JP2006516192A - Compositions and methods for tumor diagnosis and treatment - Google Patents

Abstract

The present invention relates to compositions of matter useful for the diagnosis and treatment of mammalian tumors and methods of using such compositions of matter for the same use.

Description

The present invention relates to compositions of matter useful for the diagnosis and treatment of tumors in mammals and methods of using the composition of matter for the same applications.

BACKGROUND OF THE INVENTION Malignant tumors (cancers) are the second leading cause of death following heart disease in the United States (Boring et al., CA Cancel J. Clin. 43: 7 (1993)). Cancer is derived from normal tissue and increases the number of abnormal or tumorigenic cells that form a tumor mass, the invasion of adjacent tissues by these tumorigenic tumor cells, and ultimately the blood and lymphatic system. It is characterized by the generation of malignant cells that spread through a process called metastasis to local lymph nodes and distant sites. In a cancerous state, cells proliferate under conditions in which normal cells do not grow. Cancer itself manifests in a wide variety of forms characterized by different degrees of invasiveness and aggressiveness.
In an attempt to find an effective cellular target for cancer diagnosis and treatment, researchers have specifically compared the surface of one or more specific types of cancer cells to one or more normal non-cancerous cells. We have sought to identify transmembrane or otherwise membrane-bound polypeptides that are expressed. Often, such membrane-bound polypeptides are abundantly expressed on the surface of cancer cells compared to the surface of non-cancerous cells. The identification of such tumor-associated cell surface antigen polypeptides creates the ability to specifically target cancer cells via antibody-based therapy. In this regard, it is noted that antibody-based therapy has proven very effective in the treatment of certain cancers. For example, Herceptin® and Rituxan® (both from Genentech, South San Francisco, California) are antibodies that have been successfully used to treat breast cancer and non-Hodgkin lymphoma, respectively. More specifically, Herceptin® is a recombinant DNA-derived humanized monoclonal antibody that selectively binds to the extracellular domain of human epidermal growth factor receptor 2 (HER2) proto-oncogene. Overexpression of HER2 protein is observed in 25-30% primary breast cancer. Rituxan® is a genetically engineered chimeric mouse / human monoclonal antibody against the CD20 antigen found on the surface of normal and malignant B lymphocytes. Both of these antibodies are produced recombinantly in CHO cells.

In other attempts to find an effective cellular target for cancer diagnosis and treatment, researchers have (1) one cancer cell relative to one or more specific types of non-cancerous normal cells. Or a non-membrane-bound polypeptide specifically produced by a plurality of specific types, (2) a polypeptide produced by a cancer cell with an expression level significantly higher than that of one or more normal non-cancerous cells, Or (3) the expression is specific to only one tissue type (or a very limited number of different cell types) in both cancerous and non-cancerous conditions (eg normal prostate and prostate tumor tissue) We have searched for limited polypeptides. Such polypeptides can be secreted by cancer cells or remain intracellular. Furthermore, such polypeptides may rather be expressed not by the cancer cells themselves, but by cells that produce and / or secrete polypeptides that have an enhancing or growth promoting effect on the cancer cells. Such secreted polypeptides are proteins that often give cancer cells a growth advantage over normal cells, including, for example, angiogenic factors, cell attachment factors, growth factors, and the like. Identification of antagonists of such non-membrane bound polypeptides is expected to be an effective therapeutic agent for the treatment of such cancers. Furthermore, identification of the expression pattern of such polypeptides will be useful in the diagnosis of specific cancers in mammals.
As mentioned above, genes that are up-regulated in tumor cells are attractive targets for anti-cancer therapy. The general underlying mechanism of increased expression in tumors is often unknown, but tumor-specific upregulation of some genes is a result of abnormal DNA amplification, a phenomenon common to many tumors. It is believed that there is. The availability of human genome sequences and over 4 million expressed sequence tags (ESTs) from public databases has not only allowed us to build an overall transcriptome map, Large-scale analysis of differential gene expression in tumor tissues has become possible. By using the computational methods described herein, non-random regions of a cluster of genes are found on various chromosomes that have increased expression in 10 types of tumors, and these genomic regions are often found in other methods. Corresponded to the experimentally demonstrated tumor amplicon. Thus, the present disclosure provides for the identification of the entire genome of chromosomal regions with increased tumor expression, i.e., regions with potential tumor amplicons.

  There is a great need for additional diagnostic agents that can detect the presence of tumors in mammals and therapeutic agents that effectively inhibit neoplastic cell growth, respectively. Accordingly, (1) a cell membrane-bound polypeptide that is abundantly expressed by one or more types of cancer cells compared to on normal cells or other different cancer cells, (2) one or more non-cancerous normal cells Non-specifically produced by one or more specific types of cancer cells (or by other cells that produce a polypeptide having an enhancing effect on the growth of the cancer cells) compared to the case by multiple specific types A membrane-bound polypeptide, (3) a non-membrane-bound polypeptide produced by a cancer cell with an expression level significantly higher than that of one or more normal non-cancerous cells, or (4) cancerous and non-cancerous conditions Identify polypeptides that are specifically expressed in only one (or a very limited number of different) tissue types in both (eg, normal prostate and prostate tumor tissue), and these polypeptides And it Using a nucleic acid over de, it is an object of the present invention to produce a composition useful in the therapeutic treatment and diagnostic detection of cancer in mammals. Identifies cell membrane-bound, secreted or intracellular polypeptides whose expression is restricted to a single or very limited number of tissues and uses the polypeptides and nucleic acids encoding them to detect mammalian cancer It is also an object of the present invention to produce a composition useful for therapeutic treatment and diagnostic detection.

SUMMARY OF THE INVENTION Embodiments In this specification, Applicants identify non-random regions of gene clusters of various chromosomes whose expression is significantly increased in one or more tumor types compared to normal tissues of the same tissue type. First disclosed. These non-random chromosomal regions are regions of chromosomal DNA that are not amplified in normal cells of the same tissue type but are amplified in tumor cells, and as a result are located within the amplicon when compared to the expression level in normal tissues It is thought to cause tumor-specific overexpression in the gene.
In addition, Applicants have identified various cellular polypeptides (and their expression) that are more expressed on the surface of one or more types of cancer cells compared to the surface of one or more types of normal non-cancer cells. The identification of the coding nucleic acid or fragment thereof is first described. Alternatively, such polypeptides are expressed by cells that produce and / or secrete polypeptides that have an enhancing or growth promoting effect on cancer cells. Alternatively, such polypeptides are not overexpressed by tumor cells compared to normal cells of the same tissue type, but rather a single or very limited number of tissue types (preferably It can be specifically expressed by both normal cells and tumor cells of tissues that are not essential for life, such as the prostate. Here, all of the above polypeptides are called Tumor-associated Antigenic Target polypeptides ("TAT" polypeptides) and should be effective targets for cancer therapy and diagnosis in mammals. Is expected.
Accordingly, 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 one aspect, an isolated nucleic acid molecule is disclosed herein: (a) a full-length TAT polypeptide having an amino acid sequence disclosed herein; a TAT polypeptide amino acid sequence lacking a signal peptide disclosed herein; A DNA molecule that encodes the extracellular domain of a transmembrane TAT polypeptide, with or without a signal peptide, or any other specifically defined fragment of the full-length TAT polypeptide amino acid sequence disclosed herein, Or (b) at least about 80% nucleic acid sequence identity, or at least about 81%, 82%, 83%, 84%, 85%, 86%, 87% to the complementary strand of the DNA molecule of (a) , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity Comprising a nucleotide sequence having.
In another aspect, the isolated nucleic acid molecule comprises (a) a coding sequence for a full-length TAT polypeptide cDNA disclosed herein, a coding sequence for a TAT polypeptide that lacks a signal peptide disclosed herein, A coding sequence comprising or excluding the signal peptide of the extracellular domain of the transmembrane TAT polypeptide disclosed in, or any other specifically defined fragment of the full-length TAT polypeptide amino acid sequence disclosed herein At least about 80% nucleic acid sequence identity, or at least about 81%, 82%, 83%, 84%, 85%, 86 to the coding sequence, or (b) the complementary strand of the DNA molecule of (a) %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity It comprises a nucleotide sequence having.

In a further aspect, the invention provides (a) a DNA molecule that encodes the same mature polypeptide encoded by any of the full-length coding regions of the human protein cDNA deposited with the ATCC disclosed herein, or ( b) at least about 80% nucleic acid sequence identity to the complementary strand of the DNA molecule of (a), or at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 Isolation comprising a nucleotide sequence having a nucleic acid sequence identity of%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% Nucleic acid molecules.
Another aspect of the invention is an isolation comprising a nucleotide sequence encoding a TAT polypeptide that is either transmembrane domain deficient or transmembrane domain inactivated, or is complementary to such an encoded nucleotide sequence. And the transmembrane domains of such polypeptides are disclosed herein. Accordingly, the soluble extracellular domain of the TAT polypeptides described herein is contemplated.

  In another aspect, the invention provides (a) a TAT polypeptide having the full length amino acid sequence disclosed herein, a TAT polypeptide amino acid sequence lacking a signal peptide disclosed herein, a transmembrane TAT disclosed herein. A nucleotide sequence encoding the extracellular domain of a polypeptide, with or without a signal peptide, or any other specifically defined fragment of the full-length TAT polypeptide amino acid sequence disclosed herein, or (b ) Relates to an isolated nucleic acid molecule which hybridizes with the complementary strand of the nucleotide sequence of (a). In this regard, embodiments of the present invention provide full length TAT polypeptide coding sequences (or disclosed herein) that may find use, for example, as hybridization probes useful as diagnostic probes, antisense oligonucleotide probes. A fragment of any of the amplified chromosomal regions shown in Appendix B to J), or a complementary chain thereof, or a binding site of an anti-TAT polypeptide antibody, TAT-binding oligopeptide or other small organic molecule that binds to the TAT polypeptide. It relates to a coding fragment of a full-length TAT polypeptide that can optionally encode a polypeptide comprising. Such nucleic acid fragments are usually 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, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, It is 960, 970, 980, 990, or 1000 nucleotides long, and in this context the term “about” means the displayed nucleotide sequence length plus or minus 10% of the displayed length. A novel fragment of a TAT polypeptide-encoding nucleotide sequence can be used to align any TAT polypeptide-encoding nucleotide sequence with other known nucleotide sequences using any of the well-known sequence alignment programs. It is known that it can be routinely determined by determining whether the encoded nucleotide sequence fragment is novel. All novel fragments of such TAT polypeptide-encoding nucleotide sequences are contemplated herein. Also contemplated are binding sites for TAT polypeptide fragments encoded by these nucleotide molecule fragments, preferably anti-TAT antibodies, TAT-binding oligopeptides or other small organic molecules that bind to TAT polypeptides. A TAT polypeptide fragment.

In other embodiments, the invention provides an isolated TAT polypeptide encoded by any of the isolated nucleic acid sequences identified above.
In one aspect, the present invention provides a TAT polypeptide having the full length amino acid sequence disclosed herein, a TAT polypeptide amino acid sequence lacking the signal peptide disclosed herein, having or having a signal peptide disclosed herein. Encoded by the extracellular domain of the transmembrane TAT polypeptide protein, any of the nucleic acid sequences disclosed herein, or other specifically defined fragments of the full-length TAT polypeptide amino acid sequences disclosed herein. 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 composition An isolated TAT polypeptide comprising an amino acid sequence having.

In a further aspect, the present invention provides at least about 80% amino acid sequence identity, or at least about 81% to the amino acid sequence encoded by any of the human protein cDNAs disclosed herein and deposited with the ATCC. 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 It relates to an isolated TAT polypeptide comprising an amino acid sequence having% or 99% amino acid sequence identity.
In certain aspects, the present invention provides an isolated TAT polypeptide that does not have an N-terminal signal sequence and / or initiation methionine, which is encoded by a nucleotide sequence that encodes such an amino acid sequence described above. . Methods for producing this are also disclosed herein, wherein a host cell comprising a vector containing a suitable encoding nucleic acid molecule is cultured under conditions suitable for expression of a TAT polypeptide, and a cell culture Recovering the TAT polypeptide from.

Another aspect of the invention provides an isolated TAT polypeptide in which the transmembrane domain is deleted or the transmembrane domain is inactivated. Methods for producing this are also disclosed herein, in which host cells containing a vector containing the appropriate encoding nucleic acid molecule are cultured under conditions suitable for expression of the TAT polypeptide, from the cell culture. Recovering the TAT polypeptide.
In another embodiment of the invention, the invention provides a vector comprising DNA encoding any of the polypeptides described herein. A host cell comprising any such vector is also provided. By way of example, the host cell can be a CHO cell, E. coli, or yeast. Further provided is a method for producing any of the polypeptides disclosed herein, culturing host cells under conditions suitable for expression of the desired polypeptide, and recovering the desired polypeptide from the cell culture. Comprising.

In other embodiments, the invention provides an isolated chimeric polypeptide comprising any of the TAT polypeptides disclosed herein fused to a heterologous (non-TAT) polypeptide. Examples of such chimeric molecules include any of the TAT polypeptides disclosed herein fused to a heterologous polypeptide such as, for example, an epitope tag sequence or an Fc region of an immunoglobulin.
In other embodiments, the invention provides an antibody that binds, preferably specifically, to any of the above or below described polypeptides. In some cases, the antibody is a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody, single chain antibody or anti-TAT polypeptide antibody that competitively inhibits binding to its respective antigenic epitope. The antibodies of the present invention may optionally be conjugated to growth inhibitors or cytotoxic agents such as maytansinoids or toxins including calicheamicin, antibiotics, radioisotopes, nucleolytic enzymes, etc. . The antibodies of the invention are optionally produced in CHO cells or bacterial cells and preferably induce the death of the cells to which they bind. For diagnostic purposes, the antibodies of the invention are detectably labeled or attached to a solid support.

In another embodiment of the invention, the invention provides a vector comprising DNA encoding any of the antibodies disclosed herein. A host cell comprising any such vector is also provided. By way of example, the host cell can be a CHO cell, E. coli, or yeast. Further provided is a method for producing the antibody described herein, comprising culturing host cells under conditions suitable for expression of the desired antibody and recovering the desired antibody from the cell culture.
In other embodiments, the present invention provides oligopeptides (“TAT binding oligopeptides”) that bind, preferably specifically, to any of the TAT polypeptides described above or below. In some cases, the TAT binding oligopeptides of the invention are conjugated to growth inhibitors or cytotoxic agents such as, for example, toxins including maytansinoids or calicheamicins, antibiotics, radioisotopes, nucleolytic enzymes, and the like. Can be gated. The TAT binding oligopeptides of the invention are optionally produced in CHO cells or bacterial cells and preferably induce the death of the cells to which they bind. For diagnostic purposes, the TAT binding oligopeptides of the present invention are detectably labeled or attached to a solid support or the like.
In another embodiment of the invention, the invention provides a vector comprising DNA encoding any of the TAT binding oligopeptides described herein. A host cell comprising any such vector is also provided. By way of example, the host cell can be a CHO cell, E. coli, or yeast. Further provided is a method of producing any of the TAT binding oligopeptides described herein, wherein host cells are cultured under conditions suitable for expression of the desired oligopeptide, and the desired oligopeptide is derived from the cell culture. Recovering.
In other embodiments, the present invention provides small organic molecules (“TAT binding organic molecules”) that bind, preferably specifically, to any of the TAT polypeptides described above or below. In some cases, the TAT binding organic molecules of the invention are conjugated to growth inhibitors or cytotoxic agents such as, for example, maytansinoids or toxins including calicheamicin, antibiotics, radioisotopes, nucleolytic enzymes, and the like. You can gate. The TAT binding organic molecule of the present invention preferably induces death of the cell to which it binds. For diagnostic purposes, the TAT-binding organic molecules of the invention can be detectably labeled or attached to a solid support or the like.

In a still further embodiment, the present invention, in combination with a carrier, comprises a TAT polypeptide as described herein, a chimeric TAT polypeptide as described herein, an anti-TAT antibody as described herein, a TAT binding oligopeptide as described herein. Or a composition containing a TAT-binding organic molecule as described herein. In some cases, 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 contained in the container, the composition comprising a TAT polypeptide as described herein, a chimeric TAT polypeptide as described herein, An anti-TAT antibody described herein, a TAT-binding oligopeptide described herein, or a TAT-binding organic molecule described herein can be included. The article of manufacture may further optionally include a label attached to the container, or a package insert contained within the container, which refers to the use of this composition for therapeutic treatment or diagnostic detection of tumors.
Another embodiment of the present invention is for the preparation of a medicament useful for the treatment of conditions responsive to a TAT polypeptide, chimeric TAT polypeptide, anti-TAT polypeptide antibody, TAT-binding oligopeptide, or TAT-binding organic molecule. The use of a TAT polypeptide described herein, a chimeric TAT polypeptide described herein, an anti-TAT polypeptide antibody described herein, a TAT-binding oligopeptide described herein, or a TAT-binding organic molecule described herein.

B. Further Embodiments Another embodiment of the invention relates to a method of inhibiting the growth of a cell that expresses a TAT polypeptide, said method comprising binding the cell to an antibody, oligopeptide or small organic molecule that binds to the TAT polypeptide. Contacting, wherein binding of the antibody, oligopeptide or organic molecule to the TAT polypeptide causes inhibition of the growth of cells expressing the TAT polypeptide. In a preferred embodiment, the cell is a cancer cell and the binding of an antibody, oligopeptide or organic molecule to the TAT polypeptide causes the death of the cell expressing the TAT polypeptide. In some cases, the antibody is a monoclonal antibody, an antibody fragment, a chimeric antibody, a humanized antibody, or a single chain antibody. The antibodies, TAT-binding oligopeptides and TAT-binding organic molecules used in the methods of the present invention are, for example, growth inhibitors or cytotoxic agents such as maytansinoids or toxins including calicheamicin, antibiotics, radioisotopes In some cases, it may be conjugated with a nucleolytic enzyme or the like. The antibodies and TAT-binding oligopeptides used in the methods of the invention can optionally be produced in CHO cells or bacterial cells.
Yet another embodiment of the invention relates to a method of therapeutically treating a mammal having a cancerous cell comprising a cell that expresses a TAT polypeptide, said method comprising an antibody, oligopeptide or Administering to a mammal a therapeutically effective amount of a small organic molecule, whereby an effective therapeutic treatment of the tumor is achieved. In some cases, the antibody is a monoclonal antibody, an antibody fragment, a chimeric antibody, a humanized antibody, or a single chain antibody. The antibodies, TAT-binding oligopeptides and TAT-binding organic molecules used in the methods of the present invention are, for example, growth inhibitors or cytotoxic agents such as maytansinoids or toxins including calicheamicin, antibiotics, radioisotopes In some cases, it may be conjugated with a nucleolytic enzyme or the like. The antibodies and oligopeptides used in the methods of the invention can optionally be produced in CHO cells or bacterial cells.
Yet another embodiment of the invention relates to a method for determining the presence of a TAT polypeptide in a sample suspected of containing a TAT polypeptide, said method comprising an antibody, oligopeptide or small molecule that binds the sample to the TAT polypeptide. Exposure to the organic molecule includes quantifying the binding of the antibody, oligopeptide or organic molecule to the TAT polypeptide in the sample, the presence of such binding being indicative of the presence of the TAT polypeptide in the sample. In some cases, the sample may include cells that may express a TAT polypeptide, which may be cancer cells. The antibody, TAT-binding oligopeptide or TAT-binding organic molecule used in this method is optionally detectably labeled or attached to a solid support.

A further embodiment of the invention relates to a method of diagnosing the presence of a tumor in a mammal, the method comprising (a) a test sample of tissue cells obtained from said mammal, and (b) the same tissue source or type. Detecting a level of expression of a gene encoding a TAT polypeptide in a control sample of known normal non-cancerous cells of the TAT polypeptide in the test sample as compared to the control sample. A higher level of expression indicates the presence of a tumor in the mammal from which the test sample was obtained.
Another embodiment of the present invention relates to a method for diagnosing the presence of a tumor in a mammal, the method comprising: (a) an antibody, oligo, which binds a test sample of tissue cells obtained from the mammal to a TAT polypeptide. Contacting the peptide or small organic molecule and (b) detecting a complex formed between the antibody, oligopeptide or small organic molecule and the TAT polypeptide in the test sample, Formation indicates the presence of a tumor in the mammal. In some cases, the antibody, TAT-binding oligopeptide or TAT-binding organic molecule used is detectably labeled, attached to a solid support, and / or the test sample of tissue cells has a cancerous tumor. It is obtained from an individual that seems to be.

Yet another embodiment of the present invention relates to a method of treating or preventing a cell proliferative disorder associated with a modification, preferably increased expression or activity of a TAT polypeptide, which method requires such treatment. The patient comprises administering an effective amount of an antagonist of the TAT polypeptide. Preferably, the cell proliferative disorder is cancer and the antagonist of the TAT polypeptide is an anti-TAT polypeptide antibody, a TAT-binding oligopeptide, a TAT-binding organic molecule or an antisense oligonucleotide. Effective treatment or prevention of cell proliferative disorders may be the result of direct killing or growth inhibition of cells expressing the TAT polypeptide or by antagonizing the cell growth enhancing activity of the TAT polypeptide.
Yet another embodiment of the present invention relates to a method of conjugating an antibody, oligopeptide or small organic molecule to a cell expressing TAT polypeptide, wherein the method comprises treating the cell expressing TAT polypeptide with said antibody, oligopeptide or Contacting the small organic molecule under conditions suitable for binding of the antibody, oligopeptide or small organic molecule to the TAT polypeptide to allow their binding.

Other embodiments of the invention include (a) a TAT polypeptide, (b) a nucleic acid encoding a TAT polypeptide or a vector or host cell comprising the nucleic acid, (c) an anti-TAT polypeptide antibody, (d) a TAT-binding oligo Use of peptides or (e) TAT-binding small organic molecules in the manufacture of a medicament useful for (i) therapeutic treatment or diagnostic detection of cancer or tumors, or (ii) therapeutic treatment or prevention of cell proliferative diseases About.
Another embodiment of the invention relates to a method of inhibiting the growth of cancer cells, wherein the growth of said cancer cells depends at least in part on the growth enhancing effect of the TAT polypeptide (wherein the TAT polypeptide is Which can be expressed either by the cancer cell itself or by a cell that produces a polypeptide having a growth enhancing effect on the cancer cell), wherein the method comprises the step of applying a TAT polypeptide to an antibody, oligopeptide or small organic molecule that binds to the TAT polypeptide. Contacting, thereby antagonizing the growth enhancing activity of the TAT polypeptide and then inhibiting the growth of the cancer cells. Preferably the growth of cancer cells is completely inhibited. Even more preferably, binding of the antibody, oligopeptide or small organic molecule to the TAT polypeptide induces death of the cancer cell. In some cases, the antibody is a monoclonal antibody, an antibody fragment, a chimeric antibody, a humanized antibody, or a single chain antibody. Antibodies, TAT-binding oligopeptides and TAT-binding organic molecules used in the methods of the present invention can be growth inhibitors or cytotoxic agents such as, for example, maytansinoids or toxins including calicheamicin, antibiotics, radioisotopes. It may be optionally conjugated with a body, a nucleolytic enzyme and the like. The antibodies and TAT-binding oligopeptides used in the methods of the invention can optionally be produced in CHO cells or bacterial cells.

Yet another embodiment of the invention relates to a method of therapeutically treating a tumor in a mammal, wherein the growth of said tumor depends at least in part on the growth enhancing effect of the TAT polypeptide, said method comprising: Administering to the mammal an antibody, oligopeptide or small organic molecule that binds to the TAT polypeptide, thereby antagonizing the growth enhancing activity of the TAT polypeptide and providing effective therapeutic treatment of the tumor. Bring. In some cases, the antibody is a monoclonal antibody, an antibody fragment, a chimeric antibody, a humanized antibody, or a single chain antibody. Antibodies, TAT-binding oligopeptides and TAT-binding organic molecules used in the methods of the present invention can be growth inhibitors or cytotoxic agents such as, for example, maytansinoids or toxins including calicheamicin, antibiotics, radioisotopes. It may be optionally conjugated with a body, a nucleolytic enzyme and the like. The antibodies and oligopeptides used in the methods of the invention can optionally be produced in CHO cells or bacterial cells.
Further embodiments of the present invention will become apparent to those skilled in the art upon reading this specification.

  In the accompanying drawings herein, specific cDNA sequences that are overexpressed in specific tumors compared to normal tissue counterparts were individually identified with specific letter and number combinations. These cDNA sequences were expressed from genes located within the amplified chromosomal region of tumor cells identified as shown in Example 1 below. In the cDNA sequence shown in the accompanying drawings, a start codon and / or a stop codon are shown in bold and underlined.

Detailed Description of Preferred Embodiments
I. Definitions As used herein, the terms “TAT polypeptide” and “TAT” refer to various polypeptides immediately followed by a numerical designation, and the full designation (ie, TAT / number) is described herein. Means a specific polypeptide sequence. The term “TAT / number polypeptide” and “TAT / number”, where the term “number” is provided herein as an actual numerical representation, includes native sequence polypeptides, polypeptide variants and native sequence polypeptides. And fragments of polypeptide variants (as defined further herein). The TAT polypeptides described herein may be isolated from a variety of sources, such as human tissue types or other sources, or may be prepared by recombinant or synthetic methods. The term “TAT polypeptide” refers to each individual TAT / number polypeptide described herein. All disclosures in this specification that refer to "TAT polypeptides" refer to each polypeptide individually as well as collectively. For example, the description of preparation, purification, induction, formation of antibodies, formation of TAT-binding oligopeptides, formation of TAT-binding organic molecules, administration, containing compositions, treatment of diseases, etc. relates to each polypeptide of the present invention. The term “TAT polypeptide” also includes variants of the TAT / number polypeptides described herein.

  A “native sequence TAT polypeptide” includes a polypeptide having the same amino acid sequence corresponding to a naturally occurring TAT polypeptide. Such native sequence TAT polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. The term “native sequence TAT polypeptide” specifically includes naturally occurring truncated or secreted forms (eg, extracellular domain sequences), naturally occurring mutated forms (eg, alternatively spliced) of a particular TAT polypeptide. Form) and naturally occurring allelic variants of the polypeptide. In certain embodiments of the invention, the 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 accompanying figures. Start and stop codons (if indicated) are shown in bold and underlined in the figure. Nucleic acid residues indicated by “N” in the accompanying drawings are arbitrary nucleic acid residues. However, although the TAT polypeptide disclosed in the accompanying figures is shown to begin with a methionine residue shown here as amino acid position 1 in the drawings, other TAT polypeptides located upstream or downstream of amino acid position 1 in the figures are shown. It is conceivable or possible to use a methionine residue as the starting amino acid residue of the TAT polypeptide.

  The TAT polypeptide “extracellular domain” or “ECD” means a form of a TAT polypeptide that is substantially free of transmembrane and cytoplasmic domains. Usually, TAT polypeptide ECDs have less than 1% of their transmembrane and / or cytoplasmic domains, preferably less than 0.5% of such domains. It will be understood that any transmembrane domain identified for a TAT polypeptide of the present invention is identified according to criteria routinely used in the art to identify that type of hydrophobic domain. . Although the exact boundaries of the transmembrane domain can vary, it is likely not to exceed about 5 amino acids from either end of the originally identified domain. In some cases, therefore, the extracellular domain of a TAT polypeptide may comprise no more than about 5 amino acids from either side of the transmembrane domain / extracellular domain boundary, as identified in the Examples or specification. Often, those polypeptides with and without signal peptides 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 present specification and / or the accompanying figures. However, although the C-terminal boundary of the signal peptide can vary, it is most likely less than about 5 amino acids on either side of the signal peptide C-terminal boundary, as defined herein, It is noted that boundaries can be identified according to criteria routinely used to identify such types of amino acid sequence components (eg, Nielsen et al., Prot. Eng. 10: 1-6 (1997) And von Heinje et al., Nucl. Acids. Res. 14: 4683-4690 (1986)). Furthermore, in some cases, it is also recognized that cleavage of the signal sequence from the secreted polypeptide is not completely uniform, resulting in one or more secreted species. These mature polypeptides, and the polynucleotides encoding them, that are cleaved within less than about 5 amino acids on either side of the C-terminal boundary of the signal peptide identified herein are contemplated in the present invention. .

  A “TAT polypeptide variant” is a TAT polypeptide, preferably a full-length native sequence TAT polypeptide sequence as disclosed herein, a TAT polypeptide sequence lacking a signal peptide as disclosed herein, disclosed herein. The extracellular domain of a TAT polypeptide with or without such a signal peptide or any other fragment of the full-length TAT polypeptide sequence disclosed herein (eg, only part of the complete coding sequence of a full-length TAT polypeptide) Active TAT polypeptide as defined herein having at least about 80% amino acid sequence identity to that encoded by a nucleic acid exhibiting Such TAT polypeptide variants include, for example, TAT polypeptides with one or more amino acid residues added or deleted at the N-terminus or C-terminus of the full-length natural amino acid sequence. Typically, a TAT polypeptide variant is a full-length native sequence TAT polypeptide sequence disclosed herein, a TAT polypeptide sequence lacking a signal peptide disclosed herein, a TAT polypeptide disclosed herein with or without a signal peptide. At least about 80% amino acid sequence identity to the extracellular domain, or any other specifically defined fragment of the full-length TAT polypeptide sequence disclosed herein, 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. Typically, the TAT variant polypeptide is at least about 10 amino acids long, or 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 longer. In some cases, the TAT variant polypeptide has no more than one conservative amino acid substitution compared to the native TAT polypeptide sequence, or 2, 3, 4, 5, 6, 7 compared to the native TAT polypeptide sequence. Have no more than 8, 8, or 10 conservative amino acid substitutions.

  “Percent (%) amino acid sequence identity” with respect to the TAT polypeptide sequences identified herein refers to any conservative substitutions, introducing gaps as necessary to align the sequences and obtain maximum percent sequence identity. Defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of a particular TAT polypeptide sequence after not being considered part of the sequence identity. Alignments for the purpose of determining percent amino acid sequence identity are publicly available in various ways within the skill of the art, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software This can be achieved by using simple computer software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximal alignment over the full length of the sequences being compared. However, for purposes herein,% amino acid sequence identity values can be obtained using the sequence comparison computer program ALIGN-2, the complete source code for which is provided in Table 1 below. Obtained by. The ALIGN-2 sequence comparison computer program was created by Genentech, and the source code shown in Table 1 below was submitted to the US Copyright Office, Washington DC, 20559 with user documentation and registered under US copyright registration number TXU510087 Has been. The ALIGN-2 program is publicly available from Genentech, South San Francisco, California and may be compiled from the source code provided in Table 1 below. The ALIGN-2 program is compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is used for amino acid sequence comparison, the% amino acid sequence identity of a given amino acid sequence A to, or relative to, a given amino acid sequence B (or a given amino acid sequence B A given amino acid sequence A, which has or contains some% amino acid sequence identity to, to or from it) is calculated as follows:
100 times the fraction X / Y, where X is the number of amino acid residues with a score consistent with the A and B program alignments of the sequence alignment program ALIGN-2, and Y is the total amino acid residue of B Radix. If the length of amino acid sequence A is different from the length of amino acid sequence B, it will be recognized that the% amino acid sequence identity of A to B is different from the% amino acid sequence identity of B to A. As an example of calculating% amino acid sequence identity, Tables 2 and 3 show how to calculate% amino acid sequence identity for an amino acid sequence called “TAT” of an amino acid sequence called “Comparative Protein” Where “TAT” represents the amino acid sequence of the subject virtual TAT polypeptide, and “comparison protein” represents the amino acid sequence of the polypeptide compared to the subject “TAT” polypeptide, “X”, “Y” And “Z” each represent a different hypothetical amino acid residue. Unless otherwise stated, all% amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

  A “TAT variant polynucleotide” or “TAT variant nucleic acid sequence”, as defined herein, encodes a TAT polypeptide, preferably an active TAT polypeptide, and is a full-length native sequence TAT polypeptide as disclosed herein. A peptide sequence, a full-length native sequence TAT polypeptide sequence lacking a signal peptide disclosed herein, an extracellular domain of a TAT polypeptide disclosed herein with or without a signal peptide, or a full-length TAT polypeptide disclosed herein A nucleic acid molecule having at least about 80% nucleic acid sequence identity with a nucleic acid sequence encoding any other fragment of the sequence (encoded by a nucleic acid representing only a portion of the complete coding sequence of a full-length TAT polypeptide) Means. Typically, a TAT variant polynucleotide is disclosed herein with or without a full-length native sequence TAT polypeptide sequence as disclosed herein, a full-length native sequence TAT polypeptide sequence that lacks a signal peptide as disclosed herein. At least about 80% nucleic acid sequence identity, or at least about 81%, 82% with the nucleic acid sequence encoding the extracellular domain of the TAT polypeptide, or any other fragment of the full-length TAT polypeptide sequence disclosed herein. 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or Has 99% nucleic acid sequence identity. Variants do not contain the native nucleotide sequence.

  Usually, the TAT variant polynucleotide is 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, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, The term “about” in this context means 950, 960, 970, 980, 990, or 1000 nucleotides long, plus or minus 10% of the displayed nucleotide sequence length.

  “Percent (%) nucleic acid sequence identity” relative to the TAT-encoding nucleic acid sequence identified herein introduces a gap if necessary to align the sequences and obtain maximum percent sequence identity, and Defined as the percentage of nucleotides in the candidate sequence that are identical to the nucleotides of the sequence. Alignments for the purpose of determining percent nucleic acid sequence identity can be obtained from various methods within the purview of those skilled in the art, such as publicly available computers such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. This can be achieved by using software. For purposes herein,% nucleic acid sequence identity values are obtained by using the sequence comparison computer program ALIGN-2, the complete source code for which is provided in Table 1 below. It is done. The ALIGN-2 sequence comparison computer program was created by Genentech, and the source code shown in Table 1 below was submitted to the US Copyright Office, Washington DC, 20559 along with user documentation, under US Copyright Registration Number TXU510087 It is registered with. The ALIGN-2 program is publicly available from Genentech, South San Francisco, California, and may be compiled from the source code provided in Table 1 below. The ALIGN-2 program is compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is used for nucleic acid sequence comparison, the% nucleic acid sequence identity of, or relative to, a given nucleic acid sequence C (or a given nucleic acid sequence D, or In contrast, a given nucleic acid sequence C, which has or contains a certain percentage of nucleic acid sequence identity), is calculated as follows:
100 times the fraction W / Z, where W is the number of nucleotides with a score matched by C and D alignments of the sequence alignment program ALIGN-2, and Z is the total nucleotides of D. It will be appreciated that if the length of the nucleic acid sequence C is different from the length of the nucleic acid sequence D, then the% nucleic acid sequence identity of C to D is different from the% nucleic acid sequence identity of D to C. As an example of calculating% nucleic acid sequence identity, “TAT-DNA” represents a hypothetical TAT-encoding nucleic acid sequence of interest, and “comparison DNA” of interest is compared to “TAT-DNA” nucleic acid molecules. “TAT-DNA” of nucleic acid sequences representing nucleic acid sequences, and each of “N”, “L” and “V” represents different virtual nucleotides, and Tables 4 and 5 are referred to as “comparison DNA” The calculation method of% nucleic acid sequence identity with respect to the nucleic acid sequence called is shown. Unless otherwise indicated, all% nucleic acid sequence identity values herein are obtained using the ALIGN-2 computer program as set forth in the immediately preceding paragraph.

In other embodiments, a TAT variant polynucleotide is a nucleic acid molecule that encodes a TAT polypeptide, preferably encoding a full-length TAT polypeptide described herein, under stringent hybridization and wash conditions. It can hybridize to a nucleotide sequence. A TAT variant polypeptide can be one encoded by a TAT variant polynucleotide.
The term “full-length coding region” when used in reference to a nucleic acid encoding a TAT polypeptide is the nucleotide encoding the full-length TAT polypeptide of the present invention (often indicated between the start and stop codons in the accompanying figures). Means an array. The term “full length coding region” when used in reference to an ATCC deposited nucleic acid refers to the TAT poly of the cDNA inserted into the vector deposited with the ATCC (often indicated between the start and stop codons in the accompanying figures). Means peptide coding part.

  “Isolated” as used to describe the various TAT polypeptides disclosed herein refers to polypeptides identified and separated and / or recovered from components of the natural environment. Contaminant components of the natural environment are substances that typically interfere with the diagnostic or therapeutic use of the polypeptide, including enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the polypeptide is (1) sufficient to obtain an N-terminal or internal amino acid sequence of at least 15 residues by using a spinning cup sequenator, or (2) Coomassie blue or Preferably, it is purified to homogeneity by SDS-PAGE under non-reducing or reducing conditions using silver staining. Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the TAT polypeptide natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.

Nucleic acid encoding an “isolated” TAT polypeptide or other polypeptide-encoding nucleic acid is identified and separated from at least one contaminating nucleic acid molecule normally associated with the natural source of the nucleic acid encoding the polypeptide. Nucleic acid molecules. A nucleic acid molecule encoding an isolated polypeptide is other than in the form or setting in which it is found in nature. Thus, a nucleic acid molecule that encodes an isolated polypeptide is distinguished from a nucleic acid molecule that encodes a specific polypeptide present in natural cells. However, an isolated nucleic acid molecule encoding a polypeptide includes, for example, a nucleic acid encoding a polypeptide contained in a cell that normally expresses the polypeptide where the nucleic acid molecule is in a chromosomal location different from that of natural cells. Includes molecules.
The term “control sequence” refers to a DNA sequence necessary to express an operably linked coding sequence in a particular host organism. For example, suitable control sequences for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers.

  A nucleic acid is “operably linked” when it is in a functional relationship with another nucleic acid sequence. For example, a presequence or secretory leader DNA, if expressed as a preprotein that participates in polypeptide secretion, is operably linked to the polypeptide DNA; a promoter or enhancer is responsible for transcription of the sequence. If it does, it is operably linked to the coding sequence; or the ribosome binding site is operably linked to the coding sequence if it is in a position that facilitates translation. In general, “operably linked” means that the bound DNA sequences are in close proximity and, in the case of a secretory leader, in close proximity and in the reading phase. However, enhancers do not necessarily have to be close together. Binding is achieved by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to conventional techniques.

  The “stringency” of a hybridization reaction is readily determined by those skilled in the art and is generally an empirical calculation that depends on probe length, wash temperature, and salt concentration. In general, the longer the probe, the higher the temperature required for proper annealing, and the shorter the probe, the lower the required temperature. Hybridization generally depends on the ability of denatured DNA to reanneal when the complementary strand is present in an environment below its melting point. The higher the degree of homology desired between the probe and the hybridization sequence, the higher the relative temperature that can be used. As a result, higher relative temperatures will make the reaction conditions more stringent and lower temperatures will reduce stringency. For further 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 are: (1) 0.015 M sodium chloride / 0.0015 M at low ionic strength and high temperature, eg, 50 ° C., for washing. Using sodium citrate / 0.1% sodium dodecyl sulfate; (2) denaturing agents such as formamide during hybridization, eg 50% (v / v) formamide and 0.1% bovine serum albumin at 42 ° C. /0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5 and 750 mM sodium chloride, 75 mM sodium citrate; or (3) 50% formamide at 42 ° C. 5 × SSC (0.75 M NaCl, 0.075 M citric acid Thorium), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 × Denhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS, and 42 ° C. Identified by using 10% dextran sulfate and a high stringency wash consisting of 0.2x SSC (sodium chloride / sodium citrate) for 10 minutes at 42 ° C followed by 0.1x SSC with EDTA at 55 ° C .

  “Moderate stringent conditions” were identified as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Press, 1989), Includes the use of hybridization conditions (eg, temperature, ionic strength and% SDS). Moderate stringent conditions were 20% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, and 20 mg / ml. Conditions include overnight incubation at 37 ° C. in a solution containing denatured sheared salmon sperm DNA, followed by washing the filter at 37-50 ° C. in 1 × SSC. Those skilled in the art will recognize how to adjust temperature, ionic strength, etc. as needed to accommodate factors such as probe length.

The term “epitope tag” as used herein refers to a chimeric polypeptide comprising a TAT polypeptide or anti-TAT antibody fused to a “tag polypeptide”. The tag polypeptide has sufficient residues to provide an epitope from which the antibody can be produced, and its length is short enough so as not to inhibit the activity of the fused polypeptide. Also, the tag polypeptide is preferably quite unique so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least 6 amino acid residues, usually about 8-50 amino acid residues (preferably about 10-20 residues).
“Active” or “activity” for purposes herein means a form of a TAT polypeptide that retains the biological and / or immunological activity of a naturally occurring or naturally occurring TAT, wherein “ By “biological” activity is meant a biological function (inhibition or stimulation) caused by a natural or naturally occurring TAT, other than the ability to induce the production of antibodies to an antigenic epitope carried by the natural or naturally occurring TAT, By “immunological” activity is meant the ability to elicit the production of antibodies to an antigenic epitope carried by a natural or naturally occurring TAT.

  The term “antagonist” is used in the broadest sense and includes any molecule that partially or fully blocks, inhibits or neutralizes the 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 include, in particular, agonist or antagonist antibodies or antibody fragments, fragments, or amino acid sequence variants of natural TAT polypeptides, peptides, antisense oligonucleotides, small organic molecules, and the like. A method for identifying an agonist or antagonist of a TAT polypeptide comprises contacting a TAT polypeptide with a candidate agonist or antagonist molecule and a detectable change in one or more biological activities normally associated with the TAT polypeptide. Measuring may be included.

  “Treat” or “treatment” or “palliative” refers to both therapeutic treatment and prophylactic or protective therapies, the purpose of which is to prevent or diminish the targeted pathological condition or disease ( Small). Those in need of treatment include those susceptible to the disease as well as those already suffering from the disease or those in which the disease is to be prevented. After administration of a therapeutic amount of an anti-TAT antibody, TAT-binding oligopeptide or TAT-binding organic molecule according to the method of the present invention, the patient has an observable and / or measurable decrease or disappearance of one or more of the following: If indicated, the subject or mammal has been successfully “treated” for a TAT polypeptide-expressing cancer: reduced number of cancer cells, or disappearance of cancer cells; reduced tumor size; Inhibition of cancer cell invasion into peripheral organs (ie, some deceleration and preferably cessation), including spread of cancer to soft tissue and bone; inhibition of tumor metastasis (ie, some deceleration and preferably cessation); Some inhibition of tumor growth; and / or some relief of one or more symptoms associated with a particular cancer; reduction in morbidity and mortality, and improvement in the quality of life problems. To some extent, anti-TAT antibodies or TAT-binding oligopeptides can prevent and / or kill viable cancer cell growth, which can 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 in the disease can be readily measured by routine techniques well known to physicians. In cancer therapy, efficacy can be measured, for example, by determining time to disease progression (TTP) and / or ascertaining response rate (RR). Metastases can be confirmed by staging tests, by scanning for bone and by testing for calcium levels and other enzymes to confirm spread to the bone. CT scans can also be performed by searching for the spread of the area to the pelvis and lymph nodes. Chest X-rays and measurement of liver enzyme levels by known methods are used to search for metastases to the lung and liver, respectively. Other routine methods of monitoring the disease include transrectal ultrasound tomography (TRUS) and transrectal needle biopsy (TRNB).

