AU2008202007A1 - Compositions and methods for the diagnosis and treatment of tumor - Google Patents

Description

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AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION Standard Patent Applicant(s): GENENTECH, INC.

Invention Title: COMPOSITIONS AND METHODS FOR THE DIAGNOSIS AND TREATMENT OF

TUMOR

The following statement is a full description of this invention, including the best method for performing it known to me/us: P52432 AU3 Pal-Sot FIog Appficafiw 2008-5.5 doc (M) 00

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COMPOSITIONS AND METHODS FOR THE DIAGNOSIS AND TREATMENT OF TUMOR FIBELD OP THE INOYTIN S The present invention is directed to compositions of matter useful for the diagnosis and treatment of tumor in mammals and to methods of using those compositions of matter for the same.

BACKGROUND OF THE INVENTlJ i Malignant tumors (cancers) are the second leading cause of death in the United States, after heart disease (Boring et al., CA CancelJ Clin. 43:7 (1993)). Cancer is characterized by the increase in the number of abnormal, 00 or neoplastic, cells derived from a normal tissue which proliferate to form a tumor mass, the invasion of adjacent tissues by these neoplastic tumor cells, and the generation of malignant cells which eventually spread via the blood or lymphatic system to regional lymph nodes and to distant sites via a process called metastasis. In a cancerous state, a cell proliferates under conditions in which normal cells would not grow. Cancer manifests itself in a wide variety of forms, characterized by different degrees of invasiveness and aggressiveness.

In attempts to discover effective cellular targets for cancer diagnosis and therapy, researchers have 1 5 sought to identify transmembraue or otherwise membrane-associated polypeptides that are specifically expressed on the surface of one or more particular type(s) of cancer cell as compared to on one or more normal non-cancerous cell(s). Often, such membrane-associated polypeptides are more abundantly expressed on the surface of the cancer cells as compared to on the surface of the non-cancerous cells. The identification of such tumor-associated cell surface antigen polypeptides has given rise to the ability to specifically target cancer cells for destruction via antibody-based therapies. In this regard, it is noted that antibody-based therapy has proved very effective in the treatment of certain cancers. For example, HERCBPTINO and RITUXANV (both from Genentech Inc., South San Francisco, California) are antibodies that have been used successfully to treat breast cancer and non-Hodgkin's lymphoma, respectively. More specifically, HERCEPTIN® is a recombinant DNA-derived humanized monoclonal antibody that selectively binds to the extracellular domain of the human epidermal growth factor receptor 2 (HER2) proto-oncogene. HER2 protein overexpression is observed in 25-30% of primary breast cancers. RJTUXAN® is a genetically engineered chimeiic murine/human monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes. Both these antibodies are recombinantly produced in CHO cells.

In other attempts to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify non-membrane-associated polypeptides that are specifically produced by one or more particular type(s) of cancer cell(s) as compared to by one or more particular type(s) of non-cancerous nonnal cell(s), polypeptides that are produced by cancer cells at an expression level that is significantly higher than that of one or more normal non-cancerous cell(s), or polypeptides whose expression is specifically limited to only a single (or very limited number of different) tissue type(s) in both the cancerous and non-cancerous state normal ProWat and proutate tumor tissue). Such P01ypesd may eMain lntmoeljulady located or nmay IV 00x~ by e C a cercel Mndoreosuc po lp may be expx~sed not by gm@ oanaoor Cell Itseit but znha COy viSuhch poueado secreted Ppoepdslypefdee having a Potentiating or growth iancing eact or canone Cellsa nd S n d uch e t hp l pi s are often Proteins that Provide cancer cells w ith grow th advantag e v r f o n cels a d nclde uc thi~ sfor example, angiog9 0 0 ffcto a, cellular adhesion fiwto gr o ver f ncors and the like. Iden~tificationl of antagonist of such non-membrane associated Polypeptides would be expected to serve as effective therapeutic agents for die treatment of such cancers. Furthermore, identification of the expresio INO Pattern of such polypeptides would be usefu~l for the diagnosis Of Particular canceru in mlammals.

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Despite the above identified advances in miammnalian cancer therapy, there Is a great need for additional diagnostic and therapeutic agenlts capable of detecting the presence of tumor in a mammal and for effectively inhibiting neoplastic cell growth, respectiveY. Accordingly, it is a betv ftepeetivnint dniy (1oell m i b a a s cat d lypeptides that are more abundantly expressed on one or m ore type(s) of cancer cel0s as compared toon normnal cells or on other different cancer cells, non-membrane.associated POlYPeptides 00 ~~~that ame Specifically produced by one or more particular type(s) o acrcl~)(rb te el htpodo POIYPeptides having a potentiating effect on the growth of cance cells) as cOmPared to by one or more partiocar Of non-cancerous normal cell(s), nOn-membMle-associated PolYPeptideG that are produced by cancer cells at an expression level that is significantly higher than that of one or more normal non-cancerous oell(s), or Polypeptides whose expression is specifically limited to only a single (or very limited number of different) tissue type(s) in both a cancerous and noni-cancerous state normia[ Prostate and prostate tumor tissue), and to use those Polypeptides, and their encoding nucleic acids, to producecopsiiuofmteueflithtereuc treatmient and diagnostic detection of cancer in mamimals. It is also an objective of the present invention to identify cell miembraue..associated, secreted or intracellular polypeptides whose expression Is limited to a 1.single or very limnited number of tissues, and to use those polypeptides, and their encoding nucleic acids, to produce COmnpositions of matter usefu~l in the therapeutic treatment and diagnostic detection of cancer in mammals.

A. Embodiments In the present specificationl Applicants describe for the first time the identification of various cellular POIYPePtldes (and their encoding nucleic acids or fragments thereof) which are expressed to a greater degree on the surface of or by one or more types of cancer cell(s) as compared to on the surface of or by one or more types 3 0 of normal non-cancer cells. Alternatively, such polypeptides are expressed by cells which produce and/orseet POlypeptides having a potentiating or growth-enh~anoing effect on cancer cells. Again alternatively, such PolyPeptides may not be overexpressed by tumor cells as compared to normal cells of the same tissue type, but rather may be specifically expressed by both tumor cells and normal cells of only a single or very limited number of tissue types (preferably tissues which are not essential for life, prostate, etc.). All of the above 3S~ polypeptides are herein referred to as Tumtfor-associated Antigenic Target polypeptides ("TAT' polypeptides) and are expected to serve as effective targets for cancer therapy and diagnosis in'mammals, Aoonlingy, in 011e embodiment of the preent inyention, the inyetion Pnuvdes an isolated nucio acd 00Molecule haing a nUcotide sequence that encodes a tumor_"ated~~ antigenje taije PolYpeptide or ftgret~ C) thereof (a "TAr'POlYpeptidle).

In certain aspects, the isolated nuclic acid molecule comprises a nuleotido sequence having at et about 80% nce acid sequence identity, alternatively at least about s81%, t2% 83,84,8eas%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity, to a DNA INCMolecule encoding a l-lngth TAT polypeptide haing an amino acid sequence as disclosed herein, a TAT Polypeptide amino* acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transxnmbfate TAT Polypeptide, with or without the signal peptide, as disclosed herein or any other specifically I> defined frgment of a full-length TAT Polypeptide amino acid sequence as disclosed herein, or thle complemen of the DNA molecule of (a),.en In Other aspects, the isolated nucleic acid molecule comprises a nucleotide sequence havig at least about 0080% nucleic acid sequence identity, alternatively at least about 81%, g2%,1 83%, 84%, 85%, 86%, 87%, 88%, 8904, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleie acid sequence identity, to a DNA molecule comprisig the coding sequence of a ful-lengthr TAT polypeptide cDNA as disclosed herein, the coding sequence of a TAT Polypeptide lacking the signal peptide, as disclosed herein, the coding sequence of an extracelular domain of a transmembae TAT polypeptide, with or without the signal peptide, as disclosed herein or the coding sequence of any other specifically defined fragment of the full-length TAT polypeptide amino acid sequence as disclosed herein, or the complement of the DNA molecule of In further aspects, the invention concerns an isolated nucleic acid molecule comprising a nucleotide 2o sequence having at least about 80% nucleic acid sequence identity, alternatively at least about 81,2,83%/, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity, to a DNA molecule that encodes the same mature polypepuide encoded by the full-length coding region of any of the human protein cDNAs deposited with the IATCC as disclosed herein, or the complement of the DNA molecule of Another aspect of the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a TAT polypeptide which is either transniembrane domain-deleted or transmembrane dormaininactivated, or is comnplementary to such encoding nucleotide sequence, wherein the transinerubrane domain(s) of such polypeptide(s) are disclosed herein. Therefore, soluble extracellular domains of the herein described

TAT

Polypeptides are contemplated.

34) In other aspects, the present invention is directed to isolated nucleic acid molecules which hybridize to a nucleotide sequence encoding a TAT polypeptide having a full-length amino acid sequence as disclosed heroin, a TAT polypeptide amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a transmembrane TAT Polypeptide, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of a full-length TAT polypeptide amino acid sequence as disclosed herein, or tie 3f complement of the nucleolide sequence of In this regard, an embodiment of the present invention is directed to fragments of a full-length TAT polypeptide coding sequence, or the complement thereof, as disclosed herein, that may find use as, for example, hybridization probes useful as, for example, diagnostic probes, antisense oli oai o~e ti~ prbes, or fbr encoding fzagm ents Of a ll le g u T A p ly pt d t m y opi n ly e od a0 Plptdocmrsgabidg ieoraaniAT polypetide atibodly, a TAT binding ollgopePtide or oter small organic moleule that binds to a TAT polyliePtide. Such nucleic oid fi a n saeu ull tl a nlegt, ate~iy~ytie~ bou6,7,8,9,10,11, 12, 13,14, 15,16,17, 18,19,20,21,22, 23, 24,25,26,27,28,29,30,35,40,45,50,55,60,65,7,58,8,05100,105,110,11512,125130135,140,145, 10, 55,60,65,70,75,80,85,901955 200, 210, 220, 230,240,250,260,270,280,290,300,30320,330,340, IND 350,360,370,380,390,400,410,420,430,440,450,460,470,480490,500,510, 520,530,540,550,560,570,580, 590, 600,610,620,630,640,650,660,670,680,69,700710720,730740,750,760,770,780,790,800,810,820,830, 840, 850,860,870880890,900,910,920,930,940,950,960,970,980,990, or 1000 nucleotides in lengthi, wherein in this I> context the term "about" means the referenced nucleotide sequence length plus or minus 10% of that referenced S 10 length. It is noted that novel fragments of a TAT polypeptide-encodirig nucleotide sequence may be determitned in a routine manner by aligning the TAT polypeptide-encoding nucleotide sequence with other known nuoleotide 00 sequences Using any of a number of well known sequence alignment programs and determining which TAT polypeptide~encoding nucleotide sequence fr-agment(s) are novel. All of such novel fragments of TAT polypeptideengoing nucleotide sequences ame contemplated herein. Also contemiplated are the TAT polypepticle fragments encoded by these nucleotide molecule fragments, preferably those TAT polypeptide fragments that comprise a binding site for an anti-TAT antibody, a TAT binding oligopeptide, or other small organic molecule that binds to a TAT polypeptide.

In another embodiment, the invention provides isolated TAT polypeptides encoded by any of the isolated nucleic acid sequences hereinabove Identified.

0n ma certain aspect, the invention concerns an isolated TAT polypeptide, comprising an ailno acid sequence having at least about 80%/ anmino acid sequence identity, alternatively at least about 81%, 82%, 830/, 84%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity, to a TAT polypeptide having a ftill-length amino acid sequence as disclosed herein, a TAT polypeptide amino acid sequence lacking the signal peptide as disclosed herein, an extracellular domain of a trawnmembae TAT poly'peptide protein, with or without the signal peptide, as disclosed herein, an amino acid sequence encoded by any of the nucleic acid sequences disclosed herein or any other specifically defined fragment of a full-length TAT pojypeptide amino acid sequence as disclosed herein.

In a further aspect, the inventions concerns an isolated TAT polypeptide comprising an amino acid sequence having at least about 80% amnino acid sequence identity, alterniatively at least about 8 82%, 83%, 31) 84%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequenice identity, to an amiino acid sequence encoded by any of the human protein cDNAs deposited with the ATCC as disclosed herein.

In a specific aspect, the invention provides an isolated TAT polypeptide without the N-terminal signal sequence and/or without the initiating methionine and is encoded by a nucleotide sequence that encodes such 3; an amino acid sequence as hereinbefore described. Processes for producing the same are also herein described, wherein those processes comprise culturing a host cell comprising a vector which comprises the appropijate encoding nucleic acid molecule under conditions suitable for expression of the TAT polypeptide and recovering the TAT polypeptid m tl he cell culture.

Another speo of the invention provides an isolated TAT polypeptide whioh is either transm bane Sdomanleted or traasmmbrn domain-inactite Promesses forproduoing the same are also he rain desoribe, wherein those pmoesses comprise culturing a host cell comprising a vector which comprises the appropriate encoding nucleic acid molecule under conditions suitable for expression of the TAT polypptie and recovering the TAT polypeptide from the cell culture.

I In other embodiments of the present invention, the invention provides vectors comprising DNA encoding ay of the herein described polypeptides Host cells comprising any such vector are also provided. By way of example, the host cells may be CHO cells, E. colt cells, or yeast cells. A process for producing any of the herein Sdescribed polypeptides is further provided and comprises culturing host cells under conditions suitable for 0 expression of the desired polypeptide and recovering the desired polypeptide from the cell culture, s In other embodiments, the invention provides isolated chimeric polypeptides comprising any of the herein 0 described TATpolypeptdes fused to a heterologous (non-TAT) polypeptide. Example of such chimeric molecules Ocomprise any of the herein described TAT polypeptides fused to a heterologous polypeptide such as, for example, an epitope tag sequence or a Fc region of an immunoglobulin.

1 5 In another embodment, the invention provides an antibody which binds, preferably specifically, to any of the above or below described polypeptides Optionally, the antibody is a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody, single-chain antibody or antibody that competitively inhibits the binding of an anti-TAT polypeptide antibody to its respective antigenic epitope. Antibodies of the present invention may optionally be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calichamnicin, an antibiotic, a radioactive isotope, a nucleolyti enzyme, or the like.

The antibodies of the present invention may otionaly be prduced in ells or bacteria els d prefrably induce death of a cell to which they bind. For diagnostic purposes, the antibodies of the present invention may be detectably labeled, attached to a solid support, or the like.

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

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

In other embodiments of the present invention, the invention provides vectors comprising DNA encoding any of the herein described TAT binding oligopeptides. Host cell comprising any such vector are also provided.

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'By way ofexapl, ie host cells may be CHO clls, 0 o ells, or yeast cells. A pmcess for produng any of the herein described TAT binding oligopeptides is further provided and comprises cWultulring host cells under conditions suitable for expression of the desed oligopeptide and recovering the desired oligopeptide from the cell culture.

In another embodiment, the invention provides small organic molecules ("TAT binding organic 5 molecules") which bind, preferably specifically, to any of the above or below described TAT polypeptides.

Optionally, the TAT binding organic molecules of the present invention may be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheamicin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The TAT binding organic molecules of the present invention preferably induce death of a cell to which they bind. For diagnostic purposes, the TAT binding organic molecules 10 of the present invention may be detectably labeled, attached to a solid support, or the like.

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

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

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

B. Additional Embodiments Another embodiment of the present invention is directed to a method for inhibiting the growth of a cell 3 0 that expresses a TAT polypeptide, wherein the method comprises contacting the cell with an antibody, an oligopeptide or a small organic molecule that binds to the TAT polypeptide, and wherein the binding of the antibody, oligopeptide or organic molecule to the TAT polypeptide causes inhibition of the growth of the cell expressing the TAT polypeptide. In preferred embodiments, the cell is a cancer cell and binding of the antibody, oligopeptide or organic molecule to the TAT polypeptide causes death of the cell expressing the TAT polypeptide.

Optionally, the antibody is a monoclonal antibody, antibody fragment, chimeric antibody, humanized antibody, or single-chain antibody. Antibodies, TAT binding oligopeptides and TAT binding organic molecules employed in the methods of the present invention may optionally be conjugated to a growth inhibitory agent or cytotoxic aget schas tu~,incluhgfor exaple, 'amaYtaflsfnold ormlcheant3,a "niite a radioactiveisotope, 00a nucloolytj 0 enzymeC, Or the lke, 110 antibodies and TAT binding 0o180OPtepu epoed in the mthods f the prese-ntInvention may optionally be produed in CH()y oel ormetr cl Yet another embodiment Of tile Present invention is direted to a method of therapeutically treating a MaMrnW having a cancrus tumoromprising cells that epes a TAT Polypeptide, wherein the method comprises administering to the mammal a therapeutically effective amount of an antibody, an oligopeptide or a small orgaic molecule that binds to the TAT polypeptide, thereby resulting in the effective therapeutic treatment of the tumor, Optonalytheantboy is a monoclonal antibody, antibody fragment ohimeric antibody, humanized antibody, Orsingle-chain antibody. Antibodies, TAT binding oligopeptides and TAT binding organic molecules employed in the Methods of the present invention mnay optionally be conjugated to a growth inhibitory agent or cytotoxcc agent such as a toxin, including, for example, a maytansinoid or cali heamcin, an antibiot c, ardocieiooe a nuleolytic enzyme, or the like, The antibodies and oligopeptides employed in the methods of the present invention may optionally be produced in CH0 cells or bacterial cells.

Yetanoheremodientof hePresent invention isdirected toamto fdetermrining tepresence of a TAT POlYPeptide In a sample suspectedJ Of containing the TAT po[ypeptlde, wherein the method comprises exPosing the sample to an antibody, oligopeptide or small Organic molecule that binds to the TAT polypeptide, and determining binding of the antibody, oligopeptide or organic molecule to the TAT Polypeptide in the sample, wherein the presence of such binding is indicative of the presence of the TAT Polypeptide in the sample.

Optionally, the sample may contain cells (which may be cancer cells) suspected of expressing the TAT Polypeptide. The antibody, TAT binding oligopeptide or TAT binding organic molecule employed in the method 210 may optionally be detectably labeled, attached to a solid support, or the like.

A further embodiment of the present invention is directed to a method of diagnosing the presence of a tumor in a mammral, wherein the method comprises detecting the level of expression of a gene encoding a TAT polypoptide in a test sample of tissue cells obtained from said mammal, and in a control sample of known normal non-cancerous cells of the same tissue origin or type, wherein a higher level of expression of the TAT POlypeptide in the test samnple, as compared to the control sample, is indicative of thle presence of tumor in the mamMal from which the test sample was obtained.

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

Yet another embodiment of the present invention is directed to a method .for treating or preventing a cell proliferative disorder associated with altered, preferably increased, expression or activity of a TAT polypeptide, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist POIYf id s TAT anl~ ti-TAT P OIepbtdean o y T ce C MUtVe d sordr is Cancer and the antag n is of the TA T 00yp ptd i an.11 ATp l p tde tibo TAT binding oligopeptide, A i dn z ai oe i 0 o d i e t l g m e e o f e t v e h a n e t o P re V O fi o n o f th e c e ll P r o l if e r a tiv e d is o r d e r m a y b e a r e s u l t o f diretIll*n or growth inhibition of cells that express a TAT PolYPeptide or by antagonizing the cl rwl Potentiating activity of a TAT polIypeptide. cl wt another embodiment of the present invention is directed to a method of binding ananioy IND oligopeptide or small organic molecule to a cell that expresses a TAT Polypeptide, wherein the method comprises contacting a cell that expresses a TAT polypeptide with said antibody, oligopeptide or small organic molecule under conditions which are suitable for binding of the antibody, oligopeptide or small organic molecule to said TAT Polypeptide and allowing binding therebetween, Other embodiments of the Present invention are directed to the use of a TAT polypeptide, a nucleic acid encoding a TAT Polypeptide or a vector or host cell comprising that nucleic acid,(c natTAPOYeid 00 antibody, a TAT-binding oligopeptide, or a TAT-binding small Organic Molecule in the Preparation of a anedicarnent usefiji for the therapeuto treatmient or diagnostic detection of acancer or tumor, or (11) the (71 therapeutic treatment or prevention of a cell prolifrative disorder.

Another embodiment of the-present invention is directed to a method for inhibiting the growth of a cancer cell, wherein the growth of said cancer cell is at least in part dependent upon the growth potentiating effect(s) of a TAT Polxpeptide (wherein the TAT polypeptide may be expressed either by the cancer cell itself or a cell that produces POlypeptide(s) that have a growth potentiating effect on cancer cells), wherein the method comprises conacting the TAT polypeptide with an antibody, an oligopeptide or a small organic molecule that binds to the TAT polypeptide, thereby antagonizing the growth-.potentiaig activity of the TAT polypeptide and, in turm, inhibiting the growth Of the cancer cell Preferably the growth of the cancer cell is completely inhibited. Even more preferably, binsding of the antibody, oligopeptide or small organic molecule to the TAT polypeptide induces the death of the cancer cell. Optionally, the antibody is a monoclonal antibody, antibody fr-agment, chimeric antibody, humanized antibody, or single-chain antibody. Antibodies, TAT binding oligopeptides and TAT binding organic molecules employed in the methods of the present inventioni may optionally be conjugated to a growth inhibitory agent or cytotoxic agent such as a toxin, including, for example, a maytansinoid or calicheam-icin, an antibiotic, a radioactive isotope, a nucleolytic enzyme, or the like. The antibodies and TAT binding oligopeptides employed in the methods of the present invention may optionally be produced in CH-O cells or bacterial cells. Yet another embodiment of the present invention is directed to a method of therapeutically treating a ID tumor in a manmnal, wherein the growth of said tumor is at least in part dependent upon the growth potentiating effect(s) of a TAT polypeptide, wherein the method comprises administering to the nmanmmal a therapeutically effective amount of azi antibody, an ollgopeptide or a small organic molecule that binds to the TAT polypeptide, thereby antagonizing the growth potentiating activity of said TAT Polypeptide and resulting in the effective therapeutic treatment of the tumor. Optionally, the antibody is a monoclonal antibody, antibody fhagment, chimeric 3; antibody, humanized antibody, or single-thain antibody. Antibodies, TAT binding oligopeptides and TAT binding organic molecules employed in the methods of the present invention may optionally be conjugated to a growth iniibitory~ agent or cybotoxic agent such as a toxin, including, for example, amnaytansinold or calichearnicin, an a.iitc a 'iaty soo e ulolytio ezyme, or tte i e. Te a nbodies and oligopeptides employed in the Meotods of the present nVntibn may optiotally be produced in CH0 ell or bacterial cels, Ct In further embodiments, the invention is directed to the followingseofptnilcamfr h, application: Isolated nucleic acid having a nucleotide sequence that has at least 80% nucleic acid sequenre Identity to: a DNA molecule encoding the amino acid sequence shown in any one ofFigures 57-1 12,114,116, 1 18 orl120 (SEQ ID NOS: 57 112,114, 116, 118 or 120); a DNA molecule encoding the amino acid sequence shown in any one of Figures 57-112, 114, 116, 1 18 c-i~o c120 (SEQ ID NOS:57.1 12,114,116,118 or 120), lacking its associated signal peptide; 00 it DNA molecule encoding an extracellular domain of the polypeptide shown in any one of Figures 57- 112, 114, 116, 118 or 120 (SEQ ID NOS:57.112, 114, 116, 118 or 120), with its associated signal peptide; a DNA molecule encoding an extracellular domain of thqpolypeptide shown in any one of Figures 57.

15112,114, 116, 118 orl120 (SEQ IDNOS:57..11 2 ,114,116, 118 or 120), lacking its associated signal peptide; the nucleotide sequence shown in any one of Figures 1-56,113, 115,117 or 119(SEQ IDNOS: 1-56, 113, 115, 117 cr 119); the full-length coding region of the nucleotide sequence shown in any one of Figures 1-56, 113, 115, 117 or 119 (SEQ DNOS:1.56, 113, 115, Il7or 119); or the complement of or 2. Isolated nucleic acid having: a nucleotide sequence that encodes the amino acid sequence shown in any one of Figures 57-112, 1 14,.

116,118 orl120 (SEQ IDNOS:5 7 112,114,116,118 or 120); a nucleotide sequence that encodes the amino acid sequence shown in any one of Figures 57-1 12,114, 116,118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide; a nucleotide sequence that encodes an extracellular domain of the polypeptide shown in any one of Figures 57-112,114,116, 118 or 120(SEQ ID NOS:57- 1 12,114,116, 118 or 120), with its associated signal peptide; a nucleotide sequence that encodes an extracellular domain of the polypeptide shown in any one of Figures 57-112,114, 116,118 or 120 (S3Q IDNOS: 57.1 12 114 ,1 1 6, 118 or 120), lacking its associated signal peptid; the nucleotide sequence shown in any one ofFigures 1-56,113, 115,117 or 119 (SEQ ID NOS: 1-56,113, 115,117 or 119); the full-length coding region of the nucleotide sequence shown in any one of Figures 1-56, 113, 115, 117 or 119 (SEQ ED NOS: 1-56, 113, 115,117 or 119);o the complement of or 3Si 3. Isolated nucleic acid that hybridizes to: a nucleic acid that encodes the amino acid sequence shown in any one of Figures 57-112, 114,116, 118 or 120 (SEQ ID NOS:57-11 2 ,114, 116, 118 cr 120); a tuole1o adid that Onodes the amino acid sequence Shown in any one Of Figure 57-112, 114, 116, 00118 Or 120 (SBQIDNOS:57-112,a114, 116, 118 or 120), lacking its assoatedaggal peptdo; 00 anucleic acid that encodes an extraceffulr domain of the POlYpeptde shown in any one of Figures 57-112, 114,116,118 or 120 (SEQ ID NOS;57-I 12,114, 116, 118 or 120), with it associated signal peptide; nucleic acid that encodes an extracellular domain Of the PolypeptideshwinayoefFgus 557-112,114,116,118 or 120 (SEQ ID NOS:5711 2 ,114,116, 118 or 120), lacking its associated signal peptide; IND(e) the nucleotidesequence5s 0 ow in anyoneofFiure 1-56, 113, 115, 117 or 119 (SBQIDNOS: 1-56, 113, 115, 117 or 119); (M the fi.~l-length coding region of the flucleotide sequence shown in any one of Figures 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1- 5 6 113, 115, 117 or 119); or the complement of Or 4. The nucleic acid of Claimi 3, wherein the hybridization occurs under stringent conditions.

00 5. The nucleic acid of Claim 3 which is at lenst about 5 nucleotides in length.

6. Anl expression vector comprising the nucleic acid of Claim 1, 2 6r 3.

7. 'The expression vector of Claim 6, wherein said nucleic acid is operably linked to contro~l sequences recognizedi by a hogt cell transformed with the vector.

8. A host cell Comprising the expression vector of Claim 7.

9. The host cell of Claim 8 which is a CHO cell, an col cell or a yeast celL A process for producing a Polypeptide comprising culturing the host cell of claim 8 under conditions suitable for expression of said polypeptide and recovering said polypeptide from the cell culture.

11. An isolated polypeptide having at least 80% amino acid sequence Identity to: the Poyeptde sown inayoneofFigms5 7 -112,114,116,11 lorI120(SEQ IDNOS:57 112,114, 116, 118 or 120); the polypeptide shown in any one of Figures 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-l 12, 114, 116, 118 or 120), lacking its associatedJ signal peptide; an extracellular domain of the polypeptide shown in any one of Figures 57-1 12, 114, 116, 118 or 120 (SEQ ID NOS:57-112, 114, 116, 118 or 120), with its associated signal peptide; an extracellular domain of the polypeptide shown in any one of Figures 57-112, 114, 116, 118 or 120 (SEQ UD NOS:57-1 12, 114, 116, 118 or 120), lacking its associated signal peptide; a polypeptide encoded by the nucleotide sequence shown in any one of Figures 1-56, 113, 115, 117 orI119 (SEQ IDNOS:lS 113, 11 5 ,117or 119);or a polypeptide encoded by the full.-length coding region of the nucleotide sequence shown in any one of Figures 1-56, 113, 115, 117 or119 (SEQ ID NOS:1- 5 6 ,113, 115, 117 orl119).

12, An isolated polypeptide having: the amino acid sequenceshown inanyoneofFigurts 57-112, 114,116,118 or 120 (SEQ IDNOS:57-112, 33 114, 116, 118 or 120); the amino acid sequence shlown in anyone of Figures 57-1 12,114,116, 118 or 120 (SEQIDNOS:57l 12, 114, 11 6, 118 or 120), lacking its associated signal peptide sequence; an amino acid sequence of an exaCumuar domain 0fteP~letd hw nayoeo iuv 7 112, 114,116,118S or 1 20 (SEQ ID NOS:57-11 2 ,114, 116, 118 f h oyeteson-n nO iu 7 an amino acid sequence of an extraceliular doai oth oypidsowInayo e fFiuenc 57-1 12, 114, 116, 118 or 120 (SP-Q IDN~OS:57 112,114, 116, 118 or 120), lackn t soitdsgappi sequence,;kn t soitdsga etd an amino acid sequence encoded by the nucleotide sequence shown in any one Of Fgrs15,13 IND 115,IS 117 or 119 (SEQ ID)NOS:1-56,113, 115,117 or119);D an amino acid sequence encoded by the full-length coding region of the nucleo tide sequence shown in any one ofFigures 1-56, 113, 115, 11 7 or119 (SEQ D)NOS:1s 6 113, 115,117 or 119).

13. A chinmeric polypeptide comprising the polypeptide of Claim I1I or 12 fused to a heterologous polypeptide.

14. The chimeric polypeptide of Claim 13, wherein said heterologous polypeptide is an epitope tag 00 sequence or an Pc region of an ilnmunoglobuj in.

An isolated antibody that binds to a polypeptide baying at least 80% amino acid sequence identity to: die polypeptideshown in anyone ofFigue 57-112,114,116, 118 or 120(SEQIIDNOS:57.1 12,114,116, 118 or 120); tihe polypeptide shown in any one of Figures 57-112, 114, 116, 118 or 120 (SEQ ID NOS: 57-112, 114, 116, 118 or 120), lacking its associated signal peptide; an extrucellular domain of the polypeptide shown in any one of Figures 57-112, 114, 116, 118 or 120 210 (SEQ ID NOS.57..1 2 114, 116, 118 or 120), with its associatedsignalpeptide; an oxtracellular domain of the polypeptide shown in any one of Figures 5 7-112, 114, 116, 118 or 120 (SEBQ ID NOS:57-1 12, 114, 116, 118 or 120), locking its associated signal peptide; a polypep tide encoded by the nuclootide sequence shown in any one of Figures 1-56, 113, 115, 117 or119 (SED NOS:1- 56 113, 115, 11 7 orl119); or a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one of Figures 156,13 ,1 7 or119 (SEQ ID NS:.56,113,115, 117 o 1) 16. An isolated antibody that binds to a polypeptide having: (a)theamino acid sequenceshown inany oneoffigures 57-112,114,116,1 18 or 120(SEQIIDNOS:57-112, 114, 116, 118 or 120); theamino acid sequence shownin anyone ofFpgum 57 112 ,114,116, 118 or 120(SEQDNOS:57-112, 114,116, 118 or 120), lacking its associated signal peptide sequence; an amino acid sequence of an extracellular domain of the polypeptide shown in any one of Figures 57- 112, 114, 116, 118 or 120 (SEQ ID NOS:57.1 12 114, 116, 118 or 1 2 0) with its associated signal peptide sequece an amnino acid sequence of an extracellular domain of the polypeptide shown in any one of Figures 3S 57-112, 114, 116, 118 or 120 (SEQ IDNOS:5711 2 114, 116, 118 or 120), lacking its associated signal peptide sequence; 00 115,117 or119 (SE ID NO. &56'113, 115, 117 or 119),;or itm15,13 0f an amnino acid sequence encoded by the fffl-Jengt coding region of the nucleotide sequence show,, in any One Of Figures 1-56, 113, 115,11 7 or19 (SEQ ID NOS: 1 5 6 113, 115, 17o 1) 17. The antibody of Claim 15 or 16 wh i s a mfonoclonal antibody 518. The antibody of Claim 15 or 16 which is an antibody fragment, 1-019. The antibody of Claim 15 or 16 which is a chimeric or a humnized antibody. The antibody of Claim 15 or 16 which is conjugated to a growth inhibitory agent.

21. The antibody of Claim 15 or 16 which is conjugated to a cytotoxic ag ent.

1>22. The antibody of Claim 2 1, wherein the cytotoxic agent is selected from the group consisting of toxins, antibiotics radioactive isotopes and nuoleolytic enzymes.

23. The antibody of Claim 21, wherein the cytotoxic agent is a toxin, 0024. The antibody of Claim 23, wherein the toxin is selected from the group consisting of o m~aytansinoid and callcheaicin, Te antibody of Claim 23, wherein the toxin is a maytansinoid.

1526. The antibody of Claim 15 or 16 which is produced in bacteria.

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

28. The antibody of Claim 15 or 16 which induces death of a cellI to which it binds.

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

An isolated nuocic acid having a niucleotide sequence that encodes the antibody of Claim or 16.

31. An expression vector comprising the nucleic acid of Claim 30 operably linked to control sequene recognized by a host cell transformed with the vector.

32. A host cell comprising the expression vector of Cla. i m 31.

33. The host cell of Claim 32 which is a CHO cell, an E. coi cell or a yeast cell.

34. A process for producing an antibody comprising culturing the host cell of Claim 32 under conditions suitable for expression of said antibody and recovering said antibody from the cell culturv.

An isolated oligopeptide that binds to a polypeptide having at least 80% amnino acid sequence identity to: the poypeptide shon i anyeofFige 5712114,116,118 or 120 (SEQIDNOS:57l 12,114,116, ID 1 8 or 120); the polypephide shown in any one of Figures 57-112, 114,116, 118or 120 (SBE DNOS:57.1 12,114, 116, 118 or 120), lacking its associated signal peptide; an extracellular domain of the polypeptide shown in any one of Figures 57-112, 114,116, 118 or 120 (SC3Q ID NOS:57-112,114, 116, 118 or 120), with its associated signal peptide; 3 an extracellula. domain of the polypeptide shown in any one of Figures 57-112, 114, 116, 118 or 120 (SE3Q ID NOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide; a POlYpeptide encoded lty gm~ nmoleotide sequence shownin any one of Figures 1-56, 113, 115, 117 00orll9 (SEQ IDNOS:1..561113, 115,117 or 119); or 00 a Polypeptide encoded by the full-lenot coding region of the nucleotide sequence shown in any one of Figures 1-56, 113, 115,1 17 or 119 (SEQ ID NOS: 156,113, 115, 1 17 or 119).

36. An isolated oligopeptide that binds to a polypeplide having: (athe amino acdeq enshown in aeff r 5712114,116,118 orl120(SEQ IDNOS:57..l12, IND 11 4 ,116, 118 or 120); the amino acid sequence 5 shw nayn fiu 712114, 116, 118 orl120(SEQIDNOS:57- 1 12, 114, 116, 118 or 120), lacking its associated signal peptide sequence; ()an amino acdsequence of an extracellular domain of the Polypeptide shown in any oeof Figures57 10112, 114, 116, 118 or 120 (SEQ ED N08:57-1 12, 114, 116,.118 or 120), with its associated signal peptide sequence; anamino acid sequence of an extracellular domain of the polypeptide shown in any one of Figures 00o 57-112, 114, 116, 118 or 120 (SEQ liD NQS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence; an amino acid sequence encoded by the nucleotide sequence shown In any one of Figures 1-56, 113, 115, 117or 119 (SEQ DNOS: 1- 5 6 1 13 115 1 r19;o an amino acid sequence encoded by the fllI-length coding region of the nucleotido sequence shown in anfy one of Figures 1-56, 113, 115,117 or19 (SEQ ID NOS:l- 5 6 ,113, 115,117 or119).

37. Thle oligopeptide of Claim 35 or 36 which is conjugated to a growth inhibitory agent.

38. Th6 oligopeptide of Claim 35 or 36 which is conjugated to a cytotoxic agent 39. The oligopeptide of Claimn 38, wherein the cyrtotoxic agent is selected fr'om the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.

The oligopeptide of Claim 38, wherein the cytotoxio agent Is a toxin.

41. The oligopeptide of Claim 40, wherein the toxin is selected from the group consisting of maytansinoid and calicheaicin.

42. The oligopeptide of Claim 40, wherein the toxin is a maytansinoid.

43. The oligopeptide of Claim 35 or 36 which induces death of a cell to which it binds.

44. The oligopeptide of Claim 35 or 36 which is detectably labeled.

A TAT binding organic molecule that binds to a polypeptide having at least 80% amino acid sequence identity to: 3 D thepoly eptid sh nnany one ofpigures57-11 2 1 4 1 1 6 ,118 or12 0(S BQ UDNOS:5711 2 ,ll 4 116, 118 or 120); the polypeptideshown in any one of Figures 57-112,114, 116, 118 or 120 (SEQ ED NOS:57-1 12, 114, 116, 118 or 120), lackiJng its associated signal peptide; an extraeulardoman ofthe polypeptid w.i n one of Figures 57-112, 114, 116, 118 or 120 (SEQ ED NOS:57. 112, 114, 116, 118 or 120), with its associated signal peptide; an extraceilular domain of the polypeptide shown in any one of Figures 57-112, 114, 116, 118 or 120 (SEQ ID NQS:57-112 114, 116, 118 or 120), lacking its associated signal peptide.; a polyppde encoded by the nuoleotjde sequence shown in any Oo of Figume 1-56, .113, 115, 117 00 11 9 (SQ ID NOS:1s56,113, 115, 1 17 or 119); or POlypeptide encoded by the fihll-engt coding region of the nucleotide sequence shown in aey one Of Figures 1-56, 113, 115, 117 or 119 (SEQ ID NOS: 1-56, 113, 115, 117 or 119).

46, The Organic molecule of Claim 45 that binds to a polypptide having: thie amino avid sequence shown in any one OfFigures 57-112, 114, 116, 118 or 120 (SEQ ID NOS: 57-.112, IND 114, 116, 118 or 120); the amino acid sequence shown in any one ofFigures 57-112, 114,116, 118 or 120 (SEQ IDNOS: 57-.112, 114, 116, 118 or 120), lacking its associ ated signal pepti de sequence; an amino acid sequence of an extracellular domain of the polypeptide shown in any one of igue 57.- ci 10 112, 114, 116, 118 or 120 (SEQ ID NQS:57.1 12, 114,116, 118 or 120), with its associated signal peptide sequence; an am~ino acid sequence of'an extracelular domain of the polypeptide. shown In any one of Figures 00 57-112, 114, 116, 118 cr 120 (SEQ ID NOS:57 112, 114, 116, 118 or 120), lacking its associated signal peptide sequence; an amino acid sequence encoded by the nucleotide sequence shown in any one of Figures 1-56, 113, 115, 117r 119 (SEQ ID NOS: 1-56,113, 115, 1 17 or 119);o an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of Figures 1-56,113, 115, 11 7 or119 (SHQ IDNOS:1-5 6 113, 115, 117 or 119).

47, The organic molecule of Claim 45 or 46 which is conjugated to a growth inhibitory agent.

48, The organic molecule of Claim 45 or 46 which is 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, radioactive isotopes and nucleolytio enzymes.

71e 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 maytansinoid and calicheamicin.

2552. The organic molecule of Claim 50, wherein the toxin is a maytansinoid.

53. The organic molecule of Claim 45 cr 46 which induces death of a cell to which it binds.

54. The organic molecule of Claim 45 or 46 which is detectably labeled.

A composition of matter comprising: the polypeptide of Claim 11; the polypeptide of Claim 12; the chimeric polypeptide of Claim 13; the antibody of Claim the antibody of Claim 16; the oligopeptide of Claim 3 5 the oligopeptide of Claim 36; the TAT binding organic molecule of Claim 45; or the TAT binding organic molecule of Claim 46; in comibination with a carrier.

00carir,56' M10e compIion of .matte Of Claim 55, wherei said carrier is a paramacally aceptable 57. An aztlole ofr Mnufhct=r omnprising: a Container, and thle composition of matter of Claim s5 contained within said container.

58.0 The article of manufacture of Claim 57 further comprising a label affixed to sid containr, or a 0 Package insert included with said container, rfering to the use of said composition of matter for the therapeutic treatmnent of or the diagnostic detection of a cancer.

59. A method Of inhibiting the growth of a cell that expresses a protein having at least 80% amnino acid sequence identity to: die Polypeptide shwn in any one ofFigures 57-112,114,116,118 or 120 (SEQIDNOS:s7-112,114, 116, 118 or 120); 00 0 the polypeptide shown in any one of Figures 57-112, 114, 116, 118 or 120 (SEQ D) NQS:57-1 12, 114, 0 ~116, 118 or 120), laoking its associated signal peptide; an extracellujar domain of the polypeptide shown in any one of Pigures 57-112, 114, 116, 118 or 120 ID NOS:57-1 12, 114, 116, 118 or 120), with its associated signal peptide; an extraicellular domain of the polypetide shoniayoeofFgrs512,1416,18r2 (SEQ ID NQS:57-1 12, 114, 116, 118 or 120), lacking its associated signal peptido; a polypeptide encoded by the nucleotide sequence shown in any one of Figures 1-56, 113, 115, 117 or 119 (SEQ IDNOS:1-56, 113, 115, 1 1 7 or119); or a polypeptide, encoded by the full-length coding region of the nucleotide, sequence shown in any one ofpigures 1-56, 113, 115, 117 or 119 (SEQ ID NOS: 1-56, 113, 115, 117 or 119), said method comnprising contacting said cell with an antibody, oligopeptude or organic molecule that binds to said protein, the binding of said antibody, oligopeptide or organic molecule to said protein thereby causing an inhibition of growth of said cell.

The method of Claim 59, wherein said antibody is a monoclonal antibody, 61. The method of Claim 59, wherein said antibody is an antibody fragment.

62. The method of Claim 59, wherein said antibody is a chimeric or a humanized antibody.

63. The method of Claim 59, wherein said antibody, oligopeptide or organic molecule Is conjugated to a growth inhibitory agent.

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

The method of Claim 64, wherein said cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.

66, The method of Claim 64, wherein the cytotoxio agent is a toxin.

67. The method of Claim 66, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin.

68. The method of Claim 66, wherein the toxin Is a maytansjn6id.

69. The method of Claim 59, wherein said antibody is produced in bacteria.

noe method 'If Clam 59, Wherein oadd antibody is produced in CH0 cells.

0071. The method of Claim 59, wborvln said cell is a cancer coil.

72. The method of Claim 71, wherein said cancer ceon is Ibrther exposed to rdiation treatment ora chemotheapuio agent 73. The method of Claim 71, whoi adcne elI eeted from the group consisting of a breast cancer cell, a colorectal cancer cell, a lung cancer cell, an ovarian cancer cell, a central nervous system cancer cell, IND a liver cancer cell, a bladder cancer cell, a pancreatic cancer cell, a cervical cancer cell, a melanoma cell and a leukemia cell.

74. The method of Claim 71, wherein said protein is more abundantly expressed by said cancer cell I> as compared to a normal cell of the same tissue origin, 1075. The method of Claim 59 which causes the death of said cell.

76. The method of Claim 59, wherein said protein has: the amnino acid sequence shown in anyone ofFigures 57-112,114, 116, 118 or 120 (SEQ ID NOS:57-1 12, 114, 116, 118 or 120); rKbthe aino acdsequece how i ay ne fpie 5712'14116,118 orl20(SBQIDNOS:57.

1 12 114, 116, 118 or 120), lacking its associated signal peptide sequence; an amino acid sequence of an extracellular domain of the polypeptide shown in any one of Figures 57- 112, 114, 116, 118 or 120 (SEQ I1D NOS:57-1 12,114, 116, 118 or 120), with its associated signal peptide sequence; an amino acid sequence of an extracellular domain of the polypeptide shown in any one of Figures 57-112, 114,116, 118 or 120O(SEQID NOS:57..l 12 ,114,116, 118 or 120), lacking its associated signal peptide sequence; an amino acid sequence encoded by the nucleotide sequence shown in any one of Figures 1-56, 113, 115, 117 or 119 (SEQ ID NOS:1 36,113,115,1or19;r an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown inlany oe of Figures 161131 5 1 17 1 9 (SQ IDNS1 ,15 1 7or 119).

77. A method of therapeutically treating a mammnal having a cancerous tumor comprising cells that express a protein having at least 80% amino acid sequence Identity to: (a teplppiesonianon fgue5712114,116,118 or 120 (SEQ ID NOS:57-112,114,116, 18 or 120); the polypeptide shown in any one of Figures 57-112, 114, 116,.118 or 120 (SEQ ED NOS:57-1 12,114, 3 116, 118 or 120), lacking its associated signal peptide; an extracellular domain of the polypeptide shown in any one of Figures 57-112, 114,116,118 or 120 (SEQ ID NOS:57. 112, 114, 116, 118 or 120), with its associated signal peptide; an extracellular domain of the polypeptide shown in any one of Figures 57-112, 114,116,118 or 120 (SEQ ED NOS:57.l12, 114, 116,118 or 120), lacking its associated signal peptide; a polypeptide encoded by the nucleotide sequence shown in any one of Figures 1-56, 113, 115,117 or 119 (SZ3Q DNOS:1-56, 113, 115, 117 or 119); or Wf at PolypQPtide enooded by the fuff-length codin tegion of te v=ucleo sequence sho'win any one 00 to said roanimal a therapeutially~ effective amount of an antibody, oligopeptide or organic molecule that binds to sai potenthereby effectively treating said mammal.

78. The method of Claim 77, wherein said antibody is a monoclonal antibody.

79. The method of Claim 77, wherein said antibody is an antibody fragment.

The method of Claim 77, wherein said antibody is a chimeric or a humanized antibody., 81. The method of Claim 77, wherein said antibody, oligopeptide or organic molecule is conjugated to a growth inhibitory agent.

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

83. The method of Claim 82, wherein said cytotoxic agent is selected from the group consisting of 00 toxins, antibiotics, radioactive isotopes and nucleolytic enizymies.

84. The method of Claim 82, wherein the eytotoxic agent Is a toxin, The method of Claim 84, wherein the toxin is selected from the group consisting of maytansinoid and calicheamicin, 86. The method of Claim 84, wherein the toxin is a maytansinoid.

