving misfolded forms of proteins from the desired form, the reversed phase step also removes endotoxin from the samples.

[0818] Fractions containing the desired folded TAT polypeptide are pooled and the acetonitrile removed using a gentle stream of nitrogen directed at the solution. Proteins are formulated into 20 mM Hepes, pH 6.8 with 0.14 M sodium chloride and 4% mannitol by dialysis or by gel filtration using G25 Superfme (Pharmacia) resins equilibrated in the formulation buffer and sterile filtered.

[0819] Certain of the TAT polypeptides disclosed herein have been successfully expressed and purified using this technique(s).

EXAMPLE 8

Expression of TAT in Mammalian Cells

[0820] This example illustrates preparation of a potentially glycosylated form of TAT by recombinant expression in mammalian cells.

[0821] The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employed as the expression vector. Optionally, the TAT DNA is ligated into pRK5 with selected restriction enzymes to allow insertion of the TAT DNA using ligation methods such as described in Sambrook et al., supra. The resulting vector is called pRK5-TAT.

[0822] In one embodiment, the selected host cells may be 293 cells. Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics. About 10 .mu.g pRK5-TAT DNA is mixed with about 1 .mu.g DNA encoding the VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in 500 .mu.l of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl.sub.2. To this mixture is added, dropwise, 500 .mu.l of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO.sub.4, and a precipitate is allowed to form for 10 minutes at 25.degree. C. The precipitate is suspended and added to the 293 cells and allowed to settle for about four hours at 37.degree. C. The culture medium is aspirated off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293 cells are then washed with serum free medium, fresh medium is added and the cells are incubated for about 5 days.

[0823] Approximately 24 hours after the transfections, the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 .mu.Ci/ml .sup.35S-cysteine and 200 .mu.Ci/ml .sup.35S-methionine. After a 12 hour incubation, the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15% SDS gel. The processed gel may be dried and exposed to film for a selected period of time to reveal the presence of TAT polypeptide. The cultures containing transfected cells may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays.

[0824] In an alternative technique, TAT may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are grown to maximal density in a spinner flask and 700 .mu.g pRK5-TAT DNA is added. The cells are first concentrated from the spinner flask by centrifugation and washed with PBS. The DNA-dextran precipitate is incubated on the cell pellet for four hours. The cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and re-introduced into the spinner flask containing tissue culture medium, 5 .mu.g/ml bovine insulin and 0.1 .mu.g/ml bovine transferrin. After about four days, the conditioned media is centrifuged and filtered to remove cells and debris. The sample containing expressed TAT can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography.

[0825] In another embodiment, TAT can be expressed in CHO cells. The pRK5-TAT can be transfected into CHO cells using known reagents such as CaPO.sub.4 or DEAE-dextran. As described above, the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as .sup.35S-methionine. After determining the presence of TAT polypeptide, the culture medium may be replaced with serum free medium. Preferably, the cultures are incubated for about 6 days, and then the conditioned medium is harvested. The medium containing the expressed TAT can then be concentrated and purified by any selected method.

[0826] Epitope-tagged TAT may also be expressed in host CHO cells. The TAT may be subcloned out of the pRK5 vector. The subclone insert can undergo PCR to fuse in frame with a selected epitope tag such as a poly-his tag into a Baculovirus expression vector. The poly-his tagged TAT insert can then be subcloned into a SV40 driven vector containing a selection marker such as DHFR for selection of stable clones. Finally, the CHO cells can be transfected (as described above) with the SV40 driven vector. Labeling may be performed, as described above, to verify expression. The culture medium containing the expressed poly-His tagged TAT can then be concentrated and purified by any selected method, such as by Ni.sup.2+-chelate affinity chromatography.

[0827] TAT may also be expressed in CHO and/or COS cells by a transient expression procedure or in CHO cells by another stable expression procedure.

[0828] Stable expression in CHO cells is performed using the following procedure. The proteins are expressed as an IgG construct (inmunoadhesin), in which the coding sequences for the soluble forms (e.g. extracellular domains) of the respective proteins are fused to an IgG1 constant region sequence containing the hinge, CH2 and CH2 domains and/or is a poly-His tagged form.

[0829] Following PCR amplification, the respective DNAs are subcloned in a CHO expression vector using standard techniques as described in Ausubel et al., Current Protocols of Molecular Biology, Unit 3.16, John Wiley and Sons (1997). CHO expression vectors are constructed to have compatible restriction sites 5' and 3' of the DNA of interest to allow the convenient shuttling of cDNA's. The vector used expression in CHO cells is as described in Lucas et al., Nucl. Acids Res. 24:9 (1774-1779 (1996), and uses the SV40 early promoter/enhancer to drive expression of the cDNA of interest and dihydrofolate reductase (DHFR). DHFR expression permits selection for stable maintenance of the plasmid following transfection.

[0830] Twelve micrograms of the desired plasmid DNA is introduced into approximately 10 million CHO cells using commercially available transfection reagents Superfect.RTM. (Quiagen), Dosper.RTM. or Fugene.RTM. (Boehringer Mannheim). The cells are grown as described in Lucas et al., supra. Approximately 3.times.10.sup.7 cells are frozen in an ampule for further growth and production as described below.

[0831] The ampules containing the plasmid DNA are thawed by placement into water bath and mixed by vortexing. The contents are pipetted into a centrifuge tube containing 10 mLs of media and centrifuged at 1000 rpm for 5 minutes. The supernatant is aspirated and the cells are resuspended in 10 mL of selective media (0.2 .mu.m filtered PS20 with 5% 0.2 .mu.m diafiltered fetal bovine serum). The cells are then aliquoted into a 100 mL spinner containing 90 mL of selective media. After 1-2 days, the cells are transferred into a 250 mL spinner filled with 150 mL selective growth medium and incubated at 37.degree. C. After another 2-3 days, 250 mL, 500 mL and 2000 mL spinners are seeded with 3.times.10.sup.5 cells/mL. The cell media is exchanged with fresh media by centrifugation and resuspension in production medium. Although any suitable CHO media may be employed, a production medium described in U.S. Pat. No. 5,122,469, issued Jun. 16, 1992 may actually be used. A 3 L production spinner is seeded at 1.2.times.10.sup.6 cells/mL. On day 0, the cell number pH ie determined. On day 1, the spinner is sampled and sparging with filtered air is commenced. On day 2, the spinner is sampled, the temperature shifted to 33.degree. C., and 30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (e.g., 35% polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion) taken. Throughout the production, the pH is adjusted as necessary to keep it at around 7.2. After 10 days, or until the viability dropped below 70%, the cell culture is harvested by centrifugation and filtering through a 0.22 .mu.m filter. The filtrate was either stored at 4.degree. C. or immediately loaded onto columns for purification.

[0832] For the poly-His tagged constructs, the proteins are purified using a Ni--NTA column (Qiagen). Before purification, inidazole is added to the conditioned media to a concentration of 5 mM. The conditioned media is pumped onto a 6 ml Ni--NTA column equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4.degree. C. After loading, the column is washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein is subsequently desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -80.degree. C.

[0833] Immunoadhesin (Fc-containing) constructs are purified from the conditioned media as follows. The conditioned medium is pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column is washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein is immediately neutralized by collecting 1 ml fractions into tubes containing 275 .mu.L of 1 M Tris buffer, pH 9. The highly purified protein is subsequently desalted into storage buffer as described above for the poly-His tagged proteins. The homogeneity is assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation.

[0834] Certain of the TAT polypeptides disclosed herein have been successfully expressed and purified using this technique(s).

EXAMPLE 9

Expression of TAT in Yeast

[0835] The following method describes recombinant expression of TAT in yeast.

[0836] First, yeast expression vectors are constructed for intracellular production or secretion of TAT from the ADH2/GAPDH promoter. DNA encoding TAT and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of TAT. For secretion, DNA encoding TAT can be cloned into the selected plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native TAT signal peptide or other mammalian signal peptide, or, for example, a yeast alpha-factor or invertase secretory signal/leader sequence, and linker sequences (if needed) for expression of TAT.

[0837] Yeast cells, such as yeast strain AB 110, can then be transformed with the expression plasmids described above and cultured in selected fermentation media. The transformed yeast supernatants can be analyzed by precipitation with 10% trichloroacetic acid and separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

[0838] Recombinant TAT can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters. The concentrate containing TAT may further be purified using selected column chromatography resins.

[0839] Certain of the TAT polypeptides disclosed herein have been successfully expressed and purified using this technique(s).

EXAMPLE 10

Expression of TAT in Baculovirus-Infected Insect Cells

[0840] The following method describes recombinant expression of TAT in Baculovirus-infected insect cells.

[0841] The sequence coding for TAT is fused upstream of an epitope tag contained within a baculovirus expression vector. Such epitope tags include poly-his tags and immunoglobulin tags (like Fc regions of IgG). A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen). Briefly, the sequence encoding TAT or the desired portion of the coding sequence of TAT such as the sequence encoding an extracellular domain of a transmembrane protein or the sequence encoding the mature protein if the protein is extracellular is amplified by PCR with primers complementary to the 5' and 3' regions. The 5' primer may incorporate flanking (selected) restriction enzyme sites. The product is then digested with those selected restriction enzymes and subcloned into the expression vector.

[0842] Recombinant baculovirus is generated by co-transfecting the above plasmid and BaculoGold.TM. virus DNA (Pharmingen) into Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711) using lipofectin (commercially available from GIBCO-BRL). After 4-5 days of incubation at 28.degree. C., the released viruses are harvested and used for further amplifications. Viral infection and protein expression are performed as described by O'Reilley et al., Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford University Press (1994).

[0843] Expressed poly-his tagged TAT can then be purified, for example, by Ni.sup.2+-chelate affinity chromatography as follows. Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al., Nature, 362:175-179 (1993). Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl.sub.2; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicated twice for 20 seconds on ice. The sonicates are cleared by centrifugation, and the supernatant is diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0.45 .mu.m filter. A Ni.sup.2+-NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 mL, washed with 25 mL of water and equilibrated with 25 mL of loading buffer. The filtered cell extract is loaded onto the column at 0.5 mL per minute. The column is washed to baseline A.sub.280 with loading buffer, at which point fraction collection is started. Next, the column is washed with a secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein. After reaching A.sub.280 baseline again, the column is developed with a 0 to 500 mM Imidazole gradient in the secondary wash buffer. One mL fractions are collected and analyzed by SDS-PAGE and silver staining or Western blot with Ni.sup.2+-NTA-conjugated to alkaline phosphatase (Qiagen). Fractions containing the eluted His.sub.10-tagged TAT are pooled and dialyzed against loading buffer.

[0844] Alternatively, purification of the IgG tagged (or Fc tagged) TAT can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography.

[0845] Certain of the TAT polypeptides disclosed herein have been successfully expressed and purified using this technique(s).

EXAMPLE 11

Preparation of Antibodies that Bind TAT

[0846] This example illustrates preparation of monoclonal antibodies which can specifically bind TAT.

[0847] Techniques for producing the monoclonal antibodies are known in the art and are described, for instance, in Goding, supra. Immunogens that may be employed include purified TAT, fusion proteins containing TAT, and cells expressing recombinant TAT on the cell surface. Selection of the immunogen can be made by the skilled artisan without undue experimentation.

[0848] Mice, such as Balb/c, are immunized with the TAT immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount from 1-100 micrograms. Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, Mont.) and injected into the animal's hind foot pads. The immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. Thereafter, for several weeks, the mice may also be boosted with additional immunization injections. Serum samples may be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect anti-TAT antibodies.

[0849] After a suitable antibody titer has been detected, the animals "positive" for antibodies can be injected with a final intravenous injection of TAT. Three to four days later, the mice are sacrificed and the spleen cells are harvested. The spleen cells are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU.1, available from ATCC, No. CRL 1597. The fusions generate hybridoma cells which can then be plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.

[0850] The hybridoma cells will be screened in an ELISA for reactivity against TAT. Determination of "positive" hybridoma cells secreting the desired monoclonal antibodies against TAT is within the skill in the art.

[0851] The positive hybridoma cells can be injected intraperitoneally into syngeneic Balb/c mice to produce ascites containing the anti-TAT monoclonal antibodies. Alternatively, the hybridoma cells can be grown in tissue culture flasks or roller bottles. Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed.

EXAMPLE 12

Purification of TAT Polypeptides Using Specific Antibodies

[0852] Native or recombinant TAT polypeptides may be purified by a variety of standard techniques in the art of protein purification. For example, pro-TAT polypeptide, mature TAT polypeptide, or pre-TAT polypeptide is purified by immunoaffinity chromatography using antibodies specific for the TAT polypeptide of interest. In general, an immunoaffinity column is constructed by covalently coupling the anti-TAT polypeptide antibody to an activated chromatographic resin.

[0853] Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise, monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated SEPHAROSE.TM. (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.

[0854] Such an immunoaffinity column is utilized in the purification of TAT polypeptide by preparing a fraction from cells containing TAT polypeptide in a soluble form. This preparation is derived by solubilization of the whole cell or of a subcellular fraction obtained via differential centrifugation by the addition of detergent or by other methods well known in the art. Alternatively, soluble TAT polypeptide containing a signal sequence may be secreted in useful quantity into the medium in which the cells are grown.

[0855] A soluble TAT polypeptide-containing preparation is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of TAT polypeptide (e.g., high ionic strength buffers in the presence of detergent). Then, the column is eluted under conditions that disrupt antibody/TAT polypeptide binding (e.g., a low pH buffer such as approximately pH 2-3, or a high concentration of a chaotrope such as urea or thiocyanate ion), and TAT polypeptide is collected.

EXAMPLE 13

In Vitro Tumor Cell Killing Assay

[0856] Mammalian cells expressing the TAT polypeptide of interest may be obtained using standard expression vector and cloning techniques. Alternatively, many tumor cell lines expressing TAT polypeptides of interest are publicly available, for example, through the ATCC and can be routinely identified using standard ELISA or FACS analysis. Anti-TAT polypeptide monoclonal antibodies (and toxin conjugated derivatives thereof) may then be employed in assays to determine the ability of the antibody to kill TAT polypeptide expressing cells in vitro.

[0857] For example, cells expressing the TAT polypeptide of interest are obtained as described above and plated into 96 well dishes. In one analysis, the antibody/toxin conjugate (or naked antibody) is included throughout the cell incubation for a period of 4 days. In a second independent analysis, the cells are incubated for 1 hour with the antibody/toxin conjugate (or naked antibody) and then washed and incubated in the absence of antibody/toxin conjugate for a period of 4 days. Cell viability is then measured using the CellTiter-Glo Luminescent Cell Viability Assay from Promega (Cat#G7571). Untreated cells serve as a negative control.

EXAMPLE 14

In Vivo Tumor Cell Killing Assay

[0858] To test the efficacy of conjugated or unconjugated anti-TAT polypeptide monoclonal antibodies, anti-TAT antibody is injected intraperitoneally into nude mice 24 hours prior to receiving tumor promoting cells subcutaneously in the flank. Antibody injections continue twice per week for the remainder of the study. Tumor volume is then measured twice per week.

[0859] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by the construct deposited, since the deposited embodiment is intended as a single illustration of certain aspects of the invention and any constructs that are functionally equivalent are within the scope of this invention. The deposit of material herein does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustrations that it represents. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