For bladder cancer, which is a more localized cancer, methods of confirming disease progression include urinary cell assessment by cystoscopy, monitoring of blood in the urine, ultrasound tomography or venous renal pelvis, computer tomography Visualization of the urothelial tract by radiography (CT) and magnetic resonance imaging (MRI) is included. The presence of distant metastases can be assessed by abdominal CT, chest x-ray, or skeletal radionuclide imaging.
“Chronic” administration means administration of the drug in a continuous manner as opposed to an acute manner in order to maintain the initial therapeutic effect (activity) over a long period of time. An “intermittent” administration is a treatment that is not made continuously without interruption, but rather is essentially periodic.
“Mammal” for cancer treatment, symptom alleviation or diagnosis means any animal classified as a mammal, such as humans, livestock and farm animals, zoos, sports or pet animals, such as Includes dogs, cats, cows, horses, sheep, pigs, goats, rabbits and the like. Preferably the mammal is a human.
Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

  As used herein, “carrier” includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed to them at the dosages and concentrations used. is there. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include phosphate, citrate, and other organic acid salt buffers; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; Eg serum albumin, gelatin or immunoglobulin; hydrophobic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextran; EDTA Chelating agents such as; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS® )including.

“Solid phase” or “solid support” means a non-aqueous matrix to which an antibody, TAT-binding oligopeptide or TAT-binding organic molecule of the present invention can adhere. Examples of solid phases encompassed herein are those partially or wholly formed of glass (eg, calibrated glass), polysaccharides (eg, agarose), polyacrylamide, polystyrene, polyvinyl alcohol and silicone. including. In certain embodiments, depending on the context, the solid phase can include the wells of an assay plate; otherwise, a purification column (eg, an affinity chromatography column). The term also includes a discontinuous solid phase of discrete particles as described in US Pat. No. 4,275,149.
“Liposomes” are various types of endoplasmic reticulum containing lipids, phospholipids and / or surfactants that are useful for delivery of drugs (eg, TAT polypeptides, antibodies thereto or TAT binding oligopeptides) to mammals. . The components of the liposome are usually arranged in a bilayer structure similar to the lipid orientation of the cell membrane.

As defined herein, a “small” molecule or “small” organic molecule has 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 carry out a specifically stated purpose. An “effective amount” can be determined empirically and in a routine manner in connection with the stated purpose.
The term “therapeutically effective amount” refers to the amount of an antibody, polypeptide, TAT-binding oligopeptide, TAT-binding organic molecule or other agent effective to “treat” a disease or condition in a patient or mammal. . In the case of cancer, a therapeutically effective amount of the drug reduces the number of cancer cells; reduces the size of the tumor; inhibits (ie slows down, preferably stops) cancer cell invasion to peripheral organs; Inhibit metastasis (ie slow down and preferably stop to some extent); inhibit tumor growth to some extent; and / or alleviate one or more symptoms associated with cancer to some extent. See the definition here of “treat”. To the extent that the drug prevents growth and / or kills cancer cells in which it is present, it can be mitotic and / or cytotoxic.

A “growth inhibiting amount” of an anti-TAT antibody, TAT polypeptide, TAT binding oligopeptide or TAT binding organic molecule is an amount capable of inhibiting the growth of cells, particularly tumors, eg, cancer cells, in vitro or in vivo. The “growth inhibitory amount” of an anti-TAT antibody, TAT polypeptide, TAT-binding oligopeptide or TAT-binding organic molecule for the purpose of inhibiting neoplastic cell growth can be determined empirically and routinely.
A “cytotoxic amount” of an anti-TAT antibody, TAT polypeptide, TAT binding oligopeptide or TAT binding organic molecule is an amount capable of destroying cells, particularly tumors, eg, cancer cells, in vitro or in vivo. A “cytotoxic amount” of an anti-TAT antibody, TAT polypeptide, TAT-binding oligopeptide or TAT-binding organic molecule for the purpose of inhibiting neoplastic cell growth can be determined empirically and in a routine manner. .
The term “antibody” is used in the broadest sense and includes, for example, a single anti-TAT monoclonal antibody (including agonists, antagonists, 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) are included as long as they exhibit the desired biological or immunological activity. The term “immunoglobulin” (Ig) is used interchangeably with antibody herein.

  “Isolated antibody” means one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of the natural environment are substances that interfere with the use of antibodies for diagnosis or therapy, and include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the antibody is (1) up to 95% or more, most preferably 99% or more by weight of the antibody as determined by the Lowry method, and (2) by using a spinning cup sequenator, Uniformity by SDS-PAGE under reducing or non-reducing conditions using at least 15 N-terminal or internal amino acid sequence residues or (3) Coomassie blue or preferably silver staining Until purified. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. Composed of five additional polypeptides, termed tetramer units and their accompanying J chain, thus having 10 antigen binding sites, but secreted IgA antibodies polymerize into a basic 4-chain A multivalent assembly comprising 2-5 of the unit and its associated J chain can be formed). In the case of IgG, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, but the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each H chain has three constant domains for each of the α and γ chains (C _H) is, N-terminal four of the C _H domains followed by a variable domain (V _H) with respect to μ and ε isotypes Have. Each L chain has at the N-terminus a variable domain (V _L ) followed by a constant domain (C _L ) at the other end. V _L is aligned with V _H and C _L is aligned with the first constant domain (C _H 1) of the heavy chain. Certain amino acid residues are thought to form an interface between the light and heavy chain variable domains. _VL and _VH are paired together to form a single antigen binding site. The structure and properties of different classes of antibodies are described, for example, in Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (ed.), Appleton & Lange, Norwalk, CT, 1994, page 71 and See Chapter 6.

The light chain from any vertebrate species can be assigned one of two distinct types, called kappa and lambda, based on the amino acid sequence of its constant domain. Different classes or isotypes can be assigned to immunoglobulins depending on the amino acid sequence of the heavy chain constant domain (C _H ). There are five major classes of immunoglobulins, IgA, IgD, IgE, IgG and IgM, with heavy chains called α, δ, ε, γ and μ, respectively. Class addition γ and α are divided into subclasses on the basis of relatively minor differences in _{C H} sequence and function, e.g., the following subclasses in humans: IgG1, IgG2, IgG3, IgG4 , IgA1 and IgA2 are expressed .
The term “variable” means that a portion of the variable domain varies widely in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for that particular antigen. However, variability is not evenly distributed throughout the 110-amino acid span of variable domains. Instead, the V region is a 15-30 amino acid framework region (FR) separated by shorter regions with extreme variability, referred to as “hypervariable regions”, each 9-12 amino acids long. It consists of a relatively unchanging extension called. Each of the variable domains of the natural heavy and light chains has a large β-sheet configuration and includes four FR regions connected by three hypervariable regions, which form a loop-like connection, and a β-sheet structure It may form part of. The hypervariable region of each chain is held in the immediate vicinity together with the hypervariable regions from other chains by FR, and contributes to the formation of the antigen binding site of the antibody (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ED. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not directly related to the binding of the antibody to the antigen, but show the contribution of the antibody in various effector functions, such as antibody-dependent cellular cytotoxicity (ADCC).

As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody that are responsible for antigen binding. Hypervariable regions are amino acid residues from the “complementarity determining region” or “CDR” (ie, in _VL , approximately residues 24-34 (L1), 50-56 (L2) and 89-97 (L3 ) And _VH , approximately 1-35 (H1), 50-65 (H2) and 95-102 (H3); Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health , Bethesda, MD. (1991)) and / or residues from “hypervariable loops” (eg, in _VL , residues 26-32 (L1), 50-52 (L2) and 91-96 ( L3), and in the _{V H,} approximately 26-32 (H1), 53-55 (H2) and 96-101 (H3); Chothia and Lesk J.Mol.Biol 196:. 901-917 (1987)) Comprising.

  The term “monoclonal antibody” as used herein refers to an antibody that is obtained from a substantially homogeneous population of antibodies, ie, the natural occurrence of individual antibodies that may be present in small amounts. Identical except for certain mutations. Monoclonal antibodies are highly specific and are directed against a single antigenic site. Furthermore, each monoclonal antibody is directed against a single determinant on the antigen as compared to polyclonal antibody preparations comprising different antibodies directed against different determinants (epitopes). In addition to its specificity, monoclonal antibodies are advantageous in that they are synthesized uncontaminated by other antibodies. The modifier “monoclonal” does not imply that antibodies must be produced in any particular manner. For example, monoclonal antibodies useful in the present invention can be made by the hybridoma method first described by Kohler et al., Nature 256, 495 (1975), or by recombinant DNA methods to produce bacteria, eukaryotic animals or plants. Can be made from cells (see, eg, US Pat. No. 4,816,567). A “monoclonal antibody” is a phage antibody live using the techniques described in, for example, Clackson et al., Nature 352: 624-628 (1991), and Marks et al., J. Mol. Biol. 222: 581-597 (1991). It can also be isolated from a rally.

Here, the monoclonal antibody has a part of heavy chain and / or light chain that is identical or homologous to the corresponding sequence of an antibody derived from a specific species or an antibody belonging to a specific antibody class or subclass. A "chimeric" antibody in which the remaining part of the chain is identical or homologous to an antibody from another species, or the corresponding sequence of an antibody belonging to another antibody class or subclass, as well as the desired biological activity In particular, fragments of such antibodies are included (US Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). Here, the subject chimeric antibodies include “primatized” antibodies comprising variable domain antigen-binding sequences derived from non-human primates (eg, Old World monkeys, apes, etc.) and human constant region sequences.
An “intact” antibody is one that includes an antigen-binding site and C _L and at least a heavy chain constant domain, C _H 1, C _H 2 and C _H 3. The constant domain may be a native sequence constant domain (eg, a human native sequence constant domain) or an amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.

“Antibody fragments” comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab ′, F (ab ′) ₂ , and Fv fragments; diabodies; linear antibodies (US Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8 (10): 1057-1062 [1995]); single chain antibody molecules; and multispecific antibodies formed from antibody fragments.
Papain digestion of antibodies produces two identical antibody-binding fragments, called “Fab” fragments, and a residual “Fc” fragment that is named for its ability to easily crystallize. The Fab fragment consists of a full length L chain and H chain variable region domain (V _H ) and one heavy chain first constant domain (C _H 1). Each Fab fragment is monovalent with respect to antigen binding, ie has a single antigen-binding site. Pepsin treatment of the antibody produces a single large F (ab ′) ₂ fragment, which roughly corresponds to two disulfide-linked Fab fragments with a bivalent antigen binding site and can cross-link antigen. It can be done. Fab ′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the C _H 1 domain including one or more cysteines from the antibody hinge region. Fab′-SH here represents Fab ′ in which the cysteine residue (s) of the constant domain has a free thiol group. F (ab ′) ₂ antibody fragments are usually produced as pairs of Fab ′ fragments, with a hinge cysteine between them. Other chemical couplings of antibody fragments are also known.

The Fc fragment contains the carboxy-terminal sites of both heavy chains held together by disulfides. The effector function of an antibody is determined by the sequence of the Fc region, which is the site recognized by the Fc receptor (FcR) found in certain types of cells.
“Fv” is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy chain and one light chain variable region in tight, non-covalent association. The folding of these two domains results in six hypervariable loops (three from the H and L chains, respectively) that contribute to amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for the antigen) has a lower affinity than the entire binding site, but has the ability to recognize and bind to the antigen. .

“Single-chain Fv”, also abbreviated as “sFv” or “scFv”, is an antibody fragment comprising V _H and V _L antibody domains combined within a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the _VH and _VL domains that allows the sFV to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, below.
The term “diabodies” refers to V _H so that the V domains are paired within the chain rather than between the chains, resulting in a bivalent fragment, ie a fragment having two antigen-binding sites. Means a small antibody fragment prepared by constructing an sFv fragment (see previous paragraph) with a short linker (approximately 5-10 residues) between the _VL domain and the _VL domain. A bispecific diabody is a heterodimer of two “crossover” sFv fragments, in which the V _H and V _L domains of the two antibodies reside on different polypeptide chains. Diabodies are well described, for example, in European Patent No. 404097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

  “Humanized” forms of non-human (eg, rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are non-human species in which the recipient's hypervariable region residues have the desired antibody specificity, affinity and ability, such as mouse, rat, rabbit or non-human primate ( It is a human immunoglobulin (recipient antibody) substituted by residues of the hypervariable region of the donor antibody). In some cases, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the donor antibody. These modifications are made to further refine antibody properties. Generally, a humanized antibody has at least one, typically all or almost all hypervariable loops match that of a non-human immunoglobulin and all or almost all FRs are human immunoglobulin sequences. Contains substantially all of the two variable domains. A humanized antibody optionally comprises at least part of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321, 522-525 (1986); Riechmann et al., Nature 332, 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2, 593-596 (1992). See

A “species-dependent antibody”, eg, a mammalian anti-human IgE antibody, is derived from the first mammalian species rather than the binding affinity it has for a homologue of an antigen from a second mammalian species. An antibody having a stronger binding affinity for an antigen. Typically, species-dependent antibodies are human antigens (ie, having a binding affinity (Kd) value of about 1 × 10 ⁻⁷ M or less, preferably about 1 × 10 ⁻⁸ or less, most preferably about 1 × 10 ⁻⁹ M or less) and “ Homologue of an antigen from a second non-human mammalian species that "specifically binds" but 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 Have binding affinity for. The species-dependent antibody can be any of the various types of antibodies defined above, but is preferably a humanized or human antibody.

  A “TAT binding oligopeptide” is an oligopeptide that preferably specifically binds to a TAT polypeptide as described herein. TAT-linked oligopeptides can be chemically synthesized using known oligopeptide synthesis methodologies or can be prepared and purified using recombinant techniques. TAT-binding oligopeptides are typically at least about 5 amino acids long, 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, 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 Ri, preferably against such oligopeptides TAT polypeptide as described herein is capable of specifically binding. TAT-binding oligopeptides can be identified without undue experimentation using well-known techniques. In this regard, it is noted that techniques for searching oligopeptide libraries of oligopeptides capable of specifically binding to a polypeptide target are well known in the art (eg, US Pat. No. 5,556,762, ibid. No. 5750373, No. 4,708,871, No. 4833092, No. 5,223,409, No. 5,403,484, No. 5,571,689, No. 5663143; PCT Publication Nos. WO84 / 03506, and WO84 / 03564; Geysen et al. Natl. Acad. Sci. USA, 81: 3998-4002 (1984); Geysen et al., Proc. Natl. Acad. Sci. USA, 82: 178-182 (1985); Geysen et al., In Synthetic Peptides as Antigens , 130-149 (1986); Geysen et al., J. Immunol. Meth., 102: 259-274 (1987); Schoofs et al., J. Immunol., 140: 611-616 (1988), Cwirla, SE et al. (1990). Proc. Natl. Acad. Sci. USA, 87: 6378; Lowman, HB et al. (1991) Biochemistry, 30 Clackson, T. et al. (1991) Nature, 352: 624; Marks, JD et al. (1991) J. Mol. Biol., 222: 581; Kang, AS et al. (1991) Proc. Natl. Acad. Sci. USA, 88: 8363, and Smith, GP (1991) Current Opin. Biotechnol., 2: 668).

  A “TAT binding organic molecule” is an organic molecule other than an oligopeptide or antibody as defined herein that binds, preferably specifically, to a TAT polypeptide as described herein. TAT-binding organic molecules can be identified and chemically synthesized using known methods (see, eg, PCT Publication Nos. WO00 / 00823 and WO00 / 39585). The TAT-binding organic molecule is typically less than about 2000 Daltons, or less than about 1500, 750, 500, 250, or 200 Daltons, and preferably has a TAT polypeptide as described herein, Such organic molecules capable of specifically binding can be identified without undue experimentation using well-known techniques. In this regard, it is noted that techniques for searching an organic molecular library of molecules capable of binding to a polypeptide target are well known in the art (eg, PCT Publication Nos. WO 00/00823 and WO 00/39585). reference).

An antibody, oligopeptide or other organic molecule that “binds” to an antigen of interest, eg, a tumor-associated polypeptide antigen target, targets the cell or tissue in which the antibody, oligopeptide or other organic molecule expresses the antigen It is useful as a diagnostic and / or therapeutic agent, and binds to its antigen with sufficient affinity so that it does not significantly cross-react with other proteins. In such embodiments, the extent of binding of antibodies, oligopeptides or other organic molecules to “non-target” proteins is quantified by fluorescence activated cell sorter (FACS) analysis or radioimmunoprecipitation (RIA). Less than about 10% of the binding of an antibody, oligopeptide or other organic molecule to that particular target protein. With respect to the binding of an antibody, oligopeptide or other organic molecule to a target molecule, "specifically binds" or "specifically binds" to or against a specific polypeptide or epitope on a specific polypeptide target The term “specific” means a binding that differs from a non-specific interaction as measured. Specific binding can be measured, for example, by quantifying the binding of the molecule as compared to the binding of a control molecule, which is generally a molecule of similar structure that does not have binding activity. For example, specific binding can be quantified for competition with a control molecule similar to the target, eg, excess unlabeled target. In this case, specific binding is indicated if the binding of the labeled target to the probe is competitively inhibited by excess unlabeled target. As used herein, the term “specifically binds” or “specifically binds” to, or is “specifically bound to” a particular polypeptide or epitope on a particular polypeptide target is, for example, a target At least about 10 ⁻⁴ M, alternatively at least about 10 ⁻⁵ M, alternatively at least about 10 ⁻⁶ M, alternatively at least about 10 ⁻⁷ M, alternatively at least about 10 ⁻⁸ M, alternatively at least about 10 ⁻⁹ M, Alternatively, it may be represented by a molecule having a Kd of at least about 10 ⁻¹⁰ M, alternatively at least about 10 ⁻¹¹ M, alternatively at least about 10 ⁻¹² M, or more. In one embodiment, the term “specifically binds” refers to binding where a molecule binds to a specific polypeptide or epitope of a specific polypeptide without substantially binding to any other polypeptide or polypeptide epitope. Means.

  An antibody, oligopeptide or other organic molecule that “inhibits the growth of tumor cells that express a TAT polypeptide” or “growth inhibitory” antibody, oligopeptide or other organic molecule expresses or overexpresses the appropriate TAT polypeptide It causes measurable growth inhibition of expressed cancer cells. A TAT polypeptide can be a transmembrane polypeptide expressed on the surface of a cancer cell, and can be a polypeptide produced and secreted by a cancer cell. Preferred growth inhibitory anti-TAT antibodies, oligopeptides or other organic molecules are generally suitable controls, which are controls that are tumor cells that have not been treated with the tested antibodies, oligopeptides or other organic molecules. In comparison, growth of TAT-expressing tumor cells is inhibited by more than 20%, preferably from about 20% to about 50%, and more preferably more than 50% (eg, from about 50% to about 100%). In one embodiment, growth inhibition can be measured in cell culture at an antibody concentration of about 0.1 to 30 μg / ml or about 0.5 nM to 200 nM, and growth inhibition is determined after exposure of tumor cells to the antibody. Check in 1-10 days. In vivo inhibition of tumor cell growth can be confirmed in various ways as described in the experimental examples below. Administration of about 1 μg / kg to about 100 mg / kg body weight of anti-TAT antibody decreases to tumor size or tumor cell growth within about 5 to 3 months, preferably about 5 to 30 days after the first antibody administration The antibody is growth inhibitory in vivo.

An antibody, oligopeptide or other organic molecule that “induces apoptosis” binds to annexin V, DNA fragmentation, cell contraction, endoplasmic reticulum expansion, cell fragmentation, and / or membrane vesicle formation (apoptotic body) It induces programmed cell death as determined by the The cell is usually one that overexpresses a TAT polypeptide. Preferably, the cells are tumor cells such as prostate, breast, ovary, stomach, endometrium, lung, kidney, colon, bladder cells. Various methods are available to evaluate cellular events associated with apoptosis. For example, phosphatidylserine (PS) translocation can be measured by annexin binding; DNA fragmentation can be assessed by DNA laddering; cell nucleus / chromatin aggregation associated with DNA fragmentation can be any of the diploid cells Can be evaluated by increase. Preferably, in annexin binding assays, antibodies, oligopeptides or other organic molecules that induce apoptosis are about 2 to 50 times, preferably about 5 to 50 times, most preferably about 10 to 50 times that of untreated cells. The result is that it induces annexin binding.
The “effector function” of an antibody refers to a biological activity attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and varies depending on 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; cell surface receptors (eg, B cell receptors) Down-regulation; and B cell activation.

  “Antibody-dependent cell-mediated cytotoxicity” or “ADCC” binds to Fc receptors (FcRs) present on certain cytotoxic cells (eg, natural killer (NK) cells, neutrophils and macrophages). By secreted Ig is meant a cytotoxic form that allows these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells by cytotoxin. Antibodies “comprise” cytotoxic cells, which are absolutely necessary for such killing. The primary cells that mediate ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. Expression of FcR in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). In order to assay the ADCC activity of the molecule of interest, an in vitro ADCC assay as described in US Pat. No. 5,500,362 or 5,821,337 can be performed. Effector cells useful in such assays include peripheral blood mononuclear cells (PBMC) and natural killer cells (NK cells). Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example in an animal model as disclosed in Clynes et al. (USA) 95: 652-656 (1998) It is.

  “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. Further preferred FcRs are those that bind IgG antibodies (gamma receptors) and include receptors of the FcγRI, FcγRII and FcγRIII subclasses, including allelic variants of these receptors, alternatively spliced forms . FcγRIII receptors include FcγRIIA (“active receptor”) and FcγRIIB (“inhibitory receptor”), which differ primarily in their cytoplasmic domains but have similar amino acid sequences. The activated receptor FcγRIIA contains a tyrosine-dependent immunoreceptor activation motif (ITAM) in the cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in the cytoplasmic domain (see Daeron, Annu. Rev. immunol. 15: 203-234 (1997)). ). For FcRs, see Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991); Capel et al., Immunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126 : 330-41 (1995). Other FcRs, including those identified in the future, are encompassed by the term “FcR” herein. The term also includes the neonatal receptor FcRn (Guyer et al., J. Immunol. 117: 587 (1976) Kim et al., J. Immunol. 24: 249 (1994)), which is a factor that maternal IgGs are inherited by the fetus. ) Is also included.

“Human effector cells” are leukocytes that express one or more FcRs and perform effector functions. Desirably, the cell expresses at least FcγRIII 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, with PBMC and NK cells being preferred. is there. Effector cells may be isolated from natural sources such as blood.
“Complement dependent cytotoxicity” or “CDC” means lysing a target in the presence of complement. Activation of the typical complement pathway is initiated by binding of the first complement of the complement system (Clq) to an antibody (of the appropriate subclass) that has bound the cognate antigen. To assess complement activation, a CDC assay can be performed as described, for example, in Gazzano-Santoro et al., J. Immunol. Methods 202: 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 malignancy. More specific examples of such cancers include squamous cell cancer (e.g., squamous cell carcinoma), small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous carcinoma of the lung. ) Including lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethra Cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, spawning cancer, thyroid cancer, liver cancer, Anal cancer, penile cancer, melanoma, multiple myeloma and B-cell lymphoma, brain, and head and neck cancer, and related metastases.
The terms “cell proliferative disorder” and “proliferative disorder” refer to disorders with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.
“Tumor” as used herein means all tumorigenic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.

  An antibody, oligopeptide or other organic molecule that “induces cell death” is one that renders viable cells nonviable. The cell is one that expresses a TAT polypeptide, preferably a cell that overexpresses a TAT polypeptide compared to normal cells of the same tissue type. A TAT polypeptide can be a transmembrane polypeptide expressed on the surface of a cancer cell, and can be a polypeptide produced and secreted by a cancer cell. Preferably, the cell is a cancer cell, such as a breast, ovary, stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas or bladder cell. In vitro cell death may be confirmed in the absence of complement and immune effector cells to distinguish cell death induced by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent disorder (CDC) . Thus, an assay for cell death may be performed using heat inactivated serum (ie, without complement) and in the absence of immune effector cells. To ascertain whether antibodies, oligopeptides or other organic molecules induce cell death, by incorporation of propidium iodide (PI), trypan blue (Moore et al. Cytotechnology 17: 1-11 (1995)) or 7AAD The assessed loss of membrane integrity can be assessed in connection with untreated cells. Preferred antibodies, oligopeptides or other organic molecules that induce cell death are those that induce PI uptake in a PI uptake assay in BT474 cells.

  A “TAT-expressing cell” expresses an endogenous or transfected TAT polypeptide on the surface of the cell or in a secreted form. A “TAT-expressing cancer” is a cancer comprising cells that have a TAT polypeptide present on the cell surface or that produce and secrete a TAT polypeptide. Optionally, a “TAT-expressing cancer” produces a sufficient level of TAT polypeptide on the surface of the cell, to which anti-TAT antibodies, oligopeptides or other organic molecules can bind, Has a therapeutic effect. In other embodiments, optionally a “TAT-expressing cancer” is a level sufficient to allow anti-TAT antibodies, oligopeptides or other organic molecule antagonists to bind and have a therapeutically effective amount for the cancer. Produces and secretes the TAT polypeptide. With respect to the latter, the antagonist can be an antisense oligonucleotide that reduces, suppresses or inhibits the production and secretion of secreted TAT polypeptides by tumor cells. A cancer that “overexpresses” a TAT polypeptide is one that has, or produces and secretes, significantly higher levels of TAT polypeptide on its cell surface compared to non-cancerous cells of the same tissue type. Such overexpression can occur by gene amplification or increased transcription or translation. TAT polypeptide overexpression can be quantified in diagnostic or prognostic assays by assessing increased levels of TAT protein present on or secreted by the cell (eg, encoding a TAT polypeptide) From an isolated nucleic acid via an immunohistochemical assay using an anti-TAT antibody prepared against an isolated TAT polypeptide that can be prepared using recombinant DNA technology; FACS analysis, etc.). Alternatively or additionally, for example, fluorescence in situ hybridization using a nucleic acid based probe that matches the TAT-encoding nucleic acid or its complementary strand; (FISH; see WO 98/45479 published October 1998); Cellular TAT polypeptide-encoding nucleic acid or mRNA levels may be measured via Southern blotting, Northern blotting, or polymerase chain reaction (PCR) techniques, such as real-time quantitative PCR (RT-PCR). Alternatively, TAT polypeptide overexpression may be studied, for example, by using antibody-based assays to measure antigens that are flowing in biological fluids such as serum (see also, for example, 1990 6 U.S. Pat. No. 4,933,294 issued 12 May; International Publication 91/05264 published 18 Apr. 1991; U.S. Pat. No. 5,401,638 issued Mar. 28, 1995; Sias et al., J. Immunol. Methods 132: 73-80 (1990)). Apart from the assays described above, various in vivo assays are available to skilled technicians. For example, cells in a patient's body may be exposed to an antibody that is optionally labeled with a detectable label, such as, for example, a radioactive isotope, and binding of the antibody to the patient's cells can be accomplished, for example, by radioactive By external scanning or by analyzing biopsies taken from patients previously exposed to antibodies.

As used herein, the term “immunoadhesin” refers to an antibody-like molecule that has conferred the binding specificity of a heterologous protein (“adhesin”) having the effector function of an immunoglobulin constant domain. Structurally, an immunoadhesin is a fusion of an amino acid sequence (ie, “heterologous”) with a desired binding specificity other than the antigen recognition and binding site of an antibody with an immunoglobulin constant domain sequence. The adhesin portion of an immunoadhesin molecule typically comprises a contiguous amino acid sequence that includes at least a receptor or ligand binding site. Immunoadhesin immunoglobulin constant domain sequences include IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, such as IgA (including IgA-1 and IgA-2), IgE, IgD or IgM. It can be obtained from any immunoglobulin.
The term “label” as used herein is conjugated directly or indirectly to an antibody, oligopeptide or other organic molecule to produce a “labeled” antibody, oligopeptide or other organic molecule. Detectable compound or composition. The label may be detectable by itself (eg, a radioisotope label or a fluorescent label), or in the case of an enzyme label, it may catalyze chemical conversion of the detectable substrate compound or composition.

The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. The term refers to radioisotopes (eg, radioisotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² and Lu), chemotherapeutic agents such as methotrexate. , Adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics and toxins such as It is intended to include small molecule toxins including fragments and / or variants or enzymatically active toxins of bacterial, filamentous fungal, plant or animal origin, and various anti-tumor or anti-cancer agents disclosed below. Other cytotoxic drugs are described below. Tumoricides cause destruction of tumor cells.

As used herein, “growth inhibitor” refers to a compound or composition that inhibits the growth of cells, particularly TAT-expressing cancer cells, either in vitro or in vivo. Therefore, the growth inhibitor significantly reduces the proportion of TAT-expressing cells in the S phase. Examples of growth inhibitory agents include agents that inhibit cell cycle progression (at a place other than S phase), such as agents that induce G1 arrest or M-phase arrest. Classical M-phase blockers include vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. These agents that arrest G1 also affect S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechloretamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. More information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, ed., Chapter 1, title “Cell cycle regulation, oncogene, and antineoplastic drugs”, Murakami et al. (WB Saunders: Philadelphia, 1995), especially on page 13. be able to. Taxanes (paclitaxel and docetaxel) are both anticancer agents derived from yew. Docetaxel (TAXOTERE®, Lone Pulan Roller) derived from European yew is a semi-synthetic analog of paclitaxel (TAXOL®, Bristol-Meier Squibb). Paclitaxel and docetaxel promote microtubule assembly from tubulin dimers and stabilize microtubules by preventing depolymerization that results in inhibiting cell mitosis.
“Doxorubicin” is an anthracycline antibiotic. The full chemical name of doxorubicin is (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” is a general term for proteins that are released from one cell population and act as intercellular mediators to other cells. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone ( FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and -β; Mueller inhibitor; mouse gonadotropin related Inhibin; Activin; Vascular endothelial growth factor; Integrin; Thrombopoietin (TPO); Nerve growth factor such as NGF-β; Platelet growth factor; Transforming growth factors (TGFs) such as TGF-α and TGF-β; Insulin Like growth factors-I and II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, -β, and -γ; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocytes -Macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL- 5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; tumor necrosis factors such as TNF-α and TNF-β; and others including LIF and kit ligand (KL) The polypeptide factor. As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of native sequence cytokines.
The term “package insert” refers to instructions customarily including commercial packaging of therapeutic agents, including information about efficacy, use, dosage, administration, contraindications and / or warnings regarding the use of the therapeutic agent. Point to.

II. Compositions and Methods of the Invention Anti-TAT Antibodies In one embodiment, the present invention provides anti-TAT antibodies that may find use herein as therapeutic and / or diagnostic agents. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific and heteroconjugate antibodies.

1. Polyclonal antibodies Polyclonal antibodies are preferably produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It is useful for binding relevant antigens (especially when synthetic peptides are used) to proteins that are immunogenic in the species to be immunized. For example, this antigen can be converted to keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, bifunctional or derivatizing agents such as maleimidobenzoylsulfosuccinimide ester (conjugation via cysteine residues). , N-hydroxysuccinimide (conjugation via a lysine residue), glutaraldehyde, and succinic anhydride, SOCl ₂ , or R ¹ and R ¹ may be combined using different alkyl groups R ¹ N═C═NR it can.
The animal is combined with 3 volumes of complete Freund's adjuvant, eg, 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively), and this solution is injected intradermally at multiple sites to produce antigens, immunogenic conjugates, or Immunize against derivatives. One month later, the animals are boosted by subcutaneous injection at multiple sites with an initial amount of 1/5 to 1/10 peptide or conjugate in complete Freund's adjuvant. After 7 to 14 days, animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer reaches a plateau. Conjugates can also be prepared in recombinant cell culture as protein fusions. In addition, an aggregating agent such as alum is preferably used to enhance the immune response.

2. Monoclonal antibodies Monoclonal antibodies can be made by the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or by the recombinant DNA method (US 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, and an antibody that specifically binds to the protein used for immunization is produced or can be produced. To derive. Alternatively, lymphocytes can be immunized in vitro. After immunization, lymphocytes are isolated and fused with myeloma cells using a suitable fusing agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pages 59-103 ( Academic Press, 1986)).
The hybridoma cells thus prepared are seeded in a suitable medium preferably containing one or more substances that inhibit the growth or survival of unfused parent myeloma cells (also called fusion partners). Let For example, if the parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the medium for hybridomas is typically a substance that prevents the growth of HGPRT-deficient cells. It will contain some hypoxanthine, aminopterin, and thymidine (HAT medium).

Myeloma cells, a preferred fusion partner, efficiently fuse, support stable high level expression of antibodies by selected antibody-producing cells, and select media that select against unfused parent cells. It is a sensitive cell. Among these, preferred myeloma cell lines are mouse myeloma lines such as the MOPC-21 and MPC-11 mouse tumors available from Souk Institute Cell Distribution Center, San Diego, California, USA, and , Derived from SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Manassas, Virginia, USA. Human myeloma and mouse-human heteromyeloma cell lines have also been disclosed for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications 51-63, (Marcel Dekker, Inc., New York, 1987)).
Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is measured by immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

For example, the binding affinity of a monoclonal antibody can be measured by Scatchard analysis of Munson et al., Anal. Biochem., 107: 220 (1980).
Once hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution and grown by standard methods (Goding, Monoclonal Antibodies : Principles and Practice, pages 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. The hybridoma cells can also be grown in vivo as animal ascites tumors, for example, by intraperitoneal injection of cells into mice.
Monoclonal antibodies secreted by subclones are conventional antibodies such as affinity chromatography (eg, using protein A or protein G-sepharose) or ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, etc. It is successfully separated from the culture medium, ascites or serum by purification methods.
The DNA encoding the monoclonal antibody is immediately isolated using conventional methods (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the murine antibody) Sequenced. Hybridoma cells are a preferred source of such DNA. Once isolated, the DNA is placed in an expression vector, which is then added to an E. coli cell, monkey COS cell, Chinese hamster ovary (CHO) cell, or myeloma cell that does not produce antibody protein otherwise. It can be transfected into a host cell to obtain monoclonal antibody synthesis in a recombinant host cell. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5: 256-262 (1993) and Pluckthun, Immunol. Revs. 130: 151-188 (1992). Is included.

In a further embodiment, antibodies or antibody fragments can be isolated from antibody phage libraries produced using the techniques described in McCafferty et al., Nature, 348: 552-554 (1990). Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991) describe the separation of mouse and human antibodies using phage libraries. Yes. Subsequent publications to generate high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10: 779-783 [1992]), as well as to construct very large phage libraries Describes combinatorial infection and in vivo recombination (Waterhouse et al., Nuc. Acids. Res., 21: 2265-2266 [1993]). These techniques are therefore viable alternatives to traditional monoclonal antibody hybridoma methods for the separation of monoclonal antibodies.
DNA encoding the antibody can be obtained, for example, by substituting the sequences of human heavy and light chain constant domains (C _H and C _L ) for homologous mouse sequences (US Pat. No. 4,816,567; Morrison et al., Proc. Nat. Acad. Sci., USA, 81: 6851 (1984)), or covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide (heterologous polypeptide) to an immunoglobulin coding sequence. Can be modified to produce chimeric or fusion antibody polypeptides. A non-immunoglobulin polypeptide sequence can replace the constant domain of an antibody, or a variable domain of one antigen-binding site of an antibody can be replaced to differ from one antigen-binding site with specificity for an antigen A chimeric bivalent antibody is produced comprising another antigen binding site with specificity for.

3. Human and humanized antibodies The anti-TAT antibodies of the present invention further include humanized antibodies or human antibodies. Humanized forms of non-human (eg murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (eg Fv, Fab, Fab ′, F (ab ′) ₂ or other antigen binding subsequences of antibodies). And contains the minimum sequence derived from non-human immunoglobulin. Humanized antibodies are those in which the complementarity-determining region (CDR) of the recipient contains the CDRs of a non-human species (donor antibody) that has the desired specificity, affinity and ability, such as mouse, rat or rabbit. Includes human immunoglobulins (recipient antibodies) substituted by groups. In some examples, human immunoglobulin Fv framework residues are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody has at least one, typically all or almost all CDR regions that match those of a non-human immunoglobulin and all or almost all FR regions are human immunoglobulin consensus sequences. Includes substantially all of the two variable domains. Humanized antibodies optimally comprise an immunoglobulin constant region (Fc), typically at least part of the constant region of a human immunoglobulin [Jones et al., Nature, 321: 522-525 (1986); Riechmann et al. , Nature, 332: 323-329 (1988); and Presta, Curr. Op Struct. Biol., 2: 593-596 (1992)].
Methods for humanizing non-human antibodies are well known in the art. Generally, one or more amino acid residues derived from non-human are introduced into a humanized antibody. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization is basically performed by Winter and co-workers [Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)] by replacing the rodent CDR or CDR sequence with the corresponding sequence of a human antibody. Thus, such “humanized” antibodies are chimeric antibodies (US Pat. No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted with the corresponding sequence from a non-human species. is there. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

If the antibody is intended for human therapeutic use, to reduce antigenicity and HAMA response (human anti-mouse antibody), both human light and heavy human variable domains used in generating humanized antibodies The choice is very important. In the so-called “best fit method”, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human V domain sequence closest to that of the rodent is then identified and the human framework (FR) therein is accepted for the humanized antibody (Sims et al., J. Immunol., 151: 2296 (1993) Chothia et al., J. Mol. Biol., 196: 901 (1987)). Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al., J. Immunol., 151: 2623 (1993)) .
It is further important that antibodies be humanized with retention of high binding affinity for the antigen and other favorable biological properties. To achieve this goal, a preferred method uses a three-dimensional model of the parent and humanized sequences to prepare the humanized antibody through an analysis step of the parent sequence and various conceptual humanized products. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs that illustrate and display putative three-dimensional conformational structures of selected candidate immunoglobulin sequences are commercially available. Viewing these representations allows analysis of the likely role of residues in the function of the candidate immunoglobulin sequence, ie, analysis of residues that affect the ability of the candidate immunoglobulin to bind antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen (s), is achieved. In general, hypervariable region residues directly and most substantially affect antigen binding.

Various forms of the humanized anti-TAT antibody are contemplated. For example, a humanized antibody may be an antibody fragment, such as a Fab, that may be conjugated to one or more cytotoxic agent (s) depending on the situation to produce an immunoconjugate. The humanized antibody may also 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 create transgenic animals (eg, mice) that can produce the entire repertoire of human antibodies without the production of endogenous immunoglobulin by immunization. For example, it has been described that homozygous deletion of the antibody heavy chain joining region (J _H ) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene sequence to such germline mutant mice results in the production of human antibodies upon antigen administration. Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggeman et al., Year in Immuno., 7:33 (1993); US Patent Nos. 5,545,806, 5,569,825, 5,591,669 (all GenPharm); 5,545,807; and WO 97/17852.

Alternatively, human antibodies and antibody fragments can be generated in vitro from the immunoglobulin variable (V) domain gene repertoire of non-immunized donors using phage display technology (McCafferty et al., Nature 348: 552-553 [1990]). Can be produced. According to this technique, antibody V domain genes are cloned frame-wise with filamentous bacteriophages, eg, either M13 or fd large or small coat protein genes, and displayed as functional antibody fragments on the surface of phage particles. Let Since the filamentous particle contains a single-stranded DNA copy of the phage genome, the selection of a gene encoding an antibody exhibiting these properties results as a result even based on selection based on the functional properties of the antibody. Thus, this phage mimics some properties of B cells. Phage display can be performed in a variety of formats; see, for example, Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3: 564-571 (1993). Several sources of V-gene segments can be used for phage display. Clackson et al., Nature, 352: 624-628 (1991) isolated a large variety of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. The V gene repertoire of non-immunized human donors is configurable, and antibodies against diverse and many antigens (including self-antigens) can be found in Marks et al., J. Mol. Biol. 222: 581-597 (1991), or Griffith. Et al., EMBO J. 12: 725-734 (1993). See also U.S. Pat. Nos. 5,565,332 and 5,573,905.
As mentioned above, human antibodies can be produced by in vitro activated B cells (US Pat. Nos. 5,567,610 and 5,229,275).

4). Antibody Fragments Under certain circumstances, it may be advantageous to use antibody fragments rather than whole antibodies. Smaller sized pieces allow for rapid clearance and can lead to improved access to solid tumors.
Various techniques have been developed to produce antibody fragments. Traditionally, these fragments were derived by proteolytic digestion of intact antibodies (e.g. Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) and Brennan et al., Science, 229: 81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments could all be expressed and secreted in E. coli, thus facilitating the production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab′-SH fragments can be recovered directly from E. coli and chemically combined to form F (ab ′) ₂ fragments (Carter et al., Bio / Technology 10: 163-167 (1992)). In another approach, F (ab ′) ₂ fragments can be isolated directly from recombinant host cell culture. Fab and F (ab ′) ₂ containing salvage receptor binding epitope residues with increased in vivo half-life are described in US Pat. No. 5,869,046. Other methods for generating antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; US Pat. No. 5,571,894; and US Pat. No. 5,587,458. Fv and sFv are the only species that have an intact ligation site that lacks a constant region; thus, they are suitable for reduced non-specific binding during in vivo use. The sFv fusion protein may be configured to produce a fusion of effector proteins at either the amino or carboxy terminus of the sFv. See the above-mentioned edition of Antibody Engineering, Borrebaeck. The antibody fragment may also be a “linear antibody” as described in, for example, US Pat. No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

5. Bispecific Antibodies Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes of the TAT protein. In other such antibodies, binding sites for other proteins and TAT binding sites can bind. Alternatively, the anti-TAT arm may be an Fc receptor for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16), so as to concentrate and localize cytoprotective mechanisms in TAT-expressing cells, or T It can bind to an arm that binds to a trigger molecule on leukocytes such as a cell receptor molecule (eg CD3). Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing TAT. These antibodies have a TAT binding arm and an arm that binds a cytotoxic agent (eg, saporin, anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate or radioisotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (eg F (ab ′) 2 bispecific antibodies).
WO 96/16673 describes bispecific anti-ErbB2 / anti-FcγRIII antibodies and US Pat. No. 5,837,234 discloses bispecific anti-ErbB2 / anti-FcγRI antibodies. Has been. Bispecific anti-ErbB2 / Fcα antibodies are shown in WO 98/02463. US Pat. No. 5,821,337 teaches bispecific anti-ErbB2 / anti-CD3 antibodies.
Methods for making bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305: 537-539 (1983)). Because of the random selection of immunoglobulin heavy and light chains, these hybridomas (four-part hybrids) produce a possible mixture of 10 different antibody molecules, only one of which is the correct bispecific structure. Have Usually, purification of the correct molecule, performed by an affinity chromatography step, is rather cumbersome and the product yield is low. Similar methods are disclosed in WO 93/08829 and Traunecker et al., EMBO J. 10: 3655-3659 (1991).

