ZA200602834B - Fully human antibodies against human 4-1BB (CD137) - Google Patents

Description

FULLY HUMAN ANTIBODIES AGAINST HUMAN 4-1BB(CD 137)

Field of the Invention: :

The invention is directed to fully Jhuman antibodies and, more specifically, to fmlly human antibodies to human 4-1BB (CD137).

E3ACKGROUND OF THE INVENTIONN:

An extensive body of evidence hans unequivocally demonstrated that some degree of immune response against cancesr exists in humans and animamls. In cancer’ patients, cellular components of the immune system are able to recogn_ize antigens. e=xpressed by tumor cells, such as differemtiation of oncofetal antigens or mutated g=ene products (S. Rosenberg, Nature, 41 71:380-4 (2001); P. van der Brmuggen et al.,

Tsmmunological Rev., 188:51-64 (2002)). A number of clinical studies= have shown that tumor-infiltrating lymphocytes have favorable prognostic significance (E. Halapi, : 15 Med. Oncol., 15(4):203-11 (1998); Y. Naito et al, Cancer Res., 58(16):3491-4 ( 1998); L. Zhang et al., N.E. J. Med., 348(3):203-13 (2003)). Furtherr=more, treatment vith immunomodulators, such as cytokirmes or bacterial products, cancer vaccines, or adoptive immunotherapy has led to tumo-r regression in a number of paatients (S.

Rosenberg, Cancer J. Sci. Am. 6(S):2 (20300); P. Bassi, Surg, Oncol., 1_1(1-2):77-83 (22002); S. Antonia et al., Current Opiniomn in Immunol., 16:130-6 (200-4). Despite tihese responses, immunity against cancer- frequently fails to effectively eliminate toamor cells. The causes for this failure caan be grouped into three majomr categories: (X) impaired tumor recognition by immume cells, either by variable expsression of twamor antigens or reduced expression of «lass I major histocompatibili-ty complex (EMHC); (ii) immunosuppressive tumor mmicroenvironment, as a result of secretion of imhibitory cytokines by tumor cells (e.g., TGF-B); and (iii) poor tumor irnmunogenicity due to the lack of expresssion of co-stimulatory molecmules on tumor c-ells, which results in the inability of the tumor cells to effectively stimulate T-cells.

An dvances in our understanding of the requirements for tumor antigen recognition and irmmune effector function indicate that a gpotential strategy to enhance =n anti-tumor irnmune response is to provide co-stimulaation through an auxiliary mo Jecule. Tumor amtigen-specific T-cells require costimulation to initiate and maintain e=ffector functionss. Thus, therapies that target costimulatory molecules can be applied to modulates and enhance the immune response to tumors.

T he current model for T-cell activation postualates that naive T-cells require two signals for full activation: (i) a signal provided through the binding of processed antigens ‘presented to the T-cell receptor by major hiistocompatibility complex (MHC) class I molecules; and (ii) an additional signal provided by the interaction of co- stimulatory molecules on the surface of T-cells and their ligands on antigen presenting cells. Re=cognition of an antigen by a naive T-cell is insufficient in itself to trigger T- cell activ-ation. Without a co-stimulatory signal, T-célls may be eliminated either by death or Bby induction of anergy. Signaling through the CD28 costimulatory molecule appears teo be key for the initiation of T-cell responses. However, CD137 (4-1BB) signaling has been shown to be primordial for the m aintenance and expansion of the immune mresponse to antigens, as well as, for the gen eration of memory T-cells.

CD137 (4-1BB) is a member of the tumor necrosis receptor (TNF-R) gene family, w=hich includes proteins involved in regulation of cell proliferation, differenti ation, and programmed cell death. CD137 is a 30 kDa type I membrane glycoprotein expressed as a 55 kDa homodimer. The receptor was initially described in mice (3. Kwon et al., PN.A.S. USA, 86:1963-7 ( 1989)), and later identified in humans (BM. Alderson et al., Bur. J. Immunol, 24: 2219-27 (1994); Z. Zhou et al.,

Immunol. Lett., 45:67 (1995)) (See, also, Published PCT Applications W095/07984 and WO9 6/29348, and U.S. Patent No. 6,569,997, hereby incorporated by reference (See, SEQ ID NO:2.)). The human and murine formx s of CD137 are 60% identical at the amino» acid level. Conserved sequences occur in the cytoplasmic domain, as well as 5 other regions of the molecule, indicating that these residues might be important for function of the CD137 molecule (Z. Zhou et al., Trnmunol. Lett., 45:67 (1995)).

Expression of CD137 has been shown to be predomimantly on cells of lymphoid lineage su_ch as activated T-cells, activated Natural Killer (NK) cells, NKT-cells,

CD4CD25 regulatory T-cells, and also on activated thymocytes, and intraepithelial lymphocytes. In addition, CD137 has also been showwn to be expressed on cells of myeloid origin like dendritic cells, monocytes, neutrophils, and eosinophils. Even though CID137 expression is mainly restricted to imnmme/inflammatory cells, there have been reports describing its expression on endothelial ceells associated with a small number of tissues freom inflammatory sites and tumors.

Functional activitiess of CD137 on T-cells have been. amply characterized.

Signaling through CD137 in the presence of suboptimal dosses of anti-CD3 has been ‘demonstrated to induce T—cell proliferation and cytokine symthesis (mainly IFNy), . and to inhibit activated ce Il death. These effects have been observed with both murine and human T-cells (W. Shuford et al., J. Exp. Med., 186(1):47-55 (1997); D.

Vinay et al., Semin. Immuanol., 10(6):481-9 (1998); D. Ladesrach et al, Int. Immunol., 14(10):1155-67 (2002)). Mn both humans and mice, co-stim_ulation enhances effector functions, such as IFN-y peroduction and cytotoxicity, by augementing the numbers of antigen-specific and effector CD8+ T-cells. In the absence of anti-CD3 signaling, stimulation through CD1387 does not alter T-cell function, irdicating that CD137 is a co-stimulatory molecule.

The physiological events observed following CD13 stimulation on T-cells are mediated by NF-xB ad PI3K/ERK 1/2 signals with sepaarate physiological functions. NF-kB signals trigger expression of Bcl-xy,, an a mnti-apopotic molecule, thus resulting in increased survival, whereas PI3K and ERKZ1/2 signals are ’ specifically responsible for CD137-mediated cell cycle progression (H. Lee et al., J. i Immunol., 169(9):4882-8 (2002)). The effect of CD137 activation on the inhibition of activation-induced cell death was shown in vitro by Hurt=ado et al. (J. Hurtado et al., J. Immunol., 158(6):2&600-9 (1997)), and in an ir vivo system in which anti-

CD137 monoclonal antibcodies (mabs) were shown to produ_ce long-term survival of superantigen-activated CILD8+ T-cells by preventing clonal deletion (C. Takahashi et al., J. Immunol., 162:50377 (1999)). Later, two reports demonstrated, under different experimental conditions, t-hat the CD137 signal regulated booth clonal expansion and survival of CD8+ T-cells «D. Cooper et al., Eur. J. Immunol. 32(2):521-9 (2002); M.

Maus et al., Nat. Biotechmol., 20:143 (2002)). Reduced apowptosis observed after co- stimulation correlated with increased levels of Bel-yxy, in CD®8+ T-cells, while Bel-2 expression remained unch_anged. Up-regulation of the anti-- apoptotic genes Bel. and bfl-1 via 4-1BB was shown to be mediated by NF-kB activation, since PDTC, an NF- kB-specific blocker, inhibwited 4-1BB-mediated up-regulaticon of Bel (H. Lee et al.,

J. Immunol., 169(9):4882~—8 (2002)). On the other band, clconal expansion of activated

T-cells appears to “be mediated by increased expression of «cyclins D2, D3, and E, and down-regulation-o-f the p27 protein. This effect occurs 3n both an IL-2 dependent and independent femshion (H. Lee et al., J. Immunol, 169(9-):4882-8 (2002).

Altogether, CD137 stimulation results in enhanced expansion, survival, and effector functions of newly primed CD8+ T-cells, acting, in part, directly on these cells. Both CD4+ and CD8+ T-cells have been shown to respond to CD137 stimulation, however, it appears that enhancement of T-ce 11 function is greater in

CD$+ cells (W. Shhuford et al., J. Exp. Med., 186(1):47-55 (1997); I. Gramaglia et al.,

Eur. J. Immunol., 30(2):392-402 (2000); C. Takahashi et aul, J. Immunol., 162:5037 (1999). Based or the critical role of CD137 stimulation in CD8+ T-cell function and survival, manipulation of the CD137/CD137L system prowides a plausible approach for the treatment Of tumors and viral pathogens. .

Recently, he constitutive expression of CD137 on. freshly isolated dendritic cells (DCs) was demonstrated in mice (R. Wilcox et al., J. Immunol, 169(8):4230-6 (2002); T. Futaga~wa et al., Int. Immunol., 14(3):275-86 (2002)) and humans (S. Pauly et al., J. Leukoc. Biol. 72(1):35-42 (2002)). These reportss showed that stimulation of

CD137 on DCs re=sulted in secretion of IL-6 and IL-12, amd, more importantly, it enhanced DC ability to stimulate T-cell responses to alloantigens. Furthermore, Pan et al. demonstrate d that CD137 signaling in DCs resulted in upregulation of MHC

Class I and costimulatory molecules, and produced an increased ability of DCs to : infiltrate tumors (CP. Pan et al., J. Immunol., 172(8):4779-89 (2004)). Therefore,

CD137 costimulation on DCs appears to be a novel pathway for proliferation, maturation, and nigration of DCs.