87. The method of Claim 77, wherein said antibody is producied in bacteria.

88. The method of Claim 77, wherein said antibody is produced in CHO cells.

89, The method of Claim 77, wherein said tumor is Rurther exposed to radiation treatment or a chemnotherapeutic agent.

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

91. The method of Claim 77, wherein said protein is morm abundantly expressed by the cancerous cells of said tumor as compared to a normal cell of the came tissue origin, 92. ilie method of Claim 77, whervin. said protein has: the amino acidsequence shownin any one ofFigures 57-112,114,116, 118 or 120 (SEQ IDNOS;57.l 12, 114, 116, 118 or 120); the amino acid sequence shown in any one ofFigures 57-112,114,116,118 or 120 (SEQ IDNOS:57-l 12, JO114, 116, 118 or 120), lacking its associated signal peptide sequence; an amino acid sequence of an extracellular domain of the polypeptide shown in any one of Figures 57- 112, 114, 116, 11 8 or 120 (SEQ ID NOS:57 112, 114, 116, 118 or 120), with its associated signal peptide sequence; an amino acid sequence of an extracellular domain of the polypeptide shown in any one of Figures 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-1l2, 114, 116, 118 or 120), lacking its associated signal peptide 3 5 sequence; an amidno acid sequence encoded by the nucleotide sequence shown in any one of Figures 1-56, 113, 115,117 or 119 (SEQ ED NOS:1-56, 113, 115, 117 or 119); or (an amino adid sequence encoded by the tfd1-1ength oding region of the MWIoeode sequence shown 00 'n lanyone Of Figw= 1-56, 113, 115,117 or119 (SEQIW8156,113, 115, 117 or 119), 93. A method of determining the Presence of a protein in a sample suspected of containing said Protein, wherein said protein has at least 80%/ amino acid sequence identity to: the polypeptde shown in any one ofpwgfs 57 112 114 ,116118or20(SEQED NOS:57-112,114, 116, 1 lIor 12); IND(b) the polypeptide shown in any one of Figures 57-112,114,116, 118 or 120 (SEQ ID NQS:57-l 12, 114, 116, 118 or 120), lacking its associated signal peptide; an extravellular domain of the polypeptide shown in any one of Figure 57-112, 114,116, 118 or 120 (SEQ ID NOS:S7-112, 114, 116, 118 or 120), with its assooiated signal peptide; 10(d) an extracellular domain of the polypeptide shown in anyn of Figures 57-112, 114,116,118 or 120 (SEQ ID NOS:57-1 12, 114, 116, 118 or 120), lacking its associated signal peptide; 00 a polypeptide encoded by the nueleotide sequence shown in any one of Figures 1-56, 1.13, 115, 1 17 or119 (SBQ EDNOS:1-5 6 113, 115,117 or11); or a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one ofigure 1-56,113,115,11 lor 19(SEQ IDNOS:1-56, 113,115,11 lor 11) admto oprsn xoigsi sample to an antibody, oligopeptide or organic molecule that binds to said protein and determining binding of said antibody, oligopeptide or organic molecule to said protein in said sample, wherein binding of the antibody, oligopeptide or organic molecule to said protein is indicative of the presence of said protein in said sample.

94. The method of Claim 93, wherein said sample comprises a cell suspected of expressing said protein.

The method of Claim 94, wherein said cell is a cancer cell.

96. The method of Claim 93, wherein said antibody, oligopeptide or organic molecule is detectably labeled.

197. The method of Claim 93, wherein said protein has: the amino acid sequence sown any fl~figue 5 7 -112,114, 116,118 or 120 (SEQ IDNOS:57-1 12, 114, 116,118 or 120); theamnino acid sequence shown in any one ofpjgures 57-112,114, 116, 118 or 120 (SEQ1DNOS:57-112, 114, 116,118 or 120), lacking its associated signal peptide sequence; an amidno acid sequence of an extracellular domnain of the polypeptide shown in any one of Figures 57.

ID 112, 114, 116, 118 or 120 (SBQID NOS: 57.112, 114, 116, 118 or 120), with its associated signal peptide sequence; an amino acid sequence of an extracellular domain of the polypeptide shown in any one of Figures 57-112, 114, 116, 118 or 120 (SEQ ID NOS:5711 2 114, 116, 118 or 120), lacking its associated signal peptide sequence; an amino acid sequence encoded by the nucleotide sequence shown in any one of Figures 1-56, 113, 115 11~ 7 or 119 (SEQEDNOS: 156, 113,115, 117 19);o an amnino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of Figures1 5 113, 115, 11 7 or 119 (SBQ ID NOS:1-56, 113, 115,l1 1 7or 119).

98. A method of diagnmin the presence of a tumor in a uraniial, smid method -compriig 00detonnining the level Of expession of a gene encoding a protein havyn at least, ammoai sqe identity 0K tOheI olvnetiidso.

(arhooypiden innanyoneofFigum 57-112,114,116,118 or 120(SEQ IDNOS:57-1 12,114, 116, 11 8 or 120); tie polypeptide shown in any one ofFigures 57-112,114,116, 118 orIZO (SEQ IDNOS:57-1 12,114, 116, 118 or 120), lacking its associated signal peptide; an extracellular domain of the polypeptide shown in any one of Figures 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-1 12, 114, 116,118 or 120), with its associated signal peptide; (d)an exrclu oano h oypeiesown in any one of Figures 57-112, 114,116,118 or 120 N- 10 (SEQ ID NOS:57-11 2 ,114, 116, 118 or 120), lacking its associated signal peptide; a Polypeptide encoded by the nucleotide sequence shown in any one of Figures 1-56, 113, 115, 117 00 ~or 119 (SEQ ID NcS:1-56 113, 115, 1 17 or119); or a polypeptide encoded by the flull-lengthi coding region of the nucleotide sequence shown in any one offigures1-56,113115117 or] 19 (SEQIDNOS 5,1,1517o 1),i etsmpeotsu clsotie from said mnanurial and in a control sample of known normal cells of the same tissue origin, wherein a higher level of expression of said protein in the test sample, as compared to the control sample, is indicative of the presence of tumor in the mammal ftrm which the test sample was obtained.

99. The method of Claim 98, wherein tie step of determining the level of expression of a gene encoding said protein comprises employing an oligonuoleotide in an In situ hybridization or RT-PCR analysis.

1,0100. Themethod of Claim 98, wherein the step determining the leloepressoofagnecxlg said protein comprises employing an antibody in an inmmunohistocomistry or Western blot analysis.

101. The method of Claim 98, wherein said protein has: the amino acid sequenceshown iany one ofFigures 57-112,114,116,118 or 120 (SEQIDNQS:57.1 12, 114, 116,1l18 or 120); the amino acidsequence shownin anyone ofpjgu.m 5 7 1 12,114,116,118 or 120 (SEQ IDNOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence; an amino acid sequence of an extracellular domain of the polypeptide shown in any one of Figures 57- 112, 114, 116, 118 or 120 (SEQ U)DNOS:57-112, 114, 116, 118 or 120), with its associated signal peptide sequence; an amino acid sequence of an extracellular domain of the polypeptide shown in any one, of Figures 57-112, 114, 116, 118 or 120 (SEQ DNOS:57-112, 114, 116, 118 or 120), lacking its associated signal peptide sequence; an amino acid sequence encoded by the nucleotide sequence shown in any one of Figuires 1-56, 113, 115, 117 or 119 (SEQ ID NOSA -56, 113, 115, 117 or 119); or an amino acid sequtnce encoded by the full-length coding region of the nucleotide sequence shown in any one of igures 1-56, 113, 115, 117 or 119 (SBQ ID NOS:1-56, 113, 115, 117 or 119).

102. A method of diagnosing the presence of a tumor in a mammal, said method comprihr 00 contacting a test sample of tissue cells obtained fro~m said mammal with an antibody, oligopeptide or organje molecule that binds to a protein baving at least 80% amino acid sequence identity to: C1(a) thepolypeptidesown in any oneofFigres 7 -112,114,116,118 or120 (SEQIDNOS.-57-112, 114, 116, I18 or 120); the polypeptide shown inany one of Figures 57-112, 114, 116, 118 or 120 (SEQIWDNOS: 57-112, 114, 116, 118 or 120), lacking its associated signal peptide; an exctrace11ular domain of the polypeptide shown in any one of Figures 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57.1 12, 114,116, 118 or 120), with its associated signal peptide; ()an extracellular domain of the polypeptide shown in any one of Figures 57-112, 114,116, 118 or 120) lo1 (SEQ 113 NOS: 57-1 12, 114,116, 118 or 120), lacking its associated signal peptide; a polypeptide encoded by the nucleotide sequence shown in any one of Figures 1-56, 113, 115, 117 00 orl119(SEQED NOS:1-5 6 1 3 115, 11 7 or 119); or a polypeptide encoded by the full-lengthi coding region of the nucleotide sequence shown in any one of Figures 1-56, 113, 115,117 or 119 (SEQ ID NOS: 1-56, 113, 115, 117 or 119), and detecting the formation of a 1 5 complex between said antibody, oligopeptide or organic molecule arnd said protein in the test sample, wherein the formation of a complex is indicative of the presenc of a tumor in said mammal.

103. The method of Claim 102, wherein said antibody, oligopeptide or organic molecule is detectably labeled.

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

105. The method of Claim 102, wherein said protein has: the amino acid sequenceshownnany oneofFigures 57-112,114,116, 118 or 120 (SEQ IDNOS:57-1 12, 114, 116, 118 or 120); the amino acid sequence shown in any one ofFigures 57-112, 114, 116, 118 or 120 (SEQ IDNOS:57. 112, 114, 116, 118 or 120), lacking its associated signal peptide sequence; an amino acid sequence of an extracellular domain of the polypeptide shown in any one -of Figures 57- 112, 114, 116, 118 or 120 (SEQ ID NOS:57-1 12, 114, 116, 118 or 120), with its associated signal peptide sequence; an amino acid sequence of an extracellular domain of the polypeptide shown in any one of Figures 57.112, 114, 116, 118 or 12 (SEQ ID NO:57.l11z 114, 116, 118 or 120), lacking Its associated signal peptide ID sequence; an amino acid sequence encoded by the nucleotide sequence shown in any one of Figures 1-56, 113, 115, 117 or 119 (SEQ ED NOS:1-56, 113, 115,117 or 119); or an amino acid sequence encoded by the full-length coding region of the nucleotide sequence shown in any one of Figure1 5 6, 1 1 3, 115, 117 or 119 (SEQ U)NOSA1s6,113, 115, 117 orl119).

.5106. A method for treating or preventing a cell proliferative disorder associated with increased expression or activity of a protein having at least 80% amino acid sequence identity to: (a)the~dPOlPtdshO~i 0 yo 0 0 fig 5 7 1 12 1 14 1 1 6 11 8 or 120 (SEQID)N0S:574112,114,116, 00 1 18 or 120); the polypeptide shown inany one ofFigures 57-112,114,116, 118 or 120 (SEQ ID NOS:57-1 12, 114, 11,18 or 120), lacking its associated signal peptide; an extracellular domlain) of the polypeptideb shw n*nn of Figures 57-112, 114, 116,118 or 120 (SEQ ID NOS:57-1 12, 114, 116,118 or 120), with its associated signal peptide; IND an extracellum- domain of the polypeptide shown in any one of Figures 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57.j 12, 114, 116,118 or 120), lacking its associated signal peptide; a polypeptide encoded by the nuecotide sequence shown in any one of Figures 1-56, 113, 115, 117 or 119 (SEQ ID NOS: -56, 113,115, 117 or119);o 71 10 a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one ofFigures 1-56, 113,115, 117 or 19(SI3QIDNOS:1-56,113, 115, 117 or 119), said method comprising adminstering 00 to a subject in need of such treatment an effective amount of an antagonist of said protein, thereby effectively treating or preventing said cellprlfatvdioe.

107. The method of Claim 106, wherein said ceil proliferative disorder Is cancer.

15108. The method of Claim 106, wherein said antagonist is an anti-TAT polypeptide antibody,

TAT

binding oligopeptide, TAT binding organic molecule or antisense oligonucleotide.

109. A method of binding an antibody, oligopeptide or organic molecule to a cell that expresses a protein having at least 80% amino acid sequence identity to: (a)the polypeptide shownin anyone ofFigue 57-112, 114,116, 118 or 120(SEQIDNOS:57.1 12, 114,116, 118 or 120); the polypeptide shown in any one of Figures 57-112, 114, 116, 118 or 120 (SEQ ID NOS: 57112, 114, 116, 118 or 120), lacking its associated signal peptide; an extracellular domain of the polypeptide shown in any one of Figures 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-l 12, 114, 116, 118 or 120), with its associated signal peptide; Its an extracellular domain of the polypeptide shown in any one of Figures 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57-1 12, 114, 116, 118 or 120), lacking its associated signal peptide; a polypeptide encoded by the nucleotide sequence shown in any one of Figures 1-56, 113, 115, 117 or 119 (SEQ ID NOS: 1-56, 113,115, 117 or 119); or a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one 0 of Figures 1-56, 113, 115, 11 lor 119 (Sl3QMNOS: 1-56,113, 115,117 or 119), said method comprising contacting said cell with an antibody, oligopeptide or organic molecule that binds to said protein and allowing the binding of the antibody, oligopeptide or orgaric molecule to said protein to occur, thereby binding said antibody, oligopeptide or organic molecule to said cell.

110. The method of Claim 109, wherein said antibody is a monoclonal antibody.

3S 111. The method of Claim 109, wherein said antibody is an antibody fragment.

112. The method of Claim 109, wherein said antibody is a chinmeric or a humanized antibody.

113, The method ofClaIm 109, wherin said antibody, oligopeptide or organlo molecule is coqugated 00 to a growth inhibitory agent 114, The method ofClaim 109, wherein said antibody, oligopeptide or organic molecule is coqjugated CI to a cytotoxic agent.

115. het method of Claim 114, wherein said cytotoxic agent is selected from the group consisting of toxins, antibiotics, radioactive isotopes and nucleolytic enzymes.

N 116. 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 maytansinoid and calicheamicin.

118. The method of Claim 116, wherein the toxin is a maytansinoid.

119. The method of Claim 109, wherein said antibody is produced in bacteria.

S120. The method of Claim 109, wherein said antibody is produced in CHO cells.

S121. The method of Claim 109, wherein said cell is a cancer cell.

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

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

124. The method of Claim 123, wherein said protein is more abundantly expressed by said cancer cell -O as compared to a normal cell of the same tissue origin.

125. The method of Claim 109 which causes the death of said cell.

126. Use of a nucleic acid as claimed n any of Claims I to 5 or 30 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.

127. Use ofa nucleic acid as claimed in any of Claims to 5 or 30 in thepreparation of a medicament for treating a tumor.

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

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

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

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

132. Use of a host cell as claimed in any of Claims 8, 9, 32, or 33 in the preparation ofa medicament 3 5 for the therapeutic treatment or diagnostic detection of a cancer.

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

134. Use of a host cell as claimed in any of Claims 8,9, 32 or 33 in the prparation of a medicament 0 for treatment or prevention of a cell prolifeative disorder, 135 Use of a polypeptide as claimed in any of Claims 11 to 14 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.

136. Use of a polypeptide as claimed in any of Claims 11 to 14 in the preparation of a medicament for treating a tumor.

S137. Use of a polypeptide as claimed in any of Claims 11 to 14 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder.

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

139. Use of an antibody as claimed in any of Claims 15 to 29 in the preparation of a medicament for 00 treating a tumor.

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

141. Use of an oligopeptide as claimed in any of Claims 35 to 44 in the preparation of a medicament 1 5 for the therapeutic treatment or diagnostic detection of a cancer.

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

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

:O 144. Use of a TAT binding organic molecule as claimed in any of Claims 45 to 54 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.

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

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

147. Use of a composition of matter as claimed in any of Claims 55 or 56 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer, 148. Use of a composition of matter as claimed in any of Claims 55 or 56 in the preparation of a medicament for treating a tumor.

l 0 149. Use of a composition of matter as claimed in any of Claims 55 or 56 in the preparation of a medicament for treatment or prevention of a cell proliferative disorder, 150. Use of an article of manufacture as claimed in any of Claims 57 or 58 in the preparation of a medicament for the therapeutic treatment or diagnostic detection of a cancer.

151. Use of an article of manufacture as claimed in any of Claims 57 or 58 in the preparation of a 3 5 medicament for treating a tumor, 152. Use of an article of manufacture as claimed in any of Claims 57 or 58 In the preparation of a medicament for treatment or prevention of a cell proliferative disorder.

153. A method for inblitlng the growth. of a cell, wherein th growt fsi ell tlath a d0edn upnago t~ptnitn ffoct faprtin having at least 80% amino acid sequence identity to: 0 the PIDYPOPtideshowninanyone of ligiurc 57-112,114,116,118 Or 120 (SEQIDNQS:57.112,114, 116, CI118 or120); ct(b) tle polypeptide shown in any one ofFigures 57-112,114,116, 118 or 120 (SEQ ID NOS:57.1 12, 114, 116, 118 or 120), lacking its associated signal peptide; IND an extracellular domain of the polypeptide shown in any one of Figures 57-112, 114,116, 118 or 120 (SEQ ID NOS:57-1 12, 114, 116,118 or 120), with its associated signal peptide; an extracellular domain of the Polypeptide shown in any one of Figures 5 7-112, 114, 116, 118 or 12 0 (SEQIED NOS:57- 112 114, 116, 118 or 120), 1lacking Its associ ated signal pepuide; a polypeptide encoded by the nucleotide sequence shown in any one of Figures 1-56, 113, 115, 117 or 119 (SEQ DD NO)S:1-56, 113,115,117 or119);o 00) a polypeptide encoded by the fuxll-length coding region of the nuclootide sequence shown in any one ofFigume 1-56,113,115,117 or119 (SEQ ID NOS:1-56,113,115,117 or 1 19), said methodcompiing contacting said protein with an antibody, oligopeptide or organic molecule that binds to said protein, there by inhibiting the growth of said cell.

154. The method of Claim 153, wherein said cell is a cancer cell.

155. The method of Claim 153, wherein said protein is expressed by said cell.

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

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

158, The method of Claim 153, wherein said antibody is a monoclonal antibody.

159. The method of Claim 153, wherein said antibody is an antibody fragment.

160. The method of Claim 153, wherein said antibody is a chimieric or a humanized antibody.

161. The method of Claim 153, wherein said antibody, oligopeptide or organic molecule is conjugated to a growth inhibitory agent 162. The method of Claim 153, wherein said antibody, oligopeptide or organic molecule is conjugated to a cytotoxic agent.

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

164. The method of Claim 162, wherein the cytotoxic agent is a toxin, 165. The method of Claim 164, wherein the toxin is selected from the group consisting of rnaytansinoid and calicheamnicin.

166. The method of Claim 164, wherein (he toxin is a niaytansinoid.

167. The method of Claim 153, wherein said antibody is produced in bacteria, 168, The method of Claim 153, wherein said antibody is produced in CHO cells, 169. The method of Clam 153, heonz Wad prtin, has: (a 1h4wnjno l or 120(SEQIDNOS:57.112, 114, 116, 11801or120); the amino acid sequenoshowninaly one of~jg.m 5 57-1 12, 114,116,118 or 120 (SEQ IDNS5 12 114,116,116or 10),lacking its' associated signal peptide sequence; 1214 an amino acid sequence Of an extraceluu1ar domain Of the polypeptide shown it' any one of Figures .57-.

1211,116,118 or 120 (SEQ ID NOS:57 112,114, 116,118 or 120), with itsassociated signal peptidesequece an amino acid sequence of an extracellular domain of the polypeptide soni n oeo iue 57- 12114116118or 120 (SEQ ID NOS:57-1 12, 114, 116, 116 Or 120), lacking its associated signal eptide sequence; I an amnino acid sequence encoded by tile nucleotide sequence shown in' any one of Figures 1-56, 113, c-I115, 11 7 or119 (SEQ ID NOS: 1- 56 113 ,1517or19;O 00 Mf an amino acid sequence e ncoded by the full-length coding region of the nucleotide sequence shown in aly OneOf Figures 1-56, 113, 115,117 or 119(SEQ IDNOSA156,113, 115, 117 orl119).

170. A method of therapeutically treating a tumor in a mammral, wherein the growth of said tumor is at least in part dependent upon a growth potentiating effect of a protein having at least 80% amino acid sequence identity to: the polypeptide shown in yonmffi r 5712114,116,1 1 8 or 12U (SQIDNOS:57 112,114,11 6, 118 or 120); the polypeptide shown in any one offigures 57-112,114,116, 118 or 120 (SEQ ID NOS:57-1 12,114, 20116, 118 or 120), lacking its associated signal peptide; an extracellular domain of the polypeptide shown in any one of igures 57-112, 114, 116,118 or 120 (SEQ ID HOS:57-1 12, 114, 116, 118 or 120), with its associated signal peptide; an extlcellular domain of hepolypeptd hw nan n of Figures 57-112,114,116, 118 or 120 (SEQ ID NOS:57.1 12, 114, 116,118 or 120), lacking its associated signal peptide; a polypeptide encoded by the nuclooide sequence shown in any one of Figures 1-56, 113, 115, 117 or 119 (SEQ ED NOS:1-56, 113,115, 117 or 119); or a polypeptide encoded by the full-length coding region of the nucleotide sequence shown in any one ofFigures 1-56, 113, 115,117 or119 (SEQ IDNOS:1-56,113,115,1 I7or 119), saidnmethod comprising contacting said protein with an antibody, oligopeptide or organic molecule that binds to said protein, thereby effectively treating said tumor, 171. The method of Claim 170, wherein said protein is expressed by cells of said tumor.

172. Therrmethod ofClalm 170, wherein the binding ofsaid antibody, oligopeptide or organic molecule to said protein antagonizes a cell growth.potontiating activity of said protein.

173. The method of Claim 170, wherein said antibody is a monoclonal antibody.

33 174. The method of Claim 170, wherein said antibody is an antibody fragment, 175. The method of Claim 170, wherein said antibody is a chimeric or a humanized antibody, 176. The Method of Clam 170, wherein said antibody, oligopeptide or oipao molecule is conjugated 0 to M groWth inhibitory agent 177. The method of Claim 170, wherein said antibody oligopeptlde or or l omolel s eor1jugatod to at cytotoxic agent.

178. Ihe method of Claim 177, wherein said cytotoxic agent is selected from the group consising of antibiotics, radioactive isotopes and nucleolytic. enzymes.

IND179. The method of Claim 177, wherein the cytotoxic agent is a toxin.

180. The method of Claim 179, wherein the toxin is selected from the group consisting of maytansinold and calicheanjicin.

181. The method of Claim 179, wherein the toxin is a maytansinoid.

c 10 182. The method of Claim 170, wherein said antibody is produced in bacteria.

183. The method of Claim 170, wherein said antibody is produced in CHO cells.

00184. The method of Claim 170, wherein said protein has: theamino acidsquence shownin any oneofFlgues57 1 12,114,116,118 or 120(SBQIDNOS:57.1 12, 114, 116, 11 8 or 120); 1 5(b) theaniino acid sequence shown in any one ofpigures57.

1 12,114, 116, 118 or 120 (SEQ IDNOS:57-1 12, 114, 116, 118 or 120), lacking its associated signal peptide sequence; an amino acid sequence of an extracellular domain of the polypeptide shown in any one of Figures 57- 112, 114, 116, 118 or 120 (SEQ ID NOS:57.i 12, 114, 116, 118 or 120), with its associated signal peptide sequence; an amino acid sequence of an extracellular domain of the polypeptide shown in any one of Figures 57-112, 114, 116, 118 or 120 (SEQ ID NOS:57.1 12, 114, 116, 118 or 120), lacking its associated signal peptide sequence; an amino acid sequence encoded by the nucleotide sequence shown in any one of Figures 1-56, 113, 115, 117 or 119 (SEQIDNOS: 1-56,113, 115, 117 or 11);o an amino acid sequence encoded by the full-length coding region of the nucleotide Sequence Chown in any one of Figures 1-56, 113, 115, 117 or 119 (SBQ ID NOS: 1-56, 113, 115, 117 or 119).

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

BRIEF DECRIPTION OF TPlE DFA, WINGS Figure 1 shows a nucleotide sequence (SEQ IDDNO: 1) of a TAT207 cDNA, wherein SEQ ID NO: 1 is a clone designated herein as "DNA67962".

Figure2 shows a nucleotide sequence (SEQ IDNO:2) of a TATl177 c.DNA, wherein SEQ ID NO:2 is aclone designated herein as "DNA77507'.

Figure 3 shows a nucleotide sequence (SEQ ID NO: 3) of a TAT23 5 oDNA, wherein SEQ ID NO: 3 is a clone designated herein as "DNA87993".

Figure 4 shows a nucleotide sequence (SEQ ID NO:4) ofeaTAT234 eDNA, wherein SEQID NO:4 is a Clone designated herein as "DNA92980".

Figures Shows anlucleofdese ev(SEp faA29oNwhri EJN~ 8al 00 dg1nated hi 'DTNA96792". ])N:5of&A29D1AwhtOU DN: Iacln Figure 6 shows a nucleotide sequence (SEQ ED NO:6) ofaTATI 93 CDNA, wbpreln SEQ ID NO:6 Is a clone c-I designated4 herein as "DNA96964".

Figre7 sow a uceotdeequnc (SQ DNO7)ofa TAT233 eDNA, wherein SEQ ID NO:7 is a clone designatedj herein as "DNA105792".

IND Figure 8 shows anucleotide sequence

(SEQIDNQ:

8 ofaTAT226 cDNA, wherein SEQ IDNO:8 is a clone designated herein as'"DNAI 19474".

Figure 9 shows a nucleotide sequence (SEQIDNO:9) of a TATI 99 cDNA, wherein SEQ 1iD NO:9 is a clone designated herein as "DNA142915".

Figures 1OA-B show a nuoleotide sequence (SEQ ID NO: 10) of a TAT204 eDNA, wherein SEQ ID NO: is a clone designated herein as "DNAI 50491" 00 Figures I lA-B show a nucleotide sequence (SEQ ID NO. 11) of a TAT248 oDNA, wherein SEQ I NO:II is a clone designated herein as "DNA280351".

Figure 12 shows a nucleotide sequence (SEQ ID NO: 12) of a TAT232 cDNA, wherein SEQ ID NO: 12 is a clone designatedi herein as "DNA159648".

Figure 13 shows a nucleotide sequence (SEQ ED NO:13) of aTAT219 eDNA, wherein SEQI1D NO:13 is a clone designated herein as "DNA 172500".

Figure 14 shows a nucleofide sequence(sEQ ID NO: 14 of aTAT224 cDNA, wherein SEQ ID NO: 14 is a clone designated herein as "DNA 179651 Figure 15 shows a nucleotide sequence (SEQ IDN14:15) of aTAT237 oDNA, wherein SEQ ID NO:1 5 is a clone designated herein as "DNA207698".

Figure 16 shows a nucleotide sequence (SEQ ID NO: 16) of a TAT178 cDNA, wherein SEQ ID NO:16 is a clone designated herein as 'DNA2OBSS 1".

Figures 17A-B show a nucleotide sequence (SEQ ID NO: 17) of a TATI 98 oDNA, wherein SEQ ID NO: 17 is a clone designated herein as "DNA2Ici1S9".

Figures 1 8A-B show a flucleotide sequence (SEQ ID NO: 18) of a TATI 94 eDNA, wherein SEQ ID NO: 18 is a clone designated herein as "DNA225706".

Figures 1 PA-B show a nuoleotide sequence (SEQ ID NO: 19) of a TAT223 -,DNA, wherein SEQ ID NO: 19 is a clone designated herein as "DNA225793".

Figure 20 shows a nucleotide sequence (SEQ ID NO:20) of a TATI 96 cDNA, wherein SEQ ED NO:20 is a clone designated herein as "DNA225796".

Figure 21 shows a nucleotide sequence (SEQ ID NO:21) of aTAT736 cDNA, wherein SEQ ID NO:21 is a clone designated herein as "DNA225886".

Figure 22 shows a nucleotide sequence (SEQ ID NO:22) of a TAT195 cDNA, wherein SEQ ID NO:22 is a clone designated herein as "DNA225943".

Figure 23 shows a nucleotide sequence (SEQ ID NO:23) of a TAT203 cDNA, wherein SEQ ED NO:23 Is a clone designated herein as "DNA226283".

Figuim 24A-B show anucleolddo sequence (SEQ IDNO:,24) of aTAT200 cl)MA, wherein SEQ ID NO:24 00is a clone desigaaW~ herein as "DA22,6589".

00 ~Figures 25A.B show a fuoleotide sequence (SEQ IDNO4:25) of a TA1705 oI)NA, wherein SEQM1) O:2 is a clone designatedj herein as "DNA226622".

Figures 26A..B show& a ucleotide sequence (SEQ ED NO:26) of a TATI 85 eDNA whereinSEQ1II NQ:26 is a clone designated herein as "DNA2267 17".c A, r E D IDFigures 27A-B show a nucleofide sequence (SEQ ID NO:27) of a TAnr25 cDNA, whereinSQDNO2 is a clone designated herein as sDNA227 162"l.

Figure 28 shows a nucleotide sequence (SEQ ID) NQ:28) of a TAT247 oDNA, wherein SEQ lb NO:28 is 0 ~a clone designated herein as "D)NA277804".

Figure 29 shows a nucleotide sequence (SEQ ID NQ:29) of a TAT197 cDNA wherein SE-QID NO:29 is a clone designated herein as "DNA227545".

00 Figure 30 shows a nucleotide sequence (SEQ ID NO:30) of a TAT175 cDNA, wherein SEQ ID NO:30 is a clone ,deslgnated heroin as'IDNA22761 1" Figure 31 shows a nucleotide sequence (SEQ ED 40:3 1) of a TAT708 cDNA, wherein SEQ ID NO:3 1 is a clone designated herein as "DNA261021".

Figure 32 shows a nucleotide sequence (SEQ ID NO:32) of a TAT174 eDNA, wherein SEQ ID NO:32 is a clone designated herein as "DNA233034".

Figure 33 shows a nlucleotide sequence (SEQ ID 140:33) of a TAT214 cDNA, wherein SEQ ID 140:33 is a clone designated herein as "DNA266920".

Figure 34 shows a flucleotide sequence (SEQ ED 140:34) of a TAT220 cDNA, wherein SEQ ID NO:34 is a clone designated herein as "DNA26692

I"

Figure 35 shows a nuoleotide sequence (SEQ ID 140:35) of a TAT2I cDNA, wherein SEQ ID NO:35 is clone designated herein as "DNA266922".

Figure 36 shows a nucleotide sequence (SEQ ID NO:36) of a TAT201 cDNA, wherein SEQ ID NO:36 is a clone designated herein as 'DNA23444

V".

Figures 37A-B show a nucleotide sequence (SEQ ED NO:37) of a TAT179 oDNA, wherein SEQ ID NO:37 is a clone designated herein as "DNA234834".

Figure 38 shows a nucleotide sequence (SEQ ID NO:38) of a TAT-216 cDNA, wherein SEQ ID NO:38 Is a clone designated herein as "DNA247587".

31DFigure 39 shows a flucleotide sequence (SEQ ID 140:39) of a TAT218 cDNA, wherein.SEQ ID NO:39 is a clone designated herein as "DNA255987".

Figure 40 shows a nucleotide sequence (SEQ ID 140:40) of a TAT206 eDNA, wherein SEQ ID NO0:4 is a clone designated herein as "D)NA5604

P".

Figures 41lA-13 show a nucleotjde sequence (SEQ ID NO:4 1) of a TAT3 74 eDNA, wherein SEQ ID NO.41 is a clone designated herein as "DNA257845".

Figure 42 shows a nucleotide sequence (SEQ ID 140:42) of a TAT209 cDNA, wherein SEQ ID 140:42 is a clone designated herein as "DNA260655".

Mioun 43 shovm a nucleotide sequence (SEQ rD WO:43) of a TAT, 92 eDNA, wherein SEQ ID NO,43 is 00 et. clone designated heivin as "DNA260945".

Figure 44 Shows a nucleOtidc sequence (SEQ ID NO:44) of a TATIBO 80DNA, wherein SEQ ED NO:44

JE

ri a clone designated herein as "DNA247476", Figure 45 shows a nucleotide sequence (SEQ I) '90:45) of a TAT375 cDNA, wherein SEQ ID NO:45 is a clone designated herein as "DNA260990

I!*

INDFigure 46 shows a nucleotide sequence (SEQ ID NO:46) of a TAT1 81 cDNA, wherein SEQ ID N0:46 is a clone designated herein as "DNA261001" Figure 47 shows a nucleotide sequence (SEQ D NO:47) of a TAT176 cDNA, wherein SEQ ED NO:47 is a clone designated herein as "DNA261013".

S 10 Figure 48 shows a nucleotide sequence (SEQ ID NO:48) of a TATI 84 cDNA, wherein SEQ ID) NO:48 is a clone designated herein as "DNA262144", 00 Figure 49 shows a nucleotide. sequence (SEQ ID NO:49) of a TATI 82 cDNA, wherein SEQ I1D NO:49 is a clone designated herein as "DNA266928".

Figures 50A-B show a nucleotide sequence (5 1 3Q ID 140:50) of a TAT23 oDNA, wherein SEQ ID is a clone designated herein as "DNA267342".

Figures SIA-C show a nucleotide sequence (SEQ EiD NO:5 1) of a TAM2 7 cDNA, wherein SEQ 11D NO:5 I is a clone designated herein as "DNA267626".

Figure 52 shows a nucleotide sequence (SEQ ID NQ:52) of a TAT222 cDNA, wherein SEQ lID NO:S2 is a clone designated herein as "DNA26SOS Figure 53 shows a nucleotide sequence (SEQ ID NQ:53) of a TAT2O2 'eDNA, wherein SEQ ID NO:53 is a clone designated herein as'"DNA268334".

Figure 54 shows a nucleotide sequence (SEQ ID NO:54) of a TAT215 cDNA, wherein SEQ ID NO:54 is a clone designated herein as "DNA269238".

Figure 55 shows a nucleotide sequence (SEQ ID NQ:55) of a TAT238 cDNA, wherein SEQ ID NO:55 is 2s a clone designated herein as "DNA272578".

Figure 56 shows a nucleotide sequence (SEQ ID NO: 56) of a TAT2 12 cDNA, wherein SEQ ID NO; 56 is a clone designated herein as "DNA277797".

Figure 57 shows the amiino acid sequence (SEQ ID NO:57) derived from the coding sequence of SEQ ED NO:;I shown In Figure 1, ID Figure 58 shows the amino acid sequence (SEQ II) NO:58) derived from the coding sequence of SEQ IM NO:Z shown in Figure 2.

Figure 59 shows the amnino acid sequence (SEQ ID NO: 59) derived from the coding sequence of SEQ ID) NO:3 shown in Figure 3.

Figure 60 shows the amnino acid sequence (SEQ EID NO:60) derived from the coding sequence of SEQ ED NO:4 shown in Figure4.

Figure 61 shows the an-ino acid sequence (SEQ ID NO:61) derived from the coding sequence of SE3Q ID NO: 5 shown in Figure

S.

Figur 62 ObI0DWS the amino acid sequence (SE .Q ]D HO:62) derived from the coding sequence of SEQ ID) 00 ~N0:6 sown in i, 6 C)Figure 63 shows the amino aold sequence (SEQ ID) NO:63) derived from the codigsqec fSQU NO:7 shown in Figure 7.digsqecofS

I

Figure 64 shows the amino acid sequence (SEQ ID) NO:64) derived fr m the coding sequenc of SEQ U NO:8 shown in Figures8.

I

Figure 65 shows the amino acid sequence (SEQ ID MQ:65) derived from the coding sequence of SEQ I1) NOS9 shown in Figure 9.

Figure 66 shows the amino acid sequence (SEQ ID N0:66) derived from the coding sequence of SPQ lb NO- 10 shown in Figures I OA.B.

Figure 67 shows the amino acid sequence (SEQ ID NO:67) derived frm the coding sequence of SEQI NO: II showin Figures I IA-B.

Figure 68 shows the amino acid sequence (SEQ ED) NO. 68) derived from the coding sequence of SEQ ID cINO:12 shownIn Figure12, Figure 69 shows the amino acid sequence (SEQ ID NO:69) derived from the coding sequence of SEQ ED NO: 13 shown in Fiue 3 Figure 70 shows the amnino acid sequence (SEQ ID NO: 70) derived from the coding sequence of SEQ ID NO: 14 shown in Figure 14.

Figure 71 shows the amino acid sequence (SEQ lID NO: 7 1) derived fro~m the coding sequence of SEQ ID NO: 15 shown in Figure Figure 72 shows the amino acid sequence (SEQ ID 140:72) derived from the coding sequence of SEQ II NO 6 shown in Figure 16.

Figure 73 shows the amino acid sequence (SEQ ID) 10:73) derived from the coding sequence of SEQ U)D NO: 17 shown in Figures 17A.B.

Figure 74 shows the amino acid sequence (SEQ D) N40:74) derived from the coding sequence of SEQ ED) NO: 18 shown in Figures I BA-B.

Figure 75 shows the amino acid sequence (SEQ UD 140:75) derived from the coding sequence of SEQ ID) NO: 19 shown in Figures 19A-13.

Figure 76 shows the amino acid sequence (SEQ ID NO: 76) derived from the coding sequence of SEQ ID 140:20 shown in Figure Figure 77 shows the am-ino acid sequence (SEQ EiD NO:77) derived from the coding sequence of SEQ WI 140:21 shown in Figure 21, Figure 78 shows the amino acid sequence (SEQ ED NO:78) derived from the coding sequence of SEQ ID) NO;22 shown in Figure 22.

Figure 79 shows the amino acid sequence (SEQ D) NO:79) derived from the coding sequence of SEQ HD 'S NO:23 shown in Figure 23.

Figure 80 shows the amino avid sequence (SEQ ID NO:80) derived from the coding sequence of SEQ ID 140:24 shown in Figures 24A-B.

Figure 81 shows the amino acdd sequence, (SEQ ID NO:8 1) derived fiam the coding sequence of SEQ ID 00 NO:25 shown InFigums 0 Figure 82 shows the amino acid sequence (SEQ ID NO:82) derived from the coding sequence of SEQ ED) ri NO:26 Shown in Figures 26A.B, Figure 83 shows the *amino acid sequence (SEQ ID NO: 83) derived from the coding sequence of SEQ I0 NQ:27 shown in Figures 27A..B.

IDFigure 84 shows the amino acid sequence (SEQ ID NO: 84) drvdfo h oigsqec fSQ11 NO:28 shown In Figure 28. drvdfo h oigsqec fSQE Figure 85 Show$ the amino acid sequence (SUQ ED NO: 85) derived from the coding sequence of SEQ ID) NO: 29 shown in Figure 29.

Figure 86 shows dhe amino acid sequence (SEQ ED NO:86) derived from the coding sequence of SEQ D:) shown ill Figure 0C) Figure 87 shows the amino acid sequence (SEQ ID NO:87) derived from the coding sequence of SEQ E NQ:31 shown inpigure3 1 Figure 88 shows the amino acid sequence (SEQ ED NO:88) derived fr-om the coding sequence of SEQ ID) NO:32 shown in Figure 32.

Figur 89 shows the amino acid sequence (SEQ ID NO: 89) derived from the coding sequence of SEQ ID NO:33 shown in Figure 33.

Figure 90 shows the amino acid sequence (SEQ ID NO:90) derived from the coding sequence of SEQ ID NO:34 shown in Figure 34.

Figure 91 shows the amtino acid sequence (SEQ ID NO:91) derived from the coding sequence of SEQ ID shown in Figure Figure 92 shows the amino acid sequence (SEQ ED NO.92) derived from the coding sequence of SEQ It NO:36 shown in Figure 36, Figure 93 shows the amino acid sequence (SEQ ID NO:93) derived from the coding sequence of SEQ ID) 25NO:37 shown in Figures 37A-B.

Figure 94 shows the amnino acid sequence (SEQ ID NQ:94) derived from the coding sequence of SEQ ID NO:38 shown in Figure 38, Figure 95 shows the amnino acid sequence (SEQ ID NO:95) derived from the coding sequence of SEQ ID NQ:39 shown in Figure 39.

Figure 96 shows the amino acid sequence (SEQ ID NO:96) derived from the coding sequence of SEQ MD shown in Figure Figure 97 shows the amino acid sequence (SEQ ID NO:97) derived from the coding sequence of SEQ lID NO:41 shown in Figures 4 lA-B, Figure 98 shows the amino acid sequence (SEQ D) NO:98) derived from the coding sequence of SEQ ID NO:42 shown in Figure 42.

Figure 99 shows the amino acid sequence (SEQ ID NO:99) derived from the coding sequence of SEQ ID) NO:43 shown in Figure 43.

Figure 100 shows the amino acd sequence (no( ID 110 1) derived foM the coding soquence of SEQ 00 ~ID NO:44 shOWUn Fre44 Figure 101 shows the amino add sequenc (SEQ ID NO: 101) derived from the coding sequence of SE3Q ciID) NO:45 shown in Figure Figure 102 shows the aminto acid sequence (SEQ ID NO: 102) derived from the coding sequence of SEBQ ID) NQ:46 shown in Figure 46.

IND Figure 103 shows the amitio acid sequence (SEQ NO: 103) derived from the coding sequence of S13Q ED NO:47 shown in Figure 47.

Figure 104 shows the amino acid sequence (SEQ ID NO: 104) derived from the coding sequence 6f SEQ ED NO:48 shown in Figure 48, c=KI 10Figure 105 shows the amino acid sequence (SEQ ID NO: 105) derived from the coding sequence of SEQ ID NO:49 shown in Figure 49, 0C) Figure 106 shows the amino acid sequence (SEQ ID NO: 106) derived from the coding sequence of SEQ II) NO:50 shown in Figures Figures 107A.13 show the amino acid sequence (SEQ ID NO: 107) derived from the coding sequence of SEQ ID N1O:51 shown in Figures 5 1 A-C.

Figure 108 shows the aino acid sequence (SEQ ID) NO: 108) derived from the coding sequence of SEQ ID NO:52 shown in Figure 52.

Figure 109 shows the amino acid sequence (SEQ ID NO: 109) derived from the coding sequence of SEQ ED) NO:53 shown in Figure 53, Figure 110 shows the amino acid sequence (SEQ ID NO: 110) derived from die coding sequence of SEQ II) NO:54 shown in Figure 54.

Figure I1I1 shows the amino acid sequence (SEQ ID NO: 11) derived fr-om the coding sequence of SEQ N0:55 shown in Figure Figure 112 shows the amino acid sequence (SEQ ID NO: 112) derived from the coding sequence of SEQ ED NO:S6 shown in Figure 56.

Figure 113 shows a nucleotide sequence (SEQ ID NO: 113) of a TAT376 cDNA, wherein SEQ ED NO: 113 is a clone designated herein as "DNA304853".

Figure 114 shows the amino acid sequence (SEQ ED NO:1 14) derived from the coding sequence of SEQ ID NO:l 113 shown in Figure 113.

Figure 115 shows a nucleotide sequence (SEQ ID NO: 115) of a TAT3 77 cDNA, wherein SEQ ID NO: 115 is a clone designated herein as "DNA304854', Figure 116 shows the amino acid sequence (SEQ ID NO: 116) derived from the coding sequence of SEQ ID NO: 115 shown in Figure 115.

Figure 117 shows a nucleotide sequence (SEQ ID NO: 117) of a TAT3 78 cDNA, wherein SEQ ID NO: 117 is a clone designated herein as "DNA30485".

Figure 118 shows the amino acid sequence (SEQ ED N0: 118) derived fr-om the coding sequence of SEQ ED NO.117 shown in Figure 1177.

F ig u re s 1 1 9 A B h waDc e t d e u n e l Q l g o 9 f a T 7 7 N h r i B I 0C) NO: 119 if; a clone designted herein ag "DNA287971tt.., faA7 cNAwhriSQ

I

Figure 120 shows the amino acid sequence (SEQ ID NO.:120) derived fimm the coding sequence of SEQ ID1. Definitions The terms "TAT polypeptide" and "TAT" as used herein and when immediately followed by a numerical I> designation, refer to various polypeptides, wherein the complete designation (i.e.,TAT~number) refers to specific polypeptide sequences as described herein. The terms "TAT/number polypeptide" and 'TAT/number" wherein the term "number" is provided as an actual numerical designation as used herein encompass native sequence CK1 polypeptides, polypeptide variants and fragments of native sequence polypeptides and polypeptide variants 00 (which are further defined herein). The TAT polypeptides described herein may be isolated from a variety of sources, suoh as iuom human tissue types or from another source, or prepared by recombinant or synthetic methods. The term "TAT polypeptide" refers to each individual TAT/number polypeptide disclosed herein. All disclosures in this specification which refer to the "TAT polypeptide" refer to each of the polypeptides individually as well as jointly. For example, descriptions of the preparation of, purification of, derivation of, formation of antibodies to or against formation of TAT binding oligopeptides to or against, formation of TAT binding organic molecules to or against, adnistration of, compositions containing, treatment of a disease with, etc., pertain to each polypep tide of the invention individually. The term "TAT polypeptide" also includes variants of the TAT/number polypeptides disclosed herein.

A "native sequence TAT.polypeptide" comprises a polypeptide having the same amino acid sequence as the correponding TAT polypeptide derived from nature. Such native sequence TAT polypeptjdes can be isolated from nature or can be produced by recombinant or synthetic means. The term "native sequence

TAT

polypeptide" specifically encompasses naturally-occuring truncated or secreted forms of the specific TAT polypeptidc an extracellular domain sequence), naturaliy-occurring variant forms alternatively spliced forms) and naturally-occurrjng allelic variants of the polypeptide. In certain embodiments of the invention, the native sequence TAT polylpeptides disclosed herein are mature or full-length native sequence Polypeptides comprising the full-length amino acids sequences shown in the accompanying figures. Start and stop codons (if indicated) are shown in bold font and underlined in the figures. Nucleic acid residues indicated as in the accompanying figures are any nucleic acid residue. However, while the TAT polypeptides disclosed in the accompanying Figures are shown to begin with niethionine residues designated herein as amino acid position

I

in the figures, it is conceivable and possible that other methionine residues located either upstream or downstream from thie amino acid position I in the figures may be employed as the starting amino acid residue for the TAT polypeptides.

