HK1178181A

HK1178181A - Compositions and methods for the diagnosis and treatment of tumor

Info

Publication number: HK1178181A
Application number: HK13105167.1A
Authority: HK
Inventors: Sunil Bhakta; Meredith C. Hazen; Jo-Anne S. Hongo; Jagath R. Junutula
Original assignee: 霍夫曼-拉罗奇有限公司
Priority date: 2010-05-03
Filing date: 2011-05-02
Publication date: 2013-09-06

Description

Compositions and methods for tumor diagnosis and treatment

RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patent application serial No. 61/330,698 filed on 3/5/2010, which is incorporated herein by reference in its entirety.

Technical Field

The present invention is directed to compositions of matter useful for diagnosing and treating tumors in mammals, and methods of using those compositions of matter for diagnosing and treating tumors in mammals.

Background

Malignancy (carcinoma) is the second cause of death following localized heart disease in the united states (Boring et al, CA cancer j. clin.43:7 (1993)). Cancer is characterized by an increased number of abnormal or neoplastic cells derived from normal tissue, which proliferate to form a tumor mass; the invasion of these neoplastic tumor cells into adjacent tissues; and the generation of malignant cells that eventually spread via the blood or lymphatic system to regional lymph nodes and to distant sites via a process known as metastasis. In the cancerous state, cells proliferate under conditions where normal cells do not grow. Cancer manifests itself in a variety of forms, characterized by varying degrees of invasiveness and aggressiveness.

In an attempt to find effective cellular targets for cancer diagnosis and treatment, researchers have attempted to identify transmembrane polypeptide membrane-associated polypeptides that are specifically expressed on the surface of one or more specific types of cancer cells as compared to one or more normal, noncancerous cells. Typically, such membrane-associated polypeptides are expressed in greater amounts on the surface of cancer cells than on the surface of non-cancerous cells. The identification of such tumor-associated cell surface antigen polypeptides has led to the ability to specifically target the destruction of cancer cells via antibody-based therapies. In this regard, it is noted that antibody-based therapies have proven to be very effective in the treatment of certain cancers. For example, And(all from Genentech inc., South San Francisco, California) are antibodies that have been successfully used to treat breast cancer and non-Hodgkin's (Hodgkin) lymphoma, respectively. More specifically, the present invention relates to a method for producing,is a humanized monoclonal antibody derived from recombinant DNA which selectively binds human epidermal growth factor receptor 2(HER 2)) The extracellular domain of a proto-oncogene. HER2 protein overexpression was observed in 25-30% of primary breast cancers.Are genetically engineered chimeric murine/human monoclonal antibodies directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes. Both antibodies were recombinantly produced in CHO cells.

Despite the above advances in mammalian cancer therapy, there remains a great need for additional diagnostic and therapeutic agents that are capable of detecting the presence of tumors and effectively inhibiting the growth of neoplastic cells, respectively, in mammals. Accordingly, it is an object of the present invention to identify cell membrane-associated polypeptides that are expressed in greater amounts on one or more types of cancer cells than on normal cells or on other different cancer cells, and to use those polypeptides and their encoding nucleic acids to generate compositions of matter that are useful for the therapeutic treatment and diagnostic detection of cancer in mammals.

Summary of The Invention

In this specification, applicants describe for the first time the identification of cellular polypeptides (and nucleic acids or fragments thereof encoding the same) that are expressed at a higher level on the surface of one or more types of cancer cells than they are expressed on the surface of one or more types of normal, non-cancer cells. These polypeptides, referred to herein as Tumor-associated Antigenic target polypeptides ("TAT" polypeptides), are expected to serve as effective targets for the treatment and diagnosis of cancer in mammals.

Thus, in one embodiment of the invention, the invention provides an isolated nucleic acid molecule having a nucleotide sequence encoding a tumor-associated antigenic target polypeptide or fragment thereof ("TAT" polypeptide).

In certain aspects, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, or 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) encoding

Full-length TAT polypeptides having the amino acid sequences disclosed herein,

TAT polypeptide amino acid sequences lacking signal peptides as disclosed herein,

The extracellular domain of a transmembrane TAT polypeptide disclosed herein with or without a signal peptide, or

DNA molecules of any other specifically defined fragment of a full-length TAT polypeptide amino acid sequence disclosed herein; or

(b) A complementary molecule (complement) to the DNA molecule of (a).

In other aspects, the isolated nucleic acid molecule comprises a nucleotide sequence having at least about 80% nucleic acid sequence identity, or 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) comprises

The coding sequence of the full-length TAT polypeptide cDNA disclosed in the invention,

The coding sequence for the TAT polypeptides disclosed herein lacking a signal peptide,

The coding sequence of the extracellular domain of a transmembrane TAT polypeptide with or without a signal peptide disclosed herein, or

A DNA molecule encoding a sequence of any other specifically defined fragment of a full-length TAT polypeptide amino acid sequence disclosed herein; or

(b) A complementary molecule to the DNA molecule of (a).

Another aspect of the disclosure provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding, or complementary to, a transmembrane domain deleted or transmembrane domain inactivated TAT polypeptide, wherein the transmembrane domain of such polypeptides is disclosed herein. Thus, the invention also relates to a soluble extracellular domain of a TAT polypeptide as described herein.

In other aspects, the invention is directed to isolated nucleic acid molecules that hybridize to:

(a) encoding

TAT polypeptides having the full-length amino acid sequences disclosed herein,

The nucleotide sequence of any other specifically defined fragment of the full-length TAT polypeptide amino acid sequence disclosed herein, or

(b) A complementary molecule to the nucleotide sequence of (a).

In this regard, one embodiment of the invention is directed to fragments of a full length TAT polypeptide coding sequence disclosed herein, or the complement thereof, that are useful, for example, as hybridization probes, which are useful, for example, as diagnostic probes, PCR primers, antisense oligonucleotide probes, or for encoding fragments of a full length TAT polypeptide, optionally encoding polypeptides comprising a binding site for an anti-TAT polypeptide antibody. Such nucleic acid fragments are typically 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, 530, 540, 550, 560, 570, 580, 590, 600, 640, 630, 650, 720, 650, 730, 750, 730, 700, 730, 750, 730, 700, 730, 710, 700, 710, 180, 190, 195, 180, 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, wherein the term "about" in this context means that the length of the nucleotide sequence is plus or minus 10% of the length. In addition, such nucleic acid fragments often comprise contiguous nucleotides derived from the full-length coding sequence of the TAT polypeptide, or the complement thereof. It is noted that a novel fragment of a TAT polypeptide encoding nucleotide sequence, or the complement thereof, can be determined in a conventional manner, i.e., by comparing a TAT polypeptide encoding nucleotide sequence to other known nucleotide sequences using any of a variety of well-known sequence comparison procedures, and determining which TAT polypeptide encoding nucleotide sequence(s), or the complement thereof, is novel. All such novel fragments of TAT polypeptide-encoding nucleotide sequences, or the complements thereof, are contemplated herein. Also contemplated are TAT polypeptide fragments encoded by these nucleotide molecule fragments, preferably those TAT polypeptide fragments that comprise a binding site for an anti-TAT polypeptide antibody.

In another embodiment, the invention provides an isolated TAT polypeptide encoded by any of the isolated nucleic acid sequences identified above.

In a certain aspect, the invention relates to an isolated TAT polypeptide comprising an amino acid sequence having at least about 80% amino acid sequence identity, or 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 any one selected from the group consisting of:

TAT polypeptides having the full-length amino acid sequences disclosed herein,

The extracellular domain of a transmembrane TAT polypeptide disclosed herein with or without a signal peptide,

An amino acid sequence encoded by any of the nucleic acid sequences disclosed herein,

Any other specifically defined fragment of a full-length TAT polypeptide amino acid sequence disclosed herein.

In yet another aspect, the invention relates to an isolated TAT polypeptide comprising or encoding a TAT polypeptide

(a) TAT polypeptides having the full-length amino acid sequences disclosed herein,

(b) TAT polypeptide amino acid sequences lacking signal peptides as disclosed herein,

(c) The extracellular domain of a transmembrane TAT polypeptide disclosed herein with or without a signal peptide,

(d) An amino acid sequence encoded by any of the nucleic acid sequences disclosed herein, or

(e) A nucleotide sequence encoded by a nucleotide sequence that hybridizes to the complement of a DNA molecule of any other specifically defined fragment of a full-length TAT polypeptide amino acid sequence disclosed herein.

In a particular aspect, the invention provides an isolated TAT polypeptide, which is free of an N-terminal signal sequence and/or free of an initiating methionine, and which is encoded by a nucleotide sequence encoding an amino acid sequence as described above. Also described herein is a method for producing the same, wherein the method comprises: culturing a host cell comprising a vector comprising a suitable encoding nucleic acid molecule under conditions suitable for expression of the TAT polypeptide, and recovering the TAT polypeptide from the cell culture.

Another aspect of the invention provides an isolated TAT polypeptide which is transmembrane domain deleted or transmembrane domain inactivated. Also described herein is a method for producing the same, wherein the method comprises: culturing a host cell comprising a vector comprising a suitable encoding nucleic acid molecule under conditions suitable for expression of the TAT polypeptide, and recovering the TAT polypeptide from the cell culture.

In other embodiments of the invention, the invention provides a vector comprising DNA encoding any of the polypeptides described herein. Host cells comprising any such vector are also provided. The host cell may be, for example, a CHO cell, an escherichia coli (e. Also provided are methods for producing any of the polypeptides described herein, comprising culturing a host cell under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture.

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

In another embodiment, the invention provides an antibody that binds, preferably specifically binds, any of the aforementioned or later-described polypeptides. Optionally, the antibody is a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody, single chain antibody, or an antibody that competitively inhibits the binding of an anti-TAT polypeptide antibody to its respective antigenic epitope. The antibodies of the invention may optionally be conjugated to growth inhibitors or cytotoxic agents such as toxins including, for example, maytansinoids (maytansinoids) or calicheamicins (calicheamicins), antibiotics, radioisotopes, nucleolytic enzymes (nucleolytic enzymes), or the like. The antibodies of the invention may optionally be produced in CHO cells or bacterial cells and preferably inhibit the growth or proliferation of the cells to which they bind or induce the death of the cells to which they bind. For diagnostic purposes, the antibodies of the invention can be detectably labeled, attached to a solid support, and the like.

In another aspect, the invention provides bispecific antibodies capable of binding to a first cell expressing a TAT polypeptide and a second cell expressing a cell surface target antigen. In one embodiment, the second cell is a T cell. In one embodiment, the cell surface target antigen is CD 3.

In other embodiments of the invention, the invention provides a vector comprising DNA encoding any of the antibodies described herein. Host cells comprising any such vector are also provided. For example, the host cell may be a CHO cell, an e.coli cell or a yeast cell. Also provided are methods for producing any of the antibodies described herein, comprising culturing a host cell under conditions suitable for expression of the desired antibody and recovering the desired antibody from the cell culture.

In yet another embodiment, the present invention relates to a composition of matter comprising, in combination with a carrier: a TAT polypeptide as described herein, a chimeric TAT polypeptide as described herein, or an anti-TAT antibody as described herein. Optionally, the carrier is a pharmaceutically acceptable carrier.

In yet another embodiment, the invention relates to an article of manufacture comprising a container and a composition of matter contained in the container, wherein the composition of matter can comprise a TAT polypeptide as described herein, a chimeric TAT polypeptide as described herein, or an anti-TAT antibody as described herein. The article of manufacture may optionally further comprise a label affixed to said container or a package insert comprised in said container, which relates to the use of said composition of matter in the therapeutic treatment or diagnostic detection of tumors.

Another embodiment of the invention is directed to the use of a TAT polypeptide as described herein, a chimeric TAT polypeptide as described herein, or an anti-TAT polypeptide antibody as described herein, for the preparation of a medicament useful in treating a condition responsive to said TAT polypeptide, chimeric TAT polypeptide, or anti-TAT polypeptide antibody.

Other embodiments of the invention are directed to any isolated antibody comprising one or more of the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 sequences disclosed herein, or any antibody that binds the same epitope as any such antibody.

Another embodiment of the invention is directed to a method for inhibiting the growth of a cell expressing a TAT polypeptide, wherein the method comprises contacting the cell with an antibody that binds to the TAT polypeptide, and wherein binding of the antibody to the TAT polypeptide causes growth inhibition of the cell expressing the TAT polypeptide. In a preferred embodiment, the cell is a cancer cell and binding of the antibody to the TAT polypeptide causes death of the cell expressing the TAT polypeptide. Optionally, the antibody is a monoclonal antibody, an antibody fragment, a chimeric antibody, a humanized antibody, or a single chain antibody. The antibodies employed in the methods of the invention may optionally be conjugated to growth inhibitory agents or cytotoxic agents such as toxins including, for example, maytansinoids or calicheamicins, antibiotics, radioisotopes, nucleolytic enzymes, or the like. The antibodies employed in the methods of the invention may optionally be produced in CHO cells or bacterial cells.

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 that binds to the TAT polypeptide, whereby the tumor is effectively therapeutically treated. Optionally, the antibody is a monoclonal antibody, an antibody fragment, a chimeric antibody, a humanized antibody, or a single chain antibody. The antibodies employed in the methods of the invention may optionally be conjugated to growth inhibitory agents or cytotoxic agents such as toxins including, for example, maytansinoids or calicheamicins, antibiotics, radioisotopes, nucleolytic enzymes, or the like. The antibodies employed in the methods of the invention may optionally be produced in CHO cells or bacterial cells.

Yet another embodiment of the 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 that binds to the TAT polypeptide, and determining binding of the antibody to the TAT polypeptide in the sample, wherein the presence of such binding indicates the presence of the TAT polypeptide in the sample. Optionally, the sample may comprise cells (which may be cancer cells) suspected of expressing TAT polypeptides. The antibodies employed in the methods of the invention may optionally be detectably labeled, attached to a solid support, or the like.

Yet 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 detecting the level of expression of a gene encoding a TAT polypeptide in (a) a test sample of tissue cells obtained from said mammal and (b) a control sample of known normal non-cancerous cells of the same tissue origin or type, wherein a higher level of expression of said TAT polypeptide in the test sample, as compared to the control sample, is indicative of the presence of a tumor in the mammal from which the test sample was obtained.

Yet 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 said mammal with an antibody that binds a TAT polypeptide, and (b) detecting the formation of a complex between said antibody and said TAT polypeptide in said test sample, wherein the formation of a complex indicates the presence of a tumor in said mammal. Optionally, the antibody 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.

Yet another embodiment of the invention is directed to a method of treating or preventing a cell proliferative disorder (disorder) associated with altered, preferably increased, TAT polypeptide expression or TAT polypeptide activity, comprising administering to a subject in need of such treatment an effective amount of an antagonist of TAT polypeptide. Preferably, the cell proliferative disorder is cancer and the antagonist of TAT polypeptide is an anti-TAT polypeptide antibody or an antisense oligonucleotide. Effective treatment or prevention of a TAT polypeptide in a cell proliferative disorder may be the result of killing or inhibiting the growth of cells expressing the TAT polypeptide directly, or by antagonizing the cell growth promoting activity of the TAT polypeptide.

Yet another embodiment of the invention is directed to a method of binding an antibody to a cell expressing a TAT polypeptide, wherein the method comprises contacting a cell expressing a TAT polypeptide with said antibody under conditions suitable for binding of said antibody to said TAT polypeptide and allowing binding therebetween. In a preferred embodiment, the antibody is labeled with a molecule or compound that can qualitatively and/or quantitatively determine the location and/or amount of binding of the antibody to the cell.

Other embodiments of the invention are directed to the use of a TAT polypeptide, a nucleic acid encoding a TAT polypeptide or a vector or host cell comprising the nucleic acid, or an anti-TAT polypeptide antibody in the preparation of a medicament useful in (i) the therapeutic treatment or diagnostic detection of a cancer or tumor, or (ii) the therapeutic treatment or prevention of a cell proliferative disorder (condition).

Another embodiment of the invention is directed to a method of inhibiting the growth of a cancer cell, wherein the growth of the cancer cell is at least partially dependent on the growth promoting effect of a TAT polypeptide (wherein the TAT polypeptide may be expressed by the cancer cell itself, or by a cell that produces a polypeptide having a growth promoting effect on the cancer cell), wherein the method comprises contacting the TAT polypeptide with an antibody that binds to the TAT polypeptide, thereby antagonizing the growth promoting activity of the TAT polypeptide, which in turn inhibits the growth of the cancer cell. Preferably, the growth of cancer cells is completely inhibited. Even more preferably, binding of the antibody to the TAT polypeptide induces death of the cancer cells. Optionally, the antibody is a monoclonal antibody, an antibody fragment, a chimeric antibody, a humanized antibody, or a single chain antibody. The antibodies employed in the methods of the invention may optionally be conjugated to growth inhibitory agents or cytotoxic agents such as toxins including, for example, maytansinoids or calicheamicins, antibiotics, radioisotopes, nucleolytic enzymes, or the like. The antibodies used in the methods of the invention may optionally be produced in CHO cells or bacterial cells.

Yet another embodiment of the present invention is directed to a method of therapeutically treating a tumor in a mammal, wherein the growth of the tumor is at least partially dependent on a growth promoting effect of a TAT polypeptide, wherein the method comprises administering to the mammal a therapeutically effective amount of an antibody that binds to the TAT polypeptide, thereby antagonizing the growth promoting activity of the TAT polypeptide and allowing the tumor to be therapeutically treated effectively. Optionally, the antibody is a monoclonal antibody, an antibody fragment, a chimeric antibody, a humanized antibody, or a single chain antibody. The antibodies employed in the methods of the invention may optionally be conjugated to growth inhibitory agents or cytotoxic agents such as toxins including, for example, maytansinoids or calicheamicins, antibiotics, radioisotopes, nucleolytic enzymes, or the like. The antibodies employed in the methods of the invention may optionally be produced in CHO cells or bacterial cells.

In other embodiments, the invention is directed to the following set of claims that may be filed or applied in the future:

1. an isolated nucleic acid having a nucleotide sequence with at least 80% nucleic acid sequence identity to:

(a) a DNA molecule encoding: 2, and an amino acid sequence shown in SEQ ID NO;

(b) A DNA molecule encoding: 2, lacking its related signal peptide;

(c) a DNA molecule encoding: the extracellular domain of the polypeptide shown in SEQ ID NO. 2 and related signal peptide thereof;

(d) a DNA molecule encoding: the extracellular domain of the polypeptide of SEQ ID NO. 2, lacking its associated signal peptide;

(e) 1, SEQ ID NO;

(f) 1, the full-length coding sequence of the nucleotide sequence shown in SEQ ID NO; or

(g) (ii) the complement of (a), (b), (c), (d), (e) or (f).

2. An isolated nucleic acid having:

(a) a nucleotide sequence, the DNA molecule encoding: 2, and an amino acid sequence shown in SEQ ID NO;

(b) a nucleotide sequence, the DNA molecule encoding: 2, lacking its related signal peptide;

(c) a nucleotide sequence, the DNA molecule encoding: the extracellular domain of the polypeptide shown in SEQ ID NO. 2 and related signal peptide thereof;

(d) a nucleotide sequence, the DNA molecule encoding: the extracellular domain of the polypeptide of SEQ ID NO. 2, lacking its associated signal peptide;

(e) 1, SEQ ID NO;

(f) 1, the full-length coding region of the nucleotide sequence shown in SEQ ID NO; or

(g) (ii) the complement of (a), (b), (c), (d), (e) or (f).

3. An isolated nucleic acid that hybridizes to any one of:

(a) a nucleic acid, the DNA molecule encoding: 2, and an amino acid sequence shown in SEQ ID NO;

(b) a nucleic acid, the DNA molecule encoding: 2, lacking its related signal peptide;

(c) a nucleic acid, the DNA molecule encoding: the extracellular domain of the polypeptide shown in SEQ ID NO. 2 and related signal peptide thereof;

(d) a nucleic acid, the DNA molecule encoding: the extracellular domain of the polypeptide of SEQ ID NO. 2, lacking its associated signal peptide;

(e) 1, SEQ ID NO;

(g) (ii) the complement of (a), (b), (c), (d), (e) or (f).

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

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

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

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

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

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

10. A method for producing a polypeptide comprising culturing the host cell of claim 8 under conditions suitable for expression of the polypeptide and recovering the polypeptide from the cell culture.

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

(a) polypeptide shown as SEQ ID NO. 2;

(b) 2, lacking its related signal peptide;

(c) the extracellular domain of the polypeptide shown in SEQ ID NO. 2 and related signal peptide thereof;

(d) the extracellular domain of the polypeptide of SEQ ID NO. 2, lacking its associated signal peptide;

(e) 1, or a polypeptide encoded by the nucleotide sequence shown in SEQ ID NO; or

(f) 1, and the full-length coding region of the nucleotide sequence shown in SEQ ID NO.

12. An isolated polypeptide having:

(a) 2, and an amino acid sequence shown in SEQ ID NO;

(b) 2, lacking its related signal peptide sequence;

(c) 2, and a related signal peptide sequence thereof;

(d) 2, lacking its associated signal peptide sequence;

(e) 1, and the amino acid sequence coded by the nucleotide sequence shown in SEQ ID NO; or

(f) 1, and the amino acid sequence coded by the full-length coding region of the nucleotide sequence shown in SEQ ID NO.

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

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

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

(a) polypeptide shown as SEQ ID NO. 2;

(b) 2, lacking its related signal peptide;

16. An isolated antibody that binds to a polypeptide having any one of:

(a) 2, and an amino acid sequence shown in SEQ ID NO;

(b) 2, lacking its related signal peptide sequence;

(c) 2, and a related signal peptide sequence thereof;

(d) 2, lacking its associated signal peptide sequence;

17. The antibody of claim 15 or 16, which is a monoclonal antibody.

18. The antibody of claim 15 or 16, which is an antibody fragment.

19. The antibody of claim 15 or 16, which is a chimeric or humanized antibody.

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

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

22. The antibody of claim 21, wherein the cytotoxic agent is selected from the group consisting of a toxin, an antibiotic, a radioisotope, and a nucleolytic enzyme.

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

24. The antibody of claim 23, wherein the toxin is selected from the group consisting of maytansinoids and calicheamicin.

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

26. The antibody of claim 15 or 16, which is produced in bacteria.

27. The antibody of claim 15 or 16, which is produced in CHO cells.

28. The antibody of claim 15 or 16, which induces cell death to which it binds.

29. The antibody of claim 15 or 16, which is detectably labeled.

30. An isolated nucleic acid having a nucleotide sequence encoding the antibody of claim 15 or 16.

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.

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

33. The host cell of claim 32, which is a CHO cell, an e.

34. A method for producing an antibody comprising culturing the host cell of claim 32 under conditions suitable for expression of the antibody and recovering the antibody from the cell culture.

35. A composition of matter comprising, in combination with a carrier:

(a) the polypeptide of claim 11;

(b) the polypeptide of claim 12;

(c) the chimeric polypeptide of claim 13;

(d) the antibody of claim 15; or

(e) The antibody of claim 16.

36. The composition of matter of claim 35, wherein said carrier is a pharmaceutically acceptable carrier.

37. An article of manufacture, comprising:

(a) a container; and

(b) The composition of matter of claim 35 contained in said container.

38. The article of manufacture of claim 37, further comprising a label affixed to the container or a package insert included in the container relating to the use of the composition of matter for therapeutic treatment or diagnostic detection of cancer.

39. A method of inhibiting the growth of a cell that expresses a protein having at least 80% amino acid sequence identity to any one of:

(a) polypeptide shown as SEQ ID NO. 2;

(b) 2, lacking its related signal peptide;

(f) 1, the full-length coding region of the nucleotide sequence shown in SEQ ID NO,

the method comprises contacting the cell with an antibody that binds to the protein, whereby binding of the antibody to the protein causes growth inhibition of the cell.

40. The method of claim 39, wherein the antibody is a monoclonal antibody.

41. The method of claim 39, wherein the antibody is an antibody fragment.

42. The method of claim 39, wherein the antibody is a chimeric or humanized antibody.

43. The method of claim 39, wherein the antibody is conjugated to a growth inhibitory agent.

44. The method of claim 39, wherein the antibody is conjugated to a cytotoxic agent.

45. The method of claim 44, wherein said cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioisotopes, and nucleolytic enzymes.

46. The method of claim 44, wherein said cytotoxic agent is a toxin.

47. The method of claim 46, wherein the toxin is selected from the group consisting of maytansinoids and calicheamicin.

48. The method of claim 46, wherein the toxin is a maytansinoid.

49. The method of claim 39, wherein the antibody is produced in bacteria.

50. The method of claim 39, wherein the antibody is produced in CHO cells.

51. The method of claim 39, wherein the cell is a cancer cell.

52. The method of claim 51, wherein said cancer cells are further exposed to radiation treatment or a chemotherapeutic agent.

53. The method of claim 51, wherein the 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 prostate cancer cell, a melanoma cell, and a leukemia cell.

54. The method of claim 51, wherein said cancer cells express said protein in greater amounts as compared to normal cells of the same tissue origin.

55. The method of claim 39, which causes said cell to die.

56. The method of claim 39, wherein the protein has:

(a) 2, and an amino acid sequence shown in SEQ ID NO;

(b) 2, lacking its related signal peptide sequence;

(c) 2, and a related signal peptide sequence thereof;

(d) 2, lacking its associated signal peptide sequence;

57. 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 any one of:

(a) polypeptide shown as SEQ ID NO. 2;

(b) 2, lacking its related signal peptide;

the method comprises administering to the mammal a therapeutically effective amount of an antibody that binds to the protein, thereby effectively treating the mammal.

58. The method of claim 57, wherein the antibody is a monoclonal antibody.

59. The method of claim 57, wherein the antibody is an antibody fragment.

60. The method of claim 57, wherein the antibody is a chimeric or humanized antibody.

61. The method of claim 57, wherein the antibody is conjugated to a growth inhibitory agent.

62. The method of claim 57, wherein the antibody is conjugated to a cytotoxic agent.

63. The method of claim 62, wherein said cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioisotopes, and nucleolytic enzymes.

64. The method of claim 62, wherein said cytotoxic agent is a toxin.

65. The method of claim 64, wherein the toxin is selected from the group consisting of maytansinoids and calicheamicin.

66. The method of claim 64, wherein the toxin is a maytansinoid.

67. The method of claim 57, wherein the antibody is produced in bacteria.

68. The method of claim 57, wherein the antibody is produced in CHO cells.

69. The method of claim 57, wherein the tumor is further exposed to radiation treatment or a chemotherapeutic agent.

70. The method of claim 57, wherein the tumor is a breast tumor, colorectal tumor, lung tumor, ovarian tumor, central nervous system tumor, liver tumor, bladder tumor, prostate cancer cell, pancreatic tumor, or cervical tumor.

71. The method of claim 57, wherein cancerous cells of said tumor express said protein in greater amounts than normal cells of the same tissue origin.

72. The method of claim 57, wherein the protein has:

(a) 2, and an amino acid sequence shown in SEQ ID NO;

(b) 2, lacking its related signal peptide sequence;

(c) 2, and a related signal peptide sequence thereof;

(d) 2, lacking its associated signal peptide sequence;

73. A method of determining the presence of a protein in a sample suspected of containing the protein, wherein the protein has at least 80% amino acid sequence identity to any one of:

(a) polypeptide shown as SEQ ID NO. 2;

(b) 2, lacking its related signal peptide;

the method comprises exposing the sample to an antibody that binds to the protein, and determining binding of the antibody to the protein in the sample, wherein binding of the antibody to the protein indicates the presence of the protein in the sample.

74. The method of claim 73, wherein said sample comprises a cell suspected of expressing said protein.

75. The method of claim 74, wherein the cell is a cancer cell.

76. The method of claim 73, wherein said antibody is detectably labeled.

77. The method of claim 73, wherein said protein has:

(a) 2, and an amino acid sequence shown in SEQ ID NO;

(b) 2, lacking its related signal peptide sequence;

(c) 2, and a related signal peptide sequence thereof;

(d) 2, lacking its associated signal peptide sequence;

78. A method of diagnosing the presence of a tumor in a mammal, the method comprising determining the level of expression of a gene encoding a protein having at least 80% amino acid sequence identity to any one of:

(a) polypeptide shown as SEQ ID NO. 2;

(b) 2, lacking its related signal peptide;

wherein an expression level of the protein in the test sample that is higher than the control sample indicates the presence of a tumor in the mammal from which the test sample was obtained.

79. The method of claim 78, wherein the step of determining the expression level of a gene encoding the protein comprises using an oligonucleotide in an in situ hybridization or RT-PCR assay.

80. The method of claim 78, wherein the step of determining the expression level of a gene encoding the protein comprises using an antibody in immunohistochemistry or Western blot analysis.

81. The method of claim 78, wherein the protein has:

(a) 2, and an amino acid sequence shown in SEQ ID NO;

(b) 2, lacking its related signal peptide sequence;

(c) 2, and a related signal peptide sequence thereof;

(d) 2, lacking its associated signal peptide sequence;

82. A method of diagnosing the presence of a tumor in a mammal, the method comprising contacting a test sample of tissue cells obtained from the mammal with an antibody that binds to a protein having at least 80% amino acid sequence identity to any one of:

(a) polypeptide shown as SEQ ID NO. 2;

(b) 2, lacking its related signal peptide;

and detecting the formation of a complex between the antibody and the protein in the test sample, wherein the formation of the complex indicates the presence of a tumor in the mammal.

83. The method of claim 82, wherein the antibody is detectably labeled.

84. The method of claim 82, wherein the test sample of tissue cells is obtained from an individual suspected of having a cancerous tumor.

85. The method of claim 82, wherein the protein has:

(a) 2, and an amino acid sequence shown in SEQ ID NO;

(b) 2, lacking its related signal peptide sequence;

(c) 2, and a related signal peptide sequence thereof;

(d) 2, lacking its associated signal peptide sequence;

86. A method of 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 any one of:

(a) polypeptide shown as SEQ ID NO. 2;

(b) 2, lacking its related signal peptide;

the method comprises administering to a subject in need of such treatment an effective amount of an antagonist of the protein, whereby the cell proliferative disorder is effectively treated or prevented.

87. The method of claim 86, wherein said cell proliferative disorder is cancer.

88. The method of claim 86, wherein the antagonist is an anti-TAT polypeptide antibody or an antisense oligonucleotide.

89. A method of binding an antibody to a cell that expresses a protein having at least 80% amino acid sequence identity to any one of:

(a) polypeptide shown as SEQ ID NO. 2;

(b) 2, lacking its related signal peptide;

the method comprises contacting the cell with an antibody that binds to the protein and allowing binding of the antibody to the protein to occur, thereby allowing the antibody to bind to the cell.

90. The method of claim 89, wherein said antibody is a monoclonal antibody.

91. The method of claim 89, wherein said antibody is an antibody fragment.

92. The method of claim 89, wherein said antibody is a chimeric or humanized antibody.

93. The method of claim 89, wherein said antibody is conjugated to a growth inhibitory agent.

94. The method of claim 89, wherein said antibody is conjugated to a cytotoxic agent.

95. The method of claim 94, wherein said cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioisotopes, and nucleolytic enzymes.

96. The method of claim 94, wherein said cytotoxic agent is a toxin.

97. The method of claim 96, wherein the toxin is selected from the group consisting of maytansinoids and calicheamicin.

98. The method of claim 96, wherein the toxin is a maytansinoid.

99. The method of claim 89, wherein said antibody is produced in bacteria.

100. The method of claim 89, wherein said antibody is produced in CHO cells.

101. The method of claim 89, wherein said cell is a cancer cell.

102. The method of claim 101, wherein said cancer cells are further exposed to radiation treatment or a chemotherapeutic agent.

103. The method of claim 101, wherein the 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 prostate cancer cell, a cervical cancer cell, a melanoma cell, and a leukemia cell.

104. The method of claim 103, wherein said cancer cells express said protein in greater amounts than normal cells of the same tissue origin.

105. The method of claim 89, which causes said cell to die.

106. Use of a nucleic acid according to any one of claims 1 to 5 or 30 in the manufacture of a medicament for the therapeutic treatment or diagnostic detection of cancer.

107. Use of a nucleic acid according to any one of claims 1 to 5 or 30 in the manufacture of a medicament for the treatment of a tumour.

108. Use of a nucleic acid according to any one of claims 1-5 or 30 in the manufacture of a medicament for the treatment or prevention of a cell proliferative disorder.

109. Use of an expression vector according to any one of claims 6, 7 or 31 in the manufacture of a medicament for the therapeutic treatment or diagnostic detection of cancer.

110. Use of the expression vector of any one of claims 6, 7 or 31 in the manufacture of a medicament for the treatment of a tumour.

111. Use of an expression vector according to any one of claims 6, 7 or 31 in the manufacture of a medicament for the treatment or prevention of a cell proliferative disorder.

112. Use of a host cell according to any one of claims 8, 9, 32 or 33 in the manufacture of a medicament for the therapeutic treatment or diagnostic detection of cancer.

113. Use of a host cell according to any one of claims 8, 9, 32 or 33 in the manufacture of a medicament for the treatment of a tumour.

114. Use of a host cell according to any one of claims 8, 9, 32 or 33 in the manufacture of a medicament for the treatment or prevention of a cell proliferative disorder.

115. Use of a polypeptide according to any one of claims 11 to 14 in the manufacture of a medicament for the therapeutic treatment or diagnostic detection of cancer.

116. Use of a polypeptide according to any one of claims 11 to 14 in the manufacture of a medicament for the treatment of a tumour.

117. Use of a host cell according to any one of claims 11 to 14 in the manufacture of a medicament for the treatment or prevention of a cell proliferative disorder.

118. Use of an antibody according to any one of claims 15 to 29 in the manufacture of a medicament for the therapeutic treatment or diagnostic detection of cancer.

119. Use of an antibody according to any one of claims 15 to 29 in the manufacture of a medicament for the treatment of a tumour.

120. Use of an antibody of any one of claims 15-29 in the manufacture of a medicament for treating or preventing a cell proliferative disorder.

121. Use of a composition of matter according to any one of claims 35 or 36 in the manufacture of a medicament for the therapeutic treatment or diagnostic detection of cancer.

122. Use of a composition of matter according to any one of claims 35 or 36 in the manufacture of a medicament for the treatment of a tumour.

123. Use of a composition of matter according to any one of claims 35 or 36 in the manufacture of a medicament for the treatment or prevention of a cell proliferative disorder.

124. Use of an article of manufacture according to any one of claims 37 or 38 in the manufacture of a medicament for the therapeutic treatment or diagnostic detection of cancer.

125. Use of an article of manufacture according to any one of claims 37 or 38 in the manufacture of a medicament for the treatment of a tumour.

126. Use of a preparation of any one of claims 37 or 38 in the manufacture of a medicament for the treatment or prevention of a cell proliferative disorder.

127. A method for inhibiting the growth of a cell, wherein the growth of the cell is at least partially dependent on the growth promoting effect of a protein having at least 80% amino acid sequence identity to any one of:

(a) polypeptide shown as SEQ ID NO. 2;

(b) 2, lacking its related signal peptide;

the method comprises contacting the protein with an antibody that binds to the protein, thereby inhibiting the growth of the cell.

128. The method of claim 127, wherein the cell is a cancer cell.

129. The method of claim 127, wherein the protein is expressed by the cell.

130. The method of claim 127, wherein binding of said antibody to said protein antagonizes the cell growth enhancing activity of said protein.

131. The method of claim 127, wherein binding of said antibody to said protein induces death of said cell.

132. The method of claim 127, wherein said antibody is a monoclonal antibody.

133. The method of claim 127, wherein said antibody is an antibody fragment.

134. The method of claim 127, wherein the antibody is a chimeric or humanized antibody.

135. The method of claim 127, wherein the antibody is conjugated to a growth inhibitory agent.

136. The method of claim 127, wherein said antibody is conjugated to a cytotoxic agent.

137. The method of claim 136, wherein said cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioisotopes, and nucleolytic enzymes.

138. The method of claim 136, wherein said cytotoxic agent is a toxin.

139. The method of claim 138, wherein the toxin is selected from the group consisting of maytansinoids and calicheamicin.

140. The method of claim 138, wherein the toxin is a maytansinoid.

141. The method of claim 127, wherein the antibody is produced in bacteria.

142. The method of claim 127, wherein the antibody is produced in CHO cells.

143. The method of claim 127, wherein the protein has:

(a) 2, and an amino acid sequence shown in SEQ ID NO;

(b) 2, lacking its related signal peptide sequence;

(c) 2, and a related signal peptide sequence thereof;

(d) 2, lacking its associated signal peptide sequence;

144. A method of therapeutically treating a tumor in a mammal, wherein the growth of said tumor is at least partially dependent on the growth promoting effect of a protein having at least 80% amino acid sequence identity to any one of:

(a) Polypeptide shown as SEQ ID NO. 2;

(b) 2, lacking its related signal peptide;

the method comprises contacting the protein with an antibody that binds to the protein, thereby effectively treating the tumor.

145. The method of claim 144, wherein said protein is expressed by cells of said tumor.

146. The method of claim 144, wherein binding of said antibody to said protein antagonizes the cell growth promoting activity of said protein.

147. The method of claim 144, wherein said antibody is a monoclonal antibody.

148. The method of claim 144, wherein said antibody is an antibody fragment.

149. The method of claim 144, wherein the antibody is a chimeric or humanized antibody.

150. The method of claim 144, wherein the antibody is conjugated to a growth inhibitory agent.

151. The method of claim 144, wherein said antibody is conjugated to a cytotoxic agent.

152. The method of claim 144, wherein said cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioisotopes, and nucleolytic enzymes.

153. The method of claim 151, wherein said cytotoxic agent is a toxin.

154. The method of claim 153, wherein the toxin is selected from the group consisting of maytansinoids and calicheamicin.