Sequence CWU 1

1

32 1 2442 DNA Homo sapiens 1 gagcgagagg ccgggggtgc cgagccgggc ggggagagct gggccgggag 50 agcagaacag ggaggctaga gcgcagcggg aaccggcccg gagccggagc 100 cggagcccca caggcaccta ctaaaccgcc cagccgatcg gcccccacag 150 agtggcccgc gggcctccgg ccgggcccag tcccctcccg ggccctccat 200 ggcccgggcc gctgccctcc tgccgtcgag atcgccgccg acgccgctgc 250 tgtggccgct gctgctgctg ctgctcctgg aaaccggagc ccaggatgtg 300 cgagttcaag tgctacccga ggtgcgaggc cagctcgggg gcaccgtgga 350 gctgccgtgc cacctgctgc cacctgttcc tggactgtac atctccctgg 400 tgacctggca gcgcccagat gcacctgcga accaccagaa tgtggccgcc 450 ttccacccta agatgggtcc cagcttcccc agcccgaagc ctggcagcga 500 gcggctgtcc ttcgtctctg ccaagcagag cactgggcaa gacacagagg 550 cagagctcca ggacgccacg ctggccctcc acgggctcac ggtggaggac 600 gagggcaact acacttgcga gtttgccacc ttccccaagg ggtccgtccg 650 agggatgacc tggctcagag tcatagccaa gcccaagaac caagctgagg 700 cccagaaggt cacgttcagc caggacccta cgacagtggc cctctgcatc 750 tccaaagagg gccgcccacc tgcccggatc tcctggctct catccctgga 800 ctgggaagcc aaagagactc aggtgtcagg gaccctggcc ggaactgtca 850 ctgtcaccag ccgcttcacc ttggtgccct cgggccgagc agatggtgtc 900 acggtcacct gcaaagtgga gcatgagagc ttcgaggaac cagccctgat 950 acctgtgacc ctctctgtac gctaccctcc tgaagtgtcc atctccggct 1000 atgatgacaa ctggtacctc ggccgtactg atgccaccct gagctgtgac 1050 gtccgcagca acccagagcc cacgggctat gactggagca cgacctcagg 1100 caccttcccg acctccgcag tggcccaggg ctcccagctg gtcatccacg 1150 cagtggacag tctgttcaat accaccttcg tctgcacagt caccaatgcc 1200 gtgggcatgg gccgcgctga gcaggtcatc tttgtccgag agacccccaa 1250 cacagcaggc gcaggggcca caggcggcat catcgggggc atcatcgccg 1300 ccatcattgc tactgctgtg gctgccacgg gcatccttat ctgccggcag 1350 cagcggaagg agcagacgct gcagggggca gaggaggacg aagacctgga 1400 gggacctccc tcctacaagc caccaacccc aaaagcgaag ctggaggcac 1450 aggagatgcc ctcccagctc ttcactctgg gggcctcgga gcacagccca 1500 ctcaagaccc cctactttga tgctggcgcc tcatgcactg agcaggaaat 1550 gcctcgatac catgagctgc ccaccttgga agaacggtca ggacccttgc 1600 accctggagc cacaagcctg gggtccccca tcccggtgcc tccagggcca 1650 cctgctgtgg aagacgtttc cctggatcta gaggatgagg agggggagga 1700 ggaggaagag tatctggaca agatcaaccc catctatgat gctctgtcct 1750 atagcagccc ctctgattcc taccagggca aaggctttgt catgtcccgg 1800 gccatgtatg tgtgagctgc catgcgcctg gcgtctcaca tctcacctgt 1850 tgatccctta gctttcttgc caaggatcta gtgccccctg acctctggcc 1900 aggccactgt cagttaacac atatgcattc catttgtgat gtctaccttg 1950 gtggctccac tatgacccct aacccatgag cccagagaaa ttcaccgtga 2000 taatggaatc ctggcaacct tatctcatga ggcaggaggt ggggaaggtg 2050 cttctgcaca acctctgatc ccaaggactc ctctcccaga ctgtgacctt 2100 agaccatacc tctcaccccc caatgcctcg actcccccaa aatcacaaag 2150 aagaccctag acctataatt tgtcttcagg tagtaaattc ctgcctacca 2200 agcaagcagc cccagcctag ggtcagacag ggtgagcctc atacagactg 2250 tgccttgatg gccccagcct tgggagaaga atttactgtt aacctggaag 2300 actactgaat cattttaccc ttgcccagtg gaataggacc taaacatccc 2350 ccttccgggg aaagtgggtc atctgaattg ggggtagcaa ttgatactgt 2400 tttgtaaact acatttccta caaaatatga atttatactt tg 2442 2 993 DNA Homo sapiens 2 atgatcaccc tgaacaatca agatcaacct gtcactttta acagctcaca 50 tccagatgaa tacaaaattg cagcccttgt cttctatagc tgtatcttca 100 taattggatt atttgttaac atcactgcat tatgggtttt cagttgtacc 150 accaagaaga gaaccacggt aaccatctat atgatgaatg tggcattagt 200 ggacttgata tttataatga ctttaccctt tcgaatgttt tattatgcaa 250 aagatgcatg gccatttgga gagtacttct gccagattat tggagctctc 300 acagtgtttt acccaagcat tgctttatgg cttcttgcct ttattagtgc 350 tgacagatac atggccattg tacagccgaa gtacgccaaa gaacttaaaa 400 acacgtgcaa agccgtgctg gcgtgtgtgg gagtctggat aatgaccctg 450 accacgacca cccctctgct actgctctat aaagacccag ataaagactc 500 cactcccgcc acctgcctca agatttctga catcatctat ctaaaagctg 550 tgaacgtgct gaacctcact cgactgacat tttttttctt gattcctttg 600 ttcatcatga ttgggtgcta cttggtcatt attcataatc tccttcacgg 650 caggacgtct aagctgaaac ccaaagtcaa ggagaagtcc ataaggatca 700 tcatcacgct gctggtgcag gtgctcgtct gctttatgcc cttccacatc 750 tgtttcgctt tcctgatgct gggaacgggg gagaacagtt acaatccctg 800 gggagccttt accaccttcc tcatgaacct cagcacgtgt ctggatgtga 850 ttctctacta catcgtttca aaacaatttc aggctcgagt cattagtgtc 900 atgctatacc gtaattacct tcgaagcctg cgcagaaaaa gtttccgatc 950 tggtagtcta aggtcactaa gcaatataaa cagtgaaatg tta 993 3 1107 DNA Homo sapiens 3 tgctgcaact caaactaacc aacccactgg gagaagatgc ctgggggtcc 50 aggagtcctc caagctctgc ctgccaccat cttcctcctc ttcctgctgt 100 ctgctgtcta cctgggccct gggtgccagg ccctgtggat gcacaaggtc 150 ccagcatcat tgatggtgag cctgggggaa gacgcccact tccaatgccc 200 gcacaatagc agcaacaacg ccaacgtcac ctggtggcgc gtcctccatg 250 gcaactacac gtggccccct gagttcttgg gcccgggcga ggaccccaat 300 ggtacgctga tcatccagaa tgtgaacaag agccatgggg gcatatacgt 350 gtgccgggtc caggagggca acgagtcata ccagcagtcc tgcggcacct 400 acctccgcgt gcgccagccg ccccccaggc ccttcctgga catgggggag 450 ggcaccaaga accgaatcat cacagccgag gggatcatcc tcctgttctg 500 cgcggtggtg cctgggacgc tgctgctgtt caggaaacga tggcagaacg 550 agaagctcgg gttggatgcc ggggatgaat atgaagatga aaacctttat 600 gaaggcctga acctggacga ctgctccatg tatgaggaca tctcccgggg 650 cctccagggc acctaccagg atgtgggcag cctcaacata ggagatgtcc 700 agctggagaa gccgtgacac ccctactcct gccaggctgc ccccgcctgc 750 tgtgcaccca gctccagtgt ctcagctcac ttccctggga cattctcctt 800 tcagcccttc tgggggcttc cttagtcata ttcccccagt ggggggtggg 850 agggtaacct cactcttctc caggccaggc ctccttggac tcccctgggg 900 gtgtcccact cttcttccct ctaaactgcc ccacctccta acctaatccc 950 cacgccccgc tgcctttccc aggctcccct cacccagcgg gtaatgagcc 1000 cttaatcgct gcctctaggg gagctgattg tagcagcctc gttagtgtca 1050 ccccctcctc cctgatctgt cagggccact tagtgataat aaattcttcc 1100 caactgc 1107 4 1524 DNA Homo sapiens 4 agcagacaga ggactctcat taaggaaggt gtcctgtgcc ctgaccctac 50 aagatgccaa gagaagatgc tcacttcatc tatggttacc ccaagaaggg 100 gcacggccac tcttacacca cggctgaaga ggccgctggg atcggcatcc 150 tgacagtgat cctgggagtc ttactgctca tcggctgttg gtattgtaga 200 agacgaaatg gatacagagc cttgatggat aaaagtcttc atgttggcac 250 tcaatgtgcc ttaacaagaa gatgcccaca agaagggttt gatcatcggg 300 acagcaaagt gtctcttcaa gagaaaaact gtgaacctgt ggttcccaat 350 gctccacctg cttatgagaa actctctgca gaacagtcac caccacctta 400 ttcaccttaa gagccagcga gacacctgag acatgctgaa attatttctc 450 tcacactttt gcttgaattt aatacagaca tctaatgttc tcctttggaa 500 tggtgtagga aaaatgcaag ccatctctaa taataagtca gtgttaaaat 550 tttagtaggt ccgctagcag tactaatcat gtgaggaaat gatgagaaat 600 attaaattgg gaaaactcca tcaataaatg ttgcaatgca tgatactatc 650 tgtgccagag gtaatgttag taaatccatg gtgttatttt ctgagagaca 700 gaattcaagt gggtattctg gggccatcca atttctcttt acttgaaatt 750 tggctaataa caaactagtc aggttttcga accttgaccg acatgaactg 800 tacacagaat tgttccagta ctatggagtg ctcacaaagg atacttttac 850 aggttaagac aaagggttga ctggcctatt tatctgatca agaacatgtc 900 agcaatgtct ctttgtgctc taaaattcta ttatactaca ataatatatt 950 gtaaagatcc tatagctctt tttttttgag atggagtttc gcttttgttg 1000 cccaggctgg agtgcaatgg cgcgatcttg gctcaccata acctccgcct 1050 cccaggttca agcaattctc ctgccttagc ctcctgagta gctgggatta 1100 caggcgtgcg ccactatgcc tgactaattt tgtagtttta gtagagacgg 1150 ggtttctcca tgttggtcag gctggtctca aactcctgac ctcaggtgat 1200 ctgcccgcct cagcctccca aagtgctgga attacaggcg tgagccacca 1250 cgcctggctg gatcctatat cttaggtaag acatataacg cagtctaatt 1300 acatttcact tcaaggctca atgctattct aactaatgac aagtattttc 1350 tactaaacca gaaattggta gaaggattta aataagtaaa agctactatg 1400 tactgcctta gtgctgatgc ctgtgtactg ccttaaatgt acctatggca 1450 atttagctct cttgggttcc caaatccctc tcacaagaat gtgcagaaga 1500 aatcataaag gatcagagat tctg 1524 5 1125 DNA Homo sapiens 5 gtctccccca ctgtcagcac ctcttctgtg tggtgagtgg accgcttacc 50 ccactaggtg aagatgtcag cccaggagag ctgcctcagc ctcatcaagt 100 acttcctctt cgttttcaac ctcttcttct tcgtcctcgg cagcctgatc 150 ttctgcttcg gcatctggat cctcatcgac aagaccagct tcgtgtcctt 200 tgtgggcttg gccttcgtgc ctctgcagat ctggtccaaa gtcctggcca 250 tctcaggaat cttcaccatg ggcatcgccc tcctgggttg tgtgggggcc 300 ctcaaggagc tccgctgcct cctgggcctg tattttggga tgctgctgct 350 cctgtttgcc acacagatca ccctgggaat cctcatctcc actcagcggg 400 cccagctgga gcgaagcttg cgggacgtcg tagagaaaac catccaaaag 450 tacggcacca accccgagga gaccgcggcc gaggagagct gggactatgt 500 gcagttccag ctgcgctgct gcggctggca ctacccgcag gactggttcc 550 aagtcctcat cctgagaggt aacgggtcgg aggcgcaccg cgtgccctgc 600 tcctgctaca acttgtcggc gaccaacgac tccacaatcc tagataaggt 650 gatcttgccc cagctcagca ggcttggaca cctggcgcgg tccagacaca 700 gtgcagacat ctgcgctgtc cctgcagaga gccacatcta ccgcgagggc 750 tgcgcgcagg gcctccagaa gtggctgcac aacaacctta tttccatagt 800 gggcatttgc ctgggcgtcg gcctactcga gctcgggttc atgacgctct 850 cgatattcct gtgcagaaac ctggaccacg tctacaaccg gctcgctcga 900 taccgttagg ccccgccctc cccaaagtcc cgccccgccc ccgtcacgtg 950 cgctgggcac ttccctgctg cctgtaaata tttgtttaat ccccagttcg 1000 cctggagccc tccgccttca cattcccctg gggacccacg tggctgcgtg 1050 cccctgctgc tgtcacctct cccacgggac ctggggcttt cgtccacagc 1100 ttcctgtccc catctgtcgg cctac 1125 6 4523 DNA Homo sapiens 6 ccgcggtgcc gccgggaaag atggtcgtgg cgctgcggta cgtgtggcct 50 ctcctcctct gcagcccctg cctgcttatc cagatccccg aggaatatga 100 aggacaccat gtgatggagc cacctgtcat cacggaacag tctccacggc 150 gcctggttgt cttccccaca gatgacatca gcctcaagtg tgaggccagt 200 ggcaagcccg aagtgcagtt ccgctggacg agggatggtg tccacttcaa 250 acccaaggaa gagctgggtg tgaccgtgta ccagtcgccc cactctggct 300 ccttcaccat cacgggcaac aacagcaact ttgctcagag gttccagggc 350 atctaccgct gctttgccag caataagctg ggcaccgcca tgtcccatga 400 gatccggctc atggccgagg gtgcccccaa gtggccaaag gagacagtga 450 agcccgtgga ggtggaggaa ggggagtcag tggttctgcc ttgcaaccct 500 cccccaagtg cagagcctct ccggatctac tggatgaaca gcaagatctt 550 gcacatcaag caggacgagc gggtgacgat gggccagaac ggcaacctct 600 actttgccaa tgtgctcacc tccgacaacc actcagacta catctgccac 650 gcccacttcc caggcaccag gaccatcatt cagaaggaac ccattgacct 700 ccgggtcaag gccaccaaca gcatgattga caggaagccg cgcctgctct 750 tccccaccaa ctccagcagc cacctggtgg ccttgcaggg gcagccattg 800 gtcctggagt gcatcgccga gggctttccc acgcccacca tcaaatggct 850 gcgccccagt ggccccatgc cagctgaccg tgtcacctac cagaaccaca 900 acaagaccct gcagctgctg aaagtgggcg aggaggatga tggcgagtac 950 cgctgcctgg ccgagaactc actgggcagt gcccggcatg cgtactatgt 1000 caccgtggag gctgccccgt actggctgca caagccccag agccatctat 1050 atgggccagg agagactgcc cgcctggact gccaagtcca gggcaggccc 1100 caaccagagg tcacctggag aatcaacggg atccctgtgg aggagctggc 1150 caaagaccag aagtaccgga ttcagcgtgg cgccctgatc ctgagcaacg 1200 tgcagcccag tgacacaatg gtgacccaat gtgaggcccg caaccggcac 1250 gggctcttgc tggccaatgc ctacatctac gttgtccagc tgccagccaa 1300 gatcctgact gcggacaatc agacgtacat ggctgtccag ggcagcactg 1350 cctaccttct gtgcaaggcc ttcggagcgc ctgtgcccag tgttcagtgg 1400 ctggacgagg atgggacaac agtgcttcag gacgaacgct tcttccccta 1450 tgccaatggg accctgggca ttcgagacct ccaggccaat gacaccggac 1500 gctacttctg cctggctgcc aatgaccaaa acaatgttac catcatggct 1550 aacctgaagg ttaaagatgc aactcagatc actcaggggc cccgcagcac 1600 aatcgagaag aaaggttcca gggtgacctt cacgtgccag gcctcctttg 1650 acccctcctt gcagcccagc atcacctggc gtggggacgg tcgagacctc 1700 caggagcttg gggacagtga caagtacttc atagaggatg ggcgcctggt 1750 catccacagc ctggactaca gcgaccaggg caactacagc tgcgtggcca 1800 gtaccgaact ggatgtggtg gagagtaggg cacagctctt ggtggtgggg 1850 agccctgggc cggtgccacg gctggtgctg tccgacctgc acctgctgac 1900 gcagagccag gtgcgcgtgt cctggagtcc tgcagaagac cacaatgccc 1950 ccattgagaa atatgacatt gaatttgagg acaaggaaat ggcgcctgaa 2000 aaatggtaca gtctgggcaa ggttccaggg aaccagacct ctaccaccct 2050 caagctgtcg ccctatgtcc actacacctt tagggttact gccataaaca 2100 aatatggccc cggggagccc agcccggtct ctgagactgt ggtcacacct 2150 gaggcagccc cagagaagaa ccctgtggat gtgaaggggg aaggaaatga 2200 gaccaccaat atggtcatca cgtggaagcc gctccggtgg atggactgga 2250 acgcccccca ggttcagtac cgcgtgcagt ggcgccctca ggggacacga 2300 gggccctggc aggagcagat tgtcagcgac cccttcctgg tggtgtccaa 2350 cacgtccacc ttcgtgccct atgagatcaa agtccaggcc gtcaacagcc 2400 agggcaaggg accagagccc caggtcacta tcggctactc tggagaggac 2450 tacccccagg caatccctga gctggaaggc attgaaatcc tcaactcaag 2500 tgccgtgctg gtcaagtggc ggccggtgga cctggcccag gtcaagggcc 2550 acctccgcgg atacaatgtg acgtactgga gggagggcag tcagaggaag 2600 cacagcaaga gacatatcca caaagaccat gtggtggtgc ccgccaacac 2650 caccagtgtc atcctcagtg gcttgcggcc ctatagctcc taccacctgg 2700 aggtgcaggc ctttaacggg cgaggatcgg ggcccgccag cgagttcacc 2750 ttcagcaccc cagagggagt gcctggccac cccgaggcgt tgcacctgga 2800 gtgccagtcg aacaccagcc tgctgctgcg ctggcagccc ccactcagcc 2850 acaacggcgt gctcaccggc tacgtgctct cctaccaccc cctggatgag 2900 gggggcaagg ggcaactgtc cttcaacctt cgggaccccg aacttcggac 2950 acacaacctg accgatctca gcccccacct gcggtaccgc ttccagcttc 3000 aggccaccac caaagagggc cctggtgaag ccatcgtacg ggaaggaggc 3050 actatggcct tgtctgggat ctcagatttt ggcaacatct cagccacagc 3100 gggtgaaaac tacagtgtcg tctcctgggt ccccaaggag ggccagtgca 3150 acttcaggtt ccatatcttg ttcaaagcct tgggagaaga gaagggtggg 3200 gcttcccttt cgccacagta tgtcagctac aaccagagct cctacacgca 3250 gtgggacctg cagcctgaca ctgactacga gatccacttg tttaaggaga 3300 ggatgttccg gcaccaaatg gctgtgaaga ccaatggcac aggccgcgtg 3350 aggctccctc ctgctggctt cgccactgag ggctggttca tcggctttgt 3400 gagtgccatc atcctcctgc tcctcgtcct gctcatcctc tgcttcatca 3450 agcgcagcaa gggcggcaaa tactcagtga aggataagga ggacacccag 3500 gtggactctg aggcccgacc gatgaaagat gagaccttcg gcgagtacag 3550 gtccctggag agtgacaacg aggagaaggc ctttggcagc agccagccat 3600 cgctcaacgg ggacatcaag cccctgggca gtgacgacag cctggccgat 3650 tatgggggca gcgtggatgt tcagttcaac gaggatggtt cgttcattgg 3700 ccagtacagt ggcaagaagg agaaggaggc ggcagggggc aatgacagct 3750 caggggccac ttcccccatc aaccctgccg tggccctaga atagtggagt 3800 acggacagga gatgctgtgc cccctggcct tgggatccag gcccctccct 3850 ctccagcagg cccatgggag gctggagttg gggcagagga gaacttgctg 3900 cctcggatcc ccttcctacc acccggtccc cactttattg ccaaaaccca 3950 gctgcacccc ttcctgggca cacgctgctc tgccccagct tgggcagatc 4000 tcccacatgc caggggcctt tgggtgctgt tttgccagcc catttgggca 4050 gagaggctgt ggtttggggg agaagaagta ggggtggccc gaaaggtctc 4100 cgaaatgctg tctttcttgc tccctgactg ggggcagaca tggtggggtc 4150 tcctcaggac cagggttggc accttccccc tcccccagcc accactccag 4200 cagcctggct gggactggga acagaactcg tgtccccacc atctgctgtc 4250 ttttctttgc catctctgct ccaaccggga tggcagccgg gcaaactggc 4300 cgcgggggca ggggaggcca tctggagagc ccagagtccc cccactccca 4350 gcatcgcact ctggcagcac cgcctcttcc cgccgcccag cccaccccat 4400 ggccggcttt caggagctcc atacacacgc tgccttcggt acccaccaca 4450 caacatccaa gtggcctccg tcactacctg gctgcggggc gggcacacct 4500 cctcccactg cccactggcc ggc 4523 7 4525 DNA Homo sapiens 7 gcgcggtgcc gccgggaaag atggtcgtgg cgctgcggta cgtgtggcct 50 ctcctcctct gcagcccctg cctgcttatc cagatccccg aggaatatga 100 aggacaccat gtgatggagc cacctgtcat cacggaacag tctccacggc 150 gcctggttgt cttccccaca gatgacatca gcctcaagtg tgaggccagt 200 ggcaagcccg aagtgcagtt ccgctggacg agggatggtg tccacttcaa 250 acccaaggaa gagctgggtg tgaccgtgta ccagtcgccc cactctggct 300 ccttcaccat cacgggcaac aacagcaact ttgctcagag gttccagggc 350 atctaccgct gctttgccag caataagctg ggcaccgcca tgtcccatga 400 gatccggctc atggccgagg gtgcccccaa gtggccaaag gagacagtga 450 agcccgtgga ggtggaggaa ggggagtcag tggttctgcc ttgcaaccct 500 cccccaagtg cagagcctct ccggatctac tggatgaaca gcaagatctt 550 gcacatcaag