The TATpolypeptide "extracellular domain" or 'IBCD" refers to a form of the TAT polypeptide whioh is esnilyfree of the transmeinbrxie and cytoplasmic domains. Ordinarily, a TAT polypeptide ECD will have less than of such transmembrane and/or cytoplasmic domains and preferably, will have less than 0.5% of such domains. It will be uaderstod itt any transembrane domains identified for the TAT polypeptdes of the Spresent invention are dentified pursuant to criteria routinely employed in the art for identifying that type of C hydrophobic domain. The exactboundaries of a transmembrane domain may vay but most likely by no more than about 5 amino acids at either end of the domain as initially identified herein. Optionally, therefore, an extracelular domain of a TAT polypeptide may contain from about 5 or fewer amino acids on either side of the transmembrane domainextracellular domain boundary as identified in the Examples or specification and such polypeptides, with Sor without the associated signal peptide, and nucleic acid encoding them, are contemplated by the present invention.

The approximate location of the "signal peptides" of the various TAT polypeptides disclosed herein may O be sho w n in the present specification a nd or t i e c 0 be shown in the present specification and/or te accompanying figures. It is noted, however, that the C-terminal C 10 boundary of a signal peptide may vary, but most likely by no more than about 5 amino acids on either side of the 0 signal peptide C-terminal boundary as initially identified herein, wherein the C-terminal boundary of the signal peptide may be identified pursuant to criteria routinely employed in the art for identifying that type of amino acid N\1 sequence element Nielsen etal., gkr 10:1-6(1997) and von Heinje et al., Nucl. Acids Res. 14:4683-4690 (1986)). Moreover, it is also recognized that, in some cases, cleavage of a signal sequence from a secreted polypeptide is not entirely uniform, resulting in more than one secreted species. These mature polypeptides, where the signal peptide is cleaved within no more than about 5 amino acids on either side of the C-terminal boundary of the signal peptide as identified herein, and the polynucleotides encoding them, are contemplated by the present invention.

"TAT polypeptide variant" means a TAT polypeptide, preferably an active TAT polypeptide, as defined -0 herein having at least about 80% amino acid sequence identity with a full-length native sequence TAT polypeptide sequence as disclosed herein, a TAT polypeptide sequence lacking the signal peptide as disclosed herein, an extacellular domain of a TAT polypeptide, with or without the signal peptide, as disclosed herein or any other fragment of a full-length TAT polypeptide sequence as disclosed herein (such as those encoded by a nuoleic acid that represents only a portion of the complete coding sequence for a full-length TAT polypeptide). Such TAT polypeptide variants include, for instance, TAT polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the full-length native amino acid sequence. Ordinarily, a TAT polypeptide variant will have at least about 80% amino acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% amino acid sequence identity, to a full-length native sequence TAT polypeptide sequence as disclosed herein, a TAT polypeptide sequence lacking the signal peptide as disclosed herein, an oxtracellular domain of a TAT polypeptide, with or without the signal peptide, as disclosed herein or any other specifically defined fragment of a full-length TAT polypeptide sequence as disclosed herein. Ordinarily, TAT variant polypeptides are at least about 10 amino acids in length, alternatively at leastabout 20, 30, 40, 50, 60,70,80,90,100,110, 120,130,140,150,160,170, 180, 190, 200,210,220, 230,240,250,260,270,280,290,300,310,320,330,340,350,360,370380,390,400,410,420,430,440,450,460,470 3 5 480, 490, 500, 510, 520, 530, 540, 550, 560,570, 580,590, 600 amino acids in length, or more. Optionally, TAT variant polypeptides will have no more than one conservative amino acid substitution as compared to the native TAT polypeptide sequence, alternatively no more than 2,3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitution a compared to the native TAT polypptd sequence.

o "Percent amino acid sequence identity" with respect to the TAT polypeptid sequences dentified herein is defined as the percentage of amino acid esidues in a candidate sequence that are identical with the amino acid residues in the specific TAT polypeptide sequence, after aligning the sequences and introduing gaps, if necessay, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of dithe sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithnis needed to achieve maximal aligment over the full length of the sequences being compared. For purposes herein, however, amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2, wherein the 00 complete souce code for the ALIGN-2 program is provided in Table below. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code shown in Table 1 below has been filed with user documentation in the U.S. Copyright Office, Washington 20559, where it is registered under U.S.

Copyright Registration No. TXU510087. The ALIU -2 program is publicly available through Genenteh, Inc., South San Francisco, California or may be compiled from the source code provided in Table 1 below. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequence comparisons, the amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of aino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the amino acid sequence identity of A to B will not equal the amino acid sequence identity of B to A. As examples of amino acid sequence identity calculations using this method, Tables 2 and 3 demonstrate how to calculate the amino acid sequence identity of the amino acid sequence designated "Comparison Protein" to the amino acid sequence designated "TAT", wherein "TAT" represents the amino acid sequence of a hypothetical TAT polypeptide of interest, "Comparison Protein" represents the amino acid sequence of a polypeptide against which the "TAT" polypeptide of interest is being compared, and and each represent different hypothetical amino acid residues. Unless specifically stated otherwise, all amino S acid equence identity values used herein are obtained as described in the immediately preceding paragraph using tie ALIGN-2 computer program, "TAT variant polYrnuwde e Or 'TAT vatiant nucleic aold sequence mewn a nuclejo acid moleow4e 00 Which encodes a TAT polypeptide, preferably an active TAT polypeptide, as defined heren and whichl has at leant about 80% nucle~c acid se*quence identity with a nucleotide acid sequence encoding a full-length native sequence TAT Polypeptide sequence as disclosed herein, a full-length native sequence TAT polypeptide sequence laocing the signal peptide as disclosed herein, an extacollular domain of a TAT polypeptide, with or without the signal Peptide, as disclosed herein or any other fragment of a full-length TAT polypeptide sequence as disclosed herein IND (Such as those encoded by a nucleic acid that represents only a portion of the complete coding sequence for a fulllength TAT polypeptide), Ordinarily, a TAT variant polynucleooide will have at least about 80% nucleic acid sequence identity, alternatively at leatabout81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%K, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity with a nucleic acid sequence encoding a NK 1 full-length native sequence TAT polypeptide sequence as disclosed herein, a full-length native sequence

TAT

polypeptide sequence lacking the signal peptide as disclosed herein, an extracellular domain of a TAT polypeptide, 00 with or without the signal sequence, as disclosed herein or any other fi-agmnt of a full-length TAT polypeptide sequence as disclosed herein. Variants do not encompass the native nucleotide sequence.

Ordinarily, TAT variant polynucleotides are at least about 5 nucleotides In length, alternatively at least cut 1 ,1 ,2 1 ,4 1 ,61 ,8 1 ,0 2 ,22 ,4 2 ,6 2 ,8 2 ,03 ,04 ,0 5 ,0 5, 0 75,80,85,90,gs,100,10s 110,115,120,125,130,135,140,145,150,155,160,165,170, 175,180,185,190,195,200, 210,220,230,240,250,260,270,280290300,310,320,330,340,350,360,370,380,390,400,410,420,430,440450, 460k 470, 480, 490, 500,510,520,530,540,550,560,570,580,590,600,610,620,630,640,650,660,670,680,690,7 00, 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 nucleotidos in length, wherein in this context the term "about" means the referenced nuleotide sequence length plus or minus 10% of that referenced length.

"Percent nucleic, acid sequence identity" with respect to TAT-encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with thie nucleotides in the TAT nucleic acid sequence of interest after aligning the sequences and Introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN orlMegalign (DNASTAR) software.

For purposes herein, however, nucleic acid sequence identity values pire generated using the sequence comparison computer program AL[GN-2, wherein the complete source code for the ALIGN-2 program is provided :0 in Table I below. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code shown in Table I below has been iled with user documentation in the-U,S. Copyright Office, Washington D. 20559, where it is registered under U.S. Copyright Registration No. TXU5 1008 7, The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California or may be compiled from the source code provided In Table 1 below. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital LMI V4.01), All sequence comparison parameters are set by the ALIGN-2 program and do not vary, In situations where ALIGN2 is employed for nucleic acid sequence comparisons, the nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows: 100 times the fraction W/Z where W is the number of nucleotides scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of C and D, and where Z is the total number ofnucleotides in D, It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the S 10 nucleic acid sequence identity of C to D will not equal the nucleic acid sequence identity ofD to C. As exampes "i0 of nucleic acid sequence identity calculations, Tables 4 and 5, demonstrate how to calculate the nucleic acid Ssequence identity of the nucleic acid sequence designated "Comparison DNA" to the nucleic acid sequence designated "TAT-DNA", wherein "TAT-DNA" represents a hypothetical TAT-encoding nucleic acid sequence of interest, "Comparison DNA" represents the nucleotide sequence of a nucleic acid molecule against which the AT-DNA".nuclec acid molecule of interest is being compared, nd and each represent different hypothetical nucleotides. Unless specifically stated otherwise, all nucleic acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

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

The term "fll-length coding region" when used in reference to a nucleic acid encoding a TAT polypeptide refers to the sequence of nucleotides which encode the full-length TAT polypeptide of the invention (which is often shown between start and stop codons, inclusive thereof, in the accompanying figures). The term 2 5 "full-length coding region' when used in reference to an ATCC deposited nucleic acid refers to the TAT polypeptide-encoding portion of the cDNA that is inserted into the vector deposited with the ATCC (which is often shown between start and stop codons, inclusive thereof, in the accompanying figures).

"Isolated," when used to describe the various TAT polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment.

Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the polypeptide will be purified to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassieblue or, preferably, 2- silver stain. Isolated polypeptide includes polypeptide in ltu 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.

An "isolated" TAT polypeptid"-enoding nucleic add or other olypept1de-encoding nucleo acid is a Snuclei acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the polypeptideencoding nucleic acid. An isolated polypeptide-enooding nucleic acid molecule is other than in the form or setting in which it is found in nature.

Isolated olypeptide-enoding nucleic acid molecules therefore are distinguished from the specific polypeptideencoding nucleic acid molecule as it exists in natural cells. However, an isolated polypeptide-encoding nucleic acid molecule includes polypeptide-enooding nucleic acid molecules contained in cells that ordinarily express the polypeptide where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.

The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, 00 for example, include a promoter, optionally an operator sequence, and a ribosome binding site. ukaryotio cells Sare known to utilize promoters, polyadenylation signals, and enhancers.

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

"Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration.

In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel ;0 o et al.,Cuen Protocol in Molecular Bioo Wiley Interscience Publishers, (1995).

"Stringent conditions" or "high stringency conditions", as defined herein, may be identified by those that: employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50*C; employ during hybridization a denaturing agent, such as formamide, for example, 50% formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyn'olidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42'C; or overnight hybridization in a solution that employs 50% formamide, 5 x SSC (0.75 MNaC, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 0.1% sodium pyrophosphate, 5 x Donhardt's solution, sonieated salmon sperm DNA (50 0.1% SDS, and 10% dextrn sulfateat 42'C, with a 10 minute wash at 0 42C in 0.2 x SSC (sodium chloridesodium citrate) followed by a 10 minute high-stingency wash consisting of 0.1 x SSC containing DTA at 5 5

C.

"Moderately stringent conditions" may be identified as described by Sambrook et al., Moecular Cloni A Laboratory Manua, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions temperature, ionic strength and %SDS) less stringent that those described above.

An example of moderately stringent conditions is overnight incubation at 37°C in a solution comprising: formamide, 5 x SSC (150 mMNaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 5 x Denhardt's solution, 10% dextran sulfate, and 20 mgml denatured sheared salmon spermDNA, followed by washing the filters in I x SSC at about 37-50 C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc.

S 10 as necessary to accommodate factors such as probe length and the like.

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

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

TAT.

The term "antagonist" is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native TAT polypeptide disclosed herein. In a similar manner, the term "agonist" is used in the broadest sense and includes any molecule that mimics a biological activity of a native TAT polypeptide disclosed herein. Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native TAT polypeptides, peptides, antisense oligonucleotides, small organic molecules, etc. Methods for 3 0 identifying agonists or antagonists of a TAT polypeptide may comprise contacting a TAT polypeptide with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the TAT polypeptide.

"Treating" or "treatment" or "alleviation" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. A subject or mammal is successfully "treated" for a TAT polypeptide-expressing cancer if, after receiving a therapeutic amount of an anti-TAT antibody, TAT binding Oligopeptide or TAT bindng organic molecule according to the methods of the present invention, the patient 00 shows observable and/or measuble reduction in or absence of one or mom of the following reduction in the Snumber of cancer cells or absence of the cancer cells; reduction in the tumor size; inhibition slow to some extent and preferably stop) of cancer cell infiltration into peripheral organs including the spread of cancer into soft tissue and bone; inhibition slow to some extent and preferably stop) of tumor motastasis; inhibition, to some extent, of tumor growth; and/or relief to some extent, one or more of the symptoms associated with the specific Scancer, reduced morbidity and mortality, and improvement in quality of life issues. To the extent the anti-TAT antibody or TAT.binding oligopeptide may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. Reduction of these signs or symptoms may also be felt by the patient.

SThe above parameters for assessing successful treatment and improvement in the disease are readily 10 measurable by routine procedures familiar to a physician. For cancer therapy, efficacy can be measured, for 00 example, by assessing the time to disease progression (TTP) and/or determining the response rate (RR).

Metastasis can be determined by staging tests and by bone scan and tests for calcium level and other enzymes to determine spread to the bone. CT scans can also be done to look for spread to the pelvis and lymph nodes in the area. Chest X-rays and measurement of liver enzyme levels by known methods are used to look for metastasis to the lungs and liver, respectively. Other routine methods for monitoring the disease include transrectal ultrasonography (TRUS) and transrectal needle biopsy (TRNB).

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

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

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

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

"Carriers" as used herein Include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionlo Surlhetant such as TW polyethylene glycol (PEG), and PLURONCS0.

Sy "solid phase" or "solid supporV" is meant a non-aqueous matrix to which an antibody, TAT inding oligopptide or TAT binding organic molecule of the present invention can adhere or attach. Bxamples of solid phases encompassed herein include those formed partially or entirely of glass controlled pore glass), polysaccharides agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain Sembodiments, depending on the conte the solid phase can comprise the well of an assay plate; in others it is a purification column an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Patent No. 4,275,149.

A "liposome" is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug (such as a TAT polypeptide, an antibody thereto or a TAT binding oligopeptide)toamnual. The components ofthe liposome are commonly arranged in a bilayer formation, similar 00 to the lipid arrangement of biological membranes.

D A "small" molecule or "small"organic molecule is defined herein to have a molecularweight below about 500 Daltons.

"effectve amount" of a polypeptide, antibody, TAT binding olgopeptide, TAT binding organic molecule or an agonist or antagonist thereof as disclosed heroin is an amount sufficient to carry out a specifically tated purpose. An "effective amount" may be determined empirically a a routine manner, in relation o the stated purpose.

The term "therapeutically effective amount" refers to an amount of an antibody, polypeptide,

TAT

binding oligopeptide, TAT binding organic molecule or other drug effective to "treat" a disease or disorder n a subject ormammal. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit slow to some extent and preferably stop) cancer call infiltration into peripheral organs; inhibit slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. See maythe definition herein of"tatng" To the extent the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic.

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

A "cytotoxic amount" of an anti-TAT antibody, TAT polypeptide, TAT binding ollgopeptide or TAT binding organic molecule is an amount capable of causing the destruction of a cell, especially tumor, cancer cell, either it vilr.o or in vivo. A "cytotoxic amount" of an anti-TAT antibody, TAT polypeptide, TAT binding 313 oligopeptide or TAT binding organic molecule for purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner.

41 The tem"antbody" is used in the broadest Oens and speifialy covets, for example, slz4,~ and'-TAT 00 nolOnal Antibodies (including agonist antalgonist, and neuraizing anttibodies), anti-TAT antibody Compositions with polyepltopic specificity, polyclonal antibodies, single chain adt-TAT antibodies and fiuM,,ats Of OBti-TAT antibodies (see below) as long as they exhibit the desired biological or immunological activity. The term "irnrunoglObuln?7 (1g) is used interchangeable with antibody herein.

An "isolated antibody" is 'one which has been identified and separated and/or recovered fromn a IND Component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic Or therapeutic uses for the antibody, and may include enzymes, hormones, and Other proteinaceous or nonproteinaceous solutes. In preferred embodiments# the antibody will be purified to greater than 95% by weight of antibody as determdied by the; Lowry method, and most preferably more than 99%/, by weight, to a degree sufficient to obtain at least 15 residues of N-termjinal or internal amino acid sequence by use of a spinning cup sequonator, or to homogeneity by SDS-PAOE under reducing or nonreducing conditions 0C) using Colonassle blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one puriflcation step.

The basic 4-chain antibody unit is a hoerotetramerc glycoprotein composed of two, identical light (L) chains and two identical heavy "Ii chains (an 1gM antibody consists of 5 of the basic hetertetramer unit along with an additional polypeptide called J chain, and therefore contain 10 antigen binding sites, while secreted IgA antibodies can polymerize to form polyrvalent assemblages comprising 2-5 of the basic 4-chain units along with I chain). In the case of lgC~s, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to a H by one covalent disulfide bond, while the two H chains are linked to each other by one or mome disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide lbridges. Each H chain has at the N-terminus, a variable domain followed by thre constant domains (Cu) for each of the a and T chains and four CH domains for p and e isotypes. Each L chain has at the N-terminus, a variable domain followed by a constant domain (CL) at its other end, The VL is aligned with the and the CL is aligned with the first constant domain of the heavy chain Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VR and 'VL together forms a single antigen-binding site. For the structure and properties of thre different classes of antibodies, see, Bai ajnd Clinicalmmunloa 8th edition, Daniel P. Stites, Abba 1. Ten- and Tristram G. Parslow Appleton Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amidno acid sequences of their constant domains Depending on the amidno acid sequence of the constant domain of their heavy chains imimunoglobulins can be assigned to different classes or isotypes, There are five classes of inlzunoglobulins: IgA, lgD, IgE, IgG, and 1gm, having heavy chains designated a, 6, e, and 1A, respectively. The y and a~ classes are further divided into subclasses on the basis of 3S relatively minor differences in C1, sequence and function, humans express the following subclasses: lg~l, JgG2, 1gG3, 1gG4, IgAl, and IgA2, The0 term "vatiable' refeji to the fhct that certain segmentl Of the variable domains difb extensively in 00eqnce0 among antibodies. The V domain mediates antigen binding and define specficity of a partieu1r antibody for its partcular antigeni. Howeve, the variabilityr is not evenly distriute across the I IO-amino acid span of the variable domains, Instead, the V regions consist of relatively invariant stretches called framework regions (Fl~s) of 15-30 amino acids separated by shorter regions of extrmeu variability called 'hypervariable regions" that are each 9-12 amino acids long, The variable domains of native heavy and light chains each comprise) INO four FRs largely adopting a J3-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part ot the P-sheet struicture. The hyperyariable regions in each chain are held together in close proximidty by the ERs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Se0un9Y of Prtiso u'uoo Ic Interes, 5th Ed. Public Healthj Service, National Institutes ofllealth, Bethesda, MD. (199 The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effeotor functions, such as 00 participation of the antibody In antibody dependent cellular cytotoxicity

(ADCC).

The term "hypervariable region" when used herein refers to the amrino, acid residues of an antibody which are responsible for antigen-blnding. The hypervarlable region generally comprises amino acid residues from a "icompleincentarity determnng region" or"CDR" around about residues 24-34 50-56 (L2) and 89-97 (03) in the Vi,, and around about 1-35 (H11), 50-65 (112) and 95-102 (M1) in the Vi,; Kabat et al., equence of proteL of Inirnuo Itret 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or those residues fr-om a "hypervariable, loop" residues 26-32 50-52 (L2) and 91-96 (L3) in the VL, and 26-32 (H11), 53-55 (112) and 96-101 (W1) in the Va; Chothia and Lcsk tJ Bo. 196:901-917 (1987)).

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of Substantilly homogeneous antibodies, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyolonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier 11monoclonal" is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies usefu in the present invention may be prepared by the hybridorna methodology first described by Kohler et al., Naur 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, U.S. Patent No. 4,816,567). The "rmonoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et at., Nature, 352:624-628 (1991) and Marks et at., I, ol. B 22:8-597 (1991), for example.

The monoclonal antibodies herein include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homnologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biologioal activity (se U.S. Patent No. 4,816,567; and Morrison et at, gM gRM &81:6851-6855 (1984)).

0C) Chimeric antibodies of intet~hevi inviude -Primatjd" antibodies comprising variable domain antigen-bindijng 0eune deie m annh m npi ae(~.Od W rd Monkey, Ape etc), and human constant region CK1 sequences, An "intacf' antibody is One which comprises an antigen-binding site as well as a CL and at least heavy chain constant domains, Cal, CH2 and Ca3. The constant domains may be native sequence constant domains (e.g IND human native sequence constant domains) or amino acid sequence variant thereof. Preferably, tie intact antibody has one or more effector functions.

"Antibody fragments" comprise a portion of an intact aritlbody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', and Fv fragments; c1 1 0 diabodies; linear antibodies (see U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protin En. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and mnultispecific antibodies formed from antibody fragments. 00 ~~Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" rgn ts and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of thelR chain (Vir), and the first constant domain of one heavy chain (C dl). Each F~ab fragment is monovalent with respect to antigen binding, it has a single antigen- binding site. Pepsin treatment of an antibody yields a single large fragment which roughly corresponds to two disulfide linked Pab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen. Fab' fr-agments differ from Fab fragments by having additional few residues at the carboxy term-inus of the CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a fee thiol group. F(ab') 2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them, Other chemnical couplings of antibody fragments are also known.

The Fc fragment comprises the carboxy-teinjl portions of both K chains held together by disuffides. The effector fuactions of antibodies are determined by sequences in the Fc region, which region is also the part I2t recognized by Fc receptors (FcR) found on certain types of cells.

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

"Single-chain Fv" also abbreviated as "sWy" or "scFv" are antibody fragments that comprise the VH and VrL antibody domains connected into a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptidt linker between the V, and V, domains whiich enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Plucktbun in The Pharaolnty f Monoclo_1nal Aniois vol. 113, Rosenburg and Moore eds., Springer-Ver1ag, New York, pp. 269-315 (1994); Bon'ebaeck 1995, infra.

The term "diabodieS" refera to small antibody figentgsprepared by consruting GFV fzUPMnts (UMe 00 PWcedt Paragraph) with short linkers (aboutS540 residues)botween theVH and V1, domains Such tat itec-Chaltt but not Iitra-chain pairing of theyV domains is achieved, resulting in a bivalent flagm.04 flagment haying two antigen-biding ites. Bispeific diabodles ame heteroditers of two "crossovee" s~v fagments in which the V arnd 'VL domains of the two antibodies are present on different polypeptide chains, Diabodies ame described more fully in, forexample, EP 404,097; WO 93/11161; andflollingeretal. Proc. Natl. Acad. Sol. USA, 90:6444-6448 (1993).

IND "Humanized" forms of non-human rodent) antibodies are chimreric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human irnmunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-humnan species (donor antibody) such as mouse, rat rabbit or non-human primate having the desired antibody specificity, affinity, and capability. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.

00 Furthermore, humanized antibodies may comprise residues that are not found In the recipient antibody or in the donor antibody. These modifications are made to further reline antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or ubstantially all of the hypervariable, loops correspond to those of a non-human lmrnunoglobulln and all or substantially all of the FRs are those of a human inununogiobui sequence, The humanized antibody optionally also will comprise at least a portion of an inimunoglobulin constant region typically that of a human immunoglobulin. For further details, see Jones et al.,Ngtur 321:522-52:5(1986); Riechmann etal., Nature 332-3 23- 329 (1988); and Presta, CmQ,_m~2 2:593-596 (1997).

A "species-dependent antibody," a mammalian anti-human 1gE antibody, is an antibody which has a strongerbinding affinity for an antigen from a first mammalian species than it has for a homologue of that antigen froem a second mammala species. Normally, the qpeies-dependent antibody "bind specifically" to a human antgen has a binding affinity (Kd) value of no more than about 1 x 10" M, preferably no more than about 1 x 10' and most preferably no more than about 1 xc 10'~ M) but has a binding affinity for a homologue of the antigen firomn a second non-humnan mammalian species which is at least about 50 fold, or at least about 500 fold, or at least about 1000 fold, weaker than its binding affinity for the human antigen. The species-dependent antibody can be of any of the various types of antibodies as defined above, but preferably is a humanized or human antibody.

A "TAT binding oligopeptide" is an oligopeptide that binds, preferably specifically, to a TAT polypeptide as described herein. TAT binding oligopeptides may be chien-lcally synthesized using known oligopeptide synthesis methodology or may be prepared and purified using recombinant technology.

TAT

binding oligopeptides are usually at least about 5 amino acids in length, alternatively at least about 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17,18,19,20,21,22,23,24,25,26,27,28 29,3Q 31,32,33,34,35,36,37,38,94,14, 44,4,4647,48,49,s5,23455657585966162636465,666,86,07,27,47, wherein such oligopeptides that are capable of binding, preferably specifically, to a TAT polypeptide as described herein. TATbinding oligopeptides may be identified without undue experimentation using well known teohniques, In ths regard, it is noted that techniques for screening oligopeptide libraries for oligopoptides that are capable of speffldy bindnt apolypeptide at eW eliknownintheet(e Patntos. 5,556,762,5,750,373 00 4,708,871,4,833,OM5,223,409,5,403,484, S,571, 68 9,5,663,143;PCTP~blictiofN 03 WO84A)3506 and Wo8o4/O5 Qeysen et al., Proc. Nat Acad. Sd., 81:3998-4002 (1984); Geysen et al., Proc. Nat Acad. Si 82:178-182 (198S); Geysenetal., in Syndhetleoeptides as Antigens, 130-149 (1986); Clesen eta!., muno!. W~th, Ct102:259-274 (1987); Schoofiset L imunol, 140:611-616(1988), Cwirla, S. B, et al. (l990)Proc. Nati. Acad. Soli.

USA, 87:6378; LownanH.B. eta!. (l991)Biochemiistry,30:10832; Claokson, T. eta!. (1991) Nature, 352: 624; Marks, 3. D. et (1991)i J. Mol. Biol., 222:581; Karig, A.S. etal. (1991)Proo. Natf. Acad. Sci. USA, 88:8363, and Smith, G3.

P. (1991) Current Opixi, 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 NI 10 molecules may be identified and chemically synthesized using known methodology (see, PCTPubljcafion Nos.

WOOO/00823 and W0001395 85). TAT binding organic molecules are usually less than about 2000 daltons In size, 00 alternatively lesthan about 1500, 750, 500, 250 or 200 daltons in size, wherein such organic molecules that are capbleofbinin, preferably specifically, to a TAT polypeptide as described herein may be identified without undue experimentation using well known techniques. In this regard, it is noted that techniques for screening organic molecule libraries for molecules that are capable of binding to a polypeptide, target are well known in the art (see, PCT Publication Nos. W000100823 and W000139585).

An antibody, oligopeptide or other organic molecule "which binds" an antigen of interest, e.g. a tumorassociated polypeptido antigen target, is one that binds the antigen with sufficient affinity such that the antibody, oligopeptide or other organic molecule is useflul as a diagnostic and/or therapeutic agent in targeting a cell or tissue expressing the antigen, and does not significantly cross-reaot with other proteins. In such embodiments, the extent of binding of the antibody, oligopeptide or other organic molecule to a "non-target" protein will be less than about 10% of the binding of the antibody, oligopeptide or other organic molecule to its particular target protein as determined by fluorescence activated cell sorting (FACS) analysis or radloimmunoprevlpitation

(RIA).

With regard to the binding of an antibody, oligopeptide or other organic molecule to a target molecule, the termn "4specific binding" or "specifically binds to" or is "specific for' a particular polypeptide or an epitope on a particular polypeptide target means binding that is measurably different f-rm a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be deterined by competition with a control molecule that is similar to the target, for e"ample, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target. The term "specific binding" or "specifically binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a Kd for the target of at least about 10' M, alternatively at least about 10-5 Mv, alternatively at least about 10' M, alternatively at least about 10*7 M, alternatively at least about 10'8 3 5 M, alternatively at least about 10-9 M, alternatively at least about 10-19 M, alternatively at least about 10' M, alternatively at least about 101' M, or greater. In one embodiment, the term "specific binding" refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polyeptide pitope.

00 An antibody, oligopeptide or other organic molecule that "inhibits the growth of tumor cells expressing a TAT polypeptide or a"growth inhibitory" antibody, oligopeptide or other organic molecule is one which results in measurable growth inhibition of cancer cells expressing or overexpressing the appropriate TAT polypeptide.

The TAT polypeptide may be a tansmembrano polypeptide expressed on the surface of a cancer cell or may be a polypeptide that is produced and secreted by a cancer cell. Preferred growth inhibitory anti-TAT antibodies, oligopeptides or organic molecules inhibit growth of TAT-expressing tumor cells by greater than 20%, preferably from about 20% to about 50%, and even more preferably, by greater than 50% from about 50% to about 100%) as compared to the appropriate control, the control typically being tumor cells not treated with the antibod, 1oligopeptide or other organic molecule being tested. In one embodiment, growth inhibition can be measured at C 10 an antibody concentration of about 0.1 to 30 pg/ml or about 0.5 nM to 200 nM in cell culture, where the growth inhibition is determined 1-10 days after exposure of the tumor cells to the antibody. Growth inhibition of tumor cells In vivoe can be determined in various ways such as is described in the Experimental Examples section below.

The antibody is growth inhibitory in vive If administration of the anti-TAT antibody at about I pg/kg to about 100 mg/kg body weight results in reduction in tumor size or tumor cell proliferation witlhin about 5 days to 3 months from the first administration of the antibody, preferably within about 5 to 30 days.

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

Antibody "effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fe region) of an antibody, and vary with the antibody isotype.

Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated oytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors B cell receptor); and B cell activation, "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies "arm" the cytotoxic cells and are absolutely required for such killing. The primary cells for mediating ADCC, NKce!ls, express FcyRR only, whereas monocytes express FcRI, FcRll and FcRII. FoR expression on hematopoietic cells is summarized in Table 3 on 00page 464 ofaetch and Knet AM MyjgO 9457-92 (1991). To assess ADC aRtvit of a molecle of 00 i~~nterst, anin vitrO ADOC assay, uch as that describej inUS PatentWo. 5,500,362 or 5,821,337 may be peforracd, Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (4X) cells. Alternatively, or additionally, ADOC activity of the molecule of interest may be assessed in vivo, in a animal model such as that disclosed in Clynes et al. (USA) 95:652-656 (1998).

"Tc receptor" or "FcR"' describes a receptor that binds to the Fc region of an antibody, The preferred FoR IND is a native sequence human Pd. Moreover, a prefenred FoR is ofte which binds an !gG antibody (a gamnma receptor) and includes receptors of the FoyRi, FcyRII and Fc'yRII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcryRlI receptors include FvyR.IA (an "activating receptor") and FcyRIIB (an "inhibiting receptor"), which have simailar amino acid sequences that differ primarily in the cytoplasmidc S 10 domains thereof. Activating receptor Fo-yRlA contains an iminunoreccptor tyrosine-based activation motif (ITAM in its cytoplasmnic domain. Inhibiting receptor Fc-yRIB contains an imniunoreceptor tyrosine-based 00 inhibition motif (ITI) in its cytoplasmic domain. (see review M. in Da~ron, Anan. Rv. Inimnol 15:203-234 (1997)). FeRs are revierwed in Ravetch and Kinet, Annu. ev. lniunol., 9:457-492 (1991); Capel tt al., hlmunomethods4:25-34 (1994); and del~aas etal.,1. Lab. Abn.Me. 126:330-41 (1995). OtherFuRs, inoluding those to be identified in the furture, are encompassed by the termi "FoR" herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal Ig~s to the fetus(Ouyeret al., 3. Inimuriol. 117:587 (1976) and Kim et al., 3. nunnol. 24:249 (1994)).

"Human effector cells" are leukocytes which express one or more FcRs and perform effector functions.

Preferably, the cells express at least FcTRMl and perform ADCC effector finction. Examples of human leukocytes wh~ch mediate ADCC Include peripheral blood mononuclear eellIs (PBMC), n atpzra1 killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferred. The effector cells may be Isolated from a native source, from blood.

"Complement dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in tie presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of tHe complement system (Clq) to antibodies (of the appropriate subclass) which are bound to their cognate antigen. To assess complement activation, a CDC assay, as described in Gazzano-Santorn et al., Immunol, Me!thods 202:163 (1996), may be performied.

The terms "cancer" arnd "cancerous" refer to or describe the physiological condition in mamimals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymiphoid malignancies. More particular examples of such cancers include squamous cell cancer epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the uinary tract, hepatoma, 3 5 breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, Yulval cancer, thyroid cancer, hepatic carcinoma, anal Carcinoma, penile carcinoma, melanoma, multiple inycloma arid 13-cell lymphoma, brain, as well as head and neck cancer, and assoiated metastases.

00 0 The terms "cell proliferative disorde" and "proliferative disorder refer to disorders that ar associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

"Tumor", as used herein, rferi to all neoplastic cell grwth and prolifertion, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

N 5 An antibody, oligopeptide or other organic molecule which "induces cell death" is one which causes a viable cell to become nonviable. The cell is one which expresses a TAT polypeptide, preferably a cell that overexpresses a TAT polypeptide as compared to a normal cell of the same tissue type. The TAT polypeptide may be a transmembrane polypeptide expressed on the surface of a cancer cell or may be a polypeptide that is produced and secreted by a cancer cell. Preferably, the cell is a cancer cell, a breast, ovarian, stomach, endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic or bladder cell. Cell death in vitro may be determined in 0 the absence of complement and immune effector cells to distinguish cell death induced by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). Thus, the assay for cell death may be performed using heat inactivated serum in the absence of complement) and in the absence of immune effector cells. To determine whether the antibody, oligopeptide or other organic molecule is able to induce cell death, loss of membrane integrityas evaluated by uptake of propidium iodide trypan blue (see Moore et al.

t17:1-11 (1995)) or 7AAD can be assessed relative to untreated cells. Preferred cell death-inducing antibodies, oligopeptides or other organic molecules are those which induce PI uptake in the PI uptake assay in BT474 cells.

A "TAT-expressing cell" is a cell which expresses an endogenous or transfected TAT polypeptide either on the cell surface or in a secreted form. A"TAT-expressing cancer" is a cancer comprising cells that have a TAT polypeptide present on the cell surface or that produce and secrete a TAT polypeptide. A "TAT-expressing cancer" optionally produces sufficient levels of TAT polypeptide on the surface of cells thereof, such that an anti- TAT antibody, oligopeptide ot other organic molecule can bind thereto and have a therapeutic effect with respect to the cancer. In another embodiment, a "TAT-expressing cancer" optionally produces and secretes sufficient levels of TAT polypeptide, such that an anti-TAT antibody, oligopeptide ot other organic molecule antagonist can bind thereto and have a therapeutic effect with respect to the cancer. With regard to the latter, the antagonist may be an antisense oligonucleotide which reduces, inhibits or prevents production and secretion of the secreted TAT polypeptide by tumor cells. A cancer which "overexpresses" a TAT polypeptide is one which has significantly higher levels of TAT polypeptide at the cell surface thereof, or produces and secretes, compared to ;0 a noncancerous cell of the same tissue type. Such overexpression may be caused by gene amplification or by increased transcription or translation. TAT polypeptide overexpression may be determined in a diagnostic or prognostic assay by evaluating increased levels of the TAT protein present on the surface of a cell, or secreted by the cell via an immunohistochemistry assay using anti-TAT antibodies prepared against an isolated TAT polypeptide which may be prepared using recombinant DNA technology from an isolated nucleic acid encoding 3 5 the TAT polypeptide; FACS analysis, etc.). Alternatively, or additionally, one may measure levels of TAT polypeptide-encoding nucleic acid or mRNA in the cell, via fluorescent in situ hybridization using a nucleic acid based probe corresponding to a TAT-encoding nucleic acid or the complement thereof; (FISH; see SW98/45479 published Octber, 1998), Southem blotting, Northem blotting, orpolymerase chain reaotion(PCR) techniques, such as real time quantitative PCR (RT-PCR). One may also study TAT polypoptide oveexpme sion by measuring shed antigen in a biological fluid such as serum, e.g, using antibody-based assays (see also, e.g., U.S. PatentNo. 4,933,294 issuedJune 12,1990; W091/05264 publishedApril 18,1991; U.S. Patent 5,401,638 issued March 28, 1995; and Sias et al., J. Imunol. Methods 132:73-80 (1990)). Aside fiom the above assays, various In vivo assays are available to the skilled practitioner. For example, one may expose cells within the body of the patient to an antibody which is optionally labeled with a detectable label, a radioactive isotope, and binding of the antibody to cells in the patient can be evaluated, by external scanning for radioactivity or by analyzing O a biopsy taken from a patient previously exposed to the antibody.

As used herein, the term "immunoadhesin" designates antibody-like molecules which combine the 0 10 binding specificity of a heterologous protein (an "adhesin") with the effector functions of immunoglobulin Sconstant domains. Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired Sbinding specificity which is other than the antigen recognition and binding site of an antibody is "heterologous"), and an immunoglobulin constant domain sequence. The adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand. The immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgO-l, IgG-Z, Ig-3, orIgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD orIgM.

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

The tenn "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes At 2 1 I131, I12 5 Y9, Re 8 Re 18 Sm 153 Bi 2 12 32 and radioactive isotopes of Lu), chemotherapeutic agents e.g. methotrexate, adramicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolytio enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, and the various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below. A tumoricidal agent causes destruction of tumor S0 cells.

A "growth inhibitory agent" when used herein refers to a compound or composition which inhibits growth of a cell, especially a TAT-expressing cancer cell, either in vitro or in vivo. Thus, the growth inhibitory agent may be one which significantly reduces the percentage ofTAT-expressing cells in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce 01 arrest and M-phase arrest. Classical M-phase blockers include the vincas (vinoristine and vinblastine), taxanes, and topoisomerase I inhibitors such as doxorubicin, epirubicin, daunorubloin, etoposide, and bleomycin. Those agents that arrest 01 also spill over into S-phase arrest, for example, DNA alkylating agents such 4s tMiOWifer, Ptednlsoie; d=acaijne, nlmeotloaw, cloplatin, 'ntorxt,5-fluorouail, and ara-C.

00Further frmnation can be found in The ?oeax Basisof Cair Mendelsohn and Inual, eds., Chper' enttle "Cll ycl reulaio~oncogenes, ad antielastIOU drugs" by Murakami, et al. (WB Saunders: (71 ~~~Philadelphial, 199S), especially p. 13. The taxanes (paclitaxel and do" tde) are anticancer drugs bothdrvdfo the Yew tree. Docetaxel (TAXOTEM Rlione-Poulenc Rorer), derived from the European yew, is a seinisynthetj 0

Q

analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of IND nhicrotubules from tubulin dimers and stabilize microtubules by preventing dopolymerization, which results in the inhibition of mitosis in cells.

"Doxorubicin" is an anthracycline antibiotic. The full chemical name of doxorubicin is aniiio23idealo-1xapyraohel)] O,,91-tetrahydro6,8,l-trihydroxy- lgydroxyacetyl) 1- IK Q0 iethoxy-5,12naplthacnedione.

c-iThe term "cytokine" is a generic termn for proteins refeused by one cell population which act on anoth-er 0C) cell asintercefful ar mei r. Exa ple of su harod e am l m h k n s monokines, a d traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone,

N.-

mnethionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; iflsuiinj, proinsulln; relaxin; prorelaxjn; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone hepatic growth factor, fibroblast growth factor; prolactin; placental lactogen;, tumor necrosis factor-e and mulletian-inlllbiting substance; mouse gonadotropjnassociated peptide; inhibin; actiin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-P; platelet-growth thctor, transforming growth factors (TOFs) such as TGP-cc and TGP- 0; insuUn-like growth factor-I and -11; eytliropoietin (EPO); osteo inductive factors; intorferons such as Interferon and colony stimulating factors (CSFs) such as maorophage-CSp (M-CSP); granulocytemacropag-CSp (QM-CSF); and granuocyt-CS (G-CSF); interleutkins (HAs) such as 11.4, 11 Ia, IL-2, UL-3, ILA4, 11,5, EL-6, IL-7, IL-, IL-9, L-I11 I, 12; a tumor necrosis factor such as TNF-u or TNF-13; and other polypeptide facotors including

LWF

and kit ligand As used herein, the term vythkine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

'Me term "package insert" is used to refer to Instructions custonarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.

00 00 C-C inmutd JIM 12 to 1S ,Z is everap of EQ B is averap of ND match with stop is stop-stop 1 (joker) match 0 *1 #deflne _M -8 value of a matoh with a stop 0 tnt day[26][2(6] t* A 1* B I* C 1*D*/ I* E *1 l* F 0 H *1 1** 1* K

L*

1*0*1 1* p I* R *1 S *1 /T*l

U*/

I* V 1* W *1 1* Z *1 I A B CD B FOH I J K L MN 0 P Q R S T U V W X Y Z*1 0,0-4, 0,M, I, 0), (0,31-4,3, 0, 0, 1,0, 0, 1), 0, 0, 4, 1, 0, 0,0, 2), 3, 0, 0, 0,0, 3), 1, 2, 0, 0,7,-51, 1, 1,0, 0), 0, 2,M, 0, 3, 0, 0, 2), 5, 2, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0), 0, 0, 0, 1, 3, 0, 0, 0,4, 0), 2, 6, 0, 2, 0, 0, 4, 0, 2, 0, 0, 1, 0, 1, 0, 1), LM,Y,MM,M,MMtM MMMM,

O,MJ,M_MMMMMMMMM)

M, 6, 0, 0, 1, 0, 0), 1,M, 0, 4, 0, 0, 0, 1, 6, 2, 0), 0,0, 0, 1,M, 0, 2, 1, 0), 0, 0, 0, M, 1, 3, 0, 0), 0, 0, 0, 0 0 0 0 0 0,0,0,M, 0, 0, 0, 0, 0, 0,0,0, 0,0,0), 4, 2, 0, 0, 0,-6,17, 0, o, 0, 0, 0, 0, 0, 0, 0 0 0, 0, 0, 0, 0, 0, 0, 0, 0), 0, 0, 0,10,4), 2, ID, 0, 3, 0, 0, 0,4, 4) XHNLLLn 00 00 #inlclude qstdio,h> #Include <ctypeh> #define Nfefiue #deflne #Idefine #deflne #detlue #det'lne #define ffdeflue tldeflne structjr

MAXW~

MAXOAP

JMPS

Mx

IDMAT

DMIS

DINS0 D181S

PINSO

PINS I 'p I short unsigned short 16 24 1024 4 3 0 4 Max Jumps in a diag *1 P~ don't continue ,to penalize gaps larger than this 1*Max imps In an path 1save If theres at least MX. I bases since lastimp value of matching bases *1 f* penalty for mismatched bases l* penalty for a gap penalty per base 1* penalty for agap penalty per residue *1 aLMAXJMPJ; x[MAXJW]P; struct diag lot long short struct jmp vtruct Path short n[ tat

X[

P sime ofjrnp (neg for dely) *1 base no. ofimp In seq x t* limits seq to2A16 -1 *1 score at last jmp *1 offset of prey block *1 I* current imnp index *1 list ofjmps *1 offiset ijmp; ip; 0; 1* number of leading spaces *1 :MwSI l* size ofimp (gap) -01 .JMPSI; 1* 100 OfJwp (last elem before gap) *t char char char char lot lot fat hit nt [at lot lot long struct struct *ofile; *namex[2]; *prog; *seqx[2]; dmax; drnaxo; dna; ondgaps; gapx, gapy; leno, leni; ngapx, ogapy; sniax; offset; diag *dx.

path W42J I~Output file name o/ /seq nlames: getseqsQ I* Prog name for err msgs f* seqs: getseqs() *1 1* best diag: no *1 final diag set if dna: miain()* set if penalizing end gaps *1 total gaps in seqs seq lens *1 I* total size of gaps *1 f* max score, nw() *1 bitmap for matching current offset injmp file f* holds diagonals *1 holds path for seqs *1 Cliar char *Calioc() *lnallooo, *index,c( *stropyo; *getseq() *g-callocO); 00 /*NelmuWno 1mn progragn uOR 1~5~prog fulel file2 *Where fifl and &il2 arm two dna or two protein sequences, The sequences can be in upper. or lower-mae an may contain ambiguity *Any lines beginning with 1;1, or I' are ignored *Max file length is 65535 (limited by unsigned short x in the imp struct) IND A sequence with 1/3 or more of its elements ACGTLJ is assumed to be DNA *Output is in the file 'align~out" *The program may create a tmp file in /Amp to hold info about tracebaok.

Original version developed under 13SD 4.3 on a vax 8650 c~K1 #Include "nw,h" #Incelude "day.h" 00 static _dbva[26J 1, 14,2,13,0,0,4,11l,0,0,12,0,3,5,0005,6,8,8,79,0, 10,0 static _pbval[26]( 1, 2 1(l ('DIA))jI WV 4, 8, 16, 32, 64, 1<«24, 1 «25j(I1 1(1 mairt(ac, av) m i fast so; 350 char *avO; prog If (ac 1- 3) fprinti~stderr,"usage: %s file! f11e2\n", prog); irintf(stderr,"whm tile!. and file2 arm two dna or two protein fequesa\n) frintf~stderr,"The sequences can be in upper- or lower-case\n"); fprintf(stderr,"Any lines beginning with or'< 1 are ignoredfn"); t~rintfstdlerr,"Output is in the file \"align.ou(V\n");exit(1); narnex[0] av4Ij; namex[lIIJ av[2]; veqx(Qj- getseq(narnex(O] &lenQ); veqx[lI] getseq(nam"e[l 11,len 1); xbrn (dna)? dbyel pbval; endgaps 1* 1 to penalize endgaps ofile -"align, out"; (4 output file *1 0 nwQ, 1* fill in the matrix, get the possible firps teadjmpso; f* get the actual jmps "I printo, print stats, alignment ifleanup(0); 1* unlink any bnp files *1 0C) do the alignment, retum~ best ecare.. mnaln *da: alue In Pitch and Sithl, PNAS, 8o, 138241386, 1983.