155. The method of claim 153, wherein the toxin is a maytansinoid.

156. The method of claim 144, wherein the antibody is produced in bacteria.

157. The method of claim 144, wherein the antibody is produced in CHO cells.

158. The method of claim 144, wherein the protein has:

(a) 2, and an amino acid sequence shown in SEQ ID NO;

(b) 2, lacking its related signal peptide sequence;

(c) 2, and a related signal peptide sequence thereof;

(d) 2, lacking its associated signal peptide sequence;

159. An isolated antibody that binds to the same epitope as the antibody of any one of claims 15 to 29.

160. The antibody of claim 159, which is a monoclonal antibody.

161. The antibody of claim 159, which is an antibody fragment.

162. The antibody of claim 159, which is a chimeric or humanized antibody.

163. The antibody of claim 159 coupled to a growth inhibitory agent.

164. The antibody of claim 159, conjugated to a cytotoxic agent.

165. The antibody of claim 164, wherein the cytotoxic agent is selected from the group consisting of a toxin, an antibiotic, a radioisotope, and a nucleolytic enzyme.

166. The antibody of claim 164, wherein the cytotoxic agent is a toxin.

167. The antibody of claim 166, wherein the toxin is selected from the group consisting of a maytansinoid and a calicheamicin.

168. The antibody of claim 166, wherein the toxin is a maytansinoid.

169. The antibody of claim 159, which is produced in bacteria.

170. The antibody of claim 159, which is produced in CHO cells.

171. The antibody of claim 159, which induces death of a cell to which it binds.

172. The antibody of claim 159, which is detectably labeled.

173. The antibody of claim 159, comprising at least one complementarity determining region of any antibody of claims 15-29.

174. A hybridoma cell that produces a monoclonal antibody that binds to a TAT polypeptide.

175. A method of identifying an antibody that binds to an epitope bound by any antibody of claims 15 to 29, comprising determining the ability of a test antibody to block the binding of any antibody of claims 15 to 29, wherein the ability of the test antibody to block the binding of any antibody of claims 15 to 29 to a TAT polypeptide by at least 40% at equal antibody concentrations indicates that the test antibody is capable of binding to the epitope bound by said any antibody.

Other embodiments of the invention will be apparent to those skilled in the art upon reading this specification.

Brief Description of Drawings

FIG. 1 shows the nucleotide sequence of the TAT425cDNA (SEQ ID NO:1), where SEQ ID NO:1 is the clone referred to herein as "DNA 340411".

FIG. 2 shows the amino acid sequence (SEQ ID NO:2) deduced from the coding sequence of SEQ ID NO:1 shown in FIG. 1.

Figure 3 shows the complete variable light chain amino acid sequence of the following antibodies: 2E4.6.1 and 4D11.17.2(SEQ ID NO:3), 13H2.28.2(SEQ ID NO:4), and 14E7.17.1(SEQ ID NO: 5).

Figure 4 shows the complete variable heavy chain amino acid sequence of the following antibody: 2E4.6.1(SEQ ID NO:6), 4D11.17.2(SEQ ID NO:7), 13H2.28.2(SEQ ID NO:8), and 14E7.17.1(SEQ ID NO: 9).

FIG. 5 shows various CDR-L1 sequences (SEQ ID NOs: 10-12) of selected anti-TAT 425 antibodies.

FIG. 6 shows various CDR-L2 sequences (SEQ ID NOS: 13-15) of selected anti-TAT 425 antibodies.

FIG. 7 shows various CDR-L3 sequences (SEQ ID NOs: 16-18) of selected anti-TAT 425 antibodies.

FIG. 8 shows various CDR-H1 sequences (SEQ ID NOs: 19-22) of selected anti-TAT 425 antibodies.

FIG. 9 shows various CDR-H2 sequences (SEQ ID NOs: 23-26) of selected anti-TAT 425 antibodies.

FIG. 10 shows various CDR-H3 sequences (SEQ ID NOs: 27-30) of selected anti-TAT 425 antibodies.

Detailed description of the preferred embodiments

I. Definition of

The terms "TAT polypeptide" and "TAT" as used herein and immediately following a numerical designation refer to various polypeptides, wherein the complete designation (i.e., TAT/numerical) refers to a particular polypeptide sequence described herein. The terms "TAT/numerical polypeptide" and "TAT/numerical", wherein the term "numerical" is provided as the actual numerical designation, as used herein, encompasses native sequence polypeptides, polypeptide variants, and fragments of native sequence polypeptides and polypeptide variants (further defined herein). TAT polypeptides described herein may be isolated from a variety of sources, such as human tissue types or other sources, or prepared by recombinant or synthetic methods. The term "TAT polypeptide" refers to each individual TAT/numerical polypeptide disclosed herein. All disclosures in this specification referring to "TAT polypeptides" apply to each polypeptide individually as well as collectively. For example, descriptions regarding the preparation, purification, derivatization, formation of antibodies to the polypeptide, formation of TAT binding oligopeptides to the polypeptide, formation of TAT binding organic molecules to the polypeptide, administration, compositions containing the polypeptide, treatment of diseases with the polypeptide, and the like, apply to each and every specific polypeptide of the present invention. The term "TAT polypeptide" also includes variants of TAT/numerical polypeptides disclosed herein. In one embodiment, the TAT211 polypeptide sequence is as shown in SEQ ID NO 2.

"native sequence TAT polypeptide" includes polypeptides having the same amino acid sequence as a corresponding TAT polypeptide derived from nature. Such native sequence TAT polypeptides may be isolated from nature, or may be produced by recombinant or synthetic means. The term "native sequence TAT polypeptide" specifically encompasses naturally occurring truncated or secreted forms of a particular TAT polypeptide (e.g., an extracellular domain sequence), naturally occurring variant forms of that polypeptide (e.g., alternatively spliced forms), and naturally occurring allelic variants. In certain embodiments of the invention, a native sequence TAT polypeptide disclosed herein is a mature or full-length native sequence polypeptide comprising the full-length amino acid sequence shown in the figures. The start and stop codons (if indicated) are shown in bold and underlined in the figure. The nucleic acid residues indicated as "N" or "X" in the figures are any nucleic acid residues. However, while the TAT polypeptides disclosed in the figures are shown to start with the methionine residue designated herein as amino acid 1 in the figures, it is contemplated and possible that other methionine residues located upstream or downstream of amino acid 1 in the figures may be employed as the starting amino acid residue of the TAT polypeptide.

The "extracellular domain" or "ECD" of a TAT polypeptide refers to a form of the TAT polypeptide that is substantially free of transmembrane and cytoplasmic domains. Typically, the TAT polypeptide ECD has less than 1% of the transmembrane domain and/or cytoplasmic domain, preferably less than 0.5% of the domain. It will be appreciated that any transmembrane domain identified for a TAT polypeptide of the invention is identified according to criteria conventionally used in the art to identify this type of hydrophobic domain. The precise boundaries of the transmembrane domain may vary, but most likely will be no more than about 5 amino acids at either end of the domain originally identified herein. Thus, optionally, the extracellular domain of a TAT polypeptide may contain about 5 or fewer amino acids on either side of the transmembrane domain/extracellular domain boundary identified in the examples or the specification, and such polypeptides with or without an associated signal peptide and nucleic acids encoding them are contemplated by the present invention.

The approximate location of the "signal peptide" of the various TAT polypeptides disclosed herein may be shown in the specification and/or the drawings. It should be noted, however, that the C-terminal boundary of the signal peptide may vary, but most likely is no more than about 5 amino acids either side of the C-terminal boundary of the signal peptide originally identified herein, wherein the C-terminal boundary of the signal peptide may be identified according to criteria conventional in the art for identifying this type of amino acid sequence element (e.g., Nielsen et al, prot. Eng.10:1-6(1997) and von Heinjet al, Nucl. acids. Res.14:4683-4690 (1986)). Furthermore, it is recognized that in some cases, the removal of the signal sequence from the secreted polypeptide is not completely uniform, resulting in more than one secreted species. The present invention contemplates such mature polypeptides in which 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 identified herein, and the polynucleotides encoding them.

By "TAT polypeptide variant" is meant a TAT polypeptide as defined herein, preferably an active TAT polypeptide, having at least about 80% amino acid sequence identity to a full-length native sequence TAT polypeptide sequence disclosed herein, a TAT polypeptide sequence lacking a signal peptide disclosed herein, an extracellular domain of a TAT polypeptide with or without a signal peptide disclosed herein, or any other fragment of a full-length TAT polypeptide sequence disclosed herein, such as those encoded by a nucleic acid that presents only a portion of the complete coding sequence of a full-length TAT polypeptide. Such TAT polypeptide variants include, for example, TAT polypeptides wherein one or more amino acid residues are added or deleted from the N-or C-terminus of the full-length native amino acid sequence. Typically, a TAT polypeptide variant has at least about 80% amino acid sequence identity, or 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 disclosed herein, a TAT polypeptide sequence lacking a signal peptide disclosed herein, an extracellular domain of a TAT polypeptide with or without a signal peptide disclosed herein, or any other particularly defined fragment of a full-length TAT polypeptide sequence disclosed herein. Typically, a TAT variant polypeptide is 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, the TAT variant polypeptide has no more than one conservative amino acid substitution as compared to the native TAT polypeptide sequence, or no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions as compared to the native TAT polypeptide sequence.

"percent (%) amino acid sequence identity" with respect to TAT polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in a particular 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 the purpose of determining percent amino acid sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. However, for purposes of the present invention,% amino acid sequence identity values are obtained using the sequence comparison computer program ALIGN-2, the complete source code for the program ALIGN-2 being provided in U.S. Pat. No.7,160,985 (incorporated herein by reference). The ALIGN-2 sequence comparison computer program was written by Genentech corporation and its source code shown has been submitted to the US Copyright Office (US Copyright Office, Washington d.c.,20559) along with the user document and registered with US Copyright registration number TXU 510087. The ALIGN-2 program is publicly available from Genentech corporation (South SanFrancisco, Calif.) or may be compiled from source code. The ALIGN2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters were set by the ALIGN-2 program and were not changed.

In the case of ALIGN-2 for amino acid sequence comparison, the% amino acid sequence identity of a given amino acid sequence A relative to (to), with (with), or against (against) a given amino acid sequence B (or may be stated as having or comprising a given amino acid sequence A with respect to, with, or against a given amino acid sequence B) is calculated as follows:

fractional X/Y times 100

Wherein X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the A and B alignments of this program, and wherein Y is the total number of amino acid residues in B. It will be appreciated that if the length of amino acid sequence a is not equal to the length of amino acid sequence B, then the% amino acid sequence identity of a relative to B will not be equal to the% amino acid sequence identity of B relative to a.

By "TAT variant polynucleotide" or "TAT variant nucleic acid sequence" is meant a nucleic acid molecule encoding a TAT polypeptide as defined herein, preferably an active TAT polypeptide, and having at least about 80% nucleic acid sequence identity to a nucleotide sequence encoding a full-length native sequence TAT polypeptide sequence disclosed herein, a full-length native sequence TAT polypeptide sequence disclosed herein lacking a signal peptide, a TAT polypeptide extracellular domain disclosed herein with or without a signal peptide, or any other fragment of a full-length TAT polypeptide sequence disclosed herein (such as those fragments encoded by a nucleic acid that presents only a portion of the complete coding sequence of a full-length TAT polypeptide). Typically, a TAT variant polynucleotide has at least about 80% nucleic acid sequence identity, or 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, to a nucleic acid sequence encoding a full-length native sequence TAT polypeptide sequence disclosed herein, a full-length native sequence TAT polypeptide sequence disclosed herein lacking a signal peptide, a TAT polypeptide extracellular domain disclosed herein with or without a signal sequence, or any other fragment of a full-length TAT polypeptide sequence disclosed herein. Variants do not encompass the native nucleotide sequence.

Typically, a TAT variant polynucleotide is 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, 540, 510, 520, 530, 580, 550, 560, 570, 580, 590, 640, 610, 650, 730, 700, 730, 650, 730, 700, 650, 710, 220, 500, 240, 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, wherein the term "about" in this context means that the length of the nucleotide sequence is plus or minus 10% of the length.

"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 to 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 the purpose of determining percent nucleic acid sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software. However, for purposes of the present invention,% nucleic acid sequence identity values are obtained using the sequence comparison computer program ALIGN-2, the complete source code for the ALIGN-2 program being provided in U.S. Pat. No.7,160,985 (incorporated herein by reference). The ALIGN-2 sequence comparison computer program was written by Genentech corporation and its source code has been submitted to the US Copyright Office (US Copyright Office, Washington d.c.,20559) along with the user document and registered with US Copyright registration number TXU 510087. The ALIGN-2 program is publicly available from Genentech corporation (South San Francisco, Calif.) or may be compiled from source code. The ALIGN2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters were set by the ALIGN-2 program and were not changed.

In the case of employing ALIGN-2 to compare nucleic acid sequences, the% nucleic acid sequence identity of a given nucleic acid sequence C with respect to (to), with (with), or against (against) a given amino acid sequence D (or may be stated as having or comprising a certain% nucleic acid sequence identity with respect to, with, or against a given nucleic acid sequence D) is calculated as follows:

fractional W/Z times 100

Where W is the number of nucleotides scored as identical matches by the sequence alignment program ALIGN-2 in the C and D alignments of this program, and where Z is the total number of nucleotides in D. It will be appreciated that if the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, then the% nucleic acid sequence identity of C relative to D will not be equal to the% nucleic acid sequence identity of D relative to C.

In other embodiments, a TAT variant polynucleotide is a nucleic acid molecule that encodes a TAT polypeptide and is capable of hybridizing, preferably under stringent hybridization and wash conditions, to a nucleotide sequence encoding a full-length TAT polypeptide disclosed herein. TAT variant polypeptides may be those variant polypeptides encoded by TAT variant polynucleotides.

The term "full-length coding region" when used in reference to a nucleic acid encoding a TAT polypeptide refers to a nucleotide sequence (often shown in the figures between start and stop codons (inclusive)) that encodes a full-length TAT polypeptide of the invention. The term "full-length coding region" as used in reference to ATCC deposited nucleic acids refers to the TAT polypeptide-encoding portion of the cDNA (often shown in the figures between the start and stop codons, inclusive) inserted into the vector deposited with the ATCC.

By "isolated," when used in describing the various TAT polypeptides disclosed herein, is meant a polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment refer to substances that would normally interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the polypeptide is purified (1) to an extent sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues by using a rotor sequencer, or (2) to homogeneity according to SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver staining. An isolated polypeptide includes a polypeptide in situ within a recombinant cell, since at least one component of the TAT polypeptide's natural environment will not be present. However, isolated polypeptides are typically prepared by at least one purification step.

An "isolated" TAT polypeptide-encoding nucleic acid or other polypeptide-encoding nucleic acid refers to a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule with which it is ordinarily associated in its natural source. An isolated polypeptide-encoding nucleic acid molecule differs from the form or condition in which it is found in nature. An isolated polypeptide-encoding nucleic acid molecule is thus distinguished from a particular polypeptide-encoding nucleic acid molecule when present in a native cell. However, an isolated polypeptide-encoding nucleic acid molecule includes a polypeptide-encoding nucleic acid molecule that is contained in a cell that normally expresses the polypeptide, for example, when the nucleic acid molecule has a chromosomal location in the cell that is different from its chromosomal location in the native cell.

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

A nucleic acid is "operably linked" if it is in a functional relationship with another nucleic acid sequence. For example, a DNA of a presequence (sequence) or secretory leader (secretory leader) is operably linked to a DNA of a polypeptide if it is expressed as a preprotein (preprotein) involved in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of that sequence; alternatively, if the ribosome binding site is positioned to facilitate translation, it is operably linked to a coding sequence. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading order. However, enhancers need not be contiguous. Ligation may be achieved by ligation reactions at convenient restriction sites. If there are no such sites, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

The "stringency" of hybridization reactions can be readily determined by one of ordinary skill in the art, and is generally calculated empirically based on probe length, washing temperature, and salt concentration. Generally, longer probes require higher temperatures for proper annealing, while shorter probes require 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 that can be used. As a result, it was concluded that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures are less stringent. For additional details and explanation of the stringency of hybridization reactions, see Ausubel et al, Current protocols in Molecular Biology, Wiley Interscience Publishers, 1995.

"stringent conditions" or "high stringency conditions", as defined herein, can be identified as follows: (1) washing with low ionic strength and high temperature, e.g., 0.015M sodium chloride/0.0015M sodium citrate/0.1% sodium lauryl sulfate, 50 ℃; (2) denaturing agents such as formamide, e.g., 50% (v/v) formamide and 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer pH6.5 with 750mM sodium chloride, 75mM sodium citrate, 42 ℃; or (3) hybridization overnight at 42 ℃ in a solution with 50% formamide, 5 XSSC (0.75M NaCl,0.075M sodium citrate), 50mM sodium phosphate (pH6.8),0.1% sodium pyrophosphate, 5 XDenhardt's solution, sonicated salmon sperm DNA (50. mu.g/ml), 0.1% SDS, and 10% dextran sulfate, and washing in 0.2 XSSC (sodium chloride/sodium citrate) at 42 ℃ for 10 minutes followed by a 10 minute high stringency wash in 0.1 XSSC with EDTA at 55 ℃.

"moderately stringent conditions" can be identified as described by Sambrook et al, Molecular Cloning: analytical Manual, New York, Cold Spring Harbor Press, 1989, including the use of less stringent wash solutions and hybridization conditions (e.g., temperature, ionic strength, and% SDS) than those described above. An example of moderately stringent conditions is conditions comprising: 20% formamide, 5 XSSC (150mM NaCl,15mM trisodium citrate), 50mM sodium phosphate (pH7.6),5 XDenhardt's solution, 10% dextran sulfate, and 20mg/ml denatured sheared salmon sperm DNA solution in temperature overnight, followed by 1 XSSC at about 37-50 ℃ washing filter membrane. The skilled person will recognize how to adjust the temperature, ionic strength, etc. as required to accommodate factors such as probe length.

The term "epitope tagged" as used herein refers to a chimeric polypeptide comprising a TAT polypeptide or an 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, but is short enough so that it does not interfere with the activity of the polypeptide to which it is fused. The tag polypeptide is also preferably quite unique such that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides typically have at least 6 amino acid residues and typically between about 8 and about 50 amino acid residues (preferably between about 10 and about 20 amino acid residues).

"active" or "activity" for purposes of the present invention refers to a form of a TAT polypeptide that retains the biological and/or immunological activity of native or naturally occurring TAT, wherein "biological" activity refers to a biological function (either inhibitory or stimulatory) caused by native or naturally occurring TAT other than the ability to induce antibody production against an antigenic epitope possessed by native or naturally occurring TAT, and "immunological" activity refers to the ability to induce antibody production against an antigenic epitope possessed by native or naturally occurring TAT.

The term "antagonist" is used in the broadest sense and includes any molecule that partially or completely blocks, inhibits, or neutralizes a biological activity of a native TAT polypeptide disclosed herein. Similarly, the term "agonist" is used in the broadest sense and includes any molecule that mimics the biological activity of a native TAT polypeptide disclosed herein. Suitable agonist or antagonist molecules specifically include agonistic or antagonistic antibodies or antibody fragments, fragments or amino acid sequence variants of a native TAT polypeptide, peptides, antisense oligonucleotides, small organic molecules, and the like. Methods for identifying agonist or antagonist molecules 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.

"treatment" or "treating" or "palliating" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the pathological condition or disorder being addressed. Subjects in need of treatment include subjects who have already suffered from the disorder as well as subjects who are predisposed to the disorder or who are to be prevented from the disorder. 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 an observable and/or measurable reduction or disappearance in one or more of: a decrease in the number of cancer cells or disappearance of cancer cells; reduction of tumor volume; infiltration of cancer cells into peripheral organs, including the soft tissue and bone, is inhibited (i.e., slowed to some extent, preferably stopped); tumor metastasis is inhibited (i.e., slowed to some extent, preferably halted); tumor growth is inhibited to some extent; and/or a certain reduction in one or more symptoms associated with a particular cancer; decreased morbidity and mortality; and the life quality is improved. To the extent that an anti-TAT antibody or TAT binding oligopeptide can prevent cancer cell growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. The reduction of these signs or symptoms can also be felt by the patient.

The above parameters for assessing successful treatment and improvement of a disease can be readily measured by routine procedures familiar to physicians. For cancer treatment, efficacy can be measured, for example, by assessing time to disease progression (TTP) and/or determining Response Rate (RR). Metastasis can be determined by staging test, and by bone scanning and testing for calcium levels and other enzymes to determine if transmission to bone occurs. CT scans can also be performed to see if spread to the pelvis and lymph nodes in this area. Chest X-rays and liver enzyme level measurements by known methods are used to ascertain whether metastasis to the lung and liver, respectively. Other conventional methods for monitoring disease include transrectal ultrasonography (TRUS) and transrectal needle biopsy (TRNB).

"Long-term" administration refers to administration of an agent in a continuous mode as opposed to a short-term mode, thereby maintaining the initial therapeutic effect (activity) for an extended period of time. By "intermittent" application is meant a process that is not continuous, uninterrupted, but rather is cyclic in nature.

For the treatment, alleviation of symptoms, or diagnosis of cancer, "mammal" refers to any animal classified as a mammal, including humans, domestic animals, and livestock, and zoo, sports, or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, and the like. Preferably, the mammal is a human.

Administration "in combination with" one or more other therapeutic agents includes simultaneous (co-) administration and sequential administration in any order.

"carrier" as used herein includes pharmaceutically acceptable carriers, excipients, or stabilizers which are non-toxic to the cells or mammal to which they are exposed at the dosages and concentrations employed. Typically, the physiologically acceptable carrier is an aqueous pH buffered solution. Physiologically acceptable carriers from the group consisting of buffers, such as phosphate, citrate, and other organic acid salts; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin,Gelatin or immunoglobulin; 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 asPolyethylene glycol (PEG) and

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 invention may adhere or attach. Examples of solid phases contemplated herein include those made partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamide, polystyrene, polyvinyl alcohol, and polysiloxanes (silicones). In certain embodiments, depending on the context, the solid phase may comprise the wells of an assay plate; in other embodiments, it refers to a purification column (e.g., an affinity chromatography column). This term also includes discontinuous solid phases of discrete particles, such as described in U.S. Pat. No. 4,275,149.

"liposomes" refers to vesicles composed of various types of lipids, phospholipids and/or surfactants that can be used to deliver drugs (such as TAT polypeptides, antibodies thereto, or TAT binding oligopeptides) to mammals. The components of liposomes are generally arranged in bilayer form, similar to the lipid arrangement of biological membranes.

A "small" molecule or organic "small" molecule is defined herein as having a molecular weight of less than about 500 daltons.

An "effective amount" of a polypeptide, antibody, TAT binding oligopeptide, TAT binding organic molecule, or agonist or antagonist thereof disclosed herein is an amount sufficient to achieve the stated purpose. An "effective amount" may be determined empirically and in a conventional manner, in connection with the stated purpose.

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, a therapeutically effective amount of the drug may reduce the number of cancer cells; reducing the tumor volume; inhibit (i.e., slow to some extent, preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent, preferably stop) tumor metastasis; inhibit tumor growth to some extent; and/or to reduce to some extent one or more symptoms associated with cancer. See the definition of "treatment" herein. To the extent that the drug can prevent the growth of and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic.

"growth inhibitory amount" of an anti-TAT antibody, TAT polypeptide, TAT binding oligopeptide or TAT binding organic molecule refers to an amount capable of inhibiting the growth of a cell, particularly a tumor, e.g., a cancer cell, in vitro or in vivo. The "growth inhibitory amount" of an anti-TAT antibody, TAT polypeptide, TAT binding oligopeptide or TAT binding organic molecule to inhibit growth of neoplastic cells can be determined empirically and in a routine manner.

"cytotoxic amount" of an anti-TAT antibody, TAT polypeptide, TAT binding oligopeptide or TAT binding organic molecule refers to an amount capable of causing the destruction of cells, especially tumors, e.g. cancer cells, in vitro or in vivo. The "cytotoxic amount" of an anti-TAT antibody, TAT polypeptide, TAT binding oligopeptide or TAT binding organic molecule to inhibit neoplastic cell growth can be determined empirically and in a routine manner.

The term "antibody" is used in the broadest sense and specifically covers, for example, single anti-TAT monoclonal antibodies (including agonistic, antagonistic 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) so long as they exhibit the desired biological or immunological activity. The term "immunoglobulin" (Ig) is used interchangeably herein with antibody.

An "isolated" antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment refer to substances that interfere with diagnostic or therapeutic uses of the antibody and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody is purified (1) to more than 95% by weight, most preferably more than 99% by weight of the antibody as determined by the Lowry method, (2) to an extent sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues by using a rotor sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver staining. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. However, isolated antibodies are typically prepared by at least one purification step.

The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H) (IgM antibodies are composed of 5 basic heterotetrameric units and an additional polypeptide called the J chain, and thus contain 10 antigen-binding sites; secretory IgA antibodies can be polymerized to form multivalent assemblies containing 2-5 basic 4-chain units and the J chain). In the case of IgG, the 4-chain unit is typically about 150,000 daltons. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the two heavy chains are linked to each other by one or more disulfide bonds, the number of disulfide bonds depending on the isotype of the heavy chain. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (V) at the N-terminus _H) Followed by three (for alpha and gamma chains) or four (for mu and epsilon isoforms) constant regions (C)_H). Each light chain has a variable region (V) at the N-terminus_L) Followed by a constant region (C) at its other end_L)。V_LAnd V_HAre arranged together, and C_LTo the first constant region (C) of the heavy chain_H1) Are arranged together. It is believed that specific amino acid residues are present in the light chainAnd the heavy chain variable region. One V in pair_HAnd a V_LTogether forming an antigen binding site. For the structure and properties of antibodies of different classes see, e.g., Basic and Clinical Immunology, 8 th edition, Daniel P.Stits, Abba I.Terr and Tristram G.Parslow eds, Appleton&Lange, Norwalk, CT, 1994, page 71 and chapter 6.

Light chains from any vertebrate species can be classified into one of two distinct types, called kappa (κ) and lambda (λ), depending on their constant region amino acid sequences. According to its heavy chain (C)_H) Constant region amino acid sequences, immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, have heavy chains called α, δ, ε, γ and μ, respectively. According to C_HWith minor differences in sequence and function, the γ and α classes can be further divided into subclasses, for example humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA 2.

The term "variable" refers to the fact that certain segments in the variable region differ widely in antibody sequence. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed over the 110 amino acids spanned by the variable region. In fact, the V region consists of a relatively invariant segment of 15-30 amino acids, called the framework region (KR), and extremely variant, shorter regions of 9-12 amino acids each, called "hypervariable regions", separating the framework regions. The variable regions of native heavy and light chains each comprise four FRs, which largely adopt a β -sheet conformation, connected by three hypervariable regions that form loops and, in some cases, form part of the β -sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and together with the hypervariable regions of the other chain contribute to the formation of the antigen-binding site of the antibody (see Kabat et al, Sequences of Proteins of immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, Md, 1991). The constant regions are not directly involved in binding of the antibody to the antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cell-mediated cytotoxicity (ADCC).

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. In addition, unlike polyclonal antibody preparations which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, 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, monoclonal antibodies useful in the present invention can be prepared by the hybridoma method described initially by Kohler et al, Nature,256:495(1975), or can be prepared by recombinant DNA methods in bacterial, eukaryotic animal, or plant cells (see, e.g., U.S. Pat. No. 4,816,567). "monoclonal antibodies" can also be isolated from phage antibody libraries using techniques such as those described in Clackson et al, Nature,352: 624-.

Monoclonal antibodies include herein "chimeric" antibodies, as well as fragments of such antibodies, wherein a portion of the heavy and/or light chain is identical to 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 is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, 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-. Chimeric antibodies of interest herein include "primatized" antibodies comprising variable region antigen binding sequences derived from a non-human primate (e.g., Old World Monkey, ape, etc.) and human constant region sequences.

"intact antibody" means a polypeptide comprising an antigen binding site and C_LAnd at least heavy chain constant region C_H1、C_H2 and C_H3. The constant region may be a native sequence constant region (e.g., a human native sequence constant region) or an amino acid sequence variant thereof. Preferably, the whole antibody has one or more effector functions.

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

Digestion of an antibody with papain produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, the name of which reflects its ability to crystallize readily. The Fab fragment consists of one entire light chain and one variable region of the heavy chain (V)_H) And a first constant region (C)_H1) And (4) forming. Each Fab fragment is monovalent for antigen binding, i.e. it has one antigen binding site. Pepsin treatment of the antibody produced a larger F (ab')₂A fragment, roughly equivalent to two Fab fragments linked by a disulfide bond, having bivalent antigen binding activity and still capable of cross-linking antigen. Fab' fragment is due to C_H1 domain has a few residues added to the carboxy terminus that differ from Fab fragments, including one or more cysteines from the antibody hinge region. Fab '-SH is the designation herein for Fab' in which the cysteine residues of the constant region carry a free thiol group. F (ab')₂Antibody fragments were originally produced as pairs of Fab 'fragments with hinge cysteines between the Fab' fragments. Other chemical couplings of antibody fragments are also known.

The Fc fragment comprises the carboxy-terminal portions of two heavy chains held together by disulfide bonds. The effector function of an antibody is determined by sequences in the Fc region, which is also the portion recognized by Fc receptors (fcrs) found on certain types of cells.

"Fv" is the smallest antibody fragment that contains the entire antigen recognition and binding site. This fragment consists of a dimer of one heavy chain variable region and one light chain variable region in tight, non-covalent association. Six hypervariable loops (3 loops each for the heavy and light chains) emanate from the folding of these two domains, contributing amino acid residues for antigen binding and conferring antigen binding specificity to the antibody. However, even a single variable region (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.

"Single-chain Fv", also abbreviated to "sFv" or "scFv", is an antibody V comprising a single polypeptide chain connected_HAnd V_LAntibody fragments of the domains. Preferably, the sFv polypeptide is at V_HAnd V_LPolypeptide linkers are also included between the domains, allowing the sFv to form the desired structure for antigen binding. For a review of sFv see Pl ü ckthun, The Pharmacology of Monoclonal Antibodies, vol.113, eds Rosenburg and Moore, Springer-Verlag, New York, pp.269-315, 1994, Borrebaeck1995, see below.

The term "diabodies" refers to diabodies produced by the reaction of a heavy chain variable domain at V_HAnd V_LSmall antibody fragments prepared by constructing sFv fragments (see above) using short linkers (about 5-10 residues) between domains, which allow inter-chain rather than intra-chain pairing of the V domains due to the short linkers, resulting in bivalent fragments, i.e., fragments with two antigen binding sites. Bispecific diabodies are heterodimers of two "cross" sFv fragments, where the V of both antibodies_HAnd V_LThe domains are present on different polypeptide chains. Diabodies are described more fully in, for example, EP404,097, WO93/11161, Hollinger et al, Proc. Natl. Acad. Sci. USA,90: 6444-.

"humanized" forms of non-human (e.g., rodent) antibodies refer to chimeric antibodies that contain minimal sequences derived from non-human antibodies. For the most part, humanized antibodies are those in which residues from a hypervariable region of a human immunoglobulin (recipient antibody) are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired antibody specificity, affinity, and capacity. In some instances, Framework Region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may comprise residues not found in the recipient antibody or in the donor antibody. These modifications are made to further improve the performance of the antibody. Typically, the humanized antibody will comprise substantially all of at least one, and typically two, variable regions 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 further comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details see Jones et al, Nature321:522-525(1986); Riechmann et al, Nature332:323-329(1988); Presta, curr Op, struct, biol.2:593-596 (1992).

A "species-dependent antibody," such as a mammalian anti-human IgE antibody, refers to an antibody that has a stronger binding affinity for an antigen from a first mammalian species than for a homolog of the antigen from a second mammalian species. Typically, a species-dependent antibody "specifically binds" to a human antigen (i.e., has no more than about 1x 10)^-7M, preferably not more than about 1x10^-8M, most preferably not more than about 1x10^-9M, but has a binding affinity (Kd)) for the antigen from the second non-human mammalian species that is at least about 50-fold, or at least about 500-fold, or at least about 1000-fold weaker than its binding affinity for a human antigen. The species-dependent antibody may be of the various types as defined above, but is preferably a humanized or human antibody.

The term "variable domain residue numbering according to Kabat" or "amino acid position numbering according to Kabat" and variants thereof refers to the numbering system for heavy or light chain variable domains by antibody editing as in Kabat et al, Sequences of Proteins of Immunological Interest,5th ed. Using this numbering system, the actual linear amino acid sequence may comprise fewer or additional amino acids, corresponding to a shortening or insertion of the variable domain FR or CDR. For example, the heavy chain variable domain may comprise a single amino acid insertion (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c according to Kabat, etc.) after heavy chain FR residue 82. The Kabat residue numbering of a given antibody can be determined by aligning the antibody sequences to the regions of homology with "standard" Kabat numbered sequences.

The phrase "substantially similar" or "substantially the same" as used herein means a sufficiently high degree of similarity between two numerical values (typically one relating to an antibody of the invention and the other relating to a reference/comparison antibody) such that one of skill in the art would consider the difference between the two numerical values to be of little or no biological and/or statistical significance within the context of the biological property measured by the numerical values (e.g., Kd values). The difference between the two values is preferably less than about 50%, preferably less than about 40%, preferably less than about 30%, preferably less than about 20%, preferably less than about 10%, as a function of the value of the reference/comparison antibody.

"binding affinity" generally refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies generally bind antigen slowly and tend to dissociate readily, while high affinity antibodies generally bind antigen more rapidly and tend to remain bound for a longer period of time. A variety of methods for measuring binding affinity are known in the art, any of which may be used for the purposes of the present invention. Specific exemplary embodiments are described below.

In one embodiment, the "Kd" or "Kd value" according to the present invention is measured by a radiolabelled antigen binding assay (RIA) carried out using Fab-format antibodies of interest and their antigens as described in the following assays: by using the minimum concentration of the antigen in the presence of a titration series of unlabelled antigen¹²⁵I labelling of the antigen equilibrated Fab, and then capturing the bound antigen with anti-Fab antibody coated plates to measure the binding affinity of the Fab to the antigen in solution (Chen, et al, J Mol Biol293: 865-. To determine assay conditions, microtiter plates (Dynex) were coated with anti-Fab antibodies (Cappel Labs) with 5. mu.g/ml capture in 50mM sodium carbonate (pH9.6) overnight, followed by blocking with 2% (w/v) bovine serum albumin in PBS for 2-5 hours at room temperature (about 23 ℃). In a non-adsorption plate (Nunc #269620), 100pM or 26pM [ alpha ], [ beta ]¹²⁵I]Mixing of antigen with serial dilutions of Fab of interest (e.g.in agreement with the evaluation of anti-VEGF antibodies, Fab-12, in Prestaet al, Cancer Res.57: 4593-. Then keeping the temperature of the target Fab overnight; however, the incubation may continue for a longer period of time (e.g., 65 hours) to ensure equilibrium is reached. Thereafter, the mixture is transferred to a capture plate for room temperature incubation (e.g., 1 hour). The solution was then removed and the plate washed 8 times with PBS containing 0.1% Tween-20. After the plates were dried, 150. mu.l/well scintillation fluid (MicroScint-20; Packard) was added and the plates were counted for 10 minutes on a Topcount Gamma counter (Packard). The concentration of each Fab that gave less than or equal to 20% of the maximum binding was selected for use in the competitive binding assay. According to another embodiment, the Kd or Kd value is determined by surface plasmon resonance assay using BIAcore ^TM-2000 or BIAcore^TM-3000(BIAcore, inc., Piscataway, NJ) measured at 25 ℃ using an immobilized antigen CM5 chip at about 10 Response Units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen was diluted to 5. mu.g/ml (about 0.2. mu.M) with 10mM sodium acetate pH4.8 and then injected at a flow rate of 5. mu.l/min to obtain about 10 Response Units (RU)The protein is coupled. After injection of the antigen, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) in PBS (PBST) containing 0.05% Tween-20 were injected at 25 ℃ at a flow rate of about 25. mu.l/min. The binding rate (k) was calculated by simultaneous fitting of the binding and dissociation sensorgrams using a simple one-to-one Langmuir (Langmuir) binding model (BIAcore Evaluation Software version3.2)_on) And dissociation rate (k)_off). Equilibrium dissociation constant (Kd) in the ratio k_off/k_onAnd (4) calculating. See, e.g., Chen, Y., et al, J Mol Biol293:865-881 (1999). If the binding rate is more than 10 according to the above surface plasmon resonance assay ⁶M^-1S^-1The binding rate can then be determined using fluorescence quenching techniques, i.e.measuring the increase or decrease in fluorescence emission intensity (excitation =295 nM; emission =340nM, 16nM band pass (band pass)) at 25 ℃ of 20nM anti-antigen antibody (Fab form) in PBS, pH7.2 in the presence of increasing concentrations of antigen, according to a measurement with a stirred cuvette in a spectrometer such as a spectrophotometer equipped with a flow-cut device (Aviv Instruments) or a 8000 series SLM-Aminco spectrophotometer (Thermospectronic).