Activated Natural Killer (NK) cells express CD13 <7 following stimulation with cytokines (I. Melero et al., Cell Immunol. 190(2):167-72 (1998); R. Wilcox et al., J.

Immunol., 169(8) :4230-6 (2002)). Several reports demomstrated that NK cells appear to be critical for the modulation of the antitumor immune response induced by agonistic CD137 antibodies ((I. Melero et al., Cell Immummol., 190(2):167-72 (1998);

R. Miller et al., J. Immunol., 169(4):1792-800 (2002); R. “Wilcox et al., J. Immunol., 169(8):4230-6 (2002)). Depletion of NK cells significantly reduces the antitumor activity of anti-CRD137 mabs. Ligation of CD137 on NK cells induces proliferation and IFN-y secretieon, but does not affect their cytolytic activity. Notably, in vitro,

WNO 2005/035584 PCT/UJS2004/033587

CD137-stimulated NK cells presented an immunoregulatory or "helper" activity for

CD8+ cytolytic T-cells resulting in expansion of activated T-cells. Therefore, CD137_ : signaling on NK cells may mod-ulate innate immunity to tumors.

A paradoxical effect has been described for CD137 stimulation in —that agonistic CD137 antibodies cam induce suppression of the humoral respormses to T-cell . antigens in primates and mouse models (H. Hong et al., J. Immunother., 2 3(6):613-21 (2000); R. Mittler et al., J. Exp. Med., 190(10):1535-40 (1999). Notably—, CD137 agonistic antibodies were showmn to produce significant amelioration of the= symptoms associated with antibody dependent autoimmune diseases such as systemic lupus erythematosus and experimenta 1 autoimmune encephalomyelitis (J. Foell =etal., N.Y. : Acad. Sci., 987:230-5 (2003); YZ. Sun et al., Nat. Med., 8(12):1405-13 (20 02).

Recently, Seo et al. demonstrated that, in a mouse model of rheumatoid arthritis, : treatment with an agonistic anti -CD137 antibody prevented the developmeent of the disease, and remarkably, blocked disease progression (S. K. Seo et al., Na_t. Med. 10;1099-94 (2004)). The mechanism responsible for this effect has not been well defined, but in the model of rheumatoid arthritis it was shown that treatme=nt with a

CD137 agonistic antibody resulted in the expansion of IFN-y-producing C_D11C-

CD8+ T cells. IFN-y in turn stimulated dendritic cells to produce indolammine-2,3- dioxygenase (IDO), which exerts immuno-suppressive activities. It has allso been postulated that CD137 signaling on antigen-activated CD4 + T-cells resulmts in induction of IFNy secretion whaich activates macrophages. Activated macrophages can in turn produce death signals for B cells. Continuous signaling througeh CD137 on CD4+ T-cells may subsequemtly induce activation-induced cell death (=AICD) of ) these CD4+ activated T-cells. Therefore, by eliminating antigen-activatecd T-cells and

B cells, a reduced antibody resp» onse is observed and, consequently, a drarnatic reduction of Th2-mediated inflammatory diseases is observed (B. Kwon e—tal., J.

Immunol., 168(11):5483-90 (200 02)). These studies suggest a role for the muse of agonistic CD137 antibodies for the treatment of inflammatory or autoimmune diseases, without inducing a geraeral suppression of the immune system. :

The natural ligand for CID137, CD137 ligand (CD137L), a 34kDa gglycoprotein member of the TNF superfamily, is detected mainly on activated antigen-poresenting cells (APC), such as B cells, macrophages, dendritic cells, and also on mu—rine B-cell

WO) 2005/035584 PCT/US2004/033587 lymphomas, activated T-cells, and human carcinoma lines of epithelial origin (FR.

Goodwin et al., Bur. J. Immunol, 23(10):2631-41 (1993); Z. Zhou et al., Immurol.

Lett., 45:67 (1995); H. Salih et al., J. Immunol, 165(5):2903-10 (2000)). Humaan

CD137L shares 36% homology with its murine counterpart (M. Alderson et al., Eur.

J. Immunol, 24: 2219-27 (1994)).

In addition to delivering signals to CD137-expressing cells, binding of CD137 to CD137L initiates a bidirectional sigral resulting in functional effects on CD1 37L- expressing cells. Langstein et al. demonstrated that binding of CD137-Ig fusiom. protein to CD137L on activated monocytes induced the production of IL-6, IL-8, and

TNF-a, upregulated ICAM, and inhibited IL-10, resulting in increased adherence (J.

Langstein et al., J. Inmunol., 160(5):24 88-94 (1998)). In addition, proliferatior_ of monocytes was demonstrated along with a higher rate of apoptosis (J. Langstein_ et al., — J. Leukoc. Biol,, 65(6):829-33 (1999)). These observations were confirmed by t-he studies of Ju et al. (S. Ju et al, Hybrid Hybridomics, 22(5):333-8 (2003)), whicha showed that a fimctional anti-CD137L antibody induced a high rate of proliferat—ion of peripheral blood monocytes. Blocking the ligand resulted in inhibition of T-cell activation. In addition, soluble CD137L was found in the serum of patients witlm rheumatoid arthritis and hematological malignancies (H. Salih et al., J. Immunol ., 167(7):4059-66 (2001)). Thus, the interaction of CD137 with CD137L influencees and produces functional effects on T-cells amd APC.

In another important aspect of T-cell function, it has been demonstrated that agonistic anti-CD137 antibodies rescued T-cell responses to protein antigens in =aged mice. It has been well documented that an age-related decline in the immune response to antigens occurs, a process known as immunosenescence (R. Miller,

Science, 273:70-4 (1996); R. Miller, Vaccine, 18:1654-60 (2000); F. Hakim et all.,

Curr. Opinion Immunol., 16:151-156 (2004)). This phenomenon appears to be dliue to alterations in the equilibrium between the extent of cellular expansion and cellular survival or death. Bansal-Pakala et al. tested the hypothesis that secondary costimulation through CD137 can be used to enhance T-cell responses in situaticens where T-cells do not receive sufficient stimulation, due to either reduced express ion of CD3 or CD28, or reduced quality of signals. Their studies showed that aged ranice had a deficient in vitro recall response compared to young mice (P. Bansal-Pakal aet al, J. Inmuno»l., 169(9):5005-9 (2002)). However, when aged mice were treated with anti-CD137 x.abs, the proliferative and cytokine response=s of T-cells were identical to the responses «observed in young mice. While the specifics mechanism responsible for this effect was not elucidated, it was speculated that enharcing both the expression of anti-apoptotic molecules like Bcl-y, and the promotion of IL-2 secretion in vivo may play a role in rescuing defective T-cell responses. These sstudies demonstrated the potential for agonistic anti-CD137 antibodies to rescue weak T-cell responses in elderly immumo-compromised individuals, and bas profousnd implications for the use of anti-CD137 antibodies in cancer patients.

A role for CD137 targeted therapy in the treatment of cancer was suggested by : in vivo efficacy studies in mice utilizing agonistic anti-mu rine CD137 monoclonal antibodies. In a paper by Melero et al., agonistic anti-motase CD137 antibody produced cures in P815 mastocytoma tumors, and in the low immunogenic tumor model Agl04 1. Melero et al., Nat. Med, 3(6):682-5 (1997). The anti-tumor effect required both CCD4+ and CD8+ T-cells and NK cells, since= selective in vivo depletion of each subpopulation resulted in the reduction or complet-e loss of the anti-tumor effect. It was also demonstrated that a minimal induction «of an immune response was necessary for anti-CD137 therapy to be effective. Several investigators have used anti-CD137 antibodies to demonstrate the viability of this mapproach for cancer therapy (J. Kim et al., Cancer Res., 61(5):203 1-7 (2001); O. Martiret et al., Gene Ther., 9(12):786-92 (22002); R. Miller et al., J. Immunol., 169(4): 1792-800 (2002); R. )

Wilcox et al., Cancer Res., 62(15):4413-8 (2002)).

In support of the anti-tumor efficacy data with agonistic CD137 antibodies, signals provide d by CD137L have been shown to elicit CT™L activity and anti-tumor responses (M. IDeBenedette et al., J. Inmunol., 163(9):4833-41 (1999); B. Guinn et al., J. Immunol, 162(8):5003-10 (1999)). Several reports clemonstrated that gene transfer of CD L 37 ligand into murine carcinomas resulted £n tumor rejection, demonstrating tthe requirement of costimulation in generating an efficient immune response (S. Mogi et al., Immunology, 101(4):541-7 (20007); I. Melero et al., Cell

Immunol, 190(2):167-72 (1998); B. Guinn et al., J. Immurmol., 162(8):5003-10 (1999)). Salih etal. reported the expression of CD137L in “human carcinomas and human carcinoma cell lines (H. Salih et al., J. Immunol., 1&5(5):2903-10 (2000)), and demonstrated that tumors Cells expressing the ligand were able= to deliver a co- stimulatory signal to T-cel 1s which resulted in the release of IFFN-y and IL-2, and that : this effect correlated with ghe levels of CD137L on tumors. Whether expressionof ~~ " CDI137L in human tumors could make these tumors more susceptible to agonistic

CDI137 antibodies is not kanown.

CD137L -/- mice Inave underscored the importance of the CD137/CD137L system in T-cell responses. to both viruses and tumors (M. DeBE3enedette et al., I.