Po:PAM 250 m~uco Cl When $cores are equal, we prefer mismatches to ay gap, prefer 0 new gap to extending an ongoing gap, and prefer a gap in seqx *to a gap in seq y.

IND nwo char *px, *py. 1* seqs and pirm* Int *ndely, *defy; keep track of defy *1 tnt ndeix, delx; 1* keep track of delx *1 tnt *tmp; 1* for swapping rowO, rowl1 *1 Cl tt Mis; 1* score for eaoh type *1 S 15 tnt insO, ins 1; 1* insertion penalties *1 CIregister id; 1* diagonal index *1 00register Uj; 1* imp index *1' register *0'010, *C011; f* score for curr, last row XX yy; Index Into seqa s dx (struct diag *)gCalloc("to get diags", lenO+tenp-i, siseol(struct diag)); dely- (nt ctlo("to gt dely", lenl+l, seo(nt)); CoOO (mnt caltoc("to got colO", lenl+1, stzeot~int)); col I =(int *)g__vallov("to get voil", tenl+], stseof(int)); insO DINSO:

PINSO;

inst I-(dna)? DINS I PINSI1; amax 10000; If (eudgaps)( for (colQ(0) delyfO) -imaO, yy yy len I; yy++) coIOfyyj -dely(yy] colO~yy-1] ins l; ndely[yy] yy; )colO[03 0; Waterman Bull Math Bil 84 *1 else for (yy. yy ea yy44) dely[yyj -insO; fill in mnatch unatrix for (px =seqx[Q], xx xx lenO; px4-+, x44)( initialize first entry in cot If (endgaps)( 4If t(xx col 1(0) delx =-(tns0-Ins 1); else col 1 0] delx obb[] ins 1; ndelx =xx; else delx. -intO; ndelx -O; 00 MISe ooIO[yy-lJ; ff(dna) DM TDTS else I 1 xm*xAlxm[p-'7 MT MS mls -I +-day (*Px-'At)[*pyIAI]; 1* update penalty for del In x seq; favor new del over ongong del ignore MAXCIAF if weighting endgaps If (endgaps 11 ndely[yy] MAXOJAp)( If insO dely[yy]){ S 15 dely(yyj oolO[yy] (insO+ins I); ~K1 ndeiy[yy) 1; 00 Jelse dely~yyj ins 1; adelyfyyl++; )else If (00OLQy (insO+Insl) dely~yyj)( dely[yy] =colO[yy] (InsO+ins I); else ndelylyy) 1; ndoly[yy]4-+; update penalty for del in y seq; .30 *fivor new del ovor ongong del *1 If (endgaps 11 ndeix MAXOAP)( If (0011[yy-1J insO delx)( ~dolx ollI[yy- I (inso~ins 1); 35 ndelx= 1; )else delx ins!I; ndelx++; )else if (col I[Yy-1J (insO+insl) doix) deix =olyyl (insQ+insl); ndelx 1; els ndelx44; pick the maximum soore; we're favoring *mis over any del and delx over dely id -xx -yy lni 1; SS It (mDis delx. mis dely[yyj) 0o1l[yy) mis; Table I 00 esU del d*[Y~YD I 0 ij dxf~d].fp; If (dxfidjjp,40] (I dua 11 (ndelx

H!AXJMP

xx dxridJp4[jf4 11mig> dxld.score+DlNSO)) dx4id],Ump$4; IND writjmps(id); 0 10 U dxid],ijmp -0; dx[id].offset -offset; offset slzeof(utruct jnp) slzeof(offaet); dxid.jp.nfiJ] -ndelx; C] 15 dxfid]jp.x4ij] xx; dxld. score~= deix; 00 else oolltyyJ deiyfyy]; z ij =dxfid).tnip; If (dxid.jp.n[OJ] (ldna 11 (ndoiybyyj

MAXJMP

&&xx dxidjjp.x1j4-MX) 11 mis dxid].score+D1NSO))( dxfidj.ijmpH-; If (44-U MAXJMvP)( 25 wrltejmps~id); 1U dxjjid]Ajmp 0; dx~id].offset =offset; offset slzeof(struct jnp) sizeof(offset);

I

d4idjjp.n[W -ndely~yy]; dx[1d].jp.x[OU] xx; dxld] score- dely[yy]; If (xx lenO yy <len 1) P~ last Vol *1 If (endgapp) 40 vollI yy] iftsQ~ins I *(en I yy); If (vol I [yyj smax) smatx col [yy]; dinax -id; If (endgaps xx lenQ) colI Cyy-l] Ins0+inui *(ienO xx); if (CoItI yy- SMaX) srnax colii~yy-1]; drnax -d trnp C019; 0010 col1; Col I hp; (void) ftee((char *)ndeiy); (void) free((char *)deiy); (void) free((char *)coO); (void) free((ciiar *)coil); 00 *prhitO only cudne Visible Outside this module getmatO traue back best path, count matches: plint() pirallgn() print alignment of described in away pD: printo IND dumpblockQ dump a block of lines with numbers, stars: prlaligno nums0 Put out a number line dulnpblockO ~.putlinv() put out a line (name, (numj, seq, dunipblocko tarso -put a line of stars: dutnpblockQ d sripname() strip any pathi and prefix from a seqname- M~nclude "nw.h" 00#define SPC 3 #deflne P LINE 256 1* maximum output line *f #deflue P-SPC 3 space between name or cwn and seq cI 20 extent _dayf26][26J; tnt Olen; f* set output line length PILE *fx; 1* output file pintO print tat lX, ly, firstgap, lastgap; 1* overlap,~ If ((ft fopen(ofile, iprintf(stderr,"%s: can't write %sfa", prog, ofile); Cleanup(l); 1fndntf~fx, "<first sequence: %s (length namne4Qj, lenO); Jiprintf(fk, "<second sequence: %s (length namexl], leni); Olen =60; Ix Ien0; ly -len 1; farotgap =lastgap if (dinax leni 1) 1* leading gap In x pp[O].spc firstgap= fen I*-dmax 1; ly-pplo].spc; Ielse If (dmax lea I 1) leading gap in y pp[ I Ispo firstgap =dmax (lenl 1); Ix ppr I].sp; If(r Q< lenO. 1* trailing gap In x *1 lastgap =lenO dinaxQ -1; lx lastgap, else If (dinaxQ lenO 1) trailing gap in y *1 lastgap -drnaxO (lenO 1); ly lastgap; 5getinat(Ix, ly, firstgap, laptgap); Iwotuc back the beat pah 'oount atoheg getmnatxl ly, firstgap, lastgap) let lx, ly; *"core" (minus endgaps) *1tun lot firstgap, lastgap; leading trailing overlap *1 tnt tim, jO, i I, sizO, sizi; char outx[32]; double pot; register no, nI; register char *ppl get total matches, score S 15 00 p0 seqx[01 pp[l ].epo; p1 seqx I I+ pp[0].apo; no= pp[JI-Spc 1; n I pp[o].sp 1; amn 0; whle(*PO &&*pl if (Sizo) p144; else if (eizi) sizi.-; else ~If (xbm[*PO-IAtJ&xbm[*p I -'Asp If (n044 sizo pp[Q].niO-1; sizl pp[IJnil+-]; 415 pct homology: *if Penalizing endgaps, base is the shorter seq elsie, knock off overhangs and take shorter core if(endgaps) Ix =(Ie00 lent? l080: len i1; Ix (iOx ly)? ix: ly; I 00 *(doubIe)nm/(doub~el)I fprintf(fx, "<c/od match%s in an overlap of %.2f percent siinilarity\n", nm, (am ix, pot); 00 fpIintirt, "<M8 in first sqwc dgp If(MuX) (Void) sprltt~outxy 1 %W~s)u, ngapx, (dua)? "base":resIdue", (ugapx 1 1911"") Irntl(fto gap$ In second sequence: gapy); if (gapy) (void) sprintf(outx, %iffi)", figapy, (dna)? "base":"residue", (ngapy fprlntt~fx,"l%sll, outx); If (dna) Iprinfifffx, "\n<zcore: %d (match mismatch gap penalty %d per base)\n", sanax, DMAT, DMIS, DINSO, DINS 1); else fprinff~Bt, "\n<8core:, %d (Dayhoff PAM 250 matrix, gap penalty %d per rlesldaae)\", sax, PINSO, PINS 1); If (endgups) "cendgaps penalized, left endgap: %d right endgap: %d firstgap, (dna)? "base" "residue', (firstgap l= Vil "sit, lastgap, (dna)? "base "residue", (laotgap "it"al fprintfxf, "<endgaps not penal ized~n"); stti static atic static static static atic char static char static char static chair 1* aim; Irnax; ij[2]; nc[2]; ni[2]; s14[2]; *ps[21; *po[21; Out[2J[PLMNE; staQPLINE]; matches in core for ohecking *1 f* lengths of' stripped file names *1 i*.mp index for a path *1 (number at start of current line *1 fcurrent elem number for gapping f* ptr to current element ptr to next output char slot f* output line *1 1* set by starsQ *print alignment of described in struct path pp[] static pralignQ

I

pragn mnt mnt register more; char count *1 for (I 0, linax 0; i na stdpnaine(nainex~i]); if (nnm> hmax) linax -nn; nc[i]=I nl(l]' 1 SIzUl] Ij 01 ps4l] seqxi]; po[l] outli]; 00 forQ (nn- ma mom..

c-I 5 *do "e have more of this sequence If (I *va[iD) IND mre++;continue; S 10 ff(P1,10 1* leading space pprij.spc--; c~K1 15clue If (sizli3) in a gap *Pori}++ 00else w ere puttng a sq element c-i 0 *Poll) 1f (iqlower(*ps(1])) poi4;*ps[j] toupper(*psi]); poril.++; *are we at next gap for this seq7 if' (nifiJ) pp[ji.x[jijD WO need to merge all gap$ *at this location *1 while (niti] -ppli].x(u(i)) 35 64i) 4=Pp[i.nIJ[Ij++; If Olen liore nn) dumpblockQ; Pori] out~i]; nn 0; *dump a block of lines, including numbors, stars: praig( _algn static dumpblok)umblc register I; for(i ip<W i++ 00 (Vld) ptoeW,1k);d1113pblock for 1< CK1 If (%uttiJ (*outtl] 11~ *(PORi] l I) S 5 If(0 0) nums(i); If (I Q *out1I]) IND starsQ; 110 putlineQ); If (i -0 *out[1]) i i 1)fpintf(fk, star); numnsQ); 00 1 Put out a number line: dumpblockO Cl 20 static u tnt ix; index in OUtfJ holding seq line char nline11 LD4BJ; register i, j; register char *pn, *pX, *py; for (pn nlMine, i 0; 1 lmax+pSpC; pn4+) for (i nc~ix], py ouitfixJ; *py; pnl-+) *pn=l 11 else 33 for (px -pn; j; jl 1px--) *px -j %10 101; If (i <0) else *pn 4S*pnI\~ for (pn Mne; *pn; (vold) puto(*pn, fx); So ~(void) putc('\nl, fX); *Put -Out a line (name, [num], seq, [nuin]): durnpblock() static putline(ix) putline hIt

IX;

00 'Put!Wke fat register char for (PA name'bIN, 1 0; I*px *px Y~ px44, 14+) (void) putc(*px, fx); for <max+PLSPC; S 10 (void) putoCI 1, these count from 1; 010 is current element (from 1) *ncfl is number at star of current line N- 15 for (px =outfix]; *px; px++) (void) Putc(*Px&0x7P, fx); 00 (void) putc(\nl, fx); *put a line of stars (seqq always in out(Q], outflj): dumpbiookQ *1 static s tars() stars register Char *p0, *pl, CA, *px; if 0I *out[Oj 11 (%out[o] *(Polo])

)I

I*ouI? 1 (*Qutf *(pof II return; px -star; for (I -imax+PSPC; i; i-) PX44 -11; to r (p0 out[OJ, p1I out[ I *pD *p 1; p04 4 p 144) If (iSalpha(*pQ) isalpha(*p If (xbm(*pO.IA'I&xbrnf*p I CA else If Q dna -day[*pV0W *p I -WA] 0) else

)C

else 1 00 sdpath orpTOdx fjom pN, retr len: pr-a~gaQ ct char *pfl; P* fie name (may be path) *1 tp a l INDregister char *px, *py; If(*Pj P/) py e- I; Cl 15if (py) (void) &trpy(pn, py); return(stren(pn)); 00 00 OenViO cleanup anY tap Mie gcnIeqO dl win e di onxo c-I *r poo 8d the good~jmps ftnmImp fHOif neeay *WrltemPs() write a filled airmy ofjmpS to a tZIP file: nw() #lflclude fnwbh" ID#include <y(lh char *Jnami,- 11/np~iOmgXCXAX jj;'im iefo ms* PILE *ij; tPfl oj lot cleanupo; lau m ie~ long lseekQ; *Cenptpfie* *rem"ove any tmp file if we blow 00 OleanupQi) Int Icleanup (Void) unlinkoinam); exit(*) *read, retuirn pt to seq, set dua, len, maxlen SSkip lines Starting with or'>' ONe in upper or lower case $0 char gfteq(file, lon) gte char *file, file name ge1e lt *len; 1* seq len *1 line[lO24], *pseq; register char *px, *py; lot natgc, tien; FIL13 *p If M iopen(file,"Y")) fintfqstderr,1'%s: can't read prog, file); exiq( tien nalge 0 (fgets(line, 1024, fj if (fline 1;11 Mjne 1<1 11 *ln -1 continue; for (px- line; *ps If 1 \U1; px++) If (ISupper(*px) 11islower('pQ)) 51) tien4+± If ((Pseq -malloo((unsIgned)(tlen+6))) 0 ti~rntf(stderr,"%s: malloc() failed to get %d bytes for prog, tien+-6, file); exit(l); ~pseq[O] Pseq[l] pseq[2] Py~pseq4; CI rewind(fp); while (fgeta(line, 1024, If (*line ;1 I 1~ 'ie I' contiue; for (px -line; 4 px 1= px+4) If (isupper(*Px) *X else If (lower(*px)) py4 4 touppex(*Px); If (indexV'ATO3Cu',*(p.l natgc++; 00 *py+j4 w; *py \Q1; (void) folose(fip); CI 2 dna natge (Uti 3 return(ps"q+); char g-alloo(Umeg, nx, sz) char t msg; Program, calling routine *1 gc]o lilt ax, sz; oumber and size of elements char *callooO); 100 If ((px Cloc((unsge)nx, (urnalgned)sz)) if (*msg) truintf(Mter, g eAflovO failed %S prog, insg, nx, uz); return~px); get final imps froim dx[) or tiup file, set ppOl reset dmax: mainQ readjmps()efdj p lilt fd'i int Siz,io, il; register i, j, xx; (void) folose(t); if ((fd -open(Jname, Q.-RDONLY, 0){ can't open() prog, jname); Oleanup( I); for 0i 10 i 0, danaxO drnax, xx lenO; 14+){ while for (jdxdmaxjijmp; j 0 dx~drnax]jp.xj] 5= xx; 00 61&4n~~Q1~ .radjvaps I &dxdmax]offset t) (void) Iseek4f, dxdnjof4 0); relad(fd, (char *)&dxdra]jp, ylzeOf(vtruct imp)); (void) read(fd, (char *)&dxdmalx].Offs~e size(dxfdmax]offht)); else dx[dmaxJjj! MAXIMP-l; IN f0 mp)I break; I Afintf~stder, too many gaps in aligninent~n", prog); cICOJIup(l); If siz dxdmaxjjp~n~j; XX dx~dmax),Jp,xU]; dinax 4- Sj; 00K If (sz 0)1 l* gap in second seq *1 00 pp[I.n[i I] -siz; cI 20 Id -xx -yy 1e I1 PP1lP4I1I-xx -dmax enl 1; gapy++; ngapy -~siz; i*gnore MAXGAP when doing endgaps *1 iz =(-siz MAXGAP 11 endgaps)? -siz: MAXGAp; i144; else If (sir 0) gP in first seq pf0.n~iQ] siz; PP101-410] xx; gapx+; ngapx siz; gnore MAXCJAP when doing endgaps *1 slz -(slz MAXGAP 11 endgaps)? siz: MAXGAp; else break; reverse the order ofjmps for j0,io--; j iQ; io--) ppIOD.ui; PP[0J-1ntJJ PP(1.1nPiO] pp[OJ-n[iQj i PPtOI-xt]; p[.xfioJ; pp(0).xKiO] I; IPP[ l].nU]j; pp[ Ij.nUJ pp[ IJ.n~ilIJ; pp[ I I I; IrfdN 0) (void) ciose(fd); If (fj){ (void) unlink(Jnamp); Ofia 0; 00 1*writ a fihledjmp ftrCt 0630et Of te pMe one, Of any): nW() Wliteimpsoi) rt mp Int ix; w lexp INDchar *irnctrinpo; C) If' (raktemp~jname) 0) fprintf(stden, can't mktemnpO progjnime); cleanup(l); If' ((fj =fopen(Jnaxne, "lwl)) 0 fprintf(stderr, can't write prog, mnen); 20 (void) fwdte((char x~jjp, slzeof(struct imp), 1, (void) fwrite((cbar *)&dx[ixjofot, sIzeof(dxix].ofFet), 1, fj); IAWO 00 00

TAT

Comparigon Prwteln XXU0ocD0Dy0yIy (Length -15 amino acids) amino acid sequence identity (the number of identically matching amino acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number Of amino acid residups of the TAT polypeptide) divided by 15 =33.3%

TAT

j115 Comparison Protein xxxxxYyYYYYzY (Length 10 amino acids) (Length ,15 amino acids) amino acid sequence identity= (the number of identically matching amino acid residues between the two Polypeptide sequences as determined by ALIGN-2) divided by (the total number of amino acid residues of the TAT polypeptide) divided by 10 Table

TAT-DNA

Comparison DNA

NNNNNNNNNNNNNN

NNNNNNLLLLLLLLLL

(Length =14 nucleotides) (Length -16 riuclecticles) 3/9 nuclei-. acid sequence identity= (the number of identically matching nuclootides between the two nucleic acid sequences as determined by ALION- 2) divided by (the total number of nucleotides of the TAT-DNA nucleic acid sequence) 6 divided by 14 -42.9% 00 IkQ TAT-DNA NNNN(Lngt1N 12 nucleotides) Conmparison DNA NNNNTJLYV (Length 9 nucleotides) nlucleic acid sequence identity (the number of identically matching nucleotides between the two nucleic acid sequences as determined by ALIGN..

2) divided by (the total number of nucleotides of the TAT DNA nucleic acid sequence) 4 divided byl12 =3 3 3 00 In One embodiment, the present invention provides anti-TAT antibodies which may find use herein as and/or diagnostic agents. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies.

1. Polycloal Antiis Polyolonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intTaperitone4U (ip) injections of the relevant antigen and an adjuvant It may be useful to conjugate the relevant antigen N(especially when synthetic peptides are used) to a protein that is immunogenic in the species to be inunlzec For example, the antigen can be conjugated to keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctional or derivatizing agent. malelm~idobenzoyl sulfosuccinirride ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succirtic anhydride, SOCd 2 or R'N=OC=NR 1 where R and R 1 are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, or deivatives by combining, e.g., 100 zg or 5 jAg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intraderinally at multiple sites. One month later, the animals are boosted with 115 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites, Seven to 14 days later, the animals are bled and the serum is assayed for antibody titer. Animals ame boosted until the titer plateaus. Conjugates also can be made in recombinant cell culture as protein fusions.

Also, aggregating agents such as alum are suitably used to enhance the immune response.

2. Monoclonal Antibodies Monoclonal antibodies may be made using the hybridomna method first described by Kohler et al., Naue 256:495 (1975), or may be made by recombinant DNA methods Patent No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for imimuntization. Alternatively, lymphocytes may be immninzedl In vitr. After inanunization, lyrzpho"ye am isolated and then fused with a myelona, cell fine using a guitabetbfsing egeat suoh 00 as PolyethYlene glyool, to form a hybridoma cell ((lading, l~ncoa niois rnilsadPi~t 103 (Academic Press, 1986)).

The hybridomna cells thus prepared are seeded and grown in a suitable culture medium which mediumn preferably contains one or more substances that inhibit the growth or survival of the urifused, parental myeloiaa cells (also referred to as fusion partner). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltaseae(Gi rYIteslcieu yN trnfr0 (HiP. rHPrteslc ivcuture medium for the hybridoinas typically will include hypoxanthine, aminoptesin, and thysnidine (HAT medium), which substances prevent the growth of RT-deflcient cells, Preferred fusion partner Inyeloma. cells arc those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a selective medium that selects against the unfUsed parental cells. Preferred znyeloma cell lines are murine mycloma lines, such as those derived frorn 00 MOPC-2 1 and WIC- I I mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Califoaa USA, and SP-2 and derivatives X63-Agg.653 cells available from the American Ty'pe Culture Collection, Manassas, Yirgir*a USA. Human myeloma and mouse-human heteroinyeloma cell lines also have been described for the production of human mnonoclonal antibodies (Kozbor, .Imnunol., 133:3 001 (1984); and B~rodeur et al., Monoclonal Antibody Production Technique a dApitis, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybi-idoma cells are growing is assayed forproduction ofmonoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridomna cells is determined by immunopropitation or by an In vito binding assay, such as radioimniunoassay (RIA) or enzyme-linked immnunosorbent assay (BLISA).

The binding affinity of the mnonoclonal antibody can, for example, be determined by the Scatcharrj analysis described In Munson et al., Anal. lohem., 107:220 (1980).

Once hybridoma cells that produce antibodies of the desired specificity, affinity, and/or activity are 'e 5 identified, the clones may be subeloned by limiting dilution procedures and grown by standard methods (Godirig, Monoclonal Antibodie: Prnc;ive -A Practice, pp.59-103 (Academic Press, 1986)). Suitable culturemedia for this purpose include, for example, D-MiEM or RPM- 1640 medium. In addition, the hybridonia cells may be grown in vivo as ascites tumors in an animal by i-p. injection of the cells into mice.

The mionoclonal antibodies secreted by the subolones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography using protein A or protein G-Sepharose) or ion-exchange chr-omatography, hydroxylepatite chromatography, gel electrophoresis, dialysis, etc.

DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies), The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such es coi cells, simian C0S cells, Chinese Hamster Ovary (CHO) cells, or myelonia cells that do not otherwise produce antibody protein, to obtain the syntheis of inonoclonal antibo dies in the meombinant host cells. Review amies 00 on recombinant expression in bacteria of DNA encoding the antibody include Skein et QW S5:256-262 (193 and Pltickthun, XMM I& 3:5-8 (1992).

In a ftuther embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty etal., NtUre 348:552-554 (1990). Claokson etal.,Naur 352:624-628 (1991) andMadcs etal.,J. _Mo!.Biol., 222:581-597 (1991) describe the isolation ofinurine and human antibodies, respectively, using phage libraries, Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Biotaeclology 10:779-783 (1992)), a s well as combinatorial infectioni and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Aids, Res. 21:2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of muonoclonal antibodies.

CK1 The DNA that encodes the antibody may be modified to produce chimeric or fusion antibody 00 polypoptides, for example, by substituting humnan heavy chain and light chain constant domain (C and CL) sequences for the homologous 'nurine sequences Patent No. 4,816,567; and Morrison, et al., Pro. Nal d.

JLMB& 81:6851 (1984)), or by fusing the inununoglobulin coding sequence with all or part of the coding sequence for a non-imnxunoglobulin polypeptide (heterologous polypeptide). The non-lmrnunoglobullin polypeptide sequences can substitute for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.

3. l~aadu~nzJ~ioies Ile anti-TAT antibodies of the invention may further comprise humanized antibodies or human antibodies. HumanizePi forms of non-human murine) antibodies are chimeric imxnunoglobulins, lalmunoglobulin chains or fragmnents thereof (such as Fy, Fab, Fab', F(ab%) or other antigen-binding subsequences of antibodies) which contain mainimal sequence derived from non-human immunoglobulin. Humanized antibodies include human imlnunogtobulins (recipient antibody) in 'which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immitunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, vaniable domains, in which all or substantially all of the CDR regions correspond to those of a non-human Immnunoglobulin and all or substantially all of the FR regions are those of a human imrnunoglobulln consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region typically that of a human immunoglobulin Clones et al., N'ature, 321:522-525 (1986), Riechinana et al., Nature, .332:323-329 (1988); and Presta, Cur._O,trc. iol., 2:593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These nonhuman ano' avid reidues are often zefenu to as "*mpoit residues, which ane typically taken from an -rnport" 0Variable domain. HuinanjjOu can be essentily perfbimed following the method of Winter and co-voxlcexs tlOnes et al. MRlu I2:522-525 (1986); Ricomannmet al., N-lMm 332:323-327 (1989); Vedioeyen et al., Scienee, 2_3:154-136 198)),by substituting rodent CDRs or CDR sequences for the corresponding sequences of a, human antibody. Accordingly such "humanized" antibodies are chimeric antibodies Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the coresponding IND sequence froma a nion-humall species. In practice, humanized antibodies are typically human antibodies in Which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies, The choice of human variable domains, both light and heavy, to be used in makting the humanized r-1 10 antibodies is very important to reduce antigenicity and HlAMA response (human anti-mouse antibody) when the antibody is intended for human therapeutic. use. According to the so-called "best-fit" method, the sequence of 00 the variable domain of a rodent antibody is screened against the entire libraty of known human variable domain sequences. The human V domain sequence which is closest to that of the rodent is identified and the humnan framework region (FR) within it accepted for the humanzed antibody (Sims et al., .Inuol., 151:2296 (1993); Chothia et al., 4LM9 Blol. 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 samte framework may be used for several different humanized anti bodies (Carter et al., Proc. NatL Acad. Sci. USA, 89:428 (1992); Presta et al., I munol. 151:2623 (1993)).

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

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

3 5 As an alternative to humanization, human antibodies can be generated, For example, it is now possible to produce transgel animals mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous imniunoglobulin production. For example, it has been described that the homomygous deletion of the antibody heavy.-chain joining region (4U) gene in chimeric and germ-line 00 mutant mice results in complete Inibition of endogenous antibody production, Trnsfbr of the human genii-une 0 immunogwobulin gene arry into such genn..line mutant mice will result in the production of human antibodies upon (71 antigen challenge. See, Jakobovits et al., Proc. Ni. Acd Sci. USA 90:2551 (1993); Jakobovlts et al., Fkatixr, 362:255-258 (1993); Bniggemametai. Yearinlimo 7:33 (1993); U.S. PatentNos. 5,545,806,5,569,825,5,591,669 (all of GenPharm.); 5,545,807; and WO 97117852, INO Alternatively, phage, display technology (McCafferty et at., Nature 3 48:552-553 [1990]) can be used to produce human antibodies and antibody fragments in vitro, from imnmunoglobu~in variable domain gene repertoires fromi unimmunized donors. According to this technique, antibody V domain genes are cloned in-fraine into either a major or minor coat protein gene of a filamentous bapteriophage, such as M13 or fd, and displayed (71 10 as functional antibody fragments on the surface of the phage particle. Because the filamontous particle contains a single-stranded DNA copy of the plinge genome, selections based on the functional properties of the antibody 00 ~also result in selection of the gene encoding the antibody exhibiting; those properties. Thus, the phage minaics somne of the properties of the B3-cell. Phage display can be performed in a variety of formats, reviewed in, e.g., Johnson, KevinS. and ChisweflDavid L Curregt Onion 1n Structual Biolos3:564-571 (1993). Sevenulsouces of V-gene segments can be used for phage display. Clacksonet al., Naur, 352.624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized midce. A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et a, .to io.222:581-597 (1991), or~riffith et al., &MO J. 12:725-734 (1993). See, also, U.S. Patent Nos. 5,565,332 and 5,573,905.

As discussed above, human antibodies may also be generated by it vitro activated B cells (see U. S.

Patents 5,567,610 and 5,229,275).

4.

In certain circumstances there arm advantages of using antibody fragments, rather than whole antibodies.

Mire smaller size of the fragments allows for rapid clearance, and may lead to improved access to solid tumors.

Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, Morimoto et al., iL.igal of ]Biochemical and BiphXsiCaJMethods 24: 107-1 17 (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 S0 can all be expressed in arnd secreted from E. coi, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be Isolated from the antibody phage libraries discussed above. Altematiyely, Fab'-SH fragments can be directly recovered from E. col and chemically coupled to form F(ab') 2 fragments (Carter et al., B~io/ToclmoLoUy 10:163-167 (1992)). According to another approach, F(ab') 2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab')2 fragment with increased in vivo half-life comprising 3 5 a salvage receptor binding epitope residues are described in U.S. Patent No, 5,869,046. Other teclhniques for the production of antibody fr-agments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/161 85; U.S. Patent No, 5,571,894; and U.S. PatentNo.

5.387,458, Fy and gFVuare the only Speces With intsot combining Sites tha ane devoid of constan rego ns; thus, 00they are suitable for reduced nonspecific binding during it, yivo use, s~v fulsion proteinsmybeostctdo Yield fusion of an effector protein at either the amino or the carboxy terminusofasy.Se ntbd Etwieerna e. Brrcaeck sura.The antibody fragment may also be a "linear antibody", as described in U.S. Patent 5,641,870 for example. Such linear antibody fragments May be monospecific or bispecific.

5. Bispecific Antibodies B3isPecific antibodies are antibodies that have binding specificities for at least two different epitopes.

Exermplary bispecific antibodies may bind to two different epitopes of a TAT protein as described herein. other such antibodies may combine a TAT binding site with a binding site for another protein. Alternatively, an anti-.

TAT armn may be combined with an ann which binds to a triggering molecule on a leukocyte such as a T.-cel lo 1 receptor molecule CD3), or Fe receptors for IgG (Fc-yR), such as FcrRy (CD64), FcyRIl (CD32) and FcrRMn (CDI so as to focus and localize cellular defense mechanisms to the TAT-expressing cell. Bispecific antibodies 00 may also be used to localize cytotoxic agents to cells which express TAT. Those antibodies possess a TATbinding arm and an aim which binds the cytotoxic agent saporin, anti-inteferon-v, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full length antibodies A[ 5 or antibody fragments F(ab') 2 bispecific antibodies).

WO 96/16673 describes a bispecific anti-ErbI32antiFoTRII antibody and U.S. Patent No. 5,837,234 discloses a bispecific anti-EtB2/anti..pyRj antibody. A bispecific anti-ErbB2jFca antibody is shown in W098&02463. U.S. Patent No, 5,821,337 teaocs a bispecific anti-Ertb2anti-CrD3 antibody.

Methods for making bispecific antibodies are known in the art. Traditional production of full length 2 bispeclfic antibodies is based on the co-expression of two lmnunoglobulln heavy chain-light chain pains, where the two chains have different specificities (Milstein et al., Naur 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixtutre of' 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the produot yields are low. Similar procedures are disclosed in WO 93108829, and in Traunecker et aL, "-0B 1. 10:3655-3659 (1991).

According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to imnnunoglobulin constant domain sequences, Preferably, the fusion is with an Ig heavy chain constant domain, comprising at least part of the hinge, C~j2, and CH3 regions. It 3 (D is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light chain bonding, present in at least one of the fusions. DNAs encoding the inununoglobulin heavy chain fusions and, if desired, the hmnunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host cell. This provides for greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the 3,15 construction provide the optimum yield of the desired bispecific antibody. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios have no significant affect On the Yield of ho desired Chain CombinationL hi~preered mboimnt f hisappoah, he lueciieantibodies aW composed of a hYbrid ii'nrnoglbuliu heav hain witha first binding specificity in on an,,and et hybrid hnmunoglobun eav chain- C1 light chain Pair (Providing a second binding specificity) in the other arm. It was found that this asymmxetric, struotur facilitates the separation of the desired bispecific compound from unwanited immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the *bispecific molecule IND provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al., Method in EnzMQLoQ, 121:210 (1986).

According to another approach described in US. Patent No. 5,731,168, the interface between a pair -of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered froin ci 10 recombinant cell culture. The preferred interface comprises at least a part of the C113 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 00 ~chains tyrosine. or tryptophan), Compensatory "cavities" of identical or similar size to the large side chain(s) are created on tho interface of the second antibody molecule by replacing large amino acid side chains with salalier c-i ones alanine or threonine). This provides a mechanism for increasing the yield of the beterodimer over other unwanted end-products such as hornodixners.

Bispecitic antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells Patent No. 4,676,980), and for treatment of HrI inf'ection (WO 91100360, WO 92/200373, and EP 03089). Ileteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable ctoss-inking agents are well known in the art, and are disclosed in US. Patent No. 4,676,980, along with a number of cross-Ilnicing techniques.

Techniques for generating bispecific, antibodies from antibody fragments have also been described in the literatur. For example, bispeolfic antibodies can be prepared using chemical linkage. Brennan et al., S~jcic 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') 2 fragments. These fragments are reduced in the prescnce of the ditbiol coniplexing agent, sodium arsenite, to stabilize vicinal ditliiols and prevent intermolecular disulfide formation. The Fatb' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One, of the Fab'-TNB derivatives is then reconverted to the flab'-thol by reduction with mercaptoethylan-tne and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispeciflo antibody. The bispecific antibodies produced can be used as agents for the seleotive immnobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab'-SI[ fragments from B, col, which can be chemically coupled to form bispecific antibodies, Shalaby et al., Med. 175: 2 17-225 (1992) describe the production of a fully humanized bispecific antibody F(ab') 2 molecule, Each Fab' fragment was separately secreted from E. coti and subjected to directed chemical coupling in VIrro to form the bispeoific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets, Various techniques for making and Isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bigpeiflo atibodleg have been produced wsing louoino Zlpog. Koffelny 00et at., mu-nol.148(5): 1547.1553(1992). 71w loucine zlppepeptides from theFos and Yon proteins were linked to the Fab,' portions of two different antibodies by gene fusion. The antibody homodimers were reduood at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The 'diabody" technology described by Hlollinger et al., PrLop.

Nat. Ac-ad, Sci.USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody IND fragments. The fragments comprise a VH connected to a VL by a linker which is too short to allow pairing between the two domains on the same chain, Accordingly, the 'VH and VL domains of one fi-agment are forced to pair with the complementary VL and VH domains of another fragment, iereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been c1 10 reported. See Gruber et al,, Lk unl 152:5368 (1994).

Antibodies with more than two valencies are contemplatedJ. For example, trispeciflo antibodies can be 00 prepared. Tutt et al., I. Immunpol 147:60 (199 1).

6. Htmnj te Antbodes Heteroconjugate antibodies are also within the soope, of the present invention. Heteroconiugate antibodies are composed of two covalently joined antibodies, Such antibodies have, for example, been proposed to target immune system cells to unwanted cells PatentNo. 4,676,980], and fo tramn (H neto

WO

91/00360; WO92/200373; El'03089]. It is contemplated that the antibodies may be prepared in vitiv using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, iminunotox ins may be constructed using a disulfide exchange reaction or by forming a tbiouthcr bond, Examples of suitable reagents for this purpose include kninothiolate and methiyl4.meraptobujjdat and those disclosed, for example, in U.S. Patent No, 4,676,980.

7.1 A multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind. The antibodies of the present invention can be multivalent antibodies (which are other than of the 1gM class) with three or more antigen binding sites tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody. The multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. The preferred dimerization domain comprises (or consists of) an Fe region or a hinge region. In tis scenario, the antibody will comprise an Fc region and three or mote antigen binding sites amino-terminal to the Fo region. The preferred multivalent antibody herein comprises (or consists of) th-ee to about eight but preferably four, antigen binding sites. The multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable domains. For instance, the polypeptdeo chain(s) may comprise VD 1-(X l),-VD2.(X2)Fc, wherein MDI is a first variable domain, VD2 is a second variable domain, Fo is one polypeptide chain of an Fc region, Xl and X2 represent an amnino acid or polypeptide, and nisO or 1. For instance, the polypeptide chaini(s) may comprise: -VH-Cl-I-flexible llnker-VH-CH I Fc region chain; or YR-CH -VH-CI -Fc region chain. The multivalent antibody herein preferably Auther compilses at least two (and preferably four) light chain variable domain polypeptides. The multivalent antibody herein may, for instance, comprise flm about two to about eight lWh chain variable domnain polypepides, The light chWain 0C) variable domain polypeptides contemplatcd here comprise a light chain variable domain and, optionally, further comprise a CL domain.

Itrmay be desirable to modify the antibody of the invention with respect to effector faniction, so as to enhance antigen-dependent cell-mediated cyotoxicity (ADCC) andor complement dependentcytotoxicity

(CDC)

of the antibody. This may be achieved by introducing one or more amino acid substitutions in an Po region of the antibody. Alternatively or additionally, cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disuffide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complemnent-medjated cell killing and antibody-.dependent cellular S 10 oytotoxicity (ADCC). See Caron etal.,J.EpMed. 176:1191-1 195 (1992) and Shopes, B.J. I unAl 148:2918-2922 (1992). Homodimoric antibodies with enhanced anti-tumor activity may also be prepared using heterobiflinctional cross-linkers as described in Wolffet at., Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADOC capabilities.

See Stevenson et al., AntiCne~ DmgtaDesi 3.219.230 (1989). To increase the serum half life of the antibody, one may Incorporate a salvage receptor binding epitope. into the antibody (especially an antibody fragment) as described in US. Patent 5,739,277, for example. As used herein, the term "salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule 1gGI, IgGi 2 1g6 3 or IgG 4 that is responsible for increasing the in vivo serum half-life of the IgG molecule.

invention also pertains to immuocougates comprising an antibody conjugated to a oytotoxic agent such as a chemotherapeutic agent a growth inhibitory agent, a toxin an enzymiatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (Ite., a radloconjugate).

Chemotherapeutic agents useful in the generation of such iniunoconjugates have been described above.

Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pieudornonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurifesfo,.dil proteins, dianthin proteins, Pliytolaca arnericana proteins (PAJ'1, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomyvin, and the tricothecenes. A variety of radionucl ides are available for the production of radioconjugated antibodies. Examples include 2 12Bi, 1"11,1 "DY, and 'Re. Conjugates of the 3 0 antibody and cytotoxic agent are made using a variety of bifunctional protein-coup ling agents such as Nsuccininildyl3-(2-pyridylditbiol) propionate, (SPDP), iminothiolane (MT, bifunctional derivatives of imaidoesters (such as dimethyl adipimidate HOL), active esters (such as disuccinimiddyl suberate), -aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexaitediamine), bis-diazonium derivatives (such as bls-(p-diazoriumbcnzoyl) ethylenediarain), diisocyanates (such as tolyene 2 ,6-diisooyanate), and bis- 3 5 active fluorine compounds (such as liS-difluoro-2,4.ditnitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta etaat, Science, 23: 1098 (1987). Carbon-1I4-labeled l-isotijooyanatobeMIl3methyldiethylene triarninopentaacetic acid (M-DTPA) is an exemplary chelating agent for conjugation of redlonuootido to MDe antibody, Soo W09011 1026.

00 Conjugates of an antibody and onr moresmallmolecule toxins, such as a calichemiln, mfaytansnoids, a hrichothene, and CCIO6s, and the derivatives of these toxcins that have toxin activity, are also oonte;Wlatod herein.

Mavtnsino Pad maytan-glnpjls In one preferred embodiment, an anti-TAT antibody (full length or firagments) of the invention is conjugated to one or more maytansinoid molecules.

Maytansinoids are niitototic inhibitors which act by inhibiting tubulin polymerization. Maytansine was first isolated from the east Aflican shrub Maytenus .rerrata Patent No. 3,896,111). Subsequently, It was discovered that certain microbes also produce maytansinoids, such as niaytansinol and C-3 maytansinol esters S 10 Patent No. 4,151,042). Synthetic maytansinol and derivatives and analogues thereof are disclosed, for r~~l example, in U.S. PatentNos, 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 00 4,308,269; 4,309,428;4,3 13,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; ri 4,362,663; and 4,371,533, the disclosures of which are hereby expressly incorporated by reference.

Mavtonsinoid-andbojdy QWMu~aes In an attempt to improve their therapeutic index, maytansine and niaytqrisinoids have been conjugated to antibodies specifically binding to tumor cell antigens. Inununoconjugatts containing maytansinoids and their therapeutic use are disclosed, for example, in U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 BI, the disclosures of which are hereby expressly incorporated by reference. LI.u et al., Proc. Natl, Acad. Sci.

USA 93:8619-8623 (1996) described ilnmunoconjugates comprising a maytansinold designated DM1 linked to the mnonoclonal antibody C242 directed against human colorectal cancer. The conjugate was found to be highly cytotoxio towards cultured colon cancer cellts, and showed antituimor activity in an its vivo tumor growth assay.

Chadi et al., Cancer ebc 52:127-131 (1992) describe immunoconjugates in which a maytansinold was conjugated via a disulfide linker to the murine antibody A7 binding to an antigen on human colon cancer veil lines, or to another murine monoclonal antibody TA. 1 that binds the HI3R-2lneu oncogene. The cytotoxicity of the TA. 1 maytansonoid conjugate was tested in vitro on the human breast cancer cell line SK-BR-3, which expresses 3 x 105~ HER-2 surface antigens per cell. The drug conjugate achieved a degree of cytotoxicity similar to the free maytansonid drug, which could be increased by increasing the number of maytansinoid molecules per antibody molecule. The A 7 -maytansinoid conjugate showed low systemic cytotoxicity in mice.

Anti-TAT antibody-maytansinoid conjugates are prepared by chemically linking an anti-TAT antibody to a inaytansinoid molecule without significantly diminishing the biological activity of either the antibody or the Inaytansinoid molecule. An average of 3-4 niaytansinoid molecules conjugated per antibody molecule has shown efficacy in enhancing cytotoxicity of target cells without negap.iely affecting the function or solubility of the antibody, although even one molecule of toxin/antibody would be expected to enhance cytotoxicity over the use of naked antibody, Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources. Suitable anaytansinoids are disclosed, for example, in U.S. Patent No, 5,208,020 and in the other patents and nonpatent publications referred to hereinabove. Preferred maytansinoids are maytansinul *nd mnyt2Miinol analogue. modI~ed in the aromatc ring or at other positions Oft&0 maYtalsinol moleOulc; ftch 00as Various Maytanslnol MMt.s These ame many linkinig groups known. in tho art for making antibod[Y-Maytanino~id cor~ugates, including, for example, those disclosedi in U.S. Patent No. 5,208,020 or EP Patent 0 425 235 BI, and Chad- et al., QpgM Researoh 52:127-131 (1992). The linking groups include disufide groups, thloether groups, acid labile groups, photolabile groups, peptidase labile groups, or esterase labile groups, as disclosed in the above-identified patents, IND disulfide and thioether groups being preferred, Conjugates of the antibody and maytansinoid may be made using a variety of bifunctional protein coupling agenats such as N-suocinInidy1..3-{2.pyidldtho)* propionate (SPDP), vuccinimldyl-4.{r'{.

maleimidometuzyl) cyclohexane-..carboxyiate, iminothiolane (IT, biuctional derivatives of inidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobentzoyl) hexanediamine), bis-diazoniumn derivatives (such as bis-(p- 00 diazoiumb~ yl,eylenediamne), dllsovyanates (such as toluene Z, 6 -diisocyanate), and bis-active fluorine compounds (such as ltS-dlfluoro-2,4.nirtrobezf 0 Particularly preferred coupling agents include

N-

succiidyl3(2pyidylditliio) propionate (SPDP) (Carlsson et al., BlochM, 173:723-737 (1978]) and N- 15succlnhnfidyl.4<z2pyridyluilo)penaoate (SPP) to provide for a disulfide linkage.

The linker may be attached to the maytansinoid molecule at various positions, depending on the type of the link. For examuple, an ester linkage may be formed by reaction with a hydroxyl group using conventional coupling techniques. The reaction may occur at the 0-3 position having a hydroxyl group, the C-14 position Modified with hyrdoxyrnethyl, the C-15 position modified with a hydroxyl group, and the C-20 position having a liydroxyl group. In a preferred embodiment~ the linkage is formed at the 0-3 position of maytansinol or a niaytatnslnol analogue.

Calicheanipin Another irnmunoconjugate of interest comprises an anti-TAT antibody conjugated to one or more calichean-doin molecules. The calicheamicin family of antibiotics are capable of producing double-stranded

DNA

breaks at sub-picomolar concentrations, For the preparation of conjugates of the calicheamicn family see U.S patents 5,712,374,57145865739,116,5,767,285,5,770,701,5,770,710,5,773,001,5,877,296 (all toArnericanCyananid Company). Structural analogues of calicheamicin which may be used include, but are not limited to, Ti 1 aL 2 3 N-acetyl-fi PSAG and Of I (Hinman et al., Cancer search 53:3336-3342 (1993), Lode et al., Cancer search 58:2925-2928 (1998) and the aforementioned U.S. patents to American Cyanarnid). Another anti-tumor drug that the antibody can be conjugated is QFA which is an antifolate. Both calicheamioin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Therefore, cellular uptake of these agents through antibody mediated internalization greatly enhances their cytotoxic effects.

Other cttxi ne t Other antitumor agents that can be conjugated to the anti-TAT antibodies of the invention include BCNU, 3 5 streptozoicin, vincristine and 5-fluorouracil, the family of agents known collectively LL-133328 8 omplex described in U.S. patents 5,053,394, 5,770,7 10, as well as esperamicins patent 5,877,296), Bnzyinatlly active tDxins and hl~mcnts Oiceof which can be used inolude diphthra. A chin, 00 onabinding active fragments Of diphtheria to*in exotoxin A chain (frOm Pieudomonaff aeruginsia), iin A chain, ab0 hlmoecnAcan alpha-sarin, Akeuritafordit proteins, dianthin ptoteins, PhYtoaca americana proteins (PAK1 PAPII, and PAP-S), momordica oharantia inhibitor, otircin, crotin, sapaonadla officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, WO 93/21232 Published October 28, 1993.

INDThe present invention fu~rther contemplates an inlnunoconjugate formed between an antibody and -a compound with nucleolytic activity a ribonuclease or a DNA endonuclease such as a deoxyribonuolease; DNase).