caggacgagc gggtgacgat gggccagaac ggcaacctct 600 actttgccaa tgtgctcacc tccgacaacc actcagacta catctgccac 650 gcccacttcc caggcaccag gaccatcatt cagaaggaac ccattgacct 700 ccgggtcaag gccaccaaca gcatgattga caggaagccg cgcctgctct 750 tccccaccaa ctccagcagc cacctggtgg ccttgcaggg gcagccattg 800 gtcctggagt gcatcgccga gggctttccc acgcccacca tcaaatggct 850 gcgccccagt ggccccatgc cagccgaccg tgtcacctac cagaaccaca 900 acaagaccct gcagctgctg aaagtgggcg aggaggatga tggcgagtac 950 cgctgcctgg ccgagaactc actgggcagt gcccggcatg cgtactatgt 1000 caccgtggag gctgccccgt actggctgca caagccccag agccatctat 1050 atgggccagg agagactgcc cgcctggact gccaagtcca gggcaggccc 1100 caaccagagg tcacctggag aatcaacggg atccctgtgg aggagctggc 1150 caaagaccag aagtaccgga ttcagcgtgg cgccctgatc ctgagcaacg 1200 tgcagcccag tgacacaatg gtgacccaat gtgaggcccg caaccggcac 1250 gggctcttgc tggccaatgc ctacatctac gttgtccagc tgccagccaa 1300 gatcctgact gcggacaatc agacgtacat ggctgtccag ggcagcactg 1350 cctaccttct gtgcaaggcc ttcggagcgc ctgtgcccag tgttcagtgg 1400 ctggacgagg atgggacaac agtgcttcag gacgaacgct tcttccccta 1450 tgccaatggg accctgggca ttcgagacct ccaggccaat gacaccggac 1500 gctacttctg cctggctgcc aatgaccaaa acaatgttac catcatggct 1550 aacctgaagg ttaaagatgc aactcagatc actcaggggc cccgcagcac 1600 aatcgagaag aaaggttcca gggtgacctt cacgtgccag gcctcctttg 1650 acccctcctt gcagcccagc atcacctggc gtggggacgg tcgagacctc 1700 caggagcttg gggacagtga caagtacttc atagaggatg ggcgcctggt 1750 catccacagc ctggactaca gcgaccaggg caactacagc tgcgtggcca 1800 gtaccgaact ggatgtggtg gagagtaggg cacagctctt ggtggtgggg 1850 agccctgggc cggtgccacg gctggtgctg tccgacctgc acctgctgac 1900 gcagagccag gtgcgcgtgt cctggagtcc tgcagaagac cacaatgccc 1950 ccattgagaa atatgacatt gaatttgagg acaaggaaat ggcgcctgaa 2000 aaatggtaca gtctgggcaa ggttccaggg aaccagacct ctaccaccct 2050 caagctgtcg ccctatgtcc actacacctt tagggttact gccataaaca 2100 aatatggccc cggggagccc agcccggtct ctgagactgt ggtcacacct 2150 gaggcagccc cagagaagaa ccctgtggat gtgaaggggg aaggaaatga 2200 gaccaccaat atggtcatca cgtggaagcc gctccggtgg atggactgga 2250 acgcccccca ggttcagtac cgcgtgcagt ggcgccctca ggggacacga 2300 gggccctggc aggagcagat tgtcagcgac cccttcctgg tggtgtccaa 2350 cacgtccacc ttcgtgccct atgagatcaa agtccaggcc gtcaacagcc 2400 agggcaaggg accagagccc caggtcacta tcggctactc tggagaggac 2450 tacccccagg caatccctga gctggaaggc attgaaatcc tcaactcaag 2500 tgccgtgctg gtcaagtggc ggccggtgga cctggcccag gtcaagggcc 2550 acctccgcgg atacaatgtg acgtactgga gggagggcag tcagaggaag 2600 cacagcaaga gacatatcca caaagaccat gtggtggtgc ccgccaacac 2650 caccagtgtc atcctcagtg gcttgcggcc ctatagctcc taccacctgg 2700 aggtgcaggc ctttaacggg cgaggatcgg ggcccgccag cgagttcacc 2750 ttcagcaccc cagagggagt gcctggccac cccgaggcgt tgcacctgga 2800 gtgccagtcg aacaccagcc tgctgctgcg ctggcagccc ccactcagcc 2850 acaacggcgt gctcaccggc tacgtgctct cctaccaccc cctggatgag 2900 gggggcaagg ggcaactgtc cttcaacctt cgggaccccg aacttcggac 2950 acacaacctg accgatctca gcccccacct gcggtaccgc ttccagcttc 3000 aggccaccac caaagagggc cctggtgaag ccatcgtacg ggaaggaggc 3050 actatggcct tgtctgggat ctcagatttt ggcaacatct cagccacagc 3100 gggtgaaaac tacagtgtcg tctcctgggt ccccaaggag ggccagtgca 3150 acttcaggtt ccatatcttg ttcaaagcct tgggagaaga gaagggtggg 3200 gcttcccttt cgccacagta tgtcagctac aaccagagct cctacacgca 3250 gtgggacctg cagcctgaca ctgactacga gatccacttg tttaaggaga 3300 ggatgttccg gcaccaaatg gctgtgaaga ccaatggcac aggccgcgtg 3350 aggctccctc ctgctggctt cgccactgag ggctggttca tcggctttgt 3400 gagtgccatc atcctcctgc tcctcgtcct gctcatcctc tgcttcatca 3450 agcgcagcaa gggcggcaaa tactcagtga aggataagga ggacacccag 3500 gtggactctg aggcccgacc gatgaaagat gagaccttcg gcgagtacag 3550 gtccctggag agtgacaacg aggagaaggc ctttggcagc agccagccat 3600 cgctcaacgg ggacatcaag cccctgggca gtgacgacag cctggccgat 3650 tatgggggca gcgtggatgt tcagttcaac gaggatggtt cgttcattgg 3700 ccagtacagt ggcaagaagg agaaggaggc ggcagggggc aatgacagct 3750 caggggccac ttcccccatc aaccctgccg tggccctaga atagtggagt 3800 ccaggacagg agatgctgtg cccctggcct tgggatccag gcccctccct 3850 ctccagcagg cccatgggag gctggagttg gggcagagga gaacttgctg 3900 cctcggatcc ccttcctacc acccggtccc cactttattg ccaaaaccca 3950 gctgcacccc ttcctgggca cacgctgctc tgccccagct tgggcagatc 4000 tcccacatgc caggggcctt tgggtgctgt tttgccagcc catttgggca 4050 gagaggctgt ggtttggggg agaagaagta ggggtggccc gaaagggtct 4100 ccgaaatgct gtctttcttg ctccctgact gggggcagac atggtggggt 4150 ctcctcagga ccagggttgg caccttcccc ctcccccagc cactccccag 4200 ccagcctggc tgggactggg aacagaactc ggtgtcccca ccatctgctg 4250 tcttttcttt gccatctctg ctccaaccgg gatgggagcc gggcaaactg 4300 gccgcggggg caggggaggc catctggaga gcccagagtc cccccactcc 4350 cagcatcgca ctctggcagc accgcctctt cccgccgccc agcccacccc 4400 atggccggct ttcaggagct ccatacacac gctgccttcg gtacccacca 4450 cacaacatcc aagtggcctc cgtcactacc tggctgcggg gcgggcacac 4500 ctcctcccac tgcccactgg ccggc 4525 8 768 DNA Homo sapiens 8 gatcccaccc ttccggcccc cccacggtcg cgctcctcca ggctgggcct 50 gtggccgcgg tgctttttaa ttttccccca gctcagaatc ttgctgctcg 100 gcccccagga gagcaacaac tcaacgggaa cgatgtggaa ggtgtcagct 150 ctgctcttcg ttttgggaag cgcgtcgctc tgggtcctgg cagaaggagc 200 cagcacaggc cagccagaag atgacactga gactacaggt ttggaaggcg 250 gcgttgccat gccaggtgcc gaagatgatg tggtgactcc aggaaccagc 300 gaagaccgct ataagtctgg cttgacaact ctggtggcaa caagtgtcaa 350 cagtgtaaca ggcattcgca tcgaggatct gccaacttca gaaagcacag 400 tccacgcgca agaacaaagt ccaagcgcca cagcctcaaa cgtggccacc 450 agtcactcca cggagaaagt ggatggagac acacagacaa cagttgagaa 500 agatggtttg tcaacagtga ccctggttgg aatcatagtt ggggtcttac 550 tagccatcgg tttcattggt ggaatcatcg ttgtggttat gcgaaaaatg 600 tcgggaaggt actcgcccta aagagctgaa gggttacgcc ctgctgccaa 650 cgtgcttaaa aaaagaccgt ttctgacttc tgtggccctg ttcccttgag 700 cttcgtgggg agaagattga cccgtgggaa catttgcctg ggcccattca 750 gaatccacgg tgaccttt 768 9 2163 DNA Homo sapiens Unsure 2118,2136 Unknown base 9 tttgctgtcc ggctgcctak ggtctgggaa gctcgggcac cctccctctc 50 cggggctcct gctcccaccc ctccggcccc cccaccgtcg cgctcctcca 100 ggctgggcct gtggccgcgg tgctttttaa ttttccccca gctcagaatc 150 ttgctgctcg gcccccagga gagcaacaac tcaacgggaa cgatgtggaa 200 ggtgtcagct ctgctcttcg ttttgggaag cgcgtcgctc tgggtcctgg 250 cagaaggagc cagcacaggc cagccagaag atgacactga gactacaggt 300 ttggaaggcg gcgttgccat gccaggtgcc gaagatgatg tggtgactcc 350 aggaaccagc gaagaccgct ataagtctgg cttgacaact ctggtggcaa 400 caagtgtcaa cagtgtaaca ggcattcgca tcgaggatct gccaacttca 450 gaaagcacag tccacgcgca agaacaaagt ccaagcgcca cagcctcaaa 500 cgtggccacc agtcactcca cggagaaagt ggatggagac acacagacaa 550 cagttgagaa agatggtttg tcaacagtga ccctggttgg aatcatagtt 600 ggggtcttac tagccatcgg yttcattggt ggaatcatcg ttgtggttat 650 gcgaaaaatg tcgggaaggc cctaaagagc tgaagggtta cgccctgctg 700 ccaacgtgct taaaaaaaga ccgtttctga ctctgtgccc tgtccctgag 750 ctcgtgggag aagatgaccc gtggaacact tgcctggccc actcagaatc 800 cacggtgacc tctccgcttg ccaaaataac cgaagaaaga ccgttcacca 850 gacttggctc ctctaaacat ttgctgttca aacatgtttt tgaatataca 900 ttctataaaa gattatttga aagacaaaat tcatagaaaa tggagcaaaa 950 ctgtataaac tgatttgtaa ctaacactgg accattggat cgatattaya 1000 tgctgtaacc atgtgtctcc gtctgaccat tcttgttatt gttaaaatgc 1050 agaggaatct ggaaatattt atatccacgg agtccttgga tccagtgcta 1100 cgtcagtaaa tagcaccagc attttgcaat tgctgatctg ctgaaatgta 1150 cacattctgg tctagtttgg tctatctttt aaagcctgat ctggtgtgaa 1200 taatcaacta ggaaatctaa acttggataa cacgtggtga acaactgcct 1250 ttagctggtc cagattaatc atttcaaaga catccatttt agatcacaag 1300 caggaagtcg atagtctcaa aggcactttg tttctcccaa gtaggccacc 1350 aggcagcctc tagagttgct ttacccaaat ccttctccag ccatgacttg 1400 gtgactctaa gcttgctccc acctgccccc tccacttccc tcagatgatg 1450 aggagccagg gctaaggggg cagccttctc tcttcccagt gatgcacatc 1500 cttcacattg gctgctttgt tctggaatat ggatatctca gcctggatgc 1550 cgaggaagct gctggatgct taatggtgct agaggctcaa gtgtgtttga 1600 aaccaagagc cagttgtccc ccatgcagaa agaaatcctg tgtgagcctc 1650 tggtatgaga aataaaatct gccagtttta taacattcac tttctgcctc 1700 tgaggaaaga tacagggaac aaaaatcaat ttgtacagtc ttaatattaa 1750 aagcagcttg actaaatacc tgatttaaaa atagaagaca tccccagtcc 1800 tcatgacata ccgcaaatat ctgtggggtc ctgttgaaaa gaacaaaata 1850 aaggagccca aggggtcatt ctgtctcagc accatccagc ctggcacttc 1900 tcttcccata tatccattgg attttttttt tttttttcct aaacaaagtt 1950 tttacactga gcagatgctc tgtcatgatg gcggttgtgc aattctggta 2000 tcctctaaat ttgtaagcat tcataaaaaa aaaaaaaaaa aactcgaggg 2050 ggggcccggw cccaattgcc ctatagggag tcgtattaca attcactgsc 2100 cgcgttttac aacgtcgnga ctgggaaaac cctggngtta cccaacttaa 2150 tcgccttgca gaa 2163 10 3558 DNA Homo sapiens 10 cagaccccag ttcgccgact aagcagaaga aagatcaaaa accggaaaag 50 aggagaagag caaacaggca ctttgaggaa caatcccctt taactccaag 100 ccgacagcgg tctaggaatt caagttcagt gcctaccgaa gacaaaggcg 150 ccccgaggga gtggcggtgc gaccccaggg cgtgggcccg gccgcggagc 200 ccacactgcc cggctgaccc ggtggtctcg gaccatgtct cccgccccaa 250 gacccccccg ttgtctcctg ctccccctgc tcacgctcgg caccgcgctc 300 gcctccctcg gctcggccca aagcagcagc ttcagccccg aagcctggct 350 acagcaatat ggctacctgc ctcccgggga cctacgtacc cacacacagc 400 gctcacccca gtcactctca gcggccatcg ctgccatgca gaagttttac 450 ggcttgcaag taacaggcaa agctgatgca gacaccatga aggccatgag 500 gcgcccccga tgtggtgttc cagacaagtt tggggctgag atcaaggcca 550 atgttcgaag gaagcgctac gccatccagg gtctcaaatg gcaacataat 600 gaaatcactt tctgcatcca gaattacacc cccaaggtgg gcgagtatgc 650 cacatacgag gccattcgca aggcgttccg cgtgtgggag agtgccacac 700 cactgcgctt ccgcgaggtg ccctatgcct acatccgtga gggccatgag 750 aagcaggccg acatcatgat cttctttgcc gagggcttcc atggcgacag 800 cacgcccttc gatggtgagg gcggcttcct ggcccatgcc tacttcccag 850 gccccaacat tggaggagac acccactttg actctgccga gccttggact 900 gtcaggaatg aggatctgaa tggaaatgac atcttcctgg tggctgtgca 950 cgagctgggc catgccctgg ggctcgagca ttccagtgac ccctcggcca 1000 tcatggcacc cttttaccag tggatggaca cggagaattt tgtgctgccc 1050 gatgatgacc gccggggcat ccagcaactt tatgggggtg agtcagggtt 1100 ccccaccaag atgccccctc aacccaggac tacctcccgg ccttctgttc 1150 ctgataaacc caaaaacccc acctatgggc ccaacatctg tgacgggaac 1200 tttgacaccg tggccatgct ccgaggggag atgtttgtct tcaaggagcg 1250 ctggttctgg cgggtgagga ataaccaagt gatggatgga tacccaatgc 1300 ccattggcca gttctggcgg ggcctgcctg cgtccatcaa cactgcctac 1350 gagaggaagg atggcaaatt cgtcttcttc aaaggagaca agcattgggt 1400 gtttgatgag gcgtccctgg aacctggcta ccccaagcac attaaggagc 1450 tgggccgagg gctgcctacc gacaagattg atgctgctct cttctggatg 1500 cccaatggaa agacctactt cttccgtgga aacaagtact accgtttcaa 1550 cgaagagctc agggcagtgg atagcgagta ccccaagaac atcaaagtct 1600 gggaagggat ccctgagtct cccagagggt cattcatggg cagcgatgaa 1650 gtcttcactt acttctacaa ggggaacaaa tactggaaat tcaacaacca 1700 gaagctgaag gtagaaccgg gctaccccaa gtcagccctg agggactgga 1750 tgggctgccc atcgggaggc cggccggatg aggggactga ggaggagacg 1800 gaggtgatca tcattgaggt ggacgaggag ggcggcgggg cggtgagcgc 1850 ggctgccgtg gtgctgcccg tgctgctgct gctcctggtg ctggcggtgg 1900 gccttgcagt cttcttcttc agacgccatg ggacccccag gcgactgctc 1950 tactgccagc gttccctgct ggacaaggtc tgacgcccac cgccggcccg 2000 cccactccta ccacaaggac tttgcctctg aaggccagtg gcagcaggtg 2050 gtggtgggtg ggctgctccc atcgtcccga gccccctccc cgcagcctcc 2100 ttgcttctct ctgtcccctg gctggcctcc ttcaccctga ccgcctccct 2150 ccctcctgcc ccggcattgc atcttcccta gataggtccc ctgagggctg 2200 agtgggaggg cggccctttc cagcctctgc ccctcagggg aaccctgtag 2250 ctttgtgtct gtccagcccc atctgaatgt gttgggggct ctgcacttga 2300 aggcaggacc ctcagacctc gctggtaaag gtcaaatggg gtcatctgct 2350 ccttttccat cccctgacat accttaacct ctgaactctg acctcaggag 2400 gctctgggca ctccagccct gaaagcccca ggtgtaccca attggcagcc 2450 tctcactact ctttctggct aaaaggaatc taatcttgtt gagggtagag 2500 accctgagac agtgtgaggg ggtggggact gccaagccac cctaagacct 2550 tgggaggaaa actcagagag ggtcttcgtt gctcagtcag tcaagttcct 2600 cggagatctg cctctgcctc acctacccca gggaacttcc aaggaaggag 2650 cctgagccac tggggactaa gtgggcagaa gaaacccttg gcagccctgt 2700 gcctctcgaa tgttagcctt ggatggggct ttcacagtta gaagagctga 2750 aaccaggggt gcagctgtca ggtagggtgg ggccggtggg agaggcccgg 2800 gtcagagccc tgggggtgag cctgaaggcc acagagaaag aaccttgccc 2850 aaactcaggc agctggggct gaggcccaaa ggcagaacag ccagaggggg 2900 caggagggga ccaaaaagga aaatgaggac gtgcagcagc attggaaggc 2950 tggggccggg caggccaggc caagccaagc agggggccac agggtgggct 3000 gtggagctct caggaagggc cctgaggaag gcacacttgc tcctgttggt 3050 ccctgtcctt gctgcccagg cagcgtggag gggaagggta gggcagccag 3100 agaaaggagc agagaaggca cacaaacgag gaatgagggg cttcacgaga 3150 ggccacaggg cctggctggc cacgctgtcc cggcctgctc accatctcag 3200 tgaggggcag gagctggggc tcgcttaggc tgggtccacg cttccctggt 3250 gccagcaccc ctcaagcctg tctcaccagt ggcctgccct ctcgctcccc 3300 cacccagccc acccattgaa gtctccttgg gccaccaaag gtggtggcca 3350 tggtaccggg gacttgggag agtgagaccc agtggaggga gcaagaggag 3400 agggatgtcg ggggggtggg gcacggggta ggggaaatgg ggtgaacggt 3450 gctggcagtt cggctagatt tctgtcttgt ttgttttttt gttttgttta 3500 atgtatattt ttattataat tattatatat gaattccaaa aaaaaaaaaa 3550 aaaaaaaa 3558 11 1872 DNA Homo sapiens 11 tcaggctgcc tgatctgccc agctttccag ctttcctctg gattccggcc 50 tctggtcatc cctccccacc ctctctccaa ggccctctcc tggtctccct 100 tcttctagaa ccccttcctc cacctccctc tctgcagaac ttctccttta 150 ccccccaccc cccaccactg ccccctttcc ttttctgacc tccttttgga 200 gggctcagcg ctgcccagac cataggagag atgtgggagg ctcagttcct 250 gggcttgctg tttctgcagc cgctttgggt ggctccagtg aagcctctcc 300 agccaggggc tgaggtcccg gtggtgtggg cccaggaggg ggctcctgcc 350 cagctcccct gcagccccac aatccccctc caggatctca gccttctgcg 400 aagagcaggg gtcacttggc agcatcagcc agacagtggc ccgcccgctg 450 ccgcccccgg ccatcccctg gcccccggcc ctcacccggc ggcgccctcc 500 tcctgggggc ccaggccccg ccgctacacg gtgctgagcg tgggtcccgg 550 aggcctgcgc agcgggaggc tgcccctgca gccccgcgtc cagctggatg 600 agcgcggccg gcagcgcggg gacttctcgc tatggctgcg cccagcccgg 650 cgcgcggacg ccggcgagta ccgcgccgcg gtgcacctca gggaccgcgc 700 cctctcctgc cgcctccgtc tgcgcctggg ccaggcctcg atgactgcca 750 gccccccagg atctctcaga gcctccgact gggtcatttt gaactgctcc 800 ttcagccgcc ctgaccgccc agcctctgtg cattggttcc ggaaccgggg 850 ccagggccga gtccctgtcc gggagtcccc ccatcaccac ttagcggaaa 900 gcttcctctt cctgccccaa gtcagcccca tggactctgg gccctggggc 950 tgcatcctca cctacagaga tggcttcaac gtctccatca tgtataacct 1000 cactgttctg ggtctggagc ccccaactcc cttgacagtg tacgctggag 1050 caggttccag ggtggggctg ccctgccgcc tgcctgctgg tgtggggacc 1100 cggtctttcc tcactgccaa gtggactcct cctgggggag gccctgacct 1150 cctggtgact ggagacaatg gcgactttac ccttcgacta gaggatgtga 1200 gccaggccca ggctgggacc tacacctgcc atatccatct gcaggaacag 1250 cagctcaatg ccactgtcac attggcaatc atcacagtga ctcccaaatc 1300 ctttgggtca cctggatccc tggggaagct gctttgtgag gtgactccag 1350 tatctggaca agaacgcttt gtgtggagct ctctggacac cccatcccag 1400 aggagtttct caggaccttg gctggaggca caggaggccc agctcctttc 1450 ccagccttgg caatgccagc tgtaccaggg ggagaggctt cttggagcag 1500 cagtgtactt cacagagctg tctagcccag gtgcccaacg ctctgggaga 1550 gccccaggtg ccctcccagc aggccacctc ctgctgtttc tcacccttgg 1600 tgtcctttct ctgctccttt tggtgactgg agcctttggc tttcaccttt 1650 ggagaagaca gtggcgacca agacgatttt ctgccttaga gcaagggatt 1700 caccctcgcc aggctcagag caagatagag gagctggagc aagaaccgga 1750 gccggagccg gagccggaac cggagcccga gcccgagccc gagccggagc 1800 agctctgacc tggagctgag gcagccagca gatctcagca gcccagtcca 1850 aataaacgtc ctgtctagca gc