Immunol., 163(9):4833-41 (1999); J. Tan et al., J. Immunol., W 64(5):2320-5 (2000);

B. Kwon et al, J. Inmuno-1.,, 168(11):5483-90 (2002)). Studie=s using CD137- and CDI137L~deficient mice heave demonstrated the importance of <CD137 costimulation in ~ graft-vs-host disease, and anti-viral cytolytic T-cell responses. CD137-deficient mice had an enhanced proliferation of T-cells, but a reduction in cytokine production-and cytotoxic T-cell activity (IB. Kwon et al., J. Immunol., 168( 11):5483-90 (2002); D.

Vinay et al., inmunol. Cell Biol., 81(3):176-84 (2003)). More recently, it was shown that knockout mice (CD13 7-/-) had a higher frequency of tumeor metastases (4-fold) compared to control mice. These data suggest that restoration of CD137 signaling by the use of agonistic anti-CD137 antibodies is a feasible approamch for augmenting : cellular immune responses to viral pathogens and cancers.

In addition to the data in mouse in vivo models which supports the involvement of CD137 sigenaling in antitumor immune responses, studies conducted in primary human tumor samples have confirmed the role of CCD137 in generating effector T-cells. In patients with Ewing sarcoma, Zhang et al. showed that intratumoral effector T-cells presented the CD3+/CD8+/CD288-/CD137+ phenotype.

Unexpectedly, coexistences of progressive tumor growth and amnti-tumor immunity (effector T-cells) was obsesrved. Ex vivo stimulation studies with patients’ cells demonstrated that tumor-imduced T-cell proliferation and activ=ation required / costimulation with CD137L. Stimulation of PBL with anti-CID3/CD137L, but not anti-CD3/anti-CD28, induced tumor lytic effectors. These studies provided further evidence that CD137 mediated costimulation could result in expansion of tumor reactive CTLs (H. Zhang etal., Cancer Biol. Ther., 2(5):579-8 6 (2003)).

Furthermore, expression oof CD13 7 was demonstrated in tumomr infiltrating ‘ lymphocytes in hepatocellular carcinomas (HCC) (Y. Wan et &l., World J.

VWVO 2005/035584 PCT/USS2004/033587

Gastroenterol, 10(2):195-9 (2004)). CD137 expression was detected in 1S out of 19

HCC by RT-PCR, and in 13/19 by irnmunofluorescence staining. Conversely, CD137 was not detected in the peripheral mononuclear cells of the same patients. Analyses conducted in healthy donor liver tissues failed to demonstrate expression ©f CD137.

These studies did not attempt to correlate clinical disease with CD137 expression. "Thus, studies conducted in Ewing saxcoma and hepatocellular carcinoma revealed the presence of TIL that express CD137., with concomitant disease progressiom. In Ewing sarcomas it was demonstrated that C D137+TILs were able to kill tumor ce=lls ex-vivo . suggesting that the CD137 pathway was intact in these patients, and that psethaps suppressive factors in the tumor microenvironment inhibited their function. Hence, it can be postulated that systemic admimistration of agonistic CD137 antibod—ies may provide the signal necessary for expansion of these effector T-cells.

In addition to its role in the development of immunity to cancer, experimental data supports the use of CD137 agonistic antibodies for the treatment of au_toimmune and viral diseases (B. Kwon et al., Exxp. Mol. Med., 35(1):8-16 (2003); H. Salih et al.,

J. Immunol., 167(7):4059-66 (2001); E. Kwon et al., P.N.A.S. USA, 96:15074-79 (1999); J. Foell et al., N.Y. Acad. Sci, 987:230-5 (2003); Y. Sun et al., Nat=. Med., 8(12):1405-13 (2002) S. K. Seo et al, Nat. Med. 10;1099-94 (2004)).

Consequently, based on the ro Jes of 4-1BB in modulating immune r-esponse, it would be desirable to produce anti-human 4-1BB antibodies with agonistic activities that could be used for the treatment or- prevention of human diseases such a=s cancer, infectious diseases, and autoimmune diseases.

BRIEF SUMMARY OF THE INVENTION:

The present invention provides fully human antibodies that bind to h_uman 4-

IBB (H4-1BB) and that allow binding of H4-1BB toa human 4-1BB ligand (H4- 1BBL). Thus, the invention is directed to antibodies that bind to H4-1BB ard that do not block the binding of H4-1BB to H4-{BBL, thereby permitting the bindirag of both an antibody of the invention and H4-11BBL to H4-1BB. The invention also provides antibodies with agonistic activities in that binding of the antibodies to H4-1F3B results in an enhancement and stimulation of F4-1BB mediated immune responses. These antibodies can be used as immuno-enhaancers of an anti-tumor or anti-viral immune response, or as immunornodulators of T cell mediated autoinnmune diseases. The antibodies can also be ussed as diagnostic tools for the detectiion of H4-1BB in blood or tissues of patients with cancer, autoimmune, or other dise=ases.

In one aspect, thee invention provides a monoclonal amntibody or antigen- binding portion thereof ghat specifically binds to H4-1BB, comprising a light chain : variable region and a he avy chain variable region, wherein thne light chain variable region comprises a CDR 1 (complementary determining regicon 1), a CDR2 (complementary determining region 2), and a CDR3 (compleementary determining region 3) as depicted in FIG. 4, and the heavy chain variable region comprises a

CDRI (complementary determining region 1), 2a CDR2 (complementary determining region 2), and a CDR3 (complementary determining region =3) as depicted in FIG. 3 or

FIG. 7. The monoclonal antibody (mab) can be, for example, an IgG4 antibody or

IgG1 antibody. :

In another aspect, the invention provides a monoclon_al antibody or antigen- binding portion thereof, wherein the light chain comprises a variable region as depicted in FIG. 4, and €he heavy chain comprises a variable= region as depicted in

FIG. 3 or FIG. 7. ’

In another aspect, the invention provides a monoclon_al antibody comprising a light chain and a heavy chain, wherein the light chain comprises amino acid residues 21-236 of SEQ ID NO:6 and the heavy chain comprises amimno acid residues 20-467 of SEQ ID NO:3. In another aspect, the invention provides =a monoclonal antibody comprising a light chain and a heavy chain, wherein the light chain comprises amino acid residues 21-236 of SEQ ID NO:6 and the heavy chain ceomprises amino acid residues 20-470 of SEQ ID NO:9.

The antibodies of the invention have wide therapeutic applications as immunomodulators of diseases such as cancer, autoimmune «diseases, inflammatory diseases, and infectious «liseases.

The invention fuxrther provides methods for treating cancer in a subject ] comprising administering a therapeutically effective amount of an antibody of the invention to the subject. In one aspect, this method further caomprises administering a vaccine. Suitable vaccimes include, for example, a tumor celd vaccine, a DNA vaccine, a GM-CSF-modlified tumor cell vaccine, or an antigeen-loaded dendritic cell vaccine. The cancer can be, for example, prostate cancer, melanoma, or epitheli_al cancer.

In amnother aspect, the invention providess a method for enhancing the immune response, ccomprising administration of an antit>ody of the invention and a SIV g=ag vaccine. In another aspect, the invention provides a method for enhancing the immune res -ponse, comprising administration o=f an antibody of the invention andl a

PSA vaccin e. In another aspect, the invention provides a method for enhancing #the immune response to a SIV gag vaccine, compri sing administration of an antibody of the invention. In another aspect, the invention gorovides a method for enhancing she . immune resgponse to a PSA vaccine, comprisings administration of an antibody of the invention.

The Zinvention also provides pharmaceutical compositions comprising an antibody of athe invention, or an antigen-binding: portion thereof, and a ’ pharmaceuti-cally acceptable carrier. The pharmmaceutical composition can be administerecH alone or in combination with an ageent, e.g., an agent for treating caracer such as a chemotherapeutic agent or a vaccine or other immunomodulatory agent_

The invention also provides isolated polynucleotides comprising a nucleot=ide sequence seleected from: (a) nucleotides that encode the amino acid sequence of amino acid residues 20-467 of SEQ ID NO:3; (b)) nucleotides that encode the amimro acid sequencee of SEQ ID NO:3; (¢) nucleotides #hat encode the amino acid sequerace . of amino acicd residues 21-236 of SEQ ID NO:6; (d) nucleotides that encode the amino acid sequence of SEQ ID NO:6; (e) nucleotides that encode the amino acid sequence of ammino acid residues 20-470 of SEQ ID NO:9; (f) nucleotides that enceode the amino aci_d sequence of SEQ ID NO:9; and (£2) nucleotides that encode a fragnaent of an amino acid sequence of (a) to (f), such as a variable region, constant region, <r one or more C _DRs. The isolated polynucleotides of the invention further comprise nucleotide secquences encoding at least one CDR «f FIG. 3, at least one CDR of FICS. 4, or at least o=ne CDR of FIG. 7. The invention further provides isolated polynucleotide=s that comprise the nucleotide sequeence of SEQ ID NO:1, SEQ ID

NO:4, or SEQ IDNO:7.

The inwwention also provides isolated polyp-eptides comprising an amino acicll sequence selec=ted from the group consisting of SEEQ ID NO:3, SEQ ID NO:6, and

. SEQ ID NO:9. In another aspect, the invention provides isolated polypeptides comprising the Zamino acid sequence of amino acid resiclues 20-467 of SEQ ID NO:3, isolated polypeptides comprising the amino acid sequermce of amino acid residues 21~ 236 of SEQ ID INO:6, and isolated polypeptides compri=sing the amino acid sequence of amino acid rezsidues 20-470 of SEQ ID NO:9. In anosther aspect, the invention provides isolate«d polypeptides comprising the amino ac-id sequence of at least one

CDR of FIG. 3, FIG. 4, or FIG. 7, or at least the variables or constant region of FIG. 3,

FIG. 4, or FIG. 77. :

The inve=ntion further includes an immunoglobulin having binding specificity for H4-1BB, sai«d immunoglobulin comprising an antige=n binding region. In one aspect, the immuwanoglobulin is a Fab or F(ab’), of an ant=ibody of the invention. . The invention also includes a cell line that produmces an antibody or antigen- binding portion thereof of the invention, recombinant ex=pression vectors that include : the nucleotides of the invention, and methods to make tine antibodies of the invention by culturing an aantibody-producing cell line.