For selective destruction of the tumor, tie antibody may comprise a highly radioactive atom. A variety NK 10 of radioactive isotopes are available for the production of mdioc4,njugated anti-TAT antibodies. Examples include c=K At 11 ,113, j 5 9 I Re t Re' 88 Sm 1 5 3 Bl 1

,P

2 b 1 and radioactive isotopes of Lu. When the conjugate is 00 used for diagnosis, it may comprise a radioactive atom for scintigraphic studies, for example to~ or Is, oroaspin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, ai), such as iodino-123 again, iodine-13 1, indium-I 11, fluorine-19, carbon-I 3, nWtrogen-is, oxygen-I 7, gadoli~niumn, manganese oriron.

The radio- or other labels may be incorporated in the conjugate in known ways. For example, the peptide mnay be biosynthiesized or may be synthesized by chemical amino, acid synthesis using suitable amino acid precuirsors involving, for example, fluorine-19 in place of hydrogen. Labels such astc"'orI 23.Rc 5 Re 8 and I'can be attached via a cysteine residue in the peptide. Yttrlumn-90 can be attached via a lysine, residue. The method (Piker et al (1978) Biochem, Blophys. Res. Conimun. 80: 49-57 can be used to incorporate ioditte-123. "Monoclonal Antibodies in Inlmunoscintigraphy" (Chatal,CRC Press 1989) describes other methods in detai.

Conjugates of the antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succi~niidy3(2pyridyldithio) propionate (SPDP), succinlindyl4-(Nmaleirnidomethyl) cyclohexane.I -carboxylate, iminothiolane bifunctional derivatives of itnidoesters (such as dimethyl adipimiddate HCL), active esters (such as disuccininmidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-djazonium derivatives (such as bis-,pdiazoniumbenzoyl)..ethyleflediumine), diisocyanates (such as tolyene 2 6 -diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4dinitrobenzene). For example, a ricin immunotoxin can be prepared as S0 described in Vitetta et al.,Lience 238:1098 (1987). Carbon- 14-labeled 1-stlcaaoezl3mtydehln triamldnepentaacetic acid (IvX-DTPA) is an exemplary chelating agent for conjugation of radlonuoleotide to the antibody. See W094/111026. The linker may be a "cleavable linker" facilitating release of the cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-setisitive linker, phiotolabile linker, dimethyl linker or disulfidecontaining linker (Chadi et al., CneRsarh52:127-131 (1992); U.S. Patent No. 5,208,020) may be used.

3 5 Alternatively, a fusion protein comprising the anti-TAT antibody .and cytotoxic agent may be made, by recombinant techniques or pcptide synthesis. The length of DNA may comprise respective regions encoding the two portions of t10 conjugat either adacent one another or separated bya region encodin a linker peptide 00 which Ioen not destroy the desired properties Of the Conjugate.

In Yet another embodiment, the antibody may be conjugated to a "recptor" (such streptavidin) for, utilization in tumor pro-targeting wherein the antibody-receptor conjugate is adminitered to the patient, fblowed ct by removal of unbound Conjugate fronm the circulation using a clearing agent and then administration of a "ligand" avidin.) which is conjugated to a oytotoxic agent a radionucleotide).

IND 10. homno-112osarnes 'Die anti-TAT antibodies disclosed heroin may also be formulated as immunollposomes. A "liposonle" is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug to a marrnial. The components of the liposome are commonly arranged in a bilayer formation, similar to N' 10 the lipid arrangement of biological membranes. Liposonies containing the antibody are prepared by methods N- known in the art, such as described in Epstein et al., Proc. atAcagd. i.US 82:3688 (1985); Hwang et al., Proc.

00 N- Acad. Sol. USA 77:403 0(1980), U.S. Pat. Nos. 4,485,045 and 4,544,545; and W097/3 8731 published October 23, 1997. lposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.

Particularly useful iposomes can be generated by the rverse phase evaporation method with a lipid t 5 composition comprising phosphatdylclxoline, cholesterol and P130-deivatized phosphatidylethanolamine

(PEG-

PB). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' firagments of the antibody of the present invention can be conjugated to the liposornes as described In Martin et al., I.Bo.Chem. 257:286-288 (1982) via a disulfide interchange reaction. A chemotherapeutic agent is optionally contained within the liposome. See Q3abizon et al., Q. Ntionl ncrgpLst. 81(19):1484 (1989).

B. TAT BlnOn Olietdes TAT binding oligopeptides of the present Invention are oligopeptides that bind, preferably specifically, to a TAT polypeptide as described heroin. TAT binding oligopeptides may be chemically synthesized using known oligopeptide synthesis methodology or may be prepared and purified using recombinant technology.

TAT

binding oligopeptides are usually at least about 5 amino acids in length, alternatively at least about 6, 7, 8, 9, 11, 12,13,14,15,16,17,18,19,2oD,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,4,14,3 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, 777,98,18,38,58,78,99,19,39,59,79,9 egho oe wherein such oligopeptides that are capable of binding, preferably speciically, to a TAT polypeptide as described herein. TAT binding oligopeptides may be identified without undue experimentation using well known techniques.

3 In this regard, it is noted that techniques for screening ofigopeptide libraries for oligopeptides that are capable of specifically binding to a polypeptide target are well known in the art (see, U.S. Patent Nos. 5,556,762, 5,750,373, 4,708,871,4,833,0925,223409,5,403,484,5,571,689,5,663,143; PCTPublicatjonNos. W0 84/03506 and W8403564; Geysen et al,, Proc. Natl. Acad. Soi. 8 1:3998-4002 (1984); Geysen et al., Proc. Nat]. Aced. Sci. U.S.A,, 82:178-182 (1985); Geysen etal., in Synthetic Peptides asAntigens, 130-149 (1986); Geysen etal., J. Imnunol. Moth., 1 02:259-274 (1987); Schoofs etal., 3. Ininunol., 140:611-616 (1 9 88), Cwirla, S, B. etal. (l 9 9O)Proc.Natl.Acad. Sd,.

USA, 87:6378; Lowmian, HE., etal. (1 99 l)Biochemstry, 30:10832; Clackson, T.et al. (1991)Nature, 352: 624; Marks, J. D.et al. (1991), J. Mol, Biol., 222:581; Kang, A.S. etal. (lP 9 l)Proc.Nati. Acad. Sci. USA, 88:8363, and Smith, G.

82 P- (1991) Cmnt Oph iotcowl 2:668).

ooIn this regard, bacteriopitage (piage) display is one Well known technique which allows one to soren large Oligopeptide libraries to identify member~s) of those jibrarie whic are capable of speccal bidngtr Polypeptde target Page display is a technique by which variant poypeptides ame displayed as fusion proteins ct to the coatProtein on the surface of bacteriophage particle$ (Scott, j,K and Smith, G. P. (1990) Science 249:386).

The utility of phage display lies in the fact that large libraries Of selectively randomized protein variants (or randomly cloned cDNAs) can be rapidly and efficiently sorted for those sequences that bind to a target molecule with high affinity. Display of peptide (Cwirla, S. 1B. et al. (1990) Proc. Nat!. Acad. Sci. USA, 87:6378) or protein (Lowman, H1, et al. (1991) Biochiemistry, 30:10832; Clackson, T. eta!1. (1991) Nature, 352: 624; Marks, Y. D. et al.

(1991), 1. Mo!. Biol., 222:581; Rang, A.S. eta!. (1991) Proc. Nai, Acad. Sci. USA, 88:8363) libraries onphage have (1 10 been used for screening millions of polypeptides or oligopeptides for ones with specific binding properties (Smith, (Ki P. (199 1) Current (3pm. Biotechnol., 2:668). Sorting phage libraries of random mutants requires a strategy for 00 constructing and propagating a larg number of variants, a procedure for affinity purification using the target receptor, and a means of evaluating the results of bindig erichnents. U.S. Patent Nos. 5,223,409, 5,403,484, 5,571,689, and 5,663,143, Although most phage display methods have used filamentous phage, larnbdold phage display systems (WO 95/34683; U.S. 5,627,024), T4 phage display systems (Rea, Z-J. etal. (1 99 8)Gcene 2 15:439; Zhu, Z. (1997) CAN 33:534; Jiang, 1. et (1997) can 128:44380; Ren, Z-J. et al. (1997) CAN 127:215644; Ren, Z-J. (1996) Protein Sci.

5:1833; Efinov, V. P. tal. (1995) Virus Genes 10: 173) and 177 phage display systems (Smith, P. and Scott, J.

(1993) Methods in Enzymology, 217, 228-257; U.S. 5,766,905) are also known.

Many other improvement and variations of the basic plunge display concept have now bee developed.

T1hese improvements enhance the ability of display systems to screen peptide libraries for binding to selected target molecules and to display functional proteins with the potential of scr~eening these proteins for desired properties. Combinatorial reaction devices for phage display reactions have been developed (WO 98/14277) and phage display libraries have been used to analyze and control bimiolecular interaotions (WO 98/20 169; WO 98,'20159) and properties of constrained helical peptides (WO 98/20036). WO 97/35196 describes a ethod of isolating an affinity ligand in which a phage display library is contacted with one solution In which the ligand will bind to a target molecule and a second solution in which the affinity ligand will not bind to the target molecule, to selectively isolate binding ligands, WO 97/46251 describes a method of biopanniing a random phage display library with an affinity purified antibody and then isolating binding phage, followed by a micropanning process 30(1 using microplate wells to isolate high affinity binding phage. The use of Staphlylococcus aureus protein A as an affinity tag has also been reported (Li et al. (1998) Mo! Biotech., 9:187). WO 97/47314 describes the use of substrate subtraction libraries to distinguish enzyme specificities using a combinatorial library which may be a plunge display library. A method for selecting enzymes suitable for use in detergents using phage display is described in WO 97(09446. Additional methods of selecting specific binding proteins are described in U.S. Patent Nos. 5,498,538, 5,432,018, and WO 98/11583 3.

Methods of generating peptide libraries and screening these libraries are also disclosed in U.S. Patent Nos. 5,723,286, 5,432,018, 5,580,717, 5,427,908, 5,498,530, 5,770,434, 5,734,018, 5,698,426, 5,763,192, and 5,723,32.3.

0C) TAT binding organic molecules am organic Molecules other than oligopeptides or antibodies as definedj herein that bind, preferably specifically, to a TAT polypeptide as described herein. TAT binding organic molecules may be idetified and chemically synthiesized using kcnown methodology (se, PCT Publication NSos.

WOOO/oo823 and W00013958 5 TAT binding organic molecules ame usually less Ilan about 2000 daltons in size, alternatively less than about 1500, 750, 500, 250 or 200 daltons in size, wherein such organic molecules that are IND capable of binding, preferably specifically, to a TAT polypeptide as described herein may be identified without undue experimentation using well known techniques, In this regard, it is noted that techniques for screening organic molecule libraries for molecules that are capable of binding to a polypeptide target are well known in th6 art (see, PCT Publication Nos. WOOO/0oa23 and WOOO/39585), TAT binding organic molecules may be, for (71 10 example, aldehydes, ketones, oxinies, hydrazones, semicarbazones, carbazides, primary arnines, secondary anxites, CK1 tertiary amnines, N-substituted hydrazines, hydrazides, alcohols, ethers, thiols, tiiioetliers, disulfides, carboxylic 00 acids, esters, amides, ureas, carbamnates, carbonates, ketals, thioketals, acetals, thioaoetals, aryl halides, aryl Sulfonates, alkyl halides, alkyl sulfnates, aromatic compounds, heterocyclic compounds, anilines, alkenes, alkynes, diols, amino alcohols, oxazolidines, oxazolies, thlazolidines, thiezoines, enamnines, sulfonamides, epoxldes, aziridines, isocyanates, suffontyl chlorides, diazo compounds, acid chlorides, or the like.

D, Screening o AniTA Atbodis TAT Bnding lgnyie n A idn ra Molecules~~~~ With th eiedPoees Techniques for generating antibodies, oligopeptides and organic molecules that bind to TAT polypeptides have been described above. One may futther select antibodies, oligopeptides or other organic molecules with certain biological characteristics, as desired.

.The growth inhibitory effects of an anti-TAT antiody, oligopeptide or other organic maolecule of the invention may be assessed by methods known in the art, using cells which express a TAT polypeptide either endogenously or following transfection with the TAT gene. For example, appropriate tumor cell lines and TATtratisfected cells may treated with an anti-TAT monoclonal antibody, oligopeptide or other organic molecule of the invention at various concentrations for a few days 2-7) days and stained with crystal violet or MITT or analyzed by some other colorimetic assay. Another method of measuring proliferation would be by comparing 3 H-.thytnidine uptake by the cells treated In the presence or absence an anti-TAT antibody, TAT binding oligopeptide or TAT binding organic molecule of the invention, After treatment, the cells are harvested and the amount of radioactivity incorporated into the DNA quantitated in a scintillation counter. Appropriate positive controls include treatment of a selected cell line with a growth inhibitory antibody known to inhibit growth of that cell line. Growth inhibition of tumor cells in vivo can be determined in various ways known in the art, Preferably, the tumor cell is one that overexpresses a TAT polypeptide, Preferably, the anti-TAT antibody, TAT binding oligopeptide or TAT binding organic molecule will inhibit cell proliferation of a TAT-expressing tumor cell in Wiro or in vivo by about 25-100% compared to the untreated tumor cell, more preferably, by about 30-100%, and even more preferably by about 50- 100% or 70- 100%, in one embodiment, at an antibody concentration of about 0.5 to gfnd. Growth inhibition can be measured at an antibody concentration of about 0.5 to 30 PtgWm or about ni to 200 riM in cell culture, where the growth inhibition is determined 1- 10 days afterexposure of the tumortells to OWe antibody. Mhe anibodY is gluWfh inhibitoiy In WVo if adminstration of the anti-TAT antibody at about I 00 i0g'k to about 100 m94k body wMight Meults in reduction in tumor Sime or reductionoftmrcl zifi± 0 within about 5 days to 3 months from the first administration of the antibody, preferably within about 5 to 30 days.

To select for an anti-TAT antibody, TAT binding oigopeptide or TAT binding organic molecule which induces cell doath, loss Of membrane integrity as indicated by, propidium iodide trypan blue or 7AAI) uptake may be assessed relative to controul. A P1 uptake assay can be performed in the absen ce. of complemnent INC and immune effector cells, TAT polypeptide..expressjng tumnor cells ame incubated with medium alone or mediumn containing the appropriate anti-TAT antibody at about I Oftg/nd), TAT binding oligopeptide or TAT binding organic'molecule. The cells are incubated for a 3 day time period. Following each treatment, cells are washed and aliquoted into 3 5 nun strainer-capped 12 x 75 tubes (Imad per tube, 3 tubes per treatment group) for removal of cell clumps. Tubes then receive P1 (l01tg/in), Samples may be analyzed using a FACSCAN® flow cytonieter and FACSCONVERSQ CellQuest software (Becton Dickinson). Those anti-TAT antibodies, TAT binding 00 oligopeptides or TAT binding organic molecules that induce statistically significant levels of cell death as determined by P1 uptake may be selected as cell death-inducing anti-TA'Iaitibde8, TAT binding oligopeptide or TAT binding organic molecules.

To screen for antibodies, oligopeptides or other organic molecules which bind to ani epitope on a TAT polypeptide bound by an antibody of interest, a routine cross-blocking assay such as that described In Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (198 can be performed. This assay can be used to determine if a test antibody, oligopeptide or other organic molecule bind& the same site or epitope as a knownl anti-TAT antibody. Alternatively, or additionally, epitope mapping can be 2o) performedl by methods known in the ant. For example, the antibody sequence can be mutagenizedlsc as b alanine, scanning, to identify contact residues. The mutant antibody is initailly tested for binding with polyclonal antibody to ensure proper folding. In a different method, peptides corresponding to different regions of a TAT polypeptide can be used in competition assays with the test antibodies or with a test antibody and ain antibody with a characterized or known epitope.

p. Atbd eenetEzm ediated Pdrua Thr AE

T

The antibodies of the present invention may also be used in ADEPT by conjugating the antibody to a prodrug-activating enzyme which converts a prodrug a peptidyl chemiotherapeutic agent, see W081/01 145) to an active anti-cancer drug. See, for example, WO 88/07378 and U.S. Patent No. 4,975,278.

The enzyme component of the immunoconjugate useful for ADBPT includes any enzyme capable of 3 D acting on a prodrug in such a way so as to covert it into its more active, cytotoxic fomi, Enzymes that are useful in the method of this invention include, but are not limited to, alkaline phosphatase useful for converting phosphate-contaiing prodrugs into free drugs; arylsulfatase useful for converting sulfate-containing prodrugs into free drugs; cytosine dearninase useful for converting non-toxic fluorocytosine into the anti-cancer drug, 5-fiuorouracil; proteases, such as senratia protease, thermolysin, 33 subtilisin, carboxypepudases and cathepsins (such as cathepsins 13 and that are useful for converting peptidecontaining prodrugs into free drugs; D-alanylcarboxypepfidase-s useful for converting prodrugs that contain

D-

arnino acid substituents; carbohydrate-cleaving enzymes such as P-galactosjdase and neuraminidase useful for convertinjg g1Clyood pitodrug intoD fife drugs; P-actamase usefu for converting drugsw dedvalized with53 oolatani into fr= drugs; and penicilin arnidases, such as penicillin V asnidase or penicillin 0 amidase, useflul for converting drugs derivatizeci at their amine nitrogens with phenoxyacetyl or plienylacetyl groups, respectvely, Cl into fmr drugs. Alternatively, antibodies with enzymatic activity, also known in the adt as "abzymes", can be used to convert the prodrugs of the invention into free active drugs (see, ie-g., Massey, Nature 328:457-458 (1987)).

Atitibody-abzyine conjugates can be prepared as described herein for delivery of the abzyme to a tumor cell IND population, The enzymnes Of this invention can be covalendty bound to the anti-TAT antibodies by techniques well known in the art such as the use of the hieterobifunctional crosslinking reagents discussed above. Alternatively, fusion proteins comprising at least the, antigen binding region of an antibody of the invention linked to at least 10 a functionally aotive portion of an enzyme of the invention can be constructed using recombinant DNA techniques well known In thie art (see, Neuberger et al., Naur 312:604-608 (1984).

00

F

F. ullLenthiTA PJmentdes The present invention also provides newly identified and Isolated nucleotide seqences: encoding polypeptides referred to in the present application as TAT polypeptides. In particular, cDNA9 (partial and filllength) encoding various TAT polypeptides have been identified and isolated, as disclosed in fiuher detail in the Examples below.

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

0. Anti-TAT Antibo nd TAT1 olvuptide Variants In addition to the anti-TAT antibodies and fill-length native sequence TAT polypeptides described herein, it is contemplated that anti-TAT antibody and TAT polypeptide variants can be prepared, Anti-TAT antibody and TAT polypeptide variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA, and/or by synthesis of the desired antibody or polypeptide. Those silled in the art will appreciate that amrino acid changes may alter post-translatiojial processes of the anti-TAT antibody or TAT polypeptide, such as changing the number or position of glycosylatiosi sites or altering the membrane anchoring characteristics.

Variations in the anti-TAT antibodies and TAT polypeptides described herein, can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S. Patent No. 5,364,934, Variations may be a substitution, deletion or insertion of one or more codons encoding the antibody or polypeptide that results in a change in the amino acid sequence as compared with the native sequence antibody orpolypeptide. Optionally the variation is by substitution of at least one amino 3 5 acid with any other amino acid in one or more of the domains of the anti-TAT antibody or TAT polypeptide.

Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the anti-TAT antibody or TAT Polypeptide with tha of homologous knw rtn oeue and minicmizn the number of amino acid sequence 00 Changes made in regions of high homology. Amino acid substitutions can be the result Of rnPlacing one anlino acid with another amino acid having similar stluctural and/or chemical properties, such as the rePlacement or a leucine with a serine, Le., conservative amino acid replacements. Insertions or deletions may optionally be in the range Of about 1 to 5 amino acids. The Variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the ful-length or mature native sequence.

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

ri Anti-TAT antibody and TAT polypeptide fragments may be prepared by any of a number of conventional 00 techniques. Desired peptide fragments may be chemnically synthesized. An alternative approach involves generating antibody or polypeptide fragmnents by enzymatio digestion, by treating the protein with an enzyme known to cleave proteins at sites defined by particular amidno acid residues, or by digesting the DNA with suitable restriction enzymes and isolating the desired fragment. Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired antibody or polypeptide t'igrnent, by polymerase chain reaction (PCR). Qligonuclootides that define the desired termini of the DNA fragment are employed at the 5' and 3'primers in the PCR. Preferably, anti-TAT antibody and TAT polypeptide fragments share at least one biological and/or immunological activity with the native anti-TAT antibody or TAT polypeptide disclosed herein.

In particular embodiments, conservative substitutions of interest ane shown in Table 6 under the heading of preferred substitutions. If such substitutions result in a obange in biological activity, then more substantial changes, denominated exemplary substitutions in Table 6, or as further described below in refere to amino acid classes, are, introduced and the products screened.

TAW 6 00 dla OReidiua EXWVplay *Piferivd Ala(,k) 'val; leu; le val Arg lYS; gin; asn lys Asn gin; his; lys; arg gin IDAsp glu glu Cys ser ser O~n (Q am asn Glu asp asp (fly pro; ala ala ils a9n; gin; lys; argag lieU ()luVal; mt;~ ala; phe; g CK1norleucine

IOU

S 15 Leu norleucine; ile; Yal; 00met; ala; phe ile Lys arg; gin; aga arg met(M lea; phe; ile.

IOU

Phe leu; vl; 1ui; aij;tyr

ICU

P0 ro ala ala Ser offth Thr (Te8r ser TIP MW tyr; phe tyr Tyr MY trP; phe; thr ser phe Val lIe-; feu; met; phe; ala; norleucine

ICU

Substantial modifications in function or immunological identity of the anti-TAT antibody or TAT polypetide ar accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) structure of the polypeptid-, backbone in the area of the substitution, for example, as a sheet or helical conformation, the charge or hydrophobicity of the molecule at the target site, or the bulk of the side chain.

Naturally occurring residues are divided into groups based on common uide-chain properties: hydrophobic: norleucin, met, ala, val, leu, ile; neutral hydrophilic: cys, ser, thr; 35(3) acidic: asp, glu; basic: asn, gln, his, lys, arg; residues that influence chain orientation: gly, pro; and aromnatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a memiber of one of these classes for another class.

substituted residues also may be introduced into the couseryatiYe substitution sites or, more preferably, into the remaining (non-conserved) sites, The variations can be made using methods known in the art such as oligonucleotide-modiated (sitedirected) mutagenesis, alanine scanning, and POR rnutagenesis, Site-directed mutagenesis [Carter et al., Vpol.

Acgids Res., 13:4331 1(1986); Zoller et al,,cAcds Res,, 10&:6487 (1987)], cassette mutagenesis (Wells etal., Gene, 2 :3 15 (1985)), restriction selection mutagenesis (Wells et al., Philos, Trans, R, Soc. Lndon SerA, 317-4 15 (198f)] or other known techniques can be performed on the cloned DNA to produce the anti-TAT antibody or TAT 0 polypeptide variant DNA.

Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main- 0 chain conformation of the variant [Cunningham and Wells, iene, 244:1081-1085 (1989)]. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed 1 positions [Creighton, The Proteins, Freeman Co., Chothia, J. MoBiol, 10:1 (1976)]. If alanine Ssubstitution does not yield adequate amounts of variant, an isoteric amino acid can be used.

S 10 Any cysteine residue not involved in maintaining the proper conformation of the anti-TAT antibody or t TAT polypeptlde also may be substituted, generally with serine, to improve the oxidative stability of the molecule Sand prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the anti-TAT antibody or TAT polypeptide to improve its stability (particularly whee the antibody is an antibody fragment such as an Pv fragment).

A particularly prefered type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody a humanized or human antibody). Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervarlable region sites 6-7 sites) are mutated to generate 0 all possible amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene m product of M13 packaged within each particle.

The phage-displayed variants are then screened for their biological activity binding affinity) as herein disclosed. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding.

Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and human TAT polypeptide. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.

3 0 Nucleic acid molecules encoding amino acid sequence variants of the anti-TAT antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotidemediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the anti-TAT antibody.

H. Modificatons of Anti-TAT Antibdes and TAT Polypentdes Covalent modifications of anti-TAT antibodies and TAT polypeptides are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of an anti-TAT 00anltiboy or TAT POlypeptice with an organi dervatUzn aget that is calpble Of M~actl with Selected Side 0chains or the N- or tenninal tesidues Of fihe anti-TAT antibody or TAT polypeptde. Derivatftoin with bifuinctional agents is Useful, for instance, for crossl*a anti-TAT antibody or TAT polypeptid to a ater.

insoluble Support Matrix or surface for use in the method for purifying anti-TAT antibodies, and Vice-vera.

Commonly used crosslinking agents include, ll-bls(diazoaoety).2.pheflyletliane glutaraldehyde,

N-

hydro xysuccinimide esters, for example, esters with 4 -azidosalicylic acid, homobifijnctional imidoesters, including disuccinimidyl esters such as 3 3 1 'dthobs(succitnimdylpropionate), bifinctional maleimides such as bis-Nmaleumido-1I, 8 -octane and agents such as mehl3[paiohnl~ihopoiiiae I> Other modifications include dearnidation of glutarninyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphozylation of hydroxyl (Ni 10 groups of seryl or threonyl residues, methylation of the a-amino groups of lysine, arginine, and histidine side 00ha [TE 00i chi_. riho~~j 5 tutr n~lclrrp~ Wit. Freemian& Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

(1 Another type of covalent modification of the anti-TAT antibody or TAT polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the antibody or polypeptide.

"Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence anti-TAT antibody or TAT polypeptide (either by removing the underlying glycosylation site or by deleting the glycosylation by chemicoal and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence anti-TAT antibody or TAT polypeptide. In addition, the phrase includes qualitative changes in the glycosylatilon of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.

(Glycosylation of antibodies and other polypeptides is typically either N-linked or 0-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X..seiin and asparagine..X.threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. 0linked glycosylation refers; to the attachment of one of the sugars N-aceylgalactosarine, galactose, or xylose to a hydroxyamdio acid, most comnmonly serine or threonine, although 5-hydroxyprollne or S-hydroxylysine may also be used.

Addition of glycosylation sites to the anti-TAT antibody or TAT polypeptide is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described toipeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or throonine residues to the sequence of the original anti-TAT antibody or TAT polypeptide (for 0-linked glycosylation sites). The anti-TAT antibody or TAT polypeptide amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the anti-TAT antibody or TAT polypeptide at preselected bases such that codons are generated that will translate into the desired amidno acids.

Ano*6e means Of incmwaing the number of mab*Wydrte moieties on the anti-TAT antibody or TAT 00polypeptide is by chemical or enzymatioc oupling of *lcoside to te polyete.Scmehdardsrie 0in the att in WO 87105330 published I11 September 1987, and in Aplin and Wriston, CRC sLkRe. BLocherri., pp. 259-306 (1981).

Removal Of carbohydrate moieties present on the anti-TAT antibody or TAT polypeptide may be chemically or enzymnatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and deseribed, for instance, by Rakimuddin, et al., Arch. Boc0hem.Biohys., 259~:52 (1987) and by Edge et aL, Alal Biochem., 118: 131 (981), Enzymatic cleavage of carbohydrate moieties on polypeptides can be achiev ed by the use of a variety of endo- and exo-glycosidases as desoribed by Tijotakura et al., Megth. EnzMol.,, 138:350 (1987).

C) 10 Another type of covalent modification of anti-TAT antibody or TAT polypeptide comprises linking the 00 antibody or polypeptide to one of a variety of nonprotelnaceous polymers, polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the maniner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or4,179,337. The antibody orpolypeptide also may be entrapped in microcapsules prepared, for example, by coacervation techniqdes or by interfacial polymerization (for example, hydroxymnethylcellulose or gelatin-mirocapule and poly-(methylmnetilaoylate) niorocapsules, respectively), in colloidal drug delivery systems (for eample, liposomes, albumin microspheres, microemulalons, nano-particles arnd nanocapsules), or in rnacroemulsions. Such techniques are disclosed in Remngt~on's Pharmaceutical Sciences, 1 6th edition, Oslo, Ed., (1980).

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

In one embodiment, such a chimeric molecule comprises a fusion of the anti-TAT antibody or TAT polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind.

The epitope tag is generally placed at the amino- or carboxyl- terminus of the anti-TAT antibody or TAT polypeptide. The presence of such epitope-tagged forms of the anti-TAT antibody or TAT polypeptide can be detected using an antibody against the tag polypeptide. Also, provision of the epitope tag enables the anti-TAT antibody or TAT polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. Various tag polypeptides and their respective antibodies arc well known in the art Examples include poly-histidine (poly-his) or poly-hlstidine-glycine (poly-his-gly) tags; the .0 flu HA tag polypeptide and Its antibody 12CM5 [Field et al., h1o. _CellBlol., .1:2159-2165 (1988)]; the o-myc tag and the BF9, 3C7, 6B10, 04, B7 and 9E1 0 antibodies thereto (Evan et al., Molecular and Cellular Biologv 5:36 10-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al., Protein Engineering, 1(6):547.553(1990)). Other tag polypeptides include the Flag-peptide [Hopp et al.,Bloitechnolo av f: 1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., acience, 255:192-194 (1992)]; an o-tubulin epitope 3 5 peptide [Skinner et al., L Bio. Chem. 266:15163..15166 (19Y91)]; and the T7 gene 10 protein peptide tag [Lutz- Freyermuth et aL Proc. Nat, Acad S cl, USA, 87:6393 -6397 (1990)], In an alternative embodiment, thodmeric molecule may comprise a fusion of the anti-TAT antibody or TAT polypeptide with an mmunoglobulin or a particular region of an immuoglobulin. For a bivalent form of the chimeric molecule (also referred to as an "inmunoadhesin"), such a fusion could be to the Fc region of an IgO molecule. The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of an anti-TAT antibody or TAT polypeptide in place ofat least one variable region within an Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusion includes the hinge, CH 2 and CH 3 or the hinge, CH,, CH 2 and CH3 regions of an IgGl molecule. For the production of immunoglobulin fusions see also US Patent No. 5,428,130 issued June 27,1995.

I. Preparation of Anti-TAT Antibodies and TAT Polvoentide, SThe description below relates primarily to production of anti-TAT antibodies and TAT polypeptides by culturing cells transformed or transfected with a vector containing anti-TAT antibody- and TAT polypeptide- Sencoding nucleic acid. It s, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare anti-TAT antibodies and TAT polypeptides. For instance, the appropriate amino acid sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques [see, Stewartet al., Solid-Phase Peid Snthss. W Freeman Co., San Francisco, CA (1969); Merrifield,

.A

1 5 enhn. Soc,, 85:2149-2154 (1963)]. In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using manufacture'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 ofDNA Bncodin Anti-TAT Antibody or TAT Polvpeptide DNA encoding anti-TAT antibody or TAT polypeptide may be obtained from a cDNA library prepared from tissue believed to possess the anti-TAT antibody or TAT polypeptid mRNA and to express it at a detectable level. Accordingly, human anti-TAT antibody or TAT polypeptide DNA can be conveniently obtained from a eDNA library prepared from human tissue. The anti-TAT antibody- or TAT polypeptide-encoding gone may also 2S be obtained from a genomic library or by known synthetic procedures 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 it. Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al., Molecular QCloing: A Laborato.r Manual (Now York: Cold Spring Harbor Laboratory Press, 1989). An alternative means to isolate the gene encoding anti-TAT antibody or TAT polypeptide is to use PCR methodology [Sambrook et al., guEra; Dieffenbach et al., PO Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)], Techniques for screening a cDNA library are well known in the art. The oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized.

The oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened. Methods of labeling are well known in the art, and include the use of radiolabels like "P-labeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al., .pa.

Sequences identified in such airmzy screenin methods can be compared and aligne to other known 00equences deposited and available in public databases suha nai rohrpiaesqec aaa~ Sequence identity (at either the amino acid or nucleotide level) wti eie ein ftemlcl rars CI te fll-engt se uen e c n bedetrmied I ing metodsknown in the at and as described herein.

Nucleic acid having protein coding sequence may be obtained by screening selected ODNA or genomjce libraries using the deduced amino acid sequence disclosed herein for the first time, ad fncsay sn convntinalprimer extension procedures as described in, Sanmbrook et al., Dp to detect precursors and Processing intermediates Of InMRA that may not have been reverse..transciibed into cDNA.

1>2. Selection and Tronsformatio -of Host Cells Host cells are transfected or transformed with expr-ession or cloning vectors described herein for anti-TAT 1K 10 antibody or TAT polypeptide production and cultured in conventional nutrient media modified as appropriate for C) inducing promoters, selecting transformants, or amiplifyring the genes encoding the desired sequences. The culture 00 00 conditions, such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Matnmalipan Cel1 11-technol, a Practical ApacM. Butler, ed. (rRL Press, 199 1) and Sambrook et al., ;u Methods of eukaryotic. cell transfection and prokaryotic Cell transformation are known to the ordinarily skilled artisan, for exanmple, CaCl 2 CaPO 4 liposome-mediated and electroporation. Depending on the host cell used, tranisformation is performed using standard techniques appropriate to such cells. The calcium treatment employinag calcium chloride, as described in Samnbrook et al., _mV or electroporation is generally used for prokaryotes.

Infection with 4gro bacterium twmefaclew. is used for transformation of certain plant cells, as described by Shaw et al., Gee,2:15 (1983) and WO 89105859 published 29 June 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der 131, Virolosv a:456-457 (1978) can be employed. General aspects of mammnalian cell host system transfections have been described in U.S. Patent No.

4,399,2116. Transformations into yeast are typically carried out according to the method of Van Solingen et al., L.

225 )Bact.,L30.:946 (1977) and Hsiao et al., Proc. NatI. Acd. S i, 6S) .:3829 (1979). However, other methods for introducing DNA into cells, such as by nuclear n-doroinjection, electroporation, bacterial protoplast fuasion with intact cells, or polycations, polybrene, polyornithine, may also be used. For various techniiques for transforming mammalian cells, see Keown et al., Methods in Enzymology, 185:527-537 (1990) and Mansour Ct al., Nature, 336:348-352 (1988).

3 0 Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes include but are not limited to eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriacear, such as E. coil. Various E. coil strains are publicly available, such as E. coil K12 strain MIN194 (ATCC 31,446); coil X1776 (ATCC 31,537); E coil strain W3 (ATCC 27,325) and K5 772 (ATCC 53,635). Other suitable prokaryotic, host cells include finterobacteriaceaec such Eicher-ichia, coil, Ent'ero bactr,, Erwin ia, Klebsiella, Proteus, Salmonella, Salmonella typ hinur4 cm, Serratia, Serratia inarceycangs, and Shigella, as well as Bacilli such as B. subil~s and B, ficlien(/ortnis B. lichearlorris 41 p disclosed in DD 266,710 published 12 April 1989), Pseudonionas such as P. Gell oJ, And Sfrepomycey. These examples ame illustrative rather than limiting. Strain W3110 is one 00 Particulaly Prefed host or parent host because it is a common host strain fur recombinant DNA produot fermentatIons. Preferably, the host cell secretes minimal Amounts Of protoolytic enzymes, For example, gftvain W3 110 may be modified to effeCt a genetic mutation in the genes encoding proteins endogenous to thre host, with examples of such hosts including E. col!W31 10 strain IA2, which has the complete genotype tonA E. coi W31 strain 9E4, which has the complete genotype tonA ptr3; E coll W3 110 strain 2707 (ATCC 55,244), which has the complete genotype tonA ph-3 phoA RI15 (argF1ac169 degP ormipT kanr; R. colt W31 10 stmi 37D6, which has the complete genotype tonA ptr3 phoA EIS (argF-k1,)169 cfegP onmpT rbs7 tlvG kan' E. coli W3 10 strain 40B4, which is strain 37D6 with a non-kaniamycin resistant degP deletion mutation; and an coi strain having mutant periplasmic protease disclosed in U.S. Patent No. 4,946,783 issued 7 August 1990. Alternatively, in Vio (71 10 methods of cloning, PCR or other nucleic acid polymerase reactions, are suitable.

(71 Full length antibody, antibody fragments, and antibody fusion proteins can be produced in bacteria, in 00 particular when glycosylation and Fc effector function are not needed, such as when the therapeutic antibody is corjugated to a cytotoxic agent a toxin) and the imznunoconiugate, by itself shows effectiveness in tumor cell destruction. Full length antibodies have greater half life in circulation. Production InE, coi is faster and more cost efficient. For expression of antibody fragments and polypeptides in bacteria, see, U.S. 5,f48,237 (Carter et.

U.S. 5,789,199 (Joly et and US. 5,840,523 (Simmons et al.) which describes translation initiation regio (TM.) and signal sequences for optimizing expression and secretion, these patents incorporated herein by reference.

After expression, the antibody is isolated from the E, coli cell paste in a soluble fraction and can be purified through, a protein A or G column depending on the isotype. Final purification can be carried out similar to the process for purilying antibody expressed in CHO0 cells.

In addition to prokaryotes, oukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-TAT antibody- or TAT polypeptide..noodwng vectors. Saccitaromyces cerevlslae is a commnonly used lower eukaryotic host microorganism. Others include Sclrtzosaccharomyces pombe (Beach and Nurse, Niur 290. 140 [198 EP 139,383 published 2 May 1985); K~uyverornyces hosts Patent No.

2S 4,943,529; Fleer et al., Bioi9echnoloWy 9:968-975 (199 such as, K (actis (MW98-8C, CBS683, CBS4574; Louvencowtt et al., I.Bacte lol,, 1 54(2):737-742 [1983]), Kfraglisv (ATCC 12,424), X butgaricus (ATCC 16,045), K wickeraniti (ATCC 24,178), K. wafli (ATCC 56,500), k drosopliiarwnm (ATCC 36,906; Van den Berg et al., kipjTehnay, 8:135S (1990)), K. ehermorolerans, andK inaixianus ,-yairrowia (ElP 402,226); Piclriapastors (Ell 183,070; Sreeki-isma et al., asic Microblol.,28:265-278 [1988]); Candida; 7hclihoder-ma resia (El'244,234); Necrrospora crassa (Case et al., ProcNal Aafd. S ci. USA, 76;5259-5263 [1979]); Sclnvanniomyce-, such as Schwanniomycei occidentais (EP 394,538 published 31 October 1990); and filamnentous fungi such as, NeuospraPenicjluwn, 7 'olypocladium (WO 9 1/00357 published 10 January 1991), and Aspergitlus hosts such as A. nfdula,,s (Balan-e et al., Bochem. Biophyrs. Res. Commu, 112:284-289 [1983]; Tilburn cial,, Gee 26:205.221 [1983]; Yeltonetial., Proc. ai. Acad Sd, USA, 81: 1470-1474 [1984]) and A n/ge,' (Kelly and Hynes, EMBOQ 4:475-479 [1985]). Methylotropic yeasts are suitable herein and Include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of ffartrenula, Candida, Kiceclrera, I'icla, Sacchczromycer, Torulopsr/s, and Rlrodororufa. A list of specific species that are exemplary of (his class of yeasts 00may be foundJ inC. Anthon, Tho gi NhemR*I MU*J&ft 269 (1982).

Suitable0 host ceils for the expression of glycoslated anti-TAT antibody or TAT Polypeptide ame dodveri from multiceffular organisms, Examples Ofiyrerate cells inld inet el sc s i"'hl S n Spodoptera Sf9, as well as plant cells, such as cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco. Numerous baculoviral strains and variants and corresponding permissive. insect host cells from hosts such as .podoterafruglperda (caterpillar), Aedes aegypi (mosquito), Aedev albopictus (mosquito), Dtosophila INDnelanogaster (fruitfly), and Doinbyx 'nor have been identified. A variety of viral strains fur transfiection are publicly available, the L-1 variant of Autographa caqifbiwig NPV and the Bm-5 strain of Bombyx tnort NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfeetion of Spodopterafrugipe.da cells.

C] 10 However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell linles are monkey kidney 00 CVI line transformedby SV4Q (C0S-7, ATCCCRL 165 human emblyonlokidney line (293 or293 cells subolorted C]forgrowth in suspnsionutue, Graham et al.,y.OenYiol. 36:59 (1977)); baby hamster kidney cells (BHX ATCC CCL 10); Chnesehansterovay cells/-DHYR(CHOUrlaub et at, Proc.Nadl. AcadeLi.UA77:4216 (1980)); mouse sertoli cells (TM4, Mather, 919-l Re rod. 23:243-251 (1980)); monkey kidney cells (CVI ATCC CCL 70); Aftican green monkey kidney cells (VERO-76, ATOC CR1-I 587); human cervical carcinoma cells (BELA, ATCC CCL 2); canine kidney cells (bIDOK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human llvercells(Hep G2, H1B 8065); mouse mamnmary tumor(MM'O60562, AluCC CCL5 1); TRI cells (Mather et al., Aal Y.AdS.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatorna line (llej, 02).

Host cells are transformed with the above-described expression or cloning vectors for anti-TAT antibody or TAT polypeptide production and cultured inl conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

3. SelectioDn and Use of a Reulicable Vector The nucleic acid oDNA or genomic DNA) encoding anti-TAT antibody or TAT polypeptide may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression. Various vectors are publicly available. The vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general,

DNA

is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector 0 components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan.

The TAT may be produced recombinantly not only directly, but also as a fusion polypeptide with a 5 heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or it may be a pail of the anti-TAT antibody- or TAT polypeptide-enooding DNA that is inserted into t0 vector, he "Pgal sequence may be a prkryotic Signal sequence selected, for eample, from the gro'Up 'of the lkaine phosphatase, penIcllinase, IPP, or heat.-fiUble enterotoxlii R leaders. For yews secretion the Fignaj GO~ncemaybe, the yas inveitase leader, apha fato leader (including SacarYe ad Ji1'VeroMYCM a-factor leaden, the latter described inUJS, Patent No. 5,010,182), or acid phosphatase leader, the C. albicaits gluc-oarnylase leader (E3P 362,179 published 4 April 1990), or the signal described in WO 90/1364,6 published 15 November 1990. In mammalian cell expression, mammalian signal sequences may be used to direct sretion Of the Protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders.

Both expression and cloning vectors contain it nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses.

CK 0 The origin of replication from the plasmid pBR322 Is suitable for most Gramn-negative bacteria, the 214 plasmid origin CK1 is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning 00 vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker.

T ypical selection genes encode proteins that confer resistance to antibiotics or other toxins, arepicillin, neomycin, methotrexate, or tetracycline, complement auxotrophic deficiencies, or supply critical nutients not available fromn complex media, the gene encoding D-alanlne racemase for Bacilli.

An example of suitable selectable markers for mamnmalian cells are those that enable the identification of cells competent to take up the anti-TAT antibody-. or TAT polypeptide-encoding nucleic acid, such as DHFR or thyrnldine kinase. An appropriate host cell when wild-type DHFR Is employed is the CHO cell line deficient in DFFR activity, prepared and propagated as described by Urlaub et al., Proc. NatI. Aced. Sd.SA, 77:4216 (1980).

A suitable selection gene for use in yeast Is the arpt gene present in the yeast plasmid YRp7 [Stinchoomb et al., HAWr 282:39(1979); Klngsmnanet al., Gene 7:141 (1979); Tschemperet at., Gene. 10:157 (1980)1. Theofpl gene provides a selection marker fqr a mutant strain of yeast lacking the ability to grow in tryptopban, for example, ATCC No. 44076 or PEP4-1 [Jones, goctc 85:12 (1977)].

Expression and closing vectors usually contain a promoter operably linked to the anti-TAT antibodyor TAT polypepfide-encoding nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the 1-lactamase and lactose promoter systems [Chang et al., Nature, 275:615 (1978); Goeddel at al., aure 281:544 (1979)] alkaline phosphatase, a tryptophan (tip) promoter system [Goeddel, ,uli Acd e, :07(98) P3,7] n hybrid promoters such as the tac promoter [deBoer et al,, Proc. Nat. Acd Sol. USA, 80:21-25 (1983)]. Promoters for use in bacterial systems also will contain a Shine-Dalgamro sequence operably linked to the DNA encoding andi-TAT antibody or TAT polypeptide.

Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3phosphoglycerate kinase [Hitzeman at at., Biol. Chem., 255:2073 (1980)] or otherglycolytic enzymes [Hess at al., L AdjYEnzme Re 7:149 (1968); Holland, B3iochemidstry, 17:4900 (1978)], such as enolase, glyceraidehyde.3.

phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3 -phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, pliosphoglucose isomerase, and gluookinlse, SOther yeast pmrmoters, which are inducible promoters having the additional advantage of transoription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isooytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3.

phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.

Anti-TAT antibody or TAT polypeptide transcription from vectorsin mammalian host cells is controlled for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenovirus (sch as Adenovirus bovine papilloma virus, avian sarcoma virus, cytomegalovius, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian 1 0 promoters, the actin promoter oran immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems.

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

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

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

4. Culturing the Host Cells The host cells used to produce the anti-TAT antibody or TAT polypeptide of this invention may be cultured In a variety ofmedia. Commercially available media such as Ham's Fl0 (Sigma), Minimal Essential Medium (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Moth. Enz. 58:44 (1979), Barnme et al., Anal. Biochem102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469;

WO

90/03430; WO 87/00195; or U.S. Patent Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferin, or epidemnal growth factor), salts (suoh as sodium chloride, caloium, magnesium, and phosphate), buffers (such as HEPIBS), nuoleotides (such as adenosine and thymidine), antibiotics (such as OBNTAMYCIN drug), trace elements (defined as noganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy sourcee. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the Sordinarily skilled artisan.

Detecting Oene Amplifcation/Epression Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Nat. Aad S. SA, C 10 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, N based on the sequence provided herein. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes orDNA-protein duplexes. The Santibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.

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

6. Purification of Anti-TAT Antibody and TAT PolYoptide Forms of anti-TAT antibody and TAT polypeptide may be recovered from culture medium or from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g.

Triton-X 100)orby enzymatic cleavage. Cells employed in expression of anti-TAT antibody and TAT polypeptide can be disrupted by various physical or chemical means, such as freeze-thaw cyoling, sonication, mechanical disruption, or cell lysing agents.