In a different approach, antibody variable domains with the desired binding specificities (antigen-antibody combining sites) are fused to immunoglobulin constant domain sequences. The fusion is preferably an Ig heavy chain constant domain comprising at least part of the hinge, C _H 2 and C _H 3 regions. The first heavy-chain constant region containing the site necessary for light chain bonding (C _{H 1),} it is desirable to present in at least one of the fusions. DNA encoding an immunoglobulin heavy chain fusion, if desired, an immunoglobulin light chain, is inserted into a separate expression vector and cotransfected into a suitable host organism. This allows great flexibility in adjusting the mutual proportions of the three polypeptide fragments in an embodiment where unequal proportions of the three polypeptide chains used in the assembly result in optimal yield of the desired bispecific antibody. Is given. However, if expression at an equal ratio of at least two polypeptide chains yields a high yield, or if the ratio does not significantly affect the binding of the desired chain, then all 2 or 3 polypeptide chains Can be inserted into one expression vector.
In a preferred embodiment of this approach, the bispecific antibody comprises one arm hybrid immunoglobulin heavy chain having the first binding specificity and the other arm hybrid immunoglobulin heavy chain-light chain pair (second Providing binding specificity). This asymmetric structure separates the desired bispecific compound from unwanted immunoglobulin chain combinations, since only half of the bispecific molecule has an immunoglobulin light chain, providing an easy separation method. It turns out that it makes it easier. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

According to another approach described in US Pat. No. 5,731,168, the interface between a pair of antibody molecules can be manipulated to maximize the percentage of heterodimer recovered from recombinant cell culture. A preferred interface includes at least a portion of the C _H 3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (eg tyrosine or tryptophan). A complementary “cavity” of the same or similar size as the large side chain is created at the interface of the second antibody molecule by replacing the large amino acid side chain with a small one (eg, alanine or threonine). This provides a mechanism to increase the yield of heterodimers over other unwanted end products such as homodimers.
Bispecific antibodies also include cross-linked or “heteroconjugate” antibodies. For example, one of the heteroconjugate antibodies can be bound to avidin and the other bound to biotin. Such antibodies have been proposed, for example, to target immune system cells against unwanted cells (US Pat. No. 4,676,980) and for the treatment of HIV infection (WO 91/00360, 92/92). No. 23033 and European Patent No. 03089). Heteroconjugate antibodies can be made using any convenient cross-linking method. Suitable crosslinkers are well known in the art and are disclosed in US Pat. No. 4,676,980, along with several crosslinking techniques.

Techniques for producing bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describes a procedure in which intact antibodies are proteolytically cleaved to produce F (ab ′) ₂ fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The produced Fab ′ fragment is then converted to a thionitrobenzoate (TNB) derivative. One of the Fab′-TNB derivatives is then reconverted to Fab′-thiol by reduction with mercaptoethylamine and mixed with equimolar amounts of other Fab′-TNB derivatives to form bispecific antibodies. The produced bispecific antibody can be used as a drug for selective immobilization of an enzyme.
Recent progress has facilitated the direct recovery of Fab′-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describes the preparation of _two fully humanized bispecific antibody F (ab ') molecules. Each Fab ′ fragment is secreted separately from E. coli and undergoes directed chemical coupling in vitro to form bispecific antibodies. The bispecific antibody thus formed is capable of binding to normal human T cells and cells overexpressing the ErbB2 receptor and triggers the cytolytic activity of human cytotoxic lymphocytes against human breast tumor targets. It becomes.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148 (5): 1547-1553 (1992). Leucine zipper peptides from Fos and Jun proteins are 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 reoxidized to form antibody heterodimers. This method can also be used for the production of antibody homodimers. The “diabody” technique described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993) provided an alternative mechanism for generating bispecific antibody fragments. The fragment consists of V _H attached to V _L with a linker that is short enough to allow pairing between two domains on the same strand. Thus, the V _H and V _L domains of one fragment are forced to pair with the complementary V _L and V _H domains of the other fragment, thus forming two antigen binding sites. Other strategies for producing bispecific antibody fragments by use of single chain Fv (sFv) dimers have also been reported. See Gruber et al., J. Immunol. 152: 5368 (1994).
More than bivalent antibodies are also 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 present invention. Heteroconjugate antibodies consist of two covalently bound antibodies. Such antibodies can be used, for example, to target immune system cells to unwanted cells [US Pat. No. 4,676,980] and for the treatment of HIV infection [WO 91/00360; WO 92/200373. European Patent No. 03089] has been proposed. This antibody could be prepared in vitro using known methods in synthetic protein chemistry, including those related to crosslinkers. For example, immunotoxins can be made using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in US Pat. No. 4,676,980.

7). Multivalent antibodies Multivalent antibodies can be internalized (and / or catabolized) earlier than bivalent antibodies by cells expressing the antigen to which the antibody binds. The antibody of the present invention may be a multivalent antibody (other than the IgM class) having 3 or more binding sites (eg, a tetravalent antibody), and is easily obtained by recombinant expression of a nucleic acid encoding the antibody polypeptide chain. Can be generated. Multivalent antibodies have a dimerization domain and three or more antigen binding sites. Preferred dimerization domains have (or consist of) an Fc region or a hinge region. In this scenario, the antibody will have an Fc region and three or more antigen binding sites at the amino terminus of the Fc region. Preferred multivalent antibodies here have (or consist of) 3 to 8, preferably 4 antigen binding sites. A multivalent antibody has at least one polypeptide chain (preferably two polypeptide chains), and the polypeptide chain (s) has two or more variable domains. For example, the polypeptide chain (s) has VD1- (X1) n-VD2- (X2) n-Fc, where VD1 is the first variable domain and VD2 is the second variable domain; Fc is one of the polypeptide chains of the Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. For example, the polypeptide chain (s) may have: VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fc region chain. Here, the multivalent antibody preferably further comprises at least two (preferably four) light chain variable domain polypeptides. The multivalent antibody herein has, for example, from about 2 to about 8 light chain variable domain polypeptides. The light chain variable domain polypeptides discussed herein have a light chain variable domain, and optionally further a CL domain.

8). Processing of Effector Function It is desirable to modify the antibodies of the present invention for effector function, for example to improve the antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement dependent cytotoxicity (CDC) of the antibody. This can be done by inducing one or more amino acid substitutions in the Fc region of the antibody. Alternatively or additionally, cysteine residues may be introduced into the Fc region, thereby forming an interchain disulfide bond in this region. Allogeneic dimeric antibodies so produced may have improved internalization capabilities and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp. Med. 176: 1191-1195 (1992) and Shopes, BJ Immunol. 148: 2918-2922 (1992). In addition, homodimeric antibodies with improved anti-tumor activity can be prepared using heterobifunctional cross-linking as described in Wolff et al., Cancer Research 53: 2560-2565 (1993). Alternatively, the antibody can be engineered to have two Fc regions, thereby improving complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3: 219-230 (1989).
In order to increase the serum half life of the antibody, one may introduce a salvage receptor binding epitope into the antibody (especially an antibody fragment) as described in US Pat. No. 5,739,277, for example. The term “salvage receptor binding epitope” as used herein refers to the Fc region of an IgG molecule (eg, IgG ₁ , IgG ₂ , IgG ₃ or IgG ₄ ) that is responsible for increasing the in vivo serum half-life of the IgG molecule. Means the epitope.

9. Immunoconjugates The present invention also relates to cytotoxic agents such as chemotherapeutic agents, growth inhibitors, toxins (eg, enzyme-active toxins derived from bacteria, fungi, plants or animals, or fragments thereof), or radioisotopes ( That is, the present invention relates to an immune complex comprising an antibody conjugated with a radioconjugate).
Chemotherapeutic agents useful for the generation of such immune complexes have been described above. Enzyme-active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A Chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, Curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and trichothecene ( tricothecene). A variety of radioactive nucleotides are available for the production of radioconjugated antibodies. Examples include ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y, and ¹⁸⁶ Re. The conjugates of antibodies and cytotoxic agents are various bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), imidoester bifunctional Derivatives (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- Using diazonium derivatives (such as bis- (p-diazonium benzoyl) -ethylenediamine), diisocyanates (such as triene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene) Can be created. For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an example of a chelating agent for conjugation of radionucleotide to an antibody. See WO94 / 11026.
Conjugates of antibodies and one or more small molecule toxins such as calicheamicin, maytansinoids, trichothene and CC1065, and derivatives of these toxins with toxic activity are discussed herein.

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 to inhibit tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (US Pat. No. 3,896,111). It was subsequently discovered that certain microorganisms produce maytansinoids such as maytansinol and C-3 maytansinol esters (US Pat. No. 4,115,042). Synthetic maytansinol and its derivatives and analogs are described, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,776; 4309428; 43313946; 4331529; 4317821; 43322348; 43331598; 43361650; 43364866; 44424219; 44362653; 43621663; and 43671533 The disclosure of which is expressly incorporated herein by reference.

Maytansinoid-antibody conjugates In an attempt to improve therapeutic indications, maytansin and maytansinoids are bound to antibodies that specifically bind to tumor cell antigens. Immunoconjugates containing maytansinoids and their therapeutic uses are disclosed, for example, in US Pat. Nos. 5,208,020, 5,416,064, EP 0425235B1, the disclosure of which is Is explicitly included here. Liu et al., Proc. Natl. Acad. Sci. USA 93: 8618-8623 (1996) describes an immunoconjugate containing a maytansinoid designated DM1 that binds to the monoclonal antibody C242 against human colorectal cancer. Has been. The conjugate has been found to be highly cytotoxic to cultured colon cancer cells and exhibits antitumor activity in an in vivo tumor growth assay. Chari et al., Cancer Research, 52: 127-131 (1992), describes a mouse antibody A7 in which maytansinoids bind to an antigen of a human colon cancer cell line via a disulfide bond, or a HER-2 / neu oncogene. Immunoconjugates have been described that bind to another mouse monoclonal antibody TA.1 that binds to. The cytotoxicity of the TA.1-maytansinoid conjugate was tested in vitro in the human breast cancer cell line SK-BR-3 and expressed 3 × 10 ⁵ HER-2 surface antigen per cell. Drug conjugates achieve a degree of cytotoxicity similar to that of the free maytansinoid agent, which is increased by increasing the number of maytansinoid molecules per antibody molecule. The A7-maytansinoid conjugate showed low systemic cytotoxicity in mice.

Anti-TAT polypeptide antibody-maytansinoid conjugate (immunoconjugate)
An anti-TAT antibody-maytansinoid conjugate is prepared by chemically conjugating an anti-TAT antibody to a maytansinoid molecule with little reduction in the biological activity of either the antibody or the maytansinoid molecule. . One molecule of toxin / antibody is expected to increase cytotoxicity in the use of naked antibodies, but an average of 3-4 maytansinoid molecules bound per antibody molecule is the function or lysis of the antibody. It exhibits the effect of improving cytotoxicity against target cells without adversely affecting sex. 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 US Pat. No. 5,208,020 and other patents and publications that are not the aforementioned patents. Preferred maytansinoids are maytansinol analogs, such as maytansinol, and various maytansinol esters modified at the aromatic ring or other positions of the maytansinol molecule.
For example, to make antibody-maytansinoid conjugates, including those disclosed in US Pat. No. 5,208,020 or European Patent No. 0425235B1, and those disclosed in Chari et al., Cancer Research, 52: 127-131 (1992). There are many linking groups known in the art. The linking group includes disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups, or esterase labile groups as disclosed in the above-mentioned patents, but disulfide and thioether groups. Is preferred.

Conjugates of antibodies and maytansinoids have various bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane -1-carboxylate, iminothiolane (IT), bifunctional derivatives of imide esters (eg dimethyl adipimidate HCL), active esters (eg disuccinimidyl suberate), aldehydes (eg glutaraldehyde) ), Bisazide compounds (eg, bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (eg, bis- (p-diazoniumbenzoyl) ethylenediamine), diisocyanates (eg, toluene-2,6-diisocyanate), and Diactive fluorine compounds (eg, 1,5-difluoro -2,4-dinitrobenzene). Particularly preferred coupling agents include N-succinimidyl-4- (2-pyridylthio) pentanoate (SPP) and N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) (Carlsson) provided by disulfide bonds. Et al., Biochem. J. 173: 723-737 [1978]).
The linker can be attached to the maytansinoid molecule at various positions depending on the type of linkage. For example, ester linkages can be formed by reacting with hydroxyl groups using conventional coupling techniques. The reaction occurs at the C-3 position with a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with a hydroxyl group, and the C-20 position with a hydroxyl group. In a preferred embodiment, the bond is formed at the C-3 position of maytansinol or an analogue of maytansinol.

Calicheamicin Other immunoconjugates of interest include anti-TAT antibodies conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics can cause double-stranded DNA breakage at sub-picomolar concentrations. For the preparation of conjugates of the calicheamicin family, U.S. Pat. See Company). The calicheamicin structural analogs that can be used include, but are not limited to, γ ₁ ^I , α ₂ ^I , α ₃ ^I , N-acetyl-γ ₁ ^I , PSAG, and θ ^I ₁ (Hinman et al., Cancer Research, 53: 3336-3342 (1993), Lode et al. Cancer Research, 58: 2925-2928 (1998) and the aforementioned American Cyanamid US patent). Another anti-tumor agent to which the antibody can bind is QFA, an antifolate. Both calicheamicin and QFA have a site of action in the cell and do not easily cross the plasma membrane. Thus, the uptake of these drugs into cells by antibody-mediated internalization greatly improves the cytotoxic effect.

Other cytotoxic agents Other anti-tumor agents that can bind to the anti-TAT antibodies of the present invention are described in BCNU, streptozocin, vincristine and 5-fluorouracil, US Pat. Nos. 5,053,394, 5,770,710, Included is a family of drugs collectively known as LL-E33288 conjugates, as well as esperamicine (US Pat. No. 5,877,296).
Usable enzyme active toxins and fragments thereof include diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (Pseudomonas aeruginosa), ricin A chain, abrin A chain, modecin ) A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII and PAP-S), momordica momordica Included are charantia inhibitors, curcin, crotin, sapaonaria officinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin and trichothecenes. See, for example, International Publication No. 93/21232 published October 28, 1993.
The present invention further contemplates immunoconjugates formed between an antibody and a compound having nucleolytic activity (eg, a ribonuclease or DNA endonuclease such as deoxyribonuclease; DNase).

In order to selectively destroy the tumor, the antibody may contain a highly radioactive atom. A variety of radioisotopes are utilized to generate radioconjugated anti-TAT antibodies. Examples include the radioisotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and Lu. If the conjugate is used for diagnostic purposes, it may be a radioactive atom for scintigraphic studies, such as tc ^99m or I ¹²³ , or a spin label for nuclear magnetic resonance (NMR) imaging (magnetic resonance imaging, also known as mri), For example, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron may be contained.
Radioactive or other label is introduced into the conjugate in a known manner. For example, peptides are biosynthesized or synthesized by chemical amino acid synthesis using an appropriate amino acid precursor containing fluorine-19 instead of hydrogen. Labels such as tc ^99m or I ¹²³ , Re ¹⁸⁶ , Re ¹⁸⁸ and In ¹¹¹ can be attached via the cysteine residue of the peptide. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al. (1978) Biochem. Biophys. Res. Commun. 80: 49-57) can be used to introduce iodine-123. Details of other methods are described in “Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989).

Conjugates of antibodies and cytotoxic agents are available for various bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane- 1-carboxylate, iminothiolane (IT), bifunctional derivatives of imide esters (eg dimethyl adipimidate HCL), active esters (eg disuccinimidyl suberate), aldehydes (eg glutaraldehyde) Bisazide compounds (eg bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (eg bis- (p-diazoniumbenzoyl) ethylenediamine), diisocyanates (eg triene-2,6-diisocyanate), and Active fluorine compounds (eg, 1,5-difluoro-2,4-di- Nitrobenzene) can be used. For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylene-triaminepentaacetic acid (MX-DTPA) is an example of a chelating agent for conjugating radionucleotide to an antibody. See WO94 / 11026. The linker may be a “cleavable linker” to facilitate release of the cytotoxic agent 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 Research, 52: 127-131 (1992); US Pat. No. 5,208,020).
Alternatively, a fusion protein containing the anti-TAT antibody and cytotoxic agent is made, for example, by recombinant techniques or peptide synthesis. The length of the DNA contains regions encoding the two portions of the conjugate that are separated by regions that encode linker peptides that do not destroy the desired properties of the conjugate or are adjacent to each other.
In other embodiments, an antibody is conjugated to a “receptor” (eg, streptavidin) for use in tumor pre-targeting, wherein the antibody-receptor conjugate is administered to the patient followed by a clarifying agent. The unbound conjugate is then removed from the circulation and a “ligand” (eg, avidin) conjugated to a cytotoxic agent (eg, a radionucleotide) is administered.

10. Immunoliposomes The anti-TAT antibodies disclosed herein can also be formulated as immunoliposomes. “Liposomes” are various types of endoplasmic reticulum containing lipids, phospholipids and / or surfactants that are useful for drug delivery to mammals. The components of the liposome are usually arranged in a bilayer structure similar to the lipid orientation of a biofilm. Liposomes containing antibodies are described, for example, in Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030 (1980); 4448545 and 45454545; and WO 97/38731 published Oct. 23, 1997 by methods known in the art. Liposomes with increased circulation time are disclosed in US Pat. No. 5,013,556.
Particularly useful liposomes can be made by the reverse phase evaporation method with a lipid composition containing phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through a filter with a defined pore size to obtain liposomes having a desired diameter. Fab ′ fragments of antibodies of the invention can be conjugated to liposomes via a disulfide exchange reaction as described by Martin et al., J. Biol. Chem. 257: 286-288 (1982). . In some cases, the chemotherapeutic agent is included within the liposome. See Gabizon et al., J. National Cancer Inst. 81 (19) 1484 (1989).

B. TAT binding oligopeptides The TAT binding oligopeptides of the present invention are oligopeptides that bind, preferably specifically, to a TAT polypeptide as described herein. TAT-linked oligopeptides can be chemically synthesized using known oligopeptide synthesis methods, or can be prepared and produced using recombinant techniques. TAT-binding oligopeptides are typically at least about 5 amino acids long, 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, 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 Ri, preferably against such oligopeptides TAT polypeptide as described herein is capable of specifically binding. TAT-binding oligopeptides can be identified without undue experimentation using well-known techniques. In this regard, it is noted that techniques for searching oligopeptide libraries of oligopeptides capable of specifically binding to a polypeptide target are well known in the art (eg, US Pat. No. 5,556,762, ibid. No. 5,750,373, No. 4,708,871, No. 48,33092, No. 5,223,409, No. 5,403,484, No. 5,571,689, No. 5,663,143; PCT Publication Nos. WO 84/03506, and WO 84/03564; Geysen et al. Natl. Acad. Sci. USA, 81: 3998-4002 (1984); Geysen et al., Proc. Natl. Acad. Sci. USA, 82: 178-182 (1985); Geysen et al., In Synthetic Peptides as Antigens , 130-149 (1986); Geysen et al., J. Immunol. Meth., 102: 259-274 (1987); Schoofs et al., J. Immunol., 140: 611-616 (1988), Cwirla, SE et al. (1990). Proc. Natl. Acad. Sci. USA, 87: 6378; Lowman, HB et al. (1991) Biochemistry, 30: 10832; C lackson, T. et al. (1991) Nature, 352: 624; Marks, JD et al. (1991) J. Mol. Biol., 222: 581; Kang, AS et al. (1991) Proc. Natl. Acad. Sci. USA, 88 : 8363, and Smith, GP (1991) Current Opin. Biotechnol., 2: 668).

  In this regard, bacteriophage (phage) display is well known to allow searching large oligopeptide libraries to identify those library members capable of specifically binding to a polypeptide target. Technology. Phage display is a technique by which various polypeptides are displayed as fusion proteins on coat proteins on the surface of bacteriophage particles (Scott, J.K. and Smith G. P. (1990) Science 249: 386). The usefulness of phage display can quickly and effectively classify large libraries of selectively randomized protein variants (or random clone cDNAs) for those sequences that bind to target molecules with high affinity. In the point. Peptides on phage (Cwirla, SE et al. (1990) Proc. Natl. Acad. Sci. USA, 87: 6378) or proteins (Lowman, HB et al. (1991) Biochemistry, 30: 10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, JD et al. (1991), J. Mol. Biol., 222: 581; Kang, AS et al. (1991) Proc. Natl. Acad. Sci. USA, 88: 8363) Library display Has been used to screen a myriad of polypeptides or oligopeptides for those with specific binding properties (Smith, GP (1991) Current Opin. Biotechnol., 2: 668). Classification of random mutant phage libraries requires methods to construct and propagate multiple variants, methods of affinity purification using target receptors, and means to evaluate the results of enhanced binding. U.S. Pat. Nos. 5,223,409, 5,403,484, 5,571,689, and 5,663,143.

  Most phage display methods used filamentous phage, but the λ phage display system (WO95 / 34683; US Pat. No. 5,627,024), T4 phage display system (Ren, ZJ. Et al. (1998) Gene 215: 439; Zhu, Z. (1997) CAN 33: 534; Jiang, J. et al. (1997) CAN 128: 44380; Ren, ZJ. Et al. (1997) CAN 127: 215644; Ren, ZJ. (1996) Protein Sci. 5: 1833; Efimov, VP et al. (1995) Virus Genes 10: 173) and T7 phage display system (Smith, GP and Scott, JK (1993) Methods in Enzymology, 217, 228-257; US Pat. No. 5,766,905) ing.

Currently, many other improvements and variations of the basic phage display concept have 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 with the potential for these proteins to screen for desired properties. Strengthen. Recombination reaction means for phage display reactions are described (WO 98/14277) and phage display libraries are characterized by bimolecular interactions (WO 98/20169; WO 98/20159) and restricted helix peptides (WO 98/20036). Is used to analyze and control. WO 97/35196 selectively isolates the bound ligand by contacting the phage display library with a first solution in which the ligand can bind to the target molecule and a second solution in which the affinity ligand does not bind to the target molecule. A method for isolating affinity ligands is described. WO 97/46251 describes a method of biopanning a random phage display library with affinity-purified antibodies, then isolating bound phage, followed by micropanning in the wells of a microplate to isolate high affinity binding phage. To do. The use of Staphlylococcus aureus protein A as an affinity tag has also been reported (Li et al. (1998) Mol Biotech., 9: 187). WO 97/47314 describes the use of a substrate subtraction library to identify enzyme specificity using a combinatorial library that may be a phage display library. A method for selecting enzymes suitable for use in detergents used for phage display is described in WO 97/09446. Additional methods for selecting proteins that specifically bind are described in US Pat. Nos. 5,498,538, 5,432,018, and WO 98/15833.
Methods for preparing peptide libraries and screening these libraries are described in US Pat. Nos. 5,723,286, 5,432,018, 5,580,717, 5,427,908, 5,498,530, 5,770,434 and 5,734,018. Nos. 5,698,426, 5762192, and 5723323.

C. TAT-binding organic molecule A TAT-binding organic molecule is an organic molecule other than an oligopeptide or antibody as defined herein that binds, preferably specifically, to a TAT polypeptide as described herein. TAT-binding organic molecules can be identified and chemically synthesized using known methods (see, eg, PCT Publication Nos. WO00 / 00823 and WO00 / 39585). The TAT-binding organic molecule is typically less than about 2000 Daltons in size, or about 1500, 750, 500, 250, or 200 Daltons, preferably specific for a TAT polypeptide as described herein. Such organic molecules capable of binding automatically can be identified without undue experimentation using well-known techniques. In this regard, it is noted that techniques for searching an organic molecular library of molecules capable of binding to a polypeptide target are well known in the art (see, eg, PCT Publication Nos. WO00 / 00823 and WO00 / 39585). ). TAT-binding organic molecules include, for example, aldehydes, ketones, oximes, hydrazones, semicarbazones, carbazides, primary amines, secondary amines, tertiary amines, N-substituted hydrazines, hydrazides, alcohols, ethers, thiols, thioethers, disulfides, carboxylic acids, esters Amide, urea, carbamate, carbonate, ketal, thioketal, acetal, thioacetal, aryl halide, arylsulfonic acid, halogenated alkyl, alkylsulfonic acid, aromatic compound, heterocyclic compound, aniline, alkene, alkyne Diol, amino alcohol, oxazolidine, oxazoline, thiazolidine, thiazoline, enamine, sulfonamide, epoxide, aziridine, isocyanate, sulfonyl chloride, diazo compound, acid chloride, etc. Get Ri.

D. Screening for anti-TAT antibodies, TAT-binding oligopeptides and TAT-binding organic molecules having the desired properties Techniques for generating antibodies, oligopeptides and organic molecules that bind to TAT polypeptides have been described above. One can further select antibodies, oligopeptides or organic molecules having certain biological properties, as desired.
Methods well known in the art using cells that express a TAT polypeptide, either endogenously or after transfection with a TAT gene, to inhibit the growth inhibitory effects of anti-TAT antibodies, oligopeptides or other organic molecules of the invention Can be evaluated. For example, suitable tumor cell lines and TAT transfected cells can be treated with various concentrations of the anti-TAT monoclonal antibodies, oligopeptides or other organic molecules of the invention for several days (eg, 2-7) Or stained with MTT or analyzed by some other colorimetric assay. Another method of measuring proliferation is by comparing ³ H-thymidine incorporation of cells treated in the presence or absence of anti-TAT antibodies, TAT-binding oligopeptides or TAT-binding organic molecules of the invention. After treatment, cells were collected and the radioactivity incorporated into DNA was quantified with a scintillation counter. Suitable positive controls include treating the selected cell line with a growth inhibitory antibody known to inhibit cell line growth. In vivo growth inhibition can be confirmed by various methods known in the art. Preferably, the tumor cell is one that overexpresses a TAT polypeptide. Preferably, the anti-TAT antibody, TAT-binding oligopeptide or TAT-binding organic molecule is about 25-100%, more preferably compared to untreated tumor cells, in some embodiments, at an antibody concentration of about 0.5 to 30 μg / ml. Inhibits the growth of about 30-100%, and even more preferably about 50-100% or 70-100% TAT expressing tumor cells in vitro or in vivo. Growth inhibition can be measured in cell culture at an antibody concentration of about 0.5 to 30 μg / ml or 0.5 nM to 200 nM, and the growth inhibition is 1-10 days after exposure of tumor cells to the antibody. It can be confirmed. Administration of the anti-TAT antibody at about 1 μg / kg to about 100 mg / kg body weight reduces tumor size or tumor cells within about 5 to 3 months, preferably about 5 to 30 days from the initial administration of the antibody. An antibody has a growth inhibitory effect in vivo if it causes a decrease in proliferation.

To select anti-TAT antibodies, TAT-binding oligopeptides or TAT-binding organic molecules that induce cell death, for example, compare the degree of membrane integrity loss shown by incorporation of propidium iodide (PI), trypan blue or 7AAD with controls And ask. PI uptake assays are performed in the absence of complement and immune effector cells. TAT polypeptide-expressing cell tumor cells are incubated in medium alone or medium containing an appropriate anti-TAT antibody (eg, about 10 μg / ml), a TAT-binding oligopeptide or a TAT-binding organic molecule. Incubate cells for 3 days. Following each treatment, the cells are washed and aliquoted into 35 mm strainer-capped 12 × 75 tubes (1 ml per tube, 3 tubes per treatment group) for cell clump removal. The tube is then given PI (10 μg / ml). Samples may be analyzed using a FACSCAN® flow cytometer and FACSCONVERT® CellQuest software (Becton Dickinson). Anti-TAT antibodies, TAT-binding oligopeptides or TAT-binding organic molecules that induce statistically significant levels of cell death as measured by PI uptake are cell death-inducing anti-TAT antibodies, TAT-binding oligopeptides or TAT binding It can be selected as an organic molecule.
To screen for antibodies, oligopeptides or other organic molecules that bind to an epitope on the TAT polypeptide to which the antibody of interest is bound, see Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). A conventional cross-blocking assay as described in) can be performed. This assay can be used to determine if a test antibody, oligopeptide or other organic molecule binds to the same site or epitope, as known anti-TAT antibodies. Alternatively or additionally, epitope mapping can be performed by methods well known in the art. For example, to identify contact residues, antibody sequences can be mutated, for example, by alanine scanning. This mutant antibody is first tested for binding to a polyclonal antibody to ensure proper folding. In different methods, peptides that match different regions of the TAT polypeptide can be used in competition assays with test antibody groups or test antibodies and antibodies with a characterized or known epitope.

E. Antibody-dependent enzyme-mediated prodrug therapy (ADEPT)
The antibodies of the present invention are also used in ADEPT by conjugating the antibody to a prodrug activating enzyme that converts a prodrug (eg, a peptidyl chemotherapeutic agent, see WO81 / 01145) to an active anticancer agent. be able to. See, for example, WO 88/07378 and US Pat. No. 4,975,278.
The enzyme component of the immunoconjugate useful for ADEPT includes any enzyme that can act on the prodrug to convert it to a more active cytotoxic form.
Without limitation, enzymes useful in the methods of this invention include alkaline phosphatases useful for converting phosphate-containing prodrugs to free drugs; useful for converting sulfate-containing prodrugs to free drugs Arylsulfatase; cytosine deaminase useful for converting non-toxic 5-fluorocytosine to the anticancer agent 5-fluorouracil; proteases such as serratia protease, thermolysin, subtilisin, carboxypeptidase and cathepsins (eg cathepsins B and L), peptides Useful for converting containing prodrugs to free drugs; D-alanyl carboxypeptidases useful for converting prodrugs containing D-amino acid substituents; free carbohydrate-cleaving enzymes such as glycosylated prodrugs Drug Neuraminidase and β-galactosidase useful for conversion; β-lactamase useful for converting a drug derivatized with β-lactam to the free drug; and penicillin amidases such as their phenoxyacetyl or phenylacetyl groups, respectively, with their amines Penicillin V amidase or penicillin G amidase useful for converting drugs derivatized in reactive nitrogen to free drugs is included. Alternatively, antibodies having enzymatic activity, also known as “abzymes”, can be used to convert the prodrugs of the invention into free active agents (see, eg, Massey, Nature 328: 457-458 (1987). )). Antibody-abzyme conjugates can be prepared to deliver the abzyme to a tumor cell population as described herein.
The enzyme of this invention can be covalently bound to the anti-TAT antibody by using techniques well known in the art, such as the heterobifunctional cross-linking reagents discussed above. Alternatively, a fusion protein in which at least the antigen-binding region of the antibody of the present invention is bound to at least a functionally active site of the enzyme of the present invention is prepared using recombinant DNA technology well known in the art. (Neuberger et al., Nature 312: 604-608 [1984]).

F. Full Length TAT Polypeptides The present invention provides newly identified and isolated nucleic acid sequences that encode polypeptides referred to in this application as TAT polypeptides. In particular, cDNAs (partial and full length) encoding various TAT polypeptides have been identified and isolated, as described in further detail in the Examples below.
As disclosed in the examples below, various cDNA clones have been deposited with the ATCC. The exact nucleotide sequence of these clones can be readily determined by sequencing the deposited clones using routine methods in the art. The predicted amino acid sequence can be determined from the nucleotide sequence using routine skill. For the TAT polypeptides and encoding nucleic acids described herein, Applicants have identified what is believed to be the reading frame that best matches the currently available sequence information.

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 antibodies and TAT polypeptide variants can also be prepared. 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 may alter the anti-TAT antibody post-translational processes or TAT polypeptide post-translational processes, such as changes in the number or position of glycosylation sites or changes in membrane anchoring properties.
Mutations of the anti-TAT antibodies and TAT polypeptides described herein can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations shown in US Pat. No. 5,364,934. A mutation may be a substitution, deletion or insertion of one or more codons encoding an antibody or polypeptide that results in an amino acid sequence change compared to a native sequence antibody or polypeptide. In some cases, the mutation is due to substitution of at least one amino acid with any other amino acid in one or more domains of the anti-TAT antibody or TAT polypeptide. Guidance to ascertain which amino acid residues can be inserted, substituted or deleted without adversely affecting the desired activity is compared by comparing the sequence of the anti-TAT antibody or TAT polypeptide with the sequence of a known homologous protein molecule. It is found by minimizing the number of amino acid sequence changes that occur within a highly probable region. An amino acid substitution may be the result of substituting one amino acid with another amino acid having similar structural and / or chemical properties, eg, replacement of leucine with serine, ie, a conservative amino acid substitution. Insertions and deletions can optionally be in the range of 1 to 5 amino acids. Acceptable mutations are ascertained by systematically making amino acid insertions, deletions or substitutions in the sequence and testing the resulting mutants for activity exhibited by the full-length or mature native sequence.

Anti-TAT antibody and TAT polypeptide fragments are provided herein. Such fragments may be cleaved at the N-terminus or C-terminus, or lack internal residues, for example when compared to a full-length native antibody or protein. Certain fragments lack amino acid residues that are not essential for the desired biological activity of the anti-TAT antibody or TAT polypeptide.
Anti-TAT antibody and TAT polypeptide fragments may be prepared by any of a number of conventional techniques. Desired peptide fragments may be chemically synthesized. Alternative methods include enzymatic digestion, such as treating the protein with an enzyme known to cleave the protein at a defined site at a particular amino acid residue, or digesting the DNA with an appropriate restriction enzyme. It includes the production of antibody or polypeptide fragments by isolating the desired fragment. Still other suitable techniques include 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 fragments are used in PCR 5 ′ and 3 ′ primers. Preferably, the anti-TAT antibody and TAT polypeptide fragment share at least one biological and / or immunological activity with the natural anti-TAT antibody or TAT polypeptide disclosed herein.

  In a particular embodiment, the conservative substitutions of interest are shown in Table 6 under the item of exemplary substitutions. If such a substitution results in a change in biological activity, a more substantial change is introduced and the product screened, as named in Table 6 as an exemplary substitution or as described further below in the amino acid classification. Is done.

Substantial modification of the function or immunological identity of the anti-TAT antibody or TAT polypeptide consists of (a) the structure of the polypeptide backbone of the substitution region, eg a sheet or helical arrangement, (b) the target site charge or molecular hydrophobicity Or (c) by selecting substituents that differ significantly in their effect while maintaining the bulk of the side chains. Naturally occurring residues can be grouped based on common side chain properties:
(1) Hydrophobicity: norleucine, met, ala, val, leu, ile;
(2) Neutral hydrophilicity: cys, ser, thr;
(3) Acidity: asp, glu;
(4) Basicity: asn, gln, his, lys, arg;
(5) Residues affecting chain orientation: gly, pro; and (6) Aromatics: trp, tyr, phe.
Non-conservative substitutions will require exchanging one member of these classes for another. Also, such substituted residues can be introduced at conservative substitution sites, or more preferably at remaining (non-conserved) sites.
Mutations can be made using methods known in the art such as oligonucleotide-mediated (site-specific) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl. Acids Res., 13: 4331 (1986); Zoller et al., Nucl. Acids Res., 10: 6487 (1987)], cassette mutagenesis [Wells et al., Gene, 34: 315 (1985)], restricted selective mutagenesis [Wells et al., Philos. Trans. R. Soc. London SerA, 317: 415 (1986)] or other known techniques are performed on cloned DNA. Thus, anti-TAT antibody or TAT polypeptide mutant DNA can also be prepared.

Scanning amino acid analysis can also be used to identify one or more amino acids along adjacent sequences. Preferred scanning amino acids are relatively small and neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is a typically preferred scanning amino acid in this group because it eliminates side chains beyond the beta carbon and is unlikely to change the backbone structure of the mutant [Cunningham and Wells, Science, 244: 1081-1085 (1989)]. Alanine is also typically preferred because it is the most common amino acid. Furthermore, it is often found in both buried and exposed positions [Creighton, The Proteins, (WH Freeman & Co., NY); Chothia, J. Mol. Biol., 150: 1 (1976)]. If alanine substitution does not result in a sufficient amount of mutants, isoteric amino acids can be used.
Any cysteine residues that are not involved in maintaining the proper conformation of the anti-TAT antibody or TAT polypeptide are also generally replaced with serine to improve the oxidative stability of the molecule and prevent abnormal cross-linking. obtain. Conversely, cysteine bond (s) may be added to the anti-TAT antibody or TAT polypeptide in order to improve the stability (particularly where the antibody is an antibody fragment such as an Fv fragment).

A particularly preferred type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (eg, a humanized or human antibody). In general, variants obtained for further development have improved biological properties compared to the parent antibody from which they were generated. Convenient methods for generating such substitutional variants include affinity maturation using phage display. Briefly, hypervariable region sites (eg, 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent form as a fusion from filamentous phage particles to the gene III product of M13 packed within each particle. Phage display variants are then screened for their biological activity (eg, binding affinity) as disclosed herein. In order to identify hypervariable region sites that are candidates for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively, or in addition, it may be advantageous to analyze the crystal structure of the antigen-antibody complex to identify contact points between the antibody and human TAT polypeptide. Such contact residues and adjacent residues are candidates for substitution according to the techniques described in detail herein. Once such variants are generated, a panel of variants can be screened as described herein to select antibodies with superior properties in one or more related assays for further development. .
Nucleic acid molecules encoding amino acid sequence variants of the anti-TAT antibody are prepared by a variety of methods well known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-specific) mutagenesis, PCR mutagenesis, and early prepared mutant or non-mutant forms of anti-TAT antibody cassettes. Isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or preparation by mutagenesis is included.

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 present invention. One type of covalent modification is an organic that can react with a target amino acid residue of an anti-TAT antibody or TAT polypeptide with a selected side chain or N or C-terminal residue of the anti-TAT antibody or TAT polypeptide. Reacting with a derivatizing reagent is included. Derivatization with bifunctional reagents is useful, for example, to crosslink an anti-TAT antibody or TAT polypeptide with a water-insoluble support matrix or surface used in the purification method of anti-TAT antibody, and vice versa. Commonly used crosslinking agents include, for example, 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, such as esters with 4-azidosalicylic acid, 3,3′-dithiobis ( Homobifunctional imide esters including disuccinimidyl esters such as succinimidyl propionate), bifunctional maleimides such as bis-N-maleimide-1,8-octane, and methyl-3-[(p- Reagents such as azidophenyl) -dithio] propioimidate.
Other modifications include deamination of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, lysine, arginine, and Methylation of α-amino group of histidine side chain [TE Creighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, pp. 79-86 (1983)], N-terminal amine acetylation, and optional Amidation of the C-terminal carboxyl group of

Other types of covalent modifications of anti-TAT antibodies or TAT polypeptides that are included within the scope of the invention include alteration of the natural glycosylation pattern of the antibody or polypeptide. As intended herein, “altering the native glycosylation pattern” refers to deleting one or more carbohydrate moieties found in a native sequence anti-TAT antibody or TAT polypeptide (by removing an underlying glycosylation site). Or the deletion of glycosylation by chemical and / or enzymatic means) and / or the addition of one or more glycosylation sites that are not present in the native sequence anti-TAT antibody or TAT polypeptide. Furthermore, this phrase includes qualitative changes in glycosylation of natural proteins, including changes in the nature and properties of the various carbohydrate moieties present.
Glycosylation of antibodies and other polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. A tripeptide is a sequence of asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, and is a recognition site where a carbohydrate moiety to the asparagine side chain is enzymatically conferred. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. For O-linked glycosylation, 5-hydroxyproline or 5-hydroxylysine is also used, but in most cases, one sugar of N-acetylgalactosamine, galactose, or xylose to serine or threonine is converted to a hydroxy amino acid. Refers to granting.
Addition of glycosylation sites to the anti-TAT antibody or TAT polypeptide modifies the amino acid sequence such that it contains one or more of the tripeptide sequences described above (for N-linked glycosylation sites). Can be completed easily. This modification is also generated by adding or substituting one or more serine or threonine residues to the sequence of the original anti-TAT antibody or TAT polypeptide (for O-linked glycosylation sites). The anti-TAT antibody or TAT polypeptide amino acid sequence mutates the DNA encoding the anti-TAT antibody or TAT polypeptide through a change at the DNA level, particularly at a preselected base where the codon is translated to the desired amino acid. Can be modified in some cases.

Another means of increasing the number of carbohydrate moieties on the anti-TAT antibody or TAT polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, for example, International Publication 87/05330 published September 11, 1987, and Aplin and Wriston, CRC Crit. Rev. Biochem., Pp. 259-306 (1981). It is described in.
Removal of the carbohydrate moiety present on the anti-TAT antibody or TAT polypeptide can be done chemically or enzymatically or by mutational substitution of codons encoding amino acid residues presented as targets for glucosylation. Chemical deglycosylation techniques are known in the art, for example by 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 is accomplished by using various endo and exoglycosidases as described in Thotakura et al., Meth. Enzymol. 138: 350 (1987).
Another type of anti-TAT antibody or TAT polypeptide covalent modification is disclosed in US Pat. No. 4,640,835; No. 4,496,689; No. 4,301,144; No. 4,670,417; No. 4,791,192 or No. 4,179,337. Alternatively, the antibody or polypeptide can be colloidally delivered to microcapsules prepared by, for example, coacervation or by interfacial polymerization (eg, hydroxymethylcellulose or gelatin-microcapsules and poly- (methyl methacrylate) microcapsules, respectively). Capturing with systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, edited by A. Oslo (1980).
In addition, the anti-TAT antibody or TAT polypeptide of the present invention may be modified by a method in which a chimeric molecule comprising an anti-TAT antibody or TAT polypeptide fused with another heterologous polypeptide or amino acid sequence is formed.

In one embodiment, such a chimeric molecule comprises a fusion of a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind and an anti-TAT antibody or TAT polypeptide. The epitope tag is generally located at the amino or carboxyl terminus of the anti-TAT antibody or TAT polypeptide. The presence of such an epitope tag form of an anti-TAT antibody or TAT polypeptide can be detected using an antibody against the tag polypeptide. The provision of an epitope tag also allows the anti-TAT antibody or TAT polypeptide to be readily purified by affinity purification using an anti-tag antibody or other type of affinity matrix that binds to the epitope tag. Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-His) or poly-histidine-glycine (poly-his-gly) tag; flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8: 2159. -2165 (1988)]; c-myc tag and 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and Cellular Biology, 5: 3610-3616 (1985)]; and herpes simplex virus sugars A protein D (gD) tag and its antibody [Paborsky et al., Protein Engineering, 3 (6): 547-553 (1990)]. Other tag polypeptides include flag peptides [Hopp et al., BioTechnology, 6: 1204-1210 (1988)]; KT3 epitope peptides [Martin et al., Science, 255: 192-194 (1992)]; α-tubulin epitope peptides [Skinner et al., J. Biol. Chem., 266: 15163-15166 (1991)]; and T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87: 6393-6397 ( 1990)].
In an alternative embodiment, the chimeric molecule may comprise a fusion of an anti-TAT antibody or TAT polypeptide with an immunoglobulin or a specific region of an immunoglobulin. For a bivalent form of a chimeric molecule (also referred to as “immunoadhesin”), such a fusion can be the Fc region of an IgG molecule. An Ig fusion preferably comprises a solubilized (transmembrane domain deleted or inactivated) form of an anti-TAT antibody or TAT polypeptide in place of at least one variable region within the Ig molecule. In particularly preferred embodiments, the immunoglobulin fusion comprises the hinge, CH ₂ and CH ₃ , or hinge, CH ₁ , CH ₂ and CH ₃ regions of an IgG molecule. See US Pat. No. 5,428,130 issued June 27, 1995 for the production of immunoglobulin fusions.

I. Preparation of anti-TAT antibody and TAT polypeptide The following description mainly describes anti-TAT antibody and TAT polypeptide by culturing cells transformed or transfected with a vector containing anti-TAT antibody and TAT polypeptide-encoding nucleic acid. It is related with the method of producing. Of course, it is believed that anti-TAT antibodies and TAT polypeptides can be prepared using other methods well known in the art. For example, the appropriate amino acid sequence, or portion thereof, may be generated by direct peptide synthesis using solid phase techniques [eg, Stewart et al., Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco, California (1969 ); Merrifield, J. Am. Chem. Soc., 85: 2149-2154 (1963)]. In vitro protein synthesis may be performed by using manual techniques or automation. Automated synthesis may be performed, for example, using an Applied Biosystems Peptide Synthesizer (Foster City, Calif.) According to manufacturer's instructions. Various portions of the anti-TAT antibody or TAT polypeptide may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired anti-TAT antibody or TAT polypeptide.