An "association rate" or "k" according to the present invention_on"BIAcore can also be used by the same surface plasmon resonance technique as described above^TM-2000 or BIAcore^TM-3000(BIAcore, inc., Piscataway, NJ) was determined at 25 ℃ using an immobilized antigen CM5 chip at about 10 Response Units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen was diluted to 5. mu.g/ml (about 0.2. mu.M) with 10mM sodium acetate pH4.8 and then injected at a flow rate of 5. mu.l/min to obtain about 10 Response Units (RU) of conjugated protein. After injection of the antigen, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, at 25 ℃ at about 25. mu.l/min The flow rate of the clock was injected with two-fold serial dilutions of Fab (0.78 nM to 500 nM) in PBS (PBST) containing 0.05% Tween-20. The binding rate (k) was calculated by simultaneous fitting of the binding and dissociation sensorgrams using a simple one-to-one Langmuir (Langmuir) binding model (BIAcoreevaluation Software version3.2)_on) And dissociation rate (k)_off). Equilibrium dissociation constant (Kd) in the ratio k_off/k_onAnd (4) calculating. See, e.g., Chen, Y., et al, J Mol Biol293:865-881 (1999). However, if according to the above surface plasmon resonance determination method, the binding rate exceeds 10⁶M^-1S^-1The binding rate is then preferably determined using fluorescence quenching techniques, i.e.measuring the increase or decrease in fluorescence emission intensity (excitation =295 nM; emission =340nM, 16nM bandpass) at 25 ℃ of 20nM anti-antigen antibody (Fab form) in PBS, pH7.2, in the presence of increasing concentrations of antigen, according to a measurement in a spectrometer such as a spectrophotometer equipped with a flow cut-off device (Avivinstruments) or a 8000 series SLM-Aminco spectrophotometer (Thermospectric) with a stirred cuvette. In one embodiment, the "Kd" or "Kd value" according to the present invention is measured by a radiolabelled antigen binding assay (RIA) using Fab-type antibodies and antigenic molecules as described in the following assays: by using the minimum concentration of the antigen in the presence of a titration series of unlabelled antigen ¹²⁵I labelling of the antigen equilibrated Fab, and then capturing the bound antigen with anti-Fab antibody coated plates to measure the binding affinity of the Fab to the antigen in solution (Chen, et al, J Mol Biol293: 865-. To determine assay conditions, microtiter plates (Dynex) were coated with anti-Fab antibodies (Cappel Labs) with 5. mu.g/ml capture in 50mM sodium carbonate (pH9.6) overnight, followed by blocking with 2% (w/v) bovine serum albumin in PBS for 2-5 hours at room temperature (about 23 ℃). In a non-adsorption plate (Nunc #269620), 100pM or 26pM [ alpha ], [ beta ]¹²⁵I]Mixing of antigen with serial dilutions of Fab of interest (in agreement with the evaluation of anti-VEGF antibodies, Fab-12, in Presta et al, Cancer Res.57: 4593-. Then keeping the temperature of the target Fab overnight; however, the incubation may continue for a longer period of time (e.g., 65 hours) to ensure equilibrium is reached. Thereafter, the mixture was transferred to a capture plate toThe mixture was kept at room temperature for 1 hour. The solution was then removed and the plate washed 8 times with PBS containing 0.1% Tween-20. After the plates were dried, 150. mu.l/well scintillation fluid (MicroScint-20; Packard) was added and the plates were counted for 10 minutes on a Topcount Gamma counter (Packard). The concentration of each Fab that gave less than or equal to 20% of the maximum binding was selected for use in the competitive binding assay. According to another embodiment, the Kd or Kd value is determined by surface plasmon resonance assay using BIAcore ^TM-2000 or BIAcore^TM-3000(BIAcore, inc., Piscataway, NJ) measured at 25 ℃ using an immobilized antigen CM5 chip at about 10 Response Units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen was diluted to 5. mu.g/ml (about 0.2. mu.M) with 10mM sodium acetate pH4.8 and then injected at a flow rate of 5. mu.l/min to obtain about 10 Response Units (RU) of conjugated protein. After injection of the antigen, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) in PBS (PBST) containing 0.05% Tween-20 were injected at 25 ℃ at a flow rate of about 25. mu.l/min. The binding rate (k) was calculated by simultaneous fitting of the binding and dissociation sensorgrams using a simple one-to-one Langmuir (Langmuir) binding model (BIAcoreevaluation Software version3.2)_on) And dissociation rate (k)_off). Equilibrium dissociation constant (Kd) in the ratio k_off/_onAnd (4) calculating. See, e.g., Chen, Y., et al, J Mol Biol293:865-881 (1999). If the binding rate is more than 10 according to the above surface plasmon resonance assay ⁶M^-1S^-1The binding rate can then be determined using fluorescence quenching techniques, i.e.measuring the increase or decrease in fluorescence emission intensity (excitation =295 nM; emission =340nM, 16nM bandpass) at 25 ℃ of 20nM anti-antigen antibody (Fab form) in PBS, pH7.2, in the presence of increasing concentrations of antigen, according to a measurement in a spectrometer such as a spectrophotometer equipped with a flow cut-off device (Aviv Instruments) or a 8000 series SLM-Aminco spectrophotometer (Thermospectronic) with a stirred cuvette.

In one embodiment, an "association rate" or "k" according to the present invention_on"is the use of BIAcore by the same surface plasmon resonance technique as described above^TM-2000 or BIAcore^TM-3000(BIAcore, inc., Piscataway, NJ) at 25 ℃ using an immobilized antigen CM5 chip at about 10 Response Units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen was diluted to 5. mu.g/ml (about 0.2. mu.M) with 10mM sodium acetate pH4.8 and then injected at a flow rate of 5. mu.l/min to obtain about 10 Response Units (RU) of conjugated protein. After injection of the antigen, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) in PBS (PBST) containing 0.05% Tween-20 were injected at 25 ℃ at a flow rate of about 25. mu.l/min. The binding rate (k) was calculated by simultaneous fitting of the binding and dissociation sensorgrams using a simple one-to-one Langmuir (Langmuir) binding model (BIAcore Evaluation Software version3.2) _on) And dissociation rate (k)_off). Equilibrium dissociation constant (Kd) in the ratio k_off/k_onAnd (4) calculating. See, e.g., Chen, Y., et al, J Mol Biol293:865-881 (1999). However, if according to the above surface plasmon resonance determination method, the binding rate exceeds 10⁶M^-1S^-1The binding rate is then preferably determined using fluorescence quenching techniques, i.e.measuring the increase or decrease in fluorescence emission intensity (excitation =295 nM; emission =340nM, 16nM bandpass) at 25 ℃ of 20nM anti-antigen antibody (Fab form) in PBS, pH7.2, in the presence of increasing concentrations of antigen, according to a measurement in a spectrometer such as a spectrophotometer equipped with a flow cut-off device (Aviv Instruments) or a 8000 series SLM-Aminco spectrophotometer (Thermospectric) with a stirred cuvette.

The phrase "substantially reduced" or "substantially different" as used herein means a sufficiently high degree of difference between two numerical values (typically one relating to an antibody of the invention and the other relating to a reference/comparison antibody) such that one of skill in the art would consider the difference between the two numerical values to be of statistical significance within the context of the biological property measured by the numerical values (e.g., Kd values, HAMA response). The difference between the two values is preferably greater than about 10%, preferably greater than about 20%, preferably greater than about 30%, preferably greater than about 40%, preferably greater than about 50%, as a function of the value of the reference/comparison antibody.

An "antigen" is a predetermined antigen to which an antibody can selectively bind. The target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten or other naturally occurring or synthetic compound. Preferably, the target antigen is a polypeptide. For purposes herein, an "acceptor human framework" is a framework comprising the amino acid sequence of a VL or VH framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework comprises the same amino acid sequence as it, or may comprise pre-existing amino acid sequence variations. When there are pre-existing amino acid changes, preferably there are no more than 5, preferably 4 or fewer, or 3 or fewer pre-existing amino acid changes. When pre-existing amino acid changes are present in the VH, preferably those changes are located only at three, two or one of positions 71H, 73H and 78H; for example, the amino acid residues at those positions may be 71A, 73T and/or 78A. In one embodiment, the VL acceptor human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

The antibodies of the invention may be capable of competing for binding to the same epitope bound by the second antibody. Monoclonal antibodies are considered to share "the same epitope" if they block the binding of other monoclonal antibodies by 40% or more at the same antibody concentration in a standard in vitro antibody competition binding assay.

A "human consensus framework" is a framework that represents the most common amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the human immunoglobulin VL or VH selection is from a variable region sequence subtype. Typically, the sequence subtypes are those described by Kabat et al. In one embodiment, for VL, the subtype is subtype kappa I as described in Kabat et al. In one embodiment, for the VH, the subtype is subtype III as described by Kabat et al.

A "VH subgroup III consensus framework" comprises the consensus sequence of amino acid sequences in variable heavy subgroup III obtained as described by Kabat et al.

The "VL subtype I consensus framework" comprises the consensus sequence of amino acid sequences in variable light chain kappa subtype I obtained as described by Kabat et al.

An "unmodified human framework" is a human framework having the same amino acid sequence as an acceptor human framework, e.g., no human to non-human amino acid substitutions in the acceptor human framework.

An "altered hypervariable region" for purposes herein is a hypervariable region which comprises one or more (e.g., 1 to about 16) amino acid substitutions therein.

An "unmodified hypervariable region" for purposes herein is a hypervariable region having the same amino acid sequence as the non-human antibody from which it was derived, i.e., a hypervariable region which does not have one or more amino acid substitutions therein.

The terms "hypervariable region", "HVR", "HV" or "CDR" as used herein refer to regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops. Typically, an antibody comprises 6 hypervariable regions: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). A number of hypervariable region descriptions are used herein and are included. Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al, Sequences of Proteins of immunological Interest,5th Ed. public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia instead refers to the position of the structural loops (Chothia and LeskJ. mol. biol.196:901-917 (1987)). The "contact" hypervariable regions are based on an analysis of the complex crystal structures available. The residues for each of these hypervariable regions are noted below. Kabat numbering will be used unless otherwise indicated. The positions of hypervariable regions are generally as follows: amino acids 24-34 (HVR-L1), amino acids 49-56 (HVR-L2), amino acids 89-97 (HVR-L3), amino acids 26-35A (HVR-H1), amino acids 49-65 (HVR-H2) and amino acids 93-102 (HVR-H3).

Hypervariable regions can also include "extended hypervariable regions" as follows: amino acids 24-36(L1) and amino acids 46-56(L2) in VL. For each of these definitions, the variable domain residues are numbered as described in Kabat et al (supra).

"framework" or "FR" residues are those residues in the variable region other than the hypervariable region residues as defined herein.

A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by humans and/or made using any of the techniques disclosed herein for making human antibodies. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues.

An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more of its CDRs that result in an improvement in the affinity of the antibody for the antigen as compared to a parent antibody without such alterations. Preferred affinity matured antibodies will have nanomolar or even picomolar levels of affinity for the target antigen. Affinity matured antibodies can be generated by procedures known in the art. Marks et al, Bio/Technology10:779-783(1992) describe affinity maturation via shuffling through VH and VL domains. The following documents describe random mutagenesis of CDR and/or framework residues: barbas et al, Proc.Nat.Acad.Sci.USA91: 3809-.

A "blocking" antibody or an "antagonist" antibody refers to an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Preferred blocking or antagonistic antibodies substantially inhibit or completely inhibit the biological activity of the antigen.

"TAT binding oligopeptide" refers to an oligopeptide that binds, preferably specifically binds, to a TAT polypeptide described herein. TAT binding oligopeptides may be chemically synthesized using known oligopeptide synthesis methods, or may be prepared and purified using recombinant techniques. TAT binding oligopeptides are typically 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 TAT binding oligopeptides capable of binding, preferably specifically binding, to a TAT polypeptide described herein may be identified without undue experimentation using well known techniques. In this regard, it is noted that techniques for screening libraries of oligopeptides 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 No. WO84/03506 and WO84/03564; Geysen et al, Proc. Natl. Act. Acad. Sci. U.S. A.,82: 178. 182(1985); Geysen et al, Synthetic Peptides antibodies, 130. quadrature. (1986); Geysen et al, J.Immunol. 149, Met et al, 19832; Nature J.S. 19931; Nature J. Acad. J. 1988; Nature et al., 1988: 1988; Nature J. Acad. 19932; Nature et al., 1988; Acad. S. 1984; Acad. J. 1984; Acad. 19832; Acad. 1986; Acad. 19832; Aust. J. 20; Australin. J. 19832; Aust. Acad. 20; Aust J. 20; Aust. J. 20; Aust. J. 20; Aust. J. Acad. 20; Aust. J. 20; Aust. J. 20; Aust., 102; Aust. 20; Aust. J. 20; Aust, G.P. (1991) Current Opin Biotechnol.,2: 668.

"TAT binding organic molecule" refers to an organic molecule that binds, preferably specifically binds, to a TAT polypeptide described herein, other than an oligopeptide or antibody as defined herein. TAT-binding organic molecules can be identified and chemically synthesized using known methods (see, e.g., PCT publication Nos. WO00/00823 and WO 00/39585). TAT binding organic molecules are typically less than about 2000 daltons in size, or less than about 1500, 750, 500, 250, or 200 daltons in size, wherein such organic molecules capable of binding, preferably specifically binding, a TAT polypeptide described herein can be identified using well-known techniques without undue experimentation. In this regard, it is noted that techniques for screening libraries of organic molecules for molecules capable of binding to a polypeptide target are well known in the art (see, e.g., PCT publication Nos. WO00/00823 and WO 00/39585).

An antibody, oligopeptide or other organic molecule that "binds" an antigen of interest, e.g., a tumor-associated polypeptide antigen target, refers to a molecule that binds the antigen with sufficient affinity such that the antibody, oligopeptide or other organic molecule can be used as a diagnostic and/or therapeutic agent for targeting cells or tissues expressing the antigen without significantly cross-reacting with other proteins. In such embodiments, the extent to which an antibody, oligopeptide or other organic molecule binds to a "non-target" protein will be less than about 10% of the binding of that antibody, oligopeptide or other organic molecule to its particular target protein, as determined by Fluorescence Activated Cell Sorting (FACS) analysis or Radioimmunoprecipitation (RIA). With respect to binding of an antibody, oligopeptide or other organic molecule to a target molecule, the term "specifically binds" or "specifically binds" to or to an epitope on a particular polypeptide or a particular polypeptide target means that binding is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of the molecule and comparing it to the binding of a control molecule, which is typically a molecule of similar structure but lacking binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, e.g., an excess of unlabeled target. In this case, specific binding is indicated if binding of the labeled target to the probe is competitively inhibited by an excess of 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 may, when used herein, exhibit, for example, a Kd to the target of at least about 10^-4M, or at least about 10^-5M, or at least about 10^-6M, or at least about 10^-7M, or at least about 10^-8M, or at least about 10^-9M, or at least about 10^-10M, or at least about 10^-11M, or at least about 10^-12M or 10^-12M is more than M. In one embodiment, the term "specific binding" refers to binding wherein a molecule binds to a particular polypeptide or epitope on a particular polypeptide, but does not substantially bind to any other polypeptide or polypeptide epitope.

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

An antibody, oligopeptide or other organic molecule that "induces apoptosis" refers to a molecule that induces programmed cell death as measured by annexin V binding, DNA fragmentation, cell contraction, endoplasmic reticulum expansion, cell rupture, and/or membrane vesicle formation (referred to as apoptotic bodies). The cell is typically one that overexpresses a TAT polypeptide. Preferably, the cell is a tumor cell, such as a prostate, breast, ovarian, gastric, endometrial, lung, kidney, colon, bladder tumor cell. There are a variety of methods available for assessing cellular events associated with apoptosis. For example, Phosphatidylserine (PS) translocation can be measured by annexin binding; DNA fragmentation can be assessed by DNA laddering (laddering); nuclear/chromatin condensation accompanying DNA fragmentation can be assessed by any increase in hypodiploid cells. Preferably, the antibody, oligopeptide or other organic molecule that induces apoptosis is one that results in an approximately 2 to 50 fold, preferably approximately 5 to 50 fold, most preferably approximately 10 to 50 fold, increase in induction of annexin binding relative to untreated cells in an annexin binding assay.

Antibody "effector functions" refer to those biological activities attributable to the Fc region of an antibody (either the native sequence Fc region or the amino acid sequence variant Fc region) 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 receptors); and B cell activation.

"antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cytotoxic form in which secreted Ig bound to Fc receptors (fcrs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells, followed by killing of the target cells with cytotoxins. The antibodies "arm" (arm) cytotoxic cells and are absolutely required for such killing. The main cells mediating ADCC, NK cells, express Fc γ RIII only, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. Page 464 of ravech and Kinet, annu, rev, immunol, 9:457-92(1991) summarizes FcR expression on hematopoietic cells. To assess ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed, such as described in U.S. patent 5,500,362 or 5,821,337. Effector cells useful in such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively/additionally, the ADCC activity of a molecule of interest may be assessed in vivo, for example in animal models such as disclosed in Clynes et al, PNAS (USA)95: 652-.

"Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. In addition, a preferred FcR is one that binds an IgG antibody (gamma receptor), including receptors of the Fc γ RI, Fc γ RII, and Fc γ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors. Fc γ RII receptors include Fc γ RIIA ("activating receptor") and Fc γ RIIB ("inhibiting receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor Fc γ RIIA comprises in its cytoplasmic domain an immunoreceptor tyrosine-based activation motif (ITAM). The inhibitory receptor Fc γ RIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see for review)Annu.Rev.Immunol.15:203-234 (1997)). For an overview of FcRs see ravech and Kinet, Annu.Rev.Immunol.9:457-492(1991); Capel et al, Immunomethods4:25-34(1994); de Haas et al, J.Lab.Clin.Med.126:330-341 (1995)). The term "FcR" encompasses other fcrs herein, including those that will be identified in the future. The term also includes the neonatal receptor (neonatal receptor), FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al, j.immunol.117:587(1976) and Kim et al, j.immunol.24:249 (1994)).

"human effector cells" refer to leukocytes which express one or more fcrs and perform effector functions. Preferably, the cell expresses at least Fc γ RIII and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include Peripheral Blood Mononuclear Cells (PBMC), Natural Killer (NK) cells, monocytes, cytotoxic T cells and neutrophils, preferably PBMC and NK cells. The effector cells may be isolated from a natural source, such as blood.

"complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells 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 an antibody (of the appropriate subtype) that binds to its cognate antigen. To assess complement activation, CDC assays can be performed, for example as described in Gazzano-Santoro et al, J.Immunol.methods202:163 (1996).

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 specific 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 cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma (hepatoma), breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, cancer of the liver, anal carcinoma, penile carcinoma, melanoma, multiple myeloma and B-cell lymphoma, brain cancer, and head and neck cancer, and related metastases.

The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with a degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

"tumor" as used herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous (pre-cancerous) and cancerous cells and tissues.

An antibody, oligopeptide or other organic molecule that "induces cell death" refers to a molecule that causes a viable cell to become non-viable. By cells is meant cells that express a TAT polypeptide, preferably cells that overexpress a TAT polypeptide compared to normal cells 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 produced and secreted by a cancer cell. Preferably, the cell is a cancer cell, for example a cancer cell of the breast, ovary, stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas or bladder. In vitro cell death can be determined in the absence of complement or immune effector cells to distinguish between cell death induced by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Thus, assays for cell death can be performed using heat inactivated serum (i.e., without complement) in the absence of immune effector cells. To determine whether an antibody, oligopeptide or other organic molecule is capable of inducing cell death, loss of membrane integrity can be assessed relative to untreated cells by uptake of Propidium Iodide (PI), Trypan blue (see Moore et al. cytotechnology17:1-11 (1995)), or 7 AAD. Preferred cell death-inducing antibodies, oligopeptides, or other organic molecules are those that induce PI uptake in BT474 cells in a PI uptake assay.

"cell expressing TAT" refers to a cell expressing an endogenous or transfected TAT polypeptide, either on the cell surface or in secreted form. "TAT-expressing cancer" refers to a cancer comprising cells that have a TAT polypeptide present on the surface of the cells or that produce and secrete a TAT polypeptide. A "TAT-expressing cancer" optionally produces TAT polypeptides on the cell surface at levels sufficient that anti-TAT antibodies, oligopeptides, or other organic molecules can bind thereto and have a therapeutic effect on 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 or other organic molecule antagonist can bind thereto and have a therapeutic effect on the cancer. In the latter case, the antagonist may be an antisense oligonucleotide that reduces, inhibits or prevents the production and secretion of a secreted TAT polypeptide by a tumor cell. A cancer that "overexpresses" a TAT polypeptide refers to a cancer that has significantly higher levels of TAT polypeptide on its cell surface or produces and secretes significantly higher levels of TAT polypeptide 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 can be determined in a diagnostic or prognostic assay by assessing elevated levels of TAT protein present on the surface of a cell or secreted by a cell (e.g., by immunohistochemistry assay, using anti-TAT antibodies prepared against an isolated TAT polypeptide prepared from an isolated nucleic acid encoding the TAT polypeptide using recombinant DNA techniques; FACS analysis; etc.). Alternatively, or in addition, the level of nucleic acid or mRNA encoding a TAT polypeptide in a cell can be measured, for example, by fluorescent in situ hybridization using a nucleic acid-based probe corresponding to a TAT-encoding nucleic acid or the complementary strand thereof (FISH; see WO98/45479, published in 1998 at 10 months); southern blotting; northern blotting; or Polymerase Chain Reaction (PCR) techniques such as real-time quantitative PCR (RT-PCR). TAT polypeptide overexpression can also be studied by measuring shed antigen in biological fluids such as serum using antibody-based assays (see also, e.g., U.S. Pat. No. 4,933,294, entitled 6/12/1990; WO91/05264, published 4/18/1991; U.S. Pat. No. 5,401,638, entitled 3/28/1995; Sias et al, J.Immunol.methods132:73-80 (1990)). In addition to the assays described above, a variety of in vivo assays may be utilized by the skilled practitioner. For example, cells in the patient may be exposed to an antibody optionally labeled with a detectable label, such as a radioisotope, and binding of the antibody to cells in the patient may be assessed, for example, by externally scanning for radioactivity or by analyzing a biopsy taken from a patient that has been previously exposed to the antibody.

As used herein, the term "immunoadhesin" refers to antibody-like molecules that combine the binding specificity of a heterologous protein (an "adhesin") with the effector functions of an immunoglobulin constant region. Structurally, immunoadhesins comprise fusions of amino acid sequences and immunoglobulin constant region sequences that differ from the antigen recognition and binding site of the antibody (i.e., are "heterologous"), with the desired binding specificity. The adhesin part of an immunoadhesin molecule is typically a contiguous (contiguous) amino acid sequence comprising at least the binding site for a receptor or ligand. The immunoglobulin constant region sequences in immunoadhesins can 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.

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

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of a cell and/or causes destruction of a cell. The term is intended to include: radioisotopes, e.g. At²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²And radioactive isotopes of Lu; a chemotherapeutic agent; enzymes and fragments thereof, such as nucleolytic enzymes; (ii) an antibiotic; 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 various antineoplastic or anticancer agents disclosed hereinafter. Other cytotoxic agents are described below. Tumoricidal agents cause destruction of tumor cells.

"chemotherapeutic agent" refers to a chemical compound useful for the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents (alkylating agents), such as thiotepa and thiotepaCyclophosphamide (cyclophosphamide); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines (aziridines), such as benzotepa (benzodepa), carboquone (carboquone), metoclopramide (meteredepa), and uretepa (uredepa); ethylene imines (ethylene)nimines) and methylmelamines (melamines), including altretamine (altretamine), triethylenemelamine (triethyleneemimine), triethylenephosphoramide (triethylenephosphoramide), triethylenethiophosphoramide (triethylenephosphoramide), and trimetalmetamine (trimetylomelamine); annonaceous acetogenins (especially bullatacin and bullatacin); delta-9-tetrahydrocannabinol (dronabinol), Beta-lapachone (lapachone); lapachol (lapachol); colchicines (colchicines); betulinic acid (betulinic acid); camptothecin (camptothecin) (including the synthetic analogue topotecan)CPT-11 (irinotecan),acetyl camptothecin, scopoletin (scopoletin), and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin (adozelesin), carvelesin (carzelesin), and bizelesin (bizelesin) synthetic analogs); podophyllotoxin (podophylotoxin); podophyllinic acid (podophyllic acid); teniposide (teniposide); cryptophycins (especially cryptophycins 1 and 8); dolastatin (dolastatin); duocarmycins (including synthetic analogs, KW-2189 and CB1-TM 1); eiscosahol (eleutherobin); pancratistatin; sarcodictyin; spongistatin (spongistatin); nitrogen mustards such as chlorambucil (chlorambucil), chlorambucil (chlorenaphazine), cholorophosphamide (cholorophosphamide), estramustine (estramustine), ifosfamide (ifosfamide), mechlorethamine (mechlorethamine), mechlorethamine hydrochloride (mechlorethamine oxide hydrochloride), melphalan (melphalan), neomycin (novembichin), benzene mustard cholesterol (phenylesterine), prednimustine (prednimustine), triamcinolone (trofosfamide), uracil Nitrogen mustard (uracil mustard); nitrosoureas such as carmustine (carmustine), chlorouretocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine) and ramustine (ranimustine); antibiotics such as enediynes antibiotics (enediynes) (e.g., calicheamicins, especially calicheamicin γ 1I and calicheamicin ω I1 (see, e.g., Agnew, chem. Intl. Ed. Engl.33:183-186(1994)), anthracyclines (dynemicins), including dynemicin A; epothilones (esperamicins), and neocarzinostatin (neocarzinostatin) and related chromoproteenediynes chromophores), aclacins (aclacinomycin), actinomycins (actinomycin), anidamycin (antrhramycin), azaserine (azacine), bleomycin (bleomycin), actinomycins C (cacodymycin), carbamycin, carmycin (carmycin), carcinomycin (carcinomycin), chromomycins (actinomycin L), daunomycin (daunomycin-5-D), daunomycin (daunomycin), and daunomycin,Doxorubicin (doxorubicin) (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolodoxorubicin and deoxydoxorubicin), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), marijumycin (marcellomomycin), mitomycins (mitomycins) such as mitomycin C, mycophenolic acid (mycophenolic acid), norramycin (nogalamycin), olivomycin (olivomycin), pelomycin (pelomycin), pelomycin (polyplomycin), potfiromycin, puromycin (puromycin), triiron doxorubicin (quelamycin), rodobicin (rodorubicin), streptonigrin (streptagrin), streptozocin (streptazocin), tubercidin (tunicin), ubenidin (metrizax), zocin (zostatin), zorubicin (zorubicin); antimetabolites such as methotrexate (methotrexate) and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues, such as fludara Ripariadine (fludarabine), 6-mercaptopurine (mercaptoprine), thiamiprine (thiamiprine), thioguanine (thioguanine); pyrimidine analogs such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine (azauridine), carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine (dideoxyuridine), doxifluridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine); androgens such as carotinone (calusterone), dromostanolone propionate, epitioandrostanol (epitiostanol), mepiquitane (mepiquitane), testolactone (testolactone); anti-adrenal agents such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), trilostane (trilostane); folic acid supplements such as folinic acid (folinic acid); acetoglucurolactone (acegultone); (ii) an aldophosphamide glycoside; aminolevulinic acid (aminolevulinic acid); eniluracil (eniluracil); amsacrine (amsacrine); bestrabuucil; bisantrene; edatrexate (edatraxate); desphosphamide (defosfamide); dimecorsine (demecolcine); diazaquinone (diaziqutone); elfornitine; etidinium acetate (epothilone; etoglucid (etoglucid), gallium nitrate, hydroxyurea (hydroxyurea), lentinan (lentinan), lonidamine (lonamine), maytansinoids (maytansinoids) such as maytansine (maytansine) and ansamitocins (ansamitocins), mitoguazone (mitoguazone), mitoxantrone (mitoxantrone), mopidamol, diaminenitracridine (nitacrine), pentostatin (pentostatin), mechlorethamine (phenameter), pirarubicin (pirarubicin), losoxantrone (losoxantrone), 2-ethylhydrazide (hydydrazide), procarbazine (carpronium); Polysaccharide complex (JHS natural products, Eugene, OR); razoxane (rizoxane); rhizomycin (rhizoxin); sizofuran (sizofiran); helical germanium (spirogermanium); tenuazonic acid (tenuazonic acid); triimine quinone (triaziquone); 2,2',2 "-trichlorotriethylamine; trichothecenes (trichothecenes), especially T-2 toxin, verrucaVerrucarin a, tubercidin a and snakes (anguidin)); urethane (urethan); vindesine (vindesine)Dacarbazine (dacarbazine); mannomustine (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromane (pipobroman); a polycytidysine; cytarabine (arabine) ("Ara-C"); thiotepa (thiotepa); taxoids, e.g. taxolPaclitaxel (paclitaxel) (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE^TMWithout Cremophor, albumin engineered nanoparticle dosage forms of paclitaxel (American pharmaceutical Partners, Schaumberg, Illinois) anddocetaxel (doxetaxel)Rorer, antonyy, France); chlorambucil (chlorambucil); gemcitabine (gemcitabine)6-thioguanine (thioguanine); mercaptopurine (mercaptoprine); methotrexate (methotrexate); platinum analogs such as cisplatin (cissplatin) and carboplatin (carboplatin); vinblastine (vinblastine) Platinum (platinum); etoposide (VP-16); ifosfamide (ifosfamide); mitoxantrone (mitoxantrone); vincristine (vincristine)Oxaliplatin (oxaliplatin); leucovorin (leucovorin); vinorelbine (vinorelbine)Oncostatin (novantrone); edatrexate (edatrexate); daunomycin (daunomycin); aminopterin (aminopterin); ibandronate (ibandronate); topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids (retinoids), such as retinoic acid (retinoic acid); capecitabine (capecitabine)A pharmaceutically acceptable salt, acid or derivative of any of the foregoing; and combinations of two or more of the above, such as CHOP (abbreviation for cyclophosphamide, doxorubicin, vincristine and prednisolone combination therapy) and FOLFOX (oxaliplatin)^TM) Abbreviation for treatment regimen combining 5-FU and folinic acid).

The definition also includes anti-hormonal agents that act to modulate, reduce, block or inhibit the effects of hormones that promote cancer growth, and are often in the form of systemic or systemic treatment. They may themselves be hormones. Examples include antiestrogens and Selective Estrogen Receptor Modulators (SERMs), including for example tamoxifen (tamoxifen) (including Tamoxifen) and,Raloxifene (raloxifene), droloxifene (droloxifene), 4-hydroxyttamoxifen, trioxifene (trioxifene), naloxifene (keoxifene), LY117018, onapristone (onapristone), andtoremifene (toremifene); anti-pregnenones; estrogen receptor down-regulators (ERD); agents acting to inhibit or shut down the ovary, e.g. Luteinizing Hormone Releasing Hormone (LHRH) agonists, such asAndleuprolide acetate, goserelin acetate, buserelin acetate and triptorelin acetate; anti-androgens such as flutamide (flutamide), nilutamide (nilutamide), and bicalutamide (bicalutamide); other anti-androgens, such as flutamide (flutamide), nilutamide (nilutamide), and bicalutamide (bicalutamide); and aromatase inhibitors which inhibit aromatase which regulates estrogen production in the adrenal gland, such as, for example, 4(5) -imidazole, aminoglutethimide,Megestrol acetate (megestrol acetate),Exemestane (exemestane), formestane (formestane), fadrozole (fadrozole),Vorozole (vorozole), Letrozole (letrozole) andanastrozole (anastrozole). In addition, this definition of chemotherapeutic agents includes diphosphonates such as clodronate (e.g., clodronate)OrEtidronate (etidronate), NE-58095, and,Zoledronic acid/zoledronate,Alendronate (alendronate),Pamidronate (pamidronate),Tiludronate (tiludronate) orRisedronate (risedronate); and troxacitabine (a 1, 3-dioxolane nucleoside cytosine analogue); antisense oligonucleotides, particularly those that inhibit gene expression in signaling pathways involved in adherent cell proliferation, such as, for example, PKC- α, Raf, H-Ras and epidermal growth factor receptor (EGF-R); vaccines, e.g.Vaccines and gene therapy vaccines, e.g.A vaccine,A vaccine anda vaccine;a topoisomerase 1 inhibitor;rmRH; lapatinib ditosylate (ErbB-2 and EGFR dual tyrosine kinase small molecule inhibitor, also known as GW 572016); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoingA compound (I) is provided.

"growth inhibitory agent" as used herein refers to a compound or composition that inhibits the growth of cells, particularly cancer cells expressing TAT, in vitro or in vivo. Thus, the growth inhibitory agent may be one that 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 position outside the S phase), such as agents that induce G1 arrest and M phase arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes (taxanes), and topoisomerase II inhibitors such as doxorubicin (doxorubicin), epirubicin (epirubicin), daunorubicin (daunorubicin), etoposide (etoposide), and bleomycin (bleomycin). Those agents that block G1 also spill over into S phase arrest, for example, DNA alkylating agents such as tamoxifen (tamoxifen), prednisone (prednisone), dacarbazine (dacarbazine), mechloroethylmethylamine (mechloroethylamine), cisplatin (cissplatin), methotrexate (methotrexate), 5-fluorouracil (5-fluorouracil), and ara-C. For more information see the "molecular Basis of Cancer", edited by Mendelsohn and Israel, Chapter 1, entitled "Cell cycle expansion, oncogenes, and anti-ionic drugs", Murakanii et al, WB Saunders, Philadelphia, 1995, especially page 13. Taxanes (paclitaxel and docetaxel) are both anticancer drugs derived from the yew tree. Docetaxel derived from Taxus baccata Rhone-Poulenc Rorer) is paclitaxelBristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, resulting in the inhibition of cell mitosis.

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

(8S-cis)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione

The term "cytokine" refers to the generic term for a protein released by one cell population that acts on another cell as an intercellular mediator. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; (ii) a relaxin; a prorelaxin; glycoprotein hormones such as Follicle Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH), and Luteinizing Hormone (LH); a liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and-beta; mullerian (Mullerian) inhibitory substances; mouse gonadotropin-related peptides; a statin; an activin; vascular endothelial growth factor; an integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; platelet-derived growth factor; transforming Growth Factors (TGF), such as TGF-alpha and TGF-beta; insulin-like growth factors-I and-II; erythropoietin (EPO); an osteoinductive factor; interferons such as interferon- α, - β, and- γ; colony Stimulating Factors (CSFs), such as macrophage CSFs (M-CSF), granulocyte-macrophage CSFs (GM-CSF), and granulocyte CSFs (G-CSF); interleukins (IL), 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; tumor necrosis factors such as TNF- α or TNF- β; 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.

The term "package insert" is used to refer to instructions typically included in commercial packages of therapeutic products that contain information regarding indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.

Compositions and methods of the invention

A. anti-TAT antibodies

In one embodiment, the invention provides anti-TAT antibodies useful herein as therapeutic and/or diagnostic agents. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies.

1. Polyclonal antibodies

Polyclonal antibodies are preferably generated by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant in the animal. It may be useful to couple the relevant antigen (especially when using synthetic peptides) to a protein that is immunogenic in the species to be immunized. For example, bifunctional or derivatizing reagents may be used, such as maleimidobenzoyl sulphosuccinimide ester (coupling via cysteine residues), N-hydroxysuccinimide (coupling via lysine residues), glutaraldehyde, succinic anhydride, SOCl₂Or R¹N = C = NR, wherein R and R¹Is a different hydrocarbon group, and the antigen is coupled to Keyhole Limpet Hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor.

Animals are immunized against antigens, immunogenic conjugates or derivatives by mixing, for example, 100 or 5 μ g of 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, animals were boosted with an initial amount of 1/5-1/10 of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. After 7-14 days, blood was collected from the animals, and the antibody titer of the serum was determined. The animals were boosted until titers reached a stable high level. Conjugates can also be prepared in recombinant cell culture as protein fusions. Also, a coagulant such as alum is suitably used to enhance the immune response.

2. Monoclonal antibodies

Monoclonal antibodies can be prepared by the hybridoma method originally described by Kohler et al, Nature256:495(1975), or can be prepared by recombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other suitable 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. Following immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103, Academic Press, 1986).

The hybridoma cells so prepared are seeded and cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused parent myeloma cells (also known as fusion partners). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the selective medium for the hybridomas typically will contain hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

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

The medium in which the hybridoma cells are growing can be assayed for production of monoclonal antibodies to 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).

The binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis as described in Munson et al, anal. biochem.107:220 (1980).

Once hybridoma cells producing Antibodies with the desired specificity, affinity, and/or activity are identified, the clones may be subcloned by limiting dilution procedures and cultured using standard methods (Goding, Monoclonal Antibodies: Priniplsand Practice, pp.59-103, Academic Press, 1986). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells can be cultured in vivo in animals as ascites tumors, for example, by i.p. injection of the cells into mice.

Monoclonal antibodies secreted by the subclones can be 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, hydroxyapatite chromatography, gel electrophoresis, dialysis, and the like.

DNA encoding the monoclonal antibody is readily isolated and sequenced by conventional procedures (e.g., using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells are preferred sources of such DNA. Once isolated, the DNA may be placed into an expression vector, which is then transfected into a host cell that does not otherwise produce the antibody protein, such as an escherichia coli cell, simian COS cell, Chinese Hamster Ovary (CHO) cell, or myeloma cell, to obtain synthesis of the monoclonal antibody in the recombinant host cell. A review of the recombinant expression of DNA encoding antibodies in bacteria includes Skerra et al, Curr. opinion in Immunol.5: 256-188 (1993) and Pluckthun, Immunol.Revs.130:151-188 (1992).

In another embodiment, monoclonal antibodies or antibody fragments can be isolated from phage antibody libraries constructed using the techniques described in McCafferty et al, Nature348:552-554 (1990). Clackson et al, Nature352: 624-. Subsequent publications describe the generation of high affinity (nM range) human antibodies by chain shuffling (Marks et al, Bio/Technology10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as strategies 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 isolating monoclonal antibodies.

The DNA encoding the antibody may be modified to produce chimeric or fused antibody polypeptides, for example, by using human heavy and light chain constant regions (C)_HAnd C_L) The sequence may be substituted for a homologous murine sequence (U.S. Pat. No. 4,816,567; Morrison et al, Proc. Natl. Acad. Sci. USA81:6851 (1984)), or by fusing an immunoglobulin coding sequence to the entire or part of the coding sequence of a non-immunoglobulin polypeptide (heterologous polypeptide). Non-immunoglobulin polypeptide sequences may be substituted for the constant regions of an antibody, or they may be substituted for the variable regions of one antigen-binding site of an antibody, resulting in a chimeric bivalent antibody comprising one antigen-binding site with specificity for one antigen and another antigen-binding site with specificity for a different antigen.