1872 12 1991 DNA Homo sapiens 12 acaggggtga aggcccagag accagcagaa cggcatccca gccacgacgg 50 ccactttgct ctgtctgctg tccgccacgg ccctgctctg ttccctggga 100 cacccccgcc cccacctcct caggctgcct gatctgccca gctttccagc 150 tttcctctgg attccggcct ctggtcatcc ctccccaccc tctctccaag 200 gccctctcct ggtctccctt cttctagaac cccttcctcc acctccctct 250 ctgcagaact tctcctttac cccccacccc ccaccactgc cccctttcct 300 tttctgacct ccttttggag ggctcagcgc tgcccagacc ataggagaga 350 tgtgggaggc tcagttcctg ggcttgctgt ttctgcagcc gctttgggtg 400 gctccagtga agcctctcca gccaggggct gaggtcccgg tggtgtgggc 450 ccaggagggg gctcctgccc agctcccctg cagccccaca atccccctcc 500 aggatctcag ccttctgcga agagcagggg tcacttggca gcatcagcca 550 gacagtggcc cgcccgctgc cgcccccggc catcccctgg cccccggccc 600 tcacccggcg gcgccctcct cctgggggcc caggccccgc cgctacacgg 650 tgctgagcgt gggtcccgga ggcctgcgca gcgggaggct gcccctgcag 700 ccccgcgtcc agctggatga gcgcggccgg cagcgcgggg acttctcgct 750 atggctgcgc ccagcccggc gcgcggacgc cggcgagtac cgcgccgcgg 800 tgcacctcag ggaccgcgcc ctctcctgcc gcctccgtct gcgcctgggc 850 caggcctcga tgactgccag ccccccagga tctctcagag cctccgactg 900 ggtcattttg aactgctcct tcagccgccc tgaccgccca gcctctgtgc 950 attggttccg gaaccggggc cagggccgag tccctgtccg ggagtccccc 1000 catcaccact tagcggaaag cttcctcttc ctgccccaag tcagccccat 1050 ggactctggg ccctggggct gcatcctcac ctacagagat ggcttcaacg 1100 tctccatcat gtataacctc actgttctgg gtctggagcc cccaactccc 1150 ttgacagtgt acgctggagc aggttccagg gtggggctgc cctgccgcct 1200 gcctgctggt gtggggaccc ggtctttcct cactgccaag tggactcctc 1250 ctgggggagg ccctgacctc ctggtgactg gagacaatgg cgactttacc 1300 cttcgactag aggatgtgag ccaggcccag gctgggacct acacctgcca 1350 tatccatctg caggaacagc agctcaatgc cactgtcaca ttggcaatca 1400 tcacagtgac tcccaaatcc tttgggtcac ctggatccct ggggaagctg 1450 ctttgtgagg tgactccagt atctggacaa gaacgctttg tgtggagctc 1500 tctggacacc ccatcccaga ggagtttctc aggaccttgg ctggaggcac 1550 aggaggccca gctcctttcc cagccttggc aatgccagct gtaccagggg 1600 gagaggcttc ttggagcagc agtgtacttc acagagctgt ctagcccagg 1650 tgcccaacgc tctgggagag ccccaggtgc cctcccagca ggccacctcc 1700 tgctgtttct cacccttggt gtcctttctc tgctcctttt ggtgactgga 1750 gcctttggct ttcacctttg gagaagacag tggcgaccaa gacgattttc 1800 tgccttagag caagggattc accctccgca ggctcagagc aagatagagg 1850 agctggagca agaaccggag ccggagccgg agccggaacc ggagcccgag 1900 cccgagcccg agccggagca gctctgacct ggagctgagg cagccagcag 1950 atctcagcag cccagtccaa ataaacgtcc tgtctagcag c 1991 13 7193 DNA Homo sapiens 13 agaataaggg cagggaccgc ggctcctatc tcttggtgat ccccttcccc 50 attccgcccc cgcctcaacg cccagcacag tgccctgcac acagtagtcg 100 ctcaataaat gttcgtggat gatgatgatg atgatgatga aaaaaatgca 150 gcatcaacgg cagcagcaag cggaccacgc gaacgaggca aactatgcaa 200 gaggcaccag acttcctctt tctggtgaag gaccaacttc tcagccgaat 250 agctccaagc aaactgtcct gtcttggcaa gctgcaatcg atgctgctag 300 acaggccaag gctgcccaaa ctatgagcac ctctgcaccc ccacctgtag 350 gatctctctc ccaaagaaaa cgtcagcaat acgccaagag caaaaaacag 400 ggtaactcgt ccaacagccg acctgcccgc gcccttttct gtttatcact 450 caataacccc atccgaagag cctgcattag tatagtggaa tggaaaccat 500 ttgacatatt tatattattg gctatttttg ccaattgtgt ggccttagct 550 atttacatcc cattccctga agatgattct aattcaacaa atcataactt 600 ggaaaaagta gaatatgcct tcctgattat ttttacagtc gagacatttt 650 tgaagattat agcgtatgga ttattgctac atcctaatgc ttatgttagg 700 aatggatgga atttactgga ttttgttata gtaatagtag gattgtttag 750 tgtaattttg gaacaattaa ccaaagaaac agaaggcggg aaccactcaa 800 gcggcaaatc tggaggcttt gatgtcaaag ccctccgtgc ctttcgagtg 850 ttgcgaccac ttcgactagt gtcaggggtg cccagtttac aagttgtcct 900 gaactccatt ataaaagcca tggttcccct ccttcacata gcccttttgg 950 tattatttgt aatcataatc tatgctatta taggattgga actttttatt 1000 ggaaaaatgc acaaaacatg tttttttgct gactcagata tcgtagctga 1050 agaggaccca gctccatgtg cgttctcagg gaatggacgc cagtgtactg 1100 ccaatggcac ggaatgtagg agtggctggg ttggcccgaa cggaggcatc 1150 accaactttg ataactttgc ctttgccatg cttactgtgt ttcagtgcat 1200 caccatggag ggctggacag acgtgctcta ctgggtaaat gatgcgatag 1250 gatgggaatg gccatgggtg tattttgtta gtctgatcat ccttggctca 1300 tttttcgtcc ttaacctggt tcttggtgtc cttagtggag aattctcaaa 1350 ggaaagagag aaggcaaaag cacggggaga tttccagaag ctccgggaga 1400 agcagcagct ggaggaggat ctaaagggct acttggattg gatcacccaa 1450 gctgaggaca tcgatccgga gaatgaggaa gaaggaggag aggaaggcaa 1500 acgaaatact agcatgccca ccagcgagac tgagtctgtg aacacagaga 1550 acgtcagcgg tgaaggcgag aaccgaggct gctgtggaag tctctggtgc 1600 tggtggagac ggagaggcgc ggccaaggcg gggccctctg ggtgtcggcg 1650 gtggggtcaa gccatctcaa aatccaaact cagccgacgc tggcgtcgct 1700 ggaaccgatt caatcgcaga agatgtaggg ccgccgtgaa gtctgtcacg 1750 ttttactggc tggttatcgt cctggtgttt ctgaacacct taaccatttc 1800 ctctgagcac tacaatcagc cagattggtt gacacagatt caagatattg 1850 ccaacaaagt cctcttggct ctgttcacct gcgagatgct ggtaaaaatg 1900 tacagcttgg gcctccaagc atatttcgtc tctcttttca accggtttga 1950 ttgcttcgtg gtgtgtggtg gaatcactga gacgatcctg gtggaactgg 2000 aaatcatgtc tcccctgggg atctctgtgt ttcggtgtgt gcgcctctta 2050 agaatcttca aagtgaccag gcactggact tccctgagca acttagtggc 2100 atccttatta aactccatga agtccatcgc ttcgctgttg cttctgcttt 2150 ttctcttcat tatcatcttt tccttgcttg ggatgcagct gtttggcggc 2200 aagtttaatt ttgatgaaac gcaaaccaag cggagcacct ttgacaattt 2250 ccctcaagca cttctcacag tgttccagat cctgacaggc gaagactgga 2300 atgctgtgat gtacgatggc atcatggctt acgggggccc atcctcttca 2350 ggaatgatcg tctgcatcta cttcatcatc ctcttcattt gtggtaacta 2400 tattctactg aatgtcttct tggccatcgc tgtagacaat ttggctgatg 2450 ctgaaagtct gaacactgct cagaaagaag aagcggaaga aaaggagagg 2500 aaaaagattg ccagaaaaga gagcctagaa aataaaaaga acaacaaacc 2550 agaagtcaac cagatagcca acagtgacaa caaggttaca attgatgact 2600 atagagaaga ggatgaagac aaggacccct atccgccttg cgatgtgcca 2650 gtaggggaag aggaagagga agaggaggag gatgaacctg aggttcctgc 2700 cggaccccgt cctcgaagga tctcggagtt gaacatgaag gaaaaaattg 2750 cccccatccc tgaagggagc gctttcttca ttcttagcaa gaccaacccg 2800 atccgcgtag gctgccacaa gctcatcaac caccacatct tcaccaacct 2850 catccttgtc ttcatcatgc tgagcagcgc tgccctggcc gcagaggacc 2900 ccatccgcag ccactccttc cggaacacga tactgggtta ctttgactat 2950 gccttcacag ccatctttac tgttgagatc ctgttgaaga tgacaacttt 3000 tggagctttc ctccacaaag gggccttctg caggaactac ttcaatttgc 3050 tggatatgct ggtggttggg gtgtctctgg tgtcatttgg gattcaatcc 3100 agtgccatct ccgttgtgaa gattctgagg gtcttaaggg tcctgcgtcc 3150 cctcagggcc atcaacagag caaaaggact taagcacgtg gtccagtgcg 3200 tcttcgtggc catccggacc atcggcaaca tcatgatcgt cactaccctc 3250 ctgcagttca tgtttgcctg tatcggggtc cagttgttca aggggaagtt 3300 ctatcgctgt acggatgaag ccaaaagtaa ccctgaagaa tgcaggggac 3350 ttttcatcct ctacaaggat ggggatgttg acagtcctgt ggtccgtgaa 3400 cggatctggc aaaacagtga tttcaacttc gacaacgtcc tctctgctat 3450 gatggcgctc ttcacagtct ccacgtttga gggctggcct gcgttgctgt 3500 ataaagccat cgactcgaat ggagagaaca tcggcccaat ctacaaccac 3550 cgcgtggaga tctccatctt cttcatcatc tacatcatca ttgtagcttt 3600 cttcatgatg aacatctttg tgggctttgt catcgttaca tttcaggaac 3650 aaggagaaaa agagtataag aactgtgagc tggacaaaaa tcagcgtcag 3700 tgtgttgaat acgccttgaa agcacgtccc ttgcggagat acatccccaa 3750 aaacccctac cagtacaagt tctggtacgt ggtgaactct tcgcctttcg 3800 aatacatgat gtttgtcctc atcatgctca acacactctg cttggccatg 3850 cagcactacg agcagtccaa gatgttcaat gatgccatgg acattctgaa 3900 catggtcttc accggggtgt tcaccgtcga gatggttttg aaagtcatcg 3950 catttaagcc taaggggtat tttagtgacg cctggaacac gtttgactcc 4000 ctcatcgtaa tcggcagcat tatagacgtg gccctcagcg aagcggaccc 4050 aactgaaagt gaaaatgtcc ctgtcccaac tgctacacct gggaactctg 4100 aagagagcaa tagaatctcc atcacctttt tccgtctttt ccgagtgatg 4150 cgattggtga agcttctcag caggggggaa ggcatccgga cattgctgtg 4200 gacttttatt aagtcctttc aggcgctccc gtatgtggcc ctcctcatag 4250 ccatgctgtt cttcatctat gcggtcattg gcatgcagat gtttgggaaa 4300 gttgccatga gagataacaa ccagatcaat aggaacaata acttccagac 4350 gtttccccag gcggtgctgc tgctcttcag gtgtgcaaca ggtgaggcct 4400 ggcaggagat catgctggcc tgtctcccag ggaagctctg tgaccctgag 4450 tcagattaca accccgggga ggagtataca tgtgggagca actttgccat 4500 tgtctatttc atcagttttt acatgctctg tgcatttctg atcatcaatc 4550 tgtttgtggc tgtcatcatg gataatttcg actatctgac ccgggactgg 4600 tctattttgg ggcctcacca tttagatgaa ttcaaaagaa tatggtcaga 4650 atatgaccct gaggcaaagg gaaggataaa acaccttgat gtggtcactc 4700 tgcttcgacg catccagcct cccctggggt ttgggaagtt atgtccacac 4750 agggtagcgt gcaagagatt agttgccatg aacatgcctc tcaacagtga 4800 cgggacagtc atgtttaatg caaccctgtt tgctttggtt cgaacggctc 4850 ttaagatcaa gaccgaaggg aacctggagc aagctaatga agaacttcgg 4900 gctgtgataa agaaaatttg gaagaaaacc agcatgaaat tacttgacca 4950 agttgtccct ccagctggtg atgatgaggt aaccgtgggg aagttctatg 5000 ccactttcct gatacaggac tactttagga aattcaagaa acggaaagaa 5050 caaggactgg tgggaaagta ccctgcgaag aacaccacaa ttgccctaca 5100 ggcgggatta aggacactgc atgacattgg gccagaaatc cggcgtgcta 5150 tatcgtgtga tttgcaagat gacgagcctg aggaaacaaa acgagaagaa 5200 gaagatgatg tgttcaaaag aaatggtgcc ctgcttggaa accatgtcaa 5250 tcatgttaat agtgatagga gagattccct tcagcagacc aataccaccc 5300 accgtcccct gcatgtccaa aggccttcaa ttccacctgc aagtgatact 5350 gagaaaccgc tgtttcctcc agcaggaaat tcggtgtgtc ataaccatca 5400 taaccataat tccataggaa agcaagttcc cacctcaaca aatgccaatc 5450 tcaataatgc caatatgtcc aaagctgccc atggaaagcg gcccagcatt 5500 gggaaccttg agcatgtgtc tgaaaatggg catcattctt cccacaagca 5550 tgaccgggag cctcagagaa ggtccagtgt gaaaagaacc cgctattatg 5600 aaacttacat taggtccgac tcaggagatg aacagctccc aactatttgc 5650 cgggaagacc cagagataca tggctatttc agggaccccc actgcttggg 5700 ggagcaggag tatttcagta gtgaggaatg ctacgaggat gacagctcgc 5750 ccacctggag caggcaaaac tatggctact acagcagata cccaggcaga 5800 aacatcgact ctgagaggcc ccgaggctac catcatcccc aaggattctt 5850 ggaggacgat gactcgcccg tttgctatga ttcacggaga tctccaagga 5900 gacgcctact acctcccacc ccagcatccc accggagatc ctccttcaac 5950 tttgagtgcc tgcgccggca gagcagccag gaagaggtcc cgtcgtctcc 6000 catcttcccc catcgcacgg ccctgcctct gcatctaatg cagcaacaga 6050 tcatggcagt tgccggccta gattcaagta aagcccagaa gtactcaccg 6100 agtcactcga cccggtcgtg ggccacccct ccagcaaccc ctccctaccg 6150 ggactggaca ccgtgctaca cccccctgat ccaagtggag cagtcagagg 6200 ccctggacca ggtgaacggc agcctgccgt ccctgcaccg cagctcctgg 6250 tacacagacg agcccgacat ctcctaccgg actttcacac cagccagcct 6300 gactgtcccc agcagcttcc ggaacaaaaa cagcgacaag cagaggagtg 6350 cggacagctt ggtggaggca gtcctgatat ccgaaggctt gggacgctat 6400 gcaagggacc caaaatttgt gtcagcaaca aaacacgaaa tcgctgatgc 6450 ctgtgacctc accatcgacg agatggagag tgcagccagc accctgctta 6500 atgggaacgt gcgtccccga gccaacgggg atgtgggccc cctctcacac 6550 cggcaggact atgagctaca ggactttggt cctggctaca gcgacgaaga 6600 gccagaccct gggagggatg aggaggacct ggcggatgaa atgatatgca 6650 tcaccacctt gtagccccca gcgaggggca gactggctct ggcctcaggt 6700 ggggcgcagg agagccaggg gaaaagtgcc tcatagttag gaaagtttag 6750 gcactagttg ggagtaatat tcaattaatt agacttttgt ataagagatg 6800 tcatgcctca agaaagccat aaacctggta ggaacaggtc ccaagcggtt 6850 gagcctggca gagtaccatg cgctcggccc cagctgcagg aaacagcagg 6900 ccccgccctc tcacagagga tgggtgagga ggccagacct gccctgcccc 6950 attgtccaga tgggcactgc tgtggagtct gcttctccca tgtaccaggg 7000 caccaggccc acccaactga aggcatggcg gcggggtgca ggggaaagtt 7050 aaaggtgatg acgatcatca cacctcgtgt cgttacctca gccatcggtc 7100 tagcatatca gtcactgggc ccaacatatc catttttaaa ccctttcccc 7150 caaatacact gcgtcctggt tcctgtttag ctgttctgaa ata 7193 14 1315 DNA Homo sapiens 14 tcgccatggc ctctgccgga atgcagatcc tgggagtcgt cctgacactg 50 ctgggctggg tgaatggcct ggtctcctgt gccctgccca tgtggaaggt 100 gaccgctttc atcggcaaca gcatcgtggt ggcccaggtg gtgtgggagg 150 gcctgtggat gtcctgcgtg gtgcagagca ccggccagat gcagtgcaag 200 gtgtacgact cactgctggc gctgccacag gacctgcagg ctgcacgtgc 250 cctctgtgtc atcgccctcc ttgtggccct gttcggcttg ctggtctacc 300 ttgctggggc caagtgtacc acctgtgtgg aggagaagga ttccaaggcc 350 cgcctggtgc tcacctctgg gattgtcttt gtcatctcag gggtcctgac 400 gctaatcccc gtgtgctgga cggcgcatgc catcatccgg gacttctata 450 accccctggt ggctgaggcc caaaagcggg agctgggggc ctccctctac 500 ttgggctggg cggcctcagg ccttttgttg ctgggtgggg ggttgctgtg 550 ctgcacttgc ccctcggggg ggtcccaggg ccccagccat tacatggccc 600 gctactcaac atctgcccct gccatctctc gggggccctc tgagtaccct 650 accaagaatt acgtctgacg tggaggggaa tgggggctcc gctggcgcta 700 gagccatcca gaagtggcag tgcccaacag ctttgggatg ggttcgtacc 750 ttttgtttct gcctcctgct atttttcttt tgactgagga tatttaaaat 800 tcatttgaaa actgagccaa ggtgttgact cagactctca cttaggctct 850 gctgtttctc acccttggat gatggagcca aagaggggat gctttgagat 900 tctggatctt gacatgccca tcttagaagc cagtcaagct atggaactaa 950 tgcggaggct gcttgctgtg ctggctttgc aacaagacag actgtcccca 1000 agagttcctg ctgctgctgg gggctgggct tccctagatg tcactggaca 1050 gctgcccccc atcctactca ggtctctgga gctcctctct tcacccctgg 1100 aaaaacaaat catctgttaa caaaggactg cccacctccg gaacttctga 1150 cctctgtttc ctccgtcctg ataagacgtc caccccccag ggccaggtcc 1200 cagctatgta gacccccgcc cccacctcca acactgcacc cttctgccct 1250 gcccccctcg tctcaccccc tttacactca catttttatc aaataaagca 1300 tgttttgtta gtgca 1315 15 2000 DNA Homo sapiens 15 cctgtgaaca ctatagcgct gagagagaca gtctgaaagc agaggaagac 50 atcgatcagt aacaccaaga gacaccaaag ttgaaagttt tgttttcttt 100 ccctctgttt tatttttccc ccgtgtgtcc ctactatggt cagaaagcct 150 gttgtgtcca ccatctccaa aggaggttac ctgcagggaa atgttaacgg 200 gaggctgcct tccctgggca acaaggagcc acctgggcag gagaaagtgc 250 agctgaagag gaaagtcact ttactgaggg gagtctccat tatcattggc 300 accatcattg gagcaggaat cttcatctct cctaagggcg tgctccagaa 350 cacgggcagc gtgggcatgt ctctgaccat ctggacggtg tgtggggtcc 400 tgtcactatt tggagctttg tcttatgctg aattgggaac aactataaag 450 aaatctggag gtcattacac atatattttg gaagtctttg gtccattacc 500 agcttttgta cgagtctggg tggaactcct cataatacgc cctgcagcta 550 ctgctgtgat atccctggca tttggacgct acattctgga accatttttt 600 attcaatgtg aaatccctga acttgcgatc aagctcatta cagctgtggg 650 cataactgta gtgatggtcc taaatagcat gagtgtcagc tggagcgccc 700 ggatccagat tttcttaacc ttttgcaagc tcacagcaat tctgataatt 750 atagtccctg gagttatgca gctaattaaa ggtcaaacgc agaactttaa 800 agacgccttt tcaggaagag attcaagtat tacgcggttg ccactggctt 850 tttattatgg aatgtatgca tatgctggct ggttttacct caactttgtt 900 actgaagaag tagaaaaccc tgaaaaaacc attccccttg caatatgtat 950 atccatggcc attgtcacca ttggctatgt gctgacaaat gtggcctact 1000 ttacgaccat taatgctgag gagctgctgc tttcaaatgc agtggcagtg 1050 accttttctg agcggctact gggaaatttc tcattagcag ttccgatctt 1100 tgttgccctc tcctgctttg gctccatgaa cggtggtgtg tttgctgtct 1150 ccaggttatt ctatgttgcg tctcgagagg gtcaccttcc agaaatcctc 1200 tccatgattc atgtccgcaa gcacactcct ctaccagctg ttattgtttt 1250 gcaccctttg acaatgataa tgctcttctc tggagacctc gacagtcttt 1300 tgaatttcct cagttttgcc aggtggcttt ttattgggct ggcagttgct 1350 gggctgattt atcttcgata caaatgccca gatatgcatc gtcctttcaa 1400 ggtgccactg ttcatcccag ctttgttttc cttcacatgc ctcttcatgg 1450 ttgccctttc cctctattcg gacccattta gtacagggat tggcttcgtc 1500 atcactctga ctggagtccc tgcgtattat ctctttatta tatgggacaa 1550 gaaacccagg tggtttagaa taatgtcagg gttcctagca ctgatgcctg 1600 cacaagcatg tgatatgtga aataaaatgg attcttctat agctaaatga 1650 gttccctctg gggagagttc tggtactgca atcacaatgc cagatggtgt 1700 ttatgggcta tttgtgtaag taagtggtaa gatgctatga agtaagtgtg 1750 tttgttttca tcttatggaa actcttgatg catgtgcttt tgtatggaat 1800 aaattttggt gcaatatgat gtcattcaac tttgcattga attgaatttt 1850 ggttgtattt atatgtatta tacctgtcac gcttctagtt gcttcaacca