1. Isolation of DNA encoding anti-TAT antibody or TAT polypeptide DNA encoding anti-TAT antibody or TAT polypeptide is considered to possess anti-TAT antibody or TAT polypeptide mRNA and express it at a detectable level. Obtained from a cDNA library prepared from the resulting tissue. Therefore, human anti-TAT antibody or TAT polypeptide DNA can be conveniently obtained from a cDNA library prepared from human tissue. Anti-TAT antibodies or TAT polypeptide-encoding genes can also be obtained from genomic libraries or by known synthetic methods (eg, automated nucleic acid synthesis).
Libraries can be screened with probes (such as oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by that gene. Screening of cDNA or genomic libraries with selected probes is performed using standard procedures described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). can do. Another method for isolating genes encoding anti-TAT antibodies or TAT polypeptides is to use PCR [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 selected as the probe must be long enough and sufficiently clear so that false positives are minimized. The oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened. Methods of labeling are well known in the art and include the use of radiolabels such as ³² P-labeled ATP, biotinylation or enzyme labeling. Hybridization conditions including moderate and high stringency are set forth in Sambrook et al., Supra.
The sequences identified in such library screening methods can be compared and aligned with other well-known sequences deposited and available in the public databases of GenBank et al. Or other individual sequence databases. Sequence identity (either at the amino acid or nucleotide level) within a determined region of the molecule or over the full length sequence is determined using methods known in the art and described herein. be able to.
Nucleic acids with protein coding sequences use the deduced amino acid sequences disclosed herein for the first time, and if necessary, Sambrook et al., Supra, which detects intermediates and precursors of mRNA not transcribed into cDNA. Obtained by screening selected cDNA or genomic libraries using conventional primer extension methods as described in.

2. Host cell selection and transformation Host cells are transfected or transformed with an anti-TAT antibody or TAT polypeptide production or expression vector described herein to induce a promoter, select transformants, Alternatively, the cells are cultured in a conventional nutrient medium appropriately modified to amplify the gene encoding the desired sequence. Culture conditions such as medium, temperature, pH, etc. can be selected by those skilled in the art without undue experimentation. In general, principles, protocols, and practical techniques for maximizing cell culture productivity can be found in Mammalian Cell Biotechnology: a Practical Approach, edited by M. Butler (IRL Press, 1991) and Sambrook et al. it can.
Methods of eukaryotic cell transfection and prokaryotic cell transformation, e.g., CaCl _2, CaPO _4, liposome-mediated and electroporation are known to those skilled in the art. Depending on the host cell used, transformation is done using standard methods appropriate to that cell. Calcium treatment or electroporation with calcium chloride described in Sambrook et al., Supra, is generally used for prokaryotes. Infections caused by Agrobacterium tumefaciens have been described in certain plant cells as described in Shaw et al., Gene, 23: 315 (1983) and International Publication 89/05859 published 29 June 1989. It is used for transformation. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52: 456-457 (1978) is used. General aspects of mammalian cell host system transformations are described in US Pat. No. 4,399,216. Transformation into yeast is typically performed by Van Solingen et al., J. Bact., 130: 946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76: 3829 (1979). ). However, other methods of introducing DNA into cells, such as nuclear microinjection, electroporation, intact cells, or bacterial protoplast fusion using polycations such as polybrene, polyornithine, etc. can also be used. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzymology, 185: 527-537 (1990) and Mansour et al., Nature, 336: 348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectors described herein are prokaryotic, yeast, or higher eukaryotic cells. Suitable prokaryotes include, but are not limited to, eubacteria such as Gram negative or Gram positive microorganisms such as Enterobacteriaceae such as E. coli. Various E. coli strains are publicly available, for example E. coli K12 strain MM294 (ATCC 31446); E. coli X1776 (ATCC 31537); E. coli strains W3110 (ATCC 27325) and K5772 (ATCC 53635). Other preferred prokaryotic host cells are E. coli, such as E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella Typhimurium, Serratia, For example, Serratia marcescans, and Shigella, and Neisseria gonorrhoeae, such as B. subtilis and B. licheniformis (see, for example, DD266710 issued April 12, 1989). Bacillus licheniformis 41P) described, Pseudomonas, including Enterobacteriaceae such as Pseudomonas aeruginosa and Streptomyces. These examples are illustrative rather than limiting. Strain W3110 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations. Preferably, the host cell secretes a minimal amount of proteolytic enzyme. For example, strain W3110 may be modified to result in a genetic mutation in a gene encoding a protein that is endogenous to the host, examples of such hosts include E. coli W3110 strain 1A2 with the complete genotype tonA; E. coli W3110 strain 9E4 with complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC 55,244) with complete genotype tonA ptr3 phoA E15 (argF-lac) 169 degP ompT kan ^r ; complete genotype tonA ptr3 Escherichia coli W3110 strain 37D6 with phoA E15 (argF-lac) 169 degP ompT rbs7 ilvG kan ^r ; And E. coli strains having mutant periplasmic protease disclosed in US Pat. No. 4,946,783 issued Aug. 7, Alternatively, in vitro methods of cloning such as PCR or other nucleic acid polymerase reactions are preferred.

  Full-length antibodies, antibody fragments, and antibody fusion proteins are particularly glycosylated, such as when a therapeutic antibody is conjugated to a cytotoxic agent (eg, a toxin) and the immunoconjugate itself is effective in destroying tumor cells. Bacterial and Fc effector functions can be produced in bacteria when they are not needed. Full-length antibodies have a longer half-life in the blood circulation. Production in E. coli is faster and more cost effective. For expression of antibody fragments and polypeptides in bacteria, for example, US Pat. No. 5,648,237 (Carter et al.), US Pat. No. 5,789,199 (Joly et al.), And translation initiation site (TIR) and expression and secretion are optimized. See US Pat. No. 5,840,523 (Simmons et al.) Which describes signal sequences. These patents are hereby incorporated by reference. After expression, the antibody can be separated from the E. coli cell paste into a soluble fraction and purified via, for example, a protein A or G column by isotype. Final purification can be performed, for example, in the same manner as the step for purifying an antibody expressed in CHO cells.

  In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-TAT antibody or TAT polypeptide-encoding vectors. Saccharomyces cerevisia is a commonly used lower eukaryotic host microorganism. In addition, Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 [1981]; European Patent No. 139383 published May 2, 1985); Kluyveromyces hosts ( U.S. Pat. No. 4,943,529; Fleer et al., Bio / Technology, 9: 968-975 (1991)), eg K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154 (2): 737-742 [1983]), K. fragilis (ATCC 12424), K. bulgaricus (ATCC 16045), Kluyveromyces Wickeramii (K. wickeramii) (ATCC 24178), K. waltii (ATCC 56500), K. drosophilarum (ATCC 36906; Van den Berg et al., Bio / Technology, 8: 135 (1990)), K. thermotolerans and K. marxianus; Yarrowia (European Patent No. 402226); Pichia Pastoris ( Pichia pastoris) (European Patent No. 183070; Sreekrishna et al., J. Basic Microbiol, 28: 265-278 [1988]); Candida; Trichoderma reesia (European Patent No. 244234); Proc. Natl. Acad. Sci. USA, 76: 5259-5263 [1979]); Schwanniomyces, eg Schwanniomyces occidentalis (European patent published 31 October 1990) 394538); and filamentous fungi such as Neurospora, Penicillium, Tolypocladium (International Publication 91/0 published 10 January 1991). 357); and Aspergillus hosts such as Aspergillus nidalance (Ballance et al., Biochem. Biophys. Res. Commun., 112: 284-289 [1983]; Tilburn et al., Gene, 26: 205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81: 1470-1474 [1984]) and Aspergillus niger (Kelly and Hynes, EMBO J., 4: 475-479 [1985]). Preferred methylotrophic (C1 compound assimilating, yeast) yeasts here include, but are not limited to, Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Contains yeast that can grow on methanol selected from the genus consisting of Rhodotorula. A list of specific species, illustrative of this yeast classification, is described in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).

Suitable host cells for the expression of glycosylated anti-TAT antibody or TAT polypeptide are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells such as cotton, corn, potato, soybean, petunia, tomato and tobacco cell cultures. Permissive insect hosts corresponding to many baculovirus strains and mutants and hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Drosophila mosquito, Drosophila melanogaster (Drosophila), and silkworms Cells have been identified. Various transfection virus strains are publicly available, such as the L-1 mutant of Autographa californica NPV, the Bm-5 strain of silkworm NPV, such viruses are according to the present invention. It may be used as a virus, especially for transfection of sweet potato cells.
However, the greatest interest was directed to vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) became a routine task. Examples of useful mammalian host cell lines are monkey kidney CV1 cell line transformed with SV40 (COS-7, ATCC CRL1651); human embryonic kidney cell line (293 or subcloned to grow in suspension culture) 293 cells, Graham et al., J. Gen Virol., 36:59 (1977)); Baby hamster kidney cells (BHK, ATCC CCL10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23: 243-251 (1980)); monkey kidney cells (CV1 ATCC CCL70); African green monkey kidney Cells (VERO-76, ATCC CRL-1587); human cervical tumor cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL34); buffalo rat hepatocytes (BRL 3A, ATCC CRL1442); Human lung cells (W138, ATCC CCL75); human hepatocytes (Hep G2, HB 8065); mouse breast tumor cells (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci., 383: 44-68) (1982)); MRC5 cells; FS4 cells; and human liver cancer cells (HepG2).
Host cells are transformed with the expression or cloning vectors described above to produce anti-TAT antibodies or TAT polypeptides, induce promoters, select transformants, or amplify genes encoding the desired sequences. Incubated in normal nutrient medium modified appropriately.

3. Selection and use of replicable vectors A nucleic acid (eg, cDNA or genomic DNA) encoding an anti-TAT antibody or TAT polypeptide is inserted into a replicable vector for cloning (amplification of DNA) or expression. Various vectors are publicly available. The vector can be, for example, in the form of a plasmid, cosmid, virus particle, or phage. The appropriate nucleic acid sequence is inserted into the vector by a variety of techniques. In general, DNA is inserted into an appropriate restriction endonuclease site using techniques well known in the art. Vector components generally include, but are not limited to, one or more signal sequences, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Standard ligation techniques known to those skilled in the art are used to generate suitable vectors containing one or more of these components.
TAT is not only generated directly by recombinant techniques, but as a fusion peptide with a heterologous polypeptide that is a signal sequence or a mature protein or other polypeptide having a specific cleavage site at the N-terminus of the polypeptide. Is also generated. In general, the signal sequence is a component of the vector, or it is part of the anti-TAT antibody or TAT polypeptide-encoding DNA that is inserted into the vector. The signal sequence may be, for example, a prokaryotic signal sequence selected from the group of alkaline phosphatase, penicillinase, lpp or a thermostable enterotoxin II leader. For yeast secretion, signal sequences include yeast invertase leader, alpha factor leader (Saccharomyces and Kluyveromyces alpha factor leader, the latter described in US Pat. No. 5,010,182. Or acid phosphatase leader, C. albicans glucoamylase leader (European Patent No. 362179, issued April 4, 1990) or published on November 15, 1990 It may be the signal described in WO 90/13646. In mammalian cell expression, mammalian signal sequences may be used for direct secretion of proteins such as signal sequences from secreted polypeptides of the same or related species as well as viral secretion leaders.

Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for many bacteria, yeasts and viruses. The origin of replication derived from plasmid pBR322 is suitable for most gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and the various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are suckling. Useful for cloning vectors in animal cells.
Expression and cloning vectors typically contain a selection gene, also termed a selectable marker. Typical selection genes are (a) resistant to antibiotics or other toxins such as ampicillin, neomycin, methotrexate or tetracycline, (b) supplemented with auxotrophic defects, or (c) obtained from complex media. It encodes a protein that supplies no important nutrients, such as the gene encoding Basili D-alanine racemase.
Examples of selectable markers that are suitable for mammalian cells are those that can identify cellular components that can take up anti-TAT antibodies or TAT polypeptide-encoding nucleic acids, such as DHFR or thymidine kinase. Suitable host cells when using wild-type DHFR are DHFR activity prepared and grown as described by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77: 4216 (1980). It is a defective CHO cell line. A suitable selection gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7 [Stinchcomb et al., Nature, 282: 39 (1979); Kingsman et al., Gene, 7: 141 (1979); Tschemper et al., Gene, 10: 157 (1980)]. The trp1 gene provides a selectable marker for mutant strains of yeast lacking the ability to grow with tryptophan, such as, for example, ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operably linked to the anti-TAT antibody or TAT polypeptide-encoding nucleic acid sequence and directing mRNA synthesis. Promoters recognized by a variety of competent host cells are known. Suitable promoters for use with prokaryotic hosts include β-lactamase and lactose promoter systems [Chang et al., Nature, 275: 615 (1978); Goeddel et al., Nature, 281: 544 (1979)], alkaline phosphatase, tryptophan (trp ) Promoter system [Goeddel, Nucleic Acids Res., 8: 4057 (1980); European Patent No. 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80: 21-25 (1983)]. Promoters used in bacterial systems also have a Shine-Dalgarno (SD) sequence operably linked to the DNA encoding the anti-TAT antibody or TAT polypeptide.
Examples of promoter sequences suitable for use with yeast hosts include 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem., 255: 2073 (1980)] or other glycolytic enzymes [Hess et al., J Adv. Enzyme Reg., 7: 149 (1968); Holland, Biochemistry, 17: 4900 (1978)], eg enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase Glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, trioselate isomerase, phosphoglucose isomerase, and glucokinase.
Other yeast promoters are inducible promoters that have the additional effect that transcription is controlled by growth conditions, such as alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degrading enzymes related to nitrogen metabolism, metallothionein, glycerin There are promoter regions of aldehyde-3-phosphate dehydrogenase and the enzymes that govern the utilization of maltose and galactose. Vectors and promoters that are preferably used for expression in yeast are further described in EP 73657.

Transcription of an anti-TAT antibody or TAT polypeptide from a vector in a mammalian host cell is, for example, polyomavirus, infectious epithelioma virus (UK Patent No. 2221504 published July 5, 1989), adenovirus (eg, adenovirus). 2) promoters derived from the genome of viruses such as bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and simian virus 40 (SV40), heterologous mammalian promoters such as actin promoter Alternatively, such promoters are controlled by immunoglobulin promoters and promoters obtained from heat shock promoters as long as they are compatible with the host cell system.
Transcription of DNA encoding an anti-TAT antibody or TAT polypeptide by higher eukaryotes can be enhanced by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to enhance its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include late SV40 enhancer (100-270 base pairs), cytomegalovirus early promoter enhancer, late polyoma enhancer and adenovirus enhancer of replication origin. Enhancers can be spliced into the vector at the 5 'or 3' position of the anti-TAT antibody or TAT polypeptide coding sequence, but are preferably located 5 'from the promoter.
Expression vectors used for eukaryotic host cells (nucleated cells derived from yeast, fungi, insects, plants, animals, humans, or other multicellular organisms) are sequences required for termination of transcription and stabilization of mRNA. Including. Such sequences can be obtained from the normally 5 ', sometimes 3' untranslated region of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments that are transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the anti-TAT antibody or TAT polypeptide.
Other methods, vectors and host cells suitable for adapting to the synthesis of anti-TAT antibodies or TAT polypeptides in recombinant vertebrate cell culture are described in Gething et al., Nature, 293: 620-625 (1981); Mantei Et al., Nature, 281: 40-46 (1979); European Patent No. 117060; and European Patent No. 117058.

4). Host Cell Culture Host cells used to produce the anti-TAT antibodies or TAT polypeptides of the present invention can be cultured in a variety of media. Examples of commercially available media include Ham's F10 (Sigma), minimal essential media ((MEM), Sigma), RPMI-1640 (Sigma) and Dulbecco's modified Eagle's medium ((DMEM), Sigma). It is suitable for culturing. Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102: 255 (1980), US Pat. No. 4,767,704; US Pat. No. 4,657,866; US Pat. Any medium described in US Pat. No. 5,122,469; WO 90/03430; WO 87/00195; or US Reissue No. 30985 can also be used as a culture medium for host cells. Any of these media can be hormones and / or other growth factors (eg, insulin, transferrin, or epidermal growth factor), salts (eg, sodium chloride, calcium, magnesium and phosphate), buffers (eg, HEPES), nucleosides. (Eg adenosine and thymidine), antibiotics (eg gentamicin (trade name) drugs), trace elements (defined as inorganic compounds normally present at final concentrations in the micromolar range) and glucose or equivalent energy source required Can be refilled according to. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. Culture conditions such as temperature, pH, etc. are those previously used for the host cell chosen for expression and will be apparent to those skilled in the art.

5. Gene amplification / expression detection Gene amplification and / or expression is based on the sequences provided herein, using appropriately labeled probes, eg, conventional Southern blotting, Northern to quantify mRNA transcription Can be measured directly in the sample by blotting [Thomas, Proc. Natl. Acad. Sci. USA, 77: 5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization. . Alternatively, antibodies capable of recognizing specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes, can also be used. The antibody can then be labeled and assayed, where the duplex is bound to the surface, so that the antibody bound to the duplex at the point of formation of the duplex on the surface The presence of can be detected.
Alternatively, gene expression can be measured by immunological methods that directly quantify gene product expression, such as immunohistochemical staining of cells or tissue sections and cell culture or body fluid assays. Antibodies useful for immunohistochemical staining and / or assay of sample fluids can be monoclonal or polyclonal and can be prepared in any mammal. Conveniently, the antibody is directed against a native sequence TAT polypeptide or against a synthetic peptide based on the DNA sequence provided herein, or an exogenous sequence fused to TAT DNA and encoding a specific antibody epitope. Can be prepared.

6). Purification of anti-TAT antibodies and TAT polypeptides Anti-TAT antibody and TAT polypeptide forms can be recovered from culture media or lysates of host cells. If membrane-bound, it can be detached from the membrane using a suitable wash solution (eg Triton-X100) or by enzymatic cleavage. Cells used for expression of anti-TAT antibodies and TAT polypeptides can be disrupted by various chemical or physical means such as freeze-thaw cycles, sonication, mechanical disruption, or cytolytic agents.
It is desirable to purify anti-TAT antibodies and TAT polypeptides from recombinant cell proteins or polypeptides. Purified by the following procedure, which is an example of a suitable purification procedure: fractionation on an ion exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or a cation exchange resin such as DEAE; chromatofocusing; PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A sepharose column to remove contaminants such as IgG; and metal chelation column to bind the epitope tag form of anti-TAT antibody and TAT polypeptide . It is possible to use many protein purification methods known in the art and described, for example, in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). it can. The purification process chosen 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 can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. When antibodies are produced intracellularly, as a first step, particulate debris, host cells or lysed fragments are removed, for example, by centrifugation or ultracentrifugation. Carter et al., Bio / Technology 10: 163-167 (1992) describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) over 30 minutes. Cell debris can be removed by centrifugation. If the antibody is secreted into the medium, the supernatant from such an expression system is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors such as PMSF may be included in any of the above steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of exogenous contaminants.
Antibody compositions prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of the immunoglobulin Fc region present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 [1983]). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5: 15671575 [1986]). The matrix to which the affinity ligand is bound is most often agarose, although other materials can be used. Mechanically stable matrices such as controlled pore glass and poly (styrenedivinyl) benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a C _H 3 domain, Bakerbond ABX ™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Fractionation on ion exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, heparin SEPHAROSE (trade name) chromatography on anion or cation exchange resin (polyaspartic acid column), chromatofocusing, SDS-PAGE , And other protein purification techniques such as ammonium sulfate precipitation are also available depending on the antibody recovered.
Following the optional prepurification step, the mixture containing the antibody of interest and contaminants is subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH of about 2.5-4.5. Preferably, it is carried out at low salt concentrations (eg, about 0-0.25M salt).

J. et al. Pharmaceutical preparation The therapeutic preparation of the anti-TAT antibody, TAT-binding oligopeptide, TAT-binding organic molecule and / or TAT polypeptide according to the present invention comprises an antibody, polypeptide, oligopeptide or organic molecule having a desired degree of purity. Prepared and stored by mixing with an optimal pharmaceutically acceptable carrier, excipient or stabilizer in the form of a lyophilized formulation or aqueous solution (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Hen. 1980]). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used and buffers such as acetic acid, Tris, phosphoric acid, citric acid, and other organic acids; ascorbine Antioxidants including acids and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol Resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; protein such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymer such as polyvinylpyrrolidone; Glish Amino acids such as glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; tonicifiers such as trehalose and sodium chloride; Sugars such as mannitol, trehalose or sorbitol; surfactants such as polysorbates; salt-forming counterions such as sodium; metal complexes (eg, Zn-protein complexes); and / or TWEEN®, Pluronics ( PLURONICS) or non-ionic surfactants such as polyethylene glycol (PEG). The antibody is preferably composed of antibody at a concentration between 5-200 mg / ml, preferably between 10-100 mg / ml.

The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, in addition to an anti-TAT antibody, a TAT-binding oligopeptide or a TAT-binding organic molecule, one formulation, for example, a second anti-TAT antibody that binds to a different epitope on the TAT polypeptide, or affects the growth of certain cancers It may be desirable to include antibodies to some other target, such as a growth factor that provides. Alternatively or additionally, the composition may further comprise a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal agent, and / or cardioprotectant. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
The active ingredients can also be used in colloidal drug delivery systems (eg, in microcapsules prepared by coacervation techniques or by interfacial polymerization, eg, hydroxymethylcellulose or gelatin-microcapsules and poly (methyl methacrylate) microcapsules, respectively. , Liposomes, albumin globules, microemulsions, nanoparticles and nanocapsules) or microemulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
Sustained release formulations may be prepared. Suitable examples of sustained release formulations include a semi-permeable matrix of solid hydrophobic polymer containing antibodies, which matrix is in the form of a molded article, such as a film or microcapsule. Examples of sustained release matrices include polyesters, hydrogels (eg, poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polyactides (US Pat. No. 3,773,919), L-glutamic acid and gamma ethyl-L- Degradable lactic acid-glycolic acid copolymers, such as glutamate copolymers, non-degradable ethylene-vinyl acetate, LUPRON DEPOT® (injectable globules of lactic acid-glycolic acid copolymer and leuprolide acetate), poly- (D)- (-)-3-hydroxybutyric acid is included.
Formulations used for in vivo administration must be sterile. This is easily accomplished by filtration through sterile filtration membranes.

K. Diagnosis and treatment using anti-TAT antibodies, TAT-binding oligopeptides or TAT-binding organic molecules Various diagnostic assays are available to quantify TAT expression in cancer. In one embodiment, TAT polypeptide overexpression is analyzed by immunohistochemistry (IHC). Paraffin-embedded tissue sections from tumor biopsies may be subjected to an IHC assay or matched to the following TAT protein staining intensity criteria:
Score 0-no staining is observed or membrane staining is observed in less than 10% of the tumor cells.
Score 1+-a faint / weakly perceptible membrane staining is detected in more than 10% of the tumor cells. Cells are stained only for a portion of their membrane.
Score 2+-a weak or moderate complete membrane staining is observed in more than 10% of the tumor cells.
Score 3+-moderate to strong complete membrane staining is observed in more than 10% of the tumor cells.
Tumors with a 0 or 1+ score for TAT polypeptide expression may be characterized by TAT not being overexpressed, whereas tumors with a 2+ or 3+ score are characterized by overexpression of TAT. sell.

Alternatively or additionally, a FISH assay such as INFORM® (sold by Ventana, Arizona) or PATHVISION® (Vysis, Illinois) is performed on formalin-fixed, paraffin-embedded tumor tissue. The extent of TAT overexpression in the tumor (if any) may be measured.
TAT overexpression or amplification can be assessed using in vivo diagnostic assays, e.g., molecules that bind to the molecule to be detected and have a detectable label (e.g., a radioisotope or fluorescent label) (e.g., Antibody, oligopeptide or organic molecule) and externally scanning the patient for label localization.
As described above, the anti-TAT antibodies, oligopeptides or organic molecules of the present invention have a variety of non-therapeutic uses. The anti-TAT antibodies, oligopeptides or organic molecules of the present invention are useful for the diagnosis and staining of cancers expressing TAT polypeptides (eg, in radio imaging). In order to kill and remove TAT-expressing cells from a population of mixed cells as another cell purification step, the antibody, oligopeptide or organic molecule can also be isolated in vitro, eg, in an ELISA or Western blot. Useful for the purification or immunoprecipitation of TAT polypeptides from cells for detection and quantification of peptides.

  Currently, depending on the stage of the cancer, cancer treatment includes one or a combination of the following therapies: surgical removal of cancer tissue, radiation therapy, and chemotherapy. Treatment with anti-TAT antibodies, oligopeptides or organic molecules is particularly desirable in elderly patients who are not resistant to side effects and toxins in chemotherapy and in metastatic diseases where the usefulness of radiation therapy is limited. The tumor-targeted anti-TAT antibodies, oligopeptides or organic molecules of the present invention are useful for the mitigation of TAT-expressing cancers during initial diagnosis and relapse of the disease. For therapeutic applications, anti-TAT antibodies, oligopeptides or organic molecules alone or in combination with, for example, hormones, anti-angiogenic, or radiolabeled compounds, or in combination with surgery, cryotherapy, and / or radiation therapy May be used. Treatment with anti-TAT antibodies, oligopeptides or organic molecules can be performed with other forms of conventional therapy, either consecutively before or after conventional therapy. Chemotherapeutic agents such as Taxotere® (docetaxel), Taxol® (paclitaxel), estramustine and mitoxantrone are used for the treatment of cancer, particularly in low-risk patients. In the methods of the invention for treating or alleviating cancer, an anti-TAT antibody, oligopeptide or organic molecule can be administered to a cancer patient in combination with treatment with one or more chemotherapeutic agents described above. In particular, combination therapy with paclitaxel and modified derivatives is envisaged (see for example EP 0600517). The anti-TAT antibody, oligopeptide or organic molecule will be administered with a therapeutically effective amount of the chemotherapeutic agent. In other embodiments, the anti-TAT antibody, oligopeptide or organic molecule is administered in combination with a chemotherapeutic agent, eg, chemotherapy to increase the activity and efficacy of paclitaxel. The Physician Desk Reference (PDR) discloses the doses of these drugs used in various cancer treatments. The dosage regimen and dosage of the therapeutically effective chemotherapeutic agents described above will depend on the particular cancer being treated, the extent of the disease, and other factors well known to the physician in the art. can do.

In one particular embodiment, a toxin conjugate containing 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 an improved therapeutic effect of the immunoconjugate on the killing properties of the cancer cell to which it is bound. In preferred embodiments, the cytotoxic agent targets or interferes with nucleic acid in the cancer cell. Examples of such cytotoxic agents are described above and include maytansinoids, calicheamicins, ribonucleases and DNA endonucleases.
The anti-TAT antibody, oligopeptide or organic molecule or immunoconjugate thereof can be administered in a known manner, for example, intravenously by bolus or continuous infusion over a period of time, intramuscular, intraperitoneal, intracerebral spinal, subcutaneous, intraarticular, Administered to human patients by intracapsular, intrathecal, oral, topical, or inhalation routes. Intravenous or subcutaneous administration of antibodies, oligopeptides or organic molecules is preferred.
Other treatment regimens may be combined with the administration of anti-TAT antibodies, oligopeptides or organic molecules. Combination administration includes co-administration using separate formulations or a single pharmaceutical formulation, and preferably in any order with both (or all) active agents having time to exert their biological activity simultaneously. Administration is included. Such combination therapy preferably results in a synergistic therapeutic effect.

It is also desirable to combine administration of anti-TAT antibodies or antibodies, oligopeptides or organic molecules with administration of antibodies against other tumor antigens associated with a particular cancer.
In other embodiments, the therapeutic methods of the invention comprise an anti-TAT antibody (or antibodies), oligopeptide or organic molecule and one or more chemotherapeutic agents or growth inhibitors comprising the simultaneous administration of a mixture of different chemotherapeutic agents. Combination administration with agents. Chemotherapeutic agents include estramustine phosphate, prednimustine, cisplatin, 5-fluorouracil, melphalan, cyclophosphamide, hydroxyurea and hydroxyureataxanes (eg, paclitaxel and doxetaxel) and / or anthracycline antibiotics Contains substances. The preparation and administration schedule of such chemotherapeutic agents is used according to the manufacturer's notes or determined empirically by skilled practitioners. Such chemotherapy preparation and dosing schedules are also described in Chemotherapy Service edited by MCPerry, Williams & Wilkins, Baltimore, MD (1992).
Antibodies, oligopeptide organic molecules may include antihormonal compounds; anti-estrogenic compounds such as tamoxifen; anti-progesterones such as onapristone (see EP 616812); or antiandrogens such as flutamide. Such molecules may be combined at known doses. If the cancer to be treated is an androgen-independent cancer, the patient has previously received anti-androgen treatment, and after the cancer has become androgen-independent, anti-TAT antibodies, oligopeptides or organic molecules (and possibly here) Other agents described) may be administered to the patient.

Often it is also beneficial to co-administer a cardioprotectant (to prevent or reduce myocardial dysfunction associated with therapy) or one or more cytokines to the patient. In addition to the therapeutic regimes described above, cancer cells may be surgically removed and / or radiation therapy administered prior to, simultaneously with, or following antibody, oligopeptide or organic molecule therapy. Suitable dosages for any of the above-mentioned co-administered drugs are those currently used and may be less depending on the combined action (synergism) of the drug with the anti-TAT antibody, oligopeptide or organic molecule .
The dosage and mode for prevention or treatment of the disease will be selected by a physician according to known criteria. The appropriate dose of antibody, oligopeptide or organic molecule is the type of disease to be treated, severity and process of the disease, antibody, oligopeptide or organic molecule administered for prophylactic or therapeutic purposes as described above Rather, it will depend on past treatment, patient clinical history and responsiveness of antibodies, oligopeptides or organic molecules, and the discretion of the treating physician. The antibody, oligopeptide or organic molecule is suitably administered to the patient at one time or over a series of treatments. Preferably, the antibody, oligopeptide or organic molecule is administered by intravenous infusion or subcutaneous injection. Depending on the type and severity of the disease, for example, one or more separate doses or continuous infusions, about 1 μg to 50 mg of antibody per kg of body weight (eg, 0.1-15 mg / kg / dose) to the patient. Can be candidates for the first dose. The regimen may consist of administering an initial loading dose of about 4 mg / kg followed by a maintenance dose of about 2 mg / kg of anti-TAT antibody per week. However, other dosing schedules may be effective. Depending on the factors discussed above, typical daily dosages range from about 1 μg / kg to 100 mg / kg or more. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. The progress of this therapy is easily monitored by conventional methods and assays based on criteria known to physicians or other skilled artisans.

In addition to administering antibody proteins to patients, this application discusses administration of antibodies by gene therapy. Administration of nucleic acid encoding an antibody is included in the expression “administering the antibody in a therapeutically effective amount”. See, for example, WO 96/07321 published March 14, 1996 regarding the production of intracellular antibodies using gene therapy.
There are two main ways to get the nucleic acid (optionally contained in a vector) into the patient's cells: in vivo and ex vivo. For in vivo delivery, the nucleic acid is usually injected directly into the site where the antibody is needed. In ex vivo treatment, a patient's cells are removed, nucleic acids are introduced into these isolated cells, and the modified cells are administered to the patient either directly or encapsulated in, for example, a porous membrane that is implanted in the patient (US 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 in vitro into cultured cells or in vivo into a host of interest. 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. A commonly used vector for ex vivo delivery of genes is a retroviral vector.

Currently preferred in vivo nucleic acid transfer techniques include viral vectors (eg, adenovirus, herpes simplex I virus, or adeno-associated virus), and lipid-based systems (eg, lipids useful for lipid-mediated transfer of genes include DOTMA , DOPE, and DC-Chol). For review of the currently known gene marking and gene therapy protocols, see Anderson et al., Science, 256: 808-813 (1992). See also WO 93/25673 and the references cited therein.
The anti-TAT antibody of the present invention may be in various forms encompassed by the definition of “antibody” herein. Thus, antibodies include full length or intact antibodies, antibody fragments, native sequence antibodies or amino acid variants, humanized, chimeric or fusion antibodies, immunoconjugates, and functional fragments thereof. In a fusion antibody, the antibody sequence is fused to a heterologous polypeptide sequence. The antibody can be modified in the Fc region to provide the desired effector function. As described in detail in the following paragraphs, intact antibodies bound to the cell surface with an appropriate Fc region can be complemented, for example, via antibody-dependent cytotoxicity (ADCC) or in complement-dependent cytotoxicity. Cytotoxicity can be induced by recruiting the body or by some other mechanism. Also, certain other Fc regions are used when it is desirable to remove or reduce effector function to minimize side effects and therapeutic complications.
In one embodiment, the antibody competes for or binds substantially to the same epitope as the antibody of the invention. Also contemplated are antibodies having the biological characteristics of the anti-TAT antibodies of the invention, particularly those that include in vivo tumor targeting and any cell growth inhibition or cytotoxic properties.
The above-described antibody production method will now be described in detail.

  The anti-TAT antibodies, oligopeptides or organic molecules are useful for the treatment of TAT-expressing cancers in mammals or for alleviating one or more cancer symptoms. Such cancers include prostate cancer, urethral cancer, lung cancer, breast cancer, colon cancer and ovarian cancer, in particular prostate cancer (prostate adenocarcinoma), renal cell carcinoma, colorectal adenocarcinoma, lung adenocarcinoma, squamous carcinoma of lung cells, And pleural mesothelioma. Cancer includes any of the metastatic cancers described above. The antibody, oligopeptide or organic molecule is capable of binding to at least a portion of the cancer cells expressing the TAT polypeptide in the mammal. In preferred embodiments, the antibody, oligopeptide or organic molecule binds to the TAT polypeptide of a cell in vivo or in vitro to destroy or kill TAT-expressing tumor cells or inhibit the growth of such tumor cells. It is effective to do. Such antibodies include naked anti-TAT antibodies (not conjugated to any drug). Naked antibodies having cytotoxic or cell growth inhibiting properties can more strongly destroy tumor cells when used in combination with cytotoxic agents. For example, cytotoxic properties can be imparted to an anti-TAT antibody by combining a cytotoxic agent and an antibody to form an immunoconjugate as described below. This cytotoxic agent or growth inhibitor is preferably a small molecule. Toxins such as calicheamicin or maytansinoids, and analogs or derivatives thereof are preferred.

The present invention provides a composition comprising an anti-TAT antibody, oligopeptide or organic molecule of the present invention and a carrier. For the treatment of cancer, the composition can be administered to a patient as needed for the treatment, where the composition contains one or more anti-TAT antibodies present as immunoconjugates or naked antibodies. Can do. In a further embodiment, the composition can also contain other therapeutic agents, such as growth inhibitors or cytotoxic agents including chemotherapeutic agents, in combination with these antibodies, oligopeptides or organic molecules. The present invention also provides a preparation containing the anti-TAT antibody, oligopeptide or organic molecule of the present invention and a carrier. In one embodiment, the formulation is a therapeutic formulation containing a pharmaceutically acceptable carrier.
Another aspect of the invention is an isolated nucleic acid molecule encoding an anti-TAT antibody. Includes nucleic acids encoding heavy and light chains, particularly hypervariable region residues, chains encoding native sequence antibodies and variants, modified and humanized forms of the antibodies.
The present invention treats a TAT polypeptide-expressing cancer in a mammal or alleviates one or more symptoms of cancer, comprising administering to the mammal a therapeutically effective amount of an anti-TAT antibody, oligopeptide or organic molecule. Provides a useful method. The antibody, oligopeptide or organic molecule therapeutic composition can be administered for a short period (acute) or chronically or intermittently as directed by the physician. Also provided are methods for inhibiting the growth of TAT polypeptide-expressing cells and killing the cells.
The invention also provides kits or articles of manufacture containing at least one anti-TAT antibody, oligopeptide or organic molecule. Kits containing anti-TAT antibodies, oligopeptides or organic molecules have found use in, for example, TAT cell killing assays, purification of TAT polypeptides from cells or immunoprecipitation. For example, for isolation and purification of TAT, the kit can contain an anti-TAT antibody, oligopeptide or organic molecule coupled to beads (eg, sepharose beads). Kits containing antibodies, oligopeptides or organic molecules in in vitro TAT detection and quantification, such as ELISA or Western blots, can also be provided. Such antibodies, oligopeptides or organic molecules useful for detection can be provided with a label such as a fluorescent or radiolabel.

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

  Kits useful for various purposes such as TAT-expressing cell killing assays, purification of TAT polypeptides from cells or immunoprecipitation are also provided. For isolation and purification of TAT polypeptide, the kit can include an anti-TAT antibody, oligopeptide or organic molecule bound to beads (eg, sepharose beads). Kits comprising antibodies, oligopeptides or organic molecules for detection and quantification of TAT polypeptides in vitro, such as ELISA or Western blots, can also be provided. Similar to the manufactured product, the kit comprises a container and a label or written letter attached to or attached to the container. The container contains a composition containing at least one anti-TAT antibody, oligopeptide or organic molecule of the present invention. An additional container for storing a diluent, a buffer, a control antibody, and the like may be provided. The label or written statement provides a description of the composition as well as notes regarding the intended in vitro or diagnostic use.

M.M. Uses of TAT polypeptides and TAT-polypeptide-encoding nucleic acids Nucleic acid sequences encoding TAT polypeptides (or their complementary strands) are used in the field of molecular biology, including use as hybridization probes, in chromosomes and gene mapping. And has various uses in the production of antisense RNA and DNA probes. TAT-encoding nucleic acids are also useful for preparing TAT polypeptides by the recombinant techniques described herein, and these TAT polypeptides may find use, for example, in the preparation of anti-TAT antibodies described herein.
The full-length native sequence TAT gene or a portion thereof may be isolated from the full-length TAT cDNA or other cDNAs having the desired sequence identity to the native TAT sequences disclosed herein (eg, naturally occurring variants of TAT or Can be used as hybridization probes for cDNA libraries for isolation of those encoding TAT from other species. In some cases, the length of the probe is from about 20 to about 50 bases. The hybridization probe may be derived at least in part from novel regions of the full-length natural nucleotide sequence, and these regions may contain the native sequence TAT promoter, enhancer component and intron without undue experimentation. It can be determined from the genomic sequence it contains. For example, the screening method involves synthesizing a selected probe of about 40 bases by isolating the coding region of the TAT gene using a well-known DNA sequence. Hybridization probes can be labeled with a variety of labels including radioactive nucleotides such as ³² P or ³⁵ S, or enzyme labels such as alkaline phosphatase attached to the probe via an avidin / biotin binding system. A labeled probe having a sequence complementary to the sequence of the TAT gene of the present invention screens a library of human cDNA, genomic DNA or mRNA and determines which member of the library the probe will hybridize with. Can be used to do. Hybridization techniques are described in further detail in the following examples. Any EST sequence disclosed in this application can be used as a probe in the same manner, utilizing the methods disclosed herein.

Other useful fragments of a TAT encoding nucleic acid include an antisense or sense oligonucleotide comprising a single stranded nucleic acid sequence (either RNA or DNA) that can bind to a target TAT mRNA (sense) or TAT DNA (antisense) sequence Is included. According to the present invention, the antisense or sense oligonucleotide comprises a fragment of the coding region of TAT DNA. Such fragments generally comprise at least about 14 nucleotides, preferably from about 14 to 30 nucleotides. The ability to obtain antisense or sense oligonucleotides based on a cDNA sequence encoding a given protein is described, for example, by Stein and Cohen (Cancer Res. 48: 2659, 1988) and van der Krol et al. (BioTechniques 6: 958, 1988).
Binding of the antisense or sense oligonucleotide to the target nucleic acid sequence results in the formation of a duplex, which can be one of several methods, including promoting duplex degradation, premature termination of transcription or translation, or other By this method, transcription or translation of the target sequence is blocked. Such a method is included in the present invention. Thus, antisense oligonucleotides are used to block the expression of TAT proteins, which can be responsible for cancer induction in mammals. Antisense or sense oligonucleotides further include oligonucleotides having modified sugar-phosphodiester backbones (or other sugar linkages, such as those described in WO 91/06629), where such sugar linkages are resistant to endogenous nucleases. It is. Oligonucleotides with such resistant sugar linkages are stable in vivo (ie, able to withstand enzymatic degradation), but retain sequence specificity that allows them to bind to the target nucleotide sequence.

Suitable intragenic sites for antisense binding include translation start / start codons (5′-AUG / 5′-ATG) or termination / stop codons (5′-UAA, 5 ′) of the open reading frame (ORF) of the gene. -UAG and 5-UGA / 5'-TAA, 5'-TAG and 5'-TGA) are included. These regions refer to the part of the mRNA or gene that includes from about 25 to about 50 contiguous nucleotides in either direction (ie 5 ′ or 3 ′) from the translation initiation or termination codon. Other suitable regions for antisense binding include: intron; exon; intron-exon junction; open reading frame (ORF) or “coding region” that is the region between the translation initiation codon and translation termination codon; Contains the N7-methylated guanosine residue attached to the 5'-terminal residue of the mRNA via a '-5' triphosphate linkage, including the first 50 nucleotides adjacent to the cap as well as the 5 'cap structure itself. 5 'cap of mRNA; 5' untranslated region containing a nucleotide between the translation start codon of the corresponding nucleotide on the mRNA or gene and the 5 'cap site in the 5' direction of the mRNA from the translation start codon (5 'UTR); and the portion of the mRNA in the 3' direction from the translation termination codon at the 3 'end of the corresponding nucleotide on the mRNA or gene Including nucleotides between the translation termination codon and includes 3 'untranslated region (3'UTR).
Particular examples of suitable antisense compounds useful for inhibiting the expression of TAT protein include oligonucleotides containing modified backbones or non-natural internucleoside linkages. Oligonucleotides having a modified backbone include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as often cited in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. Suitable modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothionates, phosphorodithionates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates, 3′-alkylene phosphonates, Including 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3′-aminophosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkylphosphonates Thionoalkylphosphotriesters, selenophosphates and borano-phosphates with conventional 3'-5 'linkages, their 2'-5' linkage analogs, and one or more internucleotide linkages from 3 ' 3 ', 5' to 5 'or 2' to 2 'bond With reversed polarity. Preferred oligonucleotides with reversed polarity are single 3 'to 3' linkages to the most 3 'internucleotide linkages, ie a single inverted nucleoside residue (nucleobase) that can be abasic. Or have a hydroxyl group instead). Various salts, mixed salts and free acid forms are also included. Representative US patents that teach the preparation of phosphorus-containing linkages include, but are not limited to, US Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5455939; 5455296; 5455233; 5466677; 5476925; 5519126; 5536821; 5541306; 5550111; 5563253; 5571799; 5587361; 5194599; 5555555;

Suitable modified oligonucleotide backbones that do not contain a phosphorus atom are short chain alkyl or cycloalkyl internucleoside bonds, mixed heteroatoms and alkyl or cycloalkyl internucleoside bonds, or one or more short heteroatoms or heterocyclic nucleosides. It has a skeleton formed by bonding. These have morpholino linkages (partially formed from the sugar portion of the nucleoside); siloxane skeletons; sulfides, sulfoxides and sulfone skeletons; formacetyl and thioformacetyl skeletons; methyleneformacetyl and thioformacetyl skeletons; Riboasechiru skeleton; alkene containing backbones; sulfamate backbones; Mechiren'imino and methylene hydrazino skeleton; sulfonate and sulfonamide backbones; amide backbones; and mixed N, O, include others having S and CH ₂ component parts. Representative U.S. patents that teach the preparation of such oligonucleotides include, but are not limited to, U.S. Patents 5034506; 5166315; 5185444; 5470967; 54896777; 5541307; 5561225; 5596086; 5602240; 5610289; 5602240; 568046; 5610289; 5618704; 5632070; 563312; 56633360;
In other suitable antisense oligonucleotides, both the sugar and the internucleoside linkage, ie the backbone of the nucleotide unit, are replaced with new groups. The base unit is maintained for hybridization with the appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar skeleton of the oligonucleotide is replaced with an amide-containing skeleton, in particular an aminoethylglycine skeleton. The nucleobase is retained and bound directly or indirectly to the aza nitrogen atom of the backbone amide moiety. Representative US patents that teach the preparation of PNA compounds include, but are not limited to, US 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., Science, 1991, 254, 1497-1500.
Suitable antisense oligonucleotides are phosphorothioate and / or heteroatom backbones, particularly —CH ₂ —NH—O—CH ₂ —, —CH ₂ —N (CH ₃ ) —O—CH ₂ — [methylene ( Known as methylimino) or MMI skeleton], —CH ₂ —O—N (CH ₃ ) —CH ₂ —, —CH ₂ —N (CH ₃ ) —N (CH ₃ ) —CH ₂ —, and see above -O—N (CH ₃ ) —CH ₂ —CH ₂ — [wherein the natural phosphodiester backbone is represented as —O—P—O—CH ₂ —] and above described in US Pat. No. 5,489,677 In US Pat. No. 5,602,240, which is referred to in US Pat. Also preferred are antisense oligonucleotides having a morpholino backbone structure of US Pat. No. 5,034,506 referenced above.