3. Human and humanized antibodies

anti-TAT antibodies of the invention may also include those of human originA humanized antibody or a human antibody. Humanized forms of non-human (e.g., murine) antibodies refer to chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab ', F (ab')₂Or other antigen binding subsequences of antibodies). Humanized antibodies include human immunoglobulins (recipient antibody) in which Complementarity Determining Region (CDR) residues are replaced by CDR residues from 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 with corresponding non-human residues. Humanized antibodies may also comprise residues not found in the recipient antibody or in the imported CDR or framework sequences. Typically, the humanized antibody will comprise substantially all of at least one, and typically two, variable regions 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 preferably also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al, Nature321:522-525(1986); Riechmann et al, Nature332:323-329(1988); Presta, curr. Op. struct. biol.2:593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art. Typically, humanized antibodies have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable region. Humanization can be carried out essentially according to the method of Winter and co-workers (Jones et al, Nature321:522-525(1986); Riechmann et al, Nature332:323-327(1988); Verhoeyen et al, Science239:1534-1536 (1988)), by replacing the corresponding human antibody sequences with rodent CDR sequences. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) in which substantially less than the entire human variable region is replaced with 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 replaced with residues from analogous sites in rodent antibodies.

When antibodies are intended for human therapeutic use, the choice of human variable regions, including light and heavy chains, for making humanized antibodies is important for reducing antigenicity and HAMA response (human anti-mouse antibodies). The entire library of known human variable region sequences was screened with rodent antibody variable region sequences according to the so-called "best-fit" method. The human V domain sequence closest to rodents is identified and the human Framework Region (FR) received therein for use in humanizing antibodies (Sims et al, J.Immunol.151:2296(1993); Chothia et al, J.mol.biol.196:901 (1987)). Another approach uses specific framework regions derived from the consensus sequence of all human antibodies of a specific subtype of light or heavy chain. The same framework can be used for several different humanized antibodies (Carter et al, Proc. Natl. Acad. Sci. USA89:4285(1992); Presta et al, J. Immunol.151:2623 (1993)).

More importantly, the antibodies retain high binding affinity for the antigen after humanization as well as other favorable biological properties. To achieve this, according to a preferred method, humanized antibodies are prepared by a method of analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Computer programs are also available that illustrate and display the likely three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these display images enables analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected from the recipient and import sequences and combined to obtain a desired antibody characteristic, such as increased affinity for the target antigen. In general, hypervariable region residues are directly and most substantially involved in the effect on antigen binding.

Various forms of humanized anti-TAT antibodies are envisaged. For example, the humanized antibody may be an antibody fragment, such as a Fab, optionally coupled to one or more cytotoxic agents to generate an immunoconjugate. Alternatively, the humanized antibody may be an intact antibody, such as an intact IgG1 antibody.

As an alternative to humanization, human antibodies can be generated. For example, it is now possible to generate transgenic animals (e.g., mice) that are capable of generating a complete repertoire of human antibodies upon immunization in the absence of endogenous immunoglobulin production. For example, the region of the antibody heavy chain junction (J) has been described in chimeric and germline mutant mice_H) Homozygous deletion of the gene results in complete suppression of endogenous antibody production. Transfer of the human germline immunoglobulin gene set (array) into such germline mutant mice will result in the generation of human antibodies upon antigen challenge. See, e.g., Jakobovits et al, Proc. Natl. Acad. Sci. USA90:2551(1993), Jakobovits et al, Nature362:255-258(1993), Bruggemann et al, Yeast in Immuno.7:33(1993), U.S. Pat. Nos. 5,545,806,5,569,825,5,591,669 (all Genpharm); 5,545,807; and WO 97/17852.

Alternatively, phage display technology (McCafferty et al, Nature348:552-553 (1990)) can be used to generate human antibodies and antibody fragments in vitro from a repertoire of immunoglobulin variable (V) region genes from an unimmunized donor. According to this technique, antibody V region genes are cloned in-frame to the major or minor coat protein genes of filamentous phage such as M13 or fd and displayed as functional antibody fragments on the surface of the phage particle. Since the filamentous particle comprises a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in selection of the gene encoding the antibody displaying those properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats, for review see, e.g., Johnson, Kevin S.and Chiswell, DavidJ., Current Opinion in structural Biology3:564-571 (1993). Several sources of V gene segments are available for phage display. Clackson et al, Nature352:624-628(1991) isolated from a random combinatorial library of small V genes derived from the spleen of immunized mice, a number of different anti-orooxazolone antibodies. A complete set of V genes can be constructed from non-immunized human donors and antibodies directed against a number of different antigens, including self-antigens, can be isolated essentially according to the techniques described in Markset 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.

Human antibodies can also be generated by activating B cells in vitro, as described above (see U.S. Pat. nos. 5,567,610 and 5,229,275).

4. Antibody fragments

In certain cases, it is advantageous to use 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.

Various techniques have been developed for generating antibody fragments. Traditionally, these fragments have been derived by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al, Journal of Biochemical and Biophysical Methods24:107-117(1992); Brennan et al, Science229:81 (1985)). However, these fragments can now be produced directly from recombinant host cells. Fab, Fv and scFv antibody fragments can all be expressed in and secreted by E.coli, thereby allowing easy production of large quantities of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab '-SH fragments can be recovered directly from E.coli and coupled by chemical means to form F (ab')₂Fragments (Carter et al, Bio/Technology10: 163-. According to another method, F (ab') can be isolated directly from recombinant host cell cultures ₂And (3) fragment. Fab and F (ab') with extended in vivo half-life comprising salvage receptor binding epitope residues₂Fragments are described in U.S. Pat. No. 5,869,046. Other techniques for generating 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; us patent 5,571,894; and U.S. patent 5,587,458. Fv and scFv are the only species with an intact binding site and lacking constant regions; they are therefore suitable for reducing non-specific binding during in vivo use. scFv fusion proteins can be constructed to produce effector proteinsFusions of white matter at the amino-or carboxy-terminus of scFv. See, for example, Antibody Engineering, eds. Borebaeck, supra. The antibody fragment may also be a "linear antibody," such as an antibody described in U.S. Pat. No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

5. Bispecific antibodies

Bispecific antibodies refer to antibodies having binding specificity for at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes of the TAT protein described herein. Other such antibodies may associate a TAT binding site with a binding site for another protein. Alternatively, the anti-TAT arm may be combined with an arm that binds to a triggering molecule on leukocytes, such as a T cell receptor molecule (e.g., CD 3) (see, e.g., Baeuerle, et al, curr. opin. mol. ther.11(1):22-30 (2009)) or an Fc receptor for IgG (Fc γ R), such as Fc γ RI (CD64), Fc γ RII (CD32) and Fc γ RIII (CD16), to focus and localize cellular defense mechanisms to cells expressing TAT. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing TAT. These antibodies possess a TAT binding arm and an arm that binds a cytotoxic agent (e.g., saporin (saporin), anti-interferon-alpha, vinca alkaloid (vinca alkaloid), ricin a chain, methotrexate, or radioisotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F (ab') ₂Bispecific antibodies).

Bispecific anti-ErbB 2/anti-FcyRIII antibodies are described in WO96/16673, and bispecific anti-ErbB 2/anti-FcyRII antibodies are disclosed in U.S. Pat. No.5,837,234. Bispecific anti-ErbB 2/Fc α antibodies are shown in WO 98/02463. U.S. Pat. Nos. 5,821,337 and 6,407,213 teach bispecific anti-ErbB 2/anti-CD 3 antibodies. Other bispecific antibodies that bind to an epitope on the CD3 antigen and a second epitope have been described. See, e.g., U.S. Pat. No.5,078,998 (anti-CD 3/tumor cell antigen); 5,601,819 (anti-CD 3/IL-2R; anti-CD 3/CD 28; anti-CD 3/CD 45); 6,129,914 (anti-CD 3/malignant B cell antigen); 7,112,324 (anti-CD 3/CD 19); 6,723,538 (anti-CD 3/CCR 5); 7,235,641 (anti-CD 3/EpCAM); 7,262,276 (anti-CD 3/ovarian tumor antigen); and 5,731,168 (anti-CD 3/CD4 IgG).

Methods for making bispecific antibodies are known in the art. The traditional generation of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities (Millstein et al, Nature305:537-539 (1983)). Due to the random assignment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. The purification of the correct molecule, which is usually performed by an affinity chromatography step, is rather cumbersome and the product yield is low. A similar procedure is disclosed in WO93/08829 and Travecker et al, EMBO J.10:3655-3659 (1991).

According to a different approach, antibody variable regions with the desired binding specificity (antibody-antigen binding site) are fused to immunoglobulin constant region sequences. Preferably, the fusion uses a composition comprising at least a partial hinge, C_H2 and C_H3 region, an immunoglobulin heavy chain constant region. Preferably, there is a first heavy chain constant region (C) comprising the site necessary for light chain binding in at least one of the fusions_H1). The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain is inserted into separate expression vectors and co-transfected into a suitable host cell. In embodiments where unequal ratios of the three polypeptide chains used in the construction provide the optimal yield of the desired bispecific antibody, this provides greater flexibility in adjusting the mutual ratios of the three polypeptide fragments. However, it is possible to insert the coding sequences for two or all three polypeptide chains into the same vector when expression of at least two polypeptide chains at the same ratio results in high yields or when the ratio does not have a significant effect on the yield of the desired chain combination.

In a preferred embodiment of the method, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain with a first binding specificity on one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) on the other arm. Since the presence of immunoglobulin light chains in only half of the bispecific molecule provides a convenient way of isolation, it was found that this asymmetric structure facilitates the separation of the desired bispecific complex (compound) from the unwanted immunoglobulin chain combinations. This method is disclosed in WO 94/04690. For additional details on the generation of bispecific antibodies see, e.g., Suresh et al, Methods in Enzymology121:210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. Preferred interfaces comprise at least part of C_H3 domain. In this method, one or more small amino acid side chains at the interface of the first antibody molecule are replaced with a larger side chain (e.g., tyrosine or tryptophan). Compensatory "cavities" of the same or similar size for large side chains are created at 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 heterodimers over other unwanted end products such as homodimers.

Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one antibody of the heterologous conjugate may be conjugated to avidin and the other antibody to biotin. For example, such antibodies have been proposed for targeting immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treating HIV infection (WO 91/00360, WO92/200373, and EP 03089). Heteroconjugate antibodies can be prepared using any convenient crosslinking method. Suitable crosslinking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with a number of crosslinking techniques.

Techniques for generating bispecific antibodies from antibody fragments are also described in the literature. For example, bispecific antibodies can be prepared using chemical ligation. Brennan et al, Science229:81(1985) describes proteolytic cleavage of intact antibodies to F (ab')₂A method for fragmenting. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize adjacent dithiols and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted to Thionitrobenzoate (TNB) derivatives. One of the Fab ' -TNB derivatives is then reverted back to Fab ' -thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab ' -TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as selective immobilization reagents for enzymes.

Recent advances have made it easier to recover Fab' -SH fragments directly from E.coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al, J.Exp.Med.175:217-225(1992) describe the generation of fully humanized bispecific antibodies F (ab')₂A molecule. Each Fab' fragment was separately secreted by E.coli and subjected to directed chemical coupling in vitro to form bispecific antibodies. The bispecific antibody so 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. Various techniques for the direct preparation and isolation of bispecific antibody fragments from recombinant cell cultures are also described. For example, bispecific antibodies have been generated using leucine zippers. Kostelny et al, J.Immunol.148(5): 1547-. Leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers are reduced at the hinge region to form monomers and then re-oxidized to form antibody heterodimers. This method can also be used to generate antibody homodimers. The "diabody" technology described by Hollinger et al, Proc. Natl. Acad. Sci. USA90: 6444-. The fragment comprises V connected by a linker _HAnd V_LThe linker is too short to allow pairing between the two domains on the same strand. Thus, V on a segment is forced_HAnd V_LDomain and complementary V on another fragment_LAnd V_HThe domains pair, thereby forming two antigen binding sites. Another strategy for making bispecific antibody fragments by using single chain fv (scFv) dimers has also been reported. See Gruber et al, J.Immunol.152:5368 (1994).

Antibodies with more than two titers are contemplated. For example, trispecific antibodies can be prepared. Tutt et al, J.Immunol.147:60 (1991).

6. Heteroconjugate antibodies

Heteroconjugate antibodies are also within the scope of the invention. Heteroconjugate antibodies consist of two covalently linked antibodies. Such antibodies are suggested, for example, for targeting immune system cells to unwanted cells (U.S. Pat. No. 4,676,980) and for the treatment of HIV infection (WO 91/00360; WO92/200373; EP 03089). It is contemplated that antibodies can be made in vitro using known methods of synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of agents suitable for this purpose include iminothiolate (iminothiolate) and methyl 4-mercaptobutylimidate (methyl-4-mercaptoimidate) and are disclosed, for example, in U.S. Pat. No. 4,676,980.

7. Multivalent antibodies

Multivalent antibodies may be internalized (and/or catabolized) by a cell expressing an antigen to which the antibody binds more rapidly than bivalent antibodies. The antibodies of the invention can be multivalent antibodies (other than IgM classes) that can be readily produced by recombinant expression of nucleic acids encoding the polypeptide chains of the antibodies, having three or more antigen binding sites (e.g., tetravalent antibodies). A multivalent antibody may comprise a dimerization domain and three or more antigen binding sites. Preferred dimerization domains comprise (or consist of) an Fc region or a cross-linking region. In this case, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region. Preferred multivalent antibodies herein comprise (or consist of) three to about eight, but preferably four antigen binding sites. A multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain comprises two polypeptide chainsA plurality or multiple variable regions. For example, a polypeptide chain can comprise VD1- (X1)_n-VD2-(X2)_n-Fc, wherein VD1 is a first variable region, VD2 is a second variable region, Fc is one polypeptide chain of an Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. For example, a polypeptide chain can comprise: VH-CH 1-flexible linker-VH-CH 1-Fc region chain; or VH-CH1-VH-CH1-Fc domain chain. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable region polypeptides. The multivalent antibody herein can comprise, for example, about two to about eight light chain variable region polypeptides. Light chain variable region polypeptides contemplated herein comprise a light chain variable region, and optionally further comprise a CL domain.

8. Engineering of effector functions

It may be desirable to modify an antibody of the invention with respect to effector function, for example to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. Alternatively, or in addition, cysteine residues may be introduced into the Fc region, thereby allowing interchain disulfide bonds to form in this region. The homodimeric antibody so produced may have improved internalization capacity and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al, J.Exp.Med.176: 1191-. Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al, Cancer Research53: 2560-. Alternatively, the antibody may be engineered to have dual Fc regions, and thus may have enhanced complement lysis and ADCC capabilities. See Stevenson et al, Anti-Cancer Drug Design3:219-230 (1989). To increase the serum half-life of the antibody, a salvage receptor binding epitope can be incorporated into the antibody (particularly an antibody fragment) as described, for example, in U.S. patent 5,739,277. As used herein, the term "salvage (salvage) receptor binding epitope" refers to an IgG molecule (e.g., IgG) ₁、IgG₂、IgG₃Or IgG₄) The Fc region is the epitope responsible for increasing the serum half-life of the IgG molecule in vivo.

9. Immunoconjugates

The invention also relates 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 a fragment thereof) or a radioisotope (i.e., a radioconjugate).

Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that may be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α -sarcin (sarcocin), Aleutites fordii (Aleuties fordii) toxic protein, dianthus chinensis (dianthin) toxic protein, Phytolacca americana (Phytolacca) toxic protein (PAPI, PAPII, and PAPA-S), Momordica charantia (Momordica chaetia) inhibitor, Jatropha curcin (curcin), crotin (crotin), Saponaria officinalis (saponaria officinalis) inhibitor, gelonin alba (gelonin), mitomycin (gelonin), tricin (resphin), trichothecin (triomycin), and trichothecin (enomycin). A variety of radionuclides are available for use in the production of radioconjugated antibodies. Examples include ²¹²Bi、¹³¹I、¹³¹In、⁹⁰Y and¹⁸⁶re. Conjugates of the antibody and cytotoxic agent may be prepared using a variety of bifunctional protein coupling agents, such as bifunctional derivatives of N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), Iminothiolane (IT), imidoesters (such as dimethyl adipimidate hcl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), 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). For example, can be asRicin immunotoxins are prepared as described in Vitetta et al, Science238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelator for conjugating radionucleotides to antibodies. See WO 94/11026.

Also contemplated herein are conjugates of the antibody with one or more small molecule toxins such as calicheamicin (calicheamicin), maytansinoids (maytansinoids), trichothecenes (trichothecene), and CC1065, and derivatives of these toxins that have toxin activity.

Maytansine and maytansinoids

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

Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was originally isolated from the east African shrub Maytenus serrata (Maytenus serrata) (U.S. Pat. No. 3,896,111). It was subsequently discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). The synthesis of maytansinol and its derivatives and analogues is disclosed, for example, in the following U.S. patents: 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 expressly incorporated herein by reference.

Maytansinoid-antibody conjugates

In an attempt to improve their therapeutic index, maytansine and maytansinoids have been conjugated to antibodies that specifically bind to tumor cell antigens. Immunoconjugates comprising maytansinoids and their therapeutic use are disclosed, for example, in the following patents: U.S. Pat. Nos. 5,208,020, 5,416,064, and Europe Patent EP0425235B1, the disclosure of which is expressly incorporated herein by reference. Liu et al, Proc.Natl.Acad.Sci.USA93:8618-8623(1996) describe 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 against cultured colon cancer cells and showed antitumor activity in an in vivo tumor growth assay. Chari et al, Cancer Research52: 127-. The cytotoxicity of TA.1-maytansinoid conjugates, which express 3x10 per cell, was tested in vitro on the human breast cancer cell line SK-BR-3⁵A HER-2 surface antigen. The drug conjugates achieve a similar degree of cytotoxicity as the free maytansinoid drug, which can be increased by increasing the number of maytansinoid molecules conjugated per antibody molecule. The a 7-maytansinoid conjugate showed low systemic cytotoxicity in mice.

anti-TAT polypeptide antibody-maytansinoid conjugates (immunoconjugates)

anti-TAT antibody-maytansinoid conjugates can be prepared by chemically linking an anti-TAT antibody to a maytansinoid molecule without significantly impairing the biological activity of the antibody or maytansinoid molecule. An average of 3-4 maytansinoid molecules per antibody molecule coupled showed efficacy in enhancing cytotoxicity against target cells without negatively affecting the function or solubility of the antibody, although it is expected that even one molecule of toxin/antibody will enhance cytotoxicity compared to 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 non-patent publications referred to above. Preferred maytansinoids are maytansinol and maytansinol analogs modified at aromatic rings or other positions of the maytansinol molecule, such as various maytansinol esters.

A number of linking groups are known in the art for use in the preparation of antibody-maytansinoid conjugates, including, for example, those disclosed in U.S. Pat. No. 5,208,020 or European patent 0425235B1, Chari et al, cancer research52:127-131(1992), and U.S. patent application No.10/960,602 filed on 8.10.2004, the disclosures of which are expressly incorporated herein by reference. Antibody-maytansinoid conjugates comprising the linker component SMCC can be prepared as disclosed in U.S. patent application No.10/960,602 filed on 8/10/2004. The linking group includes a disulfide group, a thioether group, an acid labile group, a photolabile group, a peptidase labile group, or an esterase labile group, as disclosed in the patents mentioned above, disulfide and thioether groups being preferred. Additional linking groups are described and exemplified herein.

Conjugates of the antibody and maytansinoid may be prepared 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), imidoesters (such as dimethyl adipimidate hcl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), 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) is used. Particularly preferred coupling agents include N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) (Carlsson et al, biochem. J.173:723-737 (1978)) and N-succinimidyl-4- (2-pyridylthio) valerate (SPP), thereby providing a disulfide linkage.

Depending on the type of linkage, linkers can be attached to various positions of the maytansinoid molecule. For example, ester linkages can be formed by reaction with hydroxyl groups using conventional coupling techniques. The reaction may occur at the C-3 position having a hydroxyl group, the C-14 position modified with a hydroxymethyl group, 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.

Auristatin and dolastatin

In some embodiments, immunoconjugates comprise an antibody of the invention conjugated to dolastatins (dolastatins) or dolastatin peptide analogs and derivatives, auristatins (U.S. Pat. Nos. 5,635,483;5,780,588). Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al (2001) antimicrob. Agents and Chemothet.45 (12):3580-3584) and to have anti-cancer (US5,663,149) and anti-fungal activity (Pettit et al (1998) antimicrob. Agents Chemothet.42: 2961-2965). Dolastatin or auristatin drug moieties (moieity) can be attached to an antibody via the N (amino) terminus or the C (carboxyl) terminus of a peptide drug moiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminally linked monomethyl auristatin drug modules DE and DF (i.e., MMAE and MMAF) as disclosed in Senter et al, Proceedings, Microsoft American Association for Cancer Research, Volume45, Abstract Number623, presented March28,2004, the disclosure of which is expressly incorporated herein in its entirety by reference.

Typically, peptide-based drug moieties can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for example, according to liquid phase synthesis methods well known in the art of peptide chemistry (see E. and K.L u bke, The Peptides, volume1, pp76-136,1965, Academic Press). The auristatin/dolastatin drug module can be prepared according to the methods in the following references: U.S. Pat. No. 5,635,483, U.S. Pat. No. 5,780588, Pettit et al (1989) J.Am.chem.Soc.111:5463-5465, Pettit et al (1998) Anti-Cancer Drug Design13:243-277, Pettit, G.R., et al.Synthesis,1996,719-725, Pettit et al (1996) J.chem.Soc.PerkinTrans.15:859-863, and Doronina (2003) Nat Biotechnol21(7): 778-784.

Calicheamicin

Another immunoconjugate of interest comprises an anti-TAT antibody conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics are capable of generating double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of calicheamicin family conjugates 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 of the company Cynamid, U.S.A.). Useful calicheamicin structural analogs include, but are not limited to, gamma₁ ^I、α₂ ^I、α₃ ^IN-acetyl-gamma₁ ^IPSAG and θ^I ₁(Hinman et al, Cancer Research53: 3336-. Another anti-tumor drug that can be conjugated to an antibody is QFA, which is an antifolate drug. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Thus, cellular uptake of these agents via antibody-mediated internalization greatly enhances their cytotoxic effects.

Other cytotoxic agents

Other anti-tumor agents that can be conjugated to the anti-TAT antibodies of the invention include BCNU, streptavidin, vincristine (vincristine), 5-fluorouracil, the family of agents collectively referred to as the LL-E33288 complex described in U.S. Pat. No. 5,053,394, U.S. Pat. No. 5,770,710, and epothilones (esperamicins) (U.S. Pat. No. 5,877,296).

Enzymatically active toxins and fragments thereof that may be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α -sarcin (sarcocin), Aleutesin (Aleutesfordii) toxic protein, dianthus chinensis (dianthin) toxic protein, Phytolacca americana (Phytolacca americana) toxic protein (PAPI, PAPII, and PAP-S), Momordica charantia (Mordicacharantia) inhibitor, Jatropha curcin (curcin), crotin (crotin), Saponaria officinalis (saponaria officinalis) inhibitor, gelonin alba (gelonin), mitomycin (gelonin), tricin (restrictocin), trichothecin (triomycin), and enomycin (enomycin). See, e.g., WO93/21232, published on month 10 and 28, 1993.

The invention also contemplates immunoconjugates formed between an antibody and a compound having nucleic acid degrading activity (e.g., a ribonuclease or a DNA endonuclease, such as a deoxyribonuclease; DNase).

For selective destruction of tumors, the antibody may comprise a highly radioactive atom. A variety of radioisotopes are available for generating radioconjugated anti-TAT antibodies. Examples include At²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹²And radioactive isotopes of Lu. Where the conjugate is to be used for diagnosis, the radioactive atom may be included for scintigraphic studies, e.g. Tc^99mOr I¹²³Or spin labels for Nuclear Magnetic Resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

Incorporation of radioactive or other labels into the conjugate can be carried out in known manner. For example, the peptides 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, may be attached via cysteine residues in the peptide^99mOr I¹²³、Re¹⁸⁶、Re¹⁸⁸And In¹¹¹. Yttrium-90 can be attached via lysine residues. The IODOGEN method (Frakeret al, biochem. Biophys. Res. Commun.80:49-57 (1978)) may be used to incorporate iodine-123. Other methods are described in detail in Monoclonal Antibodies in Immunoscintigraphy (Chatal, CRC Press, 1989).

Conjugates of the antibody and cytotoxic agent may be prepared 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), imidoesters (such as dimethyl adipimidate hcl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (p-diazoniumbenzoyl) -ethylenediamine), diisothiocyanates (such as toluene 2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene) is used. For example, a ricin immunotoxin may be prepared as described in Vitetta et al, Science238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelator for conjugating radionucleotides to antibodies. See WO 94/11026. The linker may be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al, Cancer Research52: 127-.

The compounds of the present invention specifically encompass, but are not limited to, ADCs prepared with the following crosslinking reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB, and SVSB ((4-vinylsulfone) succinimidyl benzoate, commercially available from, for example, Pierce Biotechnology, Inc. (Rockford, IL., U.S.A)). See page 467-498 of 2003-2004Applications Handbook and Catalog.

Alternatively, a fusion protein comprising an anti-TAT antibody and a cytotoxic agent may be prepared, for example, by recombinant techniques or peptide synthesis. The length of the DNA may comprise regions encoding the two parts of the conjugate, either adjacent to each other or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate.

In yet another embodiment, the antibody may be conjugated to a "receptor" (such as streptavidin) for tumor pre-targeting, wherein the antibody-receptor conjugate is administered to the patient, followed by clearance of unbound conjugate from the circulation using a clearing agent, followed by administration of a "ligand" (such as avidin) conjugated to a cytotoxic agent (such as a radionucleotide).

10. Immunoliposomes

The anti-TAT antibodies disclosed herein may also be formulated as immunoliposomes. "liposomes" refers to vesicles composed of various types of lipids, phospholipids and/or surfactants that can be used to deliver drugs to mammals. The components of liposomes are generally arranged in bilayer form, similar to the lipid arrangement of biological membranes. Liposomes containing antibodies can be prepared by methods known in the art, such as described in Epstein et al, Proc.Natl.Acad.Sci.USA82:3688(1985), Hwang et al, Proc.Natl.Acad.Sci.USA77:4030(1980), U.S. Pat. Nos. 4,485,045 and 4,544,545, and WO97/38731, published 1997 on 23/10. Liposomes with extended circulation time are disclosed in U.S. patent No. 5,013,556.

Particularly useful liposomes can be produced by reverse phase evaporation using lipid compositions comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter having a set pore size to produce liposomes having a desired diameter. Fab' fragments of the antibodies of the invention can be coupled to liposomes via a disulfide exchange reaction as described in Martin et al, J.biol.chem.257:286-288 (1982). Optionally, a chemotherapeutic agent is included in the liposomes. See Gabizon et al, J.Nationalcancer Inst.81(19):1484 (1989).

TAT binding oligopeptides

A TAT binding oligopeptide according to the present invention refers to an oligopeptide that binds, preferably specifically binds, to a TAT polypeptide as described herein. TAT binding oligopeptides may be chemically synthesized using known oligopeptide synthesis methodologies, or may be prepared and purified using recombinant techniques. TAT binding oligopeptides are typically 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 longer, wherein such oligopeptides are capable of specifically binding to a polypeptide described herein. TAT binding oligopeptides may be identified using well known techniques without undue experimentation. In this regard, it is noted that techniques for screening oligopeptide libraries for oligopeptides 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 No. WO84/03506 and WO84/03564; Geysen et al, Proc. Natl. Acl. 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, Synthetic Peptides antibodies, 130-as 149-su-ansa (1986; Geysen et al, J.n. nomol. 102, Immunol. 102, USA J.1991: 1988; Nat J.1998: Biotech. J.19931; Natl. J.1988; Nature J.1988: 1988; Biotech. J.19932; Natl. J.1998; Nature et al., Aust J.S. No. 32; Aust., Aust J.32; Aust., Aust J.S. No. D., 2000: No. D., 2000; Australian., 2000: No. D., 2000; Australian., 2000; Australin et al; Australin., 1984; Australin., EP; Aust., EP; Aust.

In this regard, phage display is a well-known technique by which large oligopeptide libraries can be screened to identify those members of the library that are capable of specifically binding to a polypeptide target. Phage display is a technique in which variant polypeptides are displayed on the surface of phage particles as fusion proteins with coat proteins (Scott, J.K. and Smith, G.P. (1990) Science249: 386). The utility of phage display is that large libraries of selectively randomized protein variants (or randomly cloned cdnas) can be rapidly and efficiently sorted for those sequences that bind to the target molecule with high affinity. The peptide (Cwirla, S.E.et al., (1990) Proc.Natl.Acad.Sci.USA87:6378) or protein (Lowman, H.B.et al., (1991) Biochemistry30:10832; Clackson, T.et al., (1991) Nature352:624; Marks, J.D.et al., (1991) J.MoI.biol.222:581; Kang, A.S.et al., (1991) Proc.Natl.Acad.Sci.USA88:8363) libraries have been used to screen millions of polypeptides or oligopeptides for those with specific binding properties (Smith, G.P. (1991) Current Opin.Biotechnol.2: 668). Phage libraries for sorting random mutants require strategies to construct and expand large numbers of variants, procedures for affinity purification using target receptors, and means to evaluate the results of binding enrichment. See U.S. Pat. nos. 5,223,409,5,403,484,5,571,689 and 5,663,143.

Although most phage display Methods have been described using filamentous phages, lambda-like (lambdoid) phage display systems (WO95/34683; US5,627,024), T4 phage display systems (Ren et al, Gene215:439(1998); Zhu et al, Cancer Research58(15):3209-3214(1998); Jiang et al, Infection & Immunity65(11):4770-4777(1997); Ren et al, Gene195(2):303-311(1997); Ren, Protein Sci.5:1833(1996); Efimov et al, Virus Genes10:173(1995)) and T7 phage display systems (Smith and Scott, Methods in Enzymology 257: 228 (1993); US5, 905) are also known.

Many other improvements and variations on the basic phage display concept have now been developed. These improvements enhance the ability of the display system to screen peptide libraries for binding to selected target molecules and to display functional proteins that have the potential to screen these proteins for desired properties. Combinatorial reaction devices for phage display reactions have been developed (WO98/14277) and phage display libraries have been used to analyse and control the properties of biomolecular interactions (WO98/20169; WO98/20159) and constrained helical peptides (WO 98/20036). WO97/35196 describes a method for isolating affinity ligands, wherein a phage display library is contacted with a first solution in which the ligand will bind to the target molecule and a second solution in which the affinity ligand will not bind to the target molecule to selectively isolate the bound ligand. WO97/46251 describes a method of biopanning a random phage display library with affinity purified antibodies, followed by isolation of bound phage, followed by a panning process using the wells of a microplate to isolate high affinity bound phage. The use of Staphylococcus aureus (Staphylococcus aureus) protein A as an affinity tag has been reported (Li et al, (1998) mol. Biotech.9: 187). WO97/47314 describes the use of substrate-subtracted libraries for discriminating between enzyme specificities, wherein combinatorial libraries are used, which may be phage display libraries. WO97/09446 describes a method of selecting enzymes suitable for use in detergents using phage display. Other methods of selecting specifically binding proteins are described in U.S. Pat. Nos. 5,498,538,5,432,018, and WO 98/15833.

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.

TAT binding organic molecules

TAT binding to organic molecules refers to organic molecules other than the oligopeptides or antibodies defined herein that bind, preferably specifically bind, to TAT polypeptides described herein. TAT-binding organic molecules can be identified and chemically synthesized using known methodologies (see, e.g., PCT publication Nos. WO00/00823 and WO 00/39585). TAT binding organic molecules are typically less than about 2000 daltons in size, or less than about 1500, 750, 500, 250, or 200 daltons in size, wherein such organic molecules capable of binding, preferably specifically binding, a TAT polypeptide described herein can be identified using well-known techniques without undue experimentation. In this regard, it is noted that the ability to screen libraries of organic molecules is usefulTechniques for molecules capable of binding to a polypeptide target are well known in the art (see, e.g., PCT publication Nos. WO00/00823 and WO 00/39585). The TAT binding organic molecule may be, for example, an aldehyde, ketone, oxime, hydrazone, semicarbazone (semicarbazone), carbazide (carbazide), primary amine, secondary amine, tertiary amine, N-substituted hydrazine, hydrazide, alcohol, ether, thiol, thioether, disulfide, carboxylic acid, ester, amide, urea, carbamate (carbamate), carbonate (carbonate), ketal (thioketal), acetal, thioacetal, aryl halide, aryl sulfonate (aryl sulfonate), alkyl halide, alkyl sulfonate (alkyl sulfonate), aromatic compound, heterocyclic compound, aniline, alkene, alkyne, diol, amino alcohol, hydrazone, semicarbazone (carbazide), carbazide, urea, carbamate (carbamate), carbonate (ketal), ketal (ketal), acetal, thioacetal, aryl halide, aryl sulfonate (aryl sulfonate), alkyl halide, alkyl sulfonate (alkyl sulfonate), aromatic compound, heterocyclic compound, aniline, alkene, alkyne, diol, amino alcohol, Oxazolidines, a,Oxazolines, thiazolidines, thiazolines, enamines, sulfonamides (sulfonamides), epoxides, aziridines (aziridines), isocyanates (isocyanates), sulfonyl chlorides, diazo compounds, acid chlorides, and the like.

D. Screening for anti-TAT antibodies, TAT binding oligopeptides, and TAT binding organic molecules having desirable properties

Techniques for generating antibodies, oligopeptides, and organic molecules that bind to TAT polypeptides have been described above. Antibodies, oligopeptides or other organic molecules may further be selected as desired to have certain biological properties.

The growth inhibitory effect of an anti-TAT antibody, oligopeptide or other organic molecule of the invention may be assessed by methods well known in the art, for example using cells that express TAT polypeptides either endogenously or following transfection with the TAT gene. For example, suitable tumor cell lines and TAT transfected cells can be treated with different concentrations of an anti-TAT monoclonal antibody, oligopeptide or other organic molecule of the invention for several 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 is by comparison in the presence or absence ofIn the absence of an anti-TAT antibody, a TAT binding oligopeptide or a cell treated when TAT binds to an organic molecule of the invention ³H-thymidine uptake. After treatment, cells were harvested and the amount of radioactivity incorporated into the DNA was measured in a scintillation counter. Suitable positive controls include treating the cell line with a growth inhibitory antibody known to inhibit growth of the selected cell line. Growth inhibition of tumor cells in vivo can be determined by a variety of methods known in the art. Preferably, the tumor cell is a cell that overexpresses a TAT polypeptide. Preferably, the anti-TAT antibody, TAT binding oligopeptide or TAT binding organic molecule will inhibit cell proliferation of TAT-expressing tumor cells in vitro or in vivo by about 25-100%, more preferably by about 30-100%, even more preferably by about 50-100% or 70-100%, and in one embodiment at a concentration of about 0.5-30 μ g/ml compared to untreated tumor cells. Growth inhibition can be measured in cell culture at antibody concentrations of about 0.5-30 μ g/ml or about 0.5nM to 200nM, where growth inhibition is measured 1-10 days after exposure of tumor cells to the antibody. An anti-TAT antibody is growth inhibitory in vivo if administration of the antibody at about 1 μ g/g to about 100mg/kg body weight results in a decrease in tumor volume or a decrease in tumor cell proliferation within about 5 days to 3 months, preferably within about 5 to 30 days, from the first administration of the antibody.

To select anti-TAT antibodies, TAT binding oligopeptides or TAT binding organic molecules that induce cell death, loss of membrane integrity can be assessed relative to controls, as indicated by, for example, Propidium Iodide (PI), trypan blue or 7AAD uptake. The PI uptake assay can be performed in the absence of complement and immune effector cells. Tumor cells expressing a TAT polypeptide are incubated with either media alone or media containing a suitable anti-TAT antibody (e.g., about 10 μ g/ml), a TAT-binding oligopeptide, or a TAT-binding organic molecule. Cells were incubated for a period of 3 days. After each treatment, the cells were washed and aliquoted (aliquot) into 35mm filter-capped (strained) 12x75 tubes (1 ml per tube, 3 tubes per treatment group) for removal of cell clumps. PI (10. mu.g/ml) was then added to the tube. Use ofFlow cytometer andthe samples were analyzed by CellQuest software (Becton Dickinson). Those anti-TAT antibodies, TAT binding oligopeptides, or TAT binding organic molecules determined by PI uptake to induce a statistically significant level of cell death may be selected as anti-TAT antibodies, TAT binding oligopeptides, or TAT binding organic molecules that induce cell death.

To screen for Antibodies, oligopeptides, or other organic molecules that bind to an epitope bound by an antibody of interest on a TAT polypeptide, a conventional cross-blocking assay can be performed, such as described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). This assay can be used to determine whether a test antibody, oligopeptide or other organic molecule binds to the same site or epitope as a known anti-TAT antibody. Alternatively, or in addition, epitope mapping (mapping) can be performed by methods known in the art. For example, antibody sequences can be mutagenized, such as by alanine scanning, to identify contact residues. Mutant antibodies were first tested for binding to polyclonal antibodies to ensure correct folding. In different methods, peptides corresponding to different regions of the TAT polypeptide can be used in competition assays, using several test antibodies or one test antibody and an antibody having a characterized or known epitope.

E. Antibody-dependent enzyme-mediated prodrug therapy (ADEPT)

The antibodies of the invention may also be used in ADEPT by coupling the antibody to a prodrug-activating enzyme which converts a prodrug (e.g. a peptidyl chemotherapeutic agent, see WO 81/01145) to an active anticancer agent. See, for example, WO88/07378 and U.S. Pat. No. 4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includes any enzyme capable of acting on a prodrug in such a way as to convert it to a more active, cytotoxic form.