BRIEF DESCRIPTION OF THE DRAWINGS:

FIG. 1 sheows a plasmid map of pD17-20H4.9.h4 a.

FIG. 2 sh ows a plasmid map of pD16gate-20H4.9.LC.

FIG. 3 (F IGS. 3A-3H) shows the nucleotide sequmence of the plasmid pD17- 20H4.9.h4a, including the coding strand (SEQ ID NO:1) , complementary strand (SEQ

ID NO:2), and armino acid sequence (leader peptide is amino acid residues 1-19 of

SEQ ID NO:3; hesavy chain is amino acid residues 20-46 7 of SEQ ID NQ:3) encoded by the coding strand.

FIG. 4 (FIGS. 4A-4F) shows the nucleotide sequence of the plasmid pD16gate-20H4.9.LC, including the coding strand (SEQ ID NO:4), complementary strand (SEQ ID MNO:5), and amino acid sequence (leader peptide is amino acid residues 1-20 of SEQ ID NO:6; light chain is amino acid residues 21-236 of SEQ ID

NO:6) encoded by the coding strand.

FIG. 5 shows a schematic of the 20H4.9-IgG1 heavy chain sequence construct.

FIG. 6 shows a schematic of the 20H4.9 light chain sequence construct.

FIG. 7 (FIGS. 7A-7D shows the nucleotide and amino acid sequences of the 20H4.9-IgG1 heavy chain construct, including the coding strand (SEQ ID NO:7), complementary strand] (SEQ ID NO:8), and amino acid sequence (leader peptide is amino acid residues 1 -19 of SEQ ID NO:9; heavy chain is amino acid mresidues 20-470 : of SEQ ID NO:9) encoded by the coding strand.

FIG. 8 (FIGS. 8A-8B) illustrates the results obtained from the b»inding of mab 20H4.9-IgG1 to human CD137 by ELISA (FIG. 84) and the effect of rmab 20H4.9-

IgGl on CD137-CD1 37L interaction (FIG. 8B).

FIG. 9 (FIGS. 9A-9B) illustrates the results obtained from the b»inding of mab 20H4.9-1gG1 to PMA -ionomycin stimulated human or cynomolgus mownkey cells.

Human CEM (FIG. 9A) or monkey PBMC (FIG. 9B) were incubated with 20H4.9-

IgG1 or human CD13 “7L fusion protein.

FIG. 10 (FIGS . 10A-10B) illustrates the results obtained by induction of IFN-y in co-stimulatory studies with anti-CD137 antibodies, which are expres sed as fold ’ increase in pg/ml over controls. Due to the variable background respon se among donors, data was normu alized relative to control treatments (=1). Mediax IFN-y baseline level for human T-cells (FIG. 10A) or monkey PBMC (FIG. 10B) stimulated with anti-CD3 alone wras 592 pg/ml and 505 pg/ml respectively.

FIG. 11 provides plasmon resonance plots of binding of mab 20¥44.9-IgG4 and mab 20H4.9-IgG1 to human CD137.

FIG. 12 illustra tes the concentration-dependent binding of 20H4 _9-IgG4 to

PMA ionomycin stimulated human CEM cells, but no binding to unstim ulated CEM cells.

FIG. 13 (FIGS. 13A-B) illustrates the induction of IFN-y in co-stimulatory studies with anti-CD13 7 antibodies. The results are expressed as fold increase in pg/ml over controls. Due to the variable background response among doenors, data was normalized relatives to control treatments (=1). Median IFN-y baseline level for human T-cells (FIG. 13 A) or monkey PBMC (FIG. 13B) stimulated witha anti-CD3 alone was 592 pg/ml and 505 pg/ml respectively.

FIG. 14 (FIGS. 14A-14B) illustrates the results obtained of dose-<lependent enhancement of IFN-y synthesis by mab 20H4.9-IgG4 (FIG. 14A), and effect of antibody crosslinking by addition of crosslinking anti-human IgG antibody (7 ug/ml) (FIG. 14B).

FIG. 15 illustrates the effect of mab 20H4.9-IgG4 on T-cell survival zand cell cycle progression. Human T-cells were costimulated with anti-CD3 (1 ug/ml) + mab 20H4.9-IgG4 at the concentrations listed. Six days after initiation of the assays, cells were collected and stained with Annexin-V and propidium iodide to determine the number of live cells (Annexin V/PI negative), or PE-conjugated cyclin D2 to detect cycling cells. Results represent the mean (£SD) of 4 lots of mab 20H4.9-Ig(54 tested in parallel.

FIG. 16 (FIGS. 16A-16D) shows in cynomolgus monkeys the antiger-specific

IFN-y response as measured by ELISPOT after treatment with a DNA vaccirae + anti- human 4-1BB antibodies. Animals were treated with a SIV gag vaccine (day 0, 28, 56; FIG. 164), SIV gag vaccine (day ©, 28, 56) and mab 20H4.9-IgG4 (day M2, 15 and 19; FIG. 16B), or SIV gag vaccine (day 0, 28, 56) and hu39E3.G4 (day E 2, 15 and 19; FIG 16C). A group of animals was left untreated (FIG. 16D). At vamrious times following treatment, blood was collected, and PBMC were separated amd evaluated for their ability to secrete IF Ny in the presence of antigen stimulat jon.

DETAILED DESCRIPTION OF THE INVENTION:

The invention is directed to the preparation and characterization of antibodies, and antigen binding fragments thereof (including fusion proteins that comprisse an antigen binding fragment of an antibody of the invention), for use in the treatment of a disease, such as a cancer, infectious disease, inflammatory disease, or autoimmmune disease. The cancer can be, for example, prostate cancer, melanoma, or epitheelial cancer.

The antibodies are capable of binding to H4-1BB, and can present highh affinity for H4-1BB and effectively enhance T cell responses. In one aspect, £he antibody induces IFN-y production in co-stimulatory assays, but does not affect the ~ binding of H4-1BB to its corresponding ligand, H4-1BBL, and does not fix complement.

. The antibodies of the invention may be produced by methods well known in the art. In one aspect, the antibodies can be produced by expression in transfected cells, such as immortalized eukaryotic cells, such as myeloma or hybridoma cells. - The antibodies of the invention amay be used alone, or together with othesr therapeutic agents such as radiotherapy (including radiation), hormonal therapy—, cytotoxic agents, vaccines, and other immunomodulatory agents, such us cytokines and biological response modifiers. These agents are particularly useful in treati ng cancer and immune-proliferative disorders. . In one aspect, the invention prowides the monoclonal antibody (mab) 20 H4.9-

IgG4. FIGS. 1 and 2 provide plasmid maps of pD17-20H4.9.h4a and pD16gate=- 20H4.9.LC, respectively, that can be used to produce mab 20H4.9-IgG4. FIG. = (FIGS. 3A-3H) provides the nucleotide sequence of the plasmid pD17-20H4.9.4a, . including the coding strand (SEQ ID NQ:1), complementary strand (SEQ ID NQD:2), and amino acid sequence (leader peptide is amino acid residues 1-19 of SEQ ID»

NO:3; heavy chain is amino acid residues 20-467 of SEQ ID NO:3) encoded by— the : coding strand. FIG. 4 (FIGS. 4A-4F) shows the nucleotide sequence of the plassmid pD16gate-20H4.9.1C, including the cocling strand (SEQ ID NO:4), complementary strand (SEQ ID NO:5), and amino acid sequence (leader peptide is amino acid residues 1-20 of SEQ ID NO:6; light chain is amino acid residues 21-236 of SE&Q ID

NO:6) encoded by the coding strand. :

In another aspect, the invention provides the monoclonal antibody (mab) 20H4.9-IgG1. FIG. 5 schematically shows a heavy chain sequence construct of mab 20H4.9-1gG1. FIG. 6 schematically shows a light chain sequence construct of mab 20H4.9, for both mab 20H4.9-1gG4 and 20 H4.9-IgG1. FIG. 7 provides the nucleotide sequence (coding strand (SEQ ID NO:7) and complementary strand (SEQ

ID NO:8)) of the heavy chain sequence construct of FIG. 5, and the amino acid sequence (leader peptide is amino acid residues 1-19 of SEQ ID NO:9; heavy ch_ain is amino acid residues 20-470 of SEQ ID INO:9) encoded by the coding strand. Th_e light chain of mab 20H4.9-IgG1 is the same as the light chain of mab 20H4.9-Ig«G4.

The invention also encompasses antibodies with conservative amino acidll substitutions from the heavy and light chr ain amino acid sequences depicted in SFEQ

ID NOS:3, 6, and 9 that have substantial 1y no effect on H4-1BB binding.

Conservative substitutions typically include tohe substitution of one amino acid for another with similar characteristics, e.g, substitutions within the following groups: valine, glycine; glycine, alanine; valine, isole=ucine, leucine; aspartic acid, glutamic - acid; assparagine, glutamine; serine, threonine=; lysine, arginine; and phenyMalanine, tyrosines, ‘The polynucleotides encoding the polypeptides of the invention typically further ~comprise an expression control sequerice operably linked to the polypeptide coding -sequences, including naturally-associamted or heterologous promotemr regions.