It may be desired to purify anti-TAT antibody and TAT polypeptide from recombinant cell proteins or polypeptides. The following procedures are exemplary of suitable purification procedures: by fractionation on an 3 0 ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cationexchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgO; and metal chelating columns to bind epitope-tagged forms of the anti-TAT antibody and TAT polypeptide. Various methods of protein purification may be employed and such methods are known in the art and described for example in Deutscher, Methods in Enzymoloy, 182 (1990); Scopes, Protein Purification: Principles nd Practice, Springer- Verlag, New York (1982). The purificationstep(s) selected will depend, for example, on the nature of the production process used and the particular anti-TAT antibody or TAT polypeptide produced.

When using recombinant techniques, the antibody can be produced itracelulmly, in the periplaarnio 00space, or directy secreted into the medium, If theantiboyI rdcdItaelld, safMsetl atclt dbieither host cells or lyed fagment, ar removed, for example, by cetrifugaion or ultrafiltration. Cator et al-Bi k2tecnIM~ 10: 163-167 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of coli. Briefly, cell Paste is thawed in the presence of sodium acetate (PH EDTA, and phenyhnethylsalfgnylfluoride (PMSF) over about 30 miii Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such eXPression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipojre Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using, for example, hydroxylapatito chromatography, gel electrophoresis, dialysis, and affinity chromiatographiy, with affinity chromatography being 00 the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype. of any iremunoglobulin Fe domain that is present in the antibody. Protein A can be used to purify antibodies that are based on humanyl1, or T4 heavy chains (Lindmark et al., L qL e 62:1-13 (1983)), Protein 0 is recommended for all mouse isotypes and for human y3 (Guss etaL, 9hOJ 5: 15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available.. Mechanically stable matrice such as controlled pore glass or poly(styrenediviny)benzone allow for faster flow rates and shorter processing timies than can be achieved with agas-ose. Where the antibody comprises a CH3 domain, the Bakerbond A13Xnhresin T. Baker, Phillipsburg, NJ) is useful forpurification. Other techiniques for protein purification such as fractionation on an ion-exchange column, ethatnol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin S13PHAROSEMa chromatography on an anion or cation exchange resin (such as a polyaspartic avid columrn), ohromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

Following any preliminay purification step(s), the mixture comprising the antibody of interest and contamilnants may be subjected to low PH hydrophobic interaction chromatography using an elution buffer at a pH1 between about 2.5-4.5, preferably performed at low salt concentrations from about 0-0.25M salt).

J. Phrcetalorutin Therapeutic formulations of tht anti-TAT antibodies, TAT binding oligopeptides, TAT binding organic mnolecules andlor TAT polypeptides used in accordance with the present invention are prepared for storage by .0 mixing the antibody, polypeptide, oligopeptide or organic molecule having the desired degree of purity with optional pharmaceutically acceptable carriers, exciplents or stabilizers (RmigosPamcuia ciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophllized formulations or aqueous solutions, Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as acetate, Tris, phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and rn methionine; preservatives (such as octadecyldimethybenzI ammoniumn chloride; hexarnethonlum chloride; benzalkoniurn chloride, benzethonium chloride; phenol, butyl or beazyl alcohol; alkyl parabens such as methyl or proDpyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 resdues) polYPeptdes; prultelas, such as serum iumin, gelatin, or immunoglobulins; hydroplijic 00 polymers Such as polyvinylpytrlidone; amino acids such as glyine, glutamine, asparagine, hitidine, azginine, or lysine; monosacobandes, disaccharides, and other carbohydrates including glucose, matmose, or dextrinq; chelating agents such as EDTA; tonicifiers such as trehalose arnd sodium chloride; sugars such as sucrose,, mannitol, trehalose or sorbitol; surfiactant such as polysorbate; salt-forming counter-ions such as sodium; inetal complexes Zn-protein complexes); and/or non-ionic surfactants such as TWIENO, PLURONICSO or polyethylene glycol (P130). The antibody preferably comprises the antibody at a concentration of between 5-200 mg/mil, preferably between 10-100 mg/mi.

The formulations herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other C) 10 For example, in addition to an anti-TAT antibody, TAT binding oligopeptide, or TAT binding organic molecule, 00 it may be desirable to include in the one formulation, an additional antibody, a second anti-TAT antibody which binds a different epitope on the TAT polypeptide, or an antibody to some other target such as a growth factor that affect the growth of the particular cancer. Alternatively, or additionally, the composition may further comprise a chemiotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent anti-hormonal agent, and/or camdioprotectant Such molecules are suitably present in combination in amounts that are effecotive for the purpose intended.

'The active ingredients may also be entrapped in micorocapsules prepared, for example, by coacez-vatjon techniques orby interfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposornes, albumin microspheres, ricroemulsions, nano-particles and nanocapsules) or in maezuemnulsions, Such techniques arm disclosed in Rpnnton's Pharnaceutia S ince 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples of suained-release preparations include semli-permneable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, films, or inicrocapsules. Examples of sustained-release matrices include polyesters, bydrogels (for example, poly( 2 .hydroxyethy..methac.ylate), or poly(viriylalcohol)), polylactides Pat No.

3,773,919), copolymers of L-glutamic acid and T' ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LTUPRONDEPO'r® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(.)-3-hydroxybutyric acid.

The formulations to be used for In vivo administration mnust be sterile. This is readily accomplished by 0 filtration through sterile filtration membranes.

K. Diagnosis and amntwith t-TAT Antibodies TATBinin Olioeoides and TAT BiJ~nding ranic Molecles To determine TAT expression in the cancer, various diagnostic assays are available. In one embodiment, TAT polypeptide overexpression may be analyzed by inimunohistochemistry (1110. Parrafin embedded tissue 3 5 sections from a tumor biopsy may be subjected to the INC assay and accorded a TAT protein staining intensity criteria as follows: Soor 0 no staning is observed or membrane staing is observed in les than 10% of tumor cells.

0 Scoe 1+ a faint/barely perceptible membrane stainng is detected in mor than 10% of the tumor cells The cells are only stained in part of their membrane.

Sce Score 2+ a weak to moderate complete membrane staining is observed in more than 10% of the tumnor cells.

core 3+ a moderate to strong complete membrane staining is observed in more than 10% of the tunor Scells.

Those tumors with 0 or 1+ scores for TAT polypeptide expression may be characterized as not overexpressing TAT, whemias those tumors with 2+ or 3+ scores may be characterized as overexpressing

TAT.

Alternatively, or additionally, FISH assays such as the INFORM (sold by Ventana, Arizona) or PATHVISION® (Vysis, Illinois) may be carried out on formalin-fixed, paraffin-embedded tumor tissue to determine Sthe extent (if any) of TAT overexpression in the tumor.

TAT overexpression or amplification may be evaluated using an in vivo diagnostic assay, by Sadministering a molecule (such as an antibody, oligopeptide or organic molecule) which binds the molecule to be detected and is tagged with a detectable label a radioactive isotope or a fluorescent label) and externally I 5 scanning the patient for localization of the label.

As described above, the anti-TAT antibodies, oligopeptides and organic molecules of the invention have various non-therapeutic applications. The anti-TAT antibodies, oligopeptides and organic molecules of the present invention can be useful for diagnosis and staging of TAT polypeptide-expressing cancers in radioimaging). The antibodies, oligopeptides and organic molecules are also useful for purification or S lnmmunaoprcipitation of TAT polypeptide from cells, for detection and quantitation of TAT polypcptide in vitro, e.g, in an ELISA or a Western blot, to Idil and eliminate TAT-expressing cells fim a population of mixed cells as a step in the purification of other cells, Currently, depending on the stage of the cancer, cancer treatment involves one or a combination of the following therapies: surgery to remove the cancerous tissue, radiation therapy, and chemotherapy. Anti-TAT antibody, oligopeptide or organic molecule therapy may be especially desirable in elderly patients who do not tolerate the toxicity and side effects of chemotherapy well and in metastatic disease where radiation therapy has limited usefulness. The tumor targeting anti-TAT antibodies, oligopeptides and organic molecules of the invention are useful to alleviate TAT-expressing cancers upon initial diagnosis of the disease or during relapse. For therapeutic applications, the anti-TAT antibody, oligopeptide or organic molecule can be used alone, or in combination therapy with, hormones, antianglogens, or radiolabelled compounds, or with surgery, cryotherapy, and/or radiotherapy. Anti-TAT antibody, oligopeptide or organic molecule treatment can be administered in conjunction with other forms of conventional therapy, either consecutively with, pre- or postconventional therapy. Chemotherapeutic drugs such as TAXOTERE® (docetaxel), TAXOL® (palictaxel), estramustine and mitoxantrone are used in treating cancer, in particular, in good risk patients. In the present method of the invention for treating or alleviating cancer, the cancer patient can be administered anti-TAT antibody, ollgopeptide or organic molecule in conjuction with treatment with the one or more of the preceding chemotherapeutic agents. In particular, combination therapy with palictaxel and modified derivatives (see, e.g., EPP600517) is contempatd The anti-TAT antibody, OligOpepid Or organic Molecule will be administered with 00 ferupctic"fY effective dose of the chetnotherpeudo agent In anothier cmbodlinen the aniti.TAT anioY oligopeptide or organic molecule is administered in oqjuinotion with chemotherapy to enhance the activity and (71 efficoacy of the chemotherapeutic agent, paclitaxel. The Physicians' Desk Reference (PDR) discloses dosages ct Of these agents that have been used in treatment of various cancers. The dosing regimen and dosages of these aforementioned chemotherapeutic drugs that are therapeutically effective will depend on the particular cance IND being treated, the e"tent of the disease and other factors faiihar to the physician of sill in the art and can be determined by the physician.

In one particular embodiment, a conjugate comprising an anti-TAT antibody, oligopeptide or organic, molecule conjugated with a cytotoxic agent is administered to the patient. Preferably, the imnrunoconjugate bound Ni 10 to the TAT protein is internalized by the cell, resulting in increased therapeutic efficacy of the imnmutoconjugate 0 00 the nucleic acid in the cancer cell. Examples of such cytotoxio agents are described above and include maytansinoids, calicheamicins, rlbomzoleases and DNA endomucleases, The anti-TAT antibodies, oligopeptides, organic molecules or toxin conjugates thereof are administered to a human patient, in accord with known methods, such as intravenous admiinistration, as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes, Intravenous or subcutaneous admiinistration of the antibody, oligopeptide or organic molecule is preferred.

Other therapeutic regimens way be combined with the administration of the anti-TAT antibody, Oligopeptide or organic molecule. The combined 'administration inludes co-administration, using separate formiulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a timne period while both (or all) active agents simultaneously exert their biological activities.

Preferably such combined therapy results in a synergistic therapeutic effect It may also be desirable to combine administration of the anti-TAT antibody or antibodies, oligopeptides or organic molecules, with administration of an antibody directed against another tumor antigen associated with the particular cancer.

In another embodiment, the therapeutic treatment methods of the present invention involves the combined administration of an anti-TAT antibody (or antibodies), oligopeptides or organic molecules and one or more chemotherapeutic agents or growth inhibitory agents, including co-administration of cocktails of different 2 0 chemnotherapeutic agents. Chemotherapeutic agents include estramustine phosphate, prednimustine, cisplatin, S-fluorouracil, melphalan, cyclophosphanilde, hydroxyurea and hydroxy-ureatanes (such as paolitaxel and doxetaxel) and/or anthracycline antibiotics, Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the silled practitioner Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service Ed., M.C.

Perry, Williams Wilkins, Baltimore, MD (1992).

The antibody, oligopeptide or orgartic molecule may be combined with an anti-hormonal compound; e.g., an anti-estrogen compound such as tamoxifen; an anti-progesterono such as onapristone (see, EP 616 812); or an antdlmgen such as flut mid in dosages kown for such moleoules. Where the cancer to be treasted is 00 androgen independent cancer, the patient may previously have been subjected to anti-androgen therapy and, after the cancer becomes androgen indepndent, the anti-TAT antibody, oligopoptide or organic molecule (and optionally other agents as described herein) may be adinistered to the patient Sometimes, t may be beneficial to also co-administer a cardioprotectant (to prevent or reduce myocardial S dysfunction associated with the therapy) or one or more cytokines to the patient. In addition to the above therapeutic regimes, the patient may be subjected to surgical removal of cancer cells and/or radiation therapy, before, simultaneously with, or post antibody, oligopeptide or organic molecule therapy. Suitable dosages for any of the above co-administered agents are those presently used and may be lowered due to the combined action S(synergy) of the agent and anti-TAT antibody, oligopeptide or organic molecule.

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

Aside from administration of the antibody protein to the patient, the present application contemplates administration of the antibody by gene therapy. Such administration of nucleic acid encoding the antibody is encompassed by the expression "administering a therapeutically effective amount of an antibody". See, for example, W096/07321 published March 14, 1996 concerning the use of gene therapy to generate Intracellular antibodies.

There are two major approaches to getting the nucleic acid (optionally contained in a vector) into the patient's cells; in vive and ex vivoe. For in vive delivery the nucleic acid is injected directly into the patient, usually at the site where the antibody is required. For ex vivo treatment, the patient's cells are removed, the nucleic acid is introduced into these isolated cells and the modified cells are administered to the patient either directly or, for example, encapsulated within porous membranes which are implanted into the patient (see, U.S. Patent Nos.

4,892,538 and 5,283,187). There are a variety of techniques available for introducing nucleic aoids into viable cells.

T techniques vary depending upon whether th nuleo acid is transferred into cultured cells In vitr, or in vtvo O in the cells of the intended host Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjetion, cell fusion, DEAE-dtran, the calium phosphate Pnreipitation method, etc. A commonly used vector for ex vivo delivery of the gene is a retroviral vector.

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

The anti-TAT antibodies of the invention can be in the different forms encompassed by the definition o 10 of "antibody" herein. Thus, the antibodies include full length or intact antibody, antibody fragments, native C sequence antibody or amino acid variants, humanized, chimeric or fusion antibodies, immunoconjugates, and Sfunctional fragments thereof. In fusion antibodies an antibody sequence is fused to a heterologous polypeptide C' sequence. The antibodies can be modified in the Fo region to provide desired effector functions. As discussed in more detail in the sections herein, with the appropriate Fo regions, the naked antibody bound on the cell surface can induce cytotoxicity, via antibody-dependent cellular cytotoxicity (ADCC) or by recruiting complement in complement dependent cytotoxicity, or some other mechanism. Alternatively, where it is desrable to eliminate or reduce effector function, so as to minimize side effects or therapeutic complications, certain other PF regions may be used.

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

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

The present anti-TAT antibodies, oligopeptides and organic molecules are useful for treating a TATexpressing cancer or alleviating one or more symptoms of the cancer in a mammal. Such a cancer includes prostate cancer, cancer of the urinary tract, lung cancer, breast cancer, colon cancer and ovarian cancer, more specifically, prostate adenocarcinoma, renal cell carcinomas, colorectal adenocarcinomas, lung adenocarcinomas, lung squamous cell carcinomas, and pleural mesothelioma. The cancers encompass metastatic cancers of any of the preceding. The antibody, oligopeptide or organic molecule is able to bind to at least a portion of the cancer cells that express TAT polypeptide in the mammal. In a preferred embodiment, the antibody, oligopeptide or organic molecule is effective to destroy or kill TAT-expressing tumor cells or inhibit the growth of such tumor cells, in vitro or In vivo, upon binding to TAT polypeptide on the cell. Such an antibody includes a naked anti-TAT antibody (not conjugated to any agent). Naked antibodies that have cytotoxic or cell growth inhibition properties can be further harnessed with a cytotoxic agent to render them even more potent in tumor cell destruction. Cytotoxic 3 S properties can be conferred to an anti-TAT antibody by, conjugating the antibody with a cytotoxic agent, to form an immunoconjugate as described herein. The cytotoxic agent or a growth inhibitory agent is preferably a small molecule. Toxins such as calicheamicln or a maytansinoid and analogs or derivatives thereof, are preferable.

The invention provides a composition comprising an anti-TAT antibody, oligopeptide or organic 00 molecule of the invention, and a carrier. For the purposes of treating cancer, compositions can be administered Sto the patient in need of such treatment, wherein the composition can comprise one or more anti-TAT antibodies present as an immunoconjugate or as the naked antibody. In a further embodiment, the compositions can comprise C these antibodies, oligopeptides or organic molecules in combination with other therapeutic agents such as cytotoxic or growth inhibitory agents, including chemotherapeutic agents. The invention also provides Sformulations comprising an anti-TAT antibody, oligopeptide or organic molecule of the invention, and a canier.

In one embodiment, the formulation is a therapeutic formulation comprising a pharmaceutically acceptable carrier.

Another aspect of the invention is isolated nucleic acids encoding the anti-TAT antibodies. Nucleic Sacids encoding both the H and L chains and especially the hypervariable region residues, chains which encode the native sequence antibody as well as variants, modifications and humanized versions of the antibody, are C00 encompassed.

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

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

L. Articles of Manufacture and Kits Another embodiment of the invention is an article of manufacture containing materials useful for the treatment of anti-TAT expressing cancer. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc.

The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for treating the cancer condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an anti-TAT antibody, oligopeptide or organic molecule of the invention. The label or package insert indicates that the composition is used for treating cancer. The label or package insert will further comprise instructions for administering the antibody, oligopeptide or organic molecule composition to the cancer patient. Additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatio water for injection (BWFI), phosphate-buffnid saline, Ringes solution and dextose solution. It may Artherinclude other materials desirable from a commercial and user standpoint, including other buffers, diluents, filteh, needles, and syringes.

O Kits are also provided that are useful for various purposes, for TAT-oxpressing cell killing assays, for purification or immunopreopitation of TAT polypeptide from cells. For isolation and purification of TAT polypeptide, the kit can contain an anti-TAT antibody, oligopeptide or organic molecule coupled to beads sepharose beads). Kits can be provided which contain the antibodies, oligopeptides or organic molecules fo Sdetection and quantitation of TAT polypeptide in vitro, in an ELISA or a Western blot. As with the article of manufacture, the kit comprises a container and a label or package insert on or associated with the container.

The container holds a composition comprising at least one anti-TAT antibody, oligopeptide or organic molecule of the invention. Additional containers may be included that contain, diluents and buffers, control antibodies. The label or package insert may provide a description of the composition as well as instructions for the intended in vitro or diagnostic use.

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

The full-length native sequence TAT gene, or portions thereof, may be used as hybridization probes for a cDNA library to isolate the full-length TAT cDNA or to isolate still other cDNAs (for instance, those encoding naturally-occung variants of TAT or TAT from other species) which have a desired sequence identity to the native TAT sequence disclosed herein. Optionally, the length of the probes will be about 20 to about 50 bases.

The hybridization probes may be derived from at least partially novel regions of the full length native nucleotide sequence wherin those regions may be determined without undue experimentation or from genomic sequences including promoters, enhancer elements and introns of native sequence TAT. By way of example, a screening method will comprise isolating the coding region of the TAT gene using the known DNA sequence to synthesize a selected probe of about 40 bases. Hybridization probes may be labeled by a variety of labels, including radionucleotides such as 32 P or 3aS, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems. Labeled probes having a sequence complementary to that of the TAT gene of the present invention can be used to screen libraries of human cDNA, genomic DNA or mRNA to determine which members of such libraries the probe hybridizes to. Hybridization techniques are described in further detail in the Examples below. Any EST sequences disclosed in the present application may similarly be employed as probes, using the methods disclosed herein.

Other useful fragments of the TAT-encoding nucleic acids include antisense or sense oligonucleotides comprising a singe-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target TAT mRNA 3 5 (sense) or TAT DNA (antisense) sequences. Antisense or sense oligonucleotides, according to the present invention, comprise a fragment of the coding region of TAT DNA. Such a fragment generally comprises at least about 14 nucleotides, preferably from about 14 to 30 nucleotides. The ability to derive an antisense or a sense 00oligomcleode, based upon a DNA squence enoding a given protein is desribd in, fr example, Stela and 00 ~Cohen (Cncer Rc-A. 48:2659, 1988) and van der Krol et al. (Bo M i nfnIM 6,958, 1988).

]Binding of antisense or sense oligonuoleotde to target nucleic acid sequences results in the formation of duplexes that block transcription or translation of the target sequence by one of severa means, including enhanced degradation of the duplexes, Premature termination of transcription or translation, or by other means.

IN Such methods are encompassed by the present invention. The antisense otigonuclootides thus may be used to block expression of TAT proteins, wherein those TAT proteins may play a tole in the induction of cancer in maruals. Antisense or sense oligotcleotides further comprise oligonucleotides having modified sugar- 17.-phosphodiester backbones (or other sugar linkages, such as those described in WO 91/06629) and wherein suoch sugar linkages are resistant to endogenous nucleases. Such oligonucleotid-es with resistant sugar linkages are S 10 stable In vivo capable of resisting enzymatic degradation) but retain sequence specificity to be able to bind 00 to taget nucleotide sequences.

Preferred intragenio sites for antisense binding include the region Incorporating the translation ri ~initiationtstart codon(5'AUQ/ 5'-ATO) or termination/stop codon (5'-UAA, 5'-UAG and 5-UGA/ 5'-TAA and 5'-TOA) of the open reading frame (ORF) of the gene. Thiese regions refer to a portion of the niRNA or gene that encompasses fr-om about 25 to about 50 contiguous nucleotides in either direction 5' or from a translation initiation or termination codon. Other preferred regions for antisense binding include: introns; exoris; intron-exon junctions; the open reading frame (ORE) or "coding region," which is the region between the translation initiation codon and the translation termination codon; the 5' cap of an mRNA which comprises an N7-imthylated guanosine residue joined to the 5'-most residue of the niRNA via a 5-5' triphosphate linkage anid includes 5' cap structure itself as well as the first 50 nucleotides adjacent to the cap; the 5' untranslated region (SUM)n, the portion of an mRNA in the 5' direction from the translation initiation oodon, and thus including nucleotides between the 5' ap site and the translation Initiation codon of an mRNA or corresponding nucioddes on the gene; and the 3'untranslated region (3'UM1), the portion of an niRNA in the 3'direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3' end of an rnMRA or corresponding nucleotides on the gene.

Specific e~eamples of preferred antisense compounds useful for inhibiting expression of TAT proteins include oligonucjeotides containing modified backbones ortnon-natural intemucleoside linkageg. Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and these that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom ini their internucleoside backbone can also be considered to be oligonuvleosides, Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral pbosphorothioates, phosphorodithioates, pho sphotri esters, aminoalkylphosplioti-ie 5 ter methyl and other alkyl phosphonates including 3-alkylene phosphoriates, phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amlno phosphoramidate and aminoalkylphosphoramidates, thionophosphoramnidates, thionoalkylphosphonates, thionoalkylphosphotiesters, selenophosphates and borano-phosphates having normal linkages, linked analogs of these, and those having inverted polarity wherein one or mco-e internucleotide linkages is a 3' to 10Q7 to 5' or 2' to 2' linkage. Preferred oligonuolectjdes having inverted polarity comprise a ~glve 3' to 3' linkage at the 00 3'-moft intemucleotide linkage ie. a single inv~erted nucleoside residue which may be abasic (the nuocobs as missing or has a hydroxyl group in place thereof. Vaious salts, mixed salts and free acid forms arm also included.

Representatiy 0 United States patents that teach the preparation of phosPhonus-containing linkages include, but Ct amnotlimited to, U.S. Pat Nos.: 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897;,5,264,423; 5,276,019g; 5,278,302; 5,286,717; 5,321,13 1; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;,5,476,925; 5,519,126; 5,536,821; IND 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899, 5,721,218; 5,672,697 and 5,625,050, each of which is herein incorporated by reference.

I> Preferred modified olig~onucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl orcycloalkyl intemuoleoside linkages, mixed hieteroatom and alkyl 1N1~ 0 or cycloalkyl inteniucleoside linkages, or one or more short chain heteroatornic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); 00 siloxane backbones; sulfide, snioxide and sulfone backbones; formacetyl and thioformactyl backbones; methylene fbnnacetyi and thioformacetyl backbones; riboacetyl backbones; alkene, containing backbones; sulfaiate backbones; methyleneirnJno and methylenehydrazino backbones; sulfonate, and sulfonamide backbones; ainide backbones; and others having mixed N, 0, S and CH.sub.2 component parts. Representative United States patents that teach the preparation of such oligonucleosides include, but arm not limited to,. U.S. Pat. Nos.: 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225;5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, cachofwh-ioh is herein incorporated by reference.

In othier preferred antisense, ollgonucleotides, both the sugar and the internuoleoside linkage, the backbone, of the nucleotide units ame replaced with novel groups. The base uisare maintained for hybridizationi with an appropriate nucleic acid target compound. One such oligoaneric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotlde is replaced with an arnide containing backbone, in particular an aninoethyiglycine backbone. The nucleobases are retained and are bound directly or Indirectly to aza nitrogen atoms of' the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos.: 5,539,082; 5,714,33 1; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254,1497-1500.

Preferred antisense oligonucleotides incorporate phosphorothioate backbones and/or heteroatom backbones, and in particular -CHI-NHi.0CH 2

-CH

2

-N(CH

3 )0.QCH.. [known as a methylene (niethylimidno) orMHl1 backbone],

-CH

2

-O-N(CH

3 )-CH2-

-CH

2 .N(CH3)..N(CH)CH.. and -O-N(CH 3

)-CH,-CH

2 [wherein the native phosphodiester backbone is represented as -0-P-0-CH 2 .J described in the above referenced U.S. Pat. No. 5,4 89,677, 2 S and the amnide backbones of the above referenced U.S, Pat. No. 5,602,240. Also preferred are antisense oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

00Modiriod oligoauclotides may also oontan one or more gubtttd suga moieties. PMrer oligOIRu0leotides comprise one of the flllowing at the 2'position: OR~ F; O-alkyl, S-alkyl orN-alkyl; 0-alkenyl,

S-

alkeyuyl, orN-akeyl; O-alkynyl, S-allcynyl orN-alkynyl; or O-alkyl-O-alkyl, whervin the alkyl, alkenyl and al kynyl may be gubstituted or unsubstituted C, to alkyl or Cq to C1. alkenyl and alkynyl. Particularly preferred are ORCHI)OLmCH3,

O(CH).OCH

3

O(CH)NR

2 O(CHJC11 3 O(CR).0NH., and O(CH2).ON[(CH 2 ).Cl 2 where n~ and m are from 1 to about 10. Other preferred antisense oligonucleotides comprise one of the fol lowing at the 2' position: C, to C, 0 loweralkyl, substituted loweralkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl orO0-aralkyl,

SIT,

SCH

3 OCN, Cl, Br, CN, CF3, 0CF 3

SOCH

3

SO

2

CH

3 0N0 2

NO

2

N

3

NH

2 heterocycloalkyl, heterocycloalkaryl, 1> aminoalkylamio, polyalkylamino, substituted silyl, an R~NA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonuecotide, or a group for improving the S 10 pharmacodynamic properties of an oligonucleotide, and oilher substituents having similar properties, A preferred c~~K1 modification includes 2'-methoxyethoxy (2'-O-CHCH 1

OCH

3 also known as 2 -O-(2-niethoxyethyl) or 2'-14E) 00 (Martin et al., Rfely. Chim. Acta, 1995, 78, 48 6-504) an alkoxyalkoxy group. A firther preferred modification includes 2 -dlnlethylamlinooxyethoxy, Ie., a O(Clf) 2 ON(CH.), group, also known as 2'-DMAOB, as described in examples hereinbelow, and 2 '-dimet11ylainoethoxyethoxy (also known in the attas 21 -O-dlmethylaminootlioxyetiiyl or 2'-DMABOE), 2 A further prefi~rd modification includes Locked Nucleic. Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or 4' carb~on atom of the sugar ring thereby forming a bicyclic sugar moiety. The linkage is preferubly a methelyne.

(-CH

2 group bridging the 2' oxygen atom and the 4' carbon atom wherein n is I or 2. LNAs and preparation thereof are described in WO 98139352 and WO 99/14226.

Other preferred modifications include 2'-methoxy 21 -amlnopropoxy (2'-OCR 2

,CH

2

C

2 21 .allyI, (2'-CHi=CH, 2'-O-allyl(2'.O-cClg i~aand 2'-fiuoo('.F). The 2'-modiflcation may be in the arabino, (up) position ornibo (down) position. A preferred 2'-arablno mnodification iv Similar modifications may also be miade at otherpositions; on the oligonucleotide, particularly the 3'position of the sugar on the 3'terminal nucleotide or in V-5 linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligornucleotides may also have sugar inimetics such as cyclobutyl moieties in place of the pentofunosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos.: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, each of which is herein incorporated by reference in its entirety.

Oligonucleotides may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine and guanine and the pyrimidine base thymine. cytosine and uracil Modified nuoleobasos include other synthetic and natural nucleobases such as S-mpthylcytosine 5-hydroxymethyl cytosine, xanthine, bypoxanthine, 2 -aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other ailkyl derivatives of adenine and guanine, 2-thiouracit, 2-thiothymine and 2 -Whocytoslne, 5-halouracil and cytosine, (-Ct=C-CH 3 or -CHI-C=CH) uracil and cytosine and other alkynyl derivatives ofpyn~midine bases, 6 -azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4 -thlouraoil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and 00h0r 84**fstutlcj adeilne and Pualme, 5-halo pamficnuy 5-bromo, 5-trifuoromefhyl ad ,,her 00 S-substitute uracils ad cytosines, 7 -methylguanine and 7-zuethladenjue, 2-F-adenine, 2-amino-adnine, B-azagunine- and 8 -azaadenine, 7 -deazaguanine and 7-deazadenine and 3-deazaguanne and 3 -deazaadenine.

Further modified nucleobases include tricyclic *pyrimidines such as phenox azine ct '~~Ytidle(lHpyrimido[5,4b1[I,4bezoxazin 2 3 H)a) phenothiazine cytidine (11-pyri~do5,4b)[,4]bnzohiain-(3H-on), -cdamps such as a substituted phoenoxazine cytidine (e.g.

9 2 -amino ethoxy)-H-pyrimi do[ 5 4

C,

4 ]benzoxazi n-2(3 H)one), carbazole cytidine 2 Fl-pyrimido[4,s..b]indol-2.ne), pyridoindole cytidine Q{.pyido[3',2':4,5Jp fflo[ 2 ,3..dpyrinildin-2-one). Modified ziucleobases may also include those in which the purine or pyrin-ldine base is replaced with other heterocycles, for example 7 -deaza-aderthne, 7 -deazaguaziosine, 2-amidnopyrldine and 2-pyridone. Further nucleobases include o 10 those disclosed in U.S. Pat No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And N0 Engineering, pages 858-859, IKroschwitz, J. ed. John Wiley Sons, 1990, and those disclosed by Englisch et al., Angewandte Chen-ie, International Edition, 1991, 30, 613. Certain of these nueceobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include pyrirnidines, 6 -azapyrimnldines and N-2, N-6 and 0-6 substituted purines, Including 2 -amiinopropyladenine, -propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nuoleic acid duplex stability by O.6-1.2.degree. C. (Sanghvi et al, Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are preferred base substitutions, even mome particularly when combined with 2 '-O-methoxyethyl sugar modifications. Represntative United States patents that teach the preparation of modified nuoceobase include, but are not limited to: U.S. Pat No. 3,687,808, as well as U.S. Pat Nos.: 4,845,205; 5,130,302; 5,134,W66 5,175,273; 5,367,066;,5,432,272; 5,457,187; 5,459,255; 5,484,98; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,09 1; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; 5,681,941 and 5,750,692, each of which is herein incorporated by reference.

Another modification of antisense oligonucleotides chemiically linking to the oligonuoleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonuoleotide.

The compounds of the invention can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intervalators, reporter molecules, polyarnines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamidc properties of oligomers, and groups that enhance the phannacokinetic properties of oligomers, Typical conjugates groups include cholesterols, lipids, cation lipids, phospholipids, cationic phospholipids, biotin, phenazine, folate, phenanthuidine, anthraquinone, acridine, fluoresceins, rhodamnines, couniarins, and dyes. Groups that enhance the pharnacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with RNA, Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excret ion. Conjugate moieties include but are not limited to lipid moieties such 3S as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994,4, 1053-1060), a thioether, hexyl-S-trltylthlol (Manoharan et al., Ann. N.Y Acad. Sd., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Obeftae t at., Nuci. Acids Me., 1992, 20, 533-53 anaihatie ch ain eg, dodocadlol or undecyl residues 00 ~(SaisonBeam t a, NMO~L, 9 9 ll 1 1011-1118; Kabanovetal., FE3BS Lemt, 1990,259,327-330; Svinarohuk, et at., Biochlmie, 1993, 75, 49-54), a phosphploid, di-hexadeoyl.rao.gycro or tiethyl-ammoniun, m)3--hshnt(ManoharanCt at., TcmttedronLen, 1995,36,3651-3654; Shea et al., Nuci. Acids Res., 1990, 18, 3777-3783), a polyaine or a polyethylene glycol chain (Manoharen et al., Nucleosides Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharajj et al., Tetrahedrn Lett., IND 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Blochim. Biophys. Acts, 1995, 1264, 229-237),. ra octadecylamie or hexylaniino-ca tonyl-oxycliol-sero moiety. Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofep., carprofen, dansylsarcosine, 2 ,3,5-trilodobeazoic acid, fiufonarnic acid, NK 10 fohic acid, a benzothiadlazide, chlorothiaide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Qligonucleoide-drug conjugates and their preparation are 00 described in U.S. patent application Scr. No. 091334,130 (filed Jun. .15, 1999) and United States patents Nos.: 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;,5,552,538; 5,578,717,5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 1 5 4,762,779; 4,789,737; 4,824,941; 4,835,263;4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;,5,272,25; 5,292,873; 5,317,098; 5,371,241,5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5M55,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,68 8,94 1, each of which is herein incorporated by reference,.

It is not necessary for all positions in a given compound to be uniformly modified, and In fact more than one of the aforementioned modifications may be Incorporated in a single compound or even at a single nucleoside within an oligonuecotide. The present invention also includes antinense compounds which ame chimeric compounds. "Chimeric" antisease compounds or "chimeras," in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemidcally distinct regions, each made up of at least one monomer unit a nucleotide in the case of an oligonucleotide compound. These oligonuoleotjdes typically contain at least one region wherein the oligonucleotido is modified so as to confer upon the oligonuoleotide increased resistance to nuclease degradation, increased cellular uptake., and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RWase Hi, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotudes are used, compared to pliosphorotioate deoxyoligonucleotides hybridizing to the same target region. Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotidermimetics as described above.

Preferred chimeric antisense ollgenucleod des incorporate at least one 2 modified sugar (preferably 2'-O-(CH 2 2 0 Cli) at the 3'termninal to confer nuclease resistance and a region with at least 4 contiguous 2'-H sugars to confer RNase H activity. Such compounds have also been referred to in the art as hybrids or gapmers. Preferred gapnais 00havq 4reion of 2' dfld suga (P ftfmbly2!0 (CM .0-C atth e3-tem itta and at the Y' tem inal s ePa zmted 00 by at least Oft teglon having at least 4 conitiguous 2!-H gugars ad prembly incorporte phosphorothioatc C)backbone linkages. Representative United States Patents that teach thepeartoofschyidtutra noldobut are not limited to, U.S. Pat Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; Ct 5,491,13.3; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference in its entirety, The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in theart may additionally or alternatively be employed. It is well known to use simnilar techniiques to prepare oligonucleotides such as the phosphorothioates and alkcylated derivatives. The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or 00 nmdxtures of compounds, as for example, lposomes, receptor targeted molecules, oral, rectal, topical or other c-i formulations, for assisting in uptake, distribution and/or absorption. Represntative United States patents that teach the preparation of such uptake, distribution and/or absorption assisting formulations include, but are not 1 5 limited to, U.S. Pat Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556;,5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.

Other examples of sense or antisense oligonucleotides include those oligonucoeotides which are vovalently linked to organic moieties, such as those described In WO 90/10048, and other moieties that increases affinity of the oligonucleotide for a target nucleic acid sequence, suchas poly{L..ysine). Further still, intercalating agents, such as ellipticine, and alkylating agents or metal complexes may be attached to sense or antisense ofigonucleotides to modify binding specificities of the antisense or sense oligonucleotide for the target nucleotide Sequence.

Antisense or sense olgonuclectides may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, CaPO, 4 -nedjated DNA transfection, electroporation, or by using gene transfer vectors such as Epstein-Barr virus. In a preferred procedure, an antisense or sense oligonucleotide is inserted into a suitable retroviral vector. A cell containing the target nucleic acid sequence is contacted with the recombinant retroviral vector, either in vivo or ex vivo. Suitable retroviral vectors include, but are not limited to, those derived from the niurine retrovirus M-MuLV, N2 (a retrovirus derived from M.MuLV), or the double copy vectors designated DCI'5A, DCTSB and DCT5C (see WO 90/13641).

Sense or antisense oligonucleotjdcs also may be Inh-oduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91104753. Suitable ligand binding molecules include, but are not limilted to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with (he ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense, oligonucleotide or its conjugated version Into the cell.

Alteratively, a sense or an anisonse oligonucleotide may be introduced into a cell ontaining the target O nucleic acid sequence by formation of an ollgonuclotide-lipid complex, as described in WO 90/10448. The sense or antisee oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase.

C Antisense or sense RNA or DNA molecules are generally at least about 5 nucleotides in length, alternatively atleast 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, 70, 780, 790, 800,810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940,950, 960,970,980,990, or 1000 nucleotides in length, wherein in this context the term "about" means S 0 the referenced nucleotide sequence length plus or minus 10% of that referenced length.

The probes may also be employed in PCR techniques to generate a pool of sequences for identification 00 of closely related TAT coding sequences.

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

When the coding sequences for TAT encode a protein which binds to another protein (example, where the TAT is a receptor), the TAT ian be used in assays to identify the other proteins or molecules involved in the binding interaction. By such methods, inhibitors of the receptorligand binding interaction can be identified.

Proteins involved in such binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction. Also, the receptor TAT can be used to isolate correlative ligand(s) Screening assays can be designed to find lead compounds that mimio the biological activity of a native TAT or a receptor for TAT. Such screening assays will include assays amenable to high-throughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates. Small molecules contemplated include synthetic organic or inorganic compounds. The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell based assays, which are well characterized in the art.

Nucleic acids which encode TAT or its modified forms can also be used to generate either transgenic animals or "knock out" animals which, in turn, are useful in the development and screening of therapeutically useful reagents. A transgenic animal a mouse or rat) is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, an embryonic stage. A transgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops. In one embodiment, cDNA encoding TAT can be used to clone genomic DNA encoding TAT in accordance with 3 sestablished techniques and the genomic sequences used to generate transgenic animals that contain cells which express DNA encoding TAT. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,0099 Typloally, particular cells would be targeted for TAT tnrnagene incoporation with issue-specio enhancer.

STrangenio animals that include a copy of a htangene encoding TAT introduced into the germ line of the animal 0 at an embryonic stage can be used to examine the effect of increased expression of DNA encoding TAT, Suoh animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression. In accordance with this facet of the invention, an animal is treated with the reagent and a reduced incidence of the pathological condition, compared to untreated animals bearing Sthe transgene, would indicate a potential therapeutic intervention for the pathological condition.

Alternatively, non-human homologues of TAT can be used to construct a TAT "knock out" animal which has a defective or altered gene encoding TAT as a result of homologous recombination between the endogenous gene encoding TAT and altered genomic DNA encoding TAT introduced into an embryonic stem cell of the animal. For example,cDNA encoding TAT can be used to clone genomio DNA encoding TAT in accordance with 00 established techniques. A portion of the genomrnic DNA encoding TAT can be deleted or replaced with another gene, such as a gone encoding a selectable marker which can be used to monitor integration. Typically, several C kilobases of unaltered flanking DNA (both at the 5' and 3' ends) ar included in the vector [see Thomas and Capecchi, Cll, 51:503 (1987) for a description of homologous recombination vectors]. The vector is introduced into an embryonic stem cell line by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected [see Li et al., ell, 69:915 (1992)]. The selected cells are then injected into a blastocyst of an animal a mouse or rat) to form aggregation chimeras [see Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, B. 3. Robertson, ed.

(IRL, Oxford, 1987), pp. 113-152. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and theembryo brought to term to create a "knock out" animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all oclls of the animal contain the homologously recombined DNA. Knockout animals can be characterized for instance, for their ability to defend against certain pathological conditions and for their development of pathological conditions due to absence of the TAT polypeptide.

Nucleic acid encoding the TAT polypeptides may also be used in gene therapy. In gene therapy applications, genes are introduced into cells in order to achieve in vive synthesis of a therapeutically effective genetic product, for example for replacement of a defective gene. "Gene therapy" includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA. Antisense :0 RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes in vivo. It has already been shown that short antisense oligonucleotides can be imported into cells where they act as inhibitors, despite their low intracellular concentrations caused by their restricted uptake by the cell membrane. (Zamecnik et roc t Acad. Sci. USA 83:4143-4146 [1986]). The oligonucleotides can be modified to enhance their uptake, e.g. by substituting their negatively charged phosphodiester groups by uncharged groups.

S There are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host. Teclniques suitable for the transfer of nucleic acid into mammalian cells (n vitro include the use ofliposomes, eleoctroporation, mlorinjection, cell fsion, DEAB-dextran, the calolum phosphatopr'eipitation Smethod, etc. The cumently prfered in vivo gene transfer techniques include transfotion with vial (typicay etroviral) vectors and viral coat protein-liposome mediated tmasfeotion Dzau ct al., nds iBtohnole 11, 205-210 [1993]). In some situations t is desirable to provide the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a reoeptor on the target cell, etc. Where liposomes are employed, proteins which bind to a cell surface membrane Sprotein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g. capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo intemalization in cycling, proteins that target intracellular localization and enhance intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, by Wu et al., Biol. Chem 262, 4429-4432 (1987); and Wagner et al., Pr at. Acd. Sci. USA 87, 3410-3414 (1990). Forreview of gene marking and gene therapy protocols see Anderson "I et al., Scien 256, 808-813 (1992).

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

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

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

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

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

In binding assays, the interaction is binding and the complex formed can be isolated or detected in the reaction mixture. In a particular embodiment, the TAT polypeptide encoded by the gene identified herein or the 3 5 drug candidate is immobilized on a solid phase, on a microtiter plate, by covalent or non-covalent attachments.

Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the TAT polypeptide and drying. Alternatively, an immobilized antibody, a monoclonal antibody, specific for the TAT polypoptide to be Immobilized can be used to anchor it to a solid surface. The assay is performd by adding the O non-immobilized component whioh may be labeled by a detectable label, to the immobilized component the Scoated surface containing the anchored component When the reaction is complete, the non-reated components are removed, by washing, and complexes anchored on the solid surface are detected. When the originally non-immobilized component carries a detectable label, the detection of label immobilized on the surface indicates that complexing occurred. Where the originally non-immobilized component does not carry a label, complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex.

If the candidate compound interacts with but does not bind to a particular TAT polypeptide encoded by a gene identified herein, its interaction with that polypeptide can be assayed by methods well known for detecting protein-protein interactions. Such assays include traditional approaches, such as, cross-linking, coimmunoprecipitation, and co-purification through gradients or chromatographic columns. In addition, protein- 0 protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers (Fieldsand Song,Nature London .340:245-246(1989);Chienetal.,roc, Natl.AcadSci, USA 88:9578-9582(1991)) as disclosed by Chevray and Nathans, Proc. Natl, Acad. Sci, USA 89: 5789-5793 (1991). Many transcriptional activators, such as yeast OAL4, consist of two physically discrete modular domains, one acting as the DNAbinding domain, the other one functioning as the transcription-activation domain. The yeast expression system described in the foregoing publications (generally referred to as the "two-hybrid system") takes advantage of this property, and employs two hybrid proteins, one in which the target protein is fused to the DNA-binding domain of GALA, and another, in which candidate activating proteins are fused to the activation domain. The expression of a GALI-lacZ reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for p-galactosidase. A complete kit (MATCHMAKERn) for identifying protein-protein interactions between two specific proteins using the two-hybrid technique is commercially available fiom Clontech.

This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions.

Compounds that interfere with the interaction of a gene encoding a TAT polypeptide identified herein and other intra- or extracellular components can be tested as follows: usually a reaction mixture is prepared containing the product of the gene and the intra- or extracellular component under conditions and for a time allowing for the interaction and binding of the two products. To test the ability of a candidate compound to inhibit binding, the reaction is run in the absence and in the presence of the test compound. In addition, a placebo may 3 0 be added to a third reaction mixture, to serve as positive control. The binding (complex formation) between the test compound and the intra- or extracellular component present in the mixture is monitored as described hereinabove. The formation of a complex in the control reaction(s) but not in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of the test compound and its reaction partner.

3 5 To assay for antagonists, the TAT polypeptide may be added to a cell along with the compound to be screened for a particular activity and the ability of the compound to inhibit the activity of interest in the presence of the TAT polypeptide indicates that the compound is an antagonist to the TAT polypeptide. Alternatively, antagonists may be detected by combining the TAT polypeptide and a potential antagonist with membrane-bound 0 TAT polypeptide receptors or recombinant receptors under apropriate conditions for a competitive inhibitio assay. The TAT polypeptide can be labeled, such as by radioactivity, such that the number of TAT polypeptide molecules bound to the reeptor can be used to determine the effectiveness of the potential antagonist. The gen encoding the receptor can be identified by numerous methods known to those of sll in the art, for example, ligand panning and FACS sorting. Coligan et al., rent Protocols in mun. Chapter 5 (1991). Preferably, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the TAT polypeptide and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that ar not responsive to the TAT polypeptide. Transfeoted cells that are grown on glass slides are exposed to labeled TAT polypeptide. The TAT polypeptide can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase. Following fixation and incubation, the slides are subjected to autoradiographic analysis. Positive pools are identified and sub-pools are prepared and retransfected using an interactive sub-pooling and re-screening process, eventually yielding a single clone that encodes the putative receptor.

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

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

More specific examples of potential antagonists include an oligonucleotide that binds to the fusions of immunoglobulin with TAT polypeptide, and, in particular, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as well as human antibodies and antibody fragments.