Enzymes useful in the methods of the invention include, but are not limited to, alkaline phosphatase enzymes that can convert a phosphate-containing prodrug into the free drug; arylsulfatase that can convert a sulfate-containing prodrug into a free drug; cytosine deaminase which converts non-toxic 5-fluorocytosine into the anticancer drug 5-fluorouracil; proteases which can convert peptide-containing prodrugs into free drugs, such as serratia protease (serratia protease), thermolysin (thermolysin), subtilisin (subtilisin), carboxypeptidases and cathepsins (such as cathepsins B and L); a D-alanylcarboxypeptidase capable of converting a prodrug containing a D-amino acid substitution; carbohydrate-cleaving enzymes that can convert glycosylated prodrugs into free drugs, such as β -galactosidase and neuraminidase; beta-lactamases which convert drugs derivatized with beta-lactams into free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, that convert drugs derivatized at their amino nitrogens with phenoxyacetyl or phenylacetyl groups, respectively, to free drugs. Alternatively, an antibody having enzymatic activity, also known in the art as an "abzyme", can be used to convert a prodrug of the invention to a free active drug (see, e.g., Massey, Nature328:457-458 (1987)). Antibody-abzyme conjugates can be prepared as described herein for delivery of abzymes to a tumor cell population.

The enzymes of the invention can be covalently bound to anti-TAT antibodies by techniques well known in the art, such as the use of heterobifunctional cross-linkers as 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, Nature312:604-608 (1984)).

F. Full-length TAT polypeptides

The invention also provides newly identified and isolated nucleotide sequences that encode polypeptides referred to herein as TAT polypeptides. Specifically, cDNAs (partial or full length) encoding a variety of TAT polypeptides have been identified and isolated, as disclosed in further detail in the examples below.

As disclosed in the examples below, a number of cDNA clones have been deposited with the ATCC. The actual nucleotide sequence of those clones can be readily determined by the skilled artisan by sequencing the deposited clones using routine methods in the art. The predicted amino acid sequence can be determined from the nucleotide sequence using conventional techniques. For the TAT polypeptides and encoding nucleic acids described herein, in some instances, applicants have identified the identifiable reading frame believed to be the best based on the sequence information available at that time.

G. anti-TAT antibodies and TAT polypeptide variants

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 may be made. anti-TAT antibodies and TAT polypeptide variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA and/or by synthesizing the desired antibody or polypeptide. One skilled in the art will appreciate that amino acid changes can alter post-translational processing of the anti-TAT antibody or TAT polypeptide, such as changing the number or position of glycosylation sites or altering membrane anchoring characteristics.

Variations may be made in the anti-TAT antibodies and TAT polypeptides described herein, for example using any of the techniques and guidelines for conservative and non-conservative mutations described, for example, in U.S. patent No. 5,364,934. The variation 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 relative to the native sequence antibody or polypeptide. Optionally, the variation is by substitution of at least one amino acid in one or more domains of the anti-TAT antibody or TAT polypeptide with any other amino acid. By comparing the sequence of an anti-TAT antibody or TAT polypeptide with the sequence of homologous known protein molecules and minimizing the number of amino acid sequence changes made in highly homologous regions, guidelines can be found for determining which amino acid residues can be inserted, substituted or deleted without adversely affecting the desired activity. Amino acid substitutions may be the result of substituting one amino acid for another with similar structural and/or chemical properties, such as substituting leucine for serine, i.e., conservative amino acid substitutions. Insertions or deletions can optionally range from about 1 to 5 amino acids. Allowable variations can be determined by systematically making amino acid insertions, substitutions, or deletions in the sequence, and testing the resulting variants for activity exhibited by the full-length or mature native sequence.

Provided herein are anti-TAT antibodies and fragments of TAT polypeptides. For example, such fragments may be truncated at the N-terminus or C-terminus, or may lack internal residues, when compared to the full-length native antibody or protein. Certain fragments lack amino acid residues that are not critical for the desired biological activity of the anti-TAT antibody or TAT polypeptide.

anti-TAT antibodies and TAT polypeptide fragments may be prepared by any of a variety of conventional techniques. The desired peptide fragment may be chemically synthesized. An alternative method involves producing antibody or polypeptide fragments by enzymatic digestion, for example by treating the protein with an enzyme known to cleave the protein at sites defined by specific 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 ends of the DNA fragment are used as 5 'and 3' primers in PCR. Preferably, the anti-TAT antibodies and TAT polypeptide fragments share at least one biological and/or immunological activity with a native anti-TAT antibody or TAT polypeptide disclosed herein.

In particular embodiments, conservative substitutions of interest are shown in table 1 under the heading "preferred substitutions". If such substitutions result in a change in biological activity, more substantial changes, referred to as "exemplary substitutions" in Table 1, or as described further below with respect to amino acid classes, can be introduced and the products screened.

TABLE 1

Original residues	Example alternatives	Preferred alternatives
			Ala(A)	val;leu;ile	val
Arg(R)	lys;gln;asn	lys
			Asn(N)	gln;his;asp;lys;arg	gln
Asp(D)	glu;asn	glu
			Cys(C)	ser;ala	ser
Gln(Q)	asn;glu	asn
			Glu(E)	asp;gln	asp

Gly(G)	pro;ala	ala
			His(H)	asn;gln;lys;arg	arg
Ile(I)	leu, val, met, ala, phe, norleucine	leu
			Leu(L)	Norleucine, ile, val, met, ala, phe	ile
Lys(K)	arg;gln;asn	arg
			Met(M)	leu;phe;ile	leu
Phe(F)	trp;leu;val;ile;ala;tyr	leu
			Pro(P)	ala	ala
Ser(S)	thr	thr
			Thr(T)	val;ser	ser
Trp(W)	tyr;phe	tyr
			Tyr(Y)	trp;phe;thr;ser	phe
Val(V)	ile, leu, met, phe, ala, norleucine	leu

Substantial modification of the functional or immunological identity of anti-TAT antibodies or TAT polypeptides is accomplished by selecting substitutions that differ significantly in their effectiveness in maintaining: (a) the structure of the polypeptide backbone of the surrogate region, e.g., as a folded sheet or helix conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain. Naturally occurring residues may be grouped as follows, according to common side chain properties:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral, hydrophilic: cys, Ser, Thr, Asn, Gln;

(3) acidic: asp and Glu;

(4) basic: his, Lys, Arg;

(5) residues that affect side chain orientation: gly, Pro; and

(6) aromatic: trp, Tyr, Phe.

Non-conservative substitutions will require the exchange of one member of one of these classes for another. Such substitute residues may also be introduced into conserved substitution sites, or more preferably, into the remaining (non-conserved) sites.

Mutagenesis can be performed using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Cloned DNA may be subjected to 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, Gene34:315(1985)), restriction selection mutagenesis (restriction mutagenesis) (Wells et al, Philos. Trans. R.Soc. London SerA317:415(1986)), or other known techniques to generate anti-TAT antibody or TAT polypeptide variant DNA.

Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Among the preferred amino acids for scanning are the relatively small neutral amino acids. Such amino acids include alanine, glycine, serine and cysteine. Alanine is generally the preferred scanning amino acid in this group because it eliminates side chains on the beta-carbon and is unlikely to alter the backbone conformation of the variant (Cunninghamand Wells, Science244:1081-1085 (1989)). Alanine is also generally preferred because it is the most common amino acid. In addition, it is often found in both concealed and exposed locations (Creighton, The Proteins, W.H.Freeman & Co., N.Y.; Chothia, J.mol.biol.150:1 (1976)). If alanine substitutions do not result in sufficient amounts of variant, isosteric amino acids may be used.

Any cysteine residues not involved in maintaining the correct conformation of the anti-TAT antibody or TAT polypeptide may also be substituted, usually with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine bonds may be added to the anti-TAT antibody or TAT polypeptide to improve its stability (particularly when the antibody is an antibody fragment such as an Fv fragment).

A particularly preferred class of surrogate variants involves replacing one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody). Typically, the resulting variants selected for further development will have improved biological properties relative to the parent antibody from which they were produced. One convenient method of generating such surrogate variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) were mutated to generate all possible amino acid substitutions at each site. The antibody variants so produced are displayed in monovalent form on filamentous phage particles as fusions to the M13 gene III product packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues which contribute significantly to antigen binding. Alternatively, or in addition, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify contact points between the antibody and the human TAT polypeptide. Such contact residues and adjacent residues are candidates for substitution according to the techniques detailed herein. Once such variants are generated, the panel of variants is screened as described herein, and antibodies with superior properties in one or more relevant assays can be selected for further development.

Nucleic acid molecules encoding amino acid sequence variants of anti-TAT antibodies can be 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 non-variant form of an anti-TAT antibody.

H. Modifications of anti-TAT antibodies and TAT polypeptides

Covalent modifications of anti-TAT antibodies and TAT polypeptides are included within the scope of the invention. One type of covalent modification includes reacting a target amino acid residue of an anti-TAT antibody or TAT polypeptide with an organic derivatizing agent that is capable of reacting with a selected side chain or N-or C-terminal residue of the anti-TAT antibody or TAT polypeptide. Derivatization with bifunctional reagents can be used, for example, to crosslink anti-TAT antibodies or TAT polypeptides with a water-insoluble supporting matrix or surface for use in a method of purification of anti-TAT antibodies, and vice versa. Commonly used cross-linking agents include, for example, 1-bis (diazo-acetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters such as the ester formed with 4-azidosalicylic acid, homobifunctional imidoesters including disuccinimidyl esters such as 3,3' -dithiobis (succinimidyl propionate), bifunctional maleimides such as bis-N-maleimide-1, 8-octane, and reagents such as methyl-3- [ (p-azidophenyl) dithio ] propionamide ester.

Other modifications include deamidation of glutaminyl and asparaginyl residues to glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the alpha-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.

Another class of covalent modifications to an anti-TAT antibody or TAT polypeptide encompassed within the scope of the invention includes altering the natural glycosylation pattern of the antibody or polypeptide. "altering the native glycosylation pattern" is intended herein to mean deleting one or more of the carbohydrate moieties (moieities) found in the native sequence anti-TAT antibody or TAT polypeptide (either by eliminating potential glycosylation sites, or by deleting glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites not present in the native sequence anti-TAT antibody or TAT polypeptide. In addition, this phrase includes qualitative changes in the glycosylation of native proteins, involving changes in the nature and proportions of the various carbohydrate modules present.

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 recognition sequences for enzymatic attachment of a carbohydrate module to an asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates potential glycosylation sites. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine may also be used.

The addition of glycosylation sites to the anti-TAT antibody or TAT polypeptide is conveniently accomplished by altering the amino acid sequence to include one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). Such alterations may also be made by adding or substituting one or more serine or threonine residues to the sequence of the original anti-TAT antibody or TAT polypeptide (for the O-linked glycosylation site). The amino acid sequence of the anti-TAT antibody or TAT polypeptide can optionally be altered by changes at the DNA level, in particular by mutating the DNA encoding the anti-TAT antibody or TAT polypeptide at preselected bases, thereby generating codons that will be translated into the desired amino acids.

Another method of increasing the number of sugar moieties on an anti-TAT antibody or TAT polypeptide is by chemically or enzymatically conjugating a glycoside to the polypeptide. Such methods are described in the art, for example, WO87/05330, published on 11.9.1987, and Aplin and Wriston, CRC Crit. Rev. biochem., pp.259-306 (1981).

Removal of the sugar module present on the anti-TAT antibody or TAT polypeptide may be achieved by chemical or enzymatic methods, or by mutational substitution of codons encoding amino acid residues serving as targets for glycosylation. Chemical deglycosylation techniques are known in the art and are described, for example, in Hakimuddin et al, Arch. biochem. Biophys.259:52(1987) and Edge et al, anal. biochem.118:131 (1981). Enzymatic cleavage of the carbohydrate moiety on the polypeptide can be achieved by using a variety of endo-and exoglycosidases, as described by Thotakura et al, meth.enzvmol.138:350 (1987).

Another class of covalent modifications to anti-TAT antibodies or TAT polypeptides involves linking the antibody or polypeptide to one of a variety of non-proteinaceous polymers, such as polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylene (polyoxysylene), in the manner described 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 may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed, for example, in Remington's Pharmaceutical Sciences,16th edition, Osol, a.ed., 1980.

An anti-TAT antibody or TAT polypeptide of the invention may also be modified in such a way as to form a chimeric molecule comprising an anti-TAT antibody or TAT polypeptide fused to another heterologous polypeptide or amino acid sequence.

In one embodiment, such chimeric molecules comprise an anti-TAT antibody or fusion of a TAT polypeptide with a tag polypeptide that provides an epitope to which the anti-tag antibody can selectively bind. Epitope tags are typically located at the amino or carboxy terminus of an anti-TAT antibody or TAT polypeptide. The presence of such epitope-tagged forms of anti-TAT antibodies or TAT polypeptides can be detected using antibodies directed against the tag polypeptide. Furthermore, the provision of an epitope tag facilitates purification of the anti-TAT antibody or TAT polypeptide by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. A variety of 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; influenza HA tag polypeptide and its antibody 12CA5(Field et al, mol.cell.biol.8:2159-2165 (1988)); the C-myc tag and its antibodies 8F9,3C7,6E10, G4, B7 and 9E10 (Evan et al, Molecular and Cellular Biology5:3610-3616 (1985)); and the herpes simplex virus glycoprotein D (gD) tag and its antibodies (Paborsky et al, protein engineering3(6):547-553 (1990)). Other tag polypeptides include the Flag peptide (Hopp et al, Biotechnology6:1204-1210 (1988)); KT3 epitope peptide (Martin et al, Science255: 192-; alpha-tubulin epitope peptide (Skinner et al, J.biol.chem.266:15163-15166 (1991)); and the T7 gene 10 protein peptide tag (Lutz-Freyrmuth et al, Proc. Natl. Acad. Sci. USA87: 6393-.

In an alternative embodiment, the chimeric molecule may comprise an anti-TAT antibody or a fusion of a TAT polypeptide with an immunoglobulin or a specific region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as an "immunoadhesin"), such a fusion may be to the Fc region of an IgG molecule. The Ig fusion preferably comprises the replacement of at least one variable region within the Ig molecule with a soluble form (transmembrane domain deleted or inactivated) of an anti-TAT antibody or TAT polypeptide. In a particularly preferred embodiment, the immunoglobulin fusion comprises the hinge region, CH, of an IgG1 molecule₂Region and CH₃Region, or hinge region, CH₁Region, CH₂Region and CH₃And (4) a zone. See also U.S. Pat. No. 5,428,130 issued on month 6 and 27 of 1995 for the preparation of immunoglobulin fusions.

I. Preparation of anti-TAT antibodies and TAT polypeptides

The following description relates generally to the preparation of anti-TAT antibodies and TAT polypeptides by culturing cells transformed or transfected with a vector comprising nucleic acids encoding the anti-TAT antibodies and TAT polypeptides. The invention of course encompasses the use of alternative methods known in the art for making anti-TAT antibodies and TAT polypeptides. For example, suitable amino acid sequences or portions thereof may be generated by direct Peptide Synthesis using Solid Phase techniques (see, e.g., Stewart et al, Solid-Phase Peptide Synthesis, W.H.Freeman Co., san Francisco, CA,1969; Merrifield, J.am.Chem.Soc.85: 2149-one 2154 (1963)). In vitro protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be accomplished, for example, using an Applied Biosystems Peptide Synthesizer (Foster City, Calif.) according to the manufacturer's instructions. Portions of an anti-TAT antibody or TAT polypeptide may be chemically synthesized separately and combined using chemical or enzymatic methods to generate the desired anti-TAT antibody and TAT polypeptide.

1. Isolation of DNA encoding anti-TAT antibodies or TAT polypeptides

DNA encoding an anti-TAT antibody or TAT polypeptide may be obtained from a cDNA library prepared from tissues thought to have the anti-TAT antibody or TAT polypeptide mRNA and express it at detectable levels. Thus, human anti-TAT antibody or TAT polypeptide DNA may be conveniently obtained from a cDNA library prepared from human tissue. anti-TAT antibodies or TAT polypeptide-encoding genes may also be obtained from genomic libraries or by known synthetic procedures (e.g., automated nucleic acid synthesis).

Libraries may 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 thereby. Screening of cDNA or genomic libraries with selected probes can be performed using standard protocols, such as those described in Sambrook et al, Molecular Cloning, A Laboratory Manual, New York, Cold Spring Harbor Laboratory Press, 1989. An alternative method of isolating the gene encoding an 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).

Techniques for screening cDNA libraries are well known in the art. The oligonucleotide sequence chosen as a probe should be long enough and unambiguous enough to minimize false positives. The oligonucleotide is preferably labeled so that it is detectable upon hybridization to the DNA in the library being screened. Methods of labeling are well known in the art and include the use of radiolabels, like ³²P-labeled ATP, biotinylation, or enzyme labeling. Hybridization conditions, including medium and high stringency, are described in Sambrook et al, supra.

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

Nucleic acids having protein coding sequences can be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequences disclosed for the first time herein, and, if necessary, using Sambrook et al, supra, for routine primer extension procedures to detect precursors and processed intermediates of mRNA that may not be reverse transcribed into cDNA.

2. Selection and transformation of host cells

Host cells are transfected or transformed with expression or cloning vectors for anti-TAT antibody or TAT polypeptide production as described herein and cultured in conventional nutrient media appropriately adjusted for induction of promoters, selection of transformants, or amplification of genes encoding the desired sequences. Culture conditions, such as medium, temperature, pH, etc., can be selected by one of skill in the art without undue experimentation. In general, the principles, protocols and Practical techniques for maximizing cell culture productivity can be found in Mammarian cell Biotechnology, a Practical Approach, M.Butler, ed., IRL Press,1991 and Sambrook et al, supra.

Methods for transfection of eukaryotic cells and transformation of prokaryotic cells are known to the skilled worker, for example CaCl₂、CaPO₄Liposome-mediated and electroporation. Depending on the host cell used, transformation is carried out using standard techniques appropriate for such cells. Calcium treatment with calcium chloride, such as Sambrook et al, supra, or electroporation is commonly used for prokaryotic cells. Infection with Agrobacterium tumefaciens (Agrobacterium tumefaciens) is used for transformation of certain plant cells, as described by Shaw et al, Gene23:315(1983) and WO89/05859, published on 29.6.1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology52: 456-. General transfection of mammalian cell host systems is described in the U.S.National patent 4,399,216. Transformation into yeast is generally carried out according to the methods 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 may be used, such as nuclear microinjection, electroporation, fusion of bacterial protoplasts with intact cells, or polycations such as polybrene, polyornithine. Various techniques for transforming mammalian cells are described in Keown et al, Methods in Enzvmology185:527-537(1990) and Mansour et al, Nature336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryotes, yeast, or higher eukaryotes. Suitable prokaryotes include, but are not limited to, eubacteria, such as gram-negative or gram-positive organisms, for example enterobacteriaceae, such as e. A variety of E.coli strains are publicly available, such as E.coli K12 strain MM294(ATCC31,446), E.coli X1776(ATCC31,537), E.coli strain W3110(ATCC27,325) and K5772(ATCC53,635). Other suitable prokaryotic host cells include the family enterobacteriaceae, such as the genera Escherichia, such as Escherichia, e.g. Escherichia coli (e.coli), Enterobacter (Enterobacter), Erwinia (Erwinia), Klebsiella (Klebsiella), Proteus (Proteus), Salmonella, such as Salmonella typhimurium (Salmonella typhimurium), Serratia, such as Serratia marcescens (Serratia), and shigella (shigella), and bacillus, such as bacillus subtilis and bacillus licheniformis (b.licheniformis) (e.g. bacillus licheniformis, disclosed in DD266,710 published on 4.12.1989), Pseudomonas (Pseudomonas aeruginosa), such as Pseudomonas aeruginosa). These examples are illustrative and not restrictive. Strain W3110 is a particularly preferred host or parent host, since it is a commonly used host strain for fermentation of recombinant DNA products. Preferably, the host cell secretes a minimal amount of proteolytic enzymes. For example Strain W3110 can be modified to produce genetic mutations in genes encoding proteins endogenous to the host, examples of such hosts include e.coli W3110 strain 1a2, which has the complete genotype tonA; coli W3110 strain 9E4, which has the complete genotype tonapt 3; escherichia coli W3110 strain 27C7(ATCC55,244) having the complete genotype tonAPr 3phoA E15(argF-lac)169degPompT kan^r(ii) a Escherichia coli W3110 strain 37D6, with the complete genotype tonA ptr3phoAE15(argF-lac)169degP ompTs rbs7ilvG kan^r(ii) a Coli W3110 strain 40B4, which is a strain 37D6 with a non-kanamycin resistant degP deletion mutation; and E.coli strains harboring the mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783, granted on 8/7/1990. Alternatively, in vitro cloning methods, such as PCR or other nucleic acid polymerase reactions, are also suitable.

Full-length antibodies, antibody fragments, and antibody fusion proteins can be prepared in bacteria, particularly when glycosylation and Fc effector function are not required, such as when therapeutic antibodies are conjugated to cytotoxic agents (e.g., toxins) and the immunoconjugate itself exhibits tumor cell destruction efficacy. Full-length antibodies have a longer half-life in circulation. Preparation in E.coli is faster and more cost-effective. 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(JoIy et al), and U.S. Pat. No. 5,840,523(Simmons et al), which describe a Translation Initiation Region (TIR) and signal sequences for optimized expression and secretion, and which are incorporated herein by reference. After expression, the antibodies are separated from the E.coli cell paste in a soluble fraction and can be purified, for example, by means of a protein A or G column, depending on the isotype. The final purification can be performed analogously to the method used for purifying antibodies expressed, for example, in CHO cells.

In addition to prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding anti-TAT antibodies or TAT polypeptides. Saccharomyces cerevisiae (Saccharomyces cerevisiae) is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe (S)chizosaccharomyces pombe）(Beach and Nurse Nature290:140(1981); published 5/2 in EP139,383,1985); kluyveromyces (Kluyveromyces) host (U.S. Pat. No. 4,943,529; Fleer et al,Bio/Technology9:968-975(1991)) such as, for example, Kluyveromyces lactis (K.lactis) (MW98-8C, CBS683, CBS4574; Louvhouse et al,J. Bacteriol.154(2), 737-742(1983)), Kluyveromyces fragilis (K.fragilis) (ATCC12,424), Kluyveromyces bulgaricus (K.bulgaricus) (ATCC16,045), Kluyveromyces vachelli (K.wickeramii) (ATCC24,178), K.wallei (ATCC56,500), Kluyveromyces drosophilus (K.drosophilus) (ATCC36,906; Van den Berg et al,Bio/Technology135(1990), Kluyveromyces thermotolerans (K.thermolerans), and Kluyveromyces marxianus (K.marxianus); yarrowia (EP402,226); pichia pastoris (Pichia pastoris) (EP183,070; Sreekrishna et al, J.Basic Microbiol.28:265-278 (1988)); candida genus (Candida); trichoderma reesei (Trichoderma reesei) (EP244,234); neurospora crassa (Neurospora crassa) (Case et al,Proc.Natl.Acad.Sci.USA76:5259-5263 (1979)); schwanniomyces (Schwanniomyces) such as Schwanniomyces occidentalis (published 10/31 in EP394,538,1990); and filamentous fungi such as, for example, Neurospora (Neurospora), Penicillium (Penicillium), Tolypocladium (WO91/00357,1991, 1.10 days published), and Aspergillus (Aspergillus) hosts such as Aspergillus nidulans (A. nidulans) (Balance et al,Biochem. Biophys.Res.Commun.112:284-289(1983);Tilburn et al.，Gene26:205-221(1983);Yelton et al.,Proc.Natl.Acad.Sci.USA81:1470-1474(1984)) and Aspergillus niger (A. niger) (Kelly and Hynes,EMBO J.4:475-479(1985)). Methylotrophic yeasts (methylotrophic yeasts) suitable for the present invention include, but are not limited to, yeasts capable of growing on methanol selected from the genera: hansenula (Hansenula), Candida (Candida), Kloeckera (Kloeckera), Pichia (Pichia), Saccharomyces (Saccharomyces), Torulopsis (Torulopsis)) And Rhodotorula (Rhodotorula). A list of specific species that are examples of such yeasts is found in C.Anthony, The Biochemistry of biology, 269 (1982).

Suitable host cells for expression of glycosylated anti-TAT antibodies or TAT polypeptides are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, and cell cultures of plant cells such as cotton, maize, potato, soybean, petunia, tomato, tobacco. . Many baculovirus strains and variants and corresponding permissive insect host cells have been identified which are derived from hosts such as Spodoptera frugiperda (caterpillars), Aedes aegypti (mosquitoes), Aedes albopictus (mosquitoes), Drosophila melanogaster (fruit flies), and Bombyx mori. A variety of viral strains are publicly available for transfection, such as the L-1 variant of Autographa californica (Autographa californica) NPV and the Bm-5 strain of Bombyx mori (Bombyx mori) NPV, and such viruses may be used in accordance with the present invention as viruses herein, particularly for transfecting Spodoptera frugiperda cells.

However, vertebrate cells are of great interest and propagation by culturing (tissue culture) vertebrate cells has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney lines (293 or 293 cells subcloned for growth in suspension culture, Graham et al, 1977, J.Gen Virol.36: 59); baby hamster kidney cells (BHK, ATCC CCL 10); chinese hamster ovary cells/-DHFR (CHO, Urlaub et al, 1980, Proc. Natl. Acad. Sci. USA77: 4216); mouse Sertoli (sertoli) cells (TM 4, Mather,1980, biol. reprod.23: 243-; monkey kidney cells (CV 1, ATCC CCL 70); vero cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); bovine rat (buffaloat) hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL 51); TRI cells (Mather et al, 1982, Annals N.Y.Acad.Sci.383: 44-68); MRC5 cells; FS4 cells; and the human hepatoma line (Hep G2).

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 appropriately adjusted for induction of promoters, selection of transformants, or amplification of genes encoding the desired sequences.

3. Selection and use of replication-competent vectors

Nucleic acids encoding anti-TAT antibodies or TAT polypeptides (e.g., cDNA or genomic DNA) can be inserted into a replicative vector for cloning (DNA amplification) or expression. A variety of vectors are publicly available. The vector may be in the form of, for example, a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence can be inserted into the vector by a variety of methods. Typically, the DNA is inserted into an appropriate restriction enzyme site using techniques known in the art. Carrier members typically include, but are not limited to, one or more of the following: 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 comprising one or more of these building blocks employs standard ligation techniques known to the skilled artisan.

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 portion of DNA encoding an anti-TAT antibody or TAT polypeptide that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected from, for example, alkaline phosphatase, penicillinase, lpp, or a heat-stable enterotoxin II leader sequence. For yeast secretion, the signal sequence may be, for example, a yeast invertase leader, an alpha factor leader (including the sugar yeast and Kluyveromyces alpha-factor leader, see U.S. Pat. No. 5,010,182), or an acid phosphatase leader, a Candida albicans glucoamylase leader (EP 362,179 published 4/1990), or the signal described in WO90/13646 published 11/15/1990. In mammalian cell expression, mammalian signal sequences can be used to direct secretion of proteins, such as signal sequences for secreted polypeptides from the same or related species, and viral secretion leader sequences.

Both expression and cloning vectors contain nucleic acid sequences that enable 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 plasmid pBR322 is suitable for most gram-negative bacteria, the 2. mu. plasmid origin is suitable for yeast, and various viral origins (SV 40, polyoma, adenovirus, VSV, or BPV) can be used for cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene, also referred to as a selectable marker. Typical selection genes encode the following proteins: (a) conferring resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline; (b) supplementing the nutritional deficiency; or (c) provide key nutrients not available from complex media, such as a gene encoding a bacillus D-alanine racemase.

An example of a suitable selectable marker for mammalian cells is one that is capable of identifying cells that have the ability to take up nucleic acid encoding an anti-TAT antibody or TAT polypeptide, such as DHFR or thymidine kinase. When wild-type DHFR is used, a suitable host cell is a CHO cell line deficient in DHFR activity, prepared and propagated as Urlaub et al, Proc.Natl.Acad.Sci.USA77:4216 (1980). A suitable selection gene for yeast is the trp1 gene present in the yeast plasmid YRp7 (Stinchcomb et al, 1979, Nature282:39; Kingsman et al, 1979,Gene7:141;Tschemper et al.,1980,Gene10:157). the trp1 gene provides a selectable marker for yeast mutants lacking the ability to grow in tryptophan, such as ATCC No.44076 or PEP4-1 (Jones,1977, Genetics85: 12).

Expression and cloning vectors typically comprise a polynucleotide encoding an anti-TAT antibodyOr a nucleic acid sequence of a TAT polypeptide, operably linked to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use in prokaryotic hosts include the beta-lactamase and lactose promoter systems (change al.,Nature275:615(1978);Goeddel et al.,Nature281:544(1979)), alkaline phosphatase, the tryptophan (trp) promoter system (Goeddel,Nucleic acids Res.8:4057(1980); EP36,776), and hybrid promoters such as the tac promoter (deBoer et al,Proc.Natl. Acad.Sci.USA80:21-25(1983)). Promoters for use in bacterial systems will also comprise a Shine-Dalgarno (s.d.) sequence operably linked to DNA encoding an anti-TAT antibody or TAT polypeptide.

Examples of promoter sequences suitable for use in yeast hosts include 3-phosphoglycerate kinase (hitzeemanet al,J.Biol.Chem.255:2073(1980)) or other glycolytic enzymes (Hess et al, J.Adv. Enzyme Reg.7:149(1968);Holland,Biochemistry4900(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.

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. Vectors and promoters suitable for yeast expression are further described in EP73,657.

Transcription of anti-TAT antibodies or TAT polypeptides from vectors in mammalian host cells is under the control of, for example, promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published at 7/5 1989), adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis b virus and simian virus 40(SV40), from heterologous mammalian promoters such as the actin promoter or immunoglobulin promoter, and from heat shock promoters, provided such promoters are compatible with the host cell system.

Transcription of DNA encoding anti-TAT antibodies or TAT polypeptides by higher eukaryotic cells can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300bp, that act on a promoter to increase transcription. Many enhancer sequences are known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). However, typically an enhancer from a eukaryotic cell virus is used. 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. Enhancers may be spliced into the vector at a position 5' or 3' to the anti-TAT antibody or TAT polypeptide coding sequence, but are preferably located at a site 5' to the promoter.

Expression vectors for 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 typically available from the 5 'and occasionally 3' ends of untranslated regions of eukaryotic or viral DNA or cDNA. These regions comprise nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding an anti-TAT antibody or TAT polypeptide.

Other methods, vectors and host cells suitable for the synthesis of anti-TAT antibodies or TAT polypeptides in recombinant vertebrate cell culture after modification are described in Gethining et al, Nature293:620-625(1981), Mantei et al, Nature281:40-46(1979), EP117,060 and EP117,058.

4. Culturing host cells

Host cells for producing an anti-TAT antibody or TAT polypeptide of the invention may be cultured in a variety of media. Commercial culture media such as Ham's F10 (Sigma), minimal essential Medium (Sigma) ((R))MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's modified Eagle's medium (DMEM, Sigma) are suitable for culturing host cells. In addition, any of the media described in the following documents may be used as the medium for the host cells: ham et al, 1979, meth.Enz.58:44, Barnes et al, 1980, anal. biochem.102: 255; us patent 4,767,704; 4,657,866, respectively; 4,927,762, respectively; 4,560,655, respectively; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. patent re.30,985. Any of these media may be supplemented as needed 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) ^TMDrugs), trace elements (defined as inorganic compounds usually present in final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements known to those skilled in the art may also be included in appropriate concentrations. Culture conditions such as temperature, pH, etc. that were previously selected for the host cell for expression will be apparent to the ordinarily skilled artisan.

5. Detecting gene amplification/expression

Amplification and/or expression of the gene can be measured directly in the sample, for example by conventional Southern blotting, Northern blotting for quantification of mRNA transcription (Thomas, Proc. Natl. Acad. Sci. USA77: 5201. 5205(1980)), dot blotting (DNA analysis) or in situ hybridization using a suitable labelled probe according to the sequences provided herein. Alternatively, antibodies that recognize specific duplexes including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes may be used. The antibody may then be labeled and an assay may be performed in which the duplex is bound to a surface such that the presence of antibody bound to the duplex is detectable when the duplex is formed on the surface.

Alternatively, for direct quantification of gene product expression, gene expression can be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and assays of cell cultures or body fluids. Antibodies useful for immunohistochemical staining and/or sample fluid assays may be monoclonal or polyclonal and may be prepared in any mammal. Conveniently, antibodies can be made against the native sequence TAT polypeptide or against synthetic peptides based on the DNA sequences provided herein or against foreign sequences fused to TAT DNA and encoding particular antibody epitopes.

6. Purification of anti-TAT antibodies and TAT polypeptides

Various forms of anti-TAT antibodies and TAT polypeptides can be recovered from the culture broth or from host cell lysates. If membrane bound, it may be released from the membrane using a suitable detergent solution (e.g., Triton-X100) or by enzymatic cleavage. Cells employed in the expression of anti-TAT antibodies and TAT polypeptides may be disrupted by a variety of physical or chemical means, such as freeze-thaw cycles, sonication, mechanical disruption, or a lytic agent.

It may be desirable to purify anti-TAT antibodies and TAT polypeptides from recombinant cellular proteins or polypeptides. The following scheme is illustrative of a suitable purification scheme: fractionation on an ion exchange column; ethanol precipitation; reversed phase HPLC; chromatography on silica or cation exchange resins such as DEAE; carrying out chromatographic focusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein aseplose columns to remove contaminants such as IgG; and a metal chelating column that binds to the anti-TAT antibody and an epitope-tagged form of the TAT polypeptide. A variety of protein purification methods can be employed, such methods are known in the art and are described, for example, in Deutscher,Methods in Enzymology,182(1990);Scopes,Protein Purification:Principes and Practice,Springer-Verlag, New York (1982). The choice of purification step will depend, for example, on the nature of the production method used and the particular anti-TAT antibody or TAT polypeptide produced.

When using recombinant techniques, the antibody may be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the antibody is produced intracellularly, as a first step, the host cells or the particulate debris of the lysed fragments are removed, for example, by centrifugation or ultrafiltration. Carter et al, Bio/Technology10: 163-. Briefly, the cell paste was thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation. If the antibody is secreted into the culture medium, the supernatant from such expression systems is typically first concentrated using a commercial protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. In any of the above steps, a protease inhibitor such as PMSF may be included to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

Antibody compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a preferred purification technique. The suitability of protein a as an affinity ligand depends on the type and isotype of any immunoglobulin Fc region present in the antibody. Protein A can be used for purification based on human gamma ¹、γ²Or gamma⁴Antibodies to heavy chains (Lindmark et al, 1983, J.Immunol. meth.62: 1-13). Protein G is recommended for all mouse isotypes and for human gamma 3(Guss et al, 1986, EMBO J.5: 1567-1575). The matrix to which the affinity ligand is attached is most commonly agarose, but other matrices may be used. Physically stable matrices such as controlled pore glass or poly (styrenedivinyl) benzene enable faster flow rates and shorter processing times than agarose. For containing C_H3 domain antibodies, Bakerbond ABX can be used^TMPurification was performed on resin (j.t. baker, phillips burg, NJ). Depending on the antibody to be recovered, other protein purification techniques may also be used, such as fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica, heparin Sepharose^TMChromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

After any preliminary purification step, the mixture comprising the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH of about 2.5-4.5, preferably at low salt concentrations (e.g., about 0-0.25M salt).

J. Pharmaceutical formulations

Therapeutic formulations of anti-TAT antibodies, TAT binding oligopeptides, TAT binding organic molecules and/or TAT polypeptides for use according to the invention may be prepared by mixing the antibody, polypeptide, oligopeptide or organic molecule of the desired purity, in lyophilized form or in aqueous solution, with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences,16th edition, Osol, a.ed., 1980). 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 acid buffers; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexane diamine chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol 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; tonicity adjusting agents (tonicifiers), such as trehalose and sodium chloride; sugars such as sucrose, mannitol, trehalose, or sorbitol; surfactants such as polysorbates; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants, such as TWEEN ^TM、PLURONICS^TMOr polyethylene glycol (PEG). The formulation of the antibody preferably comprises the antibody at a concentration of 5-200mg/ml, preferably 10-100 mg/ml.

The formulations herein may also contain more than one active compound necessary for the particular indication being treated, preferably 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 another antibody in one formulation, for example a second anti-TAT antibody that binds to a different epitope on the TAT polypeptide, or an antibody directed against some other target such as a growth factor that affects the growth of a particular cancer. Alternatively or additionally, the composition may further comprise a chemotherapeutic agent, a cytotoxic agent, a cytokine, a growth inhibitory agent, an anti-hormonal agent and/or a cardioprotective agent. Suitably, such molecules are present in combination in amounts effective for the intended purpose.

The active ingredient may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed, for example, in Remington's pharmaceutical Sciences,16th edition, Osol, a.ed., 1980.

Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable 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 (e.g., poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and L-glutamic acid gamma-ethyl ester, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRONDEPOT^TM(injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D- (-) -3-hydroxybutyric acid.

Formulations for in vivo administration must be sterile. This can be easily achieved by filtration using sterile filtration membranes.

K. Diagnostics and therapeutics using anti-TAT antibodies, TAT binding oligopeptides, and TAT binding organic molecules

To determine TAT expression in cancer, a variety of diagnostic assays are available. In one embodiment, TAT polypeptide overexpression may be assayed by Immunohistochemistry (IHC). The IHC assay can be performed on paraffin-embedded tissue sections from tumor biopsies and given the following TAT protein staining intensity criteria: score 0: no staining was observed or membrane staining was observed in less than 10% of the tumor cells.

Score 1 +: faint/barely detectable membrane staining was detected in more than 10% of tumor cells. The cells were stained only on a portion of their membrane.