Modified oligonucleotides can also include one or more substituted sugar moieties. Suitable oligonucleotides include 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; Including S-alkynyl or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl and alkynyl can be substituted or unsubstituted C ₁ -C ₁₀ alkyl or C ₂ -C ₁₀ alkenyl and alkynyl. Particularly preferred are O [(CH ₂ ) _n O] _m CH ₃ , O (CH ₂ ) _n OCH ₃ , O (CH ₂ ) _n NH ₂ , O (CH ₂ ) _n CH ₃ , O (CH ₂ ) _n ONH ₂ , and O (CH ₂ ) _n ON [(CH ₂ ) _n CH ₃ ] ₂ , where n and m are from 1 to about 10. Other suitable antisense oligonucleotides include one of the following at the 2 ′ position: C ₁ -C ₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH , SCH ₃ , OCN, Cl, Br, CN, CF ₃ , OCF ₃ , SOCH ₃ , SO ₂ CH ₃ , ONO ₂ , NO ₂ , N ₃ , NH ₂ , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino , Polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups that improve the pharmacokinetic properties of oligonucleotides, groups that improve the pharmacological properties of oligonucleotides, and others with similar properties Substituents. A preferred modification includes 2'-methoxyethoxy (2'-O- (2- methoxyethyl) or also known as _{2'-MOE-2'OCH 2 CH} 2 OCH 3) (Martin , etc., Helv. Chim. Acta., 1995, 78, 486-504), that is, it contains an alkoxyalkoxy group. Further suitable modifications are 2′-dimethylaminooxyethoxy, ie O (CH ₂ ) ₂ ON (CH ₃ ) ₂ groups, also known as 2′-DMAOE, as described in the examples below. And 2′-dimethylaminoethoxyethoxy (also known as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), ie 2′-O—CH ₂ —O—CH ₂ —N (CH ₂ ) including.
Further suitable modifications include locked nucleic acids (Locked Nucleic Acids: LNAs) in which the 2′-hydroxyl group is attached to the 3 ′ or 4 ′ carbon atom of the sugar ring to form a bicyclic sugar moiety. The bond is preferably a methylene (—CH ₂ —) _n group bridging a 2 ′ oxygen atom where n is 1 or 2 and a 4 ′ carbon atom. LNAs and their methods of manufacture are described in WO 98/39352 and WO 99/14226.
Other suitable modifications are 2'-methoxy (2'-O-CH ₃ ), 2'-aminopropoxy (2'-OCH ₂ CH ₂ CH ₂ NH ₂ ), 2'-allyl (2'-CH _2- CH = CH _2), including 2'-O- allyl _{(2'-O-CH 2 -CH} = CH 2) and 2'-fluoro and (2'-F). The 2'-modification can be in the arabino (up) position or ribo (down) position. A preferred 2'-arabino modification is 2'-F. Similar modifications can also be made at other positions of the oligonucleotide, particularly the 3 ′ position of the sugar of the 3 ′ terminal nucleotide or the 5 ′ position of the 2′-5 ′ linked oligonucleotide and the 5 ′ terminal nucleotide. Oligonucleotides can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5591722, 5597909; 5610300; 5627053; 5639873; 5646265; 5658873; 5670633; 5792747; and 5700920, each of which is incorporated herein by reference in its entirety.

Oligonucleotides can also include modifications or substitutions of nucleobases (often referred to in the art as simply “bases”). As used herein, “unmodified” or “natural” nucleobases include purine bases adenine (A) and guanine (G) and pyrimidine bases thymine (T), cytosine (C) and uracil (U). included. 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 adenines. And alkyl derivatives of guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-proponyl (—C≡C—CH ₃ or CH _2- C≡CH) uracil and alkyl derivatives of cytosine and other pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8 -Thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, 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 are included. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido [5,4-b] [1,4] benzoxazin-2 (3H) -one), phenothiazine cytidine (1H-pyrimido [5,4-b] [1,4] benzothiazin-2 (3H) -one), G-clamps such as substituted phenoxazine cytidines (eg 9- (2-aminoethoxy) -H-pyrimido [5,4- b] [1,4] benzoxazin-2 (3H) -one), carbazole cytidine (2H-pyrimido [4,5-b] indol-2-one), pyridoindole cytidine (H-pyrido [3 ′, 2 ': 4,5] pyrrolo [2,3-d] pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Additional nucleobases are those disclosed in US Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, Ed. John Wiley & Sons, 1990, and These include those disclosed in Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. . 5-Methylcytosine substitution is known to increase nucleic acid double stability by 0.6-1.2 ° C (Sanghvi et al., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp.276- 278), more particularly when combined with 2'-O-methoxyethyl sugar modification, is a suitable base substitution. Representative US patents that teach the production of modified nucleobases include, but are not limited to, US Pat. No. 3,687,808; and US Pat. Nos. 4,845,205; 5130302; 5134066; 5175273; 5367066; 5432272; 5457187; 5459255; 5484908; 5525711; 5552540; 5574469; 5594121; 556091; 5614617; 5645985; 5830653; 576588; 6005096; 5681941 and 5750692, each of which is incorporated herein by reference.

Other modifications of antisense oligonucleotides chemically attach one or more moieties or conjugates that enhance the activity, cell distribution or cellular uptake of the oligonucleotide to the oligonucleotide. The compounds of the present invention may contain a conjugate group covalently linked to a functional group such as a primary or secondary hydroxyl group. The conjugate groups of the present invention include interventions (intercalators), reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacological properties of oligomers, and enhance the pharmacokinetic properties of oligomers. A group is included. Typical conjugate groups include cholesterol, lipids, cationic lipids, phospholipids, cationic phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes . Groups that enhance pharmacological properties in the context of this invention include groups that improve oligomer uptake, enhance the resistance of the oligomer to degradation, and / or reinforce sequence-specific hybridization with RNA. Groups that enhance pharmacokinetic properties in the context of this invention include groups that improve oligomer uptake, dispersion, metabolism or excretion. Although not limited to the conjugate moiety, lipid molecules such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1063), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660-306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison -Behmoaras et al., EMBOJ., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-
330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or octadecylamine Or a hexylamino-carbonyl-oxycholesterol moiety. The oligonucleotides of the present invention are also active drug substances such as aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-planoprofen, carprofen, dansylsarcosine, 2,3, It can be conjugated to 5-triiodobenzoic acid, flufenamic acid, folinic acid, benzothiazide, chlorothiazide, diazepine, indomethicin, barbiturate, cephalosporin, sulfa drugs, antidiabetics, antibacterials or antibiotics. Oligonucleotide-drug conjugates and methods for their preparation are described in US patent application Ser. No. 09/334130 (filed Jun. 15, 1999) and US Pat. Nos. 4,828,794; ;
511263; 5241136; 5082830; 5112963; 5214136; 5214136; 524450; 5254469; 5258506; 5252536; 5272250; 5292873; 5317098; 5371241; 5559526; 5597696; 5599923; 5599928 and 5688941, each of which is incorporated herein by reference.

It is not necessary to uniformly modify all positions of a given compound, and indeed more than one of the above modifications can be introduced into a single compound or even to a single nucleoside within an oligonucleotide. The present invention also includes antisense compounds that are chimeric compounds. A “chimeric” antisense compound or “chimera” in the context of the present invention is an antisense comprising two or more chemically distinct regions each consisting of at least one monomer unit, ie, in the case of oligonucleotide compounds, nucleotides. Compounds, especially oligonucleotides. These oligonucleotides typically have at least one oligonucleotide modified to give the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity for the target nucleic acid. Including the region. Additional regions of the oligonucleotide can serve as substrates for enzymes capable of cleaving RNA: DNA or RNA: RNA hybrids. For example, RNase H is a cellular endonuclease that cleaves RNA strands of RNA: DNA duplexes. Thus, activation of RNase H results in cleavage of the RNA target, greatly improving the effectiveness of oligonucleotide inhibition of gene expression. Thus, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used compared to phosphorothioate deoxyoligonucleotides that hybridize to the same target region. The chimeric antisense compound of the present invention may be formed as a composite structure of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and / or oligonucleotide mimetics as described above. Suitable chimeric antisense oligonucleotides have at least one 2 ′ modified sugar (preferably 2′-O— (CH ₂ ) ₂ —O—CH ₃ ) and RNase H activity at the 3 ′ end to confer nuclease resistance A region with at least 4 adjacent 2′-H sugars to confer. Such compounds are also referred to in the art as hybrids or gapmers. Suitable gapmers are 3 ′ and 5 ′ terminal 2 ′ modified sugars (preferably 2′-O— (CH ₂ ) ₂ —O—, separated by at least one region with at least 4 adjacent 2′-H sugars. CH ₃ ) region, preferably including a phosphorothioate backbone bond. Representative US patents that teach the preparation of such hybrid structures include, but are not limited to, US Pat. Nos. 5013830; 5149797; 5220007; 5256775; 5366878; And 5700922, each of which is incorporated herein by reference.
Antisense compounds used in the present invention can be conveniently and routinely produced by well-known techniques of solid phase synthesis. Equipment for such synthesis is sold by several manufacturers including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be used. It is well known to use similar techniques to prepare oligonucleotides such as phosphorothioates and alkylated derivatives. The compounds of the present invention may also be mixed with other molecules, molecular structures or compound mixtures to assist in uptake, dispersion and / or absorption, such as liposomes, receptor targeting molecules, oral, rectal, topical application or other formulations. May be encapsulated, conjugated, or otherwise combined. Representative US patents that teach the preparation of formulations that assist in such uptake, dispersion and / or absorption include, but are not limited to, US Pat. Nos. 5108921; 5354844; 5416016; 5459127; 5521291; 5543158; 5549932; 5583020; 5591721; 4426330; 4535499; 5013556; 5108921; 5213804; 5227170; 5264221; 5356633; 5395619; 5416016; 5417978; Each of these is incorporated here by reference.

Other examples of sense or antisense oligonucleotides include organic moieties, such as those described in WO 90/10048, and other moieties that improve the affinity of the oligonucleotide for the target nucleic acid sequence, such as poly- Includes oligonucleotides covalently linked to (L-lysine). Furthermore, an insertion agent such as ellipticine and an alkylating agent or a metal complex may be bound to a sense or antisense oligonucleotide to modify the binding specificity of the antisense or sense oligonucleotide to the target nucleotide sequence.
The antisense or sense oligonucleotide comprises the target nucleic acid sequence by any gene transformation method including, for example, CaPO ₄ -mediated DNA transfection, electroporation, or by using a gene transformation vector such as Epstein-Barr virus. Introduced into cells. In a preferred method, the antisense or sense oligonucleotide is inserted into a suitable retroviral vector. A cell containing the target nucleic acid sequence is contacted with the recombinant retroviral vector in vivo or ex vivo. Suitable retroviral vectors include, but are not limited to, those derived from the murine retrovirus M-MuLV, N2 (retrovirus derived from M-MuLV), or double named DCT5A, DCT5B and DCT5C. Contains a copy vector (see International Publication No. 90/13641).

Sense or antisense oligonucleotides may also be introduced into cells containing the target nucleotide sequence by formation of a complex with a ligand binding molecule, as described in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, complex formation of the ligand binding molecule substantially reduces the ability of the ligand binding molecule to bind to its corresponding molecule or receptor or to prevent entry of a sense or antisense oligonucleotide or complex thereof into the cell. Does not interfere.
Alternatively, sense or antisense oligonucleotides may be introduced into cells containing the target nucleic acid sequence by formation of oligonucleotide-lipid complexes as described in WO 90/10448. The sense or antisense oligonucleotide-lipid complex is preferably degraded intracellularly by endogenous lipase.

Antisense or sense RNA or DNA molecules are usually 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, 30, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 nucleotides long, the term “about” in this context is the reference nucleotide sequence length plus or minus 10% of the reference length means.
Probes can also be used in PCR techniques to create a pool of sequences for identification of closely related TAT coding sequences.
The nucleotide sequence encoding TAT can also be used to create hybridization probes for mapping genes encoding the TAT and for gene analysis of individuals with genetic disorders. The nucleotide sequences provided herein can be mapped to specific regions of chromosomes and chromosomes using well-known techniques such as in situ hybridization, linkage analysis to known chromosomal markers, and hybridization screening in libraries. .

If the coding sequence of TAT encodes a protein that binds to another protein (eg, when TAT is a receptor), TAT can be used in assays to identify other proteins or molecules involved in binding interactions. Can be used. By such methods, inhibitors of receptor / ligand binding interactions can be identified. In addition, proteins included in such binding interactions can be used for screening peptides or small molecule inhibitors or agonists of binding interactions. Receptor TAT can also be used to isolate related ligands. Screening assays may be designed to find lead compounds that mimic the biological activity of native TAT or the receptor for TAT. Such screening assays include assays that can perform high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates. Small molecules considered include synthetic organic or inorganic compounds. Assays are performed in a variety of formats, including protein-protein binding assays, biological screening assays, immunoassays and cell-based assays that are well characterized in the art.
Also, nucleic acids encoding TAT or modified forms thereof can be used to produce either transgenic animals or “knockout” animals, which are useful for the development and screening of therapeutically useful reagents. A transgenic animal (eg, a mouse or rat) is an animal having cells that contain the transgene introduced into the animal or its ancestors before birth, eg, at the embryonic stage. A transgene is DNA that is integrated into the genome of a cell in which a transgenic animal develops. In one embodiment, the cDNA encoding TAT is a genomic DNA encoding TAT based on the genomic sequence and established techniques used to generate transgenic animals comprising cells that express DNA encoding TAT. Can be used to clone. Methods for producing transgenic animals, particularly mice or rats, are routine in the art and are described, for example, in US Pat. Nos. 4,736,866 and 4,487,0009. Typically, specific cells are targeted for introduction of the TAT transgene with a tissue-specific enhancer. Transgenic animals containing a copy of a transgene encoding TAT introduced into the germ line of the animal at the embryonic stage can be used to examine the effects of increased expression of DNA encoding TAT. Such animals can be used, for example, as tester animals for reagents that would confer protection against pathological conditions associated with their overexpression. In this aspect of the invention, if an animal is treated with a reagent and the incidence of the pathological condition is low compared to an untreated animal carrying the transgene, then a therapeutic treatment for the pathological condition is indicated. It is.

Alternatively, a non-human homologue of TAT is a defect that encodes TAT by homologous recombination between a modified genomic DNA encoding TAT introduced into an animal embryonic cell and an endogenous gene encoding TAT. Alternatively, it can be used to create TAT “knock-out” animals with altered genes. For example, cDNA encoding TAT can be used for cloning genomic DNA encoding TAT according to established techniques. A portion of the genomic DNA encoding TAT can be deleted or replaced with another gene, such as a gene encoding a selectable marker used to monitor integration. Typically, the vector contains several kilobases of intact flanking DNA (both 5 'and 3' ends) [see, eg, Thomas and Capecchi, Cell, 51: 503 (1987) for homologous recombination vectors. See also]. A vector is introduced into an embryonic stem cell line (eg, by electroporation), and cells in which the introduced DNA is homologously recombined with endogenous DNA are selected [eg, Li et al., Cell, 69: 915 (1992) reference]. The selected cells are then injected into the blastocyst of the animal (eg mouse or rat) to form an aggregate chimera [eg Bradley, Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, edited by EJ Robertson (IRL, Oxford 1987), pp. 113-152]. The chimeric embryo is then transplanted into an appropriate pseudopregnant female nanny to create a “knockout” animal over time. Offspring with DNA homologously recombined into embryonic cells are identified by standard techniques and can be used to breed animals that contain DNA in which all cells of the animal are homologously recombined . Knockout animals are characterized by their ability to defend against certain pathological conditions and progression of those pathological conditions due to a lack of TAT polypeptide.
A nucleic acid encoding a TAT polypeptide can also be used for gene therapy. In gene therapy applications, for example, to replace a defective gene, a gene is introduced into the cell to achieve in vivo synthesis of a therapeutically effective gene product. “Gene therapy” includes both conventional gene therapy in which a continuous effect is achieved by a single treatment and administration of a gene therapy drug including single or repeated administration of therapeutically effective DNA or mRNA. . Antisense RNA and DNA can be used as therapeutic agents to block in vivo expression of certain genes. It has already been shown that short antisense oligonucleotides can be transferred into cells that act as inhibitors, despite low intracellular concentrations due to limited uptake by the cell membrane (Zamecnik et al., Proc. Natl Acad. Sci. USA 83: 4143-4146 [1986]). Oligonucleotides may be modified to facilitate uptake by replacing their negatively charged phosphodiester groups with uncharged groups.

There are various techniques for introducing nucleic acids into living cells. These techniques vary depending on whether the nucleic acid is transferred to the cultured cells in vitro or in vivo in the intended host cell. Suitable techniques for transferring nucleic acids into mammalian cells in vitro include liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, calcium phosphate precipitation, and the like. Currently preferred in vivo gene transfer techniques are transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection (Dzau et al., Trends in Biotechnology 11, 205-210 (1993)). In some situations, it may be desirable to provide a source of nucleic acid with an agent that targets the target cell, such as a cell surface membrane protein or an antibody specific for the target cell, a ligand for a receptor on the target cell. When using liposomes, proteins that bind to cell surface membrane proteins with endocytosis, such as capsid proteins or fragments thereof that are specific for cell types, antibodies to proteins that undergo internalization in the cycle, intracellular localization Proteins that target targeting and improve intracellular half-life are used for targeting and / or promoting uptake. Receptor-mediated endocytosis techniques are described, for example, by Wu et al., J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990). is described. For review of gene generation and gene therapy protocols, see Anderson et al., Science 256, 808-813 (1992).
Nucleic acid molecules encoding the TAT polypeptides or fragments thereof described herein are useful for chromosome identification. In this regard, there are few chromosomal marking reagents based on actual sequence data available, so it is currently necessary to identify new chromosomal markers. Each TAT nucleic acid molecule of the present invention can be used as a chromosomal marker.
In addition, the TAT polypeptides and nucleic acid molecules of the present invention can be used for diagnosis of tissue typing, and the TAT polypeptides of the present invention are compared in one tissue, preferably in normal tissues of the same tissue type, compared to other tissues. And is specifically expressed in diseased tissues. TAT nucleic acid molecules will find use for generating probes for PCR, Northern analysis, Southern analysis and Western analysis.

The invention includes methods of screening compounds to identify those that mimic TAT polypeptides (agonists) or prevent the effects of TAT polypeptides (antagonists). Screening assays for antagonist drug candidates include compounds that bind 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; For example, it is designed to identify compounds, including those that inhibit TAT polypeptide expression from cells. Such screening assays include assays that can perform high-throughput screening of chemical libraries, making them particularly suitable for the identification of small molecule drug candidates.
This assay can be performed in a variety of formats that are well characterized in the art, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays.
All assays for antagonists require the drug candidate to be contacted with the TAT polypeptide encoded by the nucleic acid identified herein under conditions and for a time sufficient for both of these components to interact. Is common.
In binding assays, the interaction is binding and the complex formed is isolated or detected in the reaction mixture. In a particular embodiment, the TAT polypeptide or drug candidate encoded by the gene identified herein is immobilized to a solid phase, such as a microtiter plate, by covalent or non-covalent bonding. Non-covalent binding is generally achieved by coating a solid surface with a solution of TAT polypeptide and drying. Alternatively, an immobilized antibody specific for the TAT polypeptide to be immobilized, such as a monoclonal antibody, can be used to adhere to the solid surface. The assay is performed by adding a non-immobilized component, which may be labeled with a detectable label, to a coated surface containing an immobilized component, such as an anchoring component. When the reaction is completed, unreacted components are removed, for example, by washing, and the complex adhered to the solid surface is detected. If the first non-immobilized component has a detectable label, detection of the label immobilized on the surface indicates that complex formation has occurred. If the initial non-immobilized component does not have a label, complex formation can be detected, for example, by use of a labeled antibody that specifically binds to the immobilized complex.

  If a candidate compound interacts but does not bind to a particular TAT polypeptide encoded by the gene identified herein, the interaction with that polypeptide is a well-known method for detecting protein-protein interactions. Can be assayed. Such assays include traditional techniques such as cross-linking, co-immunoprecipitation, and co-purification through gradient or chromatographic columns. In addition, protein-protein interactions are described in Fields and collaborators [Fiels and Song, et al., As disclosed in Chevray and Nathans Proc. Natl. Acad. Sci. USA, 89: 5789-5793 (1991). Nature (London), 340,: 245-246 (1989); Chien et al., Proc. Natl. Acad. Sci. USA, 88: 9578-9582 (1991)]. Can be monitored. Many transcriptional activators, such as yeast GAL4, consist of two physically distinct modular domains, one that acts as a DNA binding domain and the other that functions as a transcriptional activation domain. The yeast expression system described in the previous literature (commonly referred to as the “2-hybrid system”) takes advantage of this property and uses two hybrid proteins while the target protein is the DNA binding domain of GAL4. On the other hand, the candidate activation protein is fused to the activation domain. Expression of the GAL1-lacZ reporter gene under the control of a GAL4-activated promoter depends on reconstitution of GAL4 activity through protein-protein interactions. Colonies containing interacting polypeptides are detected with a chromogenic substance for β-galactosidase. A complete kit (MATCHMAKER®) for identifying protein-protein interactions between two specific proteins using the two-hybrid technology is commercially available from Clontech. This system can also be extended to the mapping of protein domains involved in a particular protein interaction as well as the identification of amino acid residues important for this interaction.

A compound that inhibits the interaction of the gene encoding the TAT polypeptide identified herein with other intracellular or extracellular components can be tested as follows: Usually the reaction mixture consists of the gene product and the intracellular or The extracellular component is prepared under conditions and times that allow the two products to interact and bind. In order to test the ability of a candidate compound to inhibit binding, the reaction is performed in the absence and presence of the test compound. In addition, a placebo may be added to the third reaction mixture to provide a positive control. The binding (complex formation) between the test compound and the intracellular or extracellular component present in the mixture is monitored as described above. Formation of the complex in the control reaction but not the reaction mixture containing the test compound indicates that the test compound inhibits the interaction between the test compound and its reaction partner.
To assay for an antagonist, a TAT polypeptide may be added to a cell along with a compound that is screened for a particular activity, and the compound's ability to inhibit the activity of interest in the presence of the TAT polypeptide is Are antagonists of the TAT polypeptide. Alternatively, antagonists may be detected by binding the TAT polypeptide and potential antagonist with membrane-bound TAT polypeptide receptor or recombinant receptor under conditions suitable for competitive inhibition assays. The TAT polypeptide can be labeled, such as by radioactivity, and the number of TAT polypeptide molecules bound to the receptor can be used to determine the effectiveness of the potential antagonist. The gene encoding the receptor can be identified by a number of methods known to those skilled in the art, such as ligand panning and FACS sorting. Coligan et al., Current Protocols in Immun., 1 (2): Chapter 5 (1991). Preferably expression cloning is used, where polyadenylated RNA is prepared from cells reactive to TAT polypeptide, and cDNA libraries generated from this RNA are distributed into pools and reacted to COS cells or other TAT polypeptides. Used for transfection of non-sex cells. Transfected cells grown on glass slides are exposed to labeled TAT polypeptide. The TAT polypeptide can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase. After fixation and incubation, the slides are subjected to autoradiographic analysis. Positive pools are identified, subpools are prepared and retransfected using interactive subpooling and rescreening methods, and finally a single clone encoding a putative receptor is generated.

As an alternative method of receptor identification, the labeled TAT polypeptide can be photoaffinity bound to a cell membrane or extract preparation that expresses the receptor molecule. The cross-linked material is separated by PAGE and exposed to X-ray film. The labeled complex containing the receptor may be excited, separated into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from microsequencing is used to design a set of degenerate oligonucleotide probes that screens a cDNA library that identifies the gene encoding the putative receptor.
In other assays for antagonists, mammalian cells or membrane preparations expressing the receptor are incubated with labeled TAT polypeptide in the presence of the candidate compound. The ability of the compound to promote or block this interaction is then measured.
More specific examples of potential antagonists include oligonucleotides that bind to fusions of immunoglobulins and TAT polypeptides, particularly but not limited to poly- and monoclonal antibodies and antibody fragments, single chain antibodies, anti- It includes idiotype antibodies, and chimeric or humanized forms of these antibodies or fragments, as well as antibodies, including human antibodies and antibody fragments. Alternatively, a potential antagonist may be a closely related protein, eg, a mutant form of a TAT polypeptide that recognizes but does not have an effect on the receptor and thus competitively inhibits the action of the TAT polypeptide.
Other potential TAT polypeptide antagonists are antisense RNA or DNA constructs prepared using antisense technology; for example, antisense RNA or DNA molecules can hybridize to target mRNAs for protein translation. It acts to directly block the translation of mRNA by interfering. Antisense technology can be used to control gene expression through triple helix formation or antisense DNA or RNA, both of which are based on the binding of polynucleotides to DNA or RNA. For example, the 5 ′ coding portion of the polynucleotide sequence encoding the mature TAT polypeptide herein is used in the design of antisense RNA oligonucleotides of about 10 to 40 base pairs in length. DNA oligonucleotides are designed to be complementary to a region of the gene involved in transcription (Triplex-Lee et al., Nucl, Acid Res., 6: 3073 (1979); Cooney et al., Science, 241: 456 ( 1988); Dervan et al., Science, 251: 1360 (1991)), thereby preventing transcription and production of TAT polypeptides. Antisense RNA oligonucleotides hybridize to mRNA in vivo to prevent translation of mRNA molecules into TAT polypeptides (Antisense-Okano, Neurochem., 56: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression ( CRC Press: Boca Raton, FL, 1988)). The oligonucleotides described above can also be transported into cells to express antisense RNA or DNA in vivo to inhibit TAT polypeptide production. When antisense DNA is used, oligodeoxyribonucleotides derived from the translation initiation site, eg, between -10 and +10 positions of the target gene nucleotide sequence, are preferred.

Potential antagonists include small molecules that bind to the active site, receptor binding site, or growth factor or other related binding site of a 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-peptide organic or inorganic compounds.
Ribozymes are enzymatic RNA molecules that can catalyze the specific cleavage of RNA. Ribozymes act by sequence-specific hybridization to complementary target RNA, followed by nucleotide strand breaks. Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques. For further details see, for example, Rossi, Current Biology 4: 469-471 (1994) and PCT Gazette, International Publication 97/33551 (published September 18, 1997).
Nucleic acid molecules in triple helix formation used for transcription inhibition are single-stranded and composed of deoxynucleotides. The basic composition of these oligonucleotides is designed to promote triple helix formation via Hoogsteen base pairing rules, which generally require a fairly large extension of purines or pyrimidines on one strand of the duplex. To do. For further details see, for example, the above-mentioned PCT publication, WO 97/33551.
These small molecules can be identified by any one or more of the screening assays discussed above and / or by any other screening technique well known to those skilled in the art.

An isolated TAT polypeptide-encoding nucleic acid is used herein to recombinantly produce a TAT polypeptide using techniques well known in the art as described herein. Is possible. The generated TAT polypeptide can then be used to generate anti-TAT antibodies using techniques well known in the art as described herein.
Antibodies that specifically bind to the TAT polypeptide identified herein, as well as other molecules identified by the screening assays disclosed above, may be administered in the form of pharmaceutical compositions for the treatment of various diseases. Can do.
When the TAT polypeptide is intracellular and the whole antibody is used as an inhibitor, an uptake antibody is preferred. However, lipofection or liposomes can also be used to deliver antibodies, or antibody fragments, to cells. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, peptide molecules that retain the ability to bind to a target protein sequence can be designed based on the variable region sequence of the antibody. Such peptides can be synthesized chemically and / or produced by recombinant DNA techniques. See, for example, Marasco et al., Proc. Natl. Acad. Sci. USA 90, 7889-7893 (1993).
The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively or in addition, the composition may include an agent that enhances its function, such as a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitory agent. These molecules are suitably present in combination in amounts that are effective for the purpose intended.
The following examples are provided for purposes of illustration only and are not intended to limit the scope of the invention in any way.
All patents and references cited herein are hereby incorporated by reference in their entirety.

  Commercially available reagents referred to in the examples were used according to manufacturer's instructions unless otherwise indicated. The cell source identified by the ATCC accession number in the following examples and throughout the specification is American Type Culture Collection, Manassas, Virginia.

Example 1: Identification of tumor-specific amplified chromosomal regions More than 4 million EST sequences of various normal and diseased tissues were obtained from GenBank and NCBI databases. Excluding ESTs derived from the standardized library or subtractive library, EST sequences derived from normal tissues were about 1.2 million, and EST sequences derived from tumor tissues were 1.7 million. In order to quantify the expression level of each known or novel gene in the target tissue, the normalized EST abundance was calculated and expressed as a digital expression level (DEL = EST abundance per million EST) . Previous studies have shown that there is a linear correlation between standardized mRNA levels measured by real-time PCR analysis and DEL.
Here, according to the genome coordinates, the DEL values of each gene were plotted along each chromosome, and a transcriptome map was created for both normal tissue and tumor tissue in each tissue. In some specific regions, when compared to normal tissue, both genes located close to each other in tumor tissue appeared to be up-regulated. An example is the 1.8 MB region corresponding to chromosome 17q11 where 33 genes showed higher expression levels in breast tumor samples than in normal breast tissue (data not shown). This region containing the ERBB2 gene is known to be amplified in 30% of breast cancer patients and roughly corresponds to the size of a known breast tumor amplicon. Many other such tumor-related regions can be visually identified and are referred to herein as RITE (Tumor Increased Region).

To determine whether RITE is just an extreme example or a common feature of chromosomes, RITE was identified in 10 different tumors by a computational approach. For each gene, a ratio Z test between the two was performed using EST distribution data in normal and tumor samples, and the resulting Z score was used to determine the extent of information control in the tumor. To systematically identify RITE and reduce noise due to individual genes, the human genome is scanned in a 500-KB window with a 100-KB offset, and an average Z score (TMZ) is calculated for each window of genes. did. The sensory index was then empirically devised to obtain a continuous window with TMZ above the genome average. Scanning genomic regions with chest data revealed many genomic regions with unusually high sensory indices (data not shown). When the sensory index cut-off was empirically set to 3, 67 RITEs were found in breast tumors when compared to normal breast tissue. Since EST clones were obtained from multiple separate tissue libraries, these RITEs represent the entire population of breast cancer patients rather than individual patients. Next, the edges of each RITE were cleaned by trimming each side of the gene whose Z score was less than 4 times the genome average. When these putative RITEs were examined visually, a cluster of genes whose expression increased in one type of tumor was confirmed.
To address the concern that the identified RITE may simply be an accidental clustering of individual tumor-specific genes, a series of permutations analyzes the chromosomal coordinates of each gene with other available coordinates. Randomly shuffled. Ten thousand breast datasets with different orders were analyzed and the number of RITEs recorded. With the order changed, the average number of RITEs was 5.4 and the standard deviation was 1.2. This is significantly lower than that detected under natural conditions. The same analysis was performed on the other nine tissues, namely brain, large intestine, kidney, liver, lung, pancreas, prostate, stomach, and testis. In all tissues, the number of RITEs observed under natural conditions was at least 25 times the standard deviation of the average number of permutations. This indicated that the clustering of genes up-regulated by tumors was not the result of random differences in gene distribution on the genome.

The presence of these RITEs may be due to genomic changes or transcriptional control. To examine the relationship between RITE and known amplicons, cytogenetic markers were searched for each RITE and compared with known data. Identification of amplicons in tumors usually requires laborious approaches such as fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH). In brain, breast and lung tissues with the highest amount of CGH data, approximately 50% of RITE corresponds to amplicons identified by CGH analysis, which are due to genomic DNA amplification in the tumor. It was shown that. Another mechanism for the presence of RITE requires the regulation of gene expression at the transcriptional level. Although eukaryotic genes are known to be individually controlled, the eukaryotic genome has accumulated evidence for large domains of genes that are similarly expressed. Preliminary analysis of the upstream DNA sequence failed to identify promoter elements common to all genes in the RITE region (data not shown). However, these regions could be organized in some form of chromosomal scaffolds that can regulate tumor expression.
In summary, using computational approaches and available public data sources, we were able to rapidly identify non-random clusters of genes that were up-regulated in tumors. Many of the RITEs identified here appear to be the result of allelic imbalance in tumor cells. CGH based on DNA microarrays has recently been applied to high-throughput detection of chromosomal imbalances, but results are limited due to the need for large-scale microarray experiments using samples from various cancer patients In many cases, the resolution is limited by the density of the genomic DNA array. While microarray-based CGH analysis is effective in finding tumor-specific amplicons, EST-based transcriptome analysis is advantageous for identifying potential amplicons present in many cancer types and patients . The availability of these RITE regions allowed us to study the mechanism of abnormal DNA amplification in tumors and identify novel target genes for therapeutic treatment.

The results of these analyzes are shown in attached appendices BJ. In each of the appendixes B to J, it is shown on the first page of the appendix which tissue type the indicated result relates to. In any appendix, the tumor-specific chromosomal region (RITE) of the tumor tissue type is bold, and the name or number of the chromosome (eg “chr1”) starts the amplified region on the specific chromosome. It is indicated by a symbol with a coordinate number for each position and end position. The coordinate numbers are associated with the release of Build 29 of the NCBI human genome sequence (May 2002). For example, RITE indicated by “chr1.147531064.149694137” is an amplification region of chromosome 1, and its start position and stop position are 14731064 and 149694137 found in the NCBI human genome sequence Build29 release (May 2002), respectively. is there. A list of drawings showing the cDNA sequences expressed from genes located within a particular amplified chromosomal region is shown below the position of each RITE. The contents shown in the appendix are shown below.
Appendix B: 14 RITEs identified in brain tumor tissue (overexpressed cDNA sequence shown in Figure 1-682 (SEQ ID NO: 1-682))
Appendix C: 22 RITEs identified in breast tumor tissue (overexpressed cDNA sequence shown in FIGS. 683-1007 (SEQ ID NO: 683-1007))
Appendix D: Four RITEs identified in kidney tumor tissue (overexpressed cDNA sequence shown in FIGS. 1008-1162 (SEQ ID NO: 1008-1162))
Appendix E: 50 RITEs identified in liver tumor tissue (overexpressed cDNA sequence shown in FIGS. 1163-1880 (SEQ ID NO: 1163-1880))
Appendix F: 41 RITEs identified in lung tumor tissue (overexpressed cDNA sequence shown in FIGS. 1881-3158 (SEQ ID NO: 1881-3158))
Appendix G: 40 RITEs identified in pancreatic tumor tissue (overexpressed cDNA sequence shown in FIGS. 3159-4326 (SEQ ID NO: 3159-4326))
Appendix H: 12 RITEs identified in prostate tumor tissue (overexpressed cDNA sequence shown in FIGS. 4327-4457 (SEQ ID NO: 4327-4457))
Appendix I: Three RITEs identified in gastric tumor tissue (overexpressed cDNA sequence shown in FIGS. 4458-4516 (SEQ ID NO: 4458-4516))
Appendix J: 20 RITEs identified in testicular tumor tissue (overexpressed cDNA sequence shown in FIGS. 4517-4622 (SEQ ID NO: 4517-4622))

Method:
Human ESTs and mRNA Collection The collected human ESTs were NCBI's human SAGE sequences in addition to the entire GenBank EST division sequence. Using the quality information contained in the GenBank flat file, the poor quality sequences were deleted. Tissue and histology markers were added using NCI / CGAP library information (http://cgap.nci.nih.gov/Tissues/). The collected human mRNA (46515 transcript) is an mRNA sequence mapped on the human genome constructed from NCBI (May 2002 release 29), and is specified in the GenBank style flat file for each chromosome. . The position of each mRNA in the chromosome was determined using the position information contained in the flat file and information on the contig order. Pseudogenes, mRNA-labeled “ALU subfamily analogs”, and mRNAs with abnormal location specification were excluded. Of 24491 genes with EST coverage, 24388 non-overlapping genes were selected for expression characterization.

Calculation of expression levels based on ESTs The sequence alignment program BLAST19 was used to identify all EST sequences matching at least 60 base pairs with greater than 98% identity to the gene of interest. Matched ESTs were grouped by source library and further organized by library organization type. For each tissue category, the total number of matched EST clones was divided by the library size and multiplied by 1,000,000 to calculate a DEL (digital expression level) value. A typical EST library contains only thousands of clone sequences, but by integrating overlapping libraries of the same tissue type, it was possible to create an information-rich composite data source representing rare genes. These standard EST abundances were calculated for the normal and tumor tissue groups of each tissue.

Comparison of two ratios with Z test To test whether the ratios of two independent populations are identical, the Z test was applied. For all libraries, the common relative abundance (p) for any gene was calculated by summing the number of gene clones in all libraries and dividing by the total number of clones in all libraries. The relative abundance of the tumor (p1) and normal (p2) libraries was also calculated, respectively. The Z-score on the test statistics was calculated as follows:
z = (p1−p2) ÷ square root of X, where X is p (1−p) [(1 / Nt) + (1 / Nn)].
Where Nt is the total number of EST clones from any tumor tissue and Nn is the total number of EST clones from any normal tissue.

ここで、ｚ_ｉは５００ｋｂｐウィンドウ内の遺伝子のＺスコアであり、
ｍａｘはウィンドウ内の最大Ｚスコアであり、
ｍｉｎはウィンドウ内の最小Ｚスコアであり、
ｎはウィンドウ内の遺伝子の数であり、
感受性指数（Ｓ）は以下の式によって計算される。

ここで、ｍはゲノム平均値（ｇ）より大きいＴＭＺを有する連続ウィンドウのＴＭＺ平均値であり、
ｇは全てのウィンドウのＴＭＺの平均値であり、
ｎはゲノム平均値（ｇ）より大きいＴＭＺを有する連続ウィンドウの数である。
端数処理したウィンドウの平均値がゲノム平均値より小さい場合、Ｓ＝ｍ−ｇである。端数処理した平均値がゲノム平均値より大きい連続ウィンドウを有する領域については、

である。領域内の各ウィンドウは同じＳ値を持つ。 Calculation of Z Score and Sensitivity Index Rounded Average (TMZ) TMZ was calculated using a 500 kbp window moving along the chromosome at 100 kbp intervals and defined as:

Where z _i is the Z score of the gene within the 500 kbp window,
max is the maximum Z score in the window,
min is the minimum Z score in the window,
n is the number of genes in the window,
The sensitivity index (S) is calculated by the following formula.

Where m is the TMZ average value of continuous windows with TMZ greater than the genome average value (g);
g is the average value of TMZ of all windows,
n is the number of consecutive windows with TMZ greater than the genome average (g).
If the average value of the rounded window is smaller than the genome average value, S = m−g. For regions with continuous windows where the rounded average is greater than the genome average,

It is. Each window in the region has the same S value.

Example 2: Use of TAT as a hybridization probe The following method describes the use of a nucleotide sequence encoding TAT as a hybridization probe, ie for diagnosing the presence of a tumor in a mammal.
The DNA comprising the full-length or mature TAT coding sequence disclosed herein is homologous DNA in a human tissue cDNA library or a human tissue genomic library (eg, encoding a naturally occurring variant of TAT). It is also used as a probe for screening).
Hybridization and washing of the filter containing any library DNA was performed under the following high stringency conditions. Hybridization of the radiolabeled TAT-inducing probe to the filter consists of 50% formaldehyde, 5 × SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2 × denhard solution, and 20 hours at 42 ° C. in a solution of 10% dextran sulfate. The filter was washed at 42 ° C. in an aqueous solution of 0.1 × SSC and 0.1% SDS.
The DNA having the desired sequence identity with the DNA encoding the full-length native sequence TAT can then be identified using standard methods known in the art.

Example 3: Expression of TAT in E. coli This example illustrates the preparation of an unglycosylated form of TAT by recombinant expression in E. coli.
The DNA sequence encoding TAT was first amplified using selected PCR primers. The primer must have a restriction enzyme site corresponding to the restriction enzyme site of the selected expression vector. Various expression vectors are used. An example of a suitable vector is pBR322 (derived from E. coli; see Bolivar et al., Gene, 2:95 (1977)), which contains genes for ampicillin and tetracycline resistance. The vector is digested with restriction enzymes and dephosphorylated. The PCR amplified sequence is then ligated into a vector. The vector is preferably an antibiotic resistance gene, trp promoter, poly-His leader (including the first six STII codons, poly-His sequence, and enterokinase cleavage site), a TAT coding region, a lambda transcription terminator, and argU Contains genes.
The ligation mixture is then used to transform E. coli selected using the method described by Sambrook et al., Supra. Transformants are identified by their ability to grow on LB plates and then antibiotic resistant clones are selected. Plasmid DNA is isolated and confirmed by restriction analysis and DNA sequence.
Selected clones can be grown overnight in liquid media such as LB broth supplemented with antibiotics. The overnight medium is subsequently used for seeding the large-scale medium. The cells are then grown to the desired optical density, during which the expression promoter is activated.
After several hours of cell culture, collection by centrifugation is possible. Cell pellets obtained by centrifugation were solubilized using various reagents known in the art, and the solubilized TAT protein was then purified using a metal chelation column under conditions that allow the protein to bind tightly. .