1900 ttttataacc atttttgtac atattttact tgaaaatatt ttaaatggaa 1950 atttaaataa acatttgata gtttacataa taaaaaaaaa aaaaaaaaaa 2000 16 1601 DNA Homo sapiens 16 cacagaggag ccagcgaacc tctcccggcg cctgttctgg gggctttctg 50 ttccagcgtc aaggatggag gagggggaga ggagcccctt actgtcccag 100 gaaactgcag gccagaagcc cctctctgtg cacaggccac ccacctcagg 150 ctgcctaggt ccagtgccca gggaggacca ggcggaggcc tggggctgca 200 gctgctgtcc cccggagacc aagcaccagg ccttgagtgg cactcccaag 250 aaaggaccag ccccttccct ctccccaggg agcagctgcg tcaagtatct 300 gatcttcctc tccaacttcc ccttctccct gctggggctg ctggccctgg 350 ccatcgggct ctggggcctg gctgtcaagg ggtctctggg aagtgatctg 400 ggggggcccc tgcccacaga ccccatgctg gggctggcac tgggagggct 450 ggtggtcagc gcagcgagcc tggctggctg cctgggcgcc ctctgcgaga 500 acacctgcct gttacgtggc ttctccgggg gcatccttgc cttcctggtg 550 cttgaggccg tggcgggggc cctggtggtg gccctctggg gcccgctgca 600 agacagcctg gagcacaccc tgcgtgtggc catcgcccac taccaggacg 650 acccagacct gcgcttcctc ctcgaccaag tccagctcgg gctgaggtgc 700 tgcggagctg cctcctacca ggactggcag cagaacctgt actttaactg 750 cagctccccc ggggtgcagg cctgcagcct tcccgcctcc tgctgcatcg 800 acccccgcga agatggagcc tctgtcaacg accagtgcgg cttcggggtc 850 ctgcgcctgg atgcggacgc agctcagaga gtggtgtacc tggagggctg 900 cggcccgccg ctccggcggt ggctgcgcgc gaacctggct gcctcgggcg 950 gctacgcaat cgcggtggtg ctgctgcagg gggcggagct cctgctggcc 1000 gcccggctac tcggggccct cgctgcccgc agtggggcgg cgtacggccc 1050 cggagcgcac ggggaggacc gcgctggccc ccagagcccc agccccggcg 1100 ccccgcccgc tgccaaaccc gcccggggct gagcgcacgc cccgaggtcc 1150 gagaccgcca cgcacaggga tacagggggc gcctccgccc ggctaaaaag 1200 cgctgcctgc gccgccgccg ccgcctgatt tcgctcgggc ttcgggtgac 1250 ttcgccgcag gacctaccca gctcgctcac ttcgctcgct ccgcgtcccc 1300 catgccagcc cccaacgcag ggcgcccggc gaagccacgg gactggcggg 1350 aggagcacgc ggggccggag gaaatcctgg agctgaccct cacctccgag 1400 cccccactcc caccccagcc gcacagttcc cacctcctgg cacctccctc 1450 ccctggggcc gccacccctt ctgggctcgt gatggtggag ctaaggtcca 1500 ggcctctccc tcccgagtgc atttttgggg agatagtaaa tgttttattc 1550 gggtgtatca ttcatacagt aaagacacca atcttcaaaa aaaaaaaaaa 1600 a 1601 17 538 PRT Homo sapiens 17 Met Ala Arg Ala Ala Ala Leu Leu Pro Ser Arg Ser Pro Pro Thr 1 5 10 15 Pro Leu Leu Trp Pro Leu Leu Leu Leu Leu Leu Leu Glu Thr Gly 20 25 30 Ala Gln Asp Val Arg Val Gln Val Leu Pro Glu Val Arg Gly Gln 35 40 45 Leu Gly Gly Thr Val Glu Leu Pro Cys His Leu Leu Pro Pro Val 50 55 60 Pro Gly Leu Tyr Ile Ser Leu Val Thr Trp Gln Arg Pro Asp Ala 65 70 75 Pro Ala Asn His Gln Asn Val Ala Ala Phe His Pro Lys Met Gly 80 85 90 Pro Ser Phe Pro Ser Pro Lys Pro Gly Ser Glu Arg Leu Ser Phe 95 100 105 Val Ser Ala Lys Gln Ser Thr Gly Gln Asp Thr Glu Ala Glu Leu 110 115 120 Gln Asp Ala Thr Leu Ala Leu His Gly Leu Thr Val Glu Asp Glu 125 130 135 Gly Asn Tyr Thr Cys Glu Phe Ala Thr Phe Pro Lys Gly Ser Val 140 145 150 Arg Gly Met Thr Trp Leu Arg Val Ile Ala Lys Pro Lys Asn Gln 155 160 165 Ala Glu Ala Gln Lys Val Thr Phe Ser Gln Asp Pro Thr Thr Val 170 175 180 Ala Leu Cys Ile Ser Lys Glu Gly Arg Pro Pro Ala Arg Ile Ser 185 190 195 Trp Leu Ser Ser Leu Asp Trp Glu Ala Lys Glu Thr Gln Val Ser 200 205 210 Gly Thr Leu Ala Gly Thr Val Thr Val Thr Ser Arg Phe Thr Leu 215 220 225 Val Pro Ser Gly Arg Ala Asp Gly Val Thr Val Thr Cys Lys Val 230 235 240 Glu His Glu Ser Phe Glu Glu Pro Ala Leu Ile Pro Val Thr Leu 245 250 255 Ser Val Arg Tyr Pro Pro Glu Val Ser Ile Ser Gly Tyr Asp Asp 260 265 270 Asn Trp Tyr Leu Gly Arg Thr Asp Ala Thr Leu Ser Cys Asp Val 275 280 285 Arg Ser Asn Pro Glu Pro Thr Gly Tyr Asp Trp Ser Thr Thr Ser 290 295 300 Gly Thr Phe Pro Thr Ser Ala Val Ala Gln Gly Ser Gln Leu Val 305 310 315 Ile His Ala Val Asp Ser Leu Phe Asn Thr Thr Phe Val Cys Thr 320 325 330 Val Thr Asn Ala Val Gly Met Gly Arg Ala Glu Gln Val Ile Phe 335 340 345 Val Arg Glu Thr Pro Asn Thr Ala Gly Ala Gly Ala Thr Gly Gly 350 355 360 Ile Ile Gly Gly Ile Ile Ala Ala Ile Ile Ala Thr Ala Val Ala 365 370 375 Ala Thr Gly Ile Leu Ile Cys Arg Gln Gln Arg Lys Glu Gln Thr 380 385 390 Leu Gln Gly Ala Glu Glu Asp Glu Asp Leu Glu Gly Pro Pro Ser 395 400 405 Tyr Lys Pro Pro Thr Pro Lys Ala Lys Leu Glu Ala Gln Glu Met 410 415 420 Pro Ser Gln Leu Phe Thr Leu Gly Ala Ser Glu His Ser Pro Leu 425 430 435 Lys Thr Pro Tyr Phe Asp Ala Gly Ala Ser Cys Thr Glu Gln Glu 440 445 450 Met Pro Arg Tyr His Glu Leu Pro Thr Leu Glu Glu Arg Ser Gly 455 460 465 Pro Leu His Pro Gly Ala Thr Ser Leu Gly Ser Pro Ile Pro Val 470 475 480 Pro Pro Gly Pro Pro Ala Val Glu Asp Val Ser Leu Asp Leu Glu 485 490 495 Asp Glu Glu Gly Glu Glu Glu Glu Glu Tyr Leu Asp Lys Ile Asn 500 505 510 Pro Ile Tyr Asp Ala Leu Ser Tyr Ser Ser Pro Ser Asp Ser Tyr 515 520 525 Gln Gly Lys Gly Phe Val Met Ser Arg Ala Met Tyr Val 530 535 18 331 PRT Homo sapiens 18 Met Ile Thr Leu Asn Asn Gln Asp Gln Pro Val Thr Phe Asn Ser 1 5 10 15 Ser His Pro Asp Glu Tyr Lys Ile Ala Ala Leu Val Phe Tyr Ser 20 25 30 Cys Ile Phe Ile Ile Gly Leu Phe Val Asn Ile Thr Ala Leu Trp 35 40 45 Val Phe Ser Cys Thr Thr Lys Lys Arg Thr Thr Val Thr Ile Tyr 50 55 60 Met Met Asn Val Ala Leu Val Asp Leu Ile Phe Ile Met Thr Leu 65 70 75 Pro Phe Arg Met Phe Tyr Tyr Ala Lys Asp Ala Trp Pro Phe Gly 80 85 90 Glu Tyr Phe Cys Gln Ile Ile Gly Ala Leu Thr Val Phe Tyr Pro 95 100 105 Ser Ile Ala Leu Trp Leu Leu Ala Phe Ile Ser Ala Asp Arg Tyr 110 115 120 Met Ala Ile Val Gln Pro Lys Tyr Ala Lys Glu Leu Lys Asn Thr 125 130 135 Cys Lys Ala Val Leu Ala Cys Val Gly Val Trp Ile Met Thr Leu 140 145 150 Thr Thr Thr Thr Pro Leu Leu Leu Leu Tyr Lys Asp Pro Asp Lys 155 160 165 Asp Ser Thr Pro Ala Thr Cys Leu Lys Ile Ser Asp Ile Ile Tyr 170 175 180 Leu Lys Ala Val Asn Val Leu Asn Leu Thr Arg Leu Thr Phe Phe 185 190 195 Phe Leu Ile Pro Leu Phe Ile Met Ile Gly Cys Tyr Leu Val Ile 200 205 210 Ile His Asn Leu Leu His Gly Arg Thr Ser Lys Leu Lys Pro Lys 215 220 225 Val Lys Glu Lys Ser Ile Arg Ile Ile Ile Thr Leu Leu Val Gln 230 235 240 Val Leu Val Cys Phe Met Pro Phe His Ile Cys Phe Ala Phe Leu 245 250 255 Met Leu Gly Thr Gly Glu Asn Ser Tyr Asn Pro Trp Gly Ala Phe 260 265 270 Thr Thr Phe Leu Met Asn Leu Ser Thr Cys Leu Asp Val Ile Leu 275 280 285 Tyr Tyr Ile Val Ser Lys Gln Phe Gln Ala Arg Val Ile Ser Val 290 295 300 Met Leu Tyr Arg Asn Tyr Leu Arg Ser Leu Arg Arg Lys Ser Phe 305 310 315 Arg Ser Gly Ser Leu Arg Ser Leu Ser Asn Ile Asn Ser Glu Met 320 325 330 Leu 19 226 PRT Homo sapiens 19 Met Pro Gly Gly Pro Gly Val Leu Gln Ala Leu Pro Ala Thr Ile 1 5 10 15 Phe Leu Leu Phe Leu Leu Ser Ala Val Tyr Leu Gly Pro Gly Cys 20 25 30 Gln Ala Leu Trp Met His Lys Val Pro Ala Ser Leu Met Val Ser 35 40 45 Leu Gly Glu Asp Ala His Phe Gln Cys Pro His Asn Ser Ser Asn 50 55 60 Asn Ala Asn Val Thr Trp Trp Arg Val Leu His Gly Asn Tyr Thr 65 70 75 Trp Pro Pro Glu Phe Leu Gly Pro Gly Glu Asp Pro Asn Gly Thr 80 85 90 Leu Ile Ile Gln Asn Val Asn Lys Ser His Gly Gly Ile Tyr Val 95 100 105 Cys Arg Val Gln Glu Gly Asn Glu Ser Tyr Gln Gln Ser Cys Gly 110 115 120 Thr Tyr Leu Arg Val Arg Gln Pro Pro Pro Arg Pro Phe Leu Asp 125 130 135 Met Gly Glu Gly Thr Lys Asn Arg Ile Ile Thr Ala Glu Gly Ile 140 145 150 Ile Leu Leu Phe Cys Ala Val Val Pro Gly Thr Leu Leu Leu Phe 155 160 165 Arg Lys Arg Trp Gln Asn Glu Lys Leu Gly Leu Asp Ala Gly Asp 170 175 180 Glu Tyr Glu Asp Glu Asn Leu Tyr Glu Gly Leu Asn Leu Asp Asp 185 190 195 Cys Ser Met Tyr Glu Asp Ile Ser Arg Gly Leu Gln Gly Thr Tyr 200 205 210 Gln Asp Val Gly Ser Leu Asn Ile Gly Asp Val Gln Leu Glu Lys 215 220 225 Pro 20 118 PRT Homo sapiens 20 Met Pro Arg Glu Asp Ala His Phe Ile Tyr Gly Tyr Pro Lys Lys 1 5 10 15 Gly His Gly His Ser Tyr Thr Thr Ala Glu Glu Ala Ala Gly Ile 20 25 30 Gly Ile Leu Thr Val Ile Leu Gly Val Leu Leu Leu Ile Gly Cys 35 40 45 Trp Tyr Cys Arg Arg Arg Asn Gly Tyr Arg Ala Leu Met Asp Lys 50 55 60 Ser Leu His Val Gly Thr Gln Cys Ala Leu Thr Arg Arg Cys Pro 65 70 75 Gln Glu Gly Phe Asp His Arg Asp Ser Lys Val Ser Leu Gln Glu 80 85 90 Lys Asn Cys Glu Pro Val Val Pro Asn Ala Pro Pro Ala Tyr Glu 95 100 105 Lys Leu Ser Ala Glu Gln Ser Pro Pro Pro Tyr Ser Pro 110 115 21 281 PRT Homo sapiens 21 Met Ser Ala Gln Glu Ser Cys Leu Ser Leu Ile Lys Tyr Phe Leu 1 5 10 15 Phe Val Phe Asn Leu Phe Phe Phe Val Leu Gly Ser Leu Ile Phe 20 25 30 Cys Phe Gly Ile Trp Ile Leu Ile Asp Lys Thr Ser Phe Val Ser 35 40 45 Phe Val Gly Leu Ala Phe Val Pro Leu Gln Ile Trp Ser Lys Val 50 55 60 Leu Ala Ile Ser Gly Ile Phe Thr Met Gly Ile Ala Leu Leu Gly 65 70 75 Cys Val Gly Ala Leu Lys Glu Leu Arg Cys Leu Leu Gly Leu Tyr 80 85 90 Phe Gly Met Leu Leu Leu Leu Phe Ala Thr Gln Ile Thr Leu Gly 95 100 105 Ile Leu Ile Ser Thr Gln Arg Ala Gln Leu Glu Arg Ser Leu Arg 110 115 120 Asp Val Val Glu Lys Thr Ile Gln Lys Tyr Gly Thr Asn Pro Glu 125 130 135 Glu Thr Ala Ala Glu Glu Ser Trp Asp Tyr Val Gln Phe Gln Leu 140 145 150 Arg Cys Cys Gly Trp His Tyr Pro Gln Asp Trp Phe Gln Val Leu 155 160 165 Ile Leu Arg Gly Asn Gly Ser Glu Ala His Arg Val Pro Cys Ser 170 175 180 Cys Tyr Asn Leu Ser Ala Thr Asn Asp Ser Thr Ile Leu Asp Lys 185 190 195 Val Ile Leu Pro Gln Leu Ser Arg Leu Gly His Leu Ala Arg Ser 200 205 210 Arg His Ser Ala Asp Ile Cys Ala Val Pro Ala Glu Ser His Ile 215 220 225 Tyr Arg Glu Gly Cys Ala Gln Gly Leu Gln Lys Trp Leu His Asn 230 235 240 Asn Leu Ile Ser Ile Val Gly Ile Cys Leu Gly Val Gly Leu Leu 245 250 255 Glu Leu Gly Phe Met Thr Leu Ser Ile Phe Leu Cys Arg Asn Leu 260 265 270 Asp His Val Tyr Asn Arg Leu Ala Arg Tyr Arg 275 280 22 1257 PRT Homo sapiens 22 Met Val Val Ala Leu Arg Tyr Val Trp Pro Leu Leu Leu Cys Ser 1 5 10 15 Pro Cys Leu Leu Ile Gln Ile Pro Glu Glu Tyr Glu Gly His His 20 25 30 Val Met Glu Pro Pro Val Ile Thr Glu Gln Ser Pro Arg Arg Leu 35 40 45 Val Val Phe Pro Thr Asp Asp Ile Ser Leu Lys Cys Glu Ala Ser 50 55 60 Gly Lys Pro Glu Val Gln Phe Arg Trp Thr Arg Asp Gly Val His 65 70 75 Phe Lys Pro Lys Glu Glu Leu Gly Val Thr Val Tyr Gln Ser Pro 80 85 90 His Ser Gly Ser Phe Thr Ile Thr Gly Asn Asn Ser Asn Phe Ala 95 100 105 Gln Arg Phe Gln Gly Ile Tyr Arg Cys Phe Ala Ser Asn Lys Leu 110 115 120 Gly Thr Ala Met Ser His Glu Ile Arg Leu Met Ala Glu Gly Ala 125 130 135 Pro Lys Trp Pro Lys Glu Thr Val Lys Pro Val Glu Val Glu Glu 140 145 150 Gly Glu Ser Val Val Leu Pro Cys Asn Pro Pro Pro Ser Ala Glu 155 160 165 Pro Leu Arg Ile Tyr Trp Met Asn Ser Lys Ile Leu His Ile Lys 170 175 180 Gln Asp Glu Arg Val Thr Met Gly Gln Asn Gly Asn Leu Tyr Phe 185 190 195 Ala Asn Val Leu Thr Ser Asp Asn His Ser Asp Tyr Ile Cys His 200 205 210 Ala His Phe Pro Gly Thr Arg Thr Ile Ile Gln Lys Glu Pro Ile 215 220 225 Asp Leu Arg Val Lys Ala Thr Asn Ser Met Ile Asp Arg Lys Pro 230 235 240 Arg Leu Leu Phe Pro Thr Asn Ser Ser Ser His Leu Val Ala Leu 245 250 255 Gln Gly Gln Pro Leu Val Leu Glu Cys Ile Ala Glu Gly Phe Pro 260 265 270 Thr Pro Thr Ile Lys Trp Leu Arg Pro Ser Gly Pro Met Pro Ala 275 280 285 Asp Arg Val Thr Tyr Gln Asn His Asn Lys Thr Leu Gln Leu Leu 290 295 300 Lys Val Gly Glu Glu Asp Asp Gly Glu Tyr Arg Cys Leu Ala Glu 305 310 315 Asn Ser Leu Gly Ser Ala Arg His Ala Tyr Tyr Val Thr Val Glu 320 325 330 Ala Ala Pro Tyr Trp Leu His Lys Pro Gln Ser His Leu Tyr Gly 335 340 345 Pro Gly Glu Thr Ala Arg Leu Asp Cys Gln Val Gln Gly Arg Pro 350 355 360 Gln Pro Glu Val Thr Trp Arg Ile Asn Gly Ile Pro Val Glu Glu 365 370 375 Leu Ala Lys Asp Gln Lys Tyr Arg Ile Gln Arg Gly Ala Leu Ile 380 385 390 Leu Ser Asn Val Gln Pro Ser Asp Thr Met Val Thr Gln Cys Glu 395 400 405 Ala Arg Asn Arg His Gly Leu Leu Leu Ala Asn Ala Tyr Ile Tyr 410 415 420 Val Val Gln Leu Pro Ala Lys Ile Leu Thr Ala Asp Asn Gln Thr 425 430 435 Tyr Met Ala Val Gln Gly Ser Thr Ala Tyr Leu Leu Cys Lys Ala 440 445 450 Phe Gly Ala Pro Val Pro Ser Val Gln Trp Leu Asp Glu Asp Gly 455 460