Preferalbly, the expression control sequences will be eukaryotic promoter ssystems in vectors capable of transforming or transfecting eukaryotic host cells, but ceontrol sequences for prokaryotic hosts may also be 1ased. Once the vector has bee=n incorporated into an appropriate host, the hosst is maintained under conditicons suitable : for high level expression of the nucleotide secquences and, as desired, the c-ollection and purification of the light chain, heavy chaimn, light/heavy chain dimers o-r intact antibod>y, binding fragments or other immuno _globulin form may follow. (See, S.

Beycholk, Cells of Immunoglobulin Synthesis, Academic Press, N.Y. (1979)). Single chain aratibodies or minibodies (single chain a_ntibodies fused to one or momre CH domainss) may also be produced by joining nuecieic acid sequences encodingg the VL and VH regions disclosed herein with DNA eracoding a polypeptide linker.

Prokaryotic hosts, such as E. coli, and -other microbes, such as yeast, may be used to express an antibody of the invention. Mn addition to microorganism_s, mamma Mian tissue cell culture may also be use=d to express and produce the antibodies of the in—vention. Eukaryotic cells may be preferred, because a number of smuitable host cell lines capable of secreting intact immimoglobulins have been deve oped includinge, for example, CHO (chinese hamster— ovary) cell lines, COS (Afriecan green : monkey fibroblast cell line) cell lines, HeLa ce=lls, myeloma cell lines; and hybridormnas. Expression vectors for these cells can include expression control sequence=s, such as a promoter or enhancer, ancl necessary processing inforn=iation sites, suc=h as ribosome binding sites, RNA spli_ ce sites, polyadenylation site=s, and transcrip=tional terminator sequences, all well known in the art.

T~he vectors containing the DNA segme=nts of interest (e.g., the heavy and/or light chailin encoding sequences and expression control sequences) can be transferred intos the host cell by well-known methods, whaich vary depending on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, whereas calcium phosphate treatment, lipofection, or electroporation may be used for other cellulax hosts. (See, e.g., T. Maniatis et al., Mowlecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1982)).

Once expressed, the antibodies, their dimers, individual light and heavy cheains, or other immunoglobulin forms, can be purified according to standard procedures in the art, such as ammonium sullfate precipitation, affinity columns, column chromatography, gel electrophoresis, and the like. Substantially pure . 10 im-munoglobulins of at least 90 to 95% hom ©geneity are desirable, and 98 to 99% or more homogeneity are more desirable. :

The antibodies of the invention are vaseful for modulating T cell and antibeody- mediated immune responses. Typical disease states suitable for treatment includ e ca-eers, infectious diseases, inflammatory liseases, and autoimmune diseases such as multiple sclerosis, theumatoid arthritis, systemic lupus erythematosus, and m_yaesthenia gravis,

The invention also provides pharma ceutical compositions comprising at least ‘ore antibody of the invention and a pharmaceutically acceptable carrier. The pharmaceutical compositions may be sterilized by conventional well known sterilization techniques. The pharmaceutical compositions can also contain pHharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stability emnhancing agents such as mannito! or tween 80, toxicity adjusting agents and thee like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chlowride, seodium lactate, or human albumin.

The antibodies and pharmaceutical compositions of the invention are particularly useful for parenteral administration, including subcutaneous, imtramuscular, and intravenous administration. The pharmaceutical compositio ns for parenteral administration can comprise a s olution of the antibody dissolved in an amcceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be cased, all well known in the art, e.g., water, buffered water, saline, glycine and the like. "Whese solutions are sterile and generally fixee of particulate matter. It is especially advantageous to formulates parenteral compositions in dosages unit form for ease of administration and uniforsmity of dosage.

The pharmaceutical composition can further comprise an additional agent for treatment of a disease. In_ one aspect, the pharmaceutical composition includes an agent for treatment of a cancer, an infectious disease, inflarmmatory disease, or autoimmune disease. The antibody of the invention can alsso be co-administered or separately administered with an additional agent for treatmaent of a disease.

The antibodies of the invention can be used with otier agents to enhance the immune response to canceerous cells in 4 patient. In one aspoect, the antibody is used in combination with an imme unogenic agent, such as cancerous cells, purified tumor antigens (including recorrbinant proteins, peptides, and carbohydrate molecules), or cells transfected with genees encoding immune stimulating cytokines and cell surface antigens. In another aspect, the antibody is used in combination with a vaccine such as, for example, a tumor ell vaccine, a DNA vaccine, a germe-modified tumor cell vaccine, such as GM-CSF -modified tumor cell vaccine, a p eptide vaccine, or an : antigen-loaded dendritic cell vaccine.

Many experimentzal strategies for vaccination againsst tumors have been devised. In one of these sstrategies, a vaccine is prepared ussing autologous or allogeneic tumor cells. Thhese cellular vaccines have been sshown to be most effective : when the tumor cells are transduced to express GM-CSF. (GM-CSF has been shown to be a potent activator of antigen presentation for tumor va _ccination (Dranoff et al., . PN.AS,, 90:3539-43 (19993); E. Jafee et al,, J. Clin. Oncol. , 19:145-56 (2001); R.

Salgia et al, J. Clin. Oncoml, 21:624-30 (2003)).

The study of gene expression and large scale gene expression patterns in : 25 various tumors has led to whe definition of so called tumor specific antigens (S.

Rosenberg, Immunity 10:2281-7 (1999). In many cases, the=se tumor specific antigens are differentiation antigen -s expressed in the tumors and in tlhe cell from which the tumor arose, for example mmelanocyte antigens gp 100, MACGE antigens, Trp-2. Many of these antigens can be skaown to be the targets of tumor sp ecific T cells found in the host. The antibodies of th e invention may be used in conjuriction with a collection of recombinant proteins and/ or peptides expressed in a tumor i_n order to amplify and direct the immune responsse to these antigens towards a Thi response. These proteins are normally viewed by the immune system as self antigens and amre therefore tolerant to them. .

In one aspect Of the invention, the antibody is combined w ith an immunodulatory agent comprising the SIV gag antigen (as a mode] for HIV DNA vaccine) or prostate specific antigen (PSA), or a DNA vaccine comprising a nucleotide sequence that encodes the SIV gag antigen or prostate specific antigen (PSA). PSA vaccines are described in, for example, M. Pavlenko «et al., Br. J. Cancer, 91(4):688-94 (2004); I. Wolchok et al., Semin. Oncol, 30(5):659-&6 (2003); J. Kim et al., Clin. Cancer Res., 7(3 Suppl.):882s-889s (2001). SIV gag vaccines are described in, for example, B. Makitalo et al., J. Gen. Virol., 85(Pt 8):2407-15® (2004); N. Letvin et al., J. Virol., 78(14)= 7490-7 (2004); S. Mossman et al., AIDS Res. Hum.

Retroviruses., 20(4):425-34 (2004); F. Bertley et al., J. Immunol., M72(6):3745-57 (2004); L. Patterson et al., J. Virol, 78(5):2212-21 (2004); E. O'Ne=ill et al., AIDS

Res. Hum. Retroviruses, 19(10):883-90 (2003); Z. Bu et al., Virology, 309(2):272-81 (2003).

The tumor antigen may also include, for example, the protein telomerase, which is required for time synthesis of telomeres of chromosomes amd which is expressed in more than 85% of human cancers and in only a limitec number of somatic tissues (N. Kim et al., Science, 266, 2011-2013 (1994)). Tuamor antigen may also be "neo-antigens" expressed in cancer cells because of somatic mutations that alter protein sequence ovr create fusion proteins between two unrelated sequences, or idiotype from B cell turmors. Other tumor vaccines may include the proteins from viruses implicated in human cancers such a Human Papilloma Viruses (HPV), : Hepatitis Viruses (HBV and HCV), and Kaposi's Herpes Sarcoma Virus (KHSV).

Another form of tumor specific antigen which may be used with an antibody of the invention is purified heat shock proteins (HSP) isolated from the tunor tissue itself.

These heat shock proteiras contain fragments of proteins from the turmnor cells and these HSPs are highly efficient at delivery to antigen presenting cellss for eliciting tumor immunity (R. Suot et al,, Science 269: 1585-1588 (1995); Y. Tamura et al.,

Science 278: 117-120 (1997)).

The antibodies of the invention can also be used to enhance the immune response to vaccines to viral antigens, such as HIV or HCV. The ant&bodies of the invention can also be used to enhance the immune response to other= immmomodulatory agents, aand to elicit a memory immune respons«e. Examples of these agents are cytokines su<h as GM-CSF, IL-2, IL-15, 1-12, F13 ligand, CD40 ligand, adjuvants such as Cp&G-oligodeoxynucleotide (bacterial DN.~4), or antibodies to OX~40 or CTLA4.

The pharmaceutical compositions of the invention can be ad-ministered for prophylactic and/or therapeutic treatments. In therapeutic application, the a pharmaceutical composition is administered to a patient already suf¥ering from a disease, in an amount sufficient to cure or at least partially arrest or treat the disease.

An amount adequate to accomplish this is defined as a “therapeutically effective dose.” Amounts effective fox this use will depend upon the severity” of the disease state and the patient (includirag, for example, the general state of thes patient’s own immune system), and can be determined by one skilled in the art. Ira prophylactic applications, the pharmaceutical composition is administered to a patient not already inthe disease state, to enhance the patient’s resistance to the diseases state. Such an amount is defined to be a “prophylactically effective dose.” In this -wse, the precise amounts depend upon the patient’s state of health (including, for exemple, the general state of the patient’s own imrune system), and can be determined by one skilled in the art. In one aspect, the prophylactic use is for the prevention of temnor recurrence.