And the score is 2 +: weak to moderate complete membrane staining was observed in more than 10% of tumor cells.

Score 3 +: moderate to intense complete membrane staining was observed in more than 10% of tumor cells.

Those tumors that score a TAT polypeptide of 0 or 1+ may be identified as not overexpressing TAT, while those that score 2+ or 3+ may be identified as overexpressing TAT.

Alternatively, or in addition, FISH assays such as may be performed on formalin-fixed, paraffin-embedded tumor tissue(sold by Ventana, Arizona) or(Vysis, Illinois) to determine the extent of TAT overexpression, if any, in tumors.

TAT overexpression or amplification can also be assessed using in vivo diagnostic assays, for example by administering a molecule (such as an antibody, oligopeptide or organic molecule) that binds to the molecule to be detected and is labeled with a detectable label (e.g., a radioisotope or fluorescent label), and then externally scanning the patient to locate the label.

As noted above, the anti-TAT antibodies, oligopeptides, and organic molecules of the present invention have a variety of non-therapeutic applications. The anti-TAT antibodies, oligopeptides, and organic molecules of the invention are useful in the diagnosis and staging of cancers that express TAT polypeptides (e.g., in radioimaging). Antibodies, oligopeptides, and organic molecules may also be used to purify or immunoprecipitate TAT polypeptides from cells for in vitro detection and quantification of TAT polypeptides, e.g., in ELISA or Western blots, as a step in purifying other cells to kill and eliminate TAT-expressing cells from a mixed population of cells.

Now, depending on the grade of cancer, the treatment of cancer involves one or a combination of the following therapies: removing cancerous tissue by surgery, radiotherapy and chemotherapy. anti-TAT antibodies, oligopeptides, or organic molecule therapies may be particularly desirable for metastatic disease where the elderly patients and radiation therapy do not tolerate the toxicity and side effects of chemotherapy well. The tumor-targeting anti-TAT antibodies, oligopeptides, or organic molecules of the invention may be used to alleviate TAT-expressing cancers at the initial diagnosis of the disease or during relapse. For therapeutic applications, anti-TAT antibodies, oligopeptides or organic molecules may be used alone or in combination therapy with, for example, hormones, anti-angiogenic agents (anti-angiogenic) or radiolabeled compounds, or with surgery, cryotherapy and/or radiation therapy. Treatment with anti-TAT antibodies, oligopeptides, or organic molecules may be administered in combination with, sequentially to, before, or after other forms of conventional therapy. Chemotherapeutic agents such asDocetaxel (doxetaxel),Paclitaxel (paclitaxel), estramustine (estramustine) and mitoxantrone (mitoxantrone) are used for the treatment of cancer, in particular in low risk (goodrisk) patients. In the methods of treating or ameliorating cancer of the invention, a cancer patient may be administered a treatment with an anti-TAT antibody, oligopeptide or organic molecule in combination with one or more of the foregoing chemotherapeutic agents. In particular, paclitaxel is envisaged And improved derivatives (see, e.g., EP 0600517). The anti-TAT antibody, oligopeptide or organic molecule is administered with a therapeutically effective dose of a chemotherapeutic agent. In another embodiment, an anti-TAT antibody, oligopeptide or organic molecule is administered in combination with chemotherapy to enhance the activity and efficacy of a chemotherapeutic agent, such as paclitaxel. The Physicians' Desk Reference (PDR) discloses the dosages of these agents that have been used in the treatment of various cancers. The therapeutically effective dosage regimen and dosage of these aforementioned chemotherapeutic agents will depend on the particular cancer being treated, the extent of the disease, and other factors familiar to the skilled physician, and can be determined by the physician.

In a particular embodiment, a conjugate comprising an anti-TAT antibody, oligopeptide or organic molecule conjugated to 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 nucleic acid in cancer cells. Examples of such cytotoxic agents are described above and include maytansinoids, calicheamicin, ribonucleases and DNA endonucleases.

The anti-TAT antibody, oligopeptide, organic molecule or toxin conjugate thereof is administered to a human patient according to known methods, such as intravenous administration, e.g. as a bolus injection (bolus) or continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical or inhalation routes. Preferably, the antibody, oligopeptide or organic molecule is administered intravenously or subcutaneously.

Administration of anti-TAT antibodies, oligopeptides, or organic molecules may be combined with other treatment regimens. Co-administration includes co-administration using separate formulations or a single pharmaceutical formulation, as well as sequential administration in any order, wherein preferably all two (or more) active agents exert their biological activities simultaneously over a period of time. Preferably, such combination therapy results in a synergistic therapeutic effect.

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

In another embodiment, the therapeutic treatment method of the invention involves the combined administration of anti-TAT antibody(s), oligopeptide or organic molecule(s) with one or more chemotherapeutic agents or growth inhibitory agents, including the combined administration of a mixture of different chemotherapeutic agents (cocktail). Chemotherapeutic agents include estramustine phosphate (estramustine phosphate), prednimustine (prednimustine), cisplatin, 5-fluorouracil, melphalan (melphalan), cyclophosphamide, hydroxyurea and hydroxyurea taxanes (hydroxyurea taxanes), such as paclitaxel and docetaxel, and/or anthracycline (anthracycline) antibiotics. The formulation and dosing regimen for such chemotherapeutic agents can be used in accordance with the manufacturer's instructions or determined empirically by the skilled practitioner. Preparation and dosing regimens for such chemotherapies can also be found in chemotherapy service ed, m.c. perry, Williams & Wilkins, Baltimore, MD (1992).

The antibody, oligopeptide or organic molecule may be combined with an anti-hormonal compound, e.g., an anti-estrogenic compound such as tamoxifen (tamoxifen), at known doses of such molecules; antiprogestinic compounds such as onapristone (see EP616,812); or an antiandrogen compound such as flutamide. When the cancer to be treated is an androgen-independent cancer, the patient may have previously received anti-androgen therapy, and when the cancer becomes androgen-independent, an anti-TAT antibody, oligopeptide or organic molecule (optionally together with other agents described herein) may be administered to the patient.

Sometimes, it may also be beneficial to also co-administer a cardioprotective agent (to prevent or reduce myocardial dysfunction associated with therapy) or one or more cytokines to the patient. In addition to the above treatment regimens, the patient may be subjected to surgical removal of cancer cells and/or radiation therapy prior to, concurrently with, or subsequent to the antibody, oligopeptide or organic molecule therapy. Suitable dosages for any of the above co-administered agents are those presently used and may be reduced by the combined effect (synergy) of the agent with the anti-TAT antibody, oligopeptide or organic molecule.

For the prevention or treatment of disease, the dosage and mode of administration may be selected by a physician according to known criteria. The appropriate dosage of the antibody, oligopeptide or organic molecule will depend on the type of disease to be treated, the severity and course of the disease as defined above, whether the antibody, oligopeptide or organic molecule is administered for prophylactic or therapeutic purposes, previous therapy, the clinical history and response of the patient 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 once or over a series of treatments. Preferably, the antibody, oligopeptide or organic molecule is administered by intravenous infusion or by subcutaneous injection. Depending on the type and severity of the disease, about 1. mu.g/kg to about 50mg/kg body weight (e.g., about 0.1-15 mg/kg/dose) of the antibody may be administered to the patient as an initial candidate dose, whether, for example, by one or more separate administrations, or by continuous infusion. A dosing regimen may comprise administering an initial loading dose of about 4mg/kg, followed by a weekly maintenance dose of about 2mg/g of anti-TAT antibody. However, other dosage regimens may be used. Depending on the factors described above, a typical daily dose may be administered in the range of about 1 μ g/kg to 100mg/kg or more. For repeated administrations over several days or longer, depending on the condition, the treatment is maintained until the desired suppression of disease symptoms occurs. The progress of such therapy can be readily monitored by routine methods and assays, and based on criteria known to a physician or other person skilled in the art.

In addition to administering antibody proteins to patients, the present application contemplates administration of antibodies by gene therapy. Such administration of a nucleic acid encoding an antibody is encompassed in the expression "administering a therapeutically effective amount of the antibody". For the use of gene therapy to generate intrabodies see, for example, WO96/07321, published 3/14 1996.

There are two main approaches to the entry of nucleic acids (optionally contained in a vector) into the cells of a patient, i.e., in vivo and ex vivo. For in vivo delivery, the nucleic acid is typically injected directly into the patient at the site where the antibody is desired. For ex vivo treatment, the patient's cells are harvested, the nucleic acid is introduced into these isolated cells, and the modified cells are either administered directly to the patient, or, for example, are packed into a porous membrane and 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 living cells. These techniques vary depending on whether the nucleic acid is transferred to cultured cells in vitro or in vivo of the intended host. Suitable techniques for transferring nucleic acids into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, calcium phosphate precipitation, and the like. Commonly used vectors for ex vivo gene delivery are retroviral vectors.

Presently 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 (lipids that can be used for lipid-mediated gene transfer are e.g. DOTMA, DOPE and DC-Chol). For a review of currently known gene markers and gene therapy protocols, see Anderson et al, Science256:808-813 (1992). See also WO93/25673 and references cited therein.

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

In one embodiment, the antibody competes for binding to, or substantially binds to, the same epitope as an antibody of the invention. Antibodies having the biological characteristics of the anti-TAT antibodies of the invention are also envisaged, specifically including in vivo tumor targeting and any cell proliferation inhibition or cytotoxicity feature.

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

The anti-TAT antibodies, oligopeptides, and organic molecules of the present invention are useful for treating or alleviating one or more symptoms of TAT-expressing cancer in a mammal. Such cancers include prostate, urinary tract, lung, breast, colon and ovarian cancers, more particularly prostate adenocarcinoma, renal cell carcinoma, colorectal adenocarcinoma, adenocarcinoma of the lung, squamous cell carcinoma of the lung and pleural mesothelioma. Cancer encompasses any of the foregoing metastatic cancers. The antibodies, oligopeptides, or organic molecules of the invention are capable of binding to at least a portion of a cancer cell in a mammal that expresses a TAT polypeptide. In a preferred embodiment, the antibody, oligopeptide or organic molecule is effective in destroying or killing TAT-expressing tumor cells or inhibiting the growth of such tumor cells when bound to a TAT polypeptide on the cells in vitro or in vivo. Such antibodies include naked anti-TAT antibodies (not conjugated to any agent). Naked antibodies with cytotoxic or cytostatic properties may further cooperate with cytotoxic agents (harnesss) to make them more effective in destroying tumor cells. Cytotoxic properties may be imparted to an anti-TAT antibody by, for example, coupling the antibody to a cytotoxic agent to form an immunoconjugate as described herein. The cytotoxic or growth inhibitory agent is preferably a small molecule. Toxins such as calicheamicin or maytansinoids and analogs or derivatives thereof are preferred.

The invention provides compositions comprising an anti-TAT antibody, oligopeptide or organic molecule of the invention and a carrier. For the purpose of treating cancer, a composition may be administered to a patient in need of such treatment, wherein the composition may comprise one or more anti-TAT antibodies in the form of an immunoconjugate or a naked antibody. In another embodiment, the compositions may 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.

Another aspect of the invention is an isolated nucleic acid encoding an anti-TAT antibody. The invention encompasses nucleic acids encoding both heavy and light chains, particularly hypervariable region residues, chains encoding the natural sequence antibodies as well as variant, modified and humanized versions of the antibodies.

The invention also provides methods useful for treating a TAT polypeptide expressing cancer or alleviating one or more symptoms of such cancer in a mammal, comprising administering to the mammal a therapeutically effective amount of an anti-TAT antibody, oligopeptide or organic molecule. The therapeutic compositions of antibodies, oligopeptides or organic molecules may be administered, either short-term or long-term or intermittently, as directed by a physician. Methods of inhibiting growth of cells expressing TAT polypeptides and killing such cells are also provided.

The invention also provides kits and articles of manufacture comprising at least one anti-TAT antibody, oligopeptide or organic molecule. Kits comprising anti-TAT antibodies, oligopeptides, or organic molecules can be used, for example, in killing assays for cells expressing TAT, purifying or immunoprecipitating TAT polypeptides from cells. For example, for isolation and purification of TAT, the kit may comprise an anti-TAT antibody, oligopeptide or organic molecule coupled to a bead (e.g., sepharose bead). Kits comprising antibodies, oligopeptides, or organic molecules may be provided for in vitro detection and quantification of TAT, for example in ELISA or Western blot. Such antibodies, oligopeptides or organic molecules useful for detection may be provided with a label such as a fluorescent or radioactive label.

L. preparations and kits another aspect of the invention is a preparation comprising a substance useful for treating TAT-expressing cancers. The article comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be made of various materials, such as glass or plastic. The container contains a composition effective for treating a cancer condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or 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 for use in treating cancer. The label or package insert further comprises instructions for administering the antibody, oligopeptide or organic molecule composition to a cancer patient. In addition, the article of manufacture may also include a second container having a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.

Also provided are kits that can be used for a variety of purposes, such as for killing assays of cells expressing TAT, for purifying or immunoprecipitating TAT polypeptides from cells. For isolation and purification of TAT, the kit may comprise an anti-TAT antibody, oligopeptide or organic molecule coupled to a bead (e.g. sepharose bead). Kits comprising antibodies, oligopeptides, or organic molecules may be provided for in vitro detection and quantification of TAT polypeptides, for example in ELISA or Western blot. Like the article of manufacture, the kit includes a container and a label or package insert on or associated with the container. The container contains a composition comprising at least one anti-TAT antibody, oligopeptide or organic molecule of the invention. Additional containers may be included containing, for example, diluents and buffers, control antibodies. The label or package insert can provide a description of the composition as well as instructions for the intended in vitro or diagnostic use.

Use of TAT polypeptides and nucleic acids encoding TAT polypeptides

Nucleotide sequences encoding TAT polypeptides (or complements thereof) have a variety of applications in the field of molecular biology, including use as hybridization probes, for chromosome and gene mapping, and for the generation of antisense RNA and DNA probes. TAT-encoding nucleic acids may also be used to prepare TAT polypeptides by recombinant techniques described herein, wherein those TAT polypeptides may be used, for example, to prepare anti-TAT antibodies described herein.

The full-length native sequence TAT gene, or a portion thereof, can be used as a hybridization probe in cDNA libraries to isolate full-length TAT cdnas or to isolate other cdnas (e.g., those encoding naturally occurring variants of TAT or cdnas from TAT of other species) having desired sequence identity to the native TAT sequences disclosed herein. Optionally, the probe is from about 20 to about 50 bases in length. Hybridization probes can be derived from at least partially new regions of the full-length native nucleotide sequence, where those regions can be determined without undue experimentation, or from genomic sequences containing promoters, enhancer elements, and introns of the native sequence TAT. For example, the screening method will include isolating the coding region of the TAT gene using a known DNA sequence to synthesize a selected probe of about 40 bases. Hybridization probes can be labeled with a variety of labels, including radionucleotides, such as³²P or³⁵S, or an enzymatic label such as alkaline phosphatase coupled to the probe via an avidin/biotin coupling system. Labeled probes having a sequence complementary to that of the TAT gene of the invention can be used to screen human cDNA, genomic DNA, or mRNA libraries to determine which members of such libraries the probe hybridizes to. The following examples describe the hybridization techniques in more detail. Any EST sequence disclosed in the present application can similarly be used as a probe using the methods disclosed herein.

Other useful fragments of nucleic acids encoding TAT include antisense or sense oligonucleotides, including single-stranded nucleic acid sequences (either RNA or DNA) capable of binding to the target TAT mRNA (sense) or TAT DNA (antisense) sequence. In accordance with the present invention, the antisense or sense oligonucleotide comprises a fragment of a TAT DNA coding region. Such fragments typically comprise at least about 14 nucleotides, preferably about 14 to 30 nucleotides. The ability to derive antisense or sense oligonucleotides based on cDNA sequences encoding a given protein is described, for example, in Stein and Cohen, Cancer Res.48:2659,1988 and van der Krol et al, BioTechniques6:958,1988.

Binding of the antisense or sense oligonucleotide to the target nucleic acid sequence results in the formation of a duplex that blocks transcription or translation of the target sequence by one of a variety of means, including enhanced degradation of the duplex, premature termination of transcription or translation, or other means. Such methods are encompassed by the present invention. Antisense oligonucleotides are therefore useful for blocking the expression of TAT proteins, where those TAT proteins may play a role in inducing cancer in mammals. Antisense or sense oligonucleotides also include oligonucleotides having modified sugar-phosphodiester backbones (or other sugar linkages (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., are capable of resisting enzymatic degradation), but retain sequence specificity capable of binding to the target nucleotide sequence.

Preferred intragenic sites for antisense binding include regions incorporating the translation initiation codon (5' -AUG/5' -ATG) or the 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 the 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 the translation initiation or termination codon. Other preferred regions for antisense binding include: an intron; an exon; an intron-exon junction; an Open Reading Frame (ORF) or "coding region," i.e., the region between the translation initiation codon and the translation termination codon; a 5' cap of mRNA comprising N7-methylated guanosine residues linked to the 5' most residue of the mRNA via a 5' -5' triphosphate linkage, including the 5' cap structure itself and the first 50 nucleotides adjacent to the cap; a 5 'untranslated region (5' UTR), i.e., a portion of an mRNA in the 5 'direction from the translation initiation codon, thus including nucleotides between the 5' cap site and the translation initiation codon in the mRNA or corresponding nucleotides on the gene; and a 3 'untranslated region (3' UTR), i.e., a portion of the mRNA in the 3 'direction from the translation stop codon, thus including nucleotides between the translation stop codon and the 3' end in the mRNA or corresponding nucleotides on the gene.

Specific examples of preferred antisense compounds that can be used to inhibit the expression of TAT protein include oligonucleotides comprising 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 sometimes with reference to in the art, a modified oligonucleotide that does not have a phosphorus atom in its internucleoside backbone may also be considered an oligonucleotide. Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters (aminoalkylphosphotriesters), methyl and other hydrocarbyl phosphonates including 3'-alkylene phosphonates (3' -alkylene phosphates), 5'-alkylene phosphonates (5' -alkylene phosphonates) and chiral phosphonates, phosphites, phosphoramidates including 3'-amino phosphoramidates (3' -aminophosphoramidates) and aminoalkyl phosphoramidates (aminoalkylphosphoramidates), phosphorothioates (thiophosphamides), phosphorothioates (thiophospharates), phosphorothioates (thiophosphates), phosphorothioates (thioalkylphosphotriesters), phosphorothioates (thiophosphatesters), phosphorothioates (phosphorothioates), and like with normal 3'-5' linkages, and polar analogs of those with reversed polarity, wherein one or more internucleotide linkages are a 3 'to 3', 5 'to 5', or 2 'to 2' linkage. Preferred oligonucleotides with inverted polarity comprise a single 3' to 3' linkage at the 3' most internucleotide linkage, i.e., a single inverted nucleoside residue that can be abasic (nucleobase deleted or substituted with hydroxyl). Various salt, mixed salt and free acid forms are also included. Representative U.S. patents that teach the preparation of phosphorus-containing bonds 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 incorporated herein by reference.

Preferred modified oligonucleotide backbones that do not contain phosphorus atoms therein have backbones formed of short chain hydrocarbyl or cycloalkyl internucleoside linkages, mixed heteroatom and hydrocarbyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of the nucleoside); a siloxane backbone; sulfide, sulfoxide and sulfone backbones; formyl (formacetyl) and thioformyl (thioformacetyl) backbones; methylene formyl (formaceyl) and thioformyl (thioformaceyl) backbones; a ribose acetyl backbone; an alkene-containing backbone; a sulfamate (sulfamate) backbone; methylene imino and methylene hydrazino (methylene hydrazino) backbones; sulfonate and sulfonamide (sulfonamide) backbones; an amide skeleton; and others with blends N, O, S and CH₂The skeleton of the component. Representative U.S. patents that teach the preparation of such oligonucleotides 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 incorporated herein by reference.

In other preferred antisense oligonucleotides, the sugar and internucleoside linkages of the nucleotide units, i.e.the backbone, are replaced with new groups. The base unit is maintained to hybridize with a suitable nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic, has been shown to have excellent hybridization properties, and is known as Peptide Nucleic Acid (PNA). In PNA compounds, the sugar backbone of the oligonucleotide is replaced with an amide-containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and directly or indirectly bound to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. 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 incorporated herein by reference. Further teachings of PNA compounds can be found in Nielsen et al, 1991, Science254: 1497-.

Preferred antisense oligonucleotides incorporate a phosphorothioate backbone and/or a heteroatom backbone, particularly-CH₂-NH-O-CH₂-、-CH₂-N(CH₃)-O-CH₂- (known as methylene (methylimino) or MMI backbone), -CH₂-O-N(CH₃)-CH₂-CH as described in the above-mentioned U.S. Pat. No. 5,489,677₂-N(CH₃)-N(CH₃)-CH₂-and-O-N (CH)₃)-CH₂-CH₂- (wherein the natural phosphodiester backbone is represented by-O-P-O-CH)₂-) and the amide backbone of the above-mentioned U.S. Pat. No. 5,602,240. The antisense oligonucleotides having morpholino backbone structures of the above-mentioned U.S. Pat. No. 5,034,506 are also preferred.

The modified oligonucleotide may also comprise one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2' position: OH, F, O-alkyl, S-alkyl, or N-alkyl, O-alkenyl, S-alkenyl, or N-alkenyl, O-alkynyl, S-alkynyl, or N-alkynyl, or O-hydrocarbyl-O-hydrocarbyl, wherein alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C₁To C₁₀Alkyl or C₂To C₁₀Alkenyl and alkynyl groups. Particularly preferred is O [ (CH)₂)_nO]_mCH₃、O(CH₂)_nOCH₃、O(CH₂)_nNH₂、O(CH₂)_nCH₃、O(CH₂)_nONH₂And O (CH)₂)_nON[(CH₂)_nCH₃)]₂Wherein n and m are 1 to about 10. Other preferred antisense oligonucleotides comprise one of the following at the 2' position: c₁To C₁₀Lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkylaryl, arylalkyl, O-alkylaryl or O-arylalkyl radicals, SH, SCH₃，OCN，Cl，Br，CN，CF₃，OCF₃，SOCH₃，SO₂CH₃，ONO₂，NO₂，N₃，NH₂Heterocyclic hydrocarbon radicals, heterocyclic hydrocarbon aryl radicals, aminoalkyl amino radicals, polyalkyl radicalsAmino (polyalkylamino), substituted silyl, RNA cleaving group (cleavage group), reporter group (reporter group), intercalator, group for improving the pharmacokinetic properties of oligonucleotides, or group for improving the pharmacodynamic properties of nucleotides, and other substituents with similar properties. Preferred modifications include 2 '-methoxyethoxy (2' -O-CH)₂CH₂OCH₃Also known as 2'-O- (2-methoxyethyl) or 2' -MOE) (Martin et al, 1995, Heiv. Chim. acta78: 486-. Further preferred modifications include 2' -dimethylaminoyloxyethoxy, i.e. O (CH) ₂)₂ON(CH₃)₂The group, also known as 2' -DMAOE, as described in the examples below, and 2' -dimethylaminoethoxyethoxy (also known in the art as 2' -O-dimethylaminoethoxyethyl or 2' -DMAEOE), i.e. ' -O- (CH)₂)₂-O-(CH₂)₂-N(CH₃)₂。

Further preferred modifications include Locked Nucleic Acids (LNA) in which the 2' -hydroxyl group is attached to the 3' or 4' carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. The bond is preferably methylene (methlyne) (-CH) bridging the 2 'oxygen atom and the 4' carbon atom₂-)_nA group wherein n is 1 or 2. LNA and its preparation are described in WO98/39352 and WO 99/14226.

Other preferred modifications include 2 '-methoxy (2' -O-CH)₃) 2 '-Aminopropoxy (2' -OCH)₂CH₂CH₂NH₂) 2 '-allyl (2' -CH)₂-CH=CH₂) 2 '-O-allyl (2' -O-CH)₂-CH=CH₂) And 2 '-fluoro (2' -F). The 2' -modification may be at the arabinose (upper) position or ribose (lower) position. The preferred 2 '-arabinose modification is 2' -F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3 'position of the sugar at the 3' terminal nucleotide or the 5 'position of the nucleotide at the 5' terminus in 2'-5' linked oligonucleotides. Oligonucleotides may also have sugar mimetics, such as a cyclopentanosyl (pentofuranosyl) sugar substituted with a cyclobutyl moiety. Teaching representative beauty of the preparation of such modified sugar structures National patents include, but are not limited to, U.S. patents 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 hereby incorporated by reference in its entirety.

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-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other hydrocarbyl derivatives of adenine and guanine, 2-propyl and other hydrocarbyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C.ident.C-CH 3 or-CH 2-C.ident.CH) uracil and other alkynyl derivatives of cytosine and pyrimidine bases, 6-azo (azo) uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-mercapto (thiol), 8-thioalkyl (thioalkyl), 8-hydroxy and other 8-substituted adenines and guanines, 5-halo especially 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 thiophenes Oxazin cytidine (1H-pyrimidine [5, 4-b)][l,4]Styrene-acrylicOxazin-2 (3H) -ones), phenothiazine cytidine (1H-pyrimidine [5, 4-b)][l,4]Benzothiazin-2 (3H) -one), G-clip ring (G-c)lamp) such as substituted thiophenesOxazinocytidines (e.g. 9- (2-aminoethoxy) -H-pyrimidine [5, 4-b)][l,4]Styrene-acrylicOxazin-2 (3H) -one), carbazole cytidine (2H-pyrimidine [4,5-b ]]Indol-2-ones), pyridoindocytidines (H-pyrido [3',2':4, 5)]Pyrrole [2,3-d ]]Pyrimidin-2-one). Modified nucleobases may also include those wherein the purine or pyrimidine base is substituted with other heterocycles, such as 7-deaza-adenine, 7-deaza-guanine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808; the circumcise Encyclopedia Of Polymer Science and engineering, pages858-859, Kroschwitz, J.L, ed.John Wiley&Sons, 1990; and those disclosed in englishch et al, angelandandte 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-methyl cytosine substitutions have been shown to increase the stability of nucleic acid duplexes by 0.6-1.2 ℃ (Sanghvi et al, Antisense Research and Applications, CRC Press, Boca Raton,1993, pp.276-278) and are preferred base substitutions, even more preferred when combined with 2' -O-methoxyethyl sugar modifications. Representative U.S. patents that teach the preparation of modified nucleobases include, but are not limited to, U.S. Pat. No. 3,687,808, and 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 incorporated herein by reference.

Another modification of antisense oligonucleotides chemically linked to oligonucleotides is to increase the activity, cellular distribution or cellular uptake of the oligonucleotideOne or more modules or conjugates. The compounds of the invention may comprise a coupling group covalently linked to a functional group such as a primary or secondary hydroxyl group. Coupling groups of the invention include intercalators, reporters, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of the oligomers, and groups that enhance the pharmacokinetic properties of the oligomers. Typical coupling groups include cholesterol, lipids, cationic lipids, phospholipids, cationic phospholipids, biotin, phenazine, folic acid (folate), phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes. In the context of the present invention, groups that enhance pharmacodynamic properties include groups that enhance oligomer uptake, enhance oligomer resistance to degradation, and/or enhance sequence-specific hybridization to RNA. In the context of the present invention, groups that enhance pharmacokinetic properties include groups that enhance oligomer uptake, distribution, metabolism or excretion. Conjugate modules include, but are not limited to, lipid modules such as cholesterol modules (Letsinger et al, 1989, Proc. Natl. Acad. Sci. USA86:6553-6556), cholic acids (Manoharan et al, 1994, bioorg. Med. Chem. Let.4:1053-1060), thioethers such as hexyl-S-tritylmercaptan (tritylthiol) (Manoharan et al, 1992, Ann. N.Y.Acad. Sci.660:306-309; biooharan et al, 1993, bioorg. Med. Chem. Let.3:2765-2770), thiocholesterol (Ohaberuser et al, 1992, Nucl. acids Res.20:533-538), aliphatic chains such as dodecanediol or undecyl residues (ison-Behmoars. Sammor et al, Sammor. 1991, 1990, Mic. J.31, Mic. Miq. K.3, Sp. Sjohnoal. K.23-3531-54, racemic phospholipid, racemic glycerol, Sjohn-33, Mic. K.3, Mic. 3, Mic. K.3, Miq. D. K.3, Mic. D. K.3, S.3, S Phosphonates (Manohara et al, 1995, Tetrahedron Lett.36:3651-3654; Shea et al, 1990, nucleic acids Res.18:3777-3783), polyamine or polyethylene glycol chains (Manohara et al, 1995, nucleic acids) &Nucleotides14: 969-. The oligonucleotides of the invention may also be linked to activitiesDrug substance couplings, such as aspirin (aspirin), warfarin (warfarin), phenylbutazone (phenylbutazone), ibuprofen (ibuprofen), suprofen (suprofen), fenbufen (fenbufen), ketoprofen (ketoprofen), (S) - (+) -pranoprofen (pranoprofen), carprofen (carprofen), dansylsarcosine (dansylsarcosine), 2,3, 5-triiodobenzoic acid, flufenamic acid (flufenamic acid), folinic acid (folinic acid), benzothiadiazine (benzothiazide), chlorothiazide (chlothiazide), diazoxide (flufenamic acid)(diazepin), indomethacin (indomethacin), barbiturate (barbiturate), cephalosporin (cephalosporin), sulfa drug (sulfa drug), antidiabetic (antidiabic), antibacterial (antibacterial) or antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. Ser. No. 09/334,130 (filed 1999, 6/15) 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, 371, 391,723, 5,416,203, 451,463, 5,414,077, 371,391,723, 5,416,203,574, 365,574, 5,414,077, 365,697, 365,481, 365,574, 5,414,077, 365,5872, 5,414,077, 371,697,697, and 5,414,077, each of which are incorporated herein by reference, for each,597,697, 5,414,077, 371, 371,481, 5,414,077, 371, and 371,6972.

It is not necessary to make uniform modifications at all positions in a given compound, and in fact more than one of these modifications may be incorporated in a single compound, even at a single nucleoside within an oligonucleotide. The invention also includes antisense compounds that are chimeric compounds. In the context of the present invention, a "chimeric" antisense compound or "chimera" refers to an antisense compound comprising two or more chemically distinct regions, in particular oligonucleotides, each region being composed of at least one monomer unit, upon oligonucleotide acidificationIn the case of compounds, nucleotides. These oligonucleotides typically comprise at least one region in which the oligonucleotide is modified to confer to the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. The additional region of the oligonucleotide may serve as a substrate for an enzyme capable of cleaving RNA: DNA or RNA: RNA hybrids. For example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA-DNA duplex. Thus, activation of rnase H results in cleavage of the RNA target, thereby greatly increasing the efficiency of oligonucleotide inhibition of gene expression. Thus, when chimeric oligonucleotides are used, comparable results are generally obtained with shorter oligonucleotides compared to phosphorothioate deoxyoligonucleotides that hybridize to the same target region. The chimeric antisense compounds of the invention can be formed as a composite structure 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 ' -O- (CH) s) at the 3' -terminus ₂)₂-O-CH₃) To confer nuclease resistance and at least 4 regions of linked 2' -H sugars to confer RNase H activity. Such compounds are also known in the art as hybrids or conjugates. Preferred conjugates (gapmers) have a 2' modified sugar (preferably ' -O- (CH) s) at the 3' -end and the 5' -end separated by at least one region having at least 4 linked 2' -H sugars₂)₂-O-CH₃) And preferably incorporates phosphorothioate backbone linkages. Representative U.S. 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 incorporated herein by reference in its entirety.

The antisense compounds used in accordance with the present invention may be conveniently and routinely prepared by well-known solid phase synthesis techniques. There are several vendors that sell equipment for such syntheses, including, for example, applied biosystems (Foster City, Calif.). Additionally or alternatively, any other means known in the art for such synthesis may be employed. It is well known to use similar techniques for preparing oligonucleotides, such as phosphorothioates and alkylated derivatives. The compounds of the present invention may also be mixed, encapsulated, coupled or otherwise associated with other molecules, molecular structures or mixtures of compounds, for example, as liposomes, receptor targeting molecules, oral, rectal, topical or other dosage forms to aid in uptake, distribution and/or absorption. Representative U.S. patents that teach the preparation of such uptake, distribution and/or absorption aid dosage forms include, but are not limited to, U.S. patent 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 incorporated herein by reference.

Other examples of sense or antisense oligonucleotides include those covalently linked to organic moieties, such as those described in WO90/10048, and other moieties that increase the affinity of the oligonucleotide for a target nucleic acid sequence, such as poly- (L-lysine). Furthermore, an intercalating agent, such as ellipticine (ellipticine) and an alkylating agent or a metal complex may be attached to the sense or antisense oligonucleotide to modulate the binding specificity of the antisense or sense oligonucleotide to the target nucleotide sequence.

The antisense or sense oligonucleotide can be introduced into a cell containing a target nucleic acid sequence by any gene transfer method, including, for example, CaPO₄Mediated DNA transfection, electroporation, or by using gene transfer vectors such as Epstein-Barr virus. In a preferred procedure, the antisense or sense oligonucleotide is inserted into a suitable retroviral vector. Contacting a cell comprising the target nucleic acid sequence with the recombinant retroviral vector 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).

Sense or antisense oligonucleotides can also be introduced into cells containing the target nucleotide sequence by forming conjugates with ligand binding molecules, 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, the coupling to the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind its corresponding molecule or receptor or block the entry of the sense or antisense oligonucleotide or its conjugate form into the cell.

Alternatively, sense or antisense oligonucleotides can be introduced into cells comprising a target nucleic acid sequence by forming oligonucleotide-lipid complexes, as described in WO 90/10448. The sense or antisense oligonucleotide-lipid complex is preferably dissociated in the cell by an endogenous lipase.

The antisense or sense RNA or DNA molecule is typically at least about 5 nucleotides in length, or 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, 530, 580, 550, 570,580, 600, 650, 940, 900, 650, 700, 840, 700, 840, 700, 900, 520, 900, 700, 900, 700, 900.

Probes can also be used in PCR techniques to generate a pool of sequences for identifying closely related TAT coding sequences.

Nucleotide sequences encoding TAT are also useful for constructing hybridization probes for mapping the gene encoding the TAT and for genetic analysis of individuals suffering from genetic disorders. The nucleotide sequences provided herein can be mapped to chromosomes and specific regions of chromosomes using known techniques, such as in situ hybridization, linkage analysis against known chromosomal markers, and hybridization to screen libraries.

When the coding sequence for TAT encodes a protein that binds another protein (e.g., when TAT is a receptor), TAT can be used in assays to identify other proteins or molecules involved in binding interactions. By such methods, inhibitors of receptor/ligand binding interactions can be identified. Proteins involved in such binding interactions may also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction. Likewise, the receptor TAT can also be used to isolate related ligands. Screening assays can be designed to find lead compounds that mimic the biological activity of native TAT or TAT receptors. Such screening assays would include assays adapted for high throughput screening of chemical libraries, making them particularly useful for identifying small molecule drug candidates. Contemplated small molecules include synthetic organic or inorganic compounds. Assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays that are well characterized in the art.

Nucleic acids encoding TAT, or modified forms thereof, may also be used to generate transgenic animals or "knock-out" animals, which in turn may be used to develop and screen for therapeutically useful agents. A transgenic animal (e.g., a mouse or rat) refers to an animal having cells that comprise a transgene, wherein the transgene is introduced into the animal or a prenatal animal precursor (an), such as the embryonic stage. A transgene refers to DNA that is integrated into the genome of a cell that develops into a transgenic animal. In one embodiment, TAT-encoding genomic DNA can be cloned from TAT-encoding cDNA in accordance with established techniques and used to generate transgenic animals comprising cells expressing TAT-encoding DNA. Methods for generating transgenic animals, particularly animals such as mice or rats, have been conventional in the art, see, e.g., U.S. Pat. nos. 4,736,866 and 4,870,009. Typically, the incorporation of TAT transgenes is targeted to specific cells using a tissue-specific enhancer. Transgenic animals comprising transgenic copies of TAT-encoding genes introduced into the germline of the animal at the embryonic stage can be tested for the effect of increased expression of TAT-encoding DNA. Such animals may be used as test animals for testing agents believed to confer protection against pathological conditions associated with, for example, their overexpression. In accordance with this aspect of the invention, treatment of an animal with a formulation that has a reduced incidence of a pathological condition compared to an untreated animal carrying the transgene would indicate a potential therapeutic intervention for the pathological condition.

Alternatively, a non-human homolog of TAT can be used to construct TAT "knock-out" animals that have a defective or altered TAT-encoding gene due to homologous recombination between the endogenous gene encoding TAT and the altered TAT-encoding genomic DNA introduced into embryonic stem cells of the animal. For example, TAT-encoding cDNA can be used to clone TAT-encoding genomic DNA in accordance with established techniques. A portion of the genomic DNA encoding TAT may be deleted or replaced with another gene, such as a gene encoding a selectable marker that 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,1987, Cell51:503 for a description of homologous recombinant vectors). The vector is introduced into an embryonic stem Cell line (e.g., by electroporation), and cells are selected in which the introduced DNA undergoes homologous recombination with endogenous DNA (see, e.g., Li et al, 1992, Cell69: 915). The selected Cells are then injected into blastocysts of animals (e.g., mice or rats) to form aggregation chimeras (see, e.g., Bradley, in Teratoccinomas and Embryonic Stem Cells: active Approach, E.J.Robertson, ed.IRL, Oxford,1987, pp.113-152). The chimeric embryo can then be implanted into a suitable pseudopregnant female surrogate animal and the embryo generated a "knockout" animal at term. Progeny comprising 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 comprise the homologously recombined DNA. Knock-out animals can be identified, for example, by having the ability to fight certain pathological conditions and developing pathological conditions due to the lack of TAT polypeptides.