465 Thr Thr Val Leu Gln Asp Glu Arg Phe Phe Pro Tyr Ala Asn Gly 470 475 480 Thr Leu Gly Ile Arg Asp Leu Gln Ala Asn Asp Thr Gly Arg Tyr 485 490 495 Phe Cys Leu Ala Ala Asn Asp Gln Asn Asn Val Thr Ile Met Ala 500 505 510 Asn Leu Lys Val Lys Asp Ala Thr Gln Ile Thr Gln Gly Pro Arg 515 520 525 Ser Thr Ile Glu Lys Lys Gly Ser Arg Val Thr Phe Thr Cys Gln 530 535 540 Ala Ser Phe Asp Pro Ser Leu Gln Pro Ser Ile Thr Trp Arg Gly 545 550 555 Asp Gly Arg Asp Leu Gln Glu Leu Gly Asp Ser Asp Lys Tyr Phe 560 565 570 Ile Glu Asp Gly Arg Leu Val Ile His Ser Leu Asp Tyr Ser Asp 575 580 585 Gln Gly Asn Tyr Ser Cys Val Ala Ser Thr Glu Leu Asp Val Val 590 595 600 Glu Ser Arg Ala Gln Leu Leu Val Val Gly Ser Pro Gly Pro Val 605 610 615 Pro Arg Leu Val Leu Ser Asp Leu His Leu Leu Thr Gln Ser Gln 620 625 630 Val Arg Val Ser Trp Ser Pro Ala Glu Asp His Asn Ala Pro Ile 635 640 645 Glu Lys Tyr Asp Ile Glu Phe Glu Asp Lys Glu Met Ala Pro Glu 650 655 660 Lys Trp Tyr Ser Leu Gly Lys Val Pro Gly Asn Gln Thr Ser Thr 665 670 675 Thr Leu Lys Leu Ser Pro Tyr Val His Tyr Thr Phe Arg Val Thr 680 685 690 Ala Ile Asn Lys Tyr Gly Pro Gly Glu Pro Ser Pro Val Ser Glu 695 700 705 Thr Val Val Thr Pro Glu Ala Ala Pro Glu Lys Asn Pro Val Asp 710 715 720 Val Lys Gly Glu Gly Asn Glu Thr Thr Asn Met Val Ile Thr Trp 725 730 735 Lys Pro Leu Arg Trp Met Asp Trp Asn Ala Pro Gln Val Gln Tyr 740 745 750 Arg Val Gln Trp Arg Pro Gln Gly Thr Arg Gly Pro Trp Gln Glu 755 760 765 Gln Ile Val Ser Asp Pro Phe Leu Val Val Ser Asn Thr Ser Thr 770 775 780 Phe Val Pro Tyr Glu Ile Lys Val Gln Ala Val Asn Ser Gln Gly 785 790 795 Lys Gly Pro Glu Pro Gln Val Thr Ile Gly Tyr Ser Gly Glu Asp 800 805 810 Tyr Pro Gln Ala Ile Pro Glu Leu Glu Gly Ile Glu Ile Leu Asn 815 820 825 Ser Ser Ala Val Leu Val Lys Trp Arg Pro Val Asp Leu Ala Gln 830 835 840 Val Lys Gly His Leu Arg Gly Tyr Asn Val Thr Tyr Trp Arg Glu 845 850 855 Gly Ser Gln Arg Lys His Ser Lys Arg His Ile His Lys Asp His 860 865 870 Val Val Val Pro Ala Asn Thr Thr Ser Val Ile Leu Ser Gly Leu 875 880 885 Arg Pro Tyr Ser Ser Tyr His Leu Glu Val Gln Ala Phe Asn Gly 890 895 900 Arg Gly Ser Gly Pro Ala Ser Glu Phe Thr Phe Ser Thr Pro Glu 905 910 915 Gly Val Pro Gly His Pro Glu Ala Leu His Leu Glu Cys Gln Ser 920 925 930 Asn Thr Ser Leu Leu Leu Arg Trp Gln Pro Pro Leu Ser His Asn 935 940 945 Gly Val Leu Thr Gly Tyr Val Leu Ser Tyr His Pro Leu Asp Glu 950 955 960 Gly Gly Lys Gly Gln Leu Ser Phe Asn Leu Arg Asp Pro Glu Leu 965 970 975 Arg Thr His Asn Leu Thr Asp Leu Ser Pro His Leu Arg Tyr Arg 980 985 990 Phe Gln Leu Gln Ala Thr Thr Lys Glu Gly Pro Gly Glu Ala Ile 995 1000 1005 Val Arg Glu Gly Gly Thr Met Ala Leu Ser Gly Ile Ser Asp Phe 1010 1015 1020 Gly Asn Ile Ser Ala Thr Ala Gly Glu Asn Tyr Ser Val Val Ser 1025 1030 1035 Trp Val Pro Lys Glu Gly Gln Cys Asn Phe Arg Phe His Ile Leu 1040 1045 1050 Phe Lys Ala Leu Gly Glu Glu Lys Gly Gly Ala Ser Leu Ser Pro 1055 1060 1065 Gln Tyr Val Ser Tyr Asn Gln Ser Ser Tyr Thr Gln Trp Asp Leu 1070 1075 1080 Gln Pro Asp Thr Asp Tyr Glu Ile His Leu Phe Lys Glu Arg Met 1085 1090 1095 Phe Arg His Gln Met Ala Val Lys Thr Asn Gly Thr Gly Arg Val 1100 1105 1110 Arg Leu Pro Pro Ala Gly Phe Ala Thr Glu Gly Trp Phe Ile Gly 1115 1120 1125 Phe Val Ser Ala Ile Ile Leu Leu Leu Leu Val Leu Leu Ile Leu 1130 1135 1140 Cys Phe Ile Lys Arg Ser Lys Gly Gly Lys Tyr Ser Val Lys Asp 1145 1150 1155 Lys Glu Asp Thr Gln Val Asp Ser Glu Ala Arg Pro Met Lys Asp 1160 1165 1170 Glu Thr Phe Gly Glu Tyr Arg Ser Leu Glu Ser Asp Asn Glu Glu 1175 1180 1185 Lys Ala Phe Gly Ser Ser Gln Pro Ser Leu Asn Gly Asp Ile Lys 1190 1195 1200 Pro Leu Gly Ser Asp Asp Ser Leu Ala Asp Tyr Gly Gly Ser Val 1205 1210 1215 Asp Val Gln Phe Asn Glu Asp Gly Ser Phe Ile Gly Gln Tyr Ser 1220 1225 1230 Gly Lys Lys Glu Lys Glu Ala Ala Gly Gly Asn Asp Ser Ser Gly 1235 1240 1245 Ala Thr Ser Pro Ile Asn Pro Ala Val Ala Leu Glu 1250 1255 23 1257 PRT Homo sapiens 23 Met Val Val Ala Leu Arg Tyr Val Trp Pro Leu Leu Leu Cys Ser 1 5 10 15 Pro Cys Leu Leu Ile Gln Ile Pro Glu Glu Tyr Glu Gly His His 20 25 30 Val Met Glu Pro Pro Val Ile Thr Glu Gln Ser Pro Arg Arg Leu 35 40 45 Val Val Phe Pro Thr Asp Asp Ile Ser Leu Lys Cys Glu Ala Ser 50 55 60 Gly Lys Pro Glu Val Gln Phe Arg Trp Thr Arg Asp Gly Val His 65 70 75 Phe Lys Pro Lys Glu Glu Leu Gly Val Thr Val Tyr Gln Ser Pro 80 85 90 His Ser Gly Ser Phe Thr Ile Thr Gly Asn Asn Ser Asn Phe Ala 95 100 105 Gln Arg Phe Gln Gly Ile Tyr Arg Cys Phe Ala Ser Asn Lys Leu 110 115 120 Gly Thr Ala Met Ser His Glu Ile Arg Leu Met Ala Glu Gly Ala 125 130 135 Pro Lys Trp Pro Lys Glu Thr Val Lys Pro Val Glu Val Glu Glu 140 145 150 Gly Glu Ser Val Val Leu Pro Cys Asn Pro Pro Pro Ser Ala Glu 155 160 165 Pro Leu Arg Ile Tyr Trp Met Asn Ser Lys Ile Leu His Ile Lys 170 175 180 Gln Asp Glu Arg Val Thr Met Gly Gln Asn Gly Asn Leu Tyr Phe 185 190 195 Ala Asn Val Leu Thr Ser Asp Asn His Ser Asp Tyr Ile Cys His 200 205 210 Ala His Phe Pro Gly Thr Arg Thr Ile Ile Gln Lys Glu Pro Ile 215 220 225 Asp Leu Arg Val Lys Ala Thr Asn Ser Met Ile Asp Arg Lys Pro 230 235 240 Arg Leu Leu Phe Pro Thr Asn Ser Ser Ser His Leu Val Ala Leu 245 250 255 Gln Gly Gln Pro Leu Val Leu Glu Cys Ile Ala Glu Gly Phe Pro 260 265 270 Thr Pro Thr Ile Lys Trp Leu Arg Pro Ser Gly Pro Met Pro Ala 275 280 285 Asp Arg Val Thr Tyr Gln Asn His Asn Lys Thr Leu Gln Leu Leu 290 295 300 Lys Val Gly Glu Glu Asp Asp Gly Glu Tyr Arg Cys Leu Ala Glu 305 310 315 Asn Ser Leu Gly Ser Ala Arg His Ala Tyr Tyr Val Thr Val Glu 320 325 330 Ala Ala Pro Tyr Trp Leu His Lys Pro Gln Ser His Leu Tyr Gly 335 340 345 Pro Gly Glu Thr Ala Arg Leu Asp Cys Gln Val Gln Gly Arg Pro 350 355 360 Gln Pro Glu Val Thr Trp Arg Ile Asn Gly Ile Pro Val Glu Glu 365 370 375 Leu Ala Lys Asp Gln Lys Tyr Arg Ile Gln Arg Gly Ala Leu Ile 380 385 390 Leu Ser Asn Val Gln Pro Ser Asp Thr Met Val Thr Gln Cys Glu 395 400 405 Ala Arg Asn Arg His Gly Leu Leu Leu Ala Asn Ala Tyr Ile Tyr 410 415 420 Val Val Gln Leu Pro Ala Lys Ile Leu Thr Ala Asp Asn Gln Thr 425 430 435 Tyr Met Ala Val Gln Gly Ser Thr Ala Tyr Leu Leu Cys Lys Ala 440 445 450 Phe Gly Ala Pro Val Pro Ser Val Gln Trp Leu Asp Glu Asp Gly 455 460 465 Thr Thr Val Leu Gln Asp Glu Arg Phe Phe Pro Tyr Ala Asn Gly 470 475 480 Thr Leu Gly Ile Arg Asp Leu Gln Ala Asn Asp Thr Gly Arg Tyr 485 490 495 Phe Cys Leu Ala Ala Asn Asp Gln Asn Asn Val Thr Ile Met Ala 500 505 510 Asn Leu Lys Val Lys Asp Ala Thr Gln Ile Thr Gln Gly Pro Arg 515 520 525 Ser Thr Ile Glu Lys Lys Gly Ser Arg Val Thr Phe Thr Cys Gln 530 535 540 Ala Ser Phe Asp Pro Ser Leu Gln Pro Ser Ile Thr Trp Arg Gly 545 550 555 Asp Gly Arg Asp Leu Gln Glu Leu Gly Asp Ser Asp Lys Tyr Phe 560 565 570 Ile Glu Asp Gly Arg Leu Val Ile His Ser Leu Asp Tyr Ser Asp 575 580 585 Gln Gly Asn Tyr Ser Cys Val Ala Ser Thr Glu Leu Asp Val Val 590 595 600 Glu Ser Arg Ala Gln Leu Leu Val Val Gly Ser Pro Gly Pro Val 605 610 615 Pro Arg Leu Val Leu Ser Asp Leu His Leu Leu Thr Gln Ser Gln 620 625 630 Val Arg Val Ser Trp Ser Pro Ala Glu Asp His Asn Ala Pro Ile 635 640 645 Glu Lys Tyr Asp Ile Glu Phe Glu Asp Lys Glu Met Ala Pro Glu 650 655 660 Lys Trp Tyr Ser Leu Gly Lys Val Pro Gly Asn Gln Thr Ser Thr 665 670 675 Thr Leu Lys Leu Ser Pro Tyr Val His Tyr Thr Phe Arg Val Thr 680 685 690 Ala Ile Asn Lys Tyr Gly Pro Gly Glu Pro Ser Pro Val Ser Glu 695 700 705 Thr Val Val Thr Pro Glu Ala Ala Pro Glu Lys Asn Pro Val Asp 710 715 720 Val Lys Gly Glu Gly Asn Glu Thr Thr Asn Met Val Ile Thr Trp 725 730 735 Lys Pro Leu Arg Trp Met Asp Trp Asn Ala Pro Gln Val Gln Tyr 740 745 750 Arg Val Gln Trp Arg Pro Gln Gly Thr Arg Gly Pro Trp Gln Glu 755 760 765 Gln Ile Val Ser Asp Pro Phe Leu Val Val Ser Asn Thr Ser Thr 770 775 780 Phe Val Pro Tyr Glu Ile Lys Val Gln Ala Val Asn Ser Gln Gly 785 790 795 Lys Gly Pro Glu Pro Gln Val Thr Ile Gly Tyr Ser Gly Glu Asp 800 805 810 Tyr Pro Gln Ala Ile Pro Glu Leu Glu Gly Ile Glu Ile Leu Asn 815 820 825 Ser Ser Ala Val Leu Val Lys Trp Arg Pro Val Asp Leu Ala Gln 830 835 840 Val Lys Gly His Leu Arg Gly Tyr Asn Val Thr Tyr Trp Arg Glu 845 850 855 Gly Ser Gln Arg Lys His Ser Lys Arg His Ile His Lys Asp His 860 865 870 Val Val Val Pro Ala Asn Thr Thr Ser Val Ile Leu Ser Gly Leu 875 880 885 Arg Pro Tyr Ser Ser Tyr His Leu Glu Val Gln Ala Phe Asn Gly 890 895 900 Arg Gly Ser Gly Pro Ala Ser Glu Phe Thr Phe Ser Thr Pro Glu 905 910 915 Gly Val Pro Gly His Pro Glu Ala Leu His Leu Glu Cys Gln Ser 920 925 930 Asn Thr Ser Leu Leu Leu Arg Trp Gln Pro Pro Leu Ser His Asn 935 940 945 Gly Val Leu Thr Gly Tyr Val Leu Ser Tyr His Pro Leu Asp Glu 950 955 960 Gly Gly Lys Gly Gln Leu Ser Phe Asn Leu Arg Asp Pro Glu Leu 965 970 975 Arg Thr His Asn Leu Thr Asp Leu Ser Pro His Leu Arg Tyr Arg 980 985 990 Phe Gln Leu Gln Ala Thr Thr Lys Glu Gly Pro Gly Glu Ala Ile 995 1000 1005 Val Arg Glu Gly Gly Thr Met Ala Leu Ser Gly Ile Ser Asp Phe 1010 1015 1020 Gly Asn Ile Ser Ala Thr Ala Gly Glu Asn Tyr Ser Val Val Ser 1025 1030 1035 Trp Val Pro Lys Glu Gly Gln Cys Asn Phe Arg Phe His Ile Leu 1040 1045 1050 Phe Lys Ala Leu Gly Glu Glu Lys Gly Gly Ala Ser Leu Ser Pro 1055 1060 1065 Gln Tyr Val Ser Tyr Asn Gln Ser Ser Tyr Thr Gln Trp Asp Leu 1070 1075 1080 Gln Pro Asp Thr Asp Tyr Glu Ile His Leu Phe Lys Glu Arg Met 1085 1090 1095 Phe Arg His Gln Met Ala Val Lys Thr Asn Gly Thr Gly Arg Val 1100 1105 1110 Arg Leu Pro Pro Ala Gly Phe Ala Thr Glu Gly Trp Phe Ile Gly 1115 1120 1125 Phe Val Ser Ala Ile Ile Leu Leu Leu Leu Val Leu Leu Ile Leu 1130 1135 1140 Cys Phe Ile Lys Arg Ser Lys Gly Gly Lys Tyr Ser Val Lys Asp 1145 1150 1155 Lys Glu Asp Thr Gln Val Asp Ser Glu Ala Arg Pro Met Lys Asp 1160 1165 1170 Glu Thr Phe Gly Glu Tyr Arg Ser Leu Glu Ser Asp Asn Glu Glu 1175 1180 1185 Lys Ala Phe Gly Ser Ser Gln Pro Ser Leu Asn Gly Asp Ile Lys 1190 1195 1200 Pro Leu Gly Ser Asp Asp Ser Leu Ala Asp Tyr Gly Gly Ser Val 1205 1210 1215 Asp Val Gln Phe Asn Glu Asp Gly Ser Phe Ile Gly Gln Tyr Ser 1220 1225 1230 Gly Lys Lys Glu Lys Glu Ala Ala Gly Gly Asn Asp Ser Ser Gly 1235 1240 1245 Ala Thr Ser Pro Ile Asn Pro Ala Val Ala Leu Glu 1250 1255 24 162 PRT Homo sapiens 24 Met Trp Lys Val Ser Ala Leu Leu Phe Val Leu Gly Ser Ala Ser 1 5 10 15 Leu Trp Val Leu Ala Glu Gly Ala Ser Thr Gly Gln Pro Glu Asp 20 25 30 Asp Thr Glu Thr Thr Gly Leu Glu Gly Gly Val Ala Met Pro Gly 35 40 45 Ala Glu Asp Asp Val Val Thr Pro Gly Thr Ser Glu Asp Arg Tyr 50 55 60 Lys Ser Gly Leu Thr Thr Leu Val Ala Thr Ser Val Asn Ser Val 65 70 75 Thr Gly Ile Arg Ile Glu Asp Leu Pro Thr Ser Glu Ser Thr Val 80 85 90 His Ala Gln Glu Gln Ser Pro Ser Ala Thr Ala Ser Asn Val Ala 95 100 105 Thr Ser His Ser Thr Glu Lys Val Asp Gly Asp Thr Gln Thr Thr 110 115 120 Val Glu Lys Asp Gly Leu Ser Thr Val Thr Leu Val Gly Ile Ile 125 130 135 Val Gly Val Leu Leu Ala Ile Gly Phe Ile Gly Gly Ile Ile Val 140 145 150 Val Val Met Arg Lys Met Ser Gly Arg Tyr Ser Pro 155 160 25 160 PRT Homo sapiens 25 Met Trp Lys Val Ser Ala Leu Leu Phe Val Leu Gly Ser Ala Ser 1 5 10 15 Leu Trp Val Leu Ala Glu Gly Ala Ser Thr Gly Gln Pro Glu Asp 20 25 30 Asp Thr Glu Thr Thr Gly Leu Glu Gly Gly Val Ala Met Pro Gly 35 40 45 Ala Glu Asp Asp Val Val Thr Pro Gly Thr Ser Glu Asp Arg Tyr 50 55 60 Lys Ser Gly Leu Thr Thr Leu Val Ala Thr Ser Val Asn Ser Val 65