Examples:

Example 1: Generation of Amtibodies

Materials and Methods

Fully human monoclonal antibodies to the human CD137 (4—1BB) receptor were generated in the HuMA Tb-Mouse® (Medarex, Inc., Princeton, Mew Jersey).

HuMAD mice were immunized five times intraperitoneally (i.p.) andl subcutaneously (s.c.) with 25 pg of the extracellular domain of human CD137 in RIBBI adjuvant (Ribi

Immunochemical). Prior to fausion, mice were boosted intravenously (i.v.) with the same amount of antigen. Spleen cells from immunized mice with adequate titers of antibodies to huCD137 were fused to mouse myeloma cells followimg standard procedures.

Anti-human CD3 mab (clone:HIT3a), ELISA kits for human aand monkey :

IFN-y, cytometric bead axray (CBA) kits, and all conjugated antibodie=s for flow cytometry were purchased from BD Pharmingen (San Diego, California). Human

IgG A and Human IgG, x were purchased from Sigma-Aldrich (St. I_ouis, Missouri).

CEM cells (ATCC-CRL 22265) were purchased from ATCC. Culture media (RPMI), and fetal bovine serum (FBS) were purchased from Mediatech Inc. (EMerndon,

Virginia). Sheep Red BRood Alsevers was purchased from Colorado Serum Co. (Denver, Colorado).

Hybridoma screening: Detection of binding to huCD137 by BISA: To : identify hybridomas secreting anti-human CD137 antibodies, ELISA plates (Nunc

MaxiSorp) were coated with human CD137-mouse IgGy, fusion prote in at 1 pg/ml in

PBS overnight at 4°C. Plates were then washed 3 times with PBS with 0.01% Tween- 80 (PBS-T), and subsequently blocked with PBS-T plus 1% bovine sexrum albumin (BSA), for 20 min at room temperature. Fifty microliters of supernatants diluted 1:3 in PBS-T were added to the plates and incubated for 1-2 hr at ambient temperature.

Afterwards, plates were washed as before, and binding of antibodies was detected by an incubation with alkalin e phosphatase-conjugated goat F(ab’), anti-tmuman IgG antibody (Jackson Laboratories, West Grove, Pennsylvania). Plates were developed with pNPP and read at 40S nm.

Blocking assay: Twenty-six hybridomas secreting antibodies that recognized huCD137 by ELISA were evaluated for their ability to allow CD137-C=D137L interactions. These analyses were conducted initially in an ELISA forrmat. Plates were coated with human CD137-mulgGa, at 0.2 pg/ml, 100 pl/well. Serial dilutions of the mab 20H4.9-gG1, or control antibodies, diluted in PBS-T and 1 % bovine serum albumin, were added to the plate. CD137L-CDS8 fusion protein wwvas added to the wells at a concentration: of 0.2 pg/ml. Binding of antibodies was detected with a biotinylated anti-CD8 antibody (0.2 pg/ml, Ancell Corporation, Bayport, Minnesota).

After several washes, strep tavidin-alkaline phosphatase (1:2000) and pINPP for the detection of bound antibod jes were added, and the plates were read at 4 05 nm.

Toconfimn thatthe selected antibodies did not alter CD137-CD 137L binding, purified antibodies were farther characterized by BIAcore analyses. Alexperiments were carried out on a BIAc ore 3000 instrument (BIA core Inc., Piscataway, New

Jersey). Humean CD137 was immobilized covalently zat a high density on a carboxy- methylated dexxtran surface of a BIAcore sensorchip (BIAcore Inc., Piscataway, New

Jersey). Injections were conducted at 2 pg/mL in 10 mmM acetate buffer, pH 5.0. . ‘Unoccupied active esters were subsequently blocked Wby injection of an excess of ethanolamine. Regeneration of the surface was done ~with 10 mM glycine, pH 2.0.

Purifie-d samples of anti-CD137 antibodies we=re diluted to concentrations between 200 a.nd 1000 nM using HEPES buffered salJine, pH 7.4, supplemented with 0.15. M NaCl aand 0.005% surfactant P20 (HBS-EP). EHuman CD137L-CD8 fusion proteins (huCID137L) were used as source of CD137 Migand. Experiments were conducted in vwhich huCD137L was injected prior to eanti-CD137 antibodies, or vice versa. Injectiosns were performed at a flow rate of 5 pu _I/min. Bound ligand and antibodies wer-e removed by regeneration with 10 mM glycine buffer, pH 2.0.

Huma T-cell purification: T-cells or PBMCs. were obtained from healthy human donors— Blood was collected in EDTA, suspermded in elutriation buffer (RPMI containing 2.5 mM EDTA, 10 pg/ml polymyxin B), u-nderlayed with Lymphocyte

Separation Me-dium (LSM, Mediatech Inc., Herndon, ~ Virginia), and centrifuged at } 1800 rpm for 225 minutes. Cellular interfaces were collected, and centrifuged at 1500 rpm for 10 mirawutes. Afterwards, cell pellets were restaspended in elutriation buffer and washed Shaeep Red Blood Cells (SRBC, 1:10 dilution), and incubated on ice for 1 hour. Cells we=re then underlayed with LSM and centr-ifuged at 2500 rpm for 25 minutes. Interfaces were removed and SRBC were lyssed with SRBC Lysis Buffer.

Isolated T-cells were washed and resuspended in 10%S FBS/RPMIL

Flow Cytometric analyses: Binding of anti-humman CD137 antibodies to

CD137 expresssed on cells was determined by flow cytometry. A human T-cell CT leukemia cell line (CEM) or cynomolgus monkey peripheral blood monocytic cells (PBMC) were wased for these studies. These cells do neot express CD137 constitutively, but the receptor can be induced by stimwulation with phorbol myristate (PMA, 10 ng/ml) and ionomycin (1 pM) for 18 hr. Ce=1ls were then washed and incubated with various concentrations of the antibodies in staining buffer (phosphate buffer saline, P7BS, plus 1 % FCS, and 0.01 % sodium =azide). Binding of the antibodies to st®mulated or non-stimulated cells was de=tected by a fluorescein (FITC) : or phycoerithrim (PE) conjugated goat anti-human IgG (Jackson Immunoresearch,

West Grove, Penrasylvania). To confirm expression of CD137, a fusion protein consisting of the extracellular domain of CD137 ligand and mouse CD8 was used (Ancell Corporati on, Bayport, Minnesota), followed by incubation with PE- conjugated anti-mouse CDS8 (BD Pharmingen, San Diego, California). Samples were fixed in 1% formalin, kept at 4 °C, and read by flow cystometry.

Functional assays: Primary human T-cells or m_onkey PBMC obtained from healthy donors were stimulated with immobilized anti-CD3 antibody to provide the first signal for T-cell activation, and co-stimulated with human anti-human CD137 antibodies. As a ron-specific control, a humanized ant carcinoma antibody (BR96) was used at the saame antibody concentration. Plates we=re coated with anti-CD3 antibody (0.5-1 pg/ml) at 4 °C overnight. The next day- T-cells or PBMC were plated at 1-1.5 x10° /welll concentrations. Synthesis of IFN-y v=vas measured after 72 hours off culture at 37 °C either by cytometric bead array (CBA) -or by ELISA.

Cytokine a ssays ’

ELISA: A_ fier stimulation of T-cells at various tAmes, plates were centrifuged and media was removed. Cytokine levels were detected by an ELISA in accordance with the manufactearer’s instructions (BD Pharmingen, Ssan Diego, California). In brief, test samples and standards were added to anti-cytokine-coated 96-well plates.

After incubation for 2 hr at ambient temperature, plates =were washed 3 times in PBS-

T and then incubat-ed first with a working detector antibody, followed by the addition of substrate. Absorbance was read at 405 nm, and conce=ntrations were calculated based on the standard curve.

Cytometric Bead Array: Another method used to determine cytokine production in vitro was flow cytometry using the Cytomeetric Bead array (CBA) developed by BD Pharmingen. Levels of IFNy, IL-2, IT_.-5, IL-4, IL-10, and TNF-a were measured in culture supernatants following manufa_cturers’ instructions. Results were analyzed by flow cytometry with the CBA analysis software.

Results :

Hybridomass secreting antibodies that showed bineding to human CD137 were further expanded, amd subcloned. Secreted antibodies we=re purified and tested for their ability to bind to huCD137 and to allow the interaction of CD137-CD137L. Of - the panel of anti-humnan CD137 antibodies evaluated, mallb 20H4.9-IgG1 was selected

WO Z2005/035584 PC I/US2004/033587 for further evaluation based on its b& nding profile and non-blocking pr-operties. The 20H4.9-1gG1 antibody is IgG1 kapp a as determined by ELISA using amlkaline phosphatase anti-human IgGl, 2, 3, <4, and anti-kappa and lambda reag=ents (Southern : Biotech, Birmingham, Alabama). FIG. 8 (FIG. 8A - binding to humasn CD137 by

ELISA; FIG. 8B - effect of mab 20H4.9-IgG1 on CD137-CD137L interaction) provides the initial characterization ©Of mab 20H4.9-IgG1. Serial diluti_ons of mab 20H4.9-IgG1, 26G6 (a blocking anti—CD137 antibody), or tetanus toxcwid (TT, negative control) were evaluated for their ability to alter binding of CID137 to

CDI137L. Mab 20H4.9-IgG1 at conc=entrations up to 10 pg/ml did not block CD137L . 10 binding, whereas mab 26G6 inhibited binding at concentrations > 0.37 pg/ml.