TAT may be expressed in E. coli in poly-His (poly-his) tag form using the following procedure. DNA encoding TAT was first amplified using selected PCR primers. Primers provide restriction enzyme sites that correspond to the restriction enzyme sites of the selected expression vector, and efficient and reliable translation initiation, rapid purification with metal chelate columns, and proteolytic removal with enterokinase Includes other useful sequences. The PCR-amplified poly-His tag sequence was then ligated into an expression vector, which was used to transform an E. coli host based on strain 52 (W3110 fuhA (tonA) lon galE rpoHts (htpRts) clpP (lacIq)). Transformants were initially treated with 3-5 O.D. with shaking at 30 ° C. in LB containing 50 mg / ml carbenicillin. D. Grow to 600. The medium was then CRAP medium (3.57 g (NH ₄ ) ₂ SO ₄ , 0.71 g sodium citrate · 2H 2 O, 1.07 g KCl, 5.36 g Difco yeast extract, 5.36 g in 500 mL water. Diluted 50-100 times in Shefield hycase SF and 110 mM MPOS, pH 7.3, mixed with 0.55% (w / v) glucose and 7 mM MgSO ₄ ) and shaken at 30 ° C. Grow for 20-30 hours. A sample was removed and expression was confirmed by SDS-PAGE analysis, and the bulk medium was centrifuged to form a cell pellet. Cell pellets were frozen until purification and refolding.
E. coli paste from 0.5 to 1 L fermentation (6-10 g pellet) was resuspended in 10 volumes (w / v) in 7 M guanidine, 20 mM Tris, pH 8 buffer. Solid sodium sulfate and sodium tetrathionate were added to final concentrations of 0.1 M and 0.02 M, respectively, and the solution was stirred at 4 ° C. overnight. This step resulted in a denatured protein in which all cysteine residues were blocked by sulfation. The solution was concentrated in a Beckman Ultracentrifuge at 40,000 rpm for 30 minutes. The supernatant was diluted with 3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and clarified by filtration through a 0.22 micron filter. The clarified extract was loaded onto a 5 ml Qiagen Ni-NTA metal chelate column equilibrated with metal chelate column buffer. The column was washed with an additional buffer containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The protein was eluted with a buffer containing 250 mM imidazole. Fractions containing the desired protein were pooled and stored at 4 ° C. The protein concentration was estimated by its absorption at 280 nm using the extinction coefficient calculated based on its amino acid sequence.

By gradually diluting the sample into freshly prepared regeneration buffer consisting of 20 mM Tris, pH 8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. The protein was regenerated. The refolding volume was selected so that the final protein concentration was 50-100 microgram / ml. The refolding solution was slowly stirred at 4 ° C. for 12-36 hours. The refolding reaction was stopped by adding TFA at a final concentration of 0.4% (about pH 3). Prior to further purification of the protein, the solution was filtered through a 0.22 micron filter and acetonitrile was added at a final concentration of 2-10%. The renatured protein was chromatographed on a Poros R1 / H reverse phase column, eluting with a 0.1% TFA transfer buffer and a 10-80% acetonitrile gradient. Aliquots of fractions with A280 absorption were analyzed on an SDS polyacrylamide gel and fractions containing homologous regenerative proteins were pooled. In general, most correctly regenerated protein species are eluted at the lowest concentration of acetonitrile because these species are the most dense and their hydrophobic inner surface is shielded from interaction with the reverse phase resin. Aggregated species are usually eluted at higher acetonitrile concentrations. In addition to removing the incorrectly regenerated protein from the desired form, the reverse phase process also removes endotoxin from the sample.
Fractions containing the desired folded TAT polypeptide were pooled and acetonitrile was removed while directing a weak stream of nitrogen to the solution. Proteins were prepared in 20 mM Hepes, pH 6.8 containing 0.14 M sodium chloride and 4% mannitol by gel filtration and sterile filtration on G25 Superfine (Pharmacia) resin equilibrated with dialysis or preparation buffer.
Certain of the TAT polypeptides described herein have been successfully expressed and purified using this method.

Example 4: Expression of TAT in mammalian cells This example illustrates the preparation of a potential glycosylated form of TAT by recombinant expression in mammalian cells.
PRK5 (see EP307247 issued on March 15, 1989) was used as an expression vector. In some cases, TAT DNA is bound to pRK5 with the selected restriction enzyme, and TAT DNA is inserted using a ligation method such as that described by Sambrook et al. The resulting vectors are each called pRK5-TAT.
In one embodiment, the selected host cell can be a 293 cell. Human 293 cells (ATCC CCL 1573) were grown and confluent in tissue culture plates in media such as DMEM supplemented with fetal bovine serum and optionally nutrients and / or antibiotics. About 10 μg of pRK5-TAT DNA is mixed with about 1 μg of DNA encoding the VA RNA gene [Thimmappaya et al., Cell, 31: 543 (1982)] and 500 μl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 MCaCl ₂ was dissolved. To this mixture, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO ₄ was added dropwise, and a precipitate was formed at 25 ° C. for 10 minutes. The precipitate was suspended and added to 293 cells and allowed to settle at 37 ° C. for about 4 hours. The culture medium was aspirated and 2 ml of 20% glycerol in PBS 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 culture medium was removed and replaced with culture medium (alone) or culture medium containing 200 μCi / ml ³⁵ S-cysteine and 200 μCi / ml ³⁵ S-methionine. After 12 hours of incubation, the conditioned medium was collected, concentrated with a spin filter and added to a 15% SDS gel. The treated gel was dried and exposed to the film for a time selected to reveal the presence of TAT polypeptide. The medium containing the transformed cells was further incubated (in serum-free medium) and the medium was tested in the selected bioassay.

In an alternative technique, TAT is transiently introduced into 293 cells using the dextran sulfate method described in Somparyrac et al., Proc. Natl. Acad. Sci., 12: 7575 (1981). 293 cells are grown to maximal density in a spinner flask and 700 μg pRK5-TAT DNA is added. The cells were first concentrated from the spinner flask by centrifugation and washed with PBS. The DNA-dextran precipitate was incubated on the cell pellet for 4 hours. Cells were treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and reintroduced into a spinner flask containing tissue culture medium, 5 μg / ml bovine insulin and 0.1 μg / ml bovine transferrin. After about 4 days, the conditioned medium was centrifuged and filtered to remove cells and cell debris. The sample containing the expressed TAT was then concentrated and purified by selected methods such as dialysis and / or column chromatography.
In other embodiments, TAT can be expressed in CHO cells. pRK5-TAT can be transfected into CHO cells using known reagents such as CaPO ₄ or DEAE- dextran. As described above, the cell culture medium can be incubated and the culture medium can be replaced with a culture medium (alone) or a medium containing a radioactive label such as ³⁵ S-methionine. After determining the presence of the TAT polypeptide, the culture medium may be replaced with a serum-free medium. Preferably, the medium is incubated for about 6 days and then the conditioned medium is collected. The medium containing the expressed TAT can then be concentrated and purified by any selected method.

The epitope tag TAT may also be expressed in the host CHO cell. TAT was subcloned from the pRK5 vector. The subclone insert can then be subjected to PCR and fused to a frame with a selected epitope tag such as a poly-His tag in a baculovirus expression vector. The poly-His tagged TAT insert can then be subcloned into an SV40 derived vector containing a selectable marker such as DHFR for selection of stable clones. Finally, CHO cells were transfected with the SV40 derived vector (as described above). To confirm expression, labeling may be performed as described above. The culture medium containing the expressed poly-His tag TAT can then be concentrated and purified by selected methods such as Ni ²⁺ -chelate affinity chromatography.
TAT can also be expressed in CHO and / or COS cells by transient expression methods or in CHO cells by other stable expression methods.
Stable expression in CHO cells can be performed using the following method. IgG constructs (immunoadhesins) and / or poly-His in which proteins are fused to IgG1 constant region sequences in which the coding sequence (eg, extracellular domain) of each protein includes a hinge, CH2 and CH2 domains. Expressed as a tag form.
Following 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, Unit 3.16, John Wiley and Sons (1997). The CHO expression vector has restriction sites compatible with 5 ′ and 3 ′ of the DNA of interest, and is prepared so that the cDNA can be conveniently shuttled. The vector was expressed in CHO cells as described in Lucas et al., Nucl. Acids Res. 24: 9, 1774-1779 (1996) and used for expression of the target cDNA and dihydrofolate reductase (DHFR). The SV40 early promoter / enhancer is used for control. DHFR expression allows selection for stable maintenance of the plasmid following transfection.
Twelve micrograms of the desired plasmid DNA is transferred to approximately 10 million CHO cells using the commercially available transfection reagents Superfect® (Quiagen), Dosper® and Fugene® (Boehringer Mannheim). Introduced. Cells were grown as described in Lucas et al. Approximately 3 × 10 ⁷ cells were frozen in ampoules for further growth and production as described below.

An ampoule containing the plasmid DNA was placed in a water bath, thawed, and mixed by vortexing. The contents were pipetted into a centrifuge tube containing 10 mL of medium and centrifuged at 1000 rpm for 5 minutes. The supernatant was aspirated and the cells were suspended in 10 mL selective medium (0.2 μm filtered PS20, 5% 0.2 μm diafiltered fetal bovine serum added). The cells were then divided into 100 ml spinners containing 90 mL selective medium. After 1-2 days, the cells were transferred to a 250 mL spinner filled with 150 mL selective growth medium and incubated at 37 ° C. After a further 2-3 days, 250 mL, 500 mL and 2000 mL spinners were seeded at 3 × 10 ⁵ cells / mL. The cell medium was replaced with fresh medium by centrifugation and resuspended in production medium. Any suitable CHO medium may be used, but in practice the production medium described in US Pat. No. 5,122,469 issued June 16, 1992 was used. A 3 L production spinner was seeded at 1.2 × 10 ⁶ cells / mL. On day 0, cell number and pH were measured. On the first day, the spinner was sampled and sprayed with filtered air. On the second day, the spinner is sampled, the temperature is changed to 33 ° C., and 30 mL of 500 g / L glucose and 0.6 mL of 10% antifoam (eg 35% polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion) It was. Throughout production, the pH was adjusted and maintained near 7.2. After 10 days, or until the viability fell below 70%, the cell culture medium was collected by centrifugation and filtered through a 0.22 μm filter. The filtrate was stored at 4 ° C. or immediately loaded onto a purification column.
For the poly-His tag construct, the protein was purified using a Ni-NTA column (Qiagen). Prior to purification, imidazole was added to the conditioned medium to a concentration of 5 mM. Conditioned medium was pumped at 4 ° C. at a flow rate of 4-5 ml / min onto a 6 ml Ni-NTA column equilibrated with 20 mM Hepes, pH 7.4 buffer containing 0.3 M NaCl and 5 mM imidazole. After packing, the column was further washed with equilibration buffer and the protein was eluted with equilibration buffer containing 0.25M imidazole. The highly purified protein was subsequently desalted using a 25 ml G25 Superfine (Pharmacia) column in a storage buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8, and -80 Stored at 0C.
Immunoadhesin (Fc-containing) constructs were purified from conditioned media as follows. Conditioned media was pumped onto a 5 ml protein A column (Pharmacia) equilibrated with 20 mM sodium phosphate buffer, pH 6.8. After packing, the column was washed strongly with equilibration buffer and then eluted with 100 mM citric acid, pH 3.5. The eluted protein was immediately neutralized by collecting 1 ml fractions in a tube containing 275 μL of 1M Tris buffer, pH 9. The highly purified protein was subsequently desalted in the storage buffer described above for the poly-His tag protein. Homogeneity was tested on SDS polyacrylamide gel and N-terminal amino acid sequence determined by Edman degradation.
Certain of the TAT polypeptides disclosed herein have been successfully expressed and purified using this method.

Example 5: Expression of TAT in yeast The following method describes recombinant expression of TAT in yeast.
First, a yeast expression vector is produced for intracellular production or secretion of TAT from the ADH2 / GAPDH promoter. A DNA encoding TAT and a promoter are inserted into appropriate restriction enzyme sites of the selected plasmid to direct intracellular expression of TAT. For secretion, a plasmid in which DNA encoding TAT is selected is added to DNA encoding the ADH2 / GAPDH promoter, native TAT signal peptide or other mammalian signal peptide, or, for example, yeast alpha factor or convertase secretion signal / It can be cloned with a leader sequence and (if necessary) a linker sequence for expression of TAT.
Yeasts such as yeast strain AB110 can then be transformed with the expression plasmid and cultured in the selected fermentation medium. Transformed yeast supernatants can be analyzed by precipitation with 10% trichloroacetic acid and separation by SDS-PAGE, followed by staining of the gel with Coomassie blue staining.
Recombinant TAT can then be isolated and purified by removing yeast cells from the fermentation medium by centrifugation and then concentrating the medium using a selected cartridge filter. The concentrate containing TAT may be further purified using a selected column chromatography resin.
Certain of the TAT polypeptides disclosed herein have been successfully expressed and purified using this method.

Example 6: Expression of TAT in baculovirus infected insect cells The following method demonstrates recombinant expression of TAT in baculovirus infected insect cells.
A sequence encoding TAT was fused upstream of an epitope tag contained in a baculovirus expression vector. Such epitope tags include poly-His tags and immunoglobulin tags (such as the Fc region of IgG). A variety of plasmids can be used, including plasmids derived from commercially available plasmids such as pVL1393 (Navagen). Briefly, the TAT coding sequence, or a desired portion of the coding sequence of TAT, such as the sequence encoding the extracellular domain of a transmembrane protein or the sequence encoding a mature protein when the protein is extracellular, Amplified by PCR with primers complementary to the 5 'and 3' regions. The 5 ′ primer may include flanking (selected) restriction enzyme sites. The product is then digested with selected restriction enzymes and subcloned into an expression vector.
Recombinant baculoviruses should be co-transfected with the above plasmid and BaculoGold ^TM viral DNA (Pharmingen) into Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711) using lipofectin (commercially available from GIBCO-BRL). Created by. After incubation at 28 ° C. for 4-5 days, the released virus was collected and used for further amplification. Viral infection and protein expression were performed as described in O'Reilley et al., Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford University Press (1994).

The expressed poly-His tag TAT is then purified as follows, for example, by Ni ²⁺ -chelate affinity chromatography. Extracts were prepared from virus-infected recombinant Sf9 cells as described in Rupert et al., Nature, 362: 175-179 (1993). Briefly, Sf9 cells were washed and sonicated buffer (25 mM Hepes, pH 7.9; 12.5 mM MgCl ₂ ; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; Resuspended in 0.4 M KCl) and sonicated twice on ice for 20 seconds. The sonicated product was clarified by centrifugation, and the supernatant was diluted 50-fold with a loading buffer (50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0.45 μm filter. A Ni ²⁺ -NTA agarose column (commercially available from Qiagen) was prepared with a total volume of 5 mL, washed with 25 mL of water and equilibrated with 25 mL of loading buffer. The filtered cell extract was loaded onto the column at 0.5 mL per minute. The column was washed with loading buffer to A ₂₈₀ baseline where fraction collection began. The column was then washed with a secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH 6.0) that elutes non-specifically bound protein. After reaching A ₂₈₀ baseline again, the column was developed with a 0 to 500 mM imidazole gradient in the secondary wash buffer. 1 mL fractions were collected and analyzed by SDS-PAGE and silver staining or Western blot with Ni ²⁺ -NTA conjugated to alkaline phosphatase (Qiagen). Fractions containing the eluted His ₁₀ -tag TAT were pooled and dialyzed against loading buffer.
Alternatively, purification of IgG tag (or Fc tag) TAT can be performed using known chromatographic techniques including, for example, protein A or protein G column chromatography.
Certain of the TAT polypeptides disclosed herein have been successfully expressed and purified using this method.

Example 7: Preparation of antibodies that bind to TAT This example illustrates the preparation of monoclonal antibodies that can specifically bind to TAT.
Techniques for the production of monoclonal antibodies are known in the art and are described, for example, in Goding, supra. Immunogens that can be used include purified TAT, fusion proteins comprising TAT, and cells expressing recombinant TAT on the cell surface. The selection of the immunogen can be made without undue experimentation by one skilled in the art.
Mice such as Balb / c are immunized with TAT immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally at 1-100 micrograms. Alternatively, the immunogen may be emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the animal's hind footpad. Immunized mice are then boosted 10 to 12 days later with additional immunogen emulsified in the selected adjuvant. Thereafter, for several weeks, the mice are boosted with additional immunization injections. Serum samples from retroorbital hemorrhage may be taken periodically from mice for testing in an ELISA assay for detection of anti-TAT antibodies.
After a suitable antibody titer has been detected, animals “positive” for antibodies can be given a final infusion of intravenous injection of TAT. Three to four days later, mice were sacrificed and spleen cells were removed. The spleen cells were then (using 35% polyethylene glycol), P3X63AgU. Fused to 1st selected mouse myeloma cell line. Hybridoma cells were generated by fusion and then plated on 96-well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit the growth of non-fused cells, myeloma hybrids, and spleen cell hybrids.

Hybridoma cells are screened by ELISA for reactivity to TAT. The determination of “positive” hybridoma cells that secrete the desired monoclonal antibody to TAT is within the common general knowledge.
Positive hybridoma cells are injected intraperitoneally into syngeneic Balb / c mice to produce ascites containing anti-TAT monoclonal antibodies. Alternatively, hybridoma cells can be grown in tissue culture flasks or roller bottles. Purification of monoclonal antibodies produced in ascites can be performed using ammonium sulfate precipitation followed by gel exclusion chromatography. Alternatively, affinity chromatography based on the affinity of antibody for protein A or protein G can also be used.

Example 8: Purification of TAT polypeptides using specific antibodies Natural or recombinant TAT polypeptides can be purified by various standard protein purification methods in the art. For example, a pro-TAT polypeptide, mature polypeptide, or pre-TAT polypeptide is purified by immunoaffinity chromatography using an antibody specific for the TAT polypeptide of interest. In general, an immunoaffinity column is made by covalently binding an anti-TAT polypeptide antibody to an activated chromatography resin.
Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or purification with immobilized protein A (Pharmacia LKB Biotechnology, Piscataway, NJ). Similarly, monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography with immobilized protein A. The partially purified immunoglobulin is covalently coupled to a chromatographic resin such as CnBr-activated Sepharose ™ (Pharmacia LKB Biotechnology). The antibody is bound to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.
Such immunoaffinity columns are utilized in the purification of TAT polypeptides by preparing fractions from cells containing solubilized forms of TAT polypeptides. This preparation is derived by solubilization of whole cells or cell component fractions obtained via addition of detergents or differential centrifugation by methods known in the art. Alternatively, solubilized TAT polypeptide comprising a signal sequence is secreted in useful amounts into the medium in which the cells are grown.
The solubilized TAT polypeptide-containing preparation is passed through an immunoaffinity column and the column is washed under conditions that allow preferred adsorption of the TAT polypeptide (eg, a high ionic strength buffer in the presence of a detergent). The column is then eluted under conditions that degrade antibody / TAT polypeptide binding (eg, low pH, about 2-3, high concentrations of urea or chaotropes such as thiocyanate ions), and the TAT polypeptide is recovered. .

Example 9: In Vitro Tumor Cell Killing Assay Mammalian cells expressing a TAT polypeptide of interest are obtained using standard expression vectors and cloning methods. Alternatively, many tumor cell lines that express a TAT polypeptide of interest are publicly available, eg, from the ATCC, and can be routinely identified using standard ELISA or FACS analysis methods. The anti-TAT polypeptide monoclonal antibody (and its toxin conjugate derivative) can then be used in the assay to quantify the ability of the antibody in vitro to kill TAT polypeptide-expressing cells.
For example, cells expressing the TAT polypeptide of interest are obtained as described above and seeded in 96 well dishes. In one analysis example, an antibody / toxin conjugate (or naked antibody) is included during a 4 day period of cell incubation. In a second independent assay, cells are incubated with antibody / toxin conjugate (or naked antibody) for 1 hour, then washed and incubated in the absence of antibody / toxin conjugate for a period of 4 days. . Cell viability is then measured using Promega's CellTiter-Glo Luminescent Cell Viability Assay (catalog number G7571). Untreated cells serve as a negative control.

Example 10: In vivo tumor cell killing assay To test the efficacy of conjugated or unconjugated anti-TAT polypeptide monoclonal antibodies, anti-TAT antibodies were nude mice 24 hours prior to subcutaneous administration of tumor promoting cells flank. Were injected intraperitoneally. Antibody injections are continued twice weekly until the end of the experiment. The tumor volume is then measured twice weekly.

  The above written description is considered to be sufficient to enable one skilled in the art to practice the invention. The deposited implementation is intended as an illustration of one aspect of the present invention, and since any functionally equivalent construct is within the scope of the invention, the deposited construct limits the scope of the invention. Is not to be done. The deposit of material herein does not admit that the written description contained herein is insufficient to allow implementation of any aspect of the present invention, including its best mode, and that It is not to be construed as limiting the scope of the claims for the particular illustrations represented. Indeed, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