70 75 Thr Gly Ile Arg Ile Glu Asp Leu Pro Thr Ser Glu Ser Thr Val 80 85 90 His Ala Gln Glu Gln Ser Pro Ser Ala Thr Ala Ser Asn Val Ala 95 100 105 Thr Ser His Ser Thr Glu Lys Val Asp Gly Asp Thr Gln Thr Thr 110 115 120 Val Glu Lys Asp Gly Leu Ser Thr Val Thr Leu Val Gly Ile Ile 125 130 135 Val Gly Val Leu Leu Ala Ile Gly Phe Ile Gly Gly Ile Ile Val 140 145 150 Val Val Met Arg Lys Met Ser Gly Arg Pro 155 160 26 582 PRT Homo sapiens 26 Met Ser Pro Ala Pro Arg Pro Pro Arg Cys Leu Leu Leu Pro Leu 1 5 10 15 Leu Thr Leu Gly Thr Ala Leu Ala Ser Leu Gly Ser Ala Gln Ser 20 25 30 Ser Ser Phe Ser Pro Glu Ala Trp Leu Gln Gln Tyr Gly Tyr Leu 35 40 45 Pro Pro Gly Asp Leu Arg Thr His Thr Gln Arg Ser Pro Gln Ser 50 55 60 Leu Ser Ala Ala Ile Ala Ala Met Gln Lys Phe Tyr Gly Leu Gln 65 70 75 Val Thr Gly Lys Ala Asp Ala Asp Thr Met Lys Ala Met Arg Arg 80 85 90 Pro Arg Cys Gly Val Pro Asp Lys Phe Gly Ala Glu Ile Lys Ala 95 100 105 Asn Val Arg Arg Lys Arg Tyr Ala Ile Gln Gly Leu Lys Trp Gln 110 115 120 His Asn Glu Ile Thr Phe Cys Ile Gln Asn Tyr Thr Pro Lys Val 125 130 135 Gly Glu Tyr Ala Thr Tyr Glu Ala Ile Arg Lys Ala Phe Arg Val 140 145 150 Trp Glu Ser Ala Thr Pro Leu Arg Phe Arg Glu Val Pro Tyr Ala 155 160 165 Tyr Ile Arg Glu Gly His Glu Lys Gln Ala Asp Ile Met Ile Phe 170 175 180 Phe Ala Glu Gly Phe His Gly Asp Ser Thr Pro Phe Asp Gly Glu 185 190 195 Gly Gly Phe Leu Ala His Ala Tyr Phe Pro Gly Pro Asn Ile Gly 200 205 210 Gly Asp Thr His Phe Asp Ser Ala Glu Pro Trp Thr Val Arg Asn 215 220 225 Glu Asp Leu Asn Gly Asn Asp Ile Phe Leu Val Ala Val His Glu 230 235 240 Leu Gly His Ala Leu Gly Leu Glu His Ser Ser Asp Pro Ser Ala 245 250 255 Ile Met Ala Pro Phe Tyr Gln Trp Met Asp Thr Glu Asn Phe Val 260 265 270 Leu Pro Asp Asp Asp Arg Arg Gly Ile Gln Gln Leu Tyr Gly Gly 275 280 285 Glu Ser Gly Phe Pro Thr Lys Met Pro Pro Gln Pro Arg Thr Thr 290 295 300 Ser Arg Pro Ser Val Pro Asp Lys Pro Lys Asn Pro Thr Tyr Gly 305 310 315 Pro Asn Ile Cys Asp Gly Asn Phe Asp Thr Val Ala Met Leu Arg 320 325 330 Gly Glu Met Phe Val Phe Lys Glu Arg Trp Phe Trp Arg Val Arg 335 340 345 Asn Asn Gln Val Met Asp Gly Tyr Pro Met Pro Ile Gly Gln Phe 350 355 360 Trp Arg Gly Leu Pro Ala Ser Ile Asn Thr Ala Tyr Glu Arg Lys 365 370 375 Asp Gly Lys Phe Val Phe Phe Lys Gly Asp Lys His Trp Val Phe 380 385 390 Asp Glu Ala Ser Leu Glu Pro Gly Tyr Pro Lys His Ile Lys Glu 395 400 405 Leu Gly Arg Gly Leu Pro Thr Asp Lys Ile Asp Ala Ala Leu Phe 410 415 420 Trp Met Pro Asn Gly Lys Thr Tyr Phe Phe Arg Gly Asn Lys Tyr 425 430 435 Tyr Arg Phe Asn Glu Glu Leu Arg Ala Val Asp Ser Glu Tyr Pro 440 445 450 Lys Asn Ile Lys Val Trp Glu Gly Ile Pro Glu Ser Pro Arg Gly 455 460 465 Ser Phe Met Gly Ser Asp Glu Val Phe Thr Tyr Phe Tyr Lys Gly 470 475 480 Asn Lys Tyr Trp Lys Phe Asn Asn Gln Lys Leu Lys Val Glu Pro 485 490 495 Gly Tyr Pro Lys Ser Ala Leu Arg Asp Trp Met Gly Cys Pro Ser 500 505 510 Gly Gly Arg Pro Asp Glu Gly Thr Glu Glu Glu Thr Glu Val Ile 515 520 525 Ile Ile Glu Val Asp Glu Glu Gly Gly Gly Ala Val Ser Ala Ala 530 535 540 Ala Val Val Leu Pro Val Leu Leu Leu Leu Leu Val Leu Ala Val 545 550 555 Gly Leu Ala Val Phe Phe Phe Arg Arg His Gly Thr Pro Arg Arg 560 565 570 Leu Leu Tyr Cys Gln Arg Ser Leu Leu Asp Lys Val 575 580 27 525 PRT Homo sapiens 27 Met Trp Glu Ala Gln Phe Leu Gly Leu Leu Phe Leu Gln Pro Leu 1 5 10 15 Trp Val Ala Pro Val Lys Pro Leu Gln Pro Gly Ala Glu Val Pro 20 25 30 Val Val Trp Ala Gln Glu Gly Ala Pro Ala Gln Leu Pro Cys Ser 35 40 45 Pro Thr Ile Pro Leu Gln Asp Leu Ser Leu Leu Arg Arg Ala Gly 50 55 60 Val Thr Trp Gln His Gln Pro Asp Ser Gly Pro Pro Ala Ala Ala 65 70 75 Pro Gly His Pro Leu Ala Pro Gly Pro His Pro Ala Ala Pro Ser 80 85 90 Ser Trp Gly Pro Arg Pro Arg Arg Tyr Thr Val Leu Ser Val Gly 95 100 105 Pro Gly Gly Leu Arg Ser Gly Arg Leu Pro Leu Gln Pro Arg Val 110 115 120 Gln Leu Asp Glu Arg Gly Arg Gln Arg Gly Asp Phe Ser Leu Trp 125 130 135 Leu Arg Pro Ala Arg Arg Ala Asp Ala Gly Glu Tyr Arg Ala Ala 140 145 150 Val His Leu Arg Asp Arg Ala Leu Ser Cys Arg Leu Arg Leu Arg 155 160 165 Leu Gly Gln Ala Ser Met Thr Ala Ser Pro Pro Gly Ser Leu Arg 170 175 180 Ala Ser Asp Trp Val Ile Leu Asn Cys Ser Phe Ser Arg Pro Asp 185 190 195 Arg Pro Ala Ser Val His Trp Phe Arg Asn Arg Gly Gln Gly Arg 200 205 210 Val Pro Val Arg Glu Ser Pro His His His Leu Ala Glu Ser Phe 215 220 225 Leu Phe Leu Pro Gln Val Ser Pro Met Asp Ser Gly Pro Trp Gly 230 235 240 Cys Ile Leu Thr Tyr Arg Asp Gly Phe Asn Val Ser Ile Met Tyr 245 250 255 Asn Leu Thr Val Leu Gly Leu Glu Pro Pro Thr Pro Leu Thr Val 260 265 270 Tyr Ala Gly Ala Gly Ser Arg Val Gly Leu Pro Cys Arg Leu Pro 275 280 285 Ala Gly Val Gly Thr Arg Ser Phe Leu Thr Ala Lys Trp Thr Pro 290 295 300 Pro Gly Gly Gly Pro Asp Leu Leu Val Thr Gly Asp Asn Gly Asp 305 310 315 Phe Thr Leu Arg Leu Glu Asp Val Ser Gln Ala Gln Ala Gly Thr 320 325 330 Tyr Thr Cys His Ile His Leu Gln Glu Gln Gln Leu Asn Ala Thr 335 340 345 Val Thr Leu Ala Ile Ile Thr Val Thr Pro Lys Ser Phe Gly Ser 350 355 360 Pro Gly Ser Leu Gly Lys Leu Leu Cys Glu Val Thr Pro Val Ser 365 370 375 Gly Gln Glu Arg Phe Val Trp Ser Ser Leu Asp Thr Pro Ser Gln 380 385 390 Arg Ser Phe Ser Gly Pro Trp Leu Glu Ala Gln Glu Ala Gln Leu 395 400 405 Leu Ser Gln Pro Trp Gln Cys Gln Leu Tyr Gln Gly Glu Arg Leu 410 415 420 Leu Gly Ala Ala Val Tyr Phe Thr Glu Leu Ser Ser Pro Gly Ala 425 430 435 Gln Arg Ser Gly Arg Ala Pro Gly Ala Leu Pro Ala Gly His Leu 440 445 450 Leu Leu Phe Leu Thr Leu Gly Val Leu Ser Leu Leu Leu Leu Val 455 460 465 Thr Gly Ala Phe Gly Phe His Leu Trp Arg Arg Gln Trp Arg Pro 470 475 480 Arg Arg Phe Ser Ala Leu Glu Gln Gly Ile His Pro Arg Gln Ala 485 490 495 Gln Ser Lys Ile Glu Glu Leu Glu Gln Glu Pro Glu Pro Glu Pro 500 505 510 Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Gln Leu 515 520 525 28 525 PRT Homo sapiens 28 Met Trp Glu Ala Gln Phe Leu Gly Leu Leu Phe Leu Gln Pro Leu 1 5 10 15 Trp Val Ala Pro Val Lys Pro Leu Gln Pro Gly Ala Glu Val Pro 20 25 30 Val Val Trp Ala Gln Glu Gly Ala Pro Ala Gln Leu Pro Cys Ser 35 40 45 Pro Thr Ile Pro Leu Gln Asp Leu Ser Leu Leu Arg Arg Ala Gly 50 55 60 Val Thr Trp Gln His Gln Pro Asp Ser Gly Pro Pro Ala Ala Ala 65 70 75 Pro Gly His Pro Leu Ala Pro Gly Pro His Pro Ala Ala Pro Ser 80 85 90 Ser Trp Gly Pro Arg Pro Arg Arg Tyr Thr Val Leu Ser Val Gly 95 100 105 Pro Gly Gly Leu Arg Ser Gly Arg Leu Pro Leu Gln Pro Arg Val 110 115 120 Gln Leu Asp Glu Arg Gly Arg Gln Arg Gly Asp Phe Ser Leu Trp 125 130 135 Leu Arg Pro Ala Arg Arg Ala Asp Ala Gly Glu Tyr Arg Ala Ala 140 145 150 Val His Leu Arg Asp Arg Ala Leu Ser Cys Arg Leu Arg Leu Arg 155 160 165 Leu Gly Gln Ala Ser Met Thr Ala Ser Pro Pro Gly Ser Leu Arg 170 175 180 Ala Ser Asp Trp Val Ile Leu Asn Cys Ser Phe Ser Arg Pro Asp 185 190 195 Arg Pro Ala Ser Val His Trp Phe Arg Asn Arg Gly Gln Gly Arg 200 205 210 Val Pro Val Arg Glu Ser Pro His His His Leu Ala Glu Ser Phe 215 220 225 Leu Phe Leu Pro Gln Val Ser Pro Met Asp Ser Gly Pro Trp Gly 230 235 240 Cys Ile Leu Thr Tyr Arg Asp Gly Phe Asn Val Ser Ile Met Tyr 245 250 255 Asn Leu Thr Val Leu Gly Leu Glu Pro Pro Thr Pro Leu Thr Val 260 265 270 Tyr Ala Gly Ala Gly Ser Arg Val Gly Leu Pro Cys Arg Leu Pro 275 280 285 Ala Gly Val Gly Thr Arg Ser Phe Leu Thr Ala Lys Trp Thr Pro 290 295 300 Pro Gly Gly Gly Pro Asp Leu Leu Val Thr Gly Asp Asn Gly Asp 305 310 315 Phe Thr Leu Arg Leu Glu Asp Val Ser Gln Ala Gln Ala Gly Thr 320 325 330 Tyr Thr Cys His Ile His Leu Gln Glu Gln Gln Leu Asn Ala Thr 335 340 345 Val Thr Leu Ala Ile Ile Thr Val Thr Pro Lys Ser Phe Gly Ser 350 355 360 Pro Gly Ser Leu Gly Lys Leu Leu Cys Glu Val Thr Pro Val Ser 365 370 375 Gly Gln Glu Arg Phe Val Trp Ser Ser Leu Asp Thr Pro Ser Gln 380 385 390 Arg Ser Phe Ser Gly Pro Trp Leu Glu Ala Gln Glu Ala Gln Leu 395 400 405 Leu Ser Gln Pro Trp Gln Cys Gln Leu Tyr Gln Gly Glu Arg Leu 410 415 420 Leu Gly Ala Ala Val Tyr Phe Thr Glu Leu Ser Ser Pro Gly Ala 425 430 435 Gln Arg Ser Gly Arg Ala Pro Gly Ala Leu Pro Ala Gly His Leu 440 445 450 Leu Leu Phe Leu Thr Leu Gly Val Leu Ser Leu Leu Leu Leu Val 455 460 465 Thr Gly Ala Phe Gly Phe His Leu Trp Arg Arg Gln Trp Arg Pro 470 475 480 Arg Arg Phe Ser Ala Leu Glu Gln Gly Ile His Pro Pro Gln Ala 485 490 495 Gln Ser Lys Ile Glu Glu Leu Glu Gln Glu Pro Glu Pro Glu Pro 500 505 510 Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Gln Leu 515 520 525 29 2181 PRT Homo sapiens 29 Met Met Met Met Met Met Met Lys Lys Met Gln His Gln Arg Gln 1 5 10 15 Gln Gln Ala Asp His Ala Asn Glu Ala Asn Tyr Ala Arg Gly Thr 20 25 30 Arg Leu Pro Leu Ser Gly Glu Gly Pro Thr Ser Gln Pro Asn Ser 35 40 45 Ser Lys Gln Thr Val Leu Ser Trp Gln Ala Ala Ile Asp Ala Ala 50 55 60 Arg Gln Ala Lys Ala Ala Gln Thr Met Ser Thr Ser Ala Pro Pro 65 70 75 Pro Val Gly Ser Leu Ser Gln Arg Lys Arg Gln Gln Tyr Ala Lys 80 85 90 Ser Lys Lys Gln Gly Asn Ser Ser Asn Ser Arg Pro Ala Arg Ala 95 100 105 Leu Phe Cys Leu Ser Leu Asn Asn Pro Ile Arg Arg Ala Cys Ile 110 115 120 Ser Ile Val Glu Trp Lys Pro Phe Asp Ile Phe Ile Leu Leu Ala 125 130 135 Ile Phe Ala Asn Cys Val Ala Leu Ala Ile Tyr Ile Pro Phe Pro 140 145 150 Glu Asp Asp Ser Asn Ser Thr Asn His Asn Leu Glu Lys Val Glu 155 160 165 Tyr Ala Phe Leu Ile Ile Phe Thr Val Glu Thr Phe Leu Lys Ile 170 175 180 Ile Ala Tyr Gly Leu Leu Leu His Pro Asn Ala Tyr Val Arg Asn 185 190 195 Gly Trp Asn Leu Leu Asp Phe Val Ile Val Ile Val Gly Leu Phe 200 205 210 Ser Val Ile Leu Glu Gln Leu Thr Lys Glu Thr Glu Gly Gly Asn 215 220 225 His Ser Ser Gly Lys Ser Gly Gly Phe Asp Val Lys Ala Leu Arg 230 235 240 Ala Phe Arg Val Leu Arg Pro Leu Arg Leu Val Ser Gly Val Pro 245 250 255 Ser Leu Gln Val Val Leu Asn Ser Ile Ile Lys Ala Met Val Pro 260 265 270 Leu Leu His Ile Ala Leu Leu Val Leu Phe Val Ile Ile Ile Tyr 275 280 285 Ala Ile Ile Gly Leu Glu Leu Phe Ile Gly Lys Met His Lys Thr 290 295 300 Cys Phe Phe Ala Asp Ser Asp Ile Val Ala Glu Glu Asp Pro Ala 305 310 315 Pro Cys Ala Phe Ser Gly Asn Gly Arg Gln Cys Thr Ala Asn Gly 320 325 330 Thr Glu Cys Arg Ser Gly Trp Val Gly Pro Asn Gly Gly Ile Thr 335 340 345 Asn Phe Asp Asn Phe Ala Phe Ala Met Leu Thr Val Phe Gln Cys 350 355 360 Ile Thr Met Glu Gly Trp Thr Asp Val Leu Tyr Trp Val Asn Asp 365 370 375 Ala Ile Gly Trp Glu Trp Pro Trp Val Tyr Phe Val Ser Leu Ile 380 385 390 Ile Leu Gly Ser Phe Phe Val Leu Asn Leu Val Leu Gly Val Leu 395 400 405 Ser Gly Glu Phe Ser Lys Glu Arg Glu Lys Ala Lys Ala Arg Gly 410 415 420 Asp Phe Gln Lys Leu Arg Glu Lys Gln Gln Leu Glu Glu Asp Leu 425 430 435 Lys Gly Tyr Leu Asp Trp Ile Thr Gln Ala Glu Asp Ile Asp Pro 440 445 450 Glu Asn Glu Glu Glu Gly Gly Glu Glu Gly Lys Arg Asn Thr Ser 455 460 465 Met Pro Thr Ser Glu Thr Glu Ser Val Asn Thr Glu Asn Val Ser 470 475 480 Gly Glu Gly Glu Asn Arg Gly Cys Cys Gly Ser Leu Trp Cys Trp 485 490 495 Trp Arg Arg Arg Gly Ala Ala Lys Ala Gly Pro Ser Gly Cys Arg 500 505 510 Arg Trp Gly Gln Ala Ile Ser Lys Ser Lys Leu Ser Arg Arg Trp 515 520 525 Arg Arg Trp Asn Arg Phe Asn Arg Arg Arg Cys Arg Ala Ala Val 530 535 540 Lys Ser Val Thr Phe Tyr Trp Leu Val Ile Val Leu Val Phe Leu 545 550 555 Asn Thr Leu Thr Ile Ser Ser Glu His Tyr Asn Gln Pro Asp Trp 560

565 570 Leu Thr Gln Ile Gln Asp Ile Ala Asn Lys Val Leu Leu Ala Leu 575 580 585 Phe Thr Cys Glu Met Leu Val Lys Met Tyr Ser Leu Gly Leu Gln 590 595 600 Ala Tyr Phe Val Ser Leu Phe Asn Arg Phe Asp Cys Phe Val Val 605 610 615 Cys Gly Gly Ile Thr Glu Thr Ile Leu Val Glu Leu Glu Ile Met 620 625 630 Ser Pro Leu Gly Ile Ser Val Phe Arg Cys Val Arg Leu Leu Arg 635 640 645 Ile Phe Lys Val Thr Arg His Trp Thr Ser Leu Ser Asn Leu Val 650 655 660 Ala Ser Leu Leu Asn Ser Met Lys Ser Ile Ala Ser Leu Leu Leu 665 670 675 Leu Leu Phe Leu Phe Ile Ile Ile Phe Ser Leu Leu Gly Met Gln 680 685 690 Leu Phe Gly Gly Lys Phe Asn Phe Asp Glu Thr Gln Thr Lys Arg 695 700 705 Ser Thr Phe Asp Asn Phe Pro Gln Ala Leu Leu Thr Val Phe Gln 710 715 720 Ile Leu Thr Gly Glu Asp Trp Asn Ala Val Met Tyr Asp Gly Ile 725 730 735 Met Ala Tyr Gly Gly Pro Ser Ser Ser Gly Met Ile Val Cys Ile 740 745 750 Tyr Phe Ile Ile Leu Phe Ile Cys Gly Asn Tyr Ile Leu Leu Asn 755 760 765 Val Phe Leu Ala Ile Ala Val Asp Asn Leu Ala Asp Ala Glu Ser 770 775 780 Leu Asn Thr Ala Gln Lys Glu Glu Ala Glu Glu Lys Glu Arg Lys 785 790 795 Lys Ile Ala Arg Lys Glu Ser Leu Glu Asn Lys Lys Asn Asn Lys 800 805 810 Pro Glu Val Asn Gln Ile Ala Asn Ser Asp Asn Lys Val Thr Ile 815 820 825 Asp Asp Tyr Arg Glu Glu Asp Glu Asp Lys Asp Pro Tyr Pro Pro 830 835 840 Cys Asp Val Pro Val Gly Glu Glu Glu Glu Glu Glu Glu Glu Asp 845 850 855 Glu Pro Glu Val Pro Ala Gly Pro Arg Pro Arg Arg Ile Ser Glu 860 865 870 Leu Asn Met Lys Glu Lys Ile Ala Pro Ile Pro Glu Gly Ser Ala 875 880 885 Phe Phe Ile Leu Ser Lys Thr Asn Pro Ile Arg Val Gly Cys His 890 895 900 Lys Leu Ile Asn His His Ile Phe Thr Asn Leu Ile Leu Val Phe 905 910 915 Ile Met Leu Ser Ser Ala Ala Leu Ala Ala Glu Asp Pro Ile Arg 920 925 930 Ser His Ser Phe Arg Asn Thr Ile Leu Gly Tyr Phe Asp Tyr Ala 935 940 945 Phe Thr Ala Ile Phe Thr Val Glu Ile Leu Leu Lys Met Thr Thr 950 955 960 Phe Gly Ala Phe Leu His Lys Gly Ala Phe Cys Arg Asn Tyr Phe 965 970 975 Asn Leu Leu Asp Met Leu Val Val Gly Val Ser Leu Val Ser Phe 980 985 990 Gly Ile Gln Ser Ser Ala Ile Ser Val Val Lys Ile Leu Arg Val 995 1000 1005 Leu Arg Val Leu Arg Pro Leu Arg Ala Ile Asn Arg Ala Lys Gly 1010 1015 1020 Leu Lys His Val Val Gln Cys Val Phe Val Ala Ile Arg Thr Ile 1025 1030 1035 Gly Asn Ile Met Ile Val Thr Thr Leu Leu Gln Phe Met Phe Ala 1040 1045 1050 Cys Ile Gly Val Gln Leu Phe Lys Gly Lys Phe Tyr Arg Cys Thr 1055 1060 1065 Asp Glu Ala Lys Ser Asn Pro Glu Glu Cys Arg Gly Leu Phe Ile 1070 1075 1080 Leu Tyr Lys Asp Gly Asp Val Asp Ser Pro Val Val Arg Glu Arg 1085 1090 1095 Ile Trp Gln Asn Ser Asp Phe Asn Phe Asp Asn Val Leu Ser Ala 1100 1105 1110 Met Met Ala Leu Phe Thr Val Ser Thr Phe Glu Gly Trp Pro Ala 1115 1120 1125 Leu Leu Tyr Lys Ala Ile Asp Ser Asn Gly Glu Asn Ile Gly Pro 1130 1135 1140 Ile Tyr Asn His Arg Val Glu Ile Ser Ile Phe Phe Ile Ile Tyr 1145 1150 1155 Ile Ile Ile Val Ala Phe Phe Met Met Asn Ile Phe Val Gly Phe 1160 1165 1170 Val Ile Val Thr Phe Gln Glu Gln Gly Glu Lys Glu Tyr Lys Asn 1175 1180 1185 Cys Glu Leu Asp Lys Asn Gln Arg Gln Cys Val Glu Tyr Ala Leu 1190 1195 1200 Lys Ala Arg Pro Leu Arg Arg Tyr Ile Pro Lys Asn Pro Tyr Gln 1205 1210 1215 Tyr Lys Phe Trp Tyr Val Val Asn Ser Ser Pro Phe Glu Tyr Met 1220 1225 1230 Met Phe Val Leu Ile Met Leu Asn Thr Leu Cys Leu Ala Met Gln 1235 1240 1245 His Tyr Glu Gln Ser Lys Met Phe Asn Asp Ala Met Asp Ile Leu 1250 1255 1260 Asn Met Val Phe Thr Gly Val Phe Thr Val Glu Met Val Leu Lys 1265 1270 1275 Val Ile Ala Phe Lys Pro Lys Gly Tyr Phe Ser Asp Ala Trp Asn 1280 1285 1290 Thr Phe Asp Ser Leu Ile Val Ile Gly Ser Ile Ile Asp Val Ala 1295 1300 1305 Leu Ser Glu Ala Asp Pro Thr Glu Ser Glu Asn Val Pro Val Pro 1310 1315 1320 Thr Ala Thr Pro Gly Asn Ser Glu Glu Ser Asn Arg Ile Ser Ile 1325 1330 1335 Thr Phe Phe Arg Leu Phe Arg Val Met Arg Leu Val Lys Leu Leu 1340 1345 1350 Ser Arg Gly Glu Gly Ile Arg Thr Leu Leu Trp Thr Phe Ile Lys 1355 1360 1365 Ser Phe Gln Ala Leu Pro Tyr Val Ala Leu Leu Ile Ala Met Leu 1370 1375 1380 Phe Phe Ile Tyr Ala Val Ile Gly Met Gln Met Phe Gly Lys Val 1385 1390 1395 Ala Met Arg Asp Asn Asn Gln Ile Asn Arg Asn Asn Asn Phe Gln 1400 1405 1410 Thr Phe Pro Gln Ala Val Leu Leu Leu Phe Arg Cys Ala Thr Gly 1415 1420 1425 Glu Ala Trp Gln Glu Ile Met Leu Ala Cys Leu Pro Gly Lys Leu 1430 1435 1440 Cys Asp Pro Glu Ser Asp Tyr Asn Pro Gly Glu Glu Tyr Thr Cys 1445 1450 1455 Gly Ser Asn Phe Ala Ile Val Tyr Phe Ile Ser Phe Tyr Met Leu 1460 1465 1470 Cys Ala Phe Leu Ile Ile Asn Leu Phe Val Ala Val Ile Met Asp 1475 1480 1485 Asn Phe Asp Tyr Leu Thr Arg Asp Trp Ser Ile Leu Gly Pro His 1490 1495 1500 His Leu Asp Glu Phe Lys Arg Ile Trp Ser Glu Tyr Asp Pro Glu 1505 1510 1515 Ala Lys Gly Arg Ile Lys His Leu Asp Val Val Thr Leu Leu Arg 1520 1525 1530 Arg Ile Gln Pro Pro Leu Gly Phe Gly Lys Leu Cys Pro His Arg 1535 1540 1545 Val Ala Cys Lys Arg Leu Val Ala Met Asn Met Pro Leu Asn Ser 1550 1555 1560 Asp Gly Thr Val Met Phe Asn Ala Thr Leu Phe Ala Leu Val Arg 1565 1570 1575 Thr Ala Leu Lys Ile Lys Thr Glu Gly Asn Leu Glu Gln Ala Asn 1580 1585 1590 Glu Glu Leu Arg Ala Val Ile Lys Lys Ile Trp Lys Lys Thr Ser 1595 1600 1605 Met Lys Leu Leu Asp Gln Val Val Pro Pro Ala Gly Asp Asp Glu 1610 1615 1620 Val Thr Val Gly Lys Phe Tyr Ala Thr Phe Leu Ile Gln Asp Tyr 1625 1630 1635 Phe Arg Lys Phe Lys Lys Arg Lys Glu Gln Gly Leu Val Gly Lys 1640 1645 1650 Tyr Pro Ala Lys Asn Thr Thr Ile Ala Leu Gln Ala Gly Leu Arg 1655 1660 1665 Thr Leu His Asp Ile Gly Pro Glu Ile Arg Arg Ala Ile Ser Cys 1670 1675 1680 Asp Leu Gln Asp Asp Glu Pro Glu Glu Thr Lys Arg Glu Glu Glu 1685 1690 1695 Asp Asp Val Phe Lys Arg Asn Gly Ala Leu Leu Gly Asn His Val 1700 1705 1710 Asn His Val Asn Ser Asp Arg Arg Asp Ser Leu Gln Gln Thr Asn 1715 1720 1725 Thr Thr His Arg Pro Leu His Val Gln Arg Pro Ser Ile Pro Pro 1730 1735 1740 Ala Ser Asp Thr Glu Lys Pro Leu Phe Pro Pro Ala Gly Asn Ser 1745 1750 1755 Val Cys His Asn His His Asn His Asn Ser Ile Gly Lys Gln Val 1760 1765 1770 Pro Thr Ser Thr Asn Ala Asn Leu Asn Asn Ala Asn Met Ser Lys 1775 1780 1785 Ala Ala His Gly Lys Arg Pro Ser Ile Gly Asn Leu Glu His Val 1790 1795 1800 Ser Glu Asn Gly His His Ser Ser His Lys His Asp Arg Glu Pro 1805 1810 1815 Gln Arg Arg Ser Ser Val Lys Arg Thr Arg Tyr Tyr Glu Thr Tyr 1820 1825 1830 Ile Arg Ser Asp Ser Gly Asp Glu Gln Leu Pro Thr Ile Cys Arg 1835 1840 1845 Glu Asp Pro Glu Ile His Gly Tyr Phe Arg Asp Pro His Cys Leu 1850 1855 1860 Gly Glu Gln Glu Tyr Phe Ser Ser Glu Glu Cys Tyr Glu Asp Asp 1865 1870 1875 Ser Ser Pro Thr Trp Ser Arg Gln Asn Tyr Gly Tyr Tyr Ser Arg 1880 1885 1890 Tyr Pro Gly Arg Asn Ile Asp Ser Glu Arg Pro Arg Gly Tyr His 1895 1900 1905 His Pro Gln Gly Phe Leu Glu Asp Asp Asp Ser Pro Val Cys Tyr 1910 1915 1920 Asp Ser Arg Arg Ser Pro Arg Arg Arg Leu Leu Pro Pro Thr Pro 1925 1930 1935 Ala Ser His Arg Arg Ser Ser Phe Asn Phe Glu Cys Leu Arg Arg 1940 1945 1950 Gln Ser Ser Gln Glu Glu Val Pro Ser Ser Pro Ile Phe Pro His 1955 1960 1965 Arg Thr Ala Leu Pro Leu His Leu Met Gln Gln Gln Ile Met Ala 1970 1975 1980 Val Ala Gly Leu Asp Ser Ser Lys Ala Gln Lys Tyr Ser Pro Ser 1985 1990 1995 His Ser Thr Arg Ser Trp Ala Thr Pro Pro Ala Thr Pro Pro Tyr 2000 2005 2010 Arg Asp Trp Thr Pro Cys Tyr Thr Pro Leu Ile Gln Val Glu Gln 2015 2020 2025 Ser Glu Ala Leu Asp Gln Val Asn Gly Ser Leu Pro Ser Leu His 2030 2035 2040 Arg Ser Ser Trp Tyr Thr Asp Glu Pro Asp Ile Ser Tyr Arg Thr 2045 2050 2055 Phe Thr Pro Ala Ser Leu Thr Val Pro Ser Ser Phe Arg Asn Lys 2060 2065 2070 Asn Ser Asp Lys Gln Arg Ser Ala Asp Ser Leu Val Glu Ala Val 2075 2080 2085 Leu Ile Ser Glu Gly Leu Gly Arg Tyr Ala Arg Asp Pro Lys Phe 2090 2095 2100 Val Ser Ala Thr Lys His Glu Ile Ala Asp Ala Cys Asp Leu Thr 2105 2110 2115 Ile Asp Glu Met Glu Ser Ala Ala Ser Thr Leu Leu Asn Gly Asn 2120 2125 2130 Val Arg Pro Arg Ala Asn Gly Asp Val Gly Pro Leu Ser His Arg 2135 2140 2145 Gln Asp Tyr Glu Leu Gln Asp Phe Gly Pro Gly Tyr Ser Asp Glu 2150 2155 2160 Glu Pro Asp Pro Gly Arg Asp Glu Glu Asp Leu Ala Asp Glu Met 2165 2170 2175 Ile Cys Ile Thr Thr Leu 2180 30 220 PRT Homo sapiens 30 Met Ala Ser Ala Gly Met Gln Ile Leu Gly Val Val Leu Thr Leu 1 5 10 15 Leu Gly Trp Val Asn Gly Leu Val Ser Cys Ala Leu Pro Met Trp 20 25 30 Lys Val Thr Ala Phe Ile Gly Asn Ser Ile Val Val Ala Gln Val 35 40 45 Val Trp Glu Gly Leu Trp Met Ser Cys Val Val Gln Ser Thr Gly 50 55 60 Gln Met Gln Cys Lys Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln 65 70 75 Asp Leu Gln Ala Ala Arg Ala Leu Cys Val Ile Ala Leu Leu Val 80 85 90 Ala Leu Phe Gly Leu Leu Val Tyr Leu Ala Gly Ala Lys Cys Thr 95 100 105 Thr Cys Val Glu Glu Lys Asp Ser Lys Ala Arg Leu Val Leu Thr 110 115 120 Ser Gly Ile Val Phe Val Ile Ser Gly Val Leu Thr Leu Ile Pro 125 130 135 Val Cys Trp Thr Ala His Ala Ile Ile Arg Asp Phe Tyr Asn Pro 140 145 150 Leu Val Ala Glu Ala Gln Lys Arg Glu Leu Gly Ala Ser Leu Tyr 155 160 165 Leu Gly Trp Ala Ala Ser Gly Leu Leu Leu Leu Gly Gly Gly Leu 170 175 180 Leu Cys Cys Thr Cys Pro Ser Gly Gly Ser Gln Gly Pro Ser His 185 190 195 Tyr Met Ala Arg Tyr Ser Thr Ser Ala Pro Ala Ile Ser Arg Gly 200 205 210 Pro Ser Glu Tyr Pro Thr Lys Asn Tyr Val 215 220 31 494 PRT Homo sapiens 31 Met Val Arg Lys Pro Val Val Ser Thr Ile Ser Lys Gly Gly Tyr 1 5 10 15 Leu Gln Gly Asn Val Asn Gly Arg Leu Pro Ser Leu Gly Asn Lys 20 25 30 Glu Pro Pro Gly Gln Glu Lys Val Gln Leu Lys Arg Lys Val Thr 35 40 45 Leu Leu Arg Gly Val Ser Ile Ile Ile Gly Thr Ile Ile Gly Ala 50 55 60 Gly Ile Phe Ile Ser Pro Lys Gly Val Leu Gln Asn Thr Gly Ser 65 70 75 Val Gly Met Ser Leu Thr Ile Trp Thr Val Cys Gly Val Leu Ser 80 85 90 Leu Phe Gly Ala Leu Ser Tyr Ala Glu Leu Gly Thr Thr Ile Lys 95 100 105 Lys Ser Gly Gly His Tyr Thr Tyr Ile Leu Glu Val Phe Gly Pro 110 115 120 Leu Pro Ala Phe Val Arg Val Trp Val Glu Leu Leu Ile Ile Arg 125 130 135 Pro Ala Ala Thr Ala Val Ile Ser Leu Ala Phe Gly Arg Tyr Ile 140 145 150 Leu Glu Pro Phe Phe Ile Gln Cys Glu Ile Pro Glu Leu Ala Ile 155 160 165 Lys Leu Ile Thr Ala Val Gly Ile Thr Val Val Met Val Leu Asn 170 175 180 Ser Met Ser Val Ser Trp Ser Ala Arg Ile Gln Ile Phe Leu Thr 185 190 195 Phe Cys Lys Leu Thr Ala Ile Leu Ile Ile Ile Val Pro Gly Val 200 205 210 Met Gln Leu Ile Lys Gly Gln Thr Gln Asn Phe Lys Asp Ala Phe 215 220 225 Ser Gly Arg Asp Ser Ser Ile Thr Arg Leu Pro Leu Ala Phe Tyr 230 235 240 Tyr Gly Met Tyr Ala Tyr Ala Gly Trp Phe Tyr Leu Asn Phe Val 245 250 255 Thr Glu Glu Val Glu Asn Pro Glu Lys Thr Ile Pro Leu Ala Ile 260 265 270 Cys Ile Ser Met Ala Ile Val Thr Ile Gly Tyr Val Leu Thr Asn 275 280 285 Val Ala Tyr Phe Thr Thr Ile Asn Ala Glu Glu Leu Leu Leu Ser 290 295 300 Asn Ala Val Ala Val Thr Phe Ser Glu Arg Leu Leu Gly Asn Phe 305 310 315 Ser Leu Ala Val Pro Ile Phe Val Ala Leu Ser Cys Phe Gly Ser 320 325 330 Met Asn Gly Gly Val Phe Ala Val Ser Arg Leu Phe Tyr Val Ala 335 340 345 Ser Arg Glu Gly His Leu Pro Glu Ile Leu Ser Met Ile His Val 350 355 360 Arg Lys His Thr Pro Leu Pro Ala Val Ile Val Leu His Pro Leu 365 370 375 Thr Met Ile Met Leu Phe Ser Gly Asp Leu Asp Ser Leu Leu Asn 380 385 390 Phe Leu Ser Phe Ala Arg Trp Leu Phe Ile Gly Leu Ala Val Ala 395 400 405 Gly Leu Ile Tyr Leu Arg Tyr Lys Cys Pro Asp Met His Arg Pro 410 415 420 Phe Lys Val Pro Leu Phe Ile Pro Ala Leu Phe Ser Phe Thr Cys 425 430 435 Leu Phe Met Val Ala Leu Ser