Nucleic acids encoding TAT polypeptides may also be used in gene therapy. In gene therapy applications, genes are introduced into cells to achieve in vivo synthesis of therapeutically effective gene products, e.g., for replacement of defective genes. "Gene therapy" includes both conventional gene therapy, in which a sustained effect is obtained by one treatment, and administration of a gene therapeutic agent, in which one or repeated administrations of a therapeutically effective DNA or mRNA are involved. Antisense RNA and DNA are useful as therapeutic agents for blocking the expression of certain genes in vivo. It has been shown that short antisense oligonucleotides can be delivered to cells in which they act as inhibitors, although their intracellular concentration is low due to limited uptake by the cell membrane (Zamecnik et al, 1986, Proc. Natl. Acad. Sci. USA83: 4143-4146). Oligonucleotides can be modified to increase their uptake, for example by substituting their negatively charged phosphodiester groups with uncharged groups.

There are a variety of techniques available for introducing nucleic acids into living cells. These techniques vary depending on whether the nucleic acid is transferred to cultured cells in vitro, or cells of the intended host in vivo. Suitable techniques for transferring nucleic acids into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, calcium phosphate precipitation, and the like. Currently preferred in vivo gene transfer techniques include transfection using viral (usually retroviral) vectors and viral coat protein-liposome mediated transfection (Dzau et al, 1993, Trends in Biotechnology11: 205-210). In some instances, it is desirable to provide a source of nucleic acid along with an agent that targets the target cell, such as an antibody specific for a cell surface membrane protein or target cell, a ligand for a receptor on the target cell, and the like. Where liposomes are employed, proteins that bind to cell surface membrane proteins involved in endocytosis can be used to target and/or facilitate uptake, such as capsid proteins or fragments thereof that are tropic for a particular cell type, antibodies to proteins that undergo internalization in circulation, proteins that target intracellular localization and enhance intracellular half-life. Techniques for receptor-mediated endocytosis are described, for example, in Wu et al, 1987, J.biol.chem.262:4429-4432, and Wagner et al, 1990, Proc.Natl.Acad.Sci.USA87: 3410-3414. For a review of gene markers and gene therapy protocols, see Anderson et al, 1992, Science256: 808-.

Nucleic acid molecules encoding TAT polypeptides or fragments thereof described herein may be used for chromosomal identification. In this regard, the need to identify new chromosomal markers is increasing, as relatively few chromosomal marking agents are now available based on actual sequence data. Each TAT nucleic acid molecule of the invention can be used as a chromosomal marker.

The TAT polypeptides and nucleic acid molecules of the invention may also be used diagnostically for tissue typing, wherein the TAT polypeptides of the invention may be differentially expressed in one tissue compared to another, preferably in a diseased tissue compared to a normal tissue of the same tissue type. TAT nucleic acid molecules can be used to generate probes for PCR, Northern analysis, Southern analysis, and Western analysis.

Screening assays for antagonist drug candidates are designed to identify compounds that bind to or complex with the TAT polypeptide encoded by the gene identified herein, or otherwise interfere with the interaction of the encoded polypeptide with other cellular proteins, including, for example, inhibiting the expression of TAT polypeptides by cells.

Assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays that are well established in the art.

All assays for antagonists have in common that they require contacting a drug candidate with a TAT polypeptide encoded by a nucleic acid identified herein under conditions and for a time sufficient for the two components to interact.

In binding assays, interaction refers to binding, and the complex formed can be separated or detected in a reaction mixture. In a specific embodiment, the TAT polypeptide or drug candidate encoded by a gene identified herein is immobilized on a solid phase, such as a microtiter plate, by covalent or non-covalent attachment. Non-covalent attachment is typically achieved by coating the solid phase surface with a solution of TAT polypeptide and drying. Alternatively, an immobilized antibody, e.g., a monoclonal antibody, specific for the TAT polypeptide to be immobilized may be used to anchor it to the solid phase surface. The assay is performed by adding a non-immobilized component, which may be labeled with a detectable label, to an immobilized component, such as a coated surface comprising an anchoring component. Upon completion of the reaction, unreacted components are removed, for example by washing, and the complexes anchored to the solid phase surface are detected. If the initial non-immobilized component carries a detectable label, detection of the label immobilized to the surface indicates that complexation has occurred. If the initial non-immobilized component does not carry a label, complexation can be detected, for example, by using a labeled antibody that specifically binds to the immobilized complex.

If a 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 determined by well-known methods for detecting protein-protein interactions. Such assays include conventional methods such as, for example, cross-linking, co-immunoprecipitation, and co-purification by gradient or chromatography columns. In addition, protein-protein interactions can be monitored using the yeast-based genetic system described by Fields and co-workers (Fields and Song,1989, Nature (London)340: 245-. Many transcriptional activators, such as yeast GAL4, are composed of two spatially discrete modular domains, one functioning as a DNA binding domain and the other functioning as a transcriptional activation domain. The yeast expression system described in the above publication (known as the "two-hybrid system") takes advantage of this property, using two hybrid proteins, in one of which the target protein is fused to the DNA binding domain of GAL4 and in the other of which the candidate activator protein is fused to the activation domain. GAL1-lacZ reporter gene under the control of GAL 4-activated promoter Expression of the gene is dependent on reconstitution of GAL4 activity via protein-protein interactions. Colonies containing the interacting polypeptide are detected with a chromogenic substrate for beta-galactosidase. Complete kit (MATCHMAKER) for identifying protein-protein interactions between two specific proteins using two-hybrid technology^TM) Available from Clontech. This system also extends to mapping of protein domains involved in specific protein interactions and to pointing out amino acid residues that are critical for these interactions.

Compounds that interfere with the interaction of TAT polypeptides encoded by the genes identified herein with other intracellular or extracellular components can be tested as follows: the reaction mixture comprising the gene product and the intracellular or extracellular component is generally prepared under conditions and for a time sufficient to allow the two products to interact and bind. To test the ability of a candidate compound to inhibit binding, a reaction is performed in the absence and presence of the test compound. Additionally, a placebo can be added to the third reaction mixture as a positive control. Binding (complex formation) between TAT polypeptide and intracellular or extracellular components present in the mixture is monitored as described above. The formation of a complex in the control reaction but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of the TAT polypeptide with its reaction partner.

To assay for antagonists, the TAT polypeptide may be added to the cell along with a compound to be screened for a particular activity, the ability of the compound to inhibit the activity of interest in the presence of the TAT polypeptide indicating that the compound is an antagonist of the TAT polypeptide. Alternatively, antagonists can be detected by combining a TAT polypeptide and a potential antagonist with a membrane-bound TAT polypeptide receptor or recombinant receptor under conditions suitable for a competitive inhibition assay. TAT polypeptides may be labeled, such as by radiolabeling, so that the number of TAT polypeptide molecules bound to the receptor can be used to determine the efficacy of a potential antagonist. Genes encoding receptors can be identified by a variety of methods known to those skilled in the art, such as ligand panning and FACS sorting. Coligan et al, 1991, Current Protocols in Immun.1(2): Chapter 5. Preferably, expression cloning is used, wherein polyadenylated RNA is prepared from cells responsive to TAT polypeptide, and cDNA libraries constructed from this RNA are divided into several pools and used to transfect COS cells or other cells that are not responsive to TAT polypeptide. Transfected cells cultured on glass slides were exposed to labeled TAT polypeptides. TAT polypeptides can be tagged by a variety of means, including iodination or inclusion of recognition sites for site-specific protein kinases. After fixation and incubation, the slides were subjected to autoradiographic analysis. Positive pools were identified, subsets were prepared, and the interacting subsets were used for the re-transfection and re-screening process, resulting in single clones encoding putative receptors.

As an alternative to receptor identification, a labeled TAT polypeptide can be photoaffingly linked to a cell membrane expressing the receptor molecule or to an extraction preparation. The crosslinked material was resolved by PAGE and exposed to X-ray film. The labeled complex containing the receptor can be cleaved off, cleaved into peptide fragments, and subjected to protein microsequencing. The amino acid sequences obtained from microsequencing can be used to design a degenerate set of oligonucleotide probes for use in screening a cDNA library to identify genes encoding putative receptors.

In another assay for antagonists, a mammalian cell or membrane preparation expressing a receptor is incubated with a labeled TAT polypeptide in the presence of a candidate compound. The ability of the compound to enhance or block this interaction is then measured.

More specific examples of potential antagonists include polypeptides, particularly antibodies, including but not limited to polyclonal 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, that bind to a fusion of an immunoglobulin with a TAT polypeptide. Alternatively, a potential antagonist may be a closely related protein, such as a mutated form of TAT polypeptide that recognizes the receptor but does not function, thereby competitively inhibiting the action of the TAT polypeptide.

Another potential TAT polypeptide antagonist is the trans-TAT polypeptide prepared using antisense technologySense RNA or DNA constructs in which, for example, an antisense RNA or DNA molecule acts to block translation of mRNA directly by hybridizing to the target mRNA and preventing translation of the protein. Antisense technology can be used to control gene expression by forming a triple helix with antisense DNA or RNA, both based on the binding of polynucleotides to DNA or RNA. For example, the 5' coding portion of a polynucleotide sequence encoding a mature TAT polypeptide herein is used to design antisense RNA oligonucleotides of about 10-40 base pairs in length. DNA oligonucleotides are designed to be complementary to a region in the gene involved in transcription (triple helix-see Lee et al, 1979, Nucl. acids sRs.6: 3073; Cooney et al, 1988, Science241:456; Dervan et al, 1991, Science251:1360), thereby preventing transcription and production of the TAT polypeptide. Antisense RNA oligonucleotides hybridize with mRNA in vivo and block translation of mRNA molecules into TAT polypeptides (antisense-Okano, 1991, Neurochem56: 560;Oligodeoxynucleotidesas Antisense Inhibitors of Gene Expression,CRC Press Boca Raton, FL, 1988). The oligonucleotides described above can also be delivered to a cell such that antisense RNA or DNA can be expressed in vivo to inhibit production of the TAT polypeptide. When an antisense DNA is used, an oligodeoxyribonucleotide derived from the translation start site (e.g., between about-10 to +10 positions) of the nucleotide sequence of the target gene is preferred.

Potential antagonists include small molecules that bind to the active site, 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.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. Ribozymes function by sequence-specific hybridization with a complementary target RA, followed by endonucleolytic (endonucleolytic) cleavage. Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques. For more details see, e.g., Rossi,1994, Current Biology4:469-471 and PCT publication No. WO97/33551 (published 1997, 9/18).

The nucleic acid molecule in the triple helix structure 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 by the Hoogsteen base pairing principle, which usually requires a large stretch of purines or pyrimidines on one strand of the duplex. For more details see, e.g., PCT publication No. WO97/33551, supra.

These small molecules may be identified by one or more of the screening assays discussed above and/or by any other screening technique known to those of skill in the art.

Isolated nucleic acids encoding TAT polypeptides may be used herein to recombinantly produce TAT polypeptides using techniques well known in the art and as described herein. The TAT polypeptides produced may then be used to produce anti-TAT antibodies using techniques well known in the art and as described herein.

Antibodies identified herein that specifically bind to TAT polypeptides, as well as other molecules identified by the screening assays disclosed above, can be administered in the form of pharmaceutical compositions for the treatment of various disorders (conditions), including cancer.

If the TAT polypeptide is intracellular and an intact antibody is used as an inhibitor, then it is preferred that the antibody be internalized. However, the antibody or antibody fragment may also be delivered into cells using lipofection or liposomes. When antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, depending on the variable region sequence of the antibody, peptide molecules can be designed that retain the ability to bind to the target protein sequence. Such peptides may be chemically synthesized and/or produced by recombinant DNA techniques. See, e.g., Marasco et al, 1993, Proc.Natl.Acad.Sci.USA90: 7889-.

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. Alternatively, or in addition, the composition may further comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitory agent. Suitably, such molecules are present in combination in amounts effective for the intended purpose.

The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

All patents and publications cited in this specification are herein incorporated by reference in their entirety.

Examples

Unless otherwise stated, commercial reagents mentioned in the examples were used according to the manufacturer's instructions. The source of those cells identified in the examples below and throughout the specification as ATCC accession numbers is the American Type Culture Collection (Manassas, VA).

Example 1: use ofDrawing tissue expression map

Analyzing private databases containing gene expression informationGene Logic inc., Gaithersburg, MD), in an attempt to identify polypeptides (and their encoding nucleic acids) whose expression is significantly and detectably upregulated in a particular human tumor tissue of interest, as compared to other human tumors and/or normal human tissues. Specifically, a peptide obtainable by Gene Logic corporation (Gaithersburg, Md.) and Software for use with databases or written and developed by Genentech corporation andprivate software for use with databasesAnd (5) analyzing the database. The evaluation of positive hits in the analysis is based on several criteria including, for example, tissue specificity, tumor specificity, and expression levels in normal basal tissue and/or normal proliferative tissue. Using this mRNA expression analysis, it was determined that mRNA encoding TAT425 polypeptide was significantly, reproducibly, and detectably overexpressed in human prostate and kidney tumors, respectively, compared to corresponding normal human prostate and kidney tissues.

Example 2: quantitative analysis of TAT mRNA expression

In this assay, a 5' nuclease assay (e.g., a nuclease assay) is usedAnd real-time quantitative PCR (e.g., ABI Prizm7700Sequence Detection)(Perkin Elmer, applied biosystems Division, Foster City, Calif.) genes that are significantly overexpressed in one or more cancerous tumors compared to other cancerous tumors or normal non-cancerous tissues are sought. The 5 'nuclease assay reaction is a fluorescent PCR-based technique that utilizes the 5' exonuclease activity of Taq DNA polymerase to monitor gene expression in real time. Two oligonucleotide primers, the sequences of which are based on the gene or EST sequence of interest, are used to generate amplicons, typically in a PCR reaction. A third oligonucleotide or probe is designed to detect the nucleotide sequence located between the two PCR primers. The probe is not extendable by Taq DNA polymerase and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. When the two dyes are positioned close together as they are on the probe, any laser-induced emission from the reporter dye is quenched by the quencher dye. During the PCR amplification reaction, Taq DNA polymerase cleaves the probe in a template-dependent manner. The resulting probe fragments dissociate in solution, while the signal from the released reporter dye is not quenched by the second fluorophore. One molecule of reporter dye is released for each new molecule synthesized, but not Detection of the quenched reporter dye provides the basis for quantitative interpretation of the data.

The 5' nuclease procedure is performed on a real-time quantitative PCR device, such as ABI Prism7700^TMSequence Detection. The system consists of a thermal cycler, a laser, a charge-coupled device (CCD) camera, and a computer. The system amplifies samples in 96-well format on a thermal cycler. During amplification, laser-induced fluorescence signals were collected in real time by fiber optic cables for all 96 wells and detected at the CCD. The system includes software for running the device and for analyzing the data.

The starting material for screening is mRNA isolated from a variety of different cancerous tissues. The mRNA is accurately quantified, for example by fluorescence. As a negative control, RNA was isolated from a variety of normal tissues having the same tissue type as the cancerous tissue examined.

The 5' nuclease assay data was initially expressed as Ct or cycle threshold (threshold cycle). This is defined as the number of cycles that reporter signal accumulates above background fluorescence levels. The Δ Ct value is used as a quantitative measure of the relative starting copy number of a particular target sequence in a nucleic acid sample when comparing cancer mRNA results to normal human mRNA results. Since one Ct unit corresponds to 1 round of PCR cycles or an approximately 2-fold relative increase relative to normal, two units correspond to a 4-fold relative increase, 3 units correspond to an 8-fold relative increase, and so on, researchers can quantify the relative fold increase in mRNA expression between two or more different tissues.

Using this technique, the identification of TAT425 molecules that are significantly (i.e., at least 2-fold) overexpressed in human prostate tumors compared to normal, non-cancerous human prostate tissue from different human tissue donors represents an excellent polypeptide target for the diagnosis and treatment of prostate cancer in mammals.

Example 3: in situ hybridization

In situ hybridization is a powerful and versatile technique for nucleic acid sequence detection and localization in cell or tissue preparations. It can be used, for example, to identify sites of gene expression, to analyze the tissue distribution of transcription, to identify and locate viral infections, to track changes in the synthesis of specific mRNAs, and to aid in chromosome mapping.

In situ hybridization was performed following the optimized version protocol in Lu and Gillett, Cell Vision1:169-176(1994), using PCR generated³³P-labeled ribonucleic acid probes (riboprobes). Briefly, formaldehyde-fixed, paraffin-embedded human tissue sections were deparaffinized, deproteinized in proteinase K (20g/ml) at 37 ℃ for 15 minutes, and further processed as described above for in situ hybridization. From the PCR product of [ 2 ], [³³-P]UTP labeled antisense RNA probe and hybridized overnight at 55 ℃. Slides were immersed in Kodak NTB2 nuclear tracer emulsion (nuclear track emulsion) and exposed for 4 weeks. ³³P-ribonucleic acid probe synthesis

Mu.l (125mCi)³³P-UTP(Amersham BF1002,SA<2000Ci/mmol) was dried in rapid vacuum. Drying to obtain³³The following ingredients were added to each tube of P-UTP: 2.0. mu.l of 5x transcription buffer, 1.0. mu.l of DTT (100mM), 2.0. mu.l of NTP mix (2.5mM: 10. mu.l of each 10mM GTP, CTP&ATP+10μl H₂O), 1.0. mu.l UTP (50. mu.M), 1.0. mu.l Rnasin, 1.0. mu.l DNA template (1. mu.g), 1.0. mu. l H₂O, 1.0 μ l RNA polymerase (for PCR products, often T3= AS, T7= S). The tubes were incubated at 37 ℃ for 1 hour. Mu.l RQ1 DNase was added and incubated at 37 ℃ for 15 min. Mu.l of TE (10mM Tris pH7.6/1mM EDTApH8.0) was added and the mixture was transferred to DE81 paper. The remaining solution was added to a Microcon-50 ultrafiltration unit and spun for procedure 10 (6 min). The filter unit was inverted on the second tube and spun using procedure 2 (3 minutes). After the final spin recovery, 100. mu.l TE was added. Mu.l of the final product were transferred onto DE81 paper and counted in 6ml Biofluor II.

The probes were electrophoresed on a TBE/urea gel. Mu.l of probe or 5. mu.l of RNA Mrk III were added to 3. mu.l of loading buffer. After heating on a 95 ℃ heating block for 3 minutes, the probe was immediately placed on ice. Rinsing the gel wells, adding the sample, and Electrophoresis was carried out at 180 ℃ and 250 volts for 45 minutes. The gel was wrapped in saran wrap and the XAR film was exposed to light with a intensifying screen in a-70 ℃ freezer for 1 hour to overnight.³³P-hybrid

A.Pretreatment of frozen sections

The slides were removed from the freezer, placed on an aluminum tray, and thawed at room temperature for 5 minutes. The pan was placed in a 55 ℃ incubator for 5 minutes to reduce condensation. Slides were fixed in a fume hood on ice in 4% paraformaldehyde for 10 minutes and in 0.5 XSSC (25ml20x SSC +975ml SQ H)₂O) for 5 minutes at room temperature. After deproteinization at 37 ℃ for 10 min in 0.5. mu.g/ml proteinase K (12.5. mu.l 10mg/ml stock diluted in 250ml of pre-warmed RNase-free RNase buffer), sections were washed in 0.5 XSSC for 10 min at room temperature. Sections were dehydrated in 70%, 95%, 100% ethanol for 2 minutes each.

B.Pretreatment of Paraffin-Embedded sections

Paraffinizing the slides and placing them in SQ H₂O, and rinsed twice in 2x SSC at room temperature for 5 minutes each. Sections of human embryos or formalin tissues were deproteinized in 20. mu.g/ml proteinase K (500. mu.l 10mg/ml diluted in 250ml RNase-free RNase buffer, 37 ℃ 15 min.) or 8 Xproteinase K (100. mu.l diluted in 250ml RNase buffer, 37 ℃ 30 min.). Followed by rinsing in 0.5x SSC and dewatering as described above.

C.Prehybridization

Slides were placed in plastic cassettes lined with Box buffer (4x SSC,50% formamide) saturated filter paper.

D.Hybridization of

1.0x10 of each slide⁶The cpm probe and 1.0. mu.l tRNA (50mg/ml stock) were heated at 95 ℃ for 3 minutes. The slides were cooled on ice and 48. mu.l hybridization buffer was added to each slide. After vortexing, 50. mu.l of the prehybridization solution was added to 50. mu.l of the glass slide³³And (4) mixing the solution P. The slide was incubated at 55 ℃And (5) breeding over night.

E.Cleaning of

With 2 XSSC, EDTA (400ml20x SSC +16ml0.25M EDTA, V)_f4L) were washed 2 times for 10 min at room temperature and subsequently treated with rnase a (500 μ L10mg/ml diluted in 250ml rnase buffer =20 μ g/ml) for 30 min at 37 ℃. Slides were washed 2 times 10 min in 2x SSC, EDTA at room temperature. Stringent washing conditions were as follows: 55 ℃,0.1 XSSC, EDTA (20ml20x SSC +16ml EDTA, V)_f4L),2 hours.

F.Oligonucleotides

In situ analysis was performed on various DNA sequences disclosed herein. The oligonucleotides used in these assays were obtained so as to be complementary to the nucleic acids shown in the figures (or their complementary sequences).

G.Results

For TAT425, weak to moderate expression was observed in normal prostate epithelium, and other normal tissues tested were not positive. In contrast, 46 out of 64 primary prostate cancers were positive for expression and 6 out of 14 metastatic prostate cancers were positive for expression.

Example 4: validation and analysis of differential TAT polypeptide expression by GEPIS

TAT polypeptides that may have been identified as tumor antigens as described in one or more of the examples above are validated and analyzed as follows. Search of Expressed Sequence Tag (EST) DNA database by GEPISIncyte Pharmaceuticals, Palo Alto, Calif.) and identify EST sequences of interest. Computational Gene Expression Profiling (GEPIS) is a bioinformatics method developed by Genentech corporation to identify genes of interest as new cancer therapeutic targets. GEPIS uses a large amount of EST sequence and library information to determine gene expression profiles. GEPIS is able to determine gene expression profiles based on their ratio to the number of occurrences in EST databases, and it is generally based onThrough integration in a strict and statistically significant mannerA relational database and Genentech private information. In this example, GEPIS is used to identify and cross-validate novel tumor antigens, although GEPIS can be set to perform very specific analyses or extensive screening tasks. For initial screening, GEPIS was used to select fromEST sequences are identified in the database that are associated with expression in one or more particular tissues of interest, often tumor tissues of interest. The EST sequences identified in this initial screen (or consensus sequences obtained by comparing multiple related and overlapping EST sequences obtained in the initial screen) are then screened to identify the presence of at least one transmembrane domain in the encoded protein. Finally, GEPIS was used to generate complete tissue expression profiles of various sequences of interest. Using this type of screening bioinformatics, it was identified that a variety of TAT polypeptides (and nucleic acid molecules encoding them) were significantly overexpressed in a particular type of cancer or in certain cancers compared to other cancers and/or normal non-cancerous tissues. The assessment of GEPIS hits is based on several criteria including, for example, tissue specificity, tumor specificity, and expression levels in normal essential tissues and/or normal proliferative tissues.

Using this analysis, TAT425 was confirmed to be highly tissue expressed and significantly upregulated in specific tumors (i.e., prostate, kidney, and pancreas) compared to the relevant normal tissues, as determined by GEPIS. Thus, TAT425 polypeptides are excellent polypeptide targets for the diagnosis and treatment of cancer in mammals.

Example 5: preparation of antibodies that bind to TAT425 polypeptides

This example illustrates the preparation of a monoclonal antibody that specifically binds TAT 425.

Techniques for generating monoclonal antibodies are known in the art and are described, for example, in Goding, supra. Immunogens that can be used include purified TAT, TAT-containing fusion proteins, and cells expressing recombinant TAT on the cell surface. The selection of the immunogen can be made by the skilled artisan without undue experimentation.

Mice such as Balb/c are immunized by subcutaneous or intraperitoneal injection of 1-100 microgram quantities with TAT immunogen emulsified in complete Freund's adjuvant. Alternatively, the immunogen was emulsified in MPL-TDM adjuvant (Ribis immunochemical Research, Hamilton, MT) and injected into the hindpaw pad of the animal. Immunized mice are boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. After that, the mice can be boosted for several weeks by additional immunization injections. Serum samples can be taken from mice on a regular basis by post-orbital bleeds for testing in an ELISA assay to detect anti-TAT antibodies.

Animals that are "positive" for antibodies can be given a final intravenous injection of TAT after a suitable antibody titer has been detected. After 3 to 4 days, mice were sacrificed and splenocytes were harvested. Splenocytes are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line, such as p3x63agu.1, available from ATCC, No. crl1597. The fusion produces hybridoma cells, which can then be dispensed into 96-well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit proliferation of unfused cells, myeloma hybrids, and spleen cell hybrids.

Hybridoma cells were screened in ELISA for reactivity to TAT. Determination of "positive" hybridoma cells that secrete the desired monoclonal antibody against TAT is within the skill in the art.

Positive hybridoma cells can be injected intraperitoneally into syngeneic Balb/c mice to produce ascites fluid containing anti-TAT monoclonal antibodies. Alternatively, the hybridoma cells can be cultured in tissue culture flasks or roller bottles. Purification of monoclonal antibodies produced in ascites fluid can be accomplished using amine sulfate precipitation followed by gel exclusion chromatography. Alternatively, affinity chromatography based on binding of antibodies to protein a or protein G can be used.

In another technique, Balb/C mice (Charles River, Hosterli, CA) or C57BL/6/TAT425.ko mice (Genentech, south san Francisco, CA) were immunized with 50 μ g of plasmid DNA encoding TAT425 by injection via a Hydrodynamic Tail Vein (HTV) with or without mFlt3 ligand (DNA) and mGM-CSF (DNA) diluted in lactated Ringer's solution as previously described (Zhang et al, hum. Gene ther.10:1735(1999), Liu et al, Gene ther et al, 1258(1999), and Herweijer and Wolff, Gene ther The.10: 453 (2003)). Spleens were harvested 3 days after the last HTV immunization. Splenocytes from these mice, whose sera all exhibited strong binding by FACS to 293 cells overexpressing TAT425, were fused by electrofusion (Cytopulse or BTX) with X63-Ag8.653 mouse myeloma cells (American Type Culture Collection, Rockville, Md.) and fused at 37 ℃ in 7% CO₂Incubate overnight in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 4.5g/L glucose, 25mM HEPES, 0.15mg/ml oxaloacetate, 100. mu.g/ml pyruvate, 0.2U/ml insulin, 2mM L-glutamine, 100U/ml Penicillin, 100. mu.g/ml Streptomycin (Penicilin-Streptomyces, Invitrogen, Carlsbad, CA), NCTC-109(Lonza), NEAA (Invitrogen), supplemented with 5.7. mu.M azaserine and 100. mu.M inosine (HA, Sigma-Aldrich, St. Louis, Mo.) before plating into 96-well plates. Supernatants were screened for IgG production by ELISA and FACS 11 days after fusion. IgG-secreting hybridomas exhibiting strong TAT425 specific binding by FACS were amplified and subcloned by limiting dilution. For large scale production in bioreactors (Integra Biosciences, Chur, Switzerland), the final clone exhibiting the highest FACS binding after amplification of round 2 subclones. The supernatant was then purified by protein a affinity chromatography as previously described (Hongo et al, Hybridoma19:303 (2000)).

Using the techniques described above, a variety of monoclonal antibodies were generated that bind to native TAT425 polypeptides. These monoclonal antibodies were shown to bind TAT425 polypeptide expressed on the surface of a variety of cells, including 293 cells engineered to express TAT425 protein, LuCAP77 cells, LuCAP105 cells, LuCAP141 cells, and LnCAP-shTAT425 cells, using well-known and routinely employed techniques, such as Western blotting, ELISA analysis, FACS sorting analysis and/or immunohistochemical analysis of cells expressing TAT425 polypeptide. The amino acid sequences associated with these anti-TAT 425 antibodies are shown in figures 3-10. These antibodies are useful in the diagnosis and treatment of human cancers associated with TAT425 expression.

Example 6: immunohistochemical analysis

Antibodies against TAT425 were prepared as described above and immunohistochemical analysis was performed as follows using a commercially available M4I-80 rat anti-TAT 425 monoclonal antibody. Tissue sections were first fixed in acetone/ethanol for 5 minutes (frozen or paraffin embedded). Sections were then washed in PBS, followed by blocking with avidin and biotin (Vector kit) for 10 minutes, and washed in PBS after each block. The sections were then blocked with 10% serum for 20 minutes, followed by blotting to remove excess solution. Primary antibody was then added to the sections at a concentration of 10. mu.g/ml for 1 hour, followed by washing of the sections in PBS. Biotinylated secondary antibodies (anti-primary antibodies) were then added to the sections for 30 minutes, followed by washing of the sections in PBS. The sections were then exposed to the reagents of the Vector kit for 30 minutes, followed by washing of the sections in PBS. The sections were then exposed to Diaminobenzidine (Pierce) for 5 minutes, followed by washing in PBS. Sections were then counterstained with Mayers hematoxylin, coverslipped and viewed. Immunohistochemical analysis can also be performed as described in Sambrook et al, Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press,1989 and Ausubel et al, Current Protocols of Molecular Biology, Unit3.16, John Wiley and Sons 1997.

The results from these analyses demonstrated that detectable expression of TAT425 polypeptides was observed in 27 of 29 independent primary human prostate cancer samples (of which 24 showed moderate to high TAT425 expression) and 26 of 29 metastatic human prostate cancer samples (of which 13 showed moderate to high TAT425 expression).

Example 7: competitive binding assays and epitope mapping

The TAT425 epitope bound by the monoclonal antibody can be determined by standard competitive binding assays (Fendly et al, Cancer Research50: 1550-. PANDEX can be used^TMScreen Machine quantified fluorescence and cross-blocking studies of antibodies were performed by direct fluorescence on intact PC3 cells engineered to express TAT 425. Each monoclonal antibody was conjugated to Fluorescein Isothiocyanate (FITC) using established procedures (Wofsy et al, Selected Methods in cellular Immunology, p.287, Mishel and Schiigi (eds.) San Francisco: W.J.Freeman Co. (1980)). Confluent monolayers of PC3 cells expressing TAT425 were trypsinized, washed once, and resuspended at 1.75 × 106 cells/ml in a solution containing 0.5% Bovine Serum Albumin (BSA) and 0.1% NaN₃In cold PBS. Latex (latex) particles (IDC, Portland, OR) were added at a final concentration of 1% to reduce PANDEX ^TMClogging of the flat sheet membrane. Mu.l of the cell suspension and 20. mu.l of purified monoclonal antibody (100. mu.g/ml to 0.1. mu.g/ml) were added to PANDEX^TMPlate wells and incubate on ice for 30 min. Mu.l of a predetermined dilution of FITC-labeled monoclonal antibody was added to each well, incubated for 30 minutes, washed, and washed with PANDEX^TMScreen Machine quantified fluorescence. Monoclonal antibodies are considered to share an epitope if they block binding to each other by 40% or more compared to an unrelated monoclonal antibody control and at the same antibody concentration. Using this assay, one of ordinary skill in the art can identify other monoclonal antibodies that bind to the same epitope as those described above. Deletion analysis can also be performed to identify the approximate position of the antigenic epitope within the polypeptide sequence shown in SEQ ID NO. 2.

In one experiment, anti-TAT 425 monoclonal antibodies 2E4.6.1, 4D11.17.2, 13H2.28.2, and 14E7.17.1 were demonstrated to not compete with each other for epitope binding. Thus, it was determined that each of these 4 antibodies bound an independent, non-overlapping extracellular antigenic epitope in TAT425 protein.

Example 8: preparation of toxin-conjugated TAT 425-binding antibodies

Use Of Antibody-drug conjugates (ADCs), i.e. immunoconjugates, for the local delivery Of cytotoxic or cytostatic agents, i.e. drugs for killing or inhibiting tumor cells, In the treatment Of Cancer (Payne (2003) Cancer Cell3: 207. 212; Syrigos and Epeneros (1999) Anticancer Research19: 605. 614; Niculescu-Duvaz and Springer (1997) adv. drug Del. Rev.26: 151. 172; U.S. Pat. No. 4,975,278) allows for the targeted delivery Of drug moieties to tumors and the intracellular accumulation therein, whereas systemic administration Of these unconjugated drug agents may lead to unacceptable levels Of toxicity to normal cells other than tumor cells attempting to be eliminated (Baldwin et al (1986) Lancet. 15. (1986) cells, 603-05; Thorne (1985) "kinetic cells In: Clinical Applications:" Clinical antigens A84 ", pinchera et al, (eds.), pp.475-506). Thereby attempting to achieve maximum efficacy and minimal toxicity. Efforts to design and improve ADCs have focused on the selectivity of monoclonal antibodies (mabs) as well as drug conjugation and drug release characteristics. Both polyclonal and monoclonal antibodies have been reported to be useful in these strategies (Rowland et al (1986) Cancer Immunol. Immunother.,21: 183-87). Drugs used in these methods include daunomycin (daunomycin), doxorubicin (doxorubicin), methotrexate (methotrexate) and vindesine (vindesine) (Rowland et al (1986) supra). Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (Mandler et al (2000) J.of the Nat. Cancer Inst.92(19):1573-1581; Mandler et al (2000) Bioorganic & Med.Chem.Letter10: 1025-1028; Mandler et al (2002) Bioconjugate chem.13: 786-791), maytansinoids (EP 1391213; Liu et al (1996) Proc. Natl.Acad.Sci.USA93: 8-861 8623), and calicheamicin (Lode et al (1998) Cancer Res.58:2928; Hinal et al 3353: 3353).

In the antibody-drug conjugates (ADCs) of the invention, the antibody (Ab) is conjugated via a linker (L) to one or more drug moieties (D), for example from about 1 to about 20 drug moieties per antibody. Organic chemical reactions, conditions and reagents known to those skilled in the art can be used to prepare compounds having the general formula:

Ab-(L-D)_p

the ADC of (a), comprising: (1) the nucleophilic group of the antibody reacts via a covalent bond with a divalent linker reagent to form Ab-L, which then reacts with the drug moiety D; and (2) reaction of the nucleophilic group of the drug moiety with a bivalent linker reagent via a covalent bond to form D-L, followed by reaction with the nucleophilic group of the antibody. Other methods for preparing ADCs are also described herein.

The linker may be composed of one or more linker components. Exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimido 4- (2-pyridylthio) pentanoate ("SPP"), N-succinimido 4- (N-maleimidomethyl) cyclohexane-1 carboxylate ("SMCC"), and N-succinimido (4-iodo-acetyl) aminobenzoate ("SIAB"). Additional linker components are known in the art, and some are described herein.

In some embodiments, the linker may comprise amino acid residues. Exemplary amino acid linker components include dipeptides, tripeptides, tetrapeptides, or pentapeptides. Exemplary dipeptides include: valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include: glycine-valine-citrulline (gly-val-cit) and glycine-glycine (gly-gly-gly). Amino acid residues comprising the amino acid linker component include those naturally occurring amino acids, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. The amino acid linker components can be designed and optimized for their selectivity in enzymatic cleavage by specific enzymes (e.g., tumor associated proteases, cathepsin B, C and D, or plasmin proteases).

Nucleophilic groups of antibodies include, but are not limited to: (i) an N-terminal amino group; (ii) side chain amino groups, such as lysine; (iii) side chain sulfhydryl groups, such as cysteine; and (iv) glycosylating the hydroxyl or amino groups of the sugar in the antibody. Amino, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, carboxylic acid hydrazine, and aryl hydrazide are nucleophilic and capable of reacting with electrophilic groups on a linker moiety to form a covalent bond, and linker reagents include: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides, such as haloacetamides; (iii) aldehydes, ketones, carboxyl groups and maleimide groups. Some antibodies have reducible interchain disulfide bonds, i.e., cysteine bridges. The antibody may be rendered reactive for conjugation to a linker reagent by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge will theoretically form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into the antibody via reaction of lysine with 2-iminothiolane (Traut's reagent), resulting in conversion of the amine to a thiol. Reactive thiol groups may be introduced into an antibody (or fragment thereof) by introducing one, two, three, four or more cysteine residues (e.g., preparing a mutant antibody comprising one or more non-natural cysteine amino acid residues).

Antibody-drug conjugates of the invention can also be produced by modifying the antibody, i.e., introducing electrophilic moieties that can react with nucleophilic substituents on the linker reagent or drug. The sugar of the glycosylated antibody can be oxidized with, for example, a periodate oxidizing agent to form an aldehyde or ketone group that can react with the amine group of the linker reagent or drug moiety. The resulting imine Schiff base groups may form stable linkages or may be reduced, for example, with borohydride reagents to form stable amine linkages. In one embodiment, reaction of the carbohydrate moiety of a glycosylated antibody with galactose oxidase or sodium metaperiodate can generate carbonyl (aldehyde and ketone) groups in the protein that can react with appropriate groups on the drug (Hermanson, Bioconjugate technologies). In another embodiment, proteins containing an N-terminal serine or threonine residue can be reacted with sodium meta-periodate resulting in the formation of an aldehyde at the first amino acid (Geoghegan & Stroh, Bioconjugate chem.3:138-146(1992); US 5362852). Such aldehydes may react with a drug moiety or linker nucleophile.

Alternatively, fusion proteins comprising an antibody and a cytotoxic agent may be prepared, for example, by recombinant techniques or peptide synthesis. The length of the DNA may comprise regions encoding the two parts of the conjugate, either adjacent to each other or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate.

In yet another embodiment, the antibody may be conjugated to a "receptor" (such as streptavidin) for tumor pre-targeting, wherein the antibody-receptor conjugate is administered to the patient, followed by clearance of unbound conjugate from the circulation using a clearing agent, followed by administration of a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a radionucleotide).