NM_005973.3 XM_044077.3 XM_056380.2 XM_044080.2 XM_044083.5 NM_001878.2 XM_017040.3 NM_021948.2 NM_021817.1 XM_0865573.1 XM_0976566.1 XM_059219.2 NM_005920.1 NM_006365.1 XM_059216.5 XM_044217.8 XM_053245.6 NM_032323.1 XM_044148.3 NM_024897.1 NM_007221.1 XM_044155.1 XM_086562.1 NM_022367.1 NM_005572.1 XM_044166.3 XM_053243.1 XM_017041.3 XM_04128.6 NM_003145.2 XM_044172.7 XM — 010715.2 XM_04108.3 NM_032292.1 NM_017710.1 NM_033657.1 NM_018253.1 NM_01816.1 NM_0184849.1 XM_011513.3 NM_01428.1 NM_002044.1 XM_0865565.1 NM_003993.1 NM_005698.2 NM_006589.1 XM_084078.4 XM_086567.1 XM_086552.4 NM_007112.1 XM_053256.7 XM_089386.1 NM_05058.1 XM_114144.1 XM_086571.1 NM_01893.2 NM_018845.1 XM_113377.1 NM_004952.2 XM_010561.10. XM_117201.1 XM_036744.6 NM_1308098.1 NM_006694.1 NM_002870.1 NM_001030.2 XM_036829.6 NM_0155449.1 NM_0144847.1 NM_006118.2 XM_114855.1 NM_080429.1 XM_036933.7 XM_036934.4 XM_08770.2 NM_004183.1 XM_167726.1 NM_021727.2 NM_004265.2 NM_013402.3 NM_004111.4 NM_014206.1 NM_013279.1 XM_167769.1 XM_043146.5 XM_043151.7 XM_090000.4 XM_04288.6 NM_0166404.1 XM_11325.1 NM_004322.2 XM_113222.1 XM_113221.1 XM_115027.1 NM_014067.2 XM_053791.1 XM_006111.8 XM_011988.9 XM_049178.6 NM_032344.1 NM_031472.1 XM_037808.4 NM_004074.1 XM_017481.6 NM_017670.1 XM_043159.1 XM_111323.1 NM_003942.1 XM_04549.4 NM_005825.2 NM_005609.1 XM_045642.3 XM_006533.7 XM_006532.9 XM_05613.6 XM_056112.4 NM_006244.1 XM_111324.1 XM_05498.5 XM_045499.3 NM_001997.2 NM_014205.1 XM_045525.7 NM_013265.2 XM_006529.8 XM_053787.6 XM_03776.2 XM — 165896.1 XM_084617.4 XM_04533.33.3 XM_057307.6 NM_001667.1 NM_138456.1 XM_115029.1 XM_115031.1 XM_084702.4 XM_03774.6 XM_053796.5 NM_002689.2 NM_006268.2 XM_166932.1 XM_1678044.1 XM_166931.1 XM_16635.1 XM_16636.1 NM_006842.1 XM_167811.1 NM_000852.2 NM_007103.2 NM_005851.2 XM_1655598.1 XM_1655599.1 XM_166194.1 XM_1666793.1 XM_1655.94.1 XM_166190.1 NM_002496.1 NM_006019.2 XM_165601.1 XM_089954.4 NM_017635.1 NM_016028.2 NM_022338.1 NM_002335.1 XM — 166795.1 NM_018312.2 NM_001876.1 XM_166196.1 XM_165600.1 XM_166195.1 XM_167784.1 XM_16923.1 NM_005171.1 NM_0144033.1 NM_0308089.1 NM_0155416.1 XM_08961.4 XM_051166.6 XM_090501.4 XM_099664 XM_0155481.7 XM_049148.2 XM_1133726.1 XM_012230.1 NM_006121.2 XM_062616.1 XM_049141.1 XM_096612.4 NM_021934.1 XM_083884.1 XM_007464.3 NM_006925.1 NM_022137.1 XM_085065.4 XM_1656566.1 NM_032330.1 NM_001043.1 NM_000978.2 NM_017748.2 XM_008145.3 NM_002795.1 XM_008147.3 XM_113893.1 NM_005937.1 XM_085541.1 NM_018530.1 NM_016471.1 XM_058830.3 XM_117040.1 NM_002809.1 XM_04980.3 NM_007359.2 XM_1139399.1 XM_008586.5 XM_117071.1 XM_1133329.1 XM_083987.2 NM_000422.1 XM_008579.4 NM_002276.1 NM_002275.1 NM_002274.1 NM_002277.2 NM_000526.3 XM_041843.2 XM_042009.4 XM_06166.2 XM_085978.1 XM_042012.2 NM_004359.1 XM_042018.1 XM_114018.1 XM_042027.1 XM_086110.1 NM_07876.1 XM_05711.3 NM_002579.1 XM_092044.2 NM_002819.2 NM_0244888.1 XM_086035.2 XM_037560.3 NM_1387774.1 NM_005224.1 XM_03755.1 NM_033420.1 XM_092042.2 XM_037572.2 XM_03754.3 XM_009279.6 NM_002085.1 XM_009277.5 XM — 02913.4 NM_001687.1 XM_02807.2 XM_02804.3 XM_086023.2 XM_028060.4 XM_072012.3 NM_0244407.2 NM_000156.4 NM_01859.1 NM_001018.2 XM_04757.77.3 XM_086101.1 XM_086102.1 NM_138393.1 XM_05901.2 XM_0475833.1 XM_047584.2 NM_006830.2 XM_047600.2 XM_030721.1 NM_031918.1 XM_103282.1 NM_031213.1 NM_079834.1 NM_001319.4 NM_0177977.2 NM_017572.1 XM_059021.3 XM_053871.4 NM_003938.2 XM_046822.2 NM_0188049.1 NM_007165.1 XM_009274.1 NM_004152.1 XM_03698.55.3 NM_012458.1 NM_032737.1 XM_086115.1 XM_030485.3 XM_08123.2 XM_046934.3 NM_003021.2 XM_0568586 XM_055547.2 XM_086121.2 XM_086120.2 XM_052643.2 NM_024760.1 XM_009222.6 XM_052629.4 NM_003775.1 XM_009220.4 XM_072009.2 XM_052661.3 XM_08124.1 XM_0974426.1 NM_005597.1 XM_096112.2 NM_016263.1 XM_1139988.1 NM_024292.1 NM_006221.1 NM_058164.1 XM_009025.9 NM_018381.1 XM_097282.1 XM_049490.1 NM_020230.1 NM_002566.1 NM_003755.1 NM_001379.1 XM_049502.1 XM_04918.1 NM_022377.1 XM_008895.2 XM_091913.4 NM_080665.1 XM_058946.5 NM_002162.2 XM_008893.4 NM_007065.2 XM_008891.7 XM_0495561.1 NM_032885.1 XM_07204.1 NM_001800.2 NM_005498.3 XM_008887.6 XM_085862.4 NM_012218.1 XM_084014.1 NM_031209.1 XM_032710.6 NM_006858.1 XM_032719.3 NM_024029.2 XM_032724.2 NM_003072.1 XM_009082.1 XM_085924.4 XM_085920.1 XM_0719966.2 XM_032773.1 XM_05895.5 XM_032774.1 NM_000121.2 XM_032782.2 XM_032783.1 XM_057075.3 NM_1387873.1 NM_016581.2 XM_032792.1 NM_032377.1 XM_113989.1 XM — 01567.97.3 XM_064931.1 XM_032822.2 XM_064934.1 XM_064935.5 XM_032820.1 XM_032812.1 XM_091968.4 XM_032811.1 XM_091967.1 XM_032810.1 XM_032799.5 XM_009075.7 XM_08592.12.1 XM_085915.4 XM_064940.1 XM_091959.1 XM_032674.1 XM_010156.7 NM_016145.1 NM_032332.1 NM_001930.2 NM_032301.1 XM_009068.6 NM_0244038.1 XM_032041.2 XM_009065.5 NM_002229.1 XM_009063.3 NM_006397.1 NM_031429.1 XM_032034.2 NM_001375.1 NM_000159.1 NM_004461.1 XM_032020.1 NM_005053.2 XM_085916.4 XM_057080.2 XM_117130.1 NM_017722.1 NM_052876.1 NM_003765.1 XM_051364.2 NM_004647.1 NM_033256.1 XM_032281.4 XM_032285.3 NM_002812.1 XM_085987.1 XM_02955.3 NM_007181.1 XM_029450.1 NM_004924.2 XM_085989.2 XM_1155619.1 XM_009149.3 XM_056602.1 XM_008904.4 NM_001533.1 NM_012237.2 NM_002503.1 NM_01727.2 NM_021107.1 NM_024907.4 XM_059045.1 NM_005884.2 XM_103270.1 XM_04688.1 NM_0188028.1 XM_046090.2 XM_167529.1 XM_086004.2 XM_097361.2 NM_003407.1 XM_046114.3 NM_001020.2 NM_003169.2 XM_053074.4 NM_016941.1 NM_004714.1 NM_001436.2 XM_016410.2 XM_096107.1 XM_012860.4 NM_006503.1 XM_042665.2 XM_085999.1 XM_009203.6 XM_059053.5 XM_0589944.3 XM_046390.3 NM_016440.1 XM_117132.1 XM_05316.1 NM_012068.2 XM_085927.2 XM_084023.1 XM_016217.6 XM_114025.1 XM_008985.6 NM_017432.1 XM_046344.2 NM_030973.1 NM_02519.2 NM_014203.2 XM_085934.1 XM_058992.3 XM_117134.1 NM_001536.1 XM_165432.1 NM_001571.1 XM_046313.2 NM_006270.2 XM_046305.2 NM_000951.1 NM_0159593.2 XM_057199.3 XM_027904.5 XM_084026.2 NM_012423.2 XM_058991.3 NM_017916.1 NM_020309.1 NM_014441.1 NM_003598.1 NM_001774.1 XM_057188.1 XM_02783.2 NM_018111.1 NM_022165.1 XM_085942.2 NM_033142.1 NM_033183.1 NM_033043.1 NM_033377.1 NM_033378.1 NM_000894.2 NM_006666.1 XM_114024.1 NM_000146.2 XM_085957.1 NM_004324.1 NM_006184.2 XM_009097.4 NM_020904.1 NM_016246.1 NM_001190.1 NM_000511.1 XM_009103.5 NM_001217.2 XM_049974.5 NM_020126.2 NM_000979.2 XM_055866.4 XM_049963.4 NM_013348.1 NM_031485.1 XM_009108.3 NM_006801.1 XM_012808.5 NM_001425.1 XM_039938.3 XM_009118.9 XM_055864.4 XM — 113344.1 XM_046419.3 NM_002691.1 XM_052686.2 XM_058990.5 XM_119305.1 NM_13834.1 XM_058984.2 XM_091981.4 NM_032298.1 XM_053166.6 NM_002975.1 NM_3272.1 XM_085958.1 NM_001648.1 XM_055568.3 NM_007196.1 XM_009004.4 XM_031105.1 XM_091891.4 NM_001985.1 NM_013336.2 XM_067264.4 XM_114309.1 XM_084111.4 NM_004117.2 XM_166419.1 NM_007104.3 NM_021922.1 XM_165760.1 NM_022047.1 XM_166333.1 XM_1666407.1 NM_015921.1 NM_0244165.1 XM_058039.4 XM_087990.2 XM — 165743.1 XM_01657.3 NM_001350.1 NM_005453.3 XM — 165795.1 NM_004761.1 NM_014260.1 NM_005452.3 NM_003782.2 NM_022551.2 NM_080564.1 NM_002931.2 XM_1655477.1 NM_021976.1 XM_1656966.1 XM_165807.1 XM_1656988.1 XM — 165808.1 XM_165770.1 NM_006120.2 XM_1655798.1 NM_000593.3 NM_004159.1 XM_1655824.1 NM_022555.3 XM — 165717.1 XM_166480.1 XM_1657575.1 NM_022107.1 XM_165801.1 NM_006913.1 XM_1655705.1 NM_004381.2 NM_09105.2 NM_0000500.3 NM_000592.3 NM_032454.1 NM_005510.1 NM_006929.2 NM_002904.3 NM_001710.2 NM_000063.2 NM_006709.2 NM_000434.1 NM_005346.2 NM_005345.3 XM_1663488.1 NM_021177.1 NM_006295.1 NM_02559.1 NM_001288.3 NM_013974.1 NM_021160.1 NM_001320.2 NM_033177.2 XM_165712.1 NM_004639.2 ' NM_080686.1 NM_004847.2 XM_1664544.1 XM_1655823.1 XM_165771.1 NM_004640.3 NM_005931.1 XM_165832.1 XM_1655765.1 XM_087945.1 XM_166381.1 XM_166395.1 XM_168104.1 XM_165720.1 XM_166401.1 NM_001517.1 XMS65738.1 NM_003897.2 NM_005803.2 XM_087939.2 XM_1655734.1 XM_1666353.1 XM_1667376.1 XM_165782.1 XM_166409.1 XM_166398.1 NM_0144046.2 XM_1655799.1 NM_001090.1 NM_02563.1 XM_166361.1 XM — 169719.1 XM — 165764.1 XM_1655758.1 XM_066947.2 NM_024839.1 XM_1663688.1 NM_003449.2 NM_05236.1 NM_021959.1 NM_014596.2 XM_088798.2 XM_165809.1 XM_08971.2 XM_167122.1 NM_002127.2 NM_018950.1 XM — 165803.1 XM_166392.1 XM_165818.1 XM — 05516.4 XM_013042.3 NM_031206.1 XM_0886828.1 XM_093050.1 XM_002013.6 XM_086138.1 NM_000969.2 XM_059930.5 XMS005541.2 NM_0147874.1 NM_005973.3 XM_044077.3 XM_056380.2 XM_044083.5 NM_001878.2 XM_017040.3 XM_0865573.1 XM_0976566.1 XM_059219.2 NM_005920.1 XM_044217.8 XM_053245.6 XM_044148.3 NM_007221.1 XM_044155.1 NM_022367.1 NM_005572.1 XM_044166.3 XM_053243.1 XM_017041.3 XM_04128.6 NM_003145.2 XM_044172.7 XM — 010715.2 NM_006912.1 NM_025174.1 NM_032292.1 NM_017710.1 NM_033657.1 NM_018253.1 NM_01816.1 XM_017056.2 NM_0184849.1 NM_01428.1 NM_002044.1 NM_003993.1 NM_005698.2 NM_006589.1 XM_084078.4 XM_086567.1 XM_086552.4 NM_007112.1 XM_053256.7 XM_114144.1 XM_086571.1 NM_01893.2 NM_018845.1 XM_113377.1 XM_010561.10. XM_117201.1 XM_036744.6 NM_1308098.1 NM_006694.1 NM_002870.1 NM_001030.2 XM_036829.6 NM_0155449.1 NM_0144847.1 NM_006118.2 XM_036933.7 XM_036934.4 XM_05737.4 NM_017582.2 CM_03845.4 NM_006556.2 NM_020524.2 XM_034083.2 XM_034082.4 NM_001826.1 NM_025207.1 XM_05411.6 NM_003815.2 NM_002802.1 XM_0298.38.3 XM_058677.2 NM_006888.1 XM_085175.1 XM_0968813.1 XM — 017823.4 NM_006470.2 XM_051763.3 XM_0857726.1 XM_0588999.3 NM_017775.1 XM_037176.2 XM_113908.1 NM_01855.1 NM_016113.2 XM_058900.2 XM_09722.2 XM_039911.1 XM_091689.2 XM_039921.3 XM_012549.7 NM_020201.1 NM_003653.1 XM_012695.4 XM_117042.1 NM_032339.1 NM_004448.1 NM_033419.1 NM_006804.1 NM_032192.1 XM_083956.2 XM_0166544.3 NM_032875.1 XM_029046.2 XM_091460.1 NM_000981.2 NM_020405.2 XM_091455.1 XM_008150.7 NM_000978.2 NM_017748.2 XM_008145.3 NM_002795.1 XM_008147.3 XM_113893.1 NM_005937.1 NM_018530.1 NM_016471.1 XM_058830.3 XM_117040.1 NM_002809.1 XM_04980.3 NM_007359.2 XM_1139399.1 XM_1133329.1 XM_083987.2 XM_064591.1 NM_022751.1 XM_0155755.1 XM_097232.2 XM_046103.4 XM_06099.1 NM_0144649.1 NM_006012.1 NM_032306.1 NM_002096.1 NM_003685.1 XM_044564.5 XM_009015.1 NM_003811.1 XM_009010.4 XM_0445591.4 NM_004240.1 XM_044619.3 XM_044608.2 NM_0155414.2 XM_009398.4 NM_003624.1 XM_032278.5 NM_033256.1 XM_032281.4 XM_085984.2 XM_032285.3 NM_004823.1 NM_002812.1 XM_029450.1 NM_004924.2 XM_085989.2 NM_006149.2 XM_008904.4 NM_001533.1 NM_012237.2 NM_002503.1 NM_01727.2 NM_021107.1 NM_024907.4 XM_059045.1 NM_005884.2 NM_0188028.1 XM_046090.2 XM_167529.1 XM_086004.2 CM_097361.2 NM_003407.1 XM_046114.3 NM_001020.2 NM_003169.2 XM_053074.4 NM_001436.2 XM_016410.2 XM_012860.4 NM_006503.1 XM_114180.1 NM_033197.1 XM_05893.3 XM_047042.2 XM_056253.3 NM_032819.2 XM_059272.3 NM_016732.1 NM_003908.1 XM_012928.4 NM_000687.1 NM_031483.1 XM_047136.1 XM_041350.4 XM_041363.7 XM_116065.1 XM_076414.5 XM_116066.1 XM_093546.4 NM_018130.1 NM_080652.1 XM_087289.1 XM_08414.4 XM_04865.5 NM_004593.1 XM_1134061 XM_011160.5 XM_114399.1 NM_007100.1 002477.1 NM_032219.1 NM_006527.2 NM_138385.1 NM_006342.1 NM_012318.1 NM_1333334.1 XM_0358.28.2 XM_114423.1 XM_068272.1 NM_024751.1 NM_020395.1 XM_059633.5 XM_114442.1 XM_042321.4 XM_042301.2 XM_017641.3 NM_030782.1 XM_049512.2 XM_049510.3 XM_049508.3 XM_12103.1 NM_007030.1 XM_116422.1 XM_003770.4 XM_03946.2 XM_114438.1 XM_114442.1 XM_114440.1 NM_022483.2 XM_1655483.1 XM_114453.1 XM_114454.1 XM_1655484.1 NM_007255.1 XM_030771.4 XM_0307777.2 NM_024872.1 XM_030782.2 XM_003735.6 NM_030567.1 XM_094267.1 XM_003736.4 NM_006816.1 NM_013237.1 NM_130781.1 NM_022455.2 NM_002011.2 XM_055292.1 NM_012279.1 NM_016290.1 XM_030295.3 XM_055226.2 NM_014901.1 XM_030257.3 NM_016391.2 XM_055224.3 XM_113453.1 NM_003093.1 NM_0242.94.1 NM_012391.1 XM_1688084.1 XM_1666344.1 XM — 166459.1 XM_043240.1 XM_055002.1 NM_1333645.1 XM_097453.2 XM_02928.2 NM_012434.1 XM — 168627.1 NM_024653.1 XM — 168574.1 NM_006234.2 XM — 168603.1 NM_032958.1 XM_168560.1 NM_006989.1 XM_1688558.1 XM — 168635.1 NM_005824.1 NM_031905.1 NM_021039.1 NM_004279.1 XM_168590.1 NM_002803.1 XM_004720.4 NM_024033.1 XM_050288.2 XM_04584.1 XM_058073.3 XM_1145555.1 NM_001551.1 NM_0166484.1 XM_034647.1 XM_034443.3 NM_012286.1 XM_010124.3 NM_032926.1 NM_024863.2 NM_014380.1 XM_043643.1 NM_032621.1 XM_034430.2 NM_01876.2 NM_004359.1 XM_042018.1 XM_114018.1 XM_086110.1 NM_07876.1 XM_05711.3 XM_092044.2 NM_002819.2 XM_037560.3 NM_1387774.1 NM_005224.1 XM_03754.1 XM_097386.2 XM_03755.1 NM_033420.1 XM_092042.2 XM_037572.2 XM_009279.6 NM_002085.1 XM_009277.5 XM — 02913.4 NM_001687.1 XM_02807.2 XM_02804.3 XM_086023.2 NM_0244407.2 NM_01859.1 NM_001018.2 XM_04757.77.3 XM_086102.1 XM_047584.2 NM_006830.2 XM_030721.1 NM_031918.1 XM_103282.1 NM_031213.1 NM_079834.1 NM_001319.4 NM_0177977.2 NM_017572.1 XM_059021.3 NM_003938.2 XM_046822.2 NM_0188049.1 NM_007165.1 NM_004152.1 NM_016199.1 NM_012458.1 NM_032737.1 XM_086115.1 XM_030485.3 XM_046934.3 NM_003021.2 CM_046934.3 NM_003021.2 XM_052629.4 XM_072009.2 XM_052661.3 NM_005597.1 NM_016263.1 XM_059026.2 NM_006339.1 XM_047620.2 XM_059027.1 NM_001348.1 NM_001961.1 NM_0155898.1 XM_016871.6 NM_01639.1 NM_018074.1 NM_0244333.1 NM_017720.1 XM_031841.3 NM_003025.1 XM_084031.1 NM_025241.1 XM_059013.3 XM_0S9012.2 XM_035638.5 XM_05636.3 XM_05635.2 XM_035634.2 XM_012862.3 XM_065020.1 XM_05627.2 XM_035625.5 XM_032281.4 XM_085984.2 CM_032285.3 NM_002812.1 XM_085987.1 XM_02955.3 XM_029450.1 NM_004924.2 XM_008904.4 NM_001533.1 NM_012237.2 NM_01727.2 NM_021107.1 NM_024907.4 XM_059045.1 NM_005884.2 XM_009225.5 XM_046090.2 XM_167529.1 XM_086004.2 XM_097361.2 NM_003407.1 XM_046114.3 NM_001020.2 NM_003169.2 XM_053074.4 NM_001436.2 XM_096107.1 XM_012860.4 NM_006503.1 XM_092019.1 XM_009203.6 XM_059053.5 XM_047409.3 NM_000713.1 XM_04376.6 XM_047374.5 NM_004756.1 XM_059052.5 NM_004596.1 NM_017555.1 NM_0306022.4 XM_0155505.1 XM_030914.1 NM_052848.1 XM_008912.1 XM_085882.1 XM_0300901.1 NM_01835.1 XM_088574.1 XM_009171.3 XM_115603.1 NM_001022.2 NM_004706.2 NM_006423.1 NM_022752.1 NM_006494.1 XM_0298833.4 XM_041978.3 XM_017591.4 NM_006058.1 NM_002084.2 XM_117396.1 NM_0188047.1 NM_007286.1 XM_042547.3 NM_005617.2 XM_003937.5 NM_000176.1 XM_059214.2 XM_046349.5 XM_0893737. XM_096141.1 NM_015911.1 NM_006516.1 XM_0865543.4 NM_006824.1 NM_001255.1 NM_005620.1 XM_001468.5 XM_047841.5 XM_0974444.4 XM_047874.5 XM_047883.4 NM_002796.1 NM_003944.2 XM_096144.1 NM_002651.1 NM_002810.1 XM_044077.3 XM_056380.2 XM_044080.2 XM_0865573.1 XM_059223.2 XM_044217.8 XM_053245.6 XM_044148.3 XM_044155.1 XM_086562.1 NM_005572.1 XM_017041.3 XM_04128.6 NM_003145.2 XM — 010715.2 NM_025174.1 NM_032292.1 NM_017710.1 NM_033657.1 NM_018253.1 NM_01816.1 NM_0184849.1 NM_002044.1 NM_000298.1 NM_003993.1 NM_005698.2 NM_006589.1 XM_114144.1 NM_018845.1 XM_113377.1 XM_010561.10. XM_117201.1 XM_036744.6 NM_006694.1 NM_002870.1 NM_001030.2 XM_036829.6 NM_0155449.1 NM_0144847.1 NM_006118.2 XM_036934.4 NM_017582.2 XM_03645.4 NM_019058.1 XM_058400.5 XM_045140.5 XM_050993.5 XM_058343.5 XM_058602.2 XM_031866.1 NM_024541.1 NM_015062.1 XM_031890.2 NM_004193.1 XM_031926.6 NM_024040.1 XM_031949.5 NM_016169.2 NM_030912.1 XM_011921.3 XM_058359.5 XM_05835.1 NM_005566.1 NM_006292.2 XM_115081.1 XM_0877710.2 NM_004183.1 XM_167726.1 NM_021727.2 NM_004265.2 NM_013402.3 NM_014206.1 XM_043146.5 XM_043151.7 XM_04288.6 NM_0166404.1 NM_004322.2 XM_011988.9 XM_037808.4 NM_004074.1 XM_043159.1 XM_111323.1 NM_003942.1 NM_005825.2 NM_005609.1 XM_04562.32.3 XM_006533.7 XM_006532.9 XM_05613.6 XM_056112.4 XM_111324.1 XM_05498.5 XM_045499.3 NM_0019I7.2 XM_004525.7 NM_013265.2 XM_03776.2 XM_084617.4 XM_04533.33.3 XM_057307.6 XM_001667.1 XM_084702.4 XM_03774.6 NM_002689.2 NM_006268.2 XM_1678044.1 XM_16635.1 XM_16636.1 NM_006842.1 NM_000852.2 NM_007103.2 XM_033227.7 NM_001734.1 NM_005768.3 NM_006331.1 NM_007273.2 NM_080549.1 NM_138425.1 XM_032588.5 NM_000365.2 XM_032614.1 NM_031299.2 XM_006966.2 XM_165910.1 NM_016319.1 XM_033223.5 NM_005439.1 NM_001273.1 XM_033253.4 NM_002046.2 XM_033263.3 XM_006958.7 NM_016497.2 NM_018009.1 NM_002342.1 NM_001065.1 XM_0271981 XM_012272.3 XM_056481.4 NM_024056.1 NM_001659.1 XM_084866.2 XM_006826.2 XM_02862.3 NM_031157.1 NM_006163.1 NM_0166057.1 XM_006800.3 NM_005016.2 XM_113707.1 NM_032656.1 XM_0155274.6 XM_055013.4 XM — 13715.1 XM_084832.4 XM_045602.2 XM_031617.1 NM_007223.1 XM_031612.5 NM_003134.1 XM — 113826.1 XM_031561.3 NM_002225.2 XM_031555.3 NM_018145.1 NM_005258.2 NM_032850.1 XM_031510.4 NM_024111.1 XM_085313.4 XM_113821.1 NM_0166359.1 NM_007364.1 XM_091042.1 XM_044493.1 NM_006791.1 XM_060042.5 XM_085445.2 XM_058781.3 XM_1133876.1 XM_117027.1 XM_165927.1 XM_053402.2 XM_055195.2 XM_113291.1 NM_000418.1 XM_113874.1 XM_102377.2 XM_008441.5 XM_113932.1 NM_003809.1 XM_01843.3. NM_001416.1 XM_008327.3 NM_004870.1 XM_008234.3 NM_133491.1 NM_001040.2 XM_008679.2 XM_008231.2 NM_05099.1 NM_032354.1 XM_034028.1 XM_1133328.1 XM — 11399.29.1 NM_001970.2 NM_004217.1 NM_017622.1 XM_165941.1 NM_021210.1 XM — 113928.1 XM_097077.1 XM_167528.1 NM_001307.1 XM_1139394.1 XM_1655421.1 NM_0155343.1 XM_1133325.1 XM — 165422.1 XM_113931.1 XM_085689.2 XM_03669.1 XM_1133334.1 NM_001254.2 XM_036465.1 XM_055061.1 XM_036462.1 XM_083995.1 XM_008290.2 XM_04854.3 XM_008633.4 NM_02517.1 NM_002087.1 NM — 06016.1 NM_01423.1 XM_012642.4 NM_053047.1 XM_097133.2 NM_032484.1 XM_045901.4 XM_0585850.3 NM_032376.1 XM_091544.2 XM_045950.3 XM_032808.2 XM_115461.1 XM_008393.3 XM_032852.1 XM_064317.1 XM — 13918.1 NM_0263.1 NM_05233.1 XM_032817.4 XM_085635.1 XM_091549.2 XM_032781.1 NM_000151.1 XM_113917.1 XM_017557.6 XM_032766.1 NM_003766.2 NM_01419.1 NM_032353.1 XM_008401.5 XM_008402.1 NM_017595.2 XM_1168444.1 XM_085636.2 NM_000988.2 XM_008559.1 XM_115480.1 NM_005831.2 XM_117061.1 XM_1131318.1 XM_1133319.1 XM_030444.5 XM_008459.3 NM_005324.1 XM_054344.2 NM_002766.1 XM_046481.1 XM_097197.2 XM_097193.2 XM_083988.1 NM_004309.2 XM_046472.1 NM_016286.1 NM_004127.3 NM_022156.1 XM_113943.1 XM_042018.1 XM_114018.1 XM_086110.1 NM_07876.1 XM_05711.3 NM_002819.2 NM_0244888.1 NM_037560.3 NM_1387774.1 XM_03755.1 NM_033420.1 XM_092042.2 XM_037572.2 XM_03754.3 XM_009279.6 NM_002085.1 NM_001687.1 XM_02804.3 XM_028060.4 XM_072012.3 NM_000156.4 NM_01859.1 NM_001018.2 XM_086101.1 XM_086102.1 NM_138393.1 XM_047584.2 NM_006830.2 XM_047600.2 XM_030721.1 NM_031918.1 NM_079834.1 NM_001319.4 NM_0177977.2 NM_017572.1 NM_003938.2 NM_007165.1 NM_004152.1 XM_088570.1 NM_018381.1 XM_049490.1 NM_020230.1 NM_003755.1 NM_001379.1 XM_049502.1 XM_04918.1 XM_058946.5 XM_008893.4 NM_007065.2 XM_008891.7 XM_0495561.1 XM_085862.4 NM_012218.1 XM_084014.1 NM_031209.1 XM_032710.6 XM_032719.3 NM_024029.2 NM_003072.1 XM_009082.1 XM_085924.4 XM_009085.7 XM_032773.1 XM_032782.2 NM_032377.1 NM_001611.2 XM_113989.1 XM — 01567.97.3 XM_032812.1 NM_001436.2 XM_012860.4 NM_006503.1 XM_042665.2 XM_047409.3 NM_000713.1 XM_04376.6 XM_047374.5 XM_059052.5 NM_004596.1 XM_049920.1 XM_055589.2 XM_008860.6 XM_097339.1 XM_05586.5 XM_009036.3 XM_08253.1 XM_009039.1 XM_085943.4 XM_08263.1 XM_085928.1 NM_013403.1 NM_001743.3 NM_017432.1 XM_046344.2 NM_02519.2 NM_014203.2 NM_001536.1 XM_046305.2 NM_0159593.2 XM_084026.2 NM_012423.2 NM_017916.1 NM_018111.1 NM_022165.1 XM_085942.2 NM_006666.1 NM_000146.2 NM_004324.1 NM_006184.2 XM_009097.4 NM_000979.2 XM_049963.4 NM_031485.1 NM_006801.1 XM_012808.5 NM_001425.1 XM_091973.1 XM_039938.3 XM_009118.9 NM_021103.1 XM_0869920.1 XM_056752.2 XM_034215.3 XM_086916.1 XM_002568.8 NM_001747.1 NM_005911.2 XM_050962.4 NM_012100.1 XM_050638.2 NM_002601.1 NM_022730.1 XM_054226.4 NM_024085.1 XM_050885.1 XM_05808.3 XM_087157.1 XM_058100.3 XM_065804.1 NM_000784.2 XM_002591.7 NM_004328.2 XM_010866.6 XM_117275.1 NM_002712.1 NM_018635.1 XM_042326.3 NM_004404.1 NM_0144808.1 XM_1172744.1 XM_002742.4 NM_023083.1 NM_01826.2 XM_00962.5 XM_009682.3 NM_080801 NM_003091.1 NM_052881.1 NM_006899.1 NM_022760.2 NM_002836.2 XM_045394.1 NM_0308111.2 NM_016732.1 NM_003908.1 NM_000687.1 NM_014183.1 NM_080476.1 XM_0867728.1 XM_04715.2 NM_01867.1 NM_000178.1 NM_0155638.1 NM_018217.1 NM_006444.1 NM_002212.1 NM_018244.1 NM_007186.1 NM_01566.1 NM_003915.1 XM_033090.4 NM_003766.1 XM_059313.2 XM_055490.4 NM_003504.2 NM_002688.2 XM_058918.2 NM_024627.2 NM_005877.2 NM_012429.1 NM_016498.1 NM_004861.1 NM_0144303.1 XM_114209.1 NM_003661.1 NM_002473.1 NM_003753.1 XM_0993.33.3 XM_00999744 NM_138977.1 NM_016091.1 NM_012407.1 NM_012264.2 NM_001894.1 NM_006855.2 NM_006386.2 NM_0155373.1 NM_020243.2 NM_0144876.1 XM_03932.3 XM_03939.5 XM_114199.1 NM_021822.1 XM_009997.2 NM_000967.2 XM_010000.3 XM — 010003.4 XM_03372.4 NM_000001001 NM_0155705.1 XM_039410.1 NM_003932.2 NM_0144248.1 XM_086844.1 XM_010013.4 NM_002883.1 NM_017590.1 NM_016272.1 NM_001098.1 XM_03448.4 NM_002676.1 XM_04395.2 NM_001469.2 NM_005008.1 NM_004599.1 NM_024053.1 XM_010024.3 NM_002490.2 XM_040050.3 XM — 04006.33 XM_066333.4 XM_013015.3 XM_0400841 XM_084084.1 XM — 01008.3 XM_051518.6 NM_003365.1 XM_030109.5 XM — 03006.4 XM_003288.8 XM_08412.1 XM_047561.3 XM_116853.1 XM_114325.1 XM_113405.1 XM_028610.2 NM_013334.2 NM_022064.1 NM_020998.1 XM_002828.6 XM_01823.6 NM_000481.1 NM_032316.1 XM_114367.1 NM_005777.1 NM_005778.1 NM_006841.1 XM_041507.1 NM_004636.1 NM_007312.2 NM_033158.1 XM_0166345.5 XM_1164799.1 NM_002887.2 XM_047992.2 XM_114453.1 XM_1655484.1 XM_030771.4 NM_019057.1 XM_0307777.2 XM_030782.2 XM_003735.6 XM_003736.4 NM_0005055.2 NM_006816.1 NM_013237.1 NM_130781.1 NM_002011.2 NM_007355.2 XM_165772.1 XM_168123.1 XM — 166457.1 NM_018135.2 XM — 166428.1 XM_1666334.1 XM_166340.1 XM_08790.1 XM — 166404.1 NM_0153488.1 NM_080604.1 XM_1655735.1 XM_1655754.1 NM_006443.1 XM_1666408.1 NM_003131.1 NM_002821.2 NM_138343.1 XM_1655729.1 XM_165732.1 XM_166425. NM_0142623.1 NM_006245.1 XM_165830.1 NM_018960.1 NM_005510.1 NM_001710.2 NM_000063.2 NM_006709.2 NM_000434.1 NM_005346.2 NM_005345.3 NM_006295.1 NM_001288.3 NM_013974.1 NM_021160.1 NM_001320.2 NM_033177.2 XM_165571.1 NM_004639.2 NM_080686.1 NM_004847.2 XM_165771.1 NM_004640.3 NM_005931.1 XM_165832.1 XM_1655765.1 XM_087945.1 XM_166381.1 XM_165720.1 NM_001517.1 XM_1655738.1 NM_003897.2 NM_005803.2 XM_087939.2 XM_1655734.1 XM_1666353.1 XM_1667376.1 XM_165782.1 NM_0144046.2 NM_001090.1 NM_02563.1 XM_166361.1 XM — 165764.1 XM_1655758.1 XM_066947.2 NM_024839.1 NM_003449.2 NM_021959.1 NM_014596.2 XM_165809.1 XM_08971.2 XM_167122.1 XM_166392.1 XM_167128.1 XM_1666357.1 NM_001069.1 NM_004332.1 NM_000944.1 NM_004568.2 NM_030666.2 NM_020135.2 NM_004156.1 XM_0883838.1 XM_040574.5 XM_049247.2 XM_05868.2 NM_0244736.2 XM_005044.7 NM_003313.2 NM_032862.1 XM_059915.5 XM_114611.1 XM_114613.1 XM_05368.4 NM_024531.1 XM_113532.1 NM_0166458.2 NM_032687.1 XM_035370.1 XM_035341.1 NM_033301.1 XM_0987796.1 XM_05998.5 NM_013442.1 XM_048481.3 XM_04846.3 XM_114661.1 XM_04879.1 NM_003289.1 NM_006289.2 XM_048513.1 XM_048518.6 NM_000907.1 XM_005397.5 XM_048539.1 NM_007268.1 XM — 05516.4 XM_013042.3 XM_001466.8 XM_04655.1 NM_024602.1 XM_056881.6 XM_086536.4 NM_004073.1 XM_04557.1 NM_001012.1 XM_046551.2 NM_0244587.1 NM_018150.1 XM_1177688.1 XM_086370.4 XM_001605.9 NM_005727.1 XM_027983.5 XM_08634.2 NM_0166486.1 XM_055818.6 XM_086537.4 XM_032391.2 NM_006066.1 NM_002574.1 XM_032397.1 NM_05077.1 NM_012222.1 NM_032756.1 XM_114085.1 NM_006369.1 XM_097553.1 XM_059133.5 NM_0147874.1 NM_003975.1 NM_005973.3 XM_044077.3 XM_056380.2 XM_044080.2 XM_044083.5 NM_001878.2 XM_017040.3 NM_021948.2 XM_0865573.1 XM_0976566.1 XM_059219.2 NM_005920.1 XM_044217.8 XM_053245.6 XM_044148.3 NM_024897.1 NM_007221.1 XM_044155.1 XM_086562.1 NM_022367.1 NM_005572.1 XM_044166.3 XM_053243.1 XM_017041.3 XM_04128.6 NM_003145.2 XM_044172.7 XM — 010715.2 NM_006912.1 NM_032292.1 NM_017710.1 NM_033657.1 NM_018253.1 NM_01816.1 NM_0184849.1 XM_011513.3 NM_01428.1 NM_002044.1 NM_000298.1 XM_097660.1 XM_0865565.1 NM_003993.1 NM_005698.2 NM_006589.1 XM_084078.4 XM_086567.1 XM_086552.4 NM_007112.1 XM_053256.7 XM_114144.1 XM_086571.1 NM_018845.1 XM_113377.1 XM_010561'10 XM_117201.1 XM_036744.6 NM_1308098.1 NM_006694.1 NM_002870.1 NM_001030.2 XM_036829.6 NM_0155449.1 NM_0144847.1 NM_006118.2 XM_036933.7 XM_036934.4 XM_05737.4 XM_060121.4 NM_017582.2 NM_000748.1 XM_03645.4 XM_01789.1 NM_053052.1 XM_052974.5 NM_033445.1 XM_0474999.4 NM_000858.2 XM_059233.2 NM_0244319.1 XM_047545.6 XM_05684.3 XM_048859.6 NM_003739.3 XM_060976.1 NM_001818.1 XM_1655554.1 XM_1655555.1 NM_001494.2 XM_166138.1 XM_166136.1 NM_002189.1 NM_000417.1 NM_032905.1 XM_035380.1 XM_004330.4 XM_006297.4 NM_003646.1 XM_037205.4 XM_1137377.1 XM_1137378.1 NM_014342.1 NM_004551.1 NM_006560.1 XM_09647.1 XM_0946646.4 XM_006170.4 XM_037173.8 XM_006167.10. NM_003682.2 NM_005693.1 NM_001610.1 NM_000107.1 XM_089966.1 NM_01623.2 XM_037147.6 NM_02413.1 XM_1137379.1 XM_035037.1 XM_08770.2 XM_167726.1 NM_021727.2 XM_004265.2 XM_013402.3 XM_004111.4 XM_014206.1 XM_013279.1 XM_043146.5 XM_043151.7 XM_04288.6 NM_0166404.1 XM_11325.1 NM_004322.2 XM_1137375.1 XM_113222.1 NM_014067.2 XM_011988.9 XM_049178.6 NM_032344.1 NM_031472.1 XM_037808.4 NM_004074.1 XM_017481.6 NM_017670.1 XM_043159.1 XM_111323.1 NM_032251.1 NM_003942.1 XM_045642.3 XM_006533.7 XM_006532.9 XM_113676.1 XM_05613.6 XM_056112.4 NM_006244.1 XM_111324.1 XM_05498.5 XM_045499.3 NM_001997.2 XM_045525.7 NM_013265.2 XM_006529.8 XM_053787.6 XM_03776.2 XM — 165896.1 XM_084617.4 XM_04533.33.3 XM_057307.6 NM_001667.1 NM_138456.1 XM_115031.1 XM_084702.4 XM_03774.6 XM_053796.5 NM_002689.2 NM_006268.2 XM_1678044.1 XM_166931.1 XM_16635.1 XM_16636.1 NM_006842.1 NM_000852.2 NM_007103.2 XM_166192.1 NM_005851.2 XM_1655598.1 XM_1655599.1 XM_166194.1 XM_166190.1 NM_002496.1 NM_006019.2 NM_001734.1 NM_005768.3 NM_007273.2 NM_080549.1 NM_138425.1 XM_032588.5 NM_001975.2 NM_006992.1 NM_032641.1 NM_000365.2 XM_032614.1 NM_031299.2 NM_002075.1 NM_014262.1 NM_01958.1 XM_165910.1 XM_115192.1 XM_006980.5 NM_016319.1 XM_033223.5 NM_005439.1 NM_001273.1 XM_033253.4 NM_0155438.1 NM_002046.2 XM_033263.3 XM_006958.7 NM_016497.2 NM_016830.1 NM_001242.1 XM_115193.1 NM_002342.1 NM_001038.1 NM_001065.1 XM_033313.1 XM_016640.2 XM_096146.2 XM_090334.1 XM_084841.1 NM_017822.1 XM_084860.4 XM_028817.4 XM_02876.2 XM_006784.6 NM_014470.1 XM_0287760.3 NM_002733.1 XM_084866.2 XM_006826.2 XM_02862.3 XM_02373.2 NM_018319.2 NM_022054.2 NM_002802.1 XM_058677.2 NM_006888.1 XM_113759.1 NM_006427.2 NM_005163.1 XM_113760.1 XM_059346.3 XM_032235.5 XM_053817.4 XM_058640.5 NM_025100.1 NM_01795.1 XM_085103.4 XM_029340.6 XM_029344.6 XM_029342.5 XM_088588.4 XM — 060012.4 XM_115237.1 XM_115238.1 XM_090772.4 XM_115242.1 XM_085093.1 XM_0907688.1 XM_1133768.1 XM_090765.1 XM_115247.1 XM_115248.1 CM_090762.1 XM_063217.1 XM_115249.1 XM_090758.1 XM_115250.1 XM_090754.1 XM_03514.1 XM_113770.1 XM_08588587.4 XM_027520.2 NM_006428.1 XM_02749.2 NM_005009.1 NM_020664.1 NM_005632.1 XM_027458.2 NM_021168.1 XM_085422.1 NM_138418.1 XM_113865.1 NM_138769.1 XM_083939.1 NM_032259.1 XM_085426.1 NM_024042.1 XM_085413.1 NM_023933.1 XM_058770.1 NM_058192.1 XM_0274122.3 XM_0188104.2 XM_007786.7 NM_003933.3 XM_057058.3 NM_032520.1 XM — 117025.1 NM_024023.1 XM_058733.3 NM_001287.2 NM_016111.1 XM_034563.2 NM — 08018.2 XM_034570.2 XM_034571.2 NM_015133.1 XM_034586.3 NM_023936.1 XM_113869.1 XM_034588.4 XM_0345590.1 NM_005326.2 NM_031208.1 NM_016332.1 NM_004548.1 NM_002952.2 NM_006453.2 XM_05872.1 NM_004209.2 XM_034481.4 XM_0289999.4 NM_002528.3 XM_0289933.3 NM_032271.1 XM_0288833.4 XM_028881.5 XM_0969977.1 XM_083938.2 XM_028848.3 NM_006711.2 XM_102343.1 XM_028843.2 NM_017810.1 NM_003450.1 NM_005741.1 NM_003454.1 XM_036493.2 NM_003456.1 XM_064033.1 NM_004221.2 XM_0243439.1 NM_017885.1 NM_01639.1 NM_021195.2 NM_020982.1 NM_004203.1 XM_058784.3 NM_032296.1 XM_054962.3 NM_02219.1. XM_058782.2 NM_007108.1 XM_03528.2 XM_113289.1 NM_02518.1 XM_113290.1 XM_016980.4 XM_058783.3 NM_002613.1 XM_1655411.1 XM_01795.4 XM_115389.1 NM_0244793.1 XM_036660.2 NM_016292.1 NM_004380.1 XM_036671.1 XM_085445.2 XM_058781.3 XM_1133876.1 XM_117027.1 XM_165927.1 XM_053402.2 XM_055195.2 XM_113291.1 XM_03684.4 XM_1155423.1 NM_003955.1 XM_008142.6 XM_051959.3 XM_051961.3 XM_1155424.1 KM_113890.1 XM_017578.6 XM_054999.1 XM_008137.1 XM_165933.1 XM_0363.33.3 XM_058817.3 XM_008136.3 XM_113302.1 XM_113301.1 NM_006456.1 XM_036780.3 XM_091450.1 XM_03688.44.4 XM_058813.3 XM_03685.2 XM_097043.2 XM_037262.2 XM_0857717.1 NM_901168.1 XM_037196.3 XM_037195.2 XM_085533.2 XM_085706.1 XM_027039.2 XM_045104.3 NM_138973.1 XM_0857716.2 NM_020649.1 XM_115432.1 XM_008617.3 XM_113945.1 XM_012718.2 XM_04677.3 XM_119086.1 XM_096098.1 XM_046486.2 XM_085712.2 XM_046481.1 XM_097197.2 XM_097193.2 XM_008591.4 NM_004309.2 XM_046472.1 XM_097191.2 XM_113331.1 NM_016286.1 XM_113942.1 NM_004127.3 NM_022156.1 XM_113943.1 XM_113330.1 XM_117076.1 NM_006822.1 XM_08566.6 XM_08559.2 NM_004514.1 XM_113303.1 XM_117043.1 NM_031968.1 XM_083958.2 NM_022158.1 NM_005993.2 XM_165954.1 XM_041678.4 NM_003244.1 NM_003712.1 XM_041843.2 NM_0166585.1 XM_042009.4 XM_042012.2 NM_004359.1 XM_042018.1 XM_114018.1 XM_086110.1 NM_07876.1 XM_05711.3 NM_002579.1 XM_059034.1 NM_002819.2 NM_001972.1 XM_037560.3 NM_1387774.1 XM_03755.1 NM_033420.1 XM_092042.2 XM_037572.2 XM_055173.2 XM_03754.3 XM_009279.6 NM_002085.1 XM_009277.5 XM — 02913.4 NM_001687.1 XM_02807.2 XM_02804.3 XM_092031.1 XM_028060.4 XM_072012.3 NM_0244407.2 NM_000156.4 NM_01859.1 NM_001018.2 XM_04757.77.3 XM_086101.1 XM_086102.1 NM_138393.1 XM_0475833.1 XM_047584.2 NM_006830.2 XM_047600.2 NM_031918.1 NM_031213.1 NM_079834.1 NM_138422.1 NM_001319.4 NM_0177977.2 NM_017572.1 XM_059021.3 NM_003938.2 XM_046822.2 NM_0188049.1 NM_007165.1 NM_004152.1 NM_016199.1 XM_03698.55.3 NM_012458.1 Nu032737.1 XM_086115.1 XM_030485.3 XM_046934.3 NM_003021.2 XM_0568586 XM_024760.1 XM_009222.6 XM_052629.4 NM_009220.4 XM_072009.2 XM_052661.3 NM_005597.1 XM_096112.2 NM_016263.1 XM_012867.3 NM_006339.1 XM_114002.1 XM_047620.2 NM_01428.1 NM_004886.1 XM_059027.1 XM_056421.2 XM_031917.1 NM_001348.1 NM_001961.1 NM_0155898.1 XM_016871.6 NM_01639.1 NM_018074.1 NM_0244333.1 NM_017720.1 XM_031841.3 NM_003025.1 XM_084031.1 NM_025241.1 XM_059013.3 XM_092023.1 NM_052972.1 XM_059012.2 XM_035638.5 XM_010156.7 NM_016145.1 NM_032332.1 NM_001930.2 NM_032301.1 XM_009068.6 NM_0244038.1 XM_032041.2 XM_009065.5 NM_002229.1 XM_009063.3 NM_006397.1 NM_001375.1 NM_000159.1 NM_004461.1 XM_032020.1 NM_005053.2 XM_085916.4 XM_057080.2 XM_117130.1 NM_017722.1 NM_003765.1 XM_051364.2 NM_005234.2 NM_014173.1 XM_06157.1 XM_050617.1 NM_023937.1 XM_024050.1 XM_050644.1 XM_114020.1 NM_031310.1 NM_004335.2 XM_050660.3 XM_056925.1 NM_012088.1 XM — 07615.5 XM_03804.3 NM_018174.2 XM_03598.3 XM_03895.4 NM_000980.2 XM_055230.3 NM_0155683.1 XM_012179.5 XM_038150.2 XM_009293.5 XM_038146.4 ' NM_032683.1 NM_002866.1 XM_038114.4 XM_065046.1 XM_086040.1 XM_084038.1 XM_092046.1 NM_012321.1 NM_031425.1 XM_03898.1 NM_032627.1 XM_114004.1 NM_006532.1 NM_012181.2 NM_024069.1 NM_003333.2 NM_01797.1 NM_002911.2 NM_007263.1 NM_019070.1 NM_004838.1 XM_097399.1 XM_086055.2 NM_033415.2 XM_009330.7 NM_005919.1 XM_086049.1 NM_003721.2 XM_072027.3 XM_114009.1 XM_048457.1 XM_114008.1 NM_0155965.2 XM_013176.3 NM_004647.1 XM_032281.4 XM_085984.2 XM_032285.3 NM_004823.1 NM_002812.1 XM_056604.3 XM_02955.3 XM_029450.1 NM_004924.2 XM_085989.2 XM_1155619.1 NM_006149.2 XM_008904.4 NM_001533.1 NM_012237.2 NM_002503.1 NM_01727.2 NM_021107.1 NM_005884.2 XM_092066.1 XM_009225.5 NM_0188028.1 XM_046090.2 XM_167529.1 XM_086004.2 XM_097361.2 NM_003407.1 XM_046114.3 NM_001020.2 NM_003169.2 XM_053074.4 NM_016941.1 NM_004714.1 NM_001436.2 XM_016410.2 XM_096107.1 XM_012860.4 NM_006503.1 XM_042665.2 XM_009203.6 XM_059053.5 XM_047409.3 NM_013376.1 NM_013368.2 NM_000713.1 XM_04376.6 XM_047374.5 XM_097289.1 NM_004756.1 NM_024876.1 NM_0254.14.1 NM_004596.1 NM_016154.1 NM_017555.1 NM_000762.3 NM_000764.2 NM_000767.3 NM_000774.3 NM_0306022.4 XM_0155505.1 XM_030914.1 NM_052848.1 XM_008912.1 XM_085882.1 NM_030578.1 XM_058950.4 NM_020158.1 XM_0300901.1 NM_01835.1 XM_088574.1 XM_012777.2 XM_1133346.1 XM — 15603.1 NM_001022.2 NM_004706.2 NM_006423.1 NM_022752.1 NM_1333328.1 XM_057401.6 NM_006494.1 XM_064875.2 NM_015512. NM_002573.2 NM_09108.1 NM_002659.1 XM_059019.3 NM_0244327.1 XM_059018.3 XM_009200.3 XM_086014.2 NM_01427.1 NM_014400.1 XM_1133353.1 NM_003009.1 XM_049920.1 XM — 05589.2 XM_064954.1 XMF008860.6 XM_05586.5 XM_085929.4 XM_009036.3 NM_017854.1 XM_085943.4 XM_08263.1 XM_085928.1 NM_013403.1 NM_016457.2 NM_001743.3 XM_085951.1 XM_031307.4 NM_032040.1 NM_006247.2 XM_0589944.3 XM_046390.3 NM_016440.1 XM_117132.1 NM_012068.2 XM_085927.2 XM_084023.1 XM_016217.6 XM_114025.1 XM_008985.6 NM_017432.1 XM_046344.2 NM_030973.1 NM_02519.2 NM_014203.2 XM_085934.1 NM_001536.1 XM_165432.1 NM_001571.1 XM_046313.2 NM_006270.2 XM_046305.2 NM_0159593.2 XM_057199.3 XM_027904.5 XM_084026.2 NM_012423.2 XM_058991.3 NM_017916.1 NM_020309.1 XM_085941.2 003598.1 NM_001774.1 XM_057188.1 XM_02783.2 NM_018111.1 NM_022165.1 XM_085942.2 NM_006179.2 NM_006666.1 XM_114024.1 NM_000146.2 NM_004324.1 NM_006184.2 XM_009097.4 NM_020904.1 NM_001190.1 NM_017805.1 NM_000511.1 XM_009103.5 XM_049974.5 NM_020126.2 NM_000979.2 XM_009112.4 XM_09964.2 XM_049963.4 NM_031485.1 NM_006801.1 XM_012808.5 NM_001425.1 XM_04939.3 XM_039938.3 XM_009118.9 NM_138412.1 XM_009161.8 XM_092006.2 XM_085976.2 NM_003283.1 XM_012843.3 NM_014931.1 XM_035919.2 XM_035920.4 NM_032701.1 XM_008906.2 NM_000991.2 NM_014501.1 NM_013333.1 NM_007279.1 NM_01635.1 XM_097308.1 XM_034409.2 XM_034377.3 XM_05894.2 NM_0244710.1 XM_085882.1 XM_084011.1 NM_003969.1 NM_0144453.1 NM_005762.2 NM_018337.1 NM_003091.1 NM_052881.1 XM_04914.1 XM_04915.5 NM_006899.1 NM_022760.2 NM_002836.2 NM_0308111.2 NM_021826.1 XM_009582.2 NM_023935.1 XM_05418.2 XM_086711.1 XM_045431.3 XM_086708.1 NM_052970.2 NM_017874.1 XM_045451.2 XM_045460.1 XM_05893.3 NM_003098.2 XM_047042.2 XM_056253.3 XM_097772.1 NM_007238.1 NM_032819.2 XM_059272.3 NM_016732.1 NM_003908.1 XM_012928.4 NM_000687.1 NM_031483.1 NM_014183.1 NM_032514.1 XM_114179.1 XM_0867728.1 XM_04715.2 NM_052830.1 NM_01867.1 NM_000178.1 XM_047196.4 NM_0155638.1 NM_018217.1 NM_006444.1 XM_086729.1 XM_072161.3 NM_002212.1 NM_018244.1 NM_000557.2 NM_007186.1 NM_01566.1 NM_0247477.1 NM_003915.1 XM_047271.2 XM_047273.2 XM_113380.1 NM_004902.1 NM_092776.1 XM_1659988.1 NM_000100.2 XM_03599.3 NM_020132.1 NM_031487.1 XM_009794.5 XM_036011.2 NM_004649.1 XM_036042.5 NM_004928.1 XM_114196.1 XM_036087.1 XM_009805.6 NM_004339.2 XM_036107.2 XM_092854.1 XM_086781.1 NM_001112.1 XM_03165.3 XM_033017.3 XM_057166.4 XM_086807.2 XM_167538.1 XM_086802.1 NM_005675.2 XM_033048.3 NM_022719.1 XM_033090.4 XM_033096.4 NM_003776.1 XM_055490.4 NM_003504.2 NM_003277.1 NM_002688.2 NM_080647.1 NM_053004.1 XM_058918.2 XM_097833.1 NM_024627.2 NM_006440.1 NM_000754.2 KM_033900.1 XM_059310.3 NM_022727.2 NM_002882.2 XM_033828.2 NM_023004.2 NM_033257.2 XM_03629.1 XM_03670.30.3 NM_032775.1 XM_08814.2 XM_0182895.2 XM_09785.1 XM_086876.2 NM_002650.1 XM_009850.5 NM_004782.2 NM_005207.1 XM_097841.1 XM_009853.5 NM_030573.1 XM_054591.3 XM_03681.1 XM_03692.3 XM_104587.2 XM_086827.1 XM_059324.1 XM_096155.2 XM_009885.6 XM_086955.2 XM_096156.2 XM_086824.2 XM_167540.1 NM_003347.1 XM_0978886.2 NM_022044.1 NM_0144337.1 XM_059326.3 XM_097870.1 XM_03697.3. NM_0144634.1 XM_03782.3 NM_005080.2 XM_03788.77.3 NM_012265.1 NM_005243.1 NM_006478.2 XM_009905.7 XM_037942.2 XM_03795.4. XM_037947.1 NM_0268.1 XM_092894.2 NM_09103.1 NM_013387.1 NM_032204.2 NM_021090.1 XM_066291.1 XM_009915.6 NM_020530.2 NM_031937.1 NM_005877.2 XM_11724.1 NM_016498.1 NM_0144303.1 NM_000355.1 XM_086838.1 XM_086863.1 XM_097857.1 NM_014941.1 NM_017903.1 NM_134269.1 NM_080430.1 XM — 08516.4 NM_0166733.1 NM_01423.2 NM_004147.1 NM_019843.2 XM_0385520.4 NM_0144338.2 XM_097868.1 NM_014662.1 XM_056165.2 XM_01839.4 NM_020839.1 XM_114334.1 XM_059521.2 XM_047503.3 XM_047502.5 XM_08839.4 XM_08416.4 NM_002295.2 XM_076414.5 XM_116066.1 XM_093546.4 XM_114314.1 XM_093423.1 XM_002914.6 XM_02936.5 XM_08324.4 NM_006232.2 XM_029131.5 XM_029132.6 XM_0290104.4 XM_08410.1 XM_09810.1 XM_029078.5 NM_005787.1 NM_032331.1 NM_002754.2 NM_001315.1 NM_003137.2 NM_004117.2 XM_166419.1 NM_007104.3 XM_165760.1 NM_022047.1 XM_166399.1 NM_030900.1 NM_031443.1 XM_168417.1 NM_012412.2 NM_021130.1 NM_031449.1 XM_165877.1 XM_1666556.1 NM_019082.1 NM_015332.1 XM_0155258.2 XM_1666566.1 XM_165870.1 NM_000162.2 XM_1655863.1 NM_006230.1 XM_165882.1 XM_1655867.1 XM_1658583.1 NU14063.2 XM_168433.1 NM_01593.2 166557.1 NM_0320141 XM_05574.3 XM_050586.1 XM_114560.1 XM_018359.5 XM_050607.1 XM — 088246.1 XM_088240.1 XM_042927.3 NM_0188044.1 NM_032408.1 NM_001707.2 NM_032988.1 NM_032951.1 NM_032317.1 NM_017528.1 NM_004603.1 NM_031295.1 NM_001306.1 XM_05796.2 NM_0000501.1 NM_002314.2 NM_022170.1 NM_022040.2 XM_004901.3 XM_095287.2 XM_088090.2 XM_168472.1 XM_088828.1 XM_049100.4 NM_018991.1 XM_034751.1 XM_167500.1 NM_005395.1 XM_1675404.1 XM_167498.1 XM_16844.1 NM_031925.1 XM_1674999.1 NM_005918.1 XM_170385.1 NM_001540.2 XM_168538.1 NM_080744.1 NM_007155.1 XM_050377.4 NM_012230.1 XM_170389.1 XM_170390.1 XM_16847.1 XM_088067.2 XM_051298.5 XM_08837.4 XM_088374.4 NM_052963.1 NM_138465.1 XM_088298.4 XM_049247.2 XM_05868.2 XM_088829.1 NM_0244736.2 NM_003313.2 NM_023078.1 XM_1174787.1 XM_049226.6 NM_013432.2 NM_016208.1 XM_005146.8 XM_016270.8 NM_138431.1 XM_1135333.1 XM_114611.1 XM_114613.1 XM_05368.4 NM_024531.1 NM_0244555.2 XM_113532.1 NM_0166458.2 XM_035348.3 XM_117492.1 XM_03573.6 NM_032687.1 XM_035350.1 XM_035370.1 NM_030580.1 NM_033301.1 XM_070951.2 XM_070950.1 XM_1146799.1 XM_1146788.1 XM_114677.1 XM_05967.5 NM_000368.1 NM_01956.2 XM_027836.1 NM_022779.7 XM_033355.1 NM_0142.85.2 XM_033327.2 NM_054012.1 XM_0958887.1 NM_001807.1 NM_001808.1 NM_006753.2 XM_035103.4 NM_000972.2 NM_003172.2 NM_017503.2 NM_033161.2 NM_020355.1 NM_017586.1 NM_017585.2 XM_039282.2 NM_019056.2 NM_005676.2 NM_003334.1 NM_033018.1 XM_010378.4 XM_033888.3 XM_05558.3 XM_033884.2 NM_1333499.1 XM_033878.1 XM_033862.1 XM_039450.1 NM_002436.2 NM_001363.2 NM_080612.1 NM_00303639.1 XM_049337.1 NM_021806.1 NM_01425.1 NM_006014.1 XM_010150.3 NM_004699.1 XM_010152.4 NM_001183.2 XM_010154.1 XM_048420.1 NM_006013.2 XM_0484410.1 XM_048404.2 NM_004992.2 NM_001569.2 NM_003492.1 XM_048390.3 NM_003491.1 NM_001666.1 NM_000425.2 NM_006280.1 NM_004135.1 NM_014370.1 NM_0000033.2 NM_005745.3 XM_048315.1 XM_042155.3 XM_042154.3 XM_042153.4 NM_007205.2 XM_059930.5 NM_0147874.1 NM_003975.1 NM_005973.3 XM_044077.3 XM_056380.2 XM_044080.2 XM_044083.5 NM_001878.2 XM_017040.3 XM_0865573.1 XM_0976566.1 XM_059219.2 NM_005920.1 XM_044217.8 XM_053245.6 XM_044148.3 NM_024897.1 NM_007221.1 XM_044155.1 NM_022367.1 NM_005572.1 XM_053243.1 XM_017041.3 XM_04128.6 NM_003145.2 XM_044172.7 XM — 010715.2 NM_032292.1 NM_017710.1 NM_033657.1 NM_018253.1 NM_01816.1 NM_0184849.1 NM_01428.1 NM_002044.1 NM_003993.1 NM_005698.2 NM_006589.1 XM_086567.1 XM_086552.4 NM_007112.1 XM_053256.7 XM_114144.1 NM_018845.1 XM_113377.1 NM_004952.2 XM_010561.10. XM_036744.6 NM_006694.1 NM_002870.1 NM_001030.2 XM_036829.6 NM_0155449.1 NM_0144847.1 NM_006118.2 XM_036934.4 NM_017582.2 XM_03645.4 XM_1677879.1 XM_167437.1 XM_167871.1 XM_029347.7 XM_167880.1 XM_054856.2 XM_035380.1 XM_004330.4 XM_006296.1 XM_006297.4 NM_003646.1 XM_037205.4 CM_1137378.1 NM_014342.1 NM_004551.1 XM_037142.5 XM_054936.3 NM_006560.1 XM_09647.1 XM_0946646.4 XM_006170.4 XM_037173.8 NM_003682.2 NM_005693.1 NM_001610.1 NM_000107.1 XM_089966.1 NM_01623.2 XM_037147.6 NM_02413.1 XM_043146.5 XM_043151.7 XM_04288.6 NM_0166404.1 NM_004322.2 XM_113222.1 NM_014067.2 XM_011988.9 XM_049178.6 NM_032344.1 NM_031472.1 XM_037808.4 NM_004074.1 XM_017481.6 NM_017670.1 XM_043159.1 XM_111323.1 NM_003942.1 NM_005825.2 NM_005609.1 XM_045642.3 XM_006533.7 XM_006532.9 XM_05613.6 XM_056112.4 XM_111324.1 XM_05498.5 XM_045499.3 NM_001997.2 XM_045525.7 NM_013265.2 XM_006529.8 XM_053787.6 XM_03776.2 XM_084716.1 XM_084617.4 XM_04533.33.3 NM_001667.1 NM_138456.1 XM_115031.1 XM_03774.6 NM_002689.2 NM_006268.2 XM_1678044.1 XM_166931.1 XM_16635.1 XM_16636.1 NM_006842.1 NM_000852.2 NM_007105.2 NM_005851.2 XM_1655598.1 XM_1655599.1 XM_1655.94.1 XM_166190.1 NM_002496.1 NM_006019.2 XM_165601.1 NM_017635.1 NM_016028.2 NM_022338.1 NM_002335.1 NM_018312.2 XM — 165597.1 XM_006936.4 XM_006925.6 XM_099660.1 NM_002355.2 XM_051166.6 XM_099664 XM_0155481.7 XM_049148.2 XM_1133726.1 XM_049141.1 XM_096612.4 NM_021934.1 XM_083884.1 NM_007210.2 NM_016293.1 NM_020467.2 NM_002272.1 XM_058525.5 NM_002273.1 NM_000224.1 XM_029728.3 NM_01599.2 XM_0155468.1 XM_012162.4 NM_002178.1 XM_09631.2 NM_0188507.1 XM_071646.5 NM_01565.1 XM_012212.3 XM_02598.1 NM_020898.1 XM_028621.1 XM_02819.1 XM_05855.2 NM_014311.1 XM_096606.2 XM_084873.2 NM_01847.1 NM_001920.2 NM_002345.1 XM_167518.1 XM_031617.1 NM_007223.1 XM_031612.5 NM_003134.1 XM — 113826.1 XM_08530.1 XM_031561.3 NM_002225.2 NM_014952.1 NM_1304688.1 XM_031555.3 XM_031554.6 XM_031515.4 NM_018145.1 NM_005258.2 NM_018163.1 NM_032850.1 XM_031510.4 NM_13339.2 NM_080432.1 NM_024111.1 NM_032196.1 XM_113821.1 XM_031641.3 NM_0166359.1 XM_031645.1 XM_031646.3 XM_031674.5 XM_007545.3 XM_091159.1 XM_031689.6 NM_005090.1 XM_031718.4 XM_031720.6 NM_0155497.1 XM_085314.4 NM_022473.1 NM_003825.2 XM_01593.63.6 NM_033238.1 NM_004809.2 XM_012425.7 NM_005545.1 NM_032907.1 XM_058699.5 XM_115295.1 NM_016645.1 NM_006384.2 XM_044533.6 XM_011655.9 XM_054900.6 XM_035835.4 NM_032162.1 XM_040592.3 XM_091193.2 XM_0853255.1 XM_008040.7 XM_054590.1 NM_002461.1 XM_057150.3 XM_058739.2 XM_0283593 XM_0853272. NM_000485.1 XM_007803.3 NM_016209.1 NM_005187.3 XM_007909.2 NM_024043.1 NM_017702.1 XM_047436.2 NM_002386.1 NM_014972.1 XM_047462.4 NM_000135.1 XM_047520.3 XM_012545.6 XM_047525.4 NM_033251.1 NM_003119.1 XM_085409.2 XM_085408.2 XM_113861.1 XM_117012.1 XM_058830.3 NM_002809.1 XM_04980.3 NM_007359.2 XM_1139399.1 XM_008586.5 XM_1133329.1 XM_083987.2 NM_000422.1 XM_008579.4 NM_002276.1 NM_002275.1 NM_002274.1 NM_000526.3 NM_000526.3 XM_049982.1 XM_085732.2 XM_049972.4 NM_003079.2 XM_04999.1 XM_049935.2 XM_039937.1 XM_1133334.1 NM_000964.1 NM_001254.2 XM_085731.2 NM_006455.1 XM_036465.1 XM_055061.1 XM_036462.1 XM_083995.1 XM_008290.2 XM_04854.3 XM_008633.4 XM_008632.5 NM_02517.1 NM_024320.1 XM_054038.4 XM_058883.1 NM_0144726.1 NM_002265.3 NM_002145.1 XM_008561.1 XM_008560.6 XM_008559.1 NM_024017.2 XM_115480.1 NM_005831.2 XM_1131318.1 NM_031918.1 NM_031213.1 NM_079834.1 NM_001319.4 NM_0177977.2 NM_017572.1 XM_059021.3 NM_003938.2 XM_046822.2 NM_0188049.1 NM_007165.1 NM_004152.1 NM_016199.1 XM_03698.55.3 NM_012458.1 NM_032737.1 XM_086115.1 XM_030485.3 XM_08123.2 XM_046934.3 NM_003021.2 XM_0568586 XM_086121.2 XM_052629.4 XM_052629.4 XM_052661.3 NM_005597.1 NM_016263.1 XM_012867.3 NM_006339.1 XM_114002.1 XM_047620.2 NM_004886.1 XM_059027.1 NM_001348.1 NM_001961.1 NM_016149.1 XM_016871.6 NM_01639.1 NM_005755.2 XM_031841.3 NM_003025.1 XM_084031.1 NM_025241.1 XM_059013.3 XM_059012.2 XM_035638.5 XM_05636.3 XM_05635.2 XM_035634.2 XM_012862.3 XM_05627.2 XM_035625.5 XM_050561.6 XM_008930.4 XM_050564.2 XM_008854.7 NM_005234.2 NM_014173.1 XM_050617.1 NM_023937.1 NM_024050.1 NM_0313_031310.1 NM_004335.2 XM_050660.3 XM_056925.1 NM_012088.1 XM — 07615.5 NM_018174.2 XM_03598.3 NM_000980.2 XM_055230.3 NM_0155683.1 XM_012179.5 XM_009293.5 XM_038146.4 NM_032683.1 XM_086040.1 XM_038109.3 XM_092046.1 NM_012321.1 XM_03898.1 NM_032627.1 XM_114004.1 NM_006532.1 NM_012181.2 NM_024069.1 NM_003333.2 NM_01797.1 NM_012109.1 NM_018316.1 NM_025021.1 XM_009336.3 NM_002911.2 NM_021267.1 NM_007263.1 NM_019070.1 NM_004838.1 XM_086055.2 NM_033415.2 XM_009330.7 NM_005919.1 NM_003721.2 XM_072027.3 XM_114009.1 XM_048457.1 XM_114008.1 NM_0155965.2 NM_0166573.1 NM_020410.1 NM_004647.1 NM_033256.1 XM_032281.4 XM_085984.2 XM_032285.3 NM_004823.1 NM_002812.1 XM_085985.1 XM_085987.1 XM_02955.3 XM_029450.1 NM_004924.2 XM_085989.2 XM_1155619.1 NM_006149.2 XM_008904.4 NM_001533.1 XM_059046.3 NM_012237.2 NM_002503.1 NM_01727.2 NM_021107.1 NM_024907.4 XM_059045.1 NM_005884.2 XM_092067.1 XM_009225.5 NM_0188028.1 XM_046090.2 XM_167529.1 XM_086004.2 XM_097361.2 NM_003407.1 XM_046114.3 NM_001020.2 NM_003169.2 XM_053074.4 NM_016941.1 XM_0720077.2 NM_004714.1 NM_001436.2 XM_016410.2 XM_012860.4 NM_006503.1 XM_042665.2 NM_024877.1 XM_009203.6 XM_047409.3 NM_013376.1 NM_000713.1 XM_04376.6 XM_047374.5 NM_004756.1 NM_024876.1 NM_0254.14.1 XM_059052.5 NM_004596.1 NM_017555.1 XM_0155505.1 XM_030914.1 NM_052848.1 CM_008912.1 XM_085882.1 NM_020158.1 NM_01835.1 XM_088574.1 XM_009170.3 XM_012777.2 XM_1133346.1 XM_115603.1 NM_001022.2 NM_001783.1 NM_004706.2 NM_006423.1 NM_022752.1 NM_1333328.1 XM_057401.6 NM_006494.1 NM_015512. NM_002573.2 XM_0298833.4 NM_005357.1 XM_058945.2 XM_113980.1 XM_054350.3 XM — 113354.1 NM_003425.1 XM_092027.1 NM_002250.1 NM_09108.1 NM_002659.1 XM_059019.3 XM_009200.3 XM_086014.2 NM_01427.1 XM_04915.4 NM_014400.1 NM_003009.1 XM_049920.1 XM_055589.2 XM_009125.1 XM_008860.6 XM_05586.5 XM_085929.4 NM_014681.2 XM_084025.1 XM_009036.3 XM_08253.1 NM_017854.1 XM_085943.4 XM_08263.1 XM_085928.1 NM_013403.1 NM_016457.2 NM_001743.3 XM_009126.2 NM_032040.1 NM_006247.2 NM_002516.1 XM_064921.1 XM_051330.4 XM_051332.1 NM_004819.1 XM_02759.1 XM_027572.1 NM_004597.3 NM_000164.1 XM_097338.4 NM_012155.1 NM_02536.1 XM_085848.1 NM_003370.1 NM_005619.1 XM_009143.2 NM_001983.1 NM_012099.1 XM_030918.4 NM_040400.1 XM_030967.5 NM_031417.1 NM_024108.1 XM_091899.4 NM_007056.1 XM_008848.4 NM_001294.1 NM_000041.1 NM_006114.1 NM_002856.1 NM_005581.1 NM_012116.2 XM_008851.7 NM_0065055.2 XM_165962.1 NM_016440.1 NM_012068.2 XM_085927.2 XM_084023.1 XM_016217.6 XM_114025.1 XM_008985.6 NM_017432.1 XM_046344.2 NM_030973.1 NM_02519.2 NM_014203.2 NM_021733.1 XM_085934.1 NM_001536.1 XM_165432.1 NM_001571.1 XM_046313.2 NM_006270.2 XM_046305.2 NM_0159593.2 XM_027904.5 XM_084026.2 NM_012423.2 XM_02799.5 XM_058991.3 NM_017916.1 NM_003598.1 NM_001774.1 XM_057188.1 XM_02783.2 NM_022165.1 XM_085942.2 NM_006666.1 XM_114024.1 NM_000146.2 NM_004324.1 NM_006184.2 XM_009097.4 NM_020904.1 NM_001190.1 XM_049974.5 NM_020126.2 NM_000979.2 XM_009112.4 XM_09964.2 XM_049963.4 NM_031485.1 NM_006801.1 XM_012808.5 NM_001425.1 XM_039938.3 XM_009118.9 XM_055864.4 XM — 113344.1 XM_046419.3 NM_002691.1 XM_052686.2 XM_058990.5 NM_13834.1 XM_091981.4 NM_002975.1 NM_032712.1 NM_002257.1 NM_005046.1 XM_009004.4 XM_091941.1 NM_138373.1 NM_004542.1 NM_0155629.1 XM_008958.5 XM_085896.4 XM_050236.6 XM_085899.4 XM_050589.5 XM_114013.1 XM_059321.2 XM_050582.4 XM_01797.4 XM_055900.2 XM_044313.4 XM_059049.2 NM_138412.1 XM_009161.8 XM_092006.2 NM_003283.1 NM_000363.1 XM_012840.2 NM_014931.1 XM_035919.2 NM_032701.1 XM_058948.3 NM_000991.2 NM_014501.1 NM_013333.1 NM_007279.1 NM_013301.1 NM_01635.1 NM_016202.1 XM_091947.4 XM_113986.1 XM_034377.3 NM_0244710.1 NM_003650.2 XM_042765.3 XM_042770.2 NM_001247.1 XM_009436.7 XM_042805.4 XM_086723.1 XM_0422806.3 XM_042807.3 NM_06115.2 BAA20895.1 BAA2204.1 BAA2002.1 BAA2002.1 BAA20016.1 XM_037060.4 NM_006478.2 XM_03795.4. XM_037947.1 NM_0268.1 NM_09103.1 NM_013387.1 NM_032204.2 XM_066291.1 XM_009915.6 NM_031937.1 NM_005877.2 NM_012429.1 NM_016498.1 NM_0144303.1 NM_000355.1 XM_086863.1 NM_014941.1 NM_017903.1 NM_134269.1 NM_080430.1 XM — 08516.4 NM_0166733.1 NM_052880.1 XM_03805.4 NM_005488.1 NM_002133.1 XM_0997.47.4 NM_030641.1 XM_08688.2 NM_0144309.1 NM_030882.1 NM_003661.1 NM_002473.1 NM_012473.1 XM_11207.1 NM_003753.1 NM_006860.2 NM_003312.2 NM_02116.2 XM_08817.1 NM_024681.1 NM_031910.1 XM_084086.1 NM_002405.1 NM_014550.2 XM — 009967.4 NM_013365.2 NM_01857.2 NM_020315.2 NM_0023055.2 NM_02433.1 NM_007032.2 XM_0993.33.3 NM_138977.1 NM_016091.1 XM_03936.3 NM_032561.1 NM_021974.2 NM_012407.1 NM_012323.1 NM_012264.2 NM_001894.1 XM_0868599.2 NM_006855.2 NM_000001001 NM_0155705.1 NM_020831.1 NM_003932.2 NM_022098.1 NM_0144248.1 XM_086844.1 XM_010013.4 XM_114201.1 NM_002883.1 NM_017590.1 NM_003216.1 NM_001098.1 XM_03448.4 NM_002676.1 XM_04395.2 NM_001469.2 NM_005008.1 NM_04821.1 NM_004599.1 NM_024053.1 XM — 01004.3 NM_0149966.2 XM_010953.4 NM_005793.1 XM_051491.5 NM_024661.1 NM_0155933.1 NM_130384.1 XM_003301.5 XM_051518.6 NM_022911.2 NM_003365.1 XM_030109.5 XM_0300060.1 NM_004157.1 XM_003288.8 XM_08412.1 XM_047561.3 XM_116853.1 XM_114325.1 XM_113405.1 XM_028610.2 XM_002828.6 XM_002829.6 XM_01823.6 NM_022171.1 NM_000481.1 XM_011068.7 NM_032355.1 NM_005777.1 NM_005778.1 XM_041509.1 XM_041507.1 NM_004636.1 NM_006764.1 NM_003549.2 NM_012191.1 NM_018130.12 NM_013336.1 XM_114309.1 XM_030286.3 NM_0244047.1 NM_007024.2 NM_016210.1 XM_10409930.2 NM_004635.2 XM_084158.5 XM_093546.4 NM_080685. XM_032759.2 XM_067264.4 XM_041971.1 XM_084111.4 XM_041981.7 XM_1133397.1 XM_055551.3 XM_096203.4 NM_006058.2 NM_002084.1 NM_016245.1 XM_018009.5 XM_117396.1 NM_0188047.1 NM_005617.2 XM_003937.4 XM_041974.3 XM_041978.3 NM_006913.3 XM_017591.4 NM_022110.1 NM_000592.2 NM_006929.1 XM_041993.1 NM_002944.1 NM_007286.1 NM_001710.2 NM_006709.5 NM_000176.1 XM_05676.2 NM_025257.1 NM_000434.2 NM_005345.3 NM_032454.1 NM_005510.1 XM_1663488.2 NM_007312.3 NM_033158.2 NM_007275.2 NM_007182.1 NM_007022.1 NM_005346.2 NM_007024.3 XM_076414.1 NM_021177.1 NM_006295.1 NM_001288.3 NM_013974.1 NM_021160.1 NM_001320.2 NM_033177.2 XM_165712.1 NM_004639.2 NM_080686.1 NM_002341.1 XM_165771.1 NM_004640.3 NM_005931.1 XM_165832.1 XM_1655765.1 XM_087945.1 XM_166381.1 NM_080870.1 XM_167116.1 XM_166401.1 NM_001517.1 XM_1655738.1 NM_003897.2 NM_005803.2 XM_087939.2 XM_1655734.1 XM_1666353.1 XM_1667376.1 XM_165782.1 XM_166409.1 NM_0144046.2 XM_1655799.1 NM_001090.1 XM_166361.1 XM — 165764.1 XM_1655758.1 NM_003449.2 NM_05236.1 NM_021959.1 NM_014596.2 XM_165809.1 XM_167122.1 NM_018950.1 XM_166392.1 XM_167128.1 XM_166550.1 NM_001101.2 NM_003088.1 NM_019011.2 XM_1658588.1 NM_000535.2 NM_006303.2 NM_014413.2 NM_032706.1 NM_006908.2 XM_1666545.1 NM_006854.2 NM_017756.1 NM_0188106.1 NM_024067.1 XM_05724.4 XM_1655859.1 XM_055725.1 XM_030750.5 XM_1113547.1 XM_051298.5 XM_08837.4 XM_030743.7 NM_052963.1 XM_13534.1 NM_138465.1 XM_088298.4 XM_049247.2 XM_05868.2 XM_088829.1 NM_0244736.2 NM_003313.2 NM_023078.1 NM_032862.1 XM_1174787.1 XM_049226.6 NM_016208.1 XM_005146.8 XM_016270.8 NM_004260.1 XM_1135333.1 XM_059915.5 XM_114613.1 XM_05368.4 NM_024531.1 NM_0244555.2 XM_113532.1 XM_035348.3 XM_117492.1 XM_114612.1 XM_03573.6 NM_032687.1 XM_035350.1 XM_035341.1 NM_033301.1 NM_003416.1 NM_014066.2 XM_059913.5 NM_001539.1 NM_01825.1 XM_036235.1 NM_004323.2 NM_016410.2 XM_036219.5 XM_005543.8 NM_022917.2 XM_005545.4 NM_017811.1 XM_036125.2 XM_036284.5 XM_084277.1 NM_001161.1 NM_001161.1 XM_088504.1 NM_0244348.1 NM_005866.1 NM_000155.1 NM_002989.1 XM_071063.2 XM_016894.3 XM_05998.5 NM_004629.1 NM_032634.1 NM_013442.1 NM_025182.1 XM_005627.7 XM_048481.3 XM_04846.3 XM_114661.1 XM_04879.1 NM_001216.1 NM_003289.1 NM_006289.2 XM_048513.1 XM_048518.6 XM_005394.2 NM_000907.1 XM_005397.5 NM_032593.1 XM_048539.1 NM_021111.1 NM_007096.1 XM_045037.1 XM_045035.1 NM_002436.2 NM_001363.2 NM_003639.1 XM_049337.1 NM_021806.1 NM_019848.2 NM_01425.1 NM_006014.1 XM_010150.3 NM_004699.1 XM_010152.4 NM_001183.2 XM_010154.1 XM_048420.1 NM_006013.2 XM_0484410.1 XM_048404.2 NM_004992.2 NM_001569.2 NM_003492.1 XM_048390.3 NM_003491.1 NM_001666.1 NM_0010054.2 NM_000425.2 NM_006280.1 NM_004135.1 NM_0000033.2 NM_005745.3 XM_048315.1 XM_042155.3 XM_042153.4 NM_007205.2 NM_018196.1 NM_001289.3 XM_0600454.2 XM_084297.2 XM_001472.4 XM_055481.5 XM_044474.1 XM_001710.5 XM_055482.1 XM_114894.1 NM_014000.1 XM_011882.8 XM_11172.12.1 XM_0894438.4 NM_033022.1 NM_007055.1 XM_096398.1 XM_011913.3 XM_005895.7 NM_003375.1 XM_058351.5 NM_032772.1 XM_046668.4 XM_046674.4 XM_01625.4 XM_002674.7 NM_032775.1 XM_0182895.2 NM_032262.1 NM_002650.1 NM_004782.2 XM_097841.1 NM_030573.1 XM_059324.1 XM_096155.2 XM_096156.2 XM_086824.2 XM_114202.1 XM_167540.1 NM_003347.1 XM_0978886.2 NM_022044.1 NM_0144337.1 XM_059326.3 XM_097870.1 NM_0155705.1 NM_020831.1 XM_039410.1 NM_003932.2 NM_022098.1 XM_086848.1 NM_0144248.1 XM_086844.1 XM_010013.4 XM_114201.1 NM_002883.1 NM_017590.1 NM_003216.1 NM_016272.1 NM_032758.1 NM_001098.1 XM_03448.4 NM_002676.1 NM_001469.2 NM_005008.1 XM_039501.2 NM_0244588.1 NM_04821.1 NM_004599.1 NM_024053.1 XM_010024.3 NM_033318.1 NM_002490.2 XM_040050.3 XM — 04006.33 XM_013015.3 NM_014509.2 XM_084084.1 XM — 01008.3 XM_076414.5 XM_116066.1 XM_093546.4 XM_116067.1 XM_116069.1 NM_018130.1 XM_051902.4 XM_116410.1 XM_098401.1 XM_027156.3 NM_006999.2 NM_1388809.1 XM_052313.5 XM_059691.3 XM_003862.5 XM_07592.4 XM_0166170.6 XM_030424.3 XM_113451.1 XM_1655474.1 XM_1683838.1 NM_01560.10.1 XM_1666549.1 XM_166550.1 NM_001101.2 NM_003088.1 XM_1658588.1 NM_000535.2 NM_006303.2 NM_014413.2 NM_032706.1 NM_006908.2 XM_1666545.1 NM_006854.2 NM_017756.1 NM_0188106.1 XM — 166538.1 XM_037224.3 XM_1655859.1 XM_167302.1 XM_055725.1 XM_167304.1 XM_16621.1 XM — 166512.1 NM_1019059.1 XM_167279.1 XM_088260.1 NM_003406.1 XM_011657.3 NM_002568.1 XM_035046.2 XM_070950.1 XM_1146799.1 XM_1146788.1 XM_114677.1 XM_05967.5 NM_032360.1 XM_114109.1 NM_002826.2 XM_113370.1 NM_019852.1 NM_0144828.1 XM_033511.6 NM_007192.2 XM_012376.6 XM_08515.1 NM_01623.1 XM_033553.6 NM_022734.1 XM_033595.6 NM_002937.2 NM_001145.1 XM_007491.3 XM_034238.5 NM_001641.2 NM_017807.1 NM_005484.2 NM_021178.1 XM_05893.3 NM_003098.2 XM_047042.2 XM_056253.3 XM_097772.1 XM_059272.3 NM_016732.1 NM_003908.1 NM_000687.1 XM_047136.1 NM_014183.1 NM_032514.1 NM_080476.1 XM_0867728.1 XM_04715.2 NM_052830.1 NM_01867.1 NM_000178.1 NM_0155638.1 NM_018217.1 NM_006444.1 XM_086729.1 XM_072161.3 NM_002212.1 NM_018244.1 NM_007186.1 NM_01566.1 XM_016061.3 NM_003915.1 XM_047271.2 XM_047273.2 XM_113380.1 NM_004902.1 XM_092776.1 NM_01636.2 NM_0166558.2 XM_047295.1 XM_114076.1 XM_086357.1 NM_000975.2 NM_003198.1 NM_007260.2 NM_000191.1 NM_0177761.2 NM_054016.1 XM_0863737. XM_006290.5 NM_019084.1 XM_005681.1 XM_089514.1 NM_013314.1 XM_055549.3 XM_050993.5 XM_058343.5 XM_058602.2 XM_016640.2 XM_096146.2 XM_090334.1 XM_084841.1 NM_022496.1 XM_1137214.1 XM_0525421 NM_003348.1 NM_019094.1 NM_003877.2 XM_012219.4 XM_040952.1 XM_116948.1 NM_005348.1 XM_016985.2 XM_012448.9 NM_0144736.1 NM_000942.2 XM_042248.1 NM_032231.2 NM_002789.2 XM_113812.1 XM_091100.1 XM_0497970.6 NM_012170.1 NM_000126.1 XM_037176.2 NM_004278.1 NM_01855.1 XM_058900.2 NM_016428.1 NM_002634.2 XM_011118.7 NM_003563.1 NM_005827.1 NM_030802.1 NM_007067.1 XM_165954.1 XM_041678.4 NM_003244.1 XM_167362.1 XM_064591.1 XM_0155755.1 XM_097232.2 NM_022081.2 XM_0378840.4 NM_012143.1 NM_003595.1 XM_086821.1 XM_092888.1 XM_07592.4 XM_0166170.6 XM_030424.3 XM_113451.1 XM_1655474.1 XM_055002.1 NM_1333645.1 XM_097453.2 XM_02928.2 XM_05945.3 NM_004906.1 XM_087871.1 XM — 03912.1 XM_0399.10.1 NM_014161.1 XM_004237.5 XM_16621.1 XM — 166512.1 XM_167279.1 NM_032581.2 NM_002314.2 NM_022170.1 NM_022040.2 XM_004901.3 NM_003388.3 NM_01638.1 NM_032999.1 XM_113540.1 NM_015941.1 NM_006330.2 NM_014175.1 NM_005648.2 NM_000971.2 XM_088311.2 NM_0144393.1 NM_003406.1 XM_011657.3 NM_002568.1