Mab 20H4.9-IgG1 was also tested for reactivity towards CD137 expressed on buman T-cells (CEM) and in cynomolgus monkey peripheral blood mcanocytic cells (PBMC) stimulated with PMA and ionomycin. Previous studies determined that

CD137 is upregulated on T-cells following activation with PMA and icsnomycin. 15 . Control molecules consisted of an irrelevant human IgG antibody (negative control) or CD137L-CD8 fusion protein (positive control, BD Pharmingen, San Diego,

California). Results from these studies indicated that mab 20H4.9-IgG A bound to activated human CEM and PBMCs fiom cynomolgus monkeys, with minimum . binding to unstimulated cells. Similasr percentages of positive cells werse detected with either mab 20H4.9-1gG1 or CD1 37L. FIG. 9 provides the results obtained demonstrating the binding of mab 20F4.9-IgG1 to PMA-ionomycin stirmulated human or cynomolgus monkey cells. Human_ CEM (FIG. 9A) or monkey PBMC (FIG. 9B) “were incubated with 20H4.9-IgG1 or Fauman CD137L fusion protein. Secondary santibodies were added and samples were read by flow cytometry.

Next, it was determined wheth«er mab 20H4.9-IgG1 could induces enhancement <f IFN-y in costimulatory assays in thes presence of anti-CD3 stimulatiorm, the key

Munctional effect desired for an agonis€ic CD137 antibody. Mab 20H4.9-1gG1 was } evaluated for its co-stimulatory activitsy in functional studies in human ard monkey

Lymphocytes. Based on initial data, a concentration of 20 ug/ml anti-CD 137 antibody (excess antibody) was used in these stumdies. Levels of anti-CD3 antibod=y between (09.2-1 ug/ml were tested which resulted. in 10-20% CD137-positive lymp¥ocytes.

L_evels of IFN-y in supernatants were oeasured after 72 h of culture. As shown in

FIG. 10, mab 20H4.9-IgG1 enhanced [FN-y synthesis in both human and monke=y costimulatory assays to levels significantly higher than controls. Results of stud ies conducted with T-cells isolated from 8 heal thy human donors showed that in six= of them, mab 20H4.9-IgG1 enhanced IFN-y synthesis between 2.2 - 4.3-fold compamred to controls. One of the other two donors showed a 1.6-fold increase. The level of enhancement was superior to that observed with hu39E3.G4, a humanized anti- (D137 antibody provided in published PCX Application W004/010947 (herein imcorporated by reference) which showed augmentation of IFN-y in 5 out of 8 donors aand at levels lower than mab 20H4.9-1gG1 (1.5 - 2-fold increase) (FIG. 10A). Im monkey costimulatory studies, mab 20H4.9-IgG1 also demonstrated enhanced

Functional activity resulting in significant augmentation of IFN-y over controls CFIG. :

E 0B). As in the human studies, enhancemesnt of IFN-y was consistently higher than vith hu39E3.G4.

Induction of TNF-o. synthesis above control levels was also observed in

Imuman cultures, albeit at much lower levels: than IFN-y. TNF-a levels induced Eby anti-CD3 antibody alone (baseline) were about 20-50 fold lower than baseline le=vels . for IFN-y. Mab 20H4.9-IgG1 induced an iracrease of ~2 to 4.7-fold in 3 out of 8- donors. Again, hu39E3.G4 (tested in parallel) induced ~2-fold increase in the s=ame } - dllonors but at lower levels. Other cytokines tested, IL-2, IL-5, IL-10, and IL-4 d_id not change significantly with either treatment.

Together these studies demonstratec that mab 20H4.9-IgG1 presented the functional activity desired in both humans sand monkeys by inducing a Thi-type of response. Significantly, since in vivo anti-twamor activity is associated with the a bility onf anti-CD137 antibodies to induce IFN-y s-ynthesis, these results supported the s election of mab 20H4.9-IgG1 for isotype s witching. :

Example 2: In vitro characterization of mal 20H4.9-1gG4

Based on its binding kinetics, inability to block CD137-CD137L interaction, and functional effects on human T-cells, meab 20H4.9-IgG1 was selected for switching to an IgG4 form. The IgG4 form of mab 20H4.9-IgG1 is 20H4.9-1gG4 (depicted in

FIGS.3and4).

The second phase of these studies im volved the comparison of the in vitro properties of mab 20H4.9-IgG4 and mab 200H4.9-IgG1. In this section, the binding kinetics propetties, and functional effects of beoth antibodies in human and raonkey lymph_ocytes are described.

Binding kinetics

Kinetic properties of anti-buman CD 137 antibodies were evaluated by surface plasmeon resonance using a BIAcore 3000 instrument. The antigen, humarm CD137- murinee IgG,s, was immobilized covalently art a low density on the surface eof a CM5 sensor-chip. Mab 20H4.9-IgG4 and mab 20F34.9-IgG1 were injected at coracentrations _ betwesen 25 and 200 nM. FIG. 11 depicts in¥ ections at 100 nM for both meab 20H4.9-

IgGl sand mab 20H4.9-[gG4. Data calculate « using BIAevaluation software (bivalent model, global curve fit analysis) resulted in E<inetic parameters that were similar for both a ntibodies (see Table 1). Dissociation constants Kp for mab 20H4.9-JgG1 and mab 2 OH4.9-IgG4 were determined as 11.2 zand 16.6 nM, respectively. Under similar experimental conditions, mab 20H4.9-IgG4 «id not bind to murine 4-1BB.

Table | - Comparison of the binding kineticss of mab 20H4.9-1gG4 and maab 20H4.9-

IgG-1 ka ka2 Rmax Kpl 20H .9- | 3.43EH) 3.85E- 2.30E- L51E- 8.91EH) 20H4-.9- } 3.92EH0 6.51E- 6.02E+H0 oof wr a]

Flow cytometric analyses

Biotinylated mab 20H4.9-IgG4 at coracentrations ranging from 0.32 ng/ml to 5 pg/ml was tested for binding to CEM cells + PMA-ionomycin. Mab 20H4-.9-1gG4 bound to PMA-ionomycin stimulated CEM cells ina concentration-depencient mannes=r. Saturation was achieved at 0.2 pg/ml. On the other hand, as shovewn for its parentaal molecule, mab 20H4.9-IgG1, mab 2 0H4.9-1gG4 did not bind to CEEM cells that we=re not stimulated with PMA-ionomyc&n (FIG. 12). Concentration-d_ependent binding of mab 20H4-.9-IgG4 was demonstrated in PMA-ionomycin stimu: lated CEM cells (FIG. 12). Samples were read by flow cytometry.

Cellular/Functional Assays . To confirm that the process of switching the isotype of meamb 20H4.9-IgG1 did not alter the activity of the antibosdy, in vitro studies were conducted to compare the activity of mab 20H4.9-IgG4 to tthe parent mab 20H4.9-I1gG1 in mmonkey PBMC and human T-cells. The functional effects of mab 20H4.9-IgG4 on hvaman and monkey T- cells or PBMC were determined and compared to its parental moMecule, mab 20H4.9-

IgGl. Primary human T-cells or monkey PBMC obtained from hmealthy donors were stimulated with anti-CD3 antibocly (0.5 pg/ml-1 pg/ml) +- anti-bmuman CD137 ~~ antibodies. Synthesis of IFNy vwas measured after 72 h of culturee at 37°Chy cytometric bead array (CBA) for human samples or by ELISA. fo_r monkey samples.

Antibodies were tested in costimulatory assays in the presence of= suboptimal concentrations of anti-CD3 antibsody (1 pg/ml) or Concavalin A (C1 pg/ml) (donors

M5170 and 81 only). Results ares expressed as fold increase in pg/ml over controls.

Due to the variable background xesponse among donors, data wass normalized relative to control treatments (=1). FIG. 13A provides the human T-cell wresults and FIG. 13B provides the monkey PBMC restalts. As shown in FIGS. 13A-13 B, mab 20H4.9-1gG4 demonstrated costimulatory properties yielding higher levels of FN-y in human and monkey cells compared to controls. The level of enhancement of [FN-y synthesis was comparable to its parental molecule in human and monkey samp Mes. . Subsequently; the effect ©f antibody cross-linking on the —functional effect of mab 20H4.9-1gG4 was evaluatecd. Tt has been shown that cross-1&nking of antibodies may result in potentiation of their signaling ability. Thus, a studss’ was conducted to ) determine the functional activity of several batches of mab 20H4 .9-IgG4 + an anti- human IgG antibody. As shown in FIG. 14A, significant enhancement of IFN-y ) synthesis was observed for all lots tested in the absence of cross-_linking antibodies, with a plateau at concentrations «of 400 ng/ml. The augmentatiorm of IFNy synthesis by mab 20H4.9-IgG4 was further erahanced by the addition of anti-hauman IgG cross- linking antibody as shown in FIGS. 14B. Different batches of mal 20H4.9-1gG4 bad comparable cellular activities.

Thus, cross-linking of mab 20H4.9-1gG4 resulted in an erahancement of the ability of the antibody to induce IFN-y synthesis. Antibody crosss-linking in vivo may occur by cellular receptors for the Fc portion of immunoglobulins or by antiboedy dimerization. Mab 20H4.9-1gG4 is of the IgG4 isotype, which, compared to o€her

IgG isotypes, has low affinity for Fc receptors. However, IgG4 can bind to FowRI (CD64) expressed on monocytes-and meutrophils.