Altematively, a potential antagonist may be a closely related protein, for example, a mutated form of the TAT polypeptide that recognizes the receptor but imparts no effect, thereby competitively inhibiting the action of the TAT polypeptide, Another potential TAT polypeptide antagonist is an antisense RNA or DNA construct prepared using antisense technology, where, an antisense RNA or DNA molecule acts to block directly the translation of mRNA by hybridizing to targeted mnRNA and preventing protein translation. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which encodes the mature TAT polypeptides herein, is used to design an antisense

RNA

oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix see Lee et al., Nucl, Acids Res., 6:3073 S(1979); Cooney et al., Scienee 241: 456 (1988); Dervan et al., So e 251:1360 (1991)), thereby preventing transcrption and the production of the TAT polypeptide, The antisense RNA oligonuoleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the TAT polypeptide (antisense Okano, Neurocem., 56:560 (1991); Oiodo nuo ideas enseInbtor fen sin (CRC Press: Boa Raton, FL, 1988). The oligonucleotides described above can also be delivered to cells suoh that the antissene RNA or DNA may be expressed in vivo to inhibit production of the TAT polypeptide. When antisense DNA is used, oligodeoxyribonucleotides derived from the translation-initiation site, between about -10 and +10 positions of the target gene nuoleotide sequence, are preferred.

Potential antagonists include small molecules that bind to the active site, the receptor binding site, or growth factor or other relevant binding site of te 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 0 molecules, preferably soluble peptides, and synthetic non-peptidyl organic or inorganic compounds.

Ribozymes are enzymatic RNA molecules capable ofcatalyzing the specific cleavage ofRNA. Ribozyrnes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage.

Specific ribozyme cleavage sites within a potential RNA target can be identified by known teohniques. For further details see, Rossi, Current Bioloy, 4:469-471 (1994), and PCT publication No. WO 97/33551 (published September 18, 1997).

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

These small molecules can be identified by any one or more of the screening assays discussed hereinabove and/or by any other screening techniques well known for those skilled in the art Isolated TAT polypeptide-encoding nucleic acid can be used herein for recombinantly producing TAT polypeptide using techniques well known in the art and as described herein. In turn, the produced TAT polypeptides can be employed for generating anti-TAT antibodies using techniques well known in the art and as described herein.

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

If the TAT polypeptide is intracellular and whole antibodies are used as inhibitors, internalizing antibodies are preferred. However, lipofections or liposomes can also be used to deliver the antibody, or an antibody fragment, into cells. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable- 3 5 region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. See, Marasco et al, Proc, Natl. Acad. Sci. USA, 0: 7889-7893 (1993).

The formulation herein may also contain more than one active compound as necesary for the partioUja 00indication being treated, Preferably those with comsplemsentary activities that do not adversely affect each Other.

0 Alternatively, or in addition, the composition may comprise an agent that enhanoes Its function, such as, for c-I example, a ytotoxic agent, oytokine, chenotherapufic agent, or growth-inhbitory agent. Such molecules are ct suitably present in combination in amounts that ame effective for the purpose intended.

The following examples are offered for illustrative purposes only, and are not intended to limit the scope IND of the present invention In any way.

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

~iJ c-I Conmmercially available reagents referred to in the examples were used according to manufacturee~s 00 instructions unless otherwise indicated. The source of those cells identified in the following examples, and throughout the specification, by ATCC accession numbers is the American Type Culture Collection, Manassas,

VA.

EXAMLE 1: Tissue mxrsinPoiiaUiR(vexrss Dj A proprietary database containing gene expression information (GeneExpress, Gene Logic Inc., Gaithersburg, MD) was analyzed in an attempt to identify polypeptides (and their encoding nucleic acids) whose expresion is significantly upregulated in a particular tumor tissue(s) of interest as compared to other tumor~s) and/or normal tissues. Specifically, analysis of the GeneExpressVg database was conducted using either software available through Gene Logic; Inc., Gaithersburg, MDW, for use with the (3eneExpressV& database or with proprietary software written and developed at Genentech, Inc. for use with the GeneExpressOg database. The rating of positive hits in the analysis is based upon several criteria including, for example, tissue specificity, tumor specificity and expression level in normal essential and/or normal proliferating tissues. Ile following is a list of molecules whose tissue expression profile as determined firom an analysis of the GeneExpress® database evidences high tissue expression and significant upregulation of expression in a specific tumor or tumors as compared to other tumnor(s) and/or normal tissues and optionally relatively low expression in normal essential and/or normal proliferating tissues, As such, the molecules listed below are excellent polypeptide. targets for the diagnosis and therapy of cancer in mammnals.

Molecoule upreaulation of exresion in: as ompared to: DNA96792 (TAT239) colon tumor normal colon tissue DNA96792 (TAT-239) rectum tumor normal rectumn tissue DNA96792 (TAT739) pancreas tumor normal pancreas tissue DNA96792 (TAr239) lung tumor normal lung tissue DNA96792 (TAT-239) stomach tumor normal stomach tissue DNA96792 (TA'1239) esophagus tumor normal esophagus tissue DNA96792 (TAT239) breast tumor normal breast tissue DNA96792 (TAT239) uterus tumor normal uterus tissue DNA225793 (TAT-223) ovarian tumor normal ovarian tissue DNA22S793 (TAT223) kidney tumor normal kidney tissue 00 00 AMQLd DITA227611 (TATI75) DNA227611 (TAT175) DNA227611 (TATlIS) DNA261021 (TAT2O8) DNA260655 (TAP209) DNA260655 (TXI'209) DNA260655 (TAT209) DNA2 60655 ('EAI'09) DNA260655 (TAT2O9) DNA260655 ('rAT2O9) DNA260655 (TAT2O9) DNA260655 (TAnI20) DNA260655 (TAT2O9) DNA260655 CfAT209) DNA261001 (TATlB I) DNA261001 (TATIB I) DNA266928 (TAT! 82) DNA266928 (TATl82) DNA268035 (rAl222) DNA268035 (TA1722) DNA268035 (TAT722) DNA268035 (TAr222) DNA77509 (IATi 77) DNA8 7993 (TA1235) DNA87993 (TAT235) DNA87993 (TAT235) DNA87993 (TAX235) DNA87993 (TAT735) DNA87993 (TA1'235) DNA92980 (TArz34) DNA92980 MrT234) DNA92980 (TAT234) DNA92980 (TAT734) D2NA92980 (TAT234) DNA92980 (TAT234) tDNA92980 (TAT234) DNA92980 (TA1234) DNA92980 (TAT234) DNA92980 (TAT234) DNA92980 (TAT34) DNA92980 (TAT234) DNA92980 (TAT234) DNA92980 (TAT234) DNA92980 (TAT234) 4S DNA1 05792 (TAT233) DNA105792 (TAT233) DNA105792 (TAT-233) DNA105792 (fAT233) 11NA105792 CrATZ33) DNAlQS792 (TAT233) DNA105792 (TAT233) DNA1 05792 (TAT233) DNA105792 (TATZ33) DNA105792 (TAT233) DNAI 05792 (TAT233) uprmdtiuiof ~rsii n Prostate tumor colon tumor breast tumor breat tumor lung tumor colon tumor breast tumor liver tumor ovarian tumor skin tumor spleen tumor myeloid tumor muscle tumor bone tumor bone tumor lung tumor bone tumor lung tumor breast tumor colon tumor ovarian tumor uterine tumor colon tumor breast tumor pancreatic tumor lung tumor colon tumor' rectum tumor gallbladder tumor bone tumor breast tumor cervical tumor colon tumor rectum tumor eadometrial tumor liver tumor lung tumor ovarian tumor pancreatic tumor skin tumor soft tissue tumor stomach tumor bladder tumor thyroid tumor bone tumor breast tumor endomnetrial tumor esophagus tumor kidney tumor lung tumor ovarian tumor pancreatic tumor prostate tumor soft tissue tumor stomach tumor normal P~rosatte tissue niormnal colon tissue normal breast tissue normal breast tissue normal lung tissue normal colon tissue normal breast tissue normal liver tissue normal ovarian tissue normal skin tissue normal spleen tissue normal mnyclold tissue normal muscle tissue normal bone tissue normal bone tissue normal lung tissue normal bone tissue normal lung tissue normal breast tissue normal colon tissue normal Ovarian tissue normal uterine tissue normal colon tissue normal breast tissue normal pancreatic tissue normal lung tissue normal colon tissue normal rectum tissue normal gallbladder tissue normal bone timse normal breast tissue normal eervical tissue normal colon tissue normal rectum tissue normal endometrial tissue normal liver tissue normal lung tissue, normal ovarian tissue normal pancreatic tissue normal skin tissue normal soft tissue normal stomach tissue normal bladder tissue normal thyroid tissue normal bone tissue normal breast tissue normal endonietrial tissue normal esophagus tissue normal kidney tissue normal lung tissue normal ovarian tissue normal pancreatic tissue normal prostate tissue normal soft tissue normal stomach tissue 00 00 DNA105792 (TAT233) DNA 105792 (TA'r233) DNA105792 (rA 233) DNA1 19474 (TA128) DNA1 19474 (TAT228) DNA2803Sl (TAT248) DNA280351 (TA1248) DNA150648 (rAT232) DNA1 50648 (TAT232) DNA150648 (TAT232) DNA1 50648 (TAT232) DNA1 50648 (TAT232) DNA150648 (TA1232) DNA1 50648 (TAM232) DNA1 50648 (TAT232) DNA179651 (TAT224) DNA1 79651 (TAT224) DNA179651 (TAT224) DNA179651 (TA'l224) DNA179651 CI'AT-224) DNA179651 (TA7724) DNA179651 (TAT224) DNA207698 (TAT-237) DNA207698 (TAT237) DNA207698 (TAT237) DNA2Q7698 (TAT-37) DNA207698 (fAT237) 0 DNA225886 (rAm26) DNAZ25886 (TAT236) DNA225886 (TAT236) DNA225886 (TA1236) DNA225886 CrAT236) DNA225886 (TAT236) DNA225886 (TAT'236) DNA225886 (TAT236) DNA225886 (TAT236) DNA226717 (TAT 8 5) DNA226717 (TATI85) DNA227162 (TAT225) DNA227 162 (TAT225) DNA227 162 CI'AT225) DNA227 162 (TA-n25) DNA277804 (TAT247) DNA277804 (TAT247) DNA277804 (TATr247) DNA277804 (TAT247) DNA233 034 (TATI 74) S) DNA233034 (TATI 74) DNA266920 (TAT214) DNA266920 (TAT214) DNA266921I (TAT220) DNA26692 1 (TA1220) 53 DNA266922 (TAT221) ~p1O~Uatin f exprwssion in.

thyroid tumor bladder tumor brain tumor Wilm's tumor uterine tumor ovarian tumor squamous cell lung tumor colon tumor liver tumor breast tumor brain tumor lung tumor colon tumor rectum tumor kidney tumor bladder tumor breast tumor cervical tumor colon tumor rectum tumor uterine tumor lung tumor Ovarian tumor breast tumor colon tumor ovarian tumor pancreatic tumor stomach tumor breast tumor colon tumor rectum tumor endometrial tumor lung tumor ovarian tumor pancreas tumor prostate tumor bladder tumor glioma.

brain tumor breast tumor endometrial tumor lung tumor ovarian tumor breast tumor endometrial tumor lung tumor ovarian tumor glioma brain tumor glioma brain tumor gli oma brain tumor glioma normal thiyroid tissue normal bladder tissue normal brala tissue normal assooiated tissue normal uterine tissue normal ovarian tissue normal squamous cell lung tissue normal colon tissue normal liver tissue normal breast tissue normal brain tissue normal lung tissue normal colon tissue normal rectum tissue normal kidney tissue normal bladder tissue normal breast tissue normal cervical tissue normal colon tissue normal rectum tissue normal uterine tissue normal lung tissue -normal ovarian tissue normal breast tissue normal colon tissue normal ovarian tissue normal pancreatic tissue normal stomach tissue normal breast tissue normal colon tissue normal rectum tissue normal endoanetrial tissue normal lung tissue normal ovarian tissue normal pancreas tissue normal prostate tissue normal bladder tissue normal glial tissue normal brain tissue normal breast tissue normal endometrial tissue normal lung tissue normal ovarian tissue normal breast tissue normal endonietrial tissue normal lung tissue normal ovarian tissue normal glial tissue normal brain tissue normal glial tissue normal brain tissue normal glial tissue normal brain tissue normal glial tissue 00 00 Molecule D~NA266922 (TA21) DRA234441 (TA17ol) DNA234441 (TA'1701) DNA234834 (TATI79) DNA234834 (rAT179) DNA234834 (TAT179) DNA2 34 8 34 (1'ATI 79) DNA234834 (TAT! 79) DNA234834 (TAT179) DNA234834 (TAT! 79) DNA234 834 (TATI 79) DNA247587 (TAT2 16) DNA247587 (TAT2 16) DNA2475B7 (TAT216) DNA247587(TA71 6 DNA247587 (TAT2 16) DNA247587 (TAT2 16) DNA2 55 9 87 (TAT2 18) DNA56041 (TAT206) DNA257845 (rxrs74) DNA247476 (TATI 8o) DNA247476 (fAT! 80) DNA24 7476 (TAT1 8o) DNA247476 (TAT189) DNA247476 (TATI 80) IDNA24 7476 (TATI 80) IDNA247476 (TAT! 80) DNA7247476 (TAT! 80) DNA247476 (TAT! 80) DNA247476(TAT18o) IDNA247476 (TAT! 80) DNA260990 (TAT375) DNA260990 (TAT375) DNA260990 (TAT375) DNA2 60990 (TAfl 75) DNA260990 (TAT3 75) DNA260990 (TAT3 75) DNA260990 (TAT3 75) DNA260990 (TAT-375) DNA260990 (TAT375) DNA260990 (TAT3 75) DNLA260990 (TAD3 75) DNA2 61013 (TAT 7 6) DNA261013 (TATI 76) DNAz6 i o 3 (TATI 76) IDNA261013 (TAT176) DNAZ61013 (TAT176) IDNA261013 (TAT1 76) DNA262 144 (EATl84) so DNA262 144 (TAT! 84) DNA2 62 144 (TATI 84) IDNA2 62 144 (TAT! 84) DNA262144 (TATI 84) DNA262 144 (TAT! 84) DNA262 144 CI'ATI 84) WDZ~plptoi of crslon in.

brain tumor colon tumor rectum tumor breast tumor colon tumor rectum tumor Prostate tumor Pancreatic, tumor endonietrial tumor lung tumor ovarian tumor breast tumor lung tumor o'varian tumor pancreatic tumor stomach tumor urinary tumor breast tumor lyinphold tumor lymphold tumor bone tumor broaw tumor colon tumor rectum tumor kidney tumor lung tumor pancreatic tumor prostte tumor skin tumor soft tissue tumor stomach tumor bone tumor breast tumor colon tumor rectum tumor kcidney tumor lung tumor Pancreatic tumor Prostate tumor skin tumor soft tissue tumor stomach tumor breast tumor colon tumor rectum tumor lung tumor ovarian tumor stomach tumor breast tumor colon tumor rectum tumor endometrial tumor kidney tumor lung tumor ovarian tumor normal bxaia tissue noimal colon tissue normal rectum tissue normal breast tissue normal colon tissue normal rectum tissue normal Prostate tissue normal pancreatic tissue normal endometrial tissue normal lung tissue normal ovarian tissue normal breast tissue normal lung tissue normal ovarian tissue normal pancreatic tissue normal stomach tissue normal urinary tissue normal breast tissue normal lymuphoid tissue normal lymnphoid tissue normal bone tissue normal brew astisue normal colon tissue normal rectum tissue normal kidney tissue normal lung tissue normal pancreatic tissue normal prostate tissue normal skin tissue normal soft tissue normal stomach tissue normal bone tissue normal breast tissue normal colon tissue normal rectum tissue normal kidney tissue normal lung tissue normal pancreatic tissue normal prostate tissue normal skin tissue normal soft tissue normal stomach tissue normal breast tissue normal colon tissue normal rectum tissue non-nal lung tissue normal ovarian tissue normal stomach tissue normal breast tissue normal colon tissue normal rectum tissue normal endometrial tissue normal kidney tissue normal lung tissue normal ovarian tissue M91te DNA267342 (TAT2 3)) 00 00 DNA67626(TA17l) DNA267626 (TAT2 17) DNA26762 6 (TAi~i7) DNA2 67626 (rA7217) DNA26762 6 (TA1717) DNA267626 (A17 1 7 DNA268334 (TAT292) DNA2 6923 8 (TAT2 15) DNA272578 (TAT238) DNA272578 (TAT238) DNA272578 ('AT2 3 8) DNA304853 (TADh76) DNA304853 (TAT376) DNA304853 (TAT376) IDNA304853 (TAT376) DNA304853 (TAT376) DNA304853 (TAT376) DNA304853 CFAT376) DNA304853 (T1376) DNA304854 CI'AT77) DNA304854 (TAT3 7 7 DNA3 04854 (TAT3 77) DNA3 04854 (TAT3 77) DNA304854 (TAT377) DNA304854 CI'An~77) DNA304854

(TAD

77 DNA3 04854 (TAT3 77) DNA304855 (TAT378) DNA304855 CrAT378) DNA.04855 (TAT378) DNA304855 TA73 7 8) EDNA3048SS (TAT378) DNA304855 (T1378) DNA304855 (TAIT378) DNA304855 (TAT378) DNA287971 (TAT379) DNA287971 (TAT379) DNA,287971 (TAT379) DNA287971 (TAT379) DNA287971 (TAT379) DNA28797 1 (TAT379) DNA287971 (TAT379) DNA287971 (TAT379) DNA297971 (TAT379) DNA28797 1 (TAt379) S0 DNA287971 (TAT379) trOMa 'aociated with the foliowMn tumoirg bone, breast, color; rectum, lI=& ovarian, pancReas, Soft tissue, bladder breat tumor colon tumor retum tumor endometkial tumor lung tumrne Pancreatic tumor kridney tumor kidney tumor liver tumor lung tumor ovarian tumor breast tumnor colon tumor rectum tumor Prostate tumor Pancreatic tumor endomeriaj tumor lung tumor ovarian tumor breast tumor colon tumor rectum tumor prostate tumor pancreatic tumor endomnetial tumor lung tumor ovarian tumor breast tumor colon tumor rectun tumor Prostate tumor Pancreatic tumor endometrial tumor lung tumor ovarian tumor bone tumor breast tumor colon tumor rectum tumor kidney tumor lung tumor Pancreatid tumor prostate tumor skin tumor soft tissue tumor stomach tumor normal associated tissues, respectively nornal breast tissue normal colon tissue normal rectum tissue normal endometrial tissue normal lung tissue normal pancreatic tissue normal kidney tissue normal kidney tissue normal liver tissue normal lung tissue normal ovarian tissue normal breast tissue normal colon tissue normal rectum tissue normal prostate tissue.

normal pancreatic tissue noirnal endometrial tissue normal lung tissue normal ovarian tissue normal breast tissue normal colon tissue normal rectum tissue normal prostate tissue normal pancreatic tissue normal endonietrial tissue normal lung tissue normal ovarian tissue normal breast tissue normal colon tissue normal rectum tissue normal prostate tissue normal pancreatic tissue normal endonietrial tissue normal lung tissue normal ovarian tissue nornfal bone tissue normal breast tissue normal colon tissue normal rectumn tissue normal kidney tissue normal lung tissue normal pancreatic tissue normal prostate tissue normal skin tissue normal soft tissue E X A M PLE -g iS A 2 Mi r a -yA n l s st e t c p e ul t o f Aloy j i n Nucleic acid midcroarrys, often containin tZus;d oors difer nti l es, are useful r identitying diferetialyex~pressed genes in diseased tissues as compared to their normal uountorparts. Using nucleic acid microaays, test and control mNA samples from test and control tissue samples am reverse tansribed and Slabeled to generate cDNA probes. The eDNA probes are then hybridized to an arry of nucleic acids Immoblized on a solid support. The array is configured such that the seuence and position of each member of the array is known. For example, a selection of genes known to be expressed in certain disease states may be arrayed on a solid support Hybridization of a labeled probe with a particular arry member indicates that the sample from Which the probe was derived expresses that gene. If the hybridization signal of a probe from a test (disease tissue) sample 0is greater than hybridization signal of a probe from a control (normal tissue) sample, the gene or genes overexpressed in the disease tissue are identified. The implication of this result is that an overexpressed protein in a diseased tissue is useful not only as a diagnostic iarker for the presence of the disease condition, but also as a therapeutic target for treatment of the disease condition The methodology of hybridization of nucleic acids and microarray technology is well known in the art.

SIn one example, the specific preparation of nucleic acids for hybridization and probes, slides, and hybridization conditions are all detailed in PCT Patent Application Serial No. PCT/USO1/10482, filed on March 30,2001 and which is herein incorporated by reference.

In the present example, cancerous tumors derived from various human tissues were studied for upregulated gene expression relative to canerous tumors from different tissue types and/or non-cancerous human tissues in an attempt to identify those polypeptides which are overxpressed in a particular cancerous tumor(s) In certain experiments, cancerous human tumor tissue and non-cancerous human tumor tissue of the same tissue type (often from the same patient) were obtained and analyzed for TAT polypeptide expression. Additionally, cancerous human tumor tissue from any of a variety of different human tumors was obtained and compared to a "univerial" epithelial control sample which was prepared by pooling non-cancerous human tissues of epithelial origin, including liver, kidney, and lung. mnNA isolated from the pooled tissues represents a mixture of expressed gen products from these different tissues. Microarray hybridization experiments using the pooled control samples generated a linear plot in a 2-color analysis. The slope of the line generated in a 2-color analysis was then used to normalize the ratios of (test:oontrol detection) within each experiment. The normalized ratios fiom various experiments were then compared and used to identify clustering of gene expression. Thus, the pooled "universal control" sample not only allowed effective relative gene expression determinations in a simple 2 -sample comparison, it also allowed multi-sample comparisons across several experiments.

In the present experiments, nucleic acid probes derived from the herein described TAT polypeptideencoding nucleic acid sequences were used in the creation of the microarray and RNA from various tumor tissues were used for the hybridization thereto. Below is shown the results of these experiments, demonstrating that various TAT polypeptides of the present invention are significantly overexpressed in various human tumor tissues as compared to their normal counterpart tissue(s). Moreover, all of the molecules shown below are significantly overexpressed in their specific tumor tissue(s) as compared to in the "universal" epithelial control. As described above, these data demonstrate that the TAT polypeptides of the present invention are useful not only as .3 5 diagnostic markers for the presence of one or more cancerous tumors, but also serve as therapeutic targets for the treatment of those tumors.

Molme Sles son 00 DNA172500aon of eTAT21sson IR a o rd to: O D 25 2) rd carcinoma normal kidney (renal cell) tissue SXAMP 3: Quantitative Analysis of TAT mRA Expression In this assay, a 5' nuclease assay (for example, TaqMan®) and real-time quantitative PCR (for example, SABIPrizm7700 Sequence Detection System®(PerkinElmer, Applied Biosystems Division, Foster City, were Sused to find genes that are significantly overexpressed in a cancerous tumor or tumors as compared to other cancerous tumors or normal non-cancerous tissue. The 5' nuclease assay reaction is a fluorescent PCR-based technique which makes use of the 5' exonuclease activity of Taq DNA polymerase enzyme to monitor gene expression in real time. Two oligonucleotide primers (whose sequences are based upon the gene or EST sequence of interest) are used to generate an amplicon typical of a PCR reaction. A third oligonucleotide, or probe, is g designed to detect nucleotide sequence located between the two PCR primers. The probe is non-extendible by Taq DNA polymerase enzyme, and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. Any C laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are on the probe. During the PCR amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner. The resultant probe fragments disassociate in solution, and signal from the released reporter dye is free from the quenching effect of the second fluorophore. One molecule of reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.

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

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

nuclease assay data are initially expressed as Ct, or the threshold cycle. This is defined as the cycle at which the reporter signal accumulates above the background level of fluorescence. The ACt values are used as quantitative measurement of the relative number of starting copies of a particular target sequence in a nucleic acid sample when comparing cancer mRNA results to normal human mRNA results. As one Ct unit corresponds to 1 PCR cycle or approximately a 2-fold relative increase relative to normal, two units corresponds to a 4-fold relative increase, 3 units corresponds to an 8-fold relative increase and so on, one can quantitatively measure the relative fold increase in mRNA expression between two or more different tissues. Using this technique, the molecules listed below have been identified as being significantly overexpressed in a particular tumor(s) as compared to their normal non-cancerous counterpart tissue(s) (from both the same and different tissue donors) and thus, represent excellent polypeptide targets for the diagnosis and therapy of cancer in mammals.

00 00 DNA261 021 (TAT7O8) DNA77sog (rAT! 77) DNA! 19474 (TAT226) DNA179651 (TXI'224) D14A226717(TAT185) DNA227162 (T'AMS2) DNA277804 (TAT247) DNA233034 (TAT174) DNA266920 (TAT24) DN'A266921 (TAT22O) DNA266922 (Tr22 1) DNA234441 (TAT2OI) DNA234834 (TATl79) DNA247581 (TAT2 6) DNA255987 C1Anl28) DNA247476 (TATI 80) DNA260990 (TAT37S) DNA261013 (FAT176) DNA262144 (TAT184) DNA267342 (TA1213) DNA267626 (TAT2 17) DNA2 6 93 34 (TA'r202) DNA269238 (rA1215) DNA87993 (rAT23S) DNA92980, (TAT234) DNA105792 (TA1233) DNA207698 (TAT237) DNA225886 (TAT236) DNA272578 (rA '238) DNA304853 (TA 376) DNA3O04 8 54 (TAD3 77) DNA304855 (TAT378) DNA287971 (TAT-379) UIM~ultpn of exrsinin: lung tamor colon tumor ovarian tumor ovarian tumor gljoina ovarian tumror ovarian tumor glioma glioma gliorna glioma colon tumor colon tumor squamous cell lung tumor breast tumor colon tumor colon tumor breast tumor kidney tumor breast tumor breast tumor kidney tumor kidney tumor lung tumor ovarian tumor lung tumor colon tumor colon tumor ovarian tumor colon tumor colon tumor colon tumor colon tumor as COMM4lrd o normal lung tisue normal colon tissue normal ovialrin tissue normal ovarian tissue normal ovarian tissue normal ovial(rin tissue normal glial/brain tissue normal glialf rain tissue normal glial/brain tissue normal clo/bn tissue normal colon tissue normal colo isellln norissu umueel ui normlsueatisu normal breast tissue normal colon tissue normal colon tissue normal bidey tissue normal brdey tissue normal breast tissue normal bides tissue normal kidney tissue normal kine tissue normal lun tissue normal ovrng tissue normal cln tissue normal colon tissue normal olorin tissue normal coarin tissue normal colon tissue normal colon tissue normal colon tissue J3XAMEAi fIr, rftu y idization In Sitif hybridization Is a powerful and versatile technique for the detection and localization of nucleic acid sequences within cell or tissue preparations. It may be useful, for example, to identify sites of gene expression, analyze the tissue distribution of transcription, identify and localize viral infection, follow changes in specific IT0 mR1NA synthesis and aid in chromosome mapping.

In sieu hybridization was performed following an optimized version of the protocol by Lu and Gillett, Cell Viin1: 169-176 (1994), using PCR-generated 33 P-labeled riboprobes. Briefly, forrmalin-fixed, paraffin-embedded human tissues were sectioned, deparafrfinied, deproteinated in proteinase K (20 g/ml) for 15 minutes at 37TC, and further processed for In .siw hybridization as described by Lu and Gillett,supra. A (33_P] UTP-labeled antisense 'Is riboprobe was generated from a PCR product and hybridized at 55 T overnight. The slides were dipped In Kodak NTB 2 nuclear track emulsion and exposed for 4 weeks.

00 6.0 Pt1 (125 mCi) of 33 pUrp (AmershaMBp 1002, SA<~000 Clfmrnol) Were speed vao dried, To each tube cotaining dujed -urp, the following legient were added: P1 5X transcription buffer 2.0 glNTmix (25 mM 1 lu; each of10 mmGnCrp ATp +lIO 1 jItI'U? (50 pM~) 1 .0 R1 Rnasjn 1 .0 R1 DNA template (Ilpg) 1.0 p1l RNA polymerase (for PCR products T3 -AS, 7 S, usually) The tubes were incubated at 37'C for one hour, 1.0 pi RQ1 DNase were added, followed by incubation 00at37eC for 15 minutes. 90 RI TE(1 0 mM Ths pH 7,611WmvIEDTA pi 8.0) were added, and the mixture was pipetted onto DB 1 paper. The remaining solution was loaded in a Mcrocon-50 ultrafitration unit, and spun using program (6 minutes). The filtration unit was inverted over a second tube and spun using program 2 (3 minutes). After the final recovery spin, 100 up11 were added. 1 p1 of the final product was pipetted on DEBI paper and counted in 6 ml of Biofluor ML The probe was run on a TBE/urea gel. 1-3 p1l of the probe or 5 jul of RNA Mrk Ml were added to 3 g1 of loading buffer. After heating on a 95*C heat block for three minutes, the probe was immediately placed on Ice.

The wells of gel were flushed, the sample loaded, and run at 180-250 volts for 45 minutes. The gel was wrapped in saran wrap and exposed to XAR film with an intensifying screen in -70 *C freezer one hour to overnight A. Pretreatmnrt of fiizen sections The slides were removed from the freezer, placed on aluminium trays and thawed at room temperature for minutes. The trays were placed in 55 0 C incubator for five minutes to reduce condensation. The slides were fixed 10 minutes in 4% parafornialdehyde, on ice in the flume hood, and washed in 0.5 x SSC for 5 minutes, at room temperature (25 ml 20 x SSC 975 ml SQ H 2 After deproteinatlon in 0.5 ptg/mi proteinase K for 10 midnutes at 3 7 *C (12.5 pl of 10 nig/ml stock in 250 Hl prewarmed P.Nase-free RNAse buffer), the sections were washed in x SSC for 10 minutes at room temperature. The sections were dehydrated in 70%, 95%, 100% ethanol, 2 niinutes each.

U. retreatment of Para 'in-embedded ections The slides were deparaffinized, placed in SQ 1H20, and rinsed twice in 2 x SSC at room temperature, for mninutes each time. The sections were deproteinated In 20 ~g/n-d proteinase K (500 pl of 10 mg/mir in 250 ml P.Nasefree RNase bu ffer; 3 7'C, 15 minutes) human embryo, or 8 x proteinase K (100 R1 in 250 mld Rnase buffer, 3 7 C, 3 0 minutes) formalin tissues, Subsequent rinsing in 0.5 x SSC and dehydration were perfonned as described above.

Prehybridization The slides were laid out in a plastic box lined with Box buffer (4 x SSC, 50% fortnamide) saturated filter paper.

00 1.0 X lO6 cPm probe and 1. ittN"A (50 mg/m stook) per slide were heated at 950T for 3 minutes. -rhe N ~slides were coled on ice, and 48 Pd hybridization buffer were added per slide. After vortexing, 50 gp11 3 P mix were added to 50 pW Pehybridization on slide. The slides were incubated overnight a Washing was done 2 x 10 minutes with 2xSSC, EDTA at room temperature (400 ml 20 x SSC+ 16 ml 0.25M4 EDTA, VMP4'), followed by RNaseA treatment at 37 0 C for 30 minutes (500 91 of 10 rug/mI in 250 nil Rnase, buffer 20 gfm), The slides were washed 2 x 10 minutes with 2 x SSC, EDTA at room temperature. The stringency wash conditions were as follows: 2 hours at 55 OC, 0. 1 x SSC, EDTA (20 ml 20 x SSC 16 ml EDTA, Ve"4L).

F. Olionuleojde In situ analysis was performed onl a variety of DNA sequences disclosed herein. The oligonucleotides employed for these analyses were obtained so as to be complementary to the nucleic, acids (or the complemenits thereof) as shown in the accompanying figures.

I'n situ analysis was performed on a variety of DNA sequences disclosed herein. The results from these analyses ate as follows.

Positive expression is observed in 2 of 3 non-small cell lung carcinomsa, 2 of 3 pancreatic.

adenocarvinomas, 1 of 2 hepatocellular carcinomas and 2 of 3 endometrial adenocareinomas. In a separate analysis, 10 of 16 ovarian adenocarcinomas are positive and 3 of 9 endometrial adenocaminomas are positive. All normal tissues examined are negative for cxpression.

NA65 OA724 In one analysis, expression is seen in 5 of 7 uterine adenocaroinomas and in 7 of 16 ovarian adenocarsinomas. Two cases of dysgermnoma are positive as is one case of a Brenner's tumor.

In another analysis, 33 of 68 ovarian adenocarcinomas (serous, mucinous, endometrioid, clear cell) are positive for expression. Moderate to strong expression is seen in normal endometrium (no other normal tissues) and normal ovarian stroma is negative.

In yet another analysis, positi ve: expression is seen in 313 endomnetrial, 2/2 colorectal, 1/3 transitional cell, 3/3 lung and 1/2 ovarian cancers, DNA227 162r (TA12 Expression is seen in the following tumors: I of 3 lung cancers, 1 of 2 colon cancers, I of I pancreatic cancer, 2 of 3 transitional cell carcinomas, 3 of 3 endometrial carcinomnas, 2 of 2 ovarian carcinomas and 2 of 3 malignant nmelanomas.

Insa separate analysis, positive expression is seen in 6 of 9 uterine adenocarcinomas and 6 of 14 ovarian tumors.

With regard to expression in normal tissues, weak expression is seen in one core of urothelium, (superficial cell layer positive) and one core of gall bladder mucosa, All other normal tissues are negative for expression, 00

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DNA277804 0AT247 Btpression is seen in the following tumors: 1 of 3 lung canoers, 1 of 2 oolon cancers, 1 of 1 pancreatic cancer, 2 of 3 transitional cell carcinomas, 3 of 3 endometrial carcinomas, 2 of 2 ovarian carcinomas and 2 of 3 malignant melanomas.

In a separate analysis, positive expression is seen in 6 of 9 uterine adenocarcinomas and 6 of 14 ovarian 5 tumors.

With regard to expression in normal tissues, weak expression is seen in one core of urotheliurn (superficial cell layer positive) and one core of gall bladder mucosa. All other normal tissues are negative for expression.

DNA234441 (TA201) Weak (and inconsistent) expression is seen in normal kidney, normal colon mucosa and normal gallbladder. Weak to moderate, though somewhat inconsistent expression is seen in normal gastrointestinal mucosa (esophagus, stomach, small intestine, colon, anus). Significant expression in tumors is seen as follows: 11 of 12 colorectal adenooarinomas, 4 of 4 gastric adenocarinomas, 6 of 8 metastatio adenocarinomas, 4 of 4 esophageal cancers and I of 2 panreoatic adenocaroinomas.

DNA234834 fATIy79 With regard to normal tissues, it appears that there is a weak signal in colon mucosa and breast epithelium. With regard to tumor tissues, expression is seen in 1 of 2 non-small cell lung carcinomas, 2 of 2 colon cancers, 1 of 2 pancreatic cancers, I of 2 hepatocellular carcinomas, 3 of 3 endometrial carcinomas, 1 of 2 ovarian carcinomas and 2 of 3 malignant melanomas.

In a separate analysis, 12 of 16 colorectal carcinomas are positive for expression; 2 of 8 gastric adenocarvinoma are positive for expression, 2 of 4 esophageal carcinomas are positive for expression; 7 of metastatic adenocarminoma are positive for expression and 1 of 2 cholangiocareinomas are positive for expression.

Expression level is tumor tissues is consistently higher than in normal tissues.

DNA24787 (TAT216) Expression is seen in 13 of 16 non-small cell lung carcinomas. Expression is also seen in benign bronchial mucosa and occasional activated pneumocytes. Moreover, 65 of 89 cases of invasive breast cancer are positive for expression. Strong expression is seen in normal skin and normal urothelium. Moderate expression is seen in normal mammary epithelium and trophoblasts of the placenta, weak expression in normal prostate and normal gall bladder epithelium and distal renal tubules.

DNA56041 (TAT206) In non-malignant lymphoid tissue expression is seen in occasional larger lymphoid cells within germinal centers and in interfollicular regions. Positive cells account for less than 5% of all lymphoid cells. In section of spleen scattered positive cells are seen within the periarteriolar lymphoid sheath and in the marginal zone.

In four cases of Hodgkin's disease Reed-Stemberg cells are negative, positive signal is observed in scattered lymphocytes. Three of four cases of follicular lymphoma are positive (weak to moderate), four of six cases of diffuse large cell lymphoma are positive (weak to moderate). Two cases of small lymphocytic lymphoma show a weak signal in variable proportion of cells, DA27845frAT74) 00 In non-malignant lymphoid tissue expression is seen in occasional larger lymphoid cells within germinal Scenters and in interfollioular regions. Positive cells account for less than 5% of all lymphoid cells. In section of spleen scattered positive cells are seen within the periarteriolar lymphoid sheath and in the marginal zone.

In four cases of Hodgkin's disease Reed-Stemberg cells are negative, positive signal is observed in scattered lymphocytes. Three of four cases of follicular lymphoma are positive (weak to moderate), four of six Scases of diffuse large cell lymphoma are positive (weak to moderate). Two cases of small lymphocytic lymphoma show a weak signal in variable proportion of cells.

PMNA247476 AT180) With regard to normal tissues, strong expression is seen in prostatic epithelium and in a section of S 10 peripheral nerve. Moderate expression is seen in renal glomeruli, Weak expression is seen in bile duct epithelium Sand mammary epithelium. Two sections of stomach show weak expression in a subset of gastric glands. Sections 00 Sof colon and small mtestine show a signal in lamina propria and/or submucosa, most likely in small autonomic nerve fibers. Another independent ISH study fails to show expression in peripheral nerves of prostatectomy sections, despite adequate signal in prostatio epithelium.

In a separate analysis, 42 of 77 breast tumors are positive for expression.

In yet another analysis, 8 of 11 breast cancers are positive for expression.

In yet another analysis, expression is seen in 1/2 non-small cell lung carcinomas, 1/3 colorectal adenocarcinomas, 2/3 pancreatic adenocarcinomas, 1/1 prostate cancers, 1/3 transitional cell carcinomas, 3/3 renal cell carcinomas, 3/3 endometrial aden6carcinomas, 1/2 ovarian adenocarcinomas and 1/3 malignant melanomas.

In yet another analysis, expression is seen in 42 of 45 prostate cancers.

In yet another analysis, expression is seen in all of 23 primary and in 12 of 15 metastatio prostate cancers analyzed.

In yet another analysis, expression is observed in the following carcinomas as follows: pancreatic adenocaroinoma 2 of 2 cases are positive; colorectal adenocarcinoma 12 of 14 cases are positive; gastric adenocarcinoma 6 of 8 cases are positive; esophageal carcinoma 2 of 3 cases are positive; cholangiocarcinoma 1 of 1 case is positive; metastatic adenocaroinoma (ovary, liver, lymph node, diaphragm) 8 of 12 cases are positive.

(11) DNA260990 (TAT375) With regard to nonnal tissues, strong expression is seen in prostatic epithelium and in a section of peripheral nerve, Moderate expression is seen in renal glomeruli. Weak expression is seen in bile duct epithelium and mammary epithelium, Two sections of stomach show weak expression in a subset of gastric glands. Sections of colon and small intestine show a signal in lamina propria and/or submucosa, most likely in small autonomic nerve fibers. Another independent ISH study fails to show expression in peripheral nerves of prostatectomy sections, despite adequate signal in prostatic epithelium.

In a separate analysis, 42 of 77 breast tumors are positive for expression.

In yet another analysis, 8 of 11 breast cancers are positive for expression.

130 00 00 In Yet another analysis, expression is oeen in 1V2 non-smnall ceil lung carcdnomas, 1/3 colorectal caclndoas,

V/

3 panlcreatic adenocaroinomnas, III prostate cancers, 113 transitional call carcinomas, 3/3 renal Ce11 carcinomias, 313 endometria adenocarcinomas, M/ ovarian adenocarcinomas and 1/3 malignant melanowas In yet another analysis, expression is seen in 42 of 45 prostate cancers.

In yet another analysis, expression is seen in all of 23 primary and In 12 of IS5(80%/) metaftatic prostate, analyzed, In yet another analysis, expression is observed in the following carcinomas as follows: panoreatic adenocarcinoma 2 of 2 cases are positive; colorectal adenocarinoma.- 12 of 14 cases are positive; gastric adenocarcnoma 6 6f 8 cases are positive; esophageal carcinoma 2 of 3 cases are positive; chiolangocarcinona 1 of 1 case is positive; metastatic adenocarcinoma (ovary, liver, lymph node, diaphragm) 8 of 12 cases are positive.

(12) DNA261013 AjU§7) With regard to normal tissues, prostate epithelium shows a weak positive signal. Also, one core of colonic mucosa shows a weak signal in inucosal epithelium. Two cores of a testicular neoplasmn are positive.

In another analysis, 87 cases of infiltrating ductal breast cancer are available for review. 40 cases are positive forexprossion. Additionally, alltestedcenl lines (A549, SK-NMS, SKBR3, MD)A23 1, MI)A453, MDA175, MCF7) are positive for expression.

In another analysis, there is no consistent expression in benign colon, small intestinal, liver, pancreatic, gastric or esophageal tissue. In malignant tumors expression is observed as follows: colorectal1 adenocarcinomna: of 14 cases are positive, gastric adenooarcinoma: 4 of 8 cases ame positive, esophageal carcinoma: 3 of 4 cases are positive and mietastatic adenocarcinoma: 8 of I11 cases are positive.

(13) DN262144-AT184) Two of 4 cases of non-small cell lung carcinoma are positive for expression wile no signal is observed in non-neoplastic lung, In at separate analysis, three cases of non-small cell lung carcinoma are positive (14) 26329A73 Expression is not observed in any of the normal adult tissues tested. Seventy four cases of breast cancer are available for review and 30 cases give a positive signal lBxpression localizes to tumor-associated stronia.

In a separate analysis, expression is seen in a minority of sarcomas; moderate and occasionally strong expression is seen in a case of a synovial sarcoma, angiosarconia, fibrosarcoma gliosarcoma and malignant fibrohistiocytomna In most cases expression appears to localize to the malignant cell population.

(15) DN267626 (TAT217) Expression is seen in 6 of 9 invasive breast cancers. Expression is in most cases of moderate intensity, expression is also seen in benign manmmary epithelium and fibroadenoma, The large sections included in this study show expression in 1 of 1 endometrial adenocarcinomas, in 2 of 3 invasive ductal breast cancers, in benign renal tubules, in normal breast epithelium and in epidermis Sections of lung, brain, nyometrium and eye are negative.

(16) IDN268334 (TAT202) No expression is seen in any of the adult, normal tissues tested while expression is observed in 3 of 3 renal cell carcinomas, (17) M g

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00 Tunior-assocjatedj vasculature was stZr)flgy positive in all renal cell carcinomas tested in all hOPatOce11uar carcinomas tested in all gastric adenocarojuomas tested (n75), In all endonietrjal adenoarnma tested in all malignant melanomias tested in all malignant lymphomas tested In all pancreatic adenocarcinomas tested in all esophageal carcinomas tested in all Cholangiocariomas tested in 93% of all non-small cell lung cancers tested (n=1 in 86% of all invasiv~e ductal breast cancers tested in 83% of all colorectal adenocarcinomas tested in 67% of all metastaic adenocarcinomnas tested in 75% of all transitional cell carcinomas tested While TAT215 expression is also observed in endothelial components of various normal non-cancerous tissues, the expression level is significantly lower in these non-cancerous tissues as compared to their cancerous counterarts and the expression 1 0 pattern in the tumor tissues was distinct from that in the normal tissues, thereby providing a means for both 00 therapy and diagnosis of the cancerous condition.

(1)00OSSCA36 ri With regard to normal tissues, it appears that theme is a weak signal in colon muoosa and breast epithelium. With regard to tumor tissues, expression is seen in 1 of 2 non-small cell lung caroinomas, 2 of 2 colon cancers, 1 of 2 pancreatic cancers 1 of 2 hepatocellular carcinomas, 3 of 3 endometrial carcinomas, I of 2 ovarian carcinomas and 2 of 3 malignant melanomas.

In a separate analysis, 12 of 16 colorectal carcinomas are positive for exprcssionq 2 of 8 gastdc adenocaminoma are positive for expression, 2 of 4 esophageal carcinomas are positive for expression; 7 of mietastati adenocarcinoma are positive for expression and I of 2 cholanglocarcinomas are positive for expression, Expresion level is tumor tisses is consistently higher than in normal tissues.

(19) PIA085 MaDAt37 With regard to normal tissues, it appears that there is a weak signal in colon mucosa and breast epithelium, With regard to tumor tissues, expression is seen in 1 of 2 non-small cell lung carcinomas, 2 of 2 colon cancers, I of 2 panceatic cancers, 1 of 2 hepatocellular carcinomas, 3 of 3 endometrial carcinomas, I of 2 ovarian carcinomas and 2 of 3 malignant melanomas.

In a separate analysis, 12 of 16 colorectal carcinomas are positive for expression; 2 of 8 gastric adenocarcinoma are positive for expression, 2 of 4 esophageal carcinomas are positive for expression; 7 of metastatic aderiocarcinoma are positive for expression and I of 2 cholangiocarcinomas are positive for expression.

Expression level is tumor tissues is consistently higher than in normal tissues.

(20) PNA304sssT'37 With regard to normnal tissues, it appears that there is a weak signal in colon mucosa and breast epithelium. With regard to tumor tissues, expression Is seen in I of 2 non-small cell lung carcinomas, 2 of 2 colon cancers, I of 2 pancreatic cancers, I of 2 hepatocellular carcinomas, 3 of 3 endometrial carcinomas, I of 2 ovarian carcinomas and 2 of 3 malignant melanomas.

3 5 In a separate analysis, 12 of 16 colorectal carcinomas are positive for expression; 2 of 8 gastric adenocarcinoma are positive for expression, 2 of 4 esophageal carcinomas are positive for expression; 7 of mnetastatic adenocarcinoma are positive for expression and I of 2 cholangiocarcinom 85 are positive for expression.

iaxpistio level is tumor tissuev ismowtenwy highe tha in nomal tissues.