Claims (185)

(a) the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NO: 1-4622);
(b) the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NO: 1-4622); or
(c) the complementary strand of (a) or (b);
An isolated nucleic acid having a nucleotide sequence with at least 80% nucleic acid sequence identity to (a) the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NO: 1-4622);
(b) the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NO: 1-4622); or
(c) the complementary strand of (a) or (b);
An isolated nucleic acid having (a) the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NO: 1-4622);
(b) the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NO: 1-4622); or
(c) the complementary strand of (a) or (b);
An isolated nucleic acid that hybridizes to.   4. The nucleic acid of claim 3, wherein hybridization occurs under stringent conditions.   4. The nucleic acid of claim 3, which is at least about 5 nucleotides in length.   An expression vector comprising the nucleic acid according to any one of claims 1 to 3.   The expression vector of claim 6, wherein the nucleic acid is operably linked to a regulatory sequence recognized by a host cell transformed with the vector.   A host cell comprising the expression vector according to claim 7.   The host cell according to claim 8, which is a CHO cell, an E. coli cell, or a yeast cell.   A method for producing a polypeptide, comprising culturing the host cell according to claim 8 under conditions suitable for expression of the polypeptide, and recovering the polypeptide from the cell culture. (a) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622); or
(b) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622);
An isolated polypeptide having at least 80% amino acid sequence identity to. (a) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NO: 1-4622); or
(b) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622);
An isolated polypeptide having.   A chimeric polypeptide having 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 immunoglobulin epitope tag sequence or Fc region. (a) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622); or
(b) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622);
An antibody that binds to a polypeptide having at least 80% amino acid sequence identity to. (a) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NO: 1-4622); or
(b) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622);
An isolated antibody that binds to a polypeptide having   The antibody according to claim 15 or 16, which is a monoclonal antibody.   The antibody according to claim 15 or 16, which is an antibody fragment.   The antibody according to claim 15 or 16, which is a chimeric or humanized antibody.   17. An antibody according to claim 15 or 16 conjugated to a growth inhibitor.   17. A method according to claim 15 or 16 conjugated to a cytotoxic agent.   The antibody of claim 21, wherein the cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioisotopes and nucleolytic enzymes.   24. 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 calicheamicins.   24. The antibody of claim 23, wherein the toxin is a maytansinoid.   The antibody according to claim 15 or 16, which is produced in bacteria.   The antibody according to claim 15 or 16, which is produced in CHO cells.   The antibody according to claim 15 or 16, which induces death in cells to which it binds.   The antibody of claim 15 or 16, wherein the antibody is distinguishably labeled.   An isolated nucleic acid having a nucleotide sequence encoding the antibody of claim 15 or 16.   31. An expression vector having a nucleic acid according to claim 30, wherein said nucleic acid is operably linked to a regulatory sequence recognized by a host cell transformed with said vector.   32. A host cell comprising the expression vector of claim 31.   The host cell according to claim 32, which is a CHO cell, an E. coli cell, or a yeast cell.   A method for producing an antibody, comprising culturing the host cell according to claim 32 under conditions suitable for expression of the antibody, and recovering the antibody from the cell culture. (a) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622); or
(b) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622);
An isolated oligopeptide that binds to a polypeptide having at least 80% amino acid sequence identity to. (a) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NO: 1-4622); or
(b) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622);
An isolated oligopeptide that binds to a polypeptide having it.   37. The oligopeptide of claim 35 or 36 conjugated to a growth inhibitor.   37. The oligopeptide of claim 35 or 36 conjugated to a cytotoxic agent.   40. The oligopeptide of claim 38, wherein the cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioisotopes and nucleolytic enzymes.   40. The oligopeptide of claim 38, wherein the cytotoxic agent is a toxin.   41. The oligopeptide of claim 40, wherein the toxin is selected from the group consisting of maytansinoids and calicheamicins.   41. The oligopeptide of claim 40, wherein the toxin is a maytansinoid.   37. The oligopeptide of claim 35 or 36, which induces death in cells that bind.   37. The oligopeptide of claim 35 or 36, wherein the oligopeptide is distinguishably labeled. (a) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622); or
(b) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622);
A TAT-binding organic molecule that binds to a polypeptide having at least 80% amino acid sequence identity to (a) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NO: 1-4622); or
(b) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622);
46. The organic molecule of claim 45, which binds to a polypeptide having it.   47. The organic molecule of claim 45 or 46 conjugated to a growth inhibitor.   47. The organic molecule of claim 45 or 46 conjugated to a cytotoxic agent.   49. The organic molecule of claim 48, wherein the cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioisotopes and nucleolytic enzymes.   49. The organic molecule of claim 48, wherein the cytotoxic agent is a toxin.   51. The organic molecule of claim 50, wherein the toxin is selected from the group consisting of maytansinoids and calicheamicins.   51. The organic molecule of claim 50, wherein the toxin is a maytansinoid.   47. The organic molecule of claim 45 or 46 that induces death in a cell that binds.   47. The organic molecule of claim 45 or 46, wherein the organic molecule is distinguishably labeled. (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;
(E) the antibody of claim 16;
(F) the oligopeptide of claim 35;
(G) the oligopeptide of claim 36;
(H) a TAT-binding organic molecule according to claim 45; or (i) a composition of matter comprising the TAT-binding organic molecule according to claim 46 in combination with a carrier.   56. The composition of claim 55, wherein the carrier is a pharmaceutically acceptable carrier. 56. The composition of claim 55 contained in (a) a container; and (b) the container.   58. The manufacture of claim 57, further comprising a label affixed to the container, or a package insert included in the container, which refers to the use of the composition for therapeutic treatment or diagnostic detection of cancer. Goods. (a) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622); or
(b) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622);
A method for inhibiting the growth of a cell expressing a protein having an amino acid sequence identity of at least 80% relative to the protein, contacting the cell with an antibody, oligopeptide or organic molecule that binds to the protein, A method wherein the cell growth is inhibited by binding of the antibody, oligopeptide or organic molecule.   60. The method of claim 59, wherein the antibody is a monoclonal antibody.   60. The method of claim 59, wherein the antibody is an antibody fragment.   60. The method of claim 59, wherein the antibody is a chimeric or humanized antibody.   60. The method of claim 59, wherein the antibody, oligopeptide or organic molecule is conjugated to a growth inhibitor.   60. The method of claim 59, wherein the antibody, oligopeptide or organic molecule is conjugated to a cytotoxic agent.   65. The method of claim 64, wherein the cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioisotopes and nucleolytic enzymes.   65. The method of claim 64, wherein the cytotoxic agent is a toxin.   68. The method of claim 66, wherein the toxin is selected from the group consisting of maytansinoids and calicheamicins.   68. The method of claim 66, wherein the toxin is a maytansinoid.   60. The method of claim 59, wherein the antibody is produced in bacteria.   60. The method of claim 59, wherein the antibody is produced in CHO cells.   60. The method of claim 59, wherein the cell is a cancer cell.   72. The method of claim 71, wherein the cancer cells are further administered radiation therapy or a chemotherapeutic agent.   The cancer cells are breast cancer cells, colorectal cancer cells, lung cancer cells, ovarian cancer cells, central nervous system cancer cells, liver cancer cells, bladder cancer cells, pancreatic cancer cells, cervical cancer cells, melanoma cells, and leukemia cells. 72. The method of claim 71, selected from the group consisting of:   72. The method of claim 71, wherein the cancer cells express the protein in greater amounts compared to normal cells from the same tissue.   60. The method of claim 59, wherein the method induces cell death. The protein is
(a) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NO: 1-4622); or
(b) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622);
60. The method of claim 59, comprising: (a) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622); or
(b) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622);
In contrast, a method for treating a mammal having a cancerous tumor containing cells expressing a protein having at least 80% amino acid sequence identity, wherein the antibody, oligopeptide or organic molecule that binds to said protein is therapeutically effective A method of effectively treating said mammal by administering an amount to said mammal.   78. The method of claim 77, wherein the antibody is a monoclonal antibody.   78. The method of claim 77, wherein the antibody is an antibody fragment.   78. The method of claim 77, wherein the antibody is a chimeric or humanized antibody.   78. The method of claim 77, wherein the antibody, oligopeptide or organic molecule is conjugated to a growth inhibitor.   78. The method of claim 77, wherein the antibody, oligopeptide or organic molecule is conjugated to a cytotoxic agent.   83. The method of claim 82, wherein the cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioisotopes and nucleolytic enzymes.   83. The method of claim 82, wherein the cytotoxic agent is a toxin.   85. The method of claim 84, wherein the toxin is selected from the group consisting of maytansinoids and calicheamicins.   85. The method of claim 84, wherein the toxin is a maytansinoid.   78. The method of claim 77, wherein the antibody is produced in bacteria.   78. The method of claim 77, wherein the antibody is produced in CHO cells.   78. The method of claim 77, wherein the tumor is further administered with radiation therapy or a chemotherapeutic agent.   78. The method of claim 77, wherein the tumor is selected from the group consisting of a breast tumor, colorectal tumor, lung tumor, ovarian tumor, central nervous system tumor, liver tumor, bladder tumor, pancreatic tumor, or cervical tumor. .   78. The method of claim 77, wherein the cancerous cells of the tumor express the protein in greater amounts compared to normal cells from the same tissue. The protein is
(a) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NO: 1-4622); or
(b) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622);
78. The method of claim 77, comprising: (a) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622); or
(b) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622);
A method for determining the presence of the protein in a sample suspected of containing a protein having at least 80% amino acid sequence identity, wherein the antibody, oligopeptide or organic molecule that binds to the protein Exposing the sample to determine the binding of the antibody, oligopeptide or organic molecule to the protein in the sample and indicating the presence of the protein in the sample by binding of the antibody, oligopeptide or organic molecule to the protein Method.   94. The method of claim 93, wherein the sample comprises cells suspected of expressing the protein.   95. The method of claim 94, wherein the cell is a cancer cell.   94. The method of claim 93, wherein the antibody, oligopeptide or organic molecule is detectably labeled. The protein is
(a) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NO: 1-4622); or
(b) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622);
94. The method of claim 93, comprising: A method for diagnosing the presence of a tumor in a mammal, comprising:
(a) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622); or
(b) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622);
In contrast, the expression level of a gene encoding a protein having at least 80% amino acid sequence identity is expressed in a test sample of tissue cells collected from the mammal and in a control sample of known normal cells derived from the same tissue. A method in which the presence of a tumor in the mammal from which the test sample was collected indicates that the protein is expressed at a higher level in the test sample compared to a control sample.   99. The method of claim 98, wherein the step of measuring the expression level of a gene encoding the protein comprises using an oligonucleotide in situ hybridization or RT-PCR analysis.   99. The method of claim 98, wherein the step of measuring the expression level of the gene encoding the protein comprises using an antibody in immunohistochemical analysis or Western blotting analysis. The protein is
(a) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NO: 1-4622); or
(b) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622);
99. The method of claim 98, comprising: A method for diagnosing the presence of a tumor in a mammal, comprising:
The protein is
(a) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622); or
(b) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622);
A test sample of the tissue cell derived from the mammal is contacted with an antibody, oligopeptide or organic molecule that binds to a protein having at least 80% amino acid sequence identity to the antibody, Detecting the formation of a complex of said protein with a peptide or organic molecule, wherein the formation of the complex indicates the presence of a tumor in the mammal.   105. The method of claim 102, wherein the antibody, oligopeptide or organic molecule is detectably labeled.   104. The method of claim 102, wherein the tissue cell test sample is obtained from an individual suspected of having a cancerous tumor. The protein is
(a) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NO: 1-4622); or
(b) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622);
103. The method of claim 102, comprising: (a) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622); or
(b) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622);
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 said protein, wherein said protein is used for patients in need of such treatment. A method of effectively treating or preventing the cell proliferation disorder by administering a therapeutically effective amount of an antagonist of the above.   107. The method of claim 106, wherein the cell proliferative disorder is cancer.   107. The method of claim 106, wherein the antagonist is an anti-TAT polypeptide antibody, a TAT binding oligopeptide, a TAT binding organic molecule or an antisense oligonucleotide. (a) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622); or
(b) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622);
A method of binding an antibody, oligopeptide or organic molecule to a cell expressing a protein having at least 80% amino acid sequence identity to the antibody, oligopeptide or organic molecule binding to the protein A method comprising binding an antibody, oligopeptide or organic molecule to the cell by contacting the cell and binding the antibody, oligopeptide or organic molecule to the cell.   110. The method of claim 109, wherein the antibody is a monoclonal antibody.   110. The method of claim 109, wherein the antibody is an antibody fragment.   110. The method of claim 109, wherein the antibody is a chimeric or humanized antibody.   110. The method of claim 109, wherein the antibody, oligopeptide or organic molecule is conjugated to a growth inhibitor.   110. The method of claim 109, wherein the antibody, oligopeptide or organic molecule is conjugated to a cytotoxic agent.   115. The method of claim 114, wherein the cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioisotopes and nucleolytic enzymes.   115. The method of claim 114, wherein the cytotoxic agent is a toxin.   117. The method of claim 116, wherein the toxin is selected from the group consisting of maytansinoids and calicheamicins.   117. The method of claim 116, wherein the toxin is a maytansinoid.   110. The method of claim 109, wherein the antibody is produced in bacteria.   110. The method of claim 109, wherein the antibody is produced in CHO cells.   110. The method of claim 109, wherein the cell is a cancer cell.   122. The method of claim 121, wherein the cancer cells are further administered radiation therapy or a chemotherapeutic agent.   The cancer cells are breast cancer cells, colorectal cancer cells, lung cancer cells, ovarian cancer cells, central nervous system cancer cells, liver cancer cells, bladder cancer cells, pancreatic cancer cells, cervical cancer cells, melanoma cells or leukemia cells. 122. The method of claim 121, selected from the group consisting of:   124. The method of claim 123, wherein the cancerous cells express the protein in greater amounts compared to normal cells from the same tissue.   110. The method of claim 109, wherein the method induces cell death.   Use of the nucleic acid according to any one of claims 1 to 5 or 30 in the preparation of a medicament for therapeutic treatment or diagnostic detection of cancer.   Use of the nucleic acid according to any one of claims 1 to 5 or claim 30 in the preparation of a medicament for the treatment of tumors.   Use of the nucleic acid according to any one of claims 1 to 5 or 30 in the preparation of a medicament for the treatment or prevention of cell proliferation disorders.   Use of an expression vector according to claim 6, 7 or 31 in the preparation of a medicament for therapeutic treatment or diagnostic detection of cancer.   32. Use of an expression vector according to claim 6, 7 or 31 in the preparation of a medicament for the treatment of a tumor.   32. Use of an expression vector according to claim 6, 7 or 31 in the preparation of a medicament for the treatment or prevention of cell proliferation disorders.   34. Use of a host cell according to claim 8, 9, 32 or 33 in the preparation of a medicament for therapeutic treatment or diagnostic detection of cancer.   34. Use of a host cell according to claim 8, 9, 32 or 33 in the preparation of a medicament for the treatment of a tumor.   34. Use of a host cell according to claim 8, 9, 32 or 33 in the preparation of a medicament for the treatment or prevention of a cell proliferative disorder.   Use of a polypeptide according to any one of claims 11 to 14 in the preparation of a medicament for therapeutic treatment or diagnostic detection of cancer.   Use of a polypeptide according to any one of claims 11 to 14 in the preparation of a medicament for the treatment of tumors.   Use of the polypeptide according to any one of claims 11 to 14 in the preparation of a medicament for the treatment or prevention of cell proliferation disorders.   30. Use of an antibody according to any one of claims 15 to 29 in the preparation of a medicament for therapeutic treatment or diagnostic detection of cancer.   30. Use of an antibody according to any one of claims 15 to 29 in the preparation of a medicament for the treatment of a tumor.   30. Use of an antibody according to any one of claims 15 to 29 in the preparation of a medicament for the treatment or prevention of cell proliferation disorders.   45. Use of an oligopeptide according to any one of claims 35 to 44 in the preparation of a medicament for therapeutic treatment or diagnostic detection of cancer.   45. Use of an oligopeptide according to any one of claims 35 to 44 in the preparation of a medicament for the treatment of a tumor.   45. Use of the oligopeptide according to any one of 35 to 44 in the preparation of a medicament for the treatment or prevention of cell proliferation disorders.   55. Use of a TAT binding organic molecule according to any one of claims 45 to 54 in the preparation of a medicament for therapeutic treatment or diagnostic detection of cancer.   Use of a TAT binding organic molecule according to any one of claims 45 to 54 in the preparation of a medicament for the treatment of tumors.   55. Use of a TAT-binding organic molecule according to any one of claims 45 to 54 in the preparation of a medicament for the treatment or prevention of cell proliferation disorders.   57. Use of a composition according to claim 55 or 56 in the preparation of a medicament for therapeutic treatment or diagnostic detection of cancer.   57. Use of a composition according to claim 55 or 56 in the preparation of a medicament for the treatment of tumors.   57. Use of a composition according to claim 55 or 56 in the preparation of a medicament for the treatment or prevention of cell proliferation disorders.   59. Use of an article of manufacture according to claim 57 or 58 in the preparation of a medicament for therapeutic treatment or diagnostic detection of cancer.   59. Use of an article of manufacture according to claim 57 or 58 in the preparation of a medicament for the treatment of a tumor.   59. Use of an article of manufacture according to claim 57 or 58 in the preparation of a medicament for the treatment or prevention of cell proliferation disorders. (a) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622); or
(b) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622);
A method for inhibiting cell growth, wherein the growth of the cell depends at least in part on the growth-enhancing effect of a protein having at least 80% amino acid sequence identity to the antibody, oligopeptide or A method of inhibiting the growth of the cell by bringing the protein into contact with an organic molecule.   154. The method of claim 153, wherein the cell is a cancer cell.   154. The method of claim 153, wherein the cell expresses the protein.   154. The method of claim 153, wherein binding of the antibody, oligopeptide or organic molecule to the protein antagonizes cell growth enhancing activity of the protein.   154. The method of claim 153, wherein binding of the antibody, oligopeptide or organic molecule to the protein induces death of the cell.   154. The method of claim 153, wherein the antibody is a monoclonal antibody.   154. The method of claim 153, wherein the antibody is an antibody fragment.   154. The method of claim 153, wherein the antibody is a chimeric or humanized antibody.   154. The method of claim 153, wherein the antibody, oligopeptide or organic molecule is conjugated to a growth inhibitor.   154. The method of claim 153, wherein the antibody, oligopeptide or organic molecule is conjugated to a cytotoxic agent.   164. The method of claim 162, wherein the cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioisotopes and nucleolytic enzymes.   164. The method of claim 162, wherein the cytotoxic agent is a toxin.   166. The method of claim 164, wherein the toxin is selected from the group consisting of maytansinoids and calicheamicins.   166. The method of claim 164, wherein the toxin is a maytansinoid.   154. The method of claim 153, wherein the antibody is produced in bacteria.   154. The method of claim 153, wherein the antibody is produced in CHO cells. The protein is
(a) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NO: 1-4622); or
(b) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622);
154. The method of claim 153, comprising: (a) a polypeptide encoded by the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622); or
(b) a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622);
A therapeutic treatment of a mammalian tumor, wherein the tumor growth depends at least in part on the growth-enhancing effect of a protein having at least 80% amino acid sequence identity to the antibody, wherein the antibody binds to the protein; A method of effectively treating the tumor by contacting the protein with an oligopeptide or an organic molecule.   171. The method of claim 170, wherein the tumor cells express the protein.   171. The method of claim 170, wherein binding of the antibody, oligopeptide or organic molecule to the protein antagonizes cell growth enhancing activity of the protein.   171. The method of claim 170, wherein the antibody is a monoclonal antibody.   171. The method of claim 170, wherein the antibody is an antibody fragment.   171. The method of claim 170, wherein the antibody is a chimeric or humanized antibody.   171. The method of claim 170, wherein the antibody, oligopeptide or organic molecule is conjugated to a growth inhibitor.   171. The method of claim 170, wherein the antibody, oligopeptide or organic molecule is conjugated to a cytotoxic agent.   178. The method of claim 177, wherein the cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioisotopes and nucleolytic enzymes.   178. The method of claim 177, wherein the cytotoxic agent is a toxin.   179. The method of claim 179, wherein the toxin is selected from the group consisting of maytansinoids and calicheamicins.   179. The method of claim 179, wherein the toxin is a maytansinoid.   171. The method of claim 170, wherein the antibody is produced in bacteria.   171. The method of claim 170, wherein the antibody is produced in CHO cells. The protein is
(a) an amino acid sequence encoded by the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NO: 1-4622); or
(b) an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of FIGS. 1-4622 (SEQ ID NOs: 1-4622);
171. The method of claim 170, comprising:   A method for diagnosing the presence of a tumor in a mammal, wherein the presence or absence of amplification of one of the amplified chromosomal regions shown in any one of appendix B to J in a tissue sample obtained from the mammal is determined, A method wherein the presence of amplification of said chromosomal region indicates the presence of a tumor in said mammal.

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