Two other approaches were used to further characterize mab 20H4.9-Ig=G4: (i) effect on T-cell survival and (ii) effect on cyclin D2 expression. To determine - whether mab 20H4.9-IgG4 could elicit signaling through CD137 on human T-cells : and provide co-stimulatory signals to “T-cells leading to cell survival and expan_sion, human T-cells stimulated with anti-CID3 antibodies +/- mab 20H4.9-1gG4 at concentrations known to induce IFN-y synthesis were stained with annexin-V asnd : propidium iodide to determine the nurmber of live cells (Annexin V/Propidium iodide negative), and with Cyclin D2 to determine its effect on cell progression. FIG. 15 shows the average results of 4 different lots of mab 20H4.9-IgG4 on cyclin D2 * expression and survival of T-cells. Concentrations of mab 20H4.9-IgG4 of 0.4—10 pg/ml resulted in an increase in the number of live cells by approximately 1.8 - 2 fold, and yielded a significant increase in the number of cyclin D2-expressing T-cell=s (2.5 - 3 fold).

Example 3: In vivo evaluation of 4-1BB antibodies in a pharmacodynamic mociel in cynomolgus monkeys.

This example illustrates the ability of mab 20H4.9-IgG4 and mab hu39F=3.G4 ‘ to enhance the antigen specific immune response elicited by DNA vaccines.

Materials and Methods

Experimental animal groups: Female and male cynomolgus monkeys (2 .5to - 5.0 kg) were purchased from Charles River BRF (Houston, Texas) for this study~ and : 25 were housed in pairs. Each experimental group consisted of 4 males and 2 femanles which were randomized into groups by body weight. Experimental groups were= as follows: "Group 1 - SIV gag and PSA DNA vaccine (2 mg each), day 0, 28, 56, ima, plus saline control, i.v., on days 12, 15 and 19;

Group 2 ~ SIV gag and PSA DN A vaccine (2 mg each), day 0, 28, 56, i... plus mab hu39E3.G4, i.v., on days 12, 15 and 19;

Group 3 - SIV gag and PSA DNA vaccine (2 Img each), day 0, 28, 56, im., plus mab 220H4.9-IgG4, i.v., on days 12, 15 a-nd 19;

Group 4 - untreated control group. oo . Immunizations and antibody treatments: PSAa and SIV gag DNA vaccines were obtained fro-m David B. Weiner, Department of Pathology and Laboratory of

Medicine, University of Pennsylvania. (See, Kim et zal., Oncogene 20, 4497-4506 (2001); Muthumaani et al., Vaccine 21, 629-637 (200 3).)

Monkeys —were immunized by the intramuscular route with both PSA and SIV gag DNA constnumcts (2 mg/construct/immunization) ssimultaneously, followed by t=wo boosts 4 weeks apart (days 0, 28, and 56). Twelve days after the initial immunization, treatment with maab 20H4.9-[gG4 or mab hu39E3.G4- was initiated. Antibodies we=re administered i.v, at 50 mg/kg, on days 12, 15, and 19* after the first immunization.

This schedule wa_s chosen because it was shown to stappress the antibody response to : mab hu39E3.G4. 15° Clinical and Clinical pathology

Throughout the course of the study, physical examinations were conducted on . all monkeys by tine attending veterinarians. Blood sammples for hematology and serum chemistry analyses were collected prior to vaccinations and then 12, 42, 70, 97, 13-4, and 168 days afte=r immunizations.

Immunological asssays

Lo To determine the effect on the.immune respomnses induced by these therapeutic . regimens, an enzs/me-linked immune spot assay (ELISPOT) was used for the detection of IFN-=y production by antigen-specific stimmulated lymphocytes. Blood samples for ELIS POT analyses were collected prior £0 vaccinations and then 12, 422, 70,97, 134, and M68 days after immunizations. Syntlhetic peptides corresponding t—o the complete sequaences of SIV gag and the PSA antigen were used for ex-vivo stimulation of PB-MC. - Results Ce ‘

Antigen-s—pecific IFN-y secreting cells in resp=onse to PSA or SIV gag pepticies . 30 were quantitated Wby ELISPOT. FIG. 16 (FIGS. 16A—16D) illustrates the results obtained from Gr-oups 1-4, respectively. The level ofS response to PSA was very lo~w . in all groups, indii cating that the vaccine by itself did not induce a measurable and consistent imnnune response when compared to nosn-vaccinated animals. On thes other hand, SIV gag: vaccination alone resulted in signifSicant number of antigen-specific " IFN-y secretimg cells that augmented over time (FIG. 16A). Untreated animals (not vaccinated) shmowed 100-1,000 spots/10° PBMC tiaroughout the course of the stay (FIG. 16D). Mhese results established the threshold response to the vaccine; an=imals that presented. < 1,000 spots/10° PBMC were considered non-responders. In thee

Co group of animals that received vaccine, 5 out of 6 monkeys showed an increase=d response over—time, with a mean number of spots after the third immunization (day 70) - of 1,727 spotss/10° PBMC (SD=242, range=1,403—1,968 spots/10° PBMC). Onee monkey was considered a non-responder (620 spots/million PBMC). Since in these studies MHC typing was not done, it is likely that= the lack of T cell responses t=o the vaccine by so-tae monkeys may be due to MHC-nismatch. Remarkably, on da—y 70, 4 out of 6 anim -als treated with SIV gag'plus mab 2@0H4.9-IgG4 presented a significant higher number of IFN-y spots (FIG. 16C) compared to contro! animals (FIG. 1e6D) and to macaques that were immunized with DNA. vaccine alone (FIG. 16A). The mean numbem- of spots after the third immunization for the mab 20H4.9-1gG4-treated group was of ~ 3,465 spots/10° PBMC (SD=1,236, range=2,0704,780 spots/10°

PBMC), Tweo monkeys in that group did not respond to the vaccine (<800 spots/million PBMC). Following the third immu nization (day 70), treatment v=vith mab hu39E3_.G4 plus DNA vaccine resulted in 6 out of 6 animals considered a_s responders with a mean number of spots/10° PBMC of 2,348 (SD=588, range=—=1,738- 3,283) (FIG. 16B). For this group, the range of tlhe number of spots was lower— compared to —those macaques treated with mab 200H4.9-1gG4.

Treatment with both mab 20H4.9-IgG4 amd mab ku9E3.G4 was well tolerated and did not ressult in any significant changes in cRinical signs, clinical chemistr—y, or } hematological parameters relative to control morakeys.

These data show that mab 20H4.9-IgG4 treatment in combination with_ a DNA oo vaccine elicisted an in vivo enhancement of the magnitude of the specific cellular response to tthe test antigen relative to controls om to treatment with mab hu39E=3.G4, asmeasured by antigen specific IFN-y-secreting cells. Since only one dose lewwel of the antibodie=s and one dosing regimen were usec in these preliminary studies, it is unlikely that maximal responses were induced, a.nd further work to optimize conditions is required. Clearly, however, even withh this non-optimized protocol, zan enhancement= of the cellular response to test antigemns was achieved with mab 20H=1.9-

IgG4, sugges-ting that modulation of CD137 functieon may be an attractive approaczh for augmentimng the effectiveness of DNA vaccines .

Altho ugh the invention has been described in some detail by way of illustration arad example for purposes of clarity andl understanding, it will be apparent that certain chhanges and modifications may be practiced within the scope of the appended cla ims. : }

Claims (14)

1. ] P CT/US2004/033587 CLAIMS: What is claimed is:

   l. A monoclonal antibody or a ntigen-binding portion therecof that specifically binds t-0 4-1BB, comprising a light chain variable region and a heavy chuain variable region, wherein: said light chain variable region commprises a CDR1, a CDR2, andl a CDR3 as depictezd in Figure 4; and said heavy chain variable region comprises a CDR 1, a CDR2, eand a CDR3 as depictexd in Figure 3.
2. 2. The monoclonal antibody or antigen-binding portion th=ereof of claim 1, wherei n: said light chain compriscs a variabele region as depicted in figur—e 4; and said heavy chain comprises a variable region as depicted in Fig ure 3.
3. 3. A monoclonal antibody coxmprising a light chain and a heavy chain, wherei_nsaid light chain comprises amino a cid residues 21-236 of SEQ HED NO:6 and said heavy «chain comprises amino acid residues 20-467 of SEQ ID NO:3.
4. 4, A pharmaceutical composi tion comprising: the monoclonal antibody or antigen-binding portion thereof of claim 1; and a pharmaceutically acceptable carrier.
5. 5. A pharmaceutical composi tion comprising: the monoclonal antibody of claim 3; and a pharmaceutically acceptable carr ier.
6. 6. Use of the monoclonal anti body or antigen-binding port_ion thereof of claim 1. in the manufacture of a medicament for treating a cancer in a subject.
7. 7. An isolated polynucleotide comprising a nucleotide sequence that encode s the amino acid sequence of amino acid residues 20-467 of SEQ ID NO:3.
8. 8. The polynucleotide of clairm 7 that comprises the nucleotide sequence of SEQ ID NO: 1.
9. 9. An isolated polynucleotide comprising a nucleotide sequuence that cncodess the amino acid sequence of amino acid residues 21-236 of SEQ ID NO:6. 32 AMEND ED SHEET

   ® PCT/IUS20304/033587
10. 10. The polynucleotide of claim 9 that comprises the nucleotide secjuence of SEQ ID NO:4.
11. 11. An antibody according to any one of claims 1 to 3, substantiall-y as hereim described with reference to and as illustrated in any of the examples and accormpanying drawings.
12. 12. A composition according to claim 4 or claim 5, substantially ass herein descr-ibed with reference to and as illustrated in any of the examples and accompanying drawings.
13. 13. Use according to claim 6, substantially as herein described wit reference to and as illustrated in any of the examples and accompanying drawings.
14. 14. A polynucleotide according to any one of claims 7 to 10, subst=antially as hereim described with reference to and as illustrated in any of the examples and accormpanying drawings. 33 AMEN DED SHEET