00 (21) With regard to normal tissues, 8trong xpmssion is seen in prostatie epithelium ad in a section Of peripheral nerve. Moderate expression is 8s011 in renal glomeruli. Weak expresion is seen in bile duct epithpliurn and mammary epithelium. Two sec-tions Of Stomach show weak expression in a subset of gastric glands. Sectionis of colon and small intestine show a signal in lamina propria and/or submucosa, most likely in small autonon-jo IND nerve fibers. Another independent ISH study fails to show expression in peripheral nerves of prostateotomy sections, despite adequate signal in prostatic epithelium.

In a separate analysis, 42 of 77 breast tumors are positive for expression.

In yet another analysis, 8 of I1I breast cancers are positive for expression, In yet another analysis, expression is seen in 1/2 non-snial cell lung carcinomas, 1/3 colorectal adenocarclnonas, 2/3 pancreatic adenocarcinomas, 1/1 prostate cancers, 1/3 transitional cell carcinomas, 3/3 renal 00 cell carcinomas, 3/3 endornetrial adenocarcinomas, 1/2 ovarian adenocarcinomas and 1/3 malignant melanorias.

JIn yet another analysis, expression Is seen in 42 of 45 prostate oancer.

In yet another analysis, expression is seen in ali of 23 primary and in 12 of 15 mnetastatic prostate analyzedJ.

In yet another analysis, expression is observed in the following carcinomas as follows: pancreatic adenocarcinoma 2 of 2 cases ame positive; colorectal adenocarcinoma 12 of 14 cases are positive; gastric adenocarcinoma 6 of 8 cases are positive; esophiageal carcinoma 2 of 3 cases are positive; chalangocacinorn I of I case is positive; metastatic adenocarcinoma (ovary, liver, lymiph node, diaphragm).- 8 of 12 cases are positive.

EXAJM4LE Veiiain n nlsi fDferential TAT PolA etDe E ression by EPS TAT polypep tides which may have been identified as a tamor antigen as described in one or more of the above Examples were analyzed and verified as follows, An expressed sequence tag (EST) DNA database (LWEEtn Incyte Phiarmaceuticals, Palo Alto, CA) was searched and interesting EST sequences were identified by QEPIS. Gene expression profiling In sillco (GJ3PIS) is a. bioinformatics tool developed at Genentech, Inc. that characterizes genes of interest for new cancer therapeutic targets. GEPIS takes advantage of large amounts of EST sequence and library information to determine gene expression profiles. GEPIS is capable of determining the expression profile of a gene based upou its proportional correlation with the number of its occurrences in EiST databases, and it works by integrating the LWPESEQ& EiST relational database and Genentech proprietary Information In a stringent and statistically meaningfuli way. In this example, GEPIS is used to identify and cross-validate novel tumor antigens, although GEPIS can be configured to perform either very specific analyses or broad screening tasks. For the initial screen, GEPIS is used to identify EiST sequences from tile L[FESJ3Q® database that correlate to expression in a particular tissue or tissues of interest (often a tumor tissue of interest).

The 13ST sequences Identified in this initial screen (or consensus sequences obtained from aligning multiple related and overlapping EiST sequences obtained from the initial screen) were then subjected to a screen Intended to identify the presence of at least one transmembrane domain in the encoded protein, Finally, GEPIS was employed 00 tc) geam te a corM lete tisse eprmsion profile tor the 'Various sequen ces of itr s. U i gti y e o o e s bioi~je vazou~TAT polyp,epu (and their encoding nuoceic acid molecule) wee identlfied as being significatly oveexprm~ed in a particular type of caocr or certan cancers as Compared to other Cancers ancVor normal lon"Gancerostissues, The rag of GEPIS hits is based upon se-veral criteria including, for example, tissue speolficity, tumor specificity and expression level in normal essential and/or normal proliferating tissues. Th following is a list Of molecules whose tissue expression profile as determidned by GEPIS evidences high tissue expression and significant upregulation of expression in a specific tumor or turnors as compared to other tumnor(s) and/or normal tissues and optionally relatively low expression in normal essential and/or nontna! proliferating tissues. As such, the molecules listed below are excellent polypeptide targets for the diagnosis and therapy of Qancer in mnammas.

IV Molecule DNA67962 CrAT2O7) DNA67962 (TAn2O7) DNA67962 (TAT2O7) DNA67962 (TA P207) DNA67962 (TA1207) DNA96792 (TAT739) DNA96792 (T17r39) DA96792 (TA P239) DNA96792 (TA1239) DNA96792 (TAT239) DNA96792 (TA'1239) DNA9 6792 CI'AT-239) DNA96792 (TA1239) DNA96964 (TAT193) 1)NA6964 (TAT193) DNA142915 (TATI99) DNA142915 (TATI99) DNA142915 (TAT199) DI'l208551 (TAT178) DNA208551 (TAT178) DNA210159 (TATl98) DNA210159 (TAT198) DNA210159 (TAr198) DNA2 10159 (TAT198) DNA2257Q6 (TATI94) DNA225706 (TAT194) DNA225706 (TATl94) DNA225706 (TATL94) D~NA225793 (TA1223) DNA225793 (TAT223) DNA225793 (TAT223) D)NA225796 (TATI 96) DNA225943 (TATI9S) DNA225943 (TATI95) IDNA225943 (TAT195) DNA226283 (TAT2Q3) DNA226283 (TAT7O3) DNA226283 (TAT2Q3) SO DNA226283 (TAT703) upresuation Of ePression in: Colon tumoruterus tumor lung tumor prostate tumor breast tumor 0olon tumor rectum tumor Pancreas tumor lung tumor stomach tumor esophagus tumor breast tumor uterus tumor breast tutnor brain tumor breast tumor ovayW tumor brintumnor prostate tumor colon tumor prostate tumor uterus tumor breast tumor ovarian tumor adrenal tumor prostate tumor breast tumor Connective tissue tumor ovarian tumor fallopian tube tumor kidney tumor breast tumor liver tumor lung tumor breast tumor uterine tumor breast tumor squamnous cell lung tumor colon tumor as omaRqd normal colon tissue normal uters tissue normal lung tissue normal Prostate tissue normal breast tissue normal colon tissue normal rectum tissue nlormal pancreas tissue normal lung tissue normal stomach tissue normal esophagus tissue normal breast tissue normal utierus tissue normal breast tissue normal brain tissue normal breast tissue normal ovary tissue normal brain tissue normal prostate tissue normal colon tissue normal Prostate tissue normal Uterus tissue normal breast tissue normal ovarian tissue normal adrenal tissue normal prostate tissue normal breast tissue normal connective tissue normal ovarian tissue normal fallopian tube tissue normal kidney tissue normal breast tissue normal liver tissue normal lung tissue nonnal breast tissue normal uterine tissue normal breast tissue normal' squamous cell lung tissue normal Colon tissue 00 00 MkI=We DNA226283

(TAMQ)

DNA226589 CrA F200) DNA226589 (TA1700) DNA226589 (rAl:200) DNA226589 (rAT200) DNA22 6622 (TAT2OS) DNA226622 (TAT7O5) DNA22 6622 fI'ATl205) DNA226622 (TAr705) DNA226622 (TAT205) DNA227545 (TAT197) DNA227611 (TAT175) DNA227611 (TATl75) 1 5 DNA227611 (rATr175) DNA227611 (TAT175) DNA261021 (TAT2O8) DNA261021 (TAT2O)8) DNA261021 (TAT2O8) DNA261021(TAp208) DNA260655 (TA1209) DNA260655 CTA P2O9) DNA2E665 CTAt-2O9) DNA260655 (TAT2O9) DNA260655 (rAT709) DNA2i60655 (TAT2O9) DNA260655 (TAT209) DNA260,655 (TAT2Q9) DNA2G665 (TAT2O9) DNA2665 (rAnoP) DNA260945 (TATi 92) DNA260945 (TATI92) DNA260945 (l'AT192) DNA260945 (TATI 92) DNA260945 (TAT192) DNA261001

(TATIBI)

IDNA26 1001 ('rATI 8l) DNA266928 (TAfl 82) DNA266928 (TATI 82) IDNA268035 (rAT-222) DNA277797 (TA-2 12) DNA277797 (TAT2I2) DNA77509 (TAT177) DNA77509 (TATi 77) DNA97993 (TAT735) DNA87993 (TAT235) DNA87993 (TAT235) 32NA87993 (TAl235) IDNA92980 (TAT2 34) IDNA92980O(TA1234) DNA92980 (TAr234) DNA9298c, (TAT234) DNA92980 (TAT-234) DNA92980 (TAr234) DNA92980 (TA'r234) ovariant tumor brain tumor Colo tumor breas tumor Prostate tumor squamous cell lung tumor kidney tumor uterine tumor breast tumor colon tumor breast tumor prostate tumor colon tumor breast tumor uterine tumor prostate tumor colon tumor breat tumor uterine tumor lung tumor coloi tumor breast tumor liver tumor ovarian tumor sin tumor spleen tumor niyeloid tumor muscle tumor bone tumor brain tumor breast tumor colon tumor ovarian tumor pancreatic tumor bone tumor lung tumor bone tumor lung tumor ovarian tumor breast tumor pancreatic tumor colon tumor testis tumor breast tumor prostate tumor colon tumor ovarian tumor bone tumor breast tumor cervical tumor colon tumor rectum tumor enidometrlal tumor liver tumor nrmOintissu normal Ovarin tissue nornal colon tissue normal breast tissue normal prostate tissue normal squamous cell lung tissue normal kidney tissue normal laterine tissue normal breast tissue normal colon tissue normal breast tissue normal prostate tissue normal colon tissue normal breast tissue normal uterine tissue normal prostate tissue normal colon tissue normal breast tissue normal uterine tissue normal lung tissue normal colon tissue normal breast tissue normal liver tissue nomnal ovarian tissue normal skin tissue normal spleen tissue nomnal myolold tissue normal muscle tissue normal bone tissue normal brain tisse nonmal breast tissue normal colon tissue normal ovarian tissue normal pancreatic tissue normal bone tissue normal lung tissue normal bone tissue normal lung tissue normal ovarian tissue normal breast tissue normal pancreatic tissue normal colon tissue normal testis tissue normal breast tissue normal prostate tissue normal colon tissue normal ovarian tissue normal bonec tissue normal breast tissue nomnal cervical tissue normal colon tissue normal rectum tissue normal endometrial tissue normal liver tissue 00 00 DNA92980 (TAnr34) DMA92980 (rAr234) DNA92980 (TAnr34) DM92980 (TA'r234) DNA92980 (TAT234) DNA92980 t(TAT234) DIWA2980 (TAT234) DNA9z9so CI'A1234) DNA92980 (TAT234) DNA9290 (rgr734) DNA105792 (TAV233) DN{A105792 (TAT233) DNA105792 (TAnr33) DNA105792 (TrM233) DNA 105792 (TAT233) DNA105792 (TA1233) DNA.1057g2 (TAT-233) DNA105792 (TAT233) DNA105792 (TA7733) DNA105792 Crxw73) DNA105792 (TAIT233) DNA105792 (TAT233) DNA105792 (TAPZ33) DNA105792 (rAT233) DNA 105792 (TAr233) DNAI 19474 (TAT226) DNA1 19474 (TAT226) DNA1 19474 (TAT226) DNA119474 (TAT226) DNA150491 (TAr204) DNA150491 (TAT2Q4) DNA280351 (TAT248) IS DNA280351 (TAT248) DNA1 50648 (TAT232) DNA150648 (TAT-232) DNA150648 (TAT232) DNA150648 (TAT232) DNA150648 (TAT232) DNA159648 (TAT232) DNA150648 (TAT232) DNA150648 (TAT232) DNA179651 (TAT-224) DNA179651 (TAT224) DNA179651 C17AT224) DNA17965 1 (TAT224) DNA225886 (TAT-236) DNA225886 (TAT236) DNA225886 (TAT736) DNA225886 (TAT2736) DNA225886 (TAT236) DNA225886 CrAT236) DNA225886 (TAT236) DNA225886 (TAT236) ovarian tumor Pancreatic tumor akin tumor soft tissue ture stomach tumor bladder tumor thyroid tumor esophagus tumor testis, tumor adrenal tumor breast tumor enidometial tumor esophagus tumor kidney tumor lung tumor ovarian tumor pancreatic tumor prostate tumor soft tissue tumor myomlold tumor thyroid tumor bladder tumor brain tumor testis tumor kidney tumor adrenal tumor uterine tumor ovarian tumor squamous cell lung tumor colon tumor squamous cell lung tumor colon tumor Iiver tumor breast tumor brain tumor lung tumor colon tumor rectum tumor kidney tumor bladder tumor colon tumor uterine tumor lung tumor kidney tumor breast tumor cdlon tumor rectum tumor ovarian tumor pancreas tumor prostate tumor bladder tumor testis tumor norMal Ovarian tissue normal pancreatic tissue normal sin tissue normal soft tissue normal stomach tissui normal bladder tissrue normal thyroid tissue normal esophagus tissue normal testis tissue normal adrenal tissue normal breast tissue normal endornetrial tissue normal esophagus tissue normal kidney tissue normal lung tissue normal ovarian tissue normal pancreatic tissue normal prostate tissue normal soft tissue normal myeloid tissue normal thyroid tissue normal bladder tissrue normal brain tissue normal testis tissue normal kidney tissue normal adrenal tissue normal uterine tissue normal ovarian tissue normal squamous cell lung tissue normal colon tissue normal squamous cell lung tissue normal colon tissue normal liver tissue normal breast tissue normal brain tissue normal lung tissue normal colon tissue normal rectum tissue normal kidney tissue normal bladder tissue normal colon tissue normal uterine tissue normal lung tissue normal kidney tissue.

normal breast tissue normal colon tissue normal rectum tissue normal ovarian tissue normal pancreas tissue normal prostate tissue normal bladder tissue normal testis tissue 00

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0 le717 (TAT185) DNA226717 (TAT185) DNA226717 (TAT185) DNA227162 (TA1225) DNA227162 (TA225) DNA271627804 (TAT27) DNA277804 (TAT247) DNA277804 CfA'247) DNA277804 (TAT247) DNA233034 (TATI 74) DNA233034(TATl74) DNA233034 (TATI74) DNA233034 (TATI74) DNA266920 (TAT214) DNA266920 (TAT214) DNA266920 (TAT214) DNA266920 (TAT2 14) DNA266921 (TAT220) DNA266921 (TAT220) DNA266921 (TAT220) DNA266921 (TAT220) DNA266922 (TAT221) DNA266922 (TAT21) DNA266922 (TAT221) DNA266922 (TAT221) DNA234834 (TATI79) DNA234834 (TAT179) DNA234834 (TAT179) DNA234834 (TAT179) DNA247587 (TAT216) DNA247587 A 216) DNA247587 (TAT216) DNA247587 (TAT216) DNA255987 (TAT218) DNA255987 TA17 28) DNA247476 (TATI 80) DNA247476 (TAT1 80) DNA247476 (TATI 80) DNA247476 (TATi 80) DNA247476 (TATI 80) DNA247476 (TATI 80) DNA247476 (TATI 80) DNA247476 (TATI 80) DNA260990 (TAT375) DNA26099 (TA'D375) DNA260990 (TAT375) DNA260990 TAT375) DNA260990 (TAT375) DNA260990 (TAT3 75) DNA260990 (TAT375) DNA260990 (TAT375) DNA261013 (TATI 76) DNA261013 (TATi76) DNA261013 (TAT 76) DNA261013 (TAT176) DNA261013(TAT76) Uregulation of expesion in: glioma brain tmor myoloid tumor uterine tumor prostate tumor myeloid tumor uterine tumor prostate tumor glioma brain tumor kidney tumor adrenal tumor glioma brain tumor kidney tumor adrenal tumor glioma brain tumor kidney tumor adrenal tumor glioma brain tumor kidney tumor adrenal tumor colon tumor uterine tumor breast tumor prostate tumor breast tumor prostate tumor bladder tumor lymphold tumor brain tumor breast tumor prostate tumor pancreas tumor brain tumor stomach tumor bladder tumor soft tissue tumor skin tumor kidney tumor prostate tumor pancreas tumor brain tumor stomach tumor bladder tumor soft tissue tumor skin tumor kidney tumor prostate tumor colon tumor small intestine tumor pancreatic tumor uterine tumor a gomrod to: normal glial tissue normal brain tissue normal myeloid tissue normal uterine tissue normal prostate tissue normal myeloid tissue normal uterine tissue normal prostate tissue normal glial tissue normal brain tissue normal kidney tissue normal adrenal tissue normal glial tissue normal brain tissue normal kidney tissue normal adrenal tissue normal glial tissue normal brain tissue normal kidney tissue normal adrenal tissue normal glial tissue normal brain tissue normal kidney tissue normal adrenal tissue normal colon tissue normal uterine tissue normal breast tissue normal prostate tissue normal breast tissue normal prostate tissue normal bladder tissue normal ymphoid tissue normal brain tissue normal breast tissue normal prostate tissue normal pancreas tissue normal brain tissue normal stomach tissue normal bladder tissue normal soft tissue normal skin tissue normal kidney tissue normal prostate tissue normal pancreas tissue normal brain tissue normal stomach tissue normal bladder tissue normal soft tissue normal skin tissue normal kidney tissue normal prostate tissue normal colon tissue normal small intestine tissue normal pancreatic tissue normal uterine tissue 00 00 hklem PHA26 1013 (TAT176) DNA261013 (TATl76) 11TA261013 (TATI76) DNA267342 (TAT2 13) DNA267342 (TAI3) DNA267342 (TAM23) DNA267342 (TAT-213) DNIA267342 (TAT213) DNA267342 (TAT2 13) DNA267342 (TAT-13) DNA267342 (TAT213) DNA267626 (TAT 17) DNA267626 (TAT2 17) DNA267626 (TAT2 17) DNA267626 (TAT217) DNA268334 (TAT2O2) DNA269238 (TAT215) DNA269238 (TAT2JS) DNA269238 (TAT215) DNA269238 ('Ar715) DNA272578 ('rA1238) DNA272578 (TA1238) DNA272578 (TAT238) DNA272578 (TAT238) DNA304853 (rAT376) DNA304853 (TAT376) DNA304853 (TAT376) DNA304853 (rAT376) IDNA304854 (TAT377) DNA304854 (rAm7 7 DNA304854 (TAT377) DNA304854 (TAT377) DNA304855 (TAT378) DNA304 855 (TAT378) DNA304855 (TAT378) DNA304855 (TAT378) DNA287971 (TA'3 79) DNA287971 (TAT379) DNA287971 (TAT379) DWA287971 (TAT3 79) DNA287971 (TAT379) DNA287971 (TAT379) DNA287971 (TAT379) DNA287971 (TAT379) u MrMj~OLof Edueson in Ovariatumor bladder tumor stomach tumor breast tumor uterine tumor colon tumor kidney tumor bladder tumor bone tumor ovarian tumor pancreatic tumor breast tumor colon tumor pancreatic tumor ovarian tumor kidney tumor colon tumor kidney tumor adrenal tumor bladder tumor adrenal tumor lung tumor ovarian tumor uterine tumor colon tumor uterine tumor breast tumor Prostate tumor colon tumor uterine tumor breast tumor Prostate tumor colon tumor uterine tumor breast tumor prostate tumor Prostate tumor pancreas tumor brain tumor stomach tumor bladder tumor soft tissue tumor skin tumor kidney tumor normal bladder tissue normal stomach tissue nonnal breast tissue normal uterine tissue normal colon tissue normal kidney tissue riormal bladder tissue normal bone tissue normal ovarian tissue normal pancreatic, tissue normal breast tissue normal colon tissue normal pancreatic tissue normal ovarian tissue normal kidney tissue normal colon tissue normal kidney tissue normal adrenal. tissue normal bladder tissue normal adrenal tissue normal lung tissue normal ovarian timse normal uterine tissue normal colon tissue normal uterine tissue normal breast tissue normal prostate tissue normal colon tissue normal uterine tissue normal breast tissue normal prostate tissue normal colon tissue normal uterine tissue normal breast tissue normal prostate tissue normal prostate tissue normal pancreas tissue normal brain tissue normal stomach tissue normal bladder tissue normal soft tissue normal skin tissue EXAhPLIE 6: Use of TAT as a hYbrization rbe The following method describes use of a nucleotide sequence encoding TAT as a hybridization probe for) diagnosis of the presence of a tumor in a mammnal.

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

Hybrdization and wahing offilte containing eitherlibraryDNAs is performed under the following high 0 stringency conditions. Hybridization of radiolabeled TAT-derived probe to the filters is performed in a solution O of 50% formamlde, 5x SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2x Denhardts solution, and 10% dextran sulfate at 42'C for 20 hours, Washing of the filters is performed in an c aqueous solution of 0.1x SSC and 0.1% SDS at 42 0

C,

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

E XMLE Expression of TAT in E. colt This example illustrates preparation of an unglycosylated form of TAT by recombinant expression in E.

N 10 cotl.

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

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

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

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

TAT may be expressed in E. coli in a poly-His tagged form, using the following procedure. The DNA encoding TAT is initially amplified using selected PCR primers. The primers will contain restriction enzyme sites which correspond to the restriction enzyme sites on the selected expression vector, and other useful sequences providing for efficient and reliable translation initiation, rapid purification on a metal chelation column, and proteolytic removal with enterokinase. The PCR-amplified, poly-His tagged sequences are then ligated into an expression vector, which is used to transform an E. coli host based on strain 52 (W3110 fuhA(tonA) Ion galE rpoHts(htpRts) clpP(laclq). Transformants are first grown in LB containing 50 mg/ml carbenicillin at 30*C with shaking until an O.D.600 of 3-5 is reached. Cultures are then diluted 50-100 fold into CRAP media (prepared by 139 nixing3.57 g(NH4SO 4 ,0.71 gsodiumcitrate,220,1.07gKC, 536gDifooyeastextraot, 5.36gSheffildhycase 0 SF in 500 mL water, as well as 110 mM MPOS, pH7.3, 0.55% (wv) glucose and 7 mM MgSO 4 and grown for approximately 20-30 hours at30 C with shaldig. Samples areremoved to verify expressionby SDS-PAGB analysis, and the bulk culture is centrifuged to pellet the cells. Cell pellets are frozen until purification and refolding.

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

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

Aggregated species are usually eluted at higher acetonitrile concentrations. In addition to resolving misfolded forms of proteins from the desired form, the reversed phase step also removes endotoxin from the samples.

Fractions containing the desired folded TAT polypeptide are pooled and the acetonitrile removed using a gentle stream of nitrogen directed at the solution. Proteins are formulated into 20 mM Hepes, pH 6.8 with 0.14 M sodium chloride and 4% mannitol by dialysis or by gel filtration using 025 Superfine (Pharmacia) resins equilibrated in the formulation buffer and sterile filtered.

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

XAMPL 8: Empression of TAT in mammalian cells This example Illustrates preparation of a potentially glycosylated form of TAT by recombinant expression in mammalian cells.

140 The vector, pRK5 (see EP 307,247, published Mareh 15, 1989), is employed as the expression vector.

SOptionally, the TAT DNA is ligated into pRK with selected restriction enzymes to allow insertion of the TAT DNA using ligation methods such as described in Sambrook et al., W The resulting vector is called

TAT.

In one embodiment, the selected host cells may be 293 cells. Human 293 cells (ATCC CCL 1573) aregrown S 5 to confluence in tissue culture plates in medium such as DMBM supplemented with fetal calf senrm and optionally, Snutrient components and/or antibiotics. About 10 gg pRKS-TAT DNA is mixed with about 1 g DNA encoding the VA RNA gene [Thimmappaya et al., C 31:543 (1982)] and dissolved in 500 tl of 1 mM Tris-HCl, 0.1 mM EDTA, 0227 CaC. To this mixture is added, dropwise, 500 of 50 mMHEPS (pH7.35), 280 mMNaC, 1.5 mM NaPO 4 and a precipitate is allowed to form for 10 minutes at 25'C. The precipitate is suspended and added to the Cl 10 293 cells and allowed to settle for about four hours at 37'C. The culture medium is aspirated off and 2 mi of N glycerol in PBS is added for 30 seconds. The 293 cells are then washed with serum free medium, fresh medium is 0added and the cells are incubated for about 5 days.

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

In an alternative technique, TAT may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al., Proc. Natl, Acad. S 2:7575 (1981). 293 cells are grown to maximal density in a spinner flask and 700 pg pRKS-TAT DNA is added. The cells are first concentrated from the spinner flask by centrifugation and washed with PBS. The DNA-dextran precipitate is incubated on the cell pellet for four hours. The cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and reintroduced into the spinner flask containing tissue culture medium, 5 pg/ml bovine insulin and 0.1 ggml bovine transferrin. After about four days, the conditioned media is centrifuged and filtered to remove cells and debris.

The sample containing expressed TAT can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography.

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

3 Epitope-tagged TAT may also be expressed in host CHO cells. The TAT may be subcloned out of the pRKS vector, The subclone insert can undergo PCR to fuse in frame with a selected epitope tag such as a poly-his tag into a Baculovirus expression vector. The poly-his tagged TAT insert can then be suboloned into a driven Vecor Oniig at selectionl make such as DHFR for selection of "talo clones, Finally, the CH0 ells 00 02an be trlinufected (as desoribed above) with the SV40 driven vector. Labeling may be performed, as described above, to verify expression. The culture Medium contaillilg the expr"Sed poly-His tage TAT can then be N ceoncetutd and purified by any selected method, such as by N1 2 t-chelate affinity chromatography.

TAT may also be expressed in CHO1 and/or COS cells by a transient expression procedure or in CR0 cells by another stable expression procedure.

INO Stable expression in CH0 cells is performed using the following procedure. The proteins ame expressed as an IgG construct (inunoadhesin), in which the coding sequences for the soluble forms extracellular domains) of the respective proteins are fused to an Ig~l constant region sequence containing the hinge, CR2 and CR2 domains and/or is a poly-Ris tagged form.

(1 10 Following PCR amplification, the respective DNAs are subcloned in a CH0 expression vector using (7K1 ~standard techniques as described in Ausubel et Current Protocols of ole2l EilM, Unit 3.16, John Wiley 00 and Sons (1997). CH0 expression vectors are constructed to have compatible restriction sites 5' and 3' of the DNA of interest to allow the convenient shuttling of cD)NA's. The vector used expression in CHO0 cells is as described in Lucas ot al., Nuol. cdsRs 24:9 (1774-.1779 (1996), and uses the SV40 early promotedtenhanoer to drive expression of the cDNA of interet and dihydrofolate reductaise (DHFR). DHFR expression permits selection for stable maintenance of the plasmid following transfection.

Twelve micrograms of the desired plastnid DNA is introduced into approximately 10 million CR0 cells using commercially available transfection reagents Superfect!* (Quiagen), Dosper* or Fugenee (Boehdinger Mannheimn). T"he cells are grown as described in Luca et al., iupra. Approximately 3 x 10' cells are fr'ozen in an ampule for further growth and production as described below.

The ampules containing the plasmid DNA are thawed by placement into water bath and mixed by vortexing. The contents ame pipetted into a centifuge tube containing 10 mLs of media and centrifuged at 1000 rpm for 5 minutes. The supernatant is aspirated and the cells are resuspended In 10 mL of selective media (0.2 Azm filtered PS20 with 5% 0.2 pum dialfitered fetal bovine serum). The cells are then aliquoted into a 100 muL spinner containing 90 ml,. of selective media. After 1-2 days, the cells are transferred Into a 250 mL spinner filled with 150 mL seleotive growth medium and incubated at 37 9 C. After another 2-3 days, 250 mL, 500 mL and 2000 mU spinners are seeded with 3 x 105 cellsfmL. The cell media is exchanged with fresh media by centrifugation and resuspension in production medium. Although any suitable CHO media may be employed, at production medium described in U. S. Patent No. 5,122,469, issued June 16, 1992 may actually be used. A 3 Lproduction spinner is seeded at 1.2 x 10~ cells/mi,, On day 0, the cell number pH ie determined. On day 1, the spinner is sampled and sparging with filtered air is commenced. On day 2, the spinner is sampled, the temperature shifted to 33'C, and 30 ml, of 500 gfLglucose and 0.6 mL of 10% antiloamn 35% polydimethylsiloxano emulsion, Dow Comning 365 Medical Grade Emulsion) taken. Throughout (he production, the pH- is adjusted as necessary to keep it at around 7.2. After days, or until the viability dropped below 70%, the cell culture is harvested by centrifumgation and filtering through 3S a 0.22 pmr filter. The filtrate was either stored at 4'C or immediately loaded onto columns for purification.

For the poly-11is tagged constructs, the proteins are purified using a Ni-NTA column (Qiagen). Before purification, imidazole Is added to the conditioned media to a concentration of 5 nmM. The conditioned media is pumped oto 6nil -NTA coluMnNqUMli in'JaZ 20MHV, pe7.4, buffer conalaingo.3 MNaC1 and 5 n' 00 ~Itidazole at a flow rate of 4.5 mI/mm. at 4T., After loading, thie column is washed with adtonal equilibratj 0 1 00 buffer and the protein eluted with equilibration buffer containing 0.25 M imilaole, The highly purified protein is subsequently desalted Into a stiie buffer containing 10 mM-Hepes, 0. 14 M NaCI and 4% marmdtol, pH 6.8, wvith a 25 ml G25 Superfine (Pharinacia) column and stored at lImmunoadhesln (Pc-containing) constructs are purified from the conditioned media as follows, The INDconditioned medium is pumped onto a 5 nil Protein A column (Phannacla) which had been equilibrated in 20 rnMv 0 Na phosphate buffer, pH 6.8. After loading, the column is washed extensively with equilibration buffer before elution with 100 r&M citric; acid, pH 3.5. the eluted protein is immrediately neutralized by collecting 1 m! fractions into tubes containing 275 gcL of 1 M Tris buffer, pH 9. The highly purified protein is subsequently desalted into c-i 1 storage buffer as described above for the poly-His tagged proteinis. The homogeneity is assessed by SIDS polyacrylamide gels and by N-terminal amino acid sequencing by Hdman degradation.

00 Certain of the TAT polypeptides disclosed herein have been successfully expressed and puified using this technique(s).

X PL xreinofTTiYet The Mulowing method describes recombinant expression of TAT in yeast.

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

Yeast cells, such as yeast strain AB 110, can then be transformed with the expression plasnids described above and cultured in selected fermentation media. The transformed yeast supernatants can be analyzed by 2S precipitation with 1Q% trichloroacetic acid and separation by SDS-PACIE, followed by staining of the gels with Coomassie Blue stain.

Recombinant TAT carn subsequently be isolated and purified by removing the yeast cells fr-om the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters. The concentrate containing TAT may further be purified using selected column chromatography resins.

Certain of tie TAT polypeptidles disclosed herein have been successfully expressed and purified using this technique(s).

EA LE10 Extpression of TATinulvrsctdn e tCll The following method describes recombinant expression of TAT in Baculovirus-infected insect cells.

3S The sequence coding for TAT is fused upstream of an epitope tag contained within a baculovirus expression vector. Such epitope tags include poly-his tags and immunoglobulin tags (like Fc regions of IgG). A variety of plastaids may be employed, including plasmids derived from conunoecially available plasmids such as P'VL1393 (Novagen). Briefly, the sequence encodin TAT or the desirod Portion of the codin sequence of TAT 0C) Slch as the' sequence encOdin an extracellular domain Of at t(Unsmenirae protein or the sequence mnoding the 0mature protelnifthe protein is extracellularls amplfiedb Cwihrm cupentytote'ad3'ego c-i ~~The 5'primer may incorporate flanking (selected) restriction enzyme sites. The product is thn dgetdwhtoe selected restriction enzymes and subcloned into the expression vector.

Recomnbinant baculovirus is generated by co-tusfboting the above plasmidd and Baculo~old' virus IDDNA (Phariningen) into .Spodopt era frugiperda cells (ATCC CRL 1711) using lipofectin (commnercially available from GlIlCO-BRL). After 4 5 days of incubation at 28'C, the released viruses are harvested and used for further amplifications, Viral infection and protein expression are performed as described by O'Reilley et al., Baclovrusexresionvecor: A LaboratorvYManual, Oxford: Oxford University Press (1994).

N 0Expressed poly-his tagged TAT can then be purified, for "xample, by Ni 2 +-chelate affinity chromatography 0- 1 N- as follows. Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al., h~t~q 00 2362:175-179 (1993). Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 miLHepes, pH 7.9; 12.5 iM MgC 1 0. 1 raM 13DTA,~ 10% glycerol; 0.1% NP-40; 0.4 M KOI), and sonicated twice for 20 seconds on ice. The sonicates are cleared by centrifuzgation, and the supernatant is diluted 50-fold in loading buffer (50 mM phosphate, 300 mM MaCI, 10% glycerol, pH 7.8) and filtered through a 0.45 4 umn filter, A N1 2 "-NTA agarose colun (commercially available f-rm Qiagen) Is prepared with a bed volume of 5 niL, washed with 25 mL of water arnd equilibrated with 25 niL of loading buffer, The filtered cell extract is loaded onto the column at 0.5 niL per minute.

The column is washed to baseline A, 0 with loading buffer, at which point firaction collection is started. Next, the column is washed with a secondary wash buffer (SO aiM phosphate; 300 aiM NaCI, 10% glycerol, pH which elutes nionspecifically bound protein. After reaching A2, baseline again, the column is developed with a 0 to 500 M~M idazolgmdient in the secondary wash buffer. One niL fi-actions are collected and analyzed by SDS-PAGE and silver staining or Western blot with Nilt-NTA-conjugated to alkaline phosphatase (Qiagen). Fractions containing the eluted His 10 -taggod TAT are pooled and dialyzed against loading buffer.

Alternatively, purification of the IgG tagged (or Fc tagged) TAT can be performed using known chromatogrphy techniques, including for instance, Protein A or protein G column chromatography.

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

EXAIILEl Preparation of Antibodies that Bind A This example illustrates preparation of monoclonal antibodies which can specifically bind TAT, Techniques for producing the monoclonal antibodies are known in the art and are described, for instance, in Goding, supra. Imniunogens that may be employed include purified TAT, fusion proteins containing TAT, and cells expressing recombinant TAT on the cell surface. Selection of the inununogeii can be made by the skiled artisan without undue experimentation.

Mice, such as Balb/c, are immnunized with the TAT immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally In an amount ftrm 1-100 miicrograms, Alternatively, the inuiiunogen is emulsified in IPL-TDM adjuvant (Ribi hjmmunochemical Research, Hamilton, MAT) and injected into the animal18 hnd foot pads. The immunized mice ame hen boosted 10 to 12 days later with additional inunwloen 00 emulsified in the selected adjuvant Thereafter, for several weeks, the mice may also be boosted with additional imnmunization irJectious. Serum samples may be periodically obtained from the mice by retro-otbital bleeding for testing in ELISA assays to detect anti-TAT antibodies.

After a suitable antibody titer has been detected, the animals "positive" for antibodies can be 1flJe4ted with a final intravenous injection of TAT. Th=e to four days later, the mice are sacrificed and the spleen cells are INDharvested. The spleen cells are then fused (using 35% polyethylene glycol) to a selected niurine myelorna cell line such as P3X63AgJ, 1, available from ATCC, No. CR1 1597. The fusions generate hybridoma cells which can then be plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) mediumn to inhibit proliferation of non-fijsed cells, mycloma hybrids, and spleen cell hybrids, (Nl 10 The hybridoma. cells will be screened in an ELISA for reactivity against TAT. Determination of "positive" hybridoma cells secreting the desired monoclonal antibodies against TAT is within the skill in the aft, 00 ~The positive hybridoma cells can be injected intraperitonecally into synigeneic Balb/c mice to produce ascites containing the anti-TAT mionoolonal antibodies. Alternatively, the hybridoma ceils can be grown in tissue cultur flasks or roller bottles. Purification of the monoclonal antibodies produced in the ascitea can be accomplished using ammnium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed.

QXAWyILE 2: Purification of TAT Foly-tr-ie 2s Using-Spec fic Antibodies Native or recomibinant TAT polypeptides may be purified by a variety of standard techniques in the ait 210 of protein purification. For example, pro-TAT polypeptide, mature TAT polypeptide, or pie-TAT polypeptide Is purified by immunoaffinity chromatography using antibodies specific for the TAT polypeptide of interest, In general, an imrmurroaffin~ity column is constructed by ovalently coupling the anti-TAT polypeptide antibody to an activated chromatographic resin.

Polyclonal inimunoglobulins ame prepared from immune sera titherby precipitation with ammnoniumi sulfate or by purification on immobilized Protein A (Pharnnacia LKB Biotechnology, Piscataway, Likewise, monoclonal antibodies are prepared from mouse ascites fluid by anrinpug sulfate precipitation or chromatography on immobilized Protein A. Partially purified imunouglobulin is covalently attached to a chromatographiic resin such as CnBr-actjvated SEPHAROSE7hI (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is. blocked, and the derivative resin is washed according to the manufacturer's instructions.

Such an immunoaffinity column is utilized in the purification of TAT polypeptide by preparing a fraction from cells containing TATpolypeptide in a soluble form, This preparation is derived by solubilization of the whole cell or of a subcellular fraction obtained via differential centrifugation by the addition of detergent or by other methods well known in the art. Alternatively, soluble TAT polypeptide containing a signal sequence may be secreted in useful quantity into the medium in which the cells are grown.

A soluble TAT polypoptide..containing preparation is passed over the innunoafinity column, and the column is washed under conditions that allow the preferential absorbance of TAT polypeptide high ionic strength buffers in he presenc of detgent). Then, the oolumn is eluted under conditions that disrupt Santibody/TAT polypeptide binding a low pH buffer such as approximately pH 2-3, or a high concentration of a chaotrope such as urea or thiocyanate ion), and TAT polypeptide is collected.

2AMBLE In Vitro Tumor Cell Killing Assay cells expressing the TAT polypeptide of interest may be obtained using standard expression Nvector and cloning techniques. Alternatively, many tumor cell lines expressing TAT polypeptides of interest are publicly available, for example, through the ATCC and can be routinely identified using standard ELISA or FACS I analysis. Anti-TAT polypeptide monoclonal antibodies (and toxin conjugated derivatives thereof) may then be employed in assays to determine the ability of the antibody to kill TAT polypeptide expressing cells In vitro.

N 10 For example, cells expressing the TAT polypeptide of interest are obtained as described above and plated N into 96 well dishes. In one analysis, the antibody/toxin conjugate (or naked antibody) is included throughout the Scell incubation for a period of 4 days. In a second independent analysis, the cells are incubated for 1 hour with c the antibody/toxin coqugate (or naked antibody) and then washed and incubated in the absence of antibody/toxin conjugate for a period of 4 days. Cell viability is then measured using the CellTiter-Glo Luminescent Cell Viability Assay from Pomega (Cat# 07571). Untreated cells serve as a negative control.

EXAMPLE 14: In Fiv Tumor Cell Killin Assay To test the efficacy of conjugated or unconjugated anti-TAT polypeptide monoclonal antibodies, anti- TAT antibody is injected intraperitoneally into nude mice 24 hours prior to receiving tumor promoting cells subcutaneously in the flank. Antibody injections continue twice per week for the remainder of the study. Tumor volume is then measured twice per week.

The assignee of the present application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions, the materials will be promptly replaced on notification with another of the same. Availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by the construct deposited, since the deposited embodiment is intended as a single illustration of certain aspects of the invention and any constructs that are functionally equivalent are within the scope of this invention. The deposit of material herein does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustrations that it represents. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

The entire disclosure in the complete specification of our Australian Patent Application No. 2002330015 is by this cross-reference incorporated into the present specification.

Claims (19)

1. A method of inhibiting the growth of a cancer cell expressing a Tumor-associated Antigenic Target 201 (TAT 201) polypeptide of SEQ ID NO: 92, comprising administering an antagonist of said TAT 201 polypeptide, wherein the antagonist is selected from the group consisting of anti-TAT 201 antibodies and fragments thereof, fragments and variants of native TAT 201 polypeptides, peptides, IN antisense oligonucleotides, and small organic molecules.

2. Use of an antagonist of a Tumor-associated Antigenic Target 201 (TAT 201) polypeptide of SSEQ ID NO: 92 in the manufacture of a medicament for inhibiting the growth of a cancer cell expressing said TAT 201 polypeptide, wherein the antagonist is selected from the group consisting of Santi-TAT 201 antibodies and fragments thereof, fragments and variants of native TAT 201 00 polypeptides, peptides, antisense oligonucleotides, and small organic molecules.

3. A method according to claim 1 or a use according to claim 2, wherein the growth of the cancer cell is, at least in part, dependent upon the growth potentiating effect of said TAT 201 polypeptide.

4. A method or use according to any one of claims I to 3, wherein the cancer cell is a colon cancer cell. A method or use according to any one of claims 1 to 4, wherein the inhibition is complete.

6. A method or use according to claim 5, wherein said inhibition induces death of the colon cancer cell.

7. A method of prevention or treatment of a colon tumor in a mammal, comprising administering an antagonist of a Tumor-associated Antigenic Target 201 (TAT 201) polypeptide of SEQ ID NO: 92 to said mammal, wherein the antagonist is selected from the group consisting of anti-TAT 201 antibodies and fragments thereof, fragments and variants of native TAT 201 polypeptides, peptides, antisense oligonucleotides, and small organic molecules.

8. Use of an antagonist of a Tumor-associated Antigenic Target 201 (TAT 201) polypeptide of SEQ ID NO: 92 in the manufacture of a medicament for the prevention or treatment of a colon tumor in a mammal, wherein the antagonist is selected from the group consisting of anti-TAT 201 antibodies and fragments thereof, fragments and variants of native TAT 201 polypeptides, peptides, antisense oligonucleotides, and small organic molecules.

9. A method according to claim 7 or a use according to claim 8, whcrein the growth of the tumor is, at least in part, dependent upon the growth potentiating effect of said TAT 201 polypeptide. 147 A method or use according to any one of claims 7 to 9, wherein said TAT 201 polypeptide is 00 expressed by the cells of said colon tumor. O O

11. A method or use according to any one of claims 7 to 10, wherein said treatment induces a Sreduction in the size of the colon tumor. IN

12. A method or use according to any one of claims 7 to 11, wherein said treatment induces death of colon tumor cells.

13. A method of prevention or treatment of a colon proliferative disorder associated with increased expression or activity of a Tumor-associated Antigenic Target 201 (TAT 201) polypeptide of SEQ ID NO: 92, comprising administering an antagonist of said TAT 201 polypeptide, wherein the 00 antagonist is selected from the group consisting of anti-TAT 201 antibodies and fragments thereof, fragments and variants of native TAT 201 polypeptides, peptides, antisense oligonucleotides, and small Sorganic molecules.

14. Use of an antagonist of a Tumor-associated Antigenic Target 201 (TAT 201) polypeptide of SEQ ID NO: 92 in the manufacture of a medicament for the prevention or treatment of a colon proliferative disorder associated with increased expression or activity of said TAT 201 polypeptide, wherein the antagonist is selected from the group consisting of anti-TAT 201 antibodies and fragments thereof, fragments and variants of native TAT 201 polypeptides, peptides, antisense oligonucleotides, and small organic molecules. A method or use according to any one of claims I to 14, wherein said antagonist is an anti- TAT 201 antibody or a fragment thereof.

16. A method or use according to claim 15, wherein said antibody is: a monoclonal antibody; a chimeric antibody; or a human or a humanized antibody.

17. A method or use according to claim 15, wherein said antibody fragment is selected from the group consisting of Fab, Fab', F(ab') 2 Fv fragments, diabodies, linear antibodies, single-chain antibody molecules and multispecific antibodies formed from antibody fragments.

18. A method of diagnosing the presence of a tumor in a mammal, wherein the method comprises detecting the level of expression of a gene encoding a TAT 201 polypeptide of SEQ ID NO: 92 in: a test sample oftissue cells obtained from said mammal; and a control sample of known normal non-cancerous cells of the same tissue origin or type, 148 wherein a higher level of expression of the TAT 201 polypeptide in the test sample, as 00 compared to the control sample, is indicative of the presence of tumor in the mammal from which the C test sample was obtained. (N

19. A method of diagnosing the presence of a tumor in a mammal, wherein the method comprises: contacting a test sample comprising tissue cells obtained from the mammal with an 0 antibody, oligopeptide or small organic molecule which binds to a TAT 201 polypeptide of SEQ ID NO: 92; and detecting the formation of a complex between the antibody, oligopeptide or small Sorganic molecule and the TAT 201 polypeptide in the test sample, Swherein the formation of a complex is indicative of the presence of a tumor in the mammal. O 00

20. A method according to claim 19, wherein the TAT 201-binding antibody, TAT 201-binding oligopeptide or TAT 201-binding organic molecule employed is detectably labeled.

21. A method according to claim 19 or claim 20, wherein the TAT 2 01-binding antibody, TAT

201-binding oligopeptide or TAT 201-binding organic molecule is attached to a solid support. 22. A method according to any one of claims 18 to 21, wherein the tumor is a colon tumor. 23. A method according to any one of claims 18 to 22, wherein said mammal is a human. 24. A pharmaceutical composition for treating a cancerous tumor expressing a Tumor-associated Antigenic Target 201 (TAT 201) polypeptide of SEQ ID NO: 92, said composition comprising an effective amount of antagonist of said TAT polypeptide in admixture with a pharmaceutically acceptable carrier, wherein said antagonist is selected from the group consisting of anti-TAT 201 antibodies and fragments thereof, fragments and variants of native TAT 201 polypeptides, peptides, and small organic molecules. A method or use according to any one of claims I to 17, or a composition according to claim 24, wherein said antagonist is conjugated to a growth inhibitory agent or cytotoxic agent. 26. A method, use or composition according to claim 25, wherein said cytotoxic agent is a toxin. 27. A method, use or composition according to claim 26, wherein said toxin is selected from the group consisting of maytansinoid, calicheamicin, radioactive isotopes and nucleolytic enzymes. 28. A method, use or composition according to claim 27, wherein said toxin is a maytansinoid. 149 29. A kit comprising: 00 a pharmaceutical composition according to any one of claims 24 to 28; and a package insert or label indicating a beneficial use for said pharmaceutical composition in the treatment of cancer. A pharmaceutical composition according to any one of claims 24 to 28, or a kit according to IN claim 29, wherein said cancer is colon cancer. 31. A method according to any one of claims 1, 7, 13, 18 and 19, a use according to any one of claims 2, 8 and 14, a pharmaceutical composition according to claim 24, or a kit according to claim 29, C("I substantially as herein described with reference to any one or more of the examples or figures. 00 0", 0O 150