Leu Tyr Ser Asp Pro Phe Ser Thr 440 445 450 Gly Ile Gly Phe Val Ile Thr Leu Thr Gly Val Pro Ala Tyr Tyr 455 460 465 Leu Phe Ile Ile Trp Asp Lys Lys Pro Arg Trp Phe Arg Ile Met 470 475 480 Ser Gly Phe Leu Ala Leu Met Pro Ala Gln Ala Cys Asp Met 485 490 32 355 PRT Homo sapiens 32 Met Glu Glu Gly Glu Arg Ser Pro Leu Leu Ser Gln Glu Thr Ala 1 5 10 15 Gly Gln Lys Pro Leu Ser Val His Arg Pro Pro Thr Ser Gly Cys 20 25 30 Leu Gly Pro Val Pro Arg Glu Asp Gln Ala Glu Ala Trp Gly Cys 35 40 45 Ser Cys Cys Pro Pro Glu Thr Lys His Gln Ala Leu Ser Gly Thr 50 55 60 Pro Lys Lys Gly Pro Ala Pro Ser Leu Ser Pro Gly Ser Ser Cys 65 70 75 Val Lys Tyr Leu Ile Phe Leu Ser Asn Phe Pro Phe Ser Leu Leu 80 85 90 Gly Leu Leu Ala Leu Ala Ile Gly Leu Trp Gly Leu Ala Val Lys 95 100 105 Gly Ser Leu Gly Ser Asp Leu Gly Gly Pro Leu Pro Thr Asp Pro 110 115 120 Met Leu Gly Leu Ala Leu Gly Gly Leu Val Val Ser Ala Ala Ser 125 130 135 Leu Ala Gly Cys Leu Gly Ala Leu Cys Glu Asn Thr Cys Leu Leu 140 145 150 Arg Gly Phe Ser Gly Gly Ile Leu Ala Phe Leu Val Leu Glu Ala 155 160 165 Val Ala Gly Ala Leu Val Val Ala Leu Trp Gly Pro Leu Gln Asp 170 175 180 Ser Leu Glu His Thr Leu Arg Val Ala Ile Ala His Tyr Gln Asp 185 190 195 Asp Pro Asp Leu Arg Phe Leu Leu Asp Gln Val Gln Leu Gly Leu 200 205 210 Arg Cys Cys Gly Ala Ala Ser Tyr Gln Asp Trp Gln Gln Asn Leu 215 220 225 Tyr Phe Asn Cys Ser Ser Pro Gly Val Gln Ala Cys Ser Leu Pro 230 235 240 Ala Ser Cys Cys Ile Asp Pro Arg Glu Asp Gly Ala Ser Val Asn 245 250 255 Asp Gln Cys Gly Phe Gly Val Leu Arg Leu Asp Ala Asp Ala Ala 260 265 270 Gln Arg Val Val Tyr Leu Glu Gly Cys Gly Pro Pro Leu Arg Arg 275 280 285 Trp Leu Arg Ala Asn Leu Ala Ala Ser Gly Gly Tyr Ala Ile Ala 290 295 300 Val Val Leu Leu Gln Gly Ala Glu Leu Leu Leu Ala Ala Arg Leu 305 310 315 Leu Gly Ala Leu Ala Ala Arg Ser Gly Ala Ala Tyr Gly Pro Gly 320 325 330 Ala His Gly Glu Asp Arg Ala Gly Pro Gln Ser Pro Ser Pro Gly 335 340 345 Ala Pro Pro Ala Ala Lys Pro Ala Arg Gly 350 355

Claims

1. A method of inhibiting proliferation of a cancer cell that expresses a polypeptide comprising an amino acid sequence having at least 80% amino acid sequence identity to: (a) the polypeptide shown in SEQ ID NO:26, (b) the polypeptide shown in SEQ ID NO:26 lacking its associated signal peptide, or (c) a polypeptide encoded by a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO: 10, said method comprising contacting said cancer cell with an antibody that specifically binds to the polypeptide shown in SEQ ID NO:26 or the polypeptide shown in SEQ ID NO:26 lacking its associated signal peptide, thereby inhibiting the proliferation of said cancer cell.

2. The method of claim 1, wherein said antibody is a monoclonal antibody.

3. The method of claim 1, wherein said antibody is an antibody fragment.

4. The method of claim 1, wherein said antibody is a chimeric, human, or humanized antibody.

5. The method of claim 1, wherein said antibody is conjugated to a growth inhibitory agent.

6. The method of claim 1, wherein said antibody is conjugated to a cytotoxic agent.

7. The method of claim 6, wherein said cytotoxic agent is selected from the group consisting of a toxin, an antibiotic, a radioactive isotope, and a nucleolytic enzyme.

8. The method of claim 7, wherein the cytotoxic agent is a toxin.

9. The method of claim 8, wherein the toxin is selected from the group consisting of a maytansinoid and a calicheamicin.

10. The method of claim 9, wherein the toxin is a maytansinoid.

11. The method of claim 1, wherein said antibody is produced in bacteria.

12. The method of claim 1, wherein said antibody is produced in CHO cells.

13. The method of claim 1, wherein said cancer cell is further exposed to radiation treatment or a chemotherapeutic agent.

14. The method of claim 1, wherein the cancer cell expresses a polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity to: (a) the polypeptide shown in SEQ ID NO:26, (b) the polypeptide shown in SEQ ID NO:26 lacking its associated signal peptide, or (c) a polypeptide encoded by a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO:10.

15. The method of claim 1, wherein the cancer cell expresses a polypeptide comprising: (a) the polypeptide shown in SEQ ID NO:26, (b) the polypeptide shown in SEQ ID NO:26 lacking its associated signal peptide, or (c) a polypeptide encoded by a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO:10.

16. The method of claim 1, wherein the cancer cell is a breast cancer cell, lung cancer cell, colon cancer cell, rectal cancer cell, kidney cancer cell, ovarian cancer cell, pancreatic cancer cell, uterine cancer cell, bone cancer cell, skin cancer cell, bladder cancer cell, esophagus cancer cell, or soft tissue cancer cell.

17. The method of claim 16, wherein the cancer cell is a colon cancer cell.

18. A method of therapeutically treating a mammal having a tumor comprising cells that express a polypeptide comprising an amino acid sequence having at least 80% amino acid sequence identity to: (a) the polypeptide shown in SEQ ID NO: 26, (b) the polypeptide shown in SEQ ID NO:26 lacking its associated signal peptide, or (c) a polypeptide encoded by a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO:10, said method comprising administering to said mammal a therapeutically effective amount of an antibody that specifically binds to the polypeptide shown in shown in SEQ ID NO:26 or the polypeptide shown in SEQ ID NO:26 lacking its associated signal peptide, thereby effectively treating said mammal.

19. The method of claim 18, wherein said antibody is a monoclonal antibody.

20. The method of claim 18, wherein said antibody is an antibody fragment.

21. The method of claim 18, wherein said antibody is a chimeric, human, or humanized antibody.

22. The method of claim 18, wherein said antibody is conjugated to a growth inhibitory agent.

23. The method of claim 18, wherein said antibody is conjugated to a cytotoxic agent.

24. The method of claim 23, wherein said cytotoxic agent is selected from the group consisting of a toxin, an antibiotic, a radioactive isotope, and a nucleolytic enzyme.

25. The method of claim 24, wherein the cytotoxic agent is a toxin.

26. The method of claim 25, wherein the toxin is selected from the group consisting of a maytansinoid and a calicheamicin.

27. The method of claim 26, wherein the toxin is a maytansinoid.

28. The method of claim 18, wherein said antibody is produced in bacteria.

29. The method of claim 18, wherein said antibody is produced in CHO cells.

30. The method of claim 18, wherein the cells are further exposed to radiation treatment or a chemotherapeutic agent.

31. The method of claim 18, wherein the cells express a polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity to: (a) the polypeptide shown in SEQ ID NO:26, (b) the polypeptide shown in SEQ ID NO:26 lacking its associated signal peptide, or (c) a polypeptide encoded by a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO:10.

32. The method of claim 18, wherein the cells express a polypeptide comprising: (a) the polypeptide shown in SEQ ID NO:26, (b) the polypeptide shown in SEQ ID NO:26 lacking its associated signal peptide, or (c) a polypeptide encoded by a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO:10.

33. The method of claim 18, wherein the cells are breast cancer cells, lung cancer cells, colon cancer cells, rectal cancer cells, kidney cancer cells, ovarian cancer cells, pancreatic cancer cells, uterine cancer cells, bone cancer cells, skin cancer cells, bladder cancer cells, esophagus cancer cells, or soft tissue cancer cells.

34. The method of claim 33, wherein the cells are colon cancer cells.

35. A method of diagnosing the presence of a tumor in a mammal, said method comprising detecting the level of expression of a gene encoding a polypeptide comprising an amino acid sequence having at least 80% amino acid sequence identity to: (a) the polypeptide shown in SEQ ID NO:26, (b) the polypeptide shown in SEQ ID NO:26 lacking its associated signal peptide, or (c) a polypeptide encoded by a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO:10, in a test sample of tissue cells obtained from said mammal, wherein a higher level of expression of the gene encoding said polypeptide in the test sample, as compared to a control sample, is indicative of the presence of a tumor in the mammal from which the test sample was obtained.

36. The method of claim 35, wherein detecting the level of expression of a gene encoding said polypeptide comprises employing an oligonucleotide in an in situ hybridization or RT-PCR analysis.

37. The method of claim 35, wherein detecting the level of expression of a gene encoding said polypeptide comprises employing an antibody in an immunohistochemistry analysis.

38. The method of claim 35, wherein the method comprises detecting the level of expression of a gene encoding a polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity to: (a) the polypeptide shown in SEQ ID NO:26, (b) the polypeptide shown in SEQ ID NO:26 lacking its associated signal peptide, or (c) a polypeptide encoded by a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO:10.

39. The method of claim 35, wherein the method comprises detecting the level of expression of a gene encoding a polypeptide comprising: (a) the polypeptide shown in SEQ ID NO:26, (b) the polypeptide shown in SEQ ID NO:26 lacking its associated signal peptide, or (c) a polypeptide encoded by a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO:10.

40. The method of claim 35, wherein the tumor is a breast tumor, lung tumor, colon tumor, rectal tumor, kidney tumor, ovarian tumor, pancreatic tumor, uterine tumor, bone tumor, skin tumor, bladder tumor, esophagus tumor, or soft tissue tumor.

41. The method of claim 40, wherein the tumor is a colon tumor.

42. A method of diagnosing the presence of a tumor in a mammal, said method comprising contacting a test sample of tissue cells obtained from said mammal with an antibody that specifically binds to the polypeptide shown in shown in SEQ ID NO:26 or the polypeptide shown in SEQ ID NO:26 lacking its associated signal peptide, and detecting the formation of a complex between said antibody and a polypeptide in the test sample, wherein the formation of a complex is indicative of the presence of a tumor in said mammal.

43. The method of claim 42, wherein said antibody is detectably labeled.

44. The method of claim 42, wherein said test sample of tissue cells is obtained from an individual suspected of having a tumor.

45. The method of claim 42, wherein the tumor is a breast tumor, lung tumor, colon tumor, rectal tumor, kidney tumor, ovarian tumor, pancreatic tumor, uterine tumor, bone tumor, skin tumor, bladder tumor, esophagus tumor, or soft tissue tumor.

46. A method of detecting overexpression of a gene encoding a polypeptide comprising an amino acid sequence having at least 80% amino acid sequence identity to: (a) the polypeptide shown in SEQ ID NO:26, (b) the polypeptide shown in SEQ ID NO:26 lacking its associated signal peptide, or (c) a polypeptide encoded by a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO:10; wherein the method comprises detecting the level of expression of the gene encoding said polypeptide in a cancer cell, wherein a higher level of expression of the gene encoding said polypeptide in the cancer cell, as compared to a control sample, indicates that the gene encoding said polypeptide is overexpressed.

47. The method of claim 46, wherein detecting the level of expression of the gene encoding said polypeptide comprises employing an oligonucleotide in an in situ hybridization or RT-PCR analysis.

48. The method of claim 46, wherein detecting the level of expression of the gene encoding said polypeptide comprises employing an antibody in an immunohistochemistry analysis.

49. The method of claim 46, wherein the method comprises detecting overexpression of a gene encoding a polypeptide comprising an amino acid sequence having at least 95% amino acid sequence identity to: (a) the polypeptide shown in SEQ ID NO:26, (b) the polypeptide shown in SEQ ID NO:26 lacking its associated signal peptide, or (c) a polypeptide encoded by a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO:10.

50. The method of claim 46, wherein the method comprises detecting overexpression of a gene encoding a polypeptide comprising: (a) the polypeptide shown in SEQ ID NO:26, (b) the polypeptide shown in SEQ ID NO:26 lacking its associated signal peptide, or (c) a polypeptide encoded by a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO:10.

51. The method of claim 46, wherein the cancer cell is a breast cancer cell, lung cancer cell, colon cancer cell, rectal cancer cell, kidney cancer cell, ovarian cancer cell, pancreatic cancer cell, uterine cancer cell, bone cancer cell, skin cancer cell, bladder cancer cell, esophagus cancer cell, or soft tissue cancer cell.

52. The method of claim 51, wherein the cancer cell is a colon cancer cell.