Specific techniques for generating antibody-drug conjugates by linking a toxin to a purified antibody are well known in the art and are routinely employed. For example, conjugation of the purified monoclonal antibody to toxin DM1 can be achieved as follows. The purified antibody was derivatized with 4- (2-pyridylthio) pentanoic acid N-succinimidyl ester to introduce a dithiopyridyl group. 44.7mL of antibody (376.0mg,8mg/mL) in 50mM potassium sulfate buffer (pH6.5) containing NaCl (50mM) and EDTA (1mM) was treated with SPP (5.3 molar equivalents in 2.3mL ethanol). After 90 minutes of incubation at ambient temperature under argon, the reaction mixture was gel filtered through a Sephadex G25 column equilibrated with 35mM sodium citrate, 154mM NaCl and 2mM EDTA. The antibody-containing fractions are then pooled and tested. The antibody-SPP-Py (337.0mg, with releasable 2-thiopyridyl group) was diluted to a final concentration of 2.5mg/ml with the above 35mM sodium citrate buffer pH 6.5. DM1(1.7 equiv., 16.1 mol) in 3.0mM dimethylacetamide (DMA, 3% v/v in the final reaction mixture) was then added to the antibody solution. The reaction was allowed to proceed under argon at ambient temperature for 20 hours. The reaction solution was loaded on a Sephacryl S300 gel filtration column (5.0 cm. times.90.0 cm,1.77L) equilibrated with 35mM sodium citrate, 154mM NaCl, pH 6.5. The flow rate was 5.0ml/min and 65 fractions (20.0 ml each) were collected. Fractions were pooled and examined, where the number of DM1 drug molecules attached per antibody molecule (p') was determined by measuring absorbance at 252nm and 280 nm.

For illustrative purposes, conjugation of the purified monoclonal antibody to toxin DM1 was also achieved as follows. The purified antibody was derivatized with succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC, Pierce Biotechnology, Inc) to introduce SMCC linkers. 20mg/ml antibody in 50mM potassium phosphate/50 mM sodium chloride/2 mM EDTA, pH6.5 was treated with 7.5 molar equivalents of SMCC (20mM in 6.7mg/ml DMSO). After stirring at ambient temperature for 2 hours under argon, the reaction mixture was filtered through a Sephadex G25 column equilibrated with 50mM potassium phosphate/50 mM sodium chloride/2 mM EDTA, pH 6.5. Antibody-containing fractions were pooled and tested. antibody-SMCC was diluted to a final concentration of 10mg/ml with 50mM potassium phosphate/50 mM sodium chloride/2 mM EDTA, pH6.5 and reacted with a 10mM solution of DM1(1.7 equiv, assuming 5 SMCC/antibody, 7.37mg/ml) in dimethylacetamide. The reaction was stirred under argon at ambient temperature for 16.5 hours. The coupling reaction mixture was then filtered through a Sephadex G25 gel filtration column (1.5X4.9cm) equilibrated with 1 XPBS pH6.5. The DM 1/antibody ratio (p) was then measured by absorbance at 252nm and 280 nm.

In addition, the free cysteines on selected antibodies can be modified with the bismaleimide reagent bm (peo)4(Pierce Chemical), leaving unreacted maleimide groups on the surface of the antibodies. This can be achieved by dissolving BM (PEO)4 in a 50% ethanol/water mixture to a concentration of 10mM, adding 10-fold molar excess to a solution containing the antibody at a concentration of about 1.6mg/ml (10 micromolar) in phosphate buffered saline, and allowing it to react for 1 hour. Excess BM (PEO)4 was removed by gel filtration in 30mM citrate pH6 and 150mM NaCl buffer. An approximately 10-fold molar excess of DM1 was dissolved in Dimethylacetamide (DMA) and added to the antibody-BMPEO intermediate. Dimethylformamide (DMF) may also be used to dissolve the drug moiety reagent. The reaction mixture was allowed to react overnight and then gel filtered or dialyzed against PBS to remove unreacted drug. High molecular weight aggregates were removed by gel filtration on an S200 column in PBS and purified antibody-BMPEO-DM 1 conjugate was supplied.

Cytotoxic drugs are typically conjugated to antibodies through the numerous lysine residues of the antibody. Conjugation can also be achieved by the presence or engineering of an introduced thiol group in the antibody of interest. For example, cysteine residues have been introduced into proteins by genetic engineering techniques to create sites for covalent attachment of ligands (Better et al (1994) J.biol. chem.13: 9644. about.9650; Bernhard et al (1994) bioconjugate great chem.5: 126. about.132; Greenwood et al (1994) Therapeutic Immunology1: 247. about.255; Tu et al (1999) Proc.Natl.Acad.Sci USA96: 4862. about.4867; Kanno et al (2000) J.of Biotechnology,76: 207. about.214; Chmura et al (2001) Proc.Nat. Acad.Sci. USA98(15) 8480. about.8484; U.S. Pat. No.6,248,564). Once a free cysteine residue is present in the antibody of interest, the toxin can be attached to this site. For example, a drug linker reagent dissolved in DMSO, maleimidocaproyl-monomethyl auristatin E (MMAE) i.e., MC-MMAE, maleimidocaproyl-monomethyl auristatin F (MMAF) i.e., MC-MMAF, MC-val-cit-PAB-MMAE, or MC-val-cit-PAB-MMAF, is diluted to a known concentration in acetonitrile and water and added to the cysteine-derivatized antibody in chilled Phosphate Buffered Saline (PBS). After about 1 hour, an excess of maleimide was added to quench the reaction and cover any unreacted antibody thiol groups. The reaction mixture was concentrated by centrifugal ultrafiltration, purified and desalted by elution with G25 resin in PBS, filtered through a 0.2 μm filter under sterile conditions, and frozen for storage.

In addition, anti-TAT antibodies of the invention can be conjugated to auristatin and dolostatin toxins (such as MMAE and MMAF) using the techniques described below. Antibodies dissolved in 500mM sodium borate and 500mM sodium chloride pH8.0 were treated with an excess of 100mM Dithiothreitol (DTT). After incubation at 37 ℃ for about 30 minutes, the buffer was exchanged by elution on Sephadex G25 resin and eluted with PBS containing 1mM DTPA. The thiol/antibody value was checked by determining the reduced antibody concentration by absorbance of the solution at 280nm and the thiol concentration by reaction with DTNB (Aldrich, Milwaukee, Wis.) and absorbance at 412 nm. The reduced antibody dissolved in PBS was chilled on ice.

The drug linker reagent (1) maleimidocaproyl-monomethyl auristatin E (MMAE), i.e., MC-MMAE, (2) MC-MMAF, (3) MC-val-cit-PAB-MMAE, or (4) MC-val-cit-PAB-MMAF was diluted to known concentrations in acetonitrile and water and added to the reduced antibody in cooled PBS. After about 1 hour, an excess of maleimide was added to quench the reaction and cover any unreacted antibody thiol groups. The reaction mixture was concentrated by centrifugal ultrafiltration, the conjugated antibody was purified and desalted by elution from G25 resin in PBS, filtered through a 0.2 μm filter under sterile conditions, and frozen for storage.

Example 9: generation and characterization of TAT425 bispecific antibodies

The construction of TAT425 bispecific antibodies, in particular anti-CD 3/TAT425 bispecific antibodies, is described below.

A variety of bulge-into-cavity bispecific antibodies have been previously described. See, for example, U.S. Pat. Nos. 5,731,168 and 7,183,076, Ridgway et al, prot. Eng.9: 617-. In certain instances, the CH3 interface between humanized anti-CD 3/CD4-IgG chimeras previously described by Chamow et al, J.Immunol.153:4268(1994) was engineered to maximize the percentage of heteromultimers that could be recovered from mammalian cell lines co-transfected with the expression constructs. As used herein, "heteromultimer" refers to a molecule comprising at least a first polypeptide and a second polypeptide, wherein the second polypeptide differs from the first polypeptide in amino acid sequence by at least one amino acid residue, e.g., a bispecific antibody.

As described in the documents cited in the preceding paragraph, strategies for driving heteromultimer formation rely on the introduction of one or more "bulge" mutations into a first polypeptide, e.g., C of the H chain of a first antibody _H3 domain, and further introducing one or more corresponding "cavity" mutations into a second polypeptide, e.g., aC of the H chain of an antibody_HAnd 3 domains. "bulge" mutations replace smaller amino acids with larger amino acids, while "cavity" mutations replace larger amino acids with smaller amino acids. In addition, in addition to the bump and cavity mutations, in both polypeptides (e.g., both C's)_H3 domains) is introduced at the interface with a free thiol containing residue, which is shown to enhance heteromultimer formation. Various exemplary bulge-into-cavity mutations and free thiol-containing residues introduced at the polypeptide interface have been described. See, for example, U.S. Pat. Nos. 5,731,168 and 7,183,076, Ridgway et al, prot. Eng.9: 617-.

The following procedure was performed to generate an anti-CD 3/TAT425 bispecific antibody. Coli expression plasmids encoding humanized anti-CD 3 light (L) and heavy (H) chain variants of the mouse anti-CD 3 monoclonal antibody UCHT1 (U.S. Pat. nos. 5,821,337 and 6,407,213; Shalaby et al, j.exp.med.175:217[1992]; Rodrigues et al, int.j.cancer (Suppl.)7:45[1992]) were constructed. A number of suitable E.coli expression plasmids are known in the art, including pAK19(Carter et al, Bio/Tehcnomy 10:163-167 (1992)). Mutations that introduce bulges or cavities are made in the CH3 domain of the humanized anti-CD 3H chain (as described above), for example, by site-directed mutagenesis. A variety of site-directed mutagenesis methods are known in the art. See, e.g., Kunkel et al, Methods enzymol.154: 367-. In addition, E.coli expression plasmids encoding anti-TAT 425 light and heavy chains, such as the anti-TAT 425 light and heavy chains described herein, were constructed. Mutations that introduce the corresponding cavities (if the anti-CD 3CH3 domain has a protuberance mutation) or the corresponding protuberances (if the anti-CD 3CH3 domain has a cavity mutation) are made in the CH3 domain of the anti-TAT 425H chain (as described above), for example, by site-directed mutagenesis.

Bispecific anti-CD 3/TAT425 antibodies are generated by transforming the e.coli expression plasmids described above into a suitable e.g. 33B6 strain using methods well known in the art. See, e.g., Rodrigues et al, Cancer Res.55:63-70 (1995). Antibodies were secreted from transformed E.coli cultured in a 10L fermentor as previously described (Carter et al, Bio/Technology10:163-167 (1992)). Antibodies were purified and analyzed using procedures known in the art. See, e.g., Atwell et al, J.mol.biol.270:26-35 (1997).

Example 10: in vitro tumor cell killing assay

Mammalian cells expressing the TAT425 polypeptide of interest can be obtained using standard expression vectors and cloning techniques. Alternatively, many tumor cell lines expressing the TAT425 polypeptide of interest are publicly available, e.g., by ATCC, and can be routinely identified using standard ELISA or FACS analysis. Monoclonal antibodies (and toxin-conjugated derivatives thereof) against TAT425 polypeptides can then be used in assays to determine the ability of the antibodies to kill cells expressing TAT425 polypeptides in vitro.

For example, cells expressing the TAT425 polypeptide of interest are obtained as described above and dispensed into 96-well dishes. In one assay, antibody/toxin conjugates (or naked antibodies) were included throughout the 4 day cell incubation period. In a second independent assay, cells were incubated with antibody/toxin conjugates (or naked antibodies) for 1 hour, then washed and incubated for 4 days in the absence of antibody/toxin conjugates. Cell viability was then measured using the CellTiter-Glo luminescent cell viability assay from Promega (Cat # G7571). Untreated cells served as negative controls.

Example 11: use of TAT as hybridization probes

The following methods describe the use of nucleotide sequences encoding TAT as hybridization probes, for example, for diagnosing the presence of tumors in mammals.

DNA comprising the coding sequence for full-length or mature TAT as disclosed herein may also be used as a probe for screening homologous DNA (such as those encoding naturally occurring variants of TAT) in a human tissue cDNA library or a human tissue genomic library.

Hybridization and washing of the filters containing either library DNA was performed under the following high stringency conditions. Hybridization of the radiolabeled TAT derivative probe to the filter was performed in a solution of 50% formamide, 5 XSSC, 0.1% SDS,0.1% sodium pyrophosphate, 50mM sodium phosphate pH6.8,2 XDenhardt's solution, and 10% dextran sulfate at 42 ℃ for 20 hours. The washing of the filters was performed in an aqueous solution of 0.1x SSC and 0.1% SDS at 42 ℃.

The DNA having the desired sequence identity to the DNA encoding the full-length native sequence TAT can then be identified using standard techniques known in the art.

Example 12: expression of TAT in E.coli

This example illustrates the preparation of a non-glycosylated form of TAT by recombinant expression in e.

The TAT-encoding DNA sequence was first amplified using selected PCR primers. The primer should contain a restriction enzyme site that corresponds to the restriction enzyme site 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 was digested with restriction enzymes and dephosphorylated. The PCR amplification sequences were then ligated into the vector. The vector preferably comprises sequences encoding an antibiotic resistance gene, a trp promoter, a polyhistidine leader sequence (comprising the first 6 STII codons, a polyhistidine sequence and an enterokinase cleavage site), a TAT coding region, a lambda transcription terminator and an argU gene.

The selected E.coli strain was then transformed with the ligation mixture by the method described in Sambrook et al, supra. Transformants were identified by their ability to grow on LB plates and antibiotic resistant colonies were selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing.

Selected clones may be cultured overnight in liquid media, such as LB media supplemented with antibiotics. The overnight culture can then be used to inoculate a large-scale medium. The cells are then grown to the desired optical density, during which the expression promoter is turned on.

After culturing the cells for several more hours, the cells can be harvested by centrifugation. Cell pellets harvested by centrifugation can be lysed using various reagents known in the art, and the lysed TAT protein can then be purified using a metal chelating column under conditions that allow for tight binding of the protein.

Using the following scheme, TAT can be expressed in escherichia coli in a polyhistidine-tagged form. The TAT-encoding DNA was first amplified using selected PCR primers. The primers contain restriction enzyme sites corresponding to those on the selected expression vector, as well as other useful sequences that provide efficient and reliable translation initiation, rapid purification on metal chelating columns, and proteolytic cleavage of enterokinase. The PCR-amplified poly-histidine-tagged sequences were then ligated into expression vectors for transformation of E.coli hosts based on strain 52 (W3110 fuhA (tonA) lon galE rpoHts (htpRts) clpP (lacIq)). The transformants were first shake-cultured in LB medium containing 50mg/ml carbenicillin at 30 ℃ until O.D.600 reached 3-5. The culture was then incubated in CRAP medium (by mixing 3.57g (NH) in 500mL of water ₄)₂SO₄0.71g NaCitrate.2H₂O,1.07g KCl,5.36g Difco yeast extract, 5.36g Sheffield ghost SF, and 110mM MPOS pH7.3,0.55% (w/v) glucose and 7mM MgSO₄To make up) and cultured with shaking at 30 ℃ for about 20 to 30 hours. Samples were taken to verify expression by SDS-PAGE analysis and the bulk culture was centrifuged to pellet the cells. The cell pellet was frozen until purification and refolding.

Coli pellets (6-10 g pellet) from 0.5 to 1 liter of fermentation broth were resuspended in 10 volumes (w/v) of 7M guanidine, 20mM Tris pH8 buffer. Solid sodium sulfite and sodium tetrathionate were added to final concentrations of 0.1M and 0.02M, respectively, and the solution was stirred at 4 ℃ overnight. This step produces a denatured protein in which all cysteine residues are blocked by sulphitation (sulfitation). The solution was centrifuged in a Beckman ultracentrifuge at 40,000rpm for 30 minutes. The supernatant was diluted with 3-5 volumes of metal chelating column buffer (6M guanidine, 20mM Tris, pH 7.4) and filtered through a 0.22 micron filter until clear. The clarified extract was applied to a 5ml Qiagen Ni-NTA metal chelating column equilibrated in metal chelating column buffer. The column was washed with additional buffer containing 50mM imidazole (Calbiochem, Utrol grade) pH 7.4. The protein was eluted with a buffer containing 250mM imidazole. Fractions containing the desired protein were pooled and stored at 4 ℃. The concentration of the protein was estimated by its absorbance at 280nm using an extinction coefficient calculated from the amino acid sequence of the protein.

Proteins were refolded by diluting the protein sample in freshly prepared refolding buffer consisting of: 20mM Tris, pH8.6,0.3M NaCl,2.5M urea, 5mM cysteine, 20mM glycine and 1mM EDTA. The refolding volume was chosen so that the final protein concentration was between 50 and 100. mu.g/ml. The refolding solution was gently agitated at 4 ℃ for 12-36 hours. The refolding reaction was terminated by the addition of TFA to a final concentration of 0.4% (pH. about.3). Prior to further purification of the protein, the solution was filtered through a 0.22 micron filter and acetonitrile was added to a final concentration of 2-10%. The refolded protein was chromatographed on a Poros R1/H reverse phase column using 0.1% TFA as the flow buffer and a gradient of 10 to 80% acetonitrile. Aliquots of the fractions with a280 absorbance were analyzed on SDS polyacrylamide gel and fractions containing homogeneously refolded proteins were pooled. Generally, the correct refolded forms of most proteins are eluted at the lowest acetonitrile concentrations, since those forms are the most compact, with the hydrophobic interior masked from interaction with the reverse phase resin. The aggregated form is often eluted at higher acetonitrile concentrations. In addition to releasing the misfolded form of the protein from the desired form, the reverse phase step also removes endotoxin from the sample.

Fractions containing the desired folded TAT polypeptide were combined and acetonitrile was removed with a gentle nitrogen stream directed at the solution. Proteins were formulated into 20mM hepes pH6.8 containing 0.14M sodium chloride and 4% mannose by dialysis or by gel filtration using G25Superfine (Pharmacia) resin equilibrated in formulation buffer and sterile filtered.

Certain TAT polypeptides disclosed herein have been successfully expressed and purified using this technique.

Example 13: expression of TAT in mammalian cells

This example illustrates the preparation of potentially glycosylated forms of TAT by recombinant expression in mammalian cells.

Vector pRK5 (see EP307,247 published on 3/15 of 1989) was used as an expression vector. Optionally, the TAT DNA is ligated into pRK5 digested with selected restriction enzymes to allow insertion of the TAT DNA, using a ligation method such as described in Sambrook et al, supra. The resulting vector was designated pRK 5-TAT.

In one embodiment, the selected host cell may be a 293 cell. Human 293 cells (ATCC CCL 1573) are cultured to confluence in tissue culture dishes in a medium such as DMEM supplemented with fetal bovine serum, optionally together with nutrients and/or antibiotics. About 10. mu.g of pRK5-TAT DNA was mixed with about 1. mu.g of DNA encoding the VA RNA gene (Thimmappaya et al, Cell,31:543 (1982)), and dissolved in 500. mu.l of 1mM Tris-HCl,0.1mM EDTA,0.227M CaCl ₂. To the mixture was added dropwise 500. mu.l of 50mM HEPES (pH7.35),280mM NaCl,1.5mM NaPO₄And precipitated at 25 ℃ for 10 minutes to form a precipitate. The precipitate was suspended and added to 293 cells, which were allowed to stand at 37 ℃ for about 4 hours. The medium was aspirated off and 2ml of PBS containing 20% glycerol was added for 30 seconds. The 293 cells were then washed with serum-free medium, fresh medium was added, and the cells were incubated for about 5 days.

Approximately 24 hours after transfection, the medium was removed and used either with medium (alone) or with 200. mu. Ci/ml³⁵S-cysteine and 200. mu. Ci/ml³⁵Medium replacement of S-methionine. After 12 hours of incubation, conditioned media was collected, concentrated on a rotary filter, and applied to a 15% SDS gel. The treated gel may be dried and the film exposed for a selected period of time to reveal the presence of the TAT polypeptide. The cultures containing transfected cells can be further incubated (in serum-free medium) and the medium tested in the selected bioassay.

In an alternative technique, TAT can be transiently introduced into 293 cells using the dextran sulfate method described by Somparyrac et al, Proc.Natl.Acad.Sci.,12:7575 (1981). 293 cells were grown to maximum density in rotary shake flasks and 700. mu.g pRK5-TAT DNA was added. Cells were first concentrated by centrifugation from a rotating flask and washed with PBS. The DNA-dextran pellet was incubated on the cell pellet for 4 hours. Cells were treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and again placed in a rotating shaker flask containing tissue culture medium, 5. mu.g/ml bovine insulin, and 0.1. mu.g/ml bovine transferrin. After about 4 days, the conditioned medium was centrifuged and filtered to remove cells and debris. The sample containing the expressed TAT can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography.

In another embodiment, TAT may be expressed in CHO cells. Known reagents such as CaPO can be used₄Or DEAE-dextran, pRK5-TAT was transfected into CHO cells. As described above, the cell culture may be incubated and incubated with culture medium (alone) or with a radioactive label such as³⁵The medium for S-methionine replaced the medium described above. Following determination of the presence of TAT polypeptide, the medium can be replaced with serum-free medium. Preferably, the culture is incubated for about 6 days and then the conditioned medium is harvested. The culture broth containing the expressed TAT can then be concentrated and purified by any chosen method.

Epitope-tagged TAT can also be expressed in host CHO cells. TAT can be subcloned out of pRK5 vector. The subcloned insert may be PCR fused into a baculovirus expression vector in frame with a selected epitope tag, such as a polyhistidine tag. The poly-histidine-tagged TAT insert can then be subcloned into an SV40 driven vector containing a selection marker such as DHFR to select for stable clones. Finally, CHO cells can be transfected with SV40 driven vectors (as described above). The labeling can be performed as described above to verify expression. And then may be passed through any selected method such as Ni ²⁺-chelate affinity chromatography to concentrate and purify the culture broth containing the expressed polyhistidine-tagged TAT.

TAT may also be expressed in CHO and/or COS cells by transient expression protocols, or in CHO cells by other stable expression protocols.

Stable expression in CHO cells was performed using the following protocol. The proteins were expressed as IgG constructs (immunoadhesins) in which the coding sequence for the soluble form of the corresponding protein (e.g. the extracellular domain) was fused to the IgG1 constant region sequence containing the hinge, CH2 and CH3 domains, and/or carrying a poly-histidine tag.

After PCR amplification, each DNA was subcloned into a CHO expression vector using standard techniques as described in Ausubel et al, Current Protocols of Molecular Biology, Unit3.16, John Wiley and Sons (1997). The CHO expression vector was constructed with compatible restriction sites 5 'and 3' to the DNA of interest to allow for convenient shuttling of the cDNA. Vectors for expression in CHO cells use the SV40 early promoter/enhancer to drive expression of the cDNA of interest and dihydrofolate reductase (DHFR) as described in Lucas et al, Nucl. acids Res.24:9(1774-1779) (1996). DHFR expression allows stable maintenance of the selection plasmid after transfection.

Using commercial transfection reagents(Quiagen)、Or(Boehringer Mannheim) 12. mu.g of the desired plasmid DNA was introduced into approximately 1X10⁷And (4) CHO cells. Cells were cultured as described in Lucas et al, supra. Will be about 3x10⁷The individual cells were cryopreserved in ampoules for further culture and production as described below.

Melting plasmid DNA containing ampoules by placing in a water bath and mixing with shaking. The contents were transferred to a centrifuge tube containing 10mL of medium and centrifuged at 1000rpm for 5 minutesA clock. The supernatant was aspirated and the cells were resuspended in 10mL selection medium (0.2 μm filtered PS20 containing 5%0.2 μm diafiltered fetal bovine serum). The cells were then aliquoted into 100mL roller bottles containing 90mL selection medium. After 1-2 days, cells were transferred to 250mL spinner flasks containing 150mL selective growth medium and incubated at 37 ℃. After a further 2-3 days, at 3X10⁵cells/mL were inoculated in 250mL, 500mL and 2000mL roller bottles. The cell culture medium was replaced with fresh medium by centrifugation and re-suspension in production medium. Although any suitable CHO medium may be used, in practice the production medium described in U.S. Pat. No. 5,122,469, issued 6, 16 1992, may be used. At 1.2x10⁶Individual cells/mL were inoculated into 3L production roller bottles. On day 0, cell number and pH were determined. On day 1, the flasks were sampled and the sparging of filtered air was started. On day 2, the flasks were sampled, the temperature was changed to 33 ℃, and 30mL of 500g/L glucose and 0.6mL of 10% antifoam (e.g., 35% dimethicone emulsion, Dow Corning365 medical emulsion) were added. During the whole production process, the pH is adjusted as required to be kept at about 7.2. After 10 days, or until viability drops below 70%, cell cultures were harvested by centrifugation and filtered through 0.22 μm filters. The filtrate was stored at 4 ℃ or immediately applied to a column for purification.

For the poly-histidine-tagged constructs, proteins were purified using Ni-NTA columns (Qiagen). Imidazole was added to the conditioned medium to a concentration of 5mM before purification. Conditioned medium was pumped at 4 ℃ to a 6ml Ni-NTA column equilibrated in 20mM Hepes pH7.4 containing 0.3M NaCl and 5mM imidazole at a flow rate of 4-5 ml/min. After loading, the column was washed with additional equilibration buffer and the protein was eluted with equilibration buffer containing 0.25M imidazole. The highly purified protein was then desalted using a 25ml G25Superfine (Pharmacia) column into a storage buffer containing 10mM Hepes,0.14M NaCl and 4% mannitol, pH6.8, and stored at-80 ℃.

Immunoadhesin (Fc containing) constructs were purified from conditioned media as follows. The conditioned culture was applied by pump to a 5ml protein A column (Pharmacia) equilibrated in 20mM sodium phosphate buffer, pH 6.8. After loading, the column was washed thoroughly with equilibration buffer and then eluted with 100mM citric acid, pH 3.5. The eluted protein was immediately neutralized by collecting 1ml fractions into tubes containing 275. mu.L of 1M Tris buffer, pH 9. The highly purified protein was then desalted into storage buffer as described above for the polyhistidine-tagged proteins. Homogeneity was assessed by SDS polyacrylamide gel and N-terminal amino acid sequencing by Edman degradation.

Example 14: expression of TAT in Yeast

The following method describes recombinant expression of TAT in yeast.

First, a yeast expression vector was constructed for intracellular production or secretion of TAT initiated by ADH2/GAPDH promoter. DNA encoding TAT and the promoter is inserted into the appropriate restriction enzyme sites of selected plasmids to direct intracellular expression of TAT. For secretion, the DNA encoding TAT can be cloned into a selected plasmid together with DNA encoding ADH2/GAPDH promoter for TAT expression, a native TAT signal peptide or other mammalian signal peptide, or, for example, a yeast alpha factor or invertase secretion signal/leader sequence, and a linker sequence, if desired.

Yeast cells such as yeast strain AB110 can then be transformed with the expression plasmids described above and cultured in the selected fermentation medium. The supernatant of the transformed yeast can be analyzed by precipitation with 10% trichloroacetic acid and separation by SDS-PAGE, followed by gel staining with Coomassie blue.

Recombinant TAT can then be isolated and purified by removing yeast cells from the fermentation broth by centrifugation and concentrating the broth using a selected cartridge filter. The TAT-containing concentrate can be further purified using selected column chromatography resins.

Example 15: expression of TAT in baculovirus infected insect cells

The following method describes recombinant expression of TAT in baculovirus infected insect cells.

The sequence encoding TAT is fused upstream of the epitope tag contained within the baculovirus expression vector. Such epitope tags include polyhistidine tags and immunoglobulin tags (like the Fc region of IgG). A variety of plasmids may be employed, including plasmids derived from commercial plasmids such as pVL1393 (Novagen). Briefly, the TAT-encoding sequence or a desired portion of the TAT-encoding sequence, such as the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding a mature protein (if the protein is extracellular), is amplified by PCR using primers complementary to the 5 'and 3' regions. The 5' primer will incorporate flanking (selected) restriction enzyme sites. The product is then digested with those selected restriction enzymes and subcloned into an expression vector.

The above plasmid and BACULOGOLD were transfected with lipofectin (commercially available from GIBCO-BRL)^TMViral DNA (Pharmingen) was co-transfected into Spodoptera frugiperda ("Sf 9") cells (ATCC CRL 1711) to produce recombinant baculovirus. After 4-5 days of incubation at 28 ℃, the released virus was harvested and used for further amplification. Viral infection and protein expression was performed as described in O' Reilley et al, Baculoviral expression vectors: A Laboratory Manual, Oxford: Oxford University Press (1994).

The expressed polyhistidine-tagged TAT can then be purified, for example, by Ni as follows²⁺-chelate affinity chromatography. Extracts were prepared from recombinant virus-infected Sf9 cells as described by Rupert et al, Nature,362:175-179 (1993). Briefly, Sf9 cells were washed and resuspended in sonication buffer (25 mLHepes pH7.9,12.5mM MgCl)₂0.1mM EDTA,10% glycerol, 0.1% NP-40,0.4 MKCl), sonicated 2 times for 20 seconds on ice. The sonicate was clarified by centrifugation and the supernatant was placed in loading buffer (50 mM phosphate, 300mM NaCl,10% glycerol)Oil, ph 7.8) and filtered through a 0.45 μm filter. Preparation of Ni in a bed volume of 5mL²⁺NTA agarose columns (available from Qiagen), washed with 25mL water and equilibrated with 25mL loading buffer. The filtered cell extract was applied to the column at 0.5 mL/min. Wash column to baseline A with loading buffer₂₈₀At this point, collection of fractions begins. Next, the column was washed with a second washing buffer (50 mM phosphate, 300mM NaCl,10% glycerol, pH 6.0) to elute the non-specifically bound proteins. Again reach A₂₈₀After baseline, the column was washed with a gradient of 0 to 500mM imidazole in a second wash buffer. Fractions of 1mL were collected and stained by SDS-PAGE and silver staining or by using Ni coupled with alkaline phosphatase ²⁺Western blot analysis of NTA (Qiagen). Mixing the eluates with His₁₀Fraction of labeled TAT and dialyzed against loading buffer.

Alternatively, purification of IgG-tagged (or Fc-tagged) TAT can be performed using known chromatographic techniques, including, for example, protein a or protein G column chromatography.

Example 16: purification of TAT polypeptides using specific antibodies

Native or recombinant TAT polypeptides can be purified by a variety of standard techniques in the art of protein purification. For example, a pro-TAT polypeptide, a mature TAT polypeptide, or a pre-TAT polypeptide (pre-TAT polypeptide) is purified by immunoaffinity chromatography using an antibody specific for the TAT polypeptide of interest. Generally, immunoaffinity columns are constructed by covalently coupling anti-TAT polypeptide antibodies to activated chromatography resins.

Polyclonal immunoglobulins were prepared from immune sera by precipitation with ammonium sulfate or by purification of immobilized protein a (pharmacilkb Biotechnology, Piscataway, n.j.). Similarly, monoclonal antibodies were prepared from mouse ascites fluid by ammonium sulfate precipitation or immobilized protein a chromatography. Covalent attachment of partially purified immunoglobulins to chromatography trees Lipid such as CnBr activated SEPHAROSE^TM(Pharmacia LKB Biotechnology). The antibody was coupled to the resin according to the manufacturer's instructions, the resin was blocked, and the derivatized resin was washed.

Such immunoaffinity chromatography columns are used for the purification of TAT polypeptides by preparing a fraction containing TAT polypeptides in soluble form from cells. This preparation is solubilized by adding detergent to whole cells or a subcellular fraction obtained by differential centrifugation or derivatized by other methods well known in the art. Alternatively, a soluble TAT polypeptide comprising a signal sequence may be secreted into the medium in which the cells are cultured in useful amounts.

The preparation containing soluble TAT polypeptide is passed through an immunoaffinity column and the column is washed (e.g., a high ionic strength buffer in the presence of a detergent) under conditions that allow preferential adsorption of the TAT polypeptide. The column is then eluted under conditions that disrupt antibody/TAT polypeptide binding (e.g., a low pH buffer, such as about pH2-3, or a high concentration of chaotropic agent, such as urea or thiocyanate ions) and the TAT polypeptide is collected.

The foregoing written description is considered to be sufficient to enable those skilled in the art to practice the invention. The invention is not to be limited in scope by the deposited constructs, as the deposited embodiments are intended as single illustrations of certain aspects of the invention, and any constructs that are functionally equivalent are within the scope of the invention. The material deposits herein are not to be construed as an admission that the written description contained herein is not sufficient to enable 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 exemplifications thereof described. 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 are within the scope of the appended claims.

Claims

1. An isolated antibody comprising at least one CDR sequence selected from the group consisting of:

(a) 10-12 of any CDR-L1 sequence of SEQ ID NO;

(b) a CDR-L2 sequence of any one of SEQ ID NOS 13-15;

(c) a CDR-L3 sequence of any one of SEQ ID NOs 16-18;

(d) a CDR-H1 sequence of any one of SEQ ID NOs 19-22;

(e) a CDR-H2 sequence of any one of SEQ ID NOS 23-26; and

(f) the CDR-H3 sequence of any one of SEQ ID NOs 27-30.

2. The antibody of claim 1 which is an antibody fragment.

3. The antibody of claim 1, which is a chimeric or humanized antibody.

4. The antibody of claim 1, which is a bispecific antibody.

5. The antibody of claim 4 which binds to a protein expressed on the surface of a T cell.

6. The antibody of claim 5, wherein the protein expressed on the surface of a T cell is CD 3.

7. The antibody of claim 1, which is conjugated to a growth inhibitory agent.

8. The antibody of claim 1, which is conjugated to a cytotoxic agent.

9. The antibody of claim 8, wherein the cytotoxic agent is selected from the group consisting of a toxin, an antibiotic, a radioisotope, and a nucleolytic enzyme.

10. The antibody of claim 8, wherein the cytotoxic agent is a toxin.

11. The antibody of claim 10, wherein the toxin is selected from the group consisting of maytansinoids and calicheamicin.

12. The antibody of claim 10, wherein the toxin is a maytansinoid.

13. The antibody of claim 1, which is produced in bacteria.

14. The antibody of claim 1, which is produced in CHO cells.

15. The antibody of claim 1 which induces death of a cell to which it binds.

16. The antibody of claim 15, wherein the cell is a prostate cancer cell.

17. The antibody of claim 1, which is detectably labeled.

18. An isolated antibody comprising any of the VL sequences set forth in SEQ ID NOS: 3-5.

19. An isolated antibody comprising any of the VH sequences shown in SEQ ID NOS: 6-9.

20. An isolated antibody comprising the VL sequence SEQ ID NO 3 and the VH sequence SEQ ID NO 6.

21. An isolated antibody comprising the VL sequence SEQ ID NO 3 and the VH sequence SEQ ID NO 7.

22. An isolated antibody comprising the VL sequence SEQ ID NO 4 and the VH sequence SEQ ID NO 8.

23. An isolated antibody comprising the VL sequence SEQ ID NO 5 and the VH sequence SEQ ID NO 9.

24. A cell that produces the antibody of claim 1.

25. An isolated nucleic acid encoding the antibody of claim 1.

26. A method of identifying a first antibody that binds to a TAT425 antigenic epitope bound by a second antibody, wherein said second antibody is an antibody according to claim 1, said method comprising determining the ability of said first antibody to block the binding of said second antibody to a TAT425 polypeptide, wherein the ability of said first antibody to block the binding of said second antibody to said TAT425 polypeptide by at least 40% at equal antibody concentrations indicates that said first antibody is capable of binding to an epitope bound by said second antibody.

27. A method of inhibiting growth of a cell expressing a TAT425 polypeptide, the method comprising contacting the cell with an antibody of claim 1, wherein binding of the antibody to the TAT425 polypeptide causes growth inhibition of the cell.

28. The method of claim 27, wherein the TAT425 polypeptide comprises the amino acid sequence SEQ id No. 2 or an extracellular domain thereof.

29. The method of claim 27, wherein the cell is a prostate cancer cell.

30. A method of therapeutically treating a mammal having a cancerous tumor comprising cells that express a TAT425 polypeptide, said method comprising administering to said mammal a therapeutically effective amount of an antibody of claim 1, thereby effectively treating said mammal.

31. The method of claim 30, wherein the TAT425 polypeptide comprises the amino acid sequence SEQ id No. 2 or an extracellular domain thereof.

32. The method of claim 30, wherein the cell is a prostate cancer cell.

33. A method of determining the presence of a TAT425 protein in a sample suspected of containing said protein, said method comprising exposing said sample to an antibody of claim 1 and determining binding of said antibody to said protein in said sample, wherein binding of said antibody to said protein is indicative of the presence of said protein in said sample.

34. The method of claim 33, wherein said sample comprises cells suspected of expressing said protein.

35. The method of claim 34, wherein the cell is a prostate cancer cell.

36. The method of claim 33, wherein the antibody is detectably labeled.

37. 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 TAT425 polypeptide 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 TAT425 polypeptide in the test sample as compared to the control sample is indicative of the presence of a tumor in the mammal from which the test sample was obtained.

38. The method of claim 37, wherein the step of determining the expression level of a gene encoding said polypeptide comprises using an oligonucleotide in an in situ hybridization or RT-PCR assay.

39. The method of claim 37, wherein the step of determining the expression level of a gene encoding said protein comprises using an antibody in immunohistochemistry or Western blot analysis.

40. The method of claim 37, wherein the tumor is a prostate tumor.

41. The method of claim 37, wherein the TAT211 polypeptide comprises the amino acid sequence of SEQ id No. 2 or an extracellular domain thereof.

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 of claim 1 and detecting the formation of a complex between said antibody and TAT425 protein in the test sample, wherein the formation of a complex indicates the presence of a tumor in said mammal.

43. The method of claim 42, wherein the test sample of tissue cells is obtained from an individual suspected of having a cancerous tumor.

44. The method of claim 43, wherein the cancerous tumor is a prostate tumor.

45. The method of claim 42, wherein the TAT425 polypeptide comprises an amino acid sequence of SEQ ID NO 2 or an extracellular domain thereof.

46. An antibody that binds to the same TAT425 epitope as an antibody selected from the group consisting of: 2E4.6.1, 4D11.17.2, 13H2.28.2, and 14 E7.17.1.