Human momoclonal antibodies to ctla-4

<u>BACKGROUND OF THE INVENTION</u>

1. <i><u>Cross-Reference to Related Applications</u></i>

The present application claims priority to <patcit id="pcit0001" dnum="US11364798P"><text>U.S. Provisional Patent Application, Serial No. 60/113,647, filed December 23, 1998</text></patcit>, the disclosure of which is hereby incorporated in its entirety herein.

2<i>. <u>Summary of the Invention</u></i>

In accordance with the present invention, there are provided fully human monoclonal antibodies against human cytotoxic T-lymphocyte antigen 4 (CTLA-4). Nucleotide sequences encoding and amino acid sequences comprising heavy and light chain immunoglobulin molecules, particularly contiguous heavy and light chain sequences spanning the complementarity determining regions (CDRs), specifically from within FR1 and/or CDR1 through CDR3 and/or within FR4, are provided. Further provided are antibodies having similar binding properties and antibodies (or other antagonists) having similar functionality as antibodies disclosed herein.

3. <i><u>Background of the Technology</u></i>

Regulation of immune response in patients would provide a desirable treatment of many human diseases that could lead to a specificity of action that is rarely found through the use of conventional drugs. Both up-regulation and down-regulation of responses of the immune system would be possible. The roles of T cells and B cells have been extensively studied and characterized in connection with the regulation of immune response. From these studies, the role of T cells appear, in many cases, to be particularly important in disease prevention and treatment.

T cells possess very complex systems for controlling their interactions. Interactions between T cells utilize numerous receptors and soluble factors for the process. Thus, what effect any particular signal may have on the immune response generally varies and depends on the particular factors, receptors and counter-receptors that are involved in the pathway. The pathways for down-regulating responses are as important as those required for activation. Thymic education leading to T-cell tolerance is one mechanism for preventing an immune response to a particular antigen. Other mechanisms, such as secretion of suppressive cytokines, are also known.

Activation of T cells requires not only stimulation through the antigen receptor (T cell receptor (TCR)), but additional signaling through co-stimulatory surface molecules such as CD28. The ligands for CD28 are the B7-1 (CD80) and B7-2 (CD86) proteins, which are expressed on antigen-presenting cells such as dendritic cells, activated B-cells or monocytes that interact with T-cell CD28 or CTLA-4 to deliver a costimulatory signal. The role of costimulatory signaling was studied in experimental allergic encephalomyelitis (EAE) by <nplcit id="ncit0001" npl-type="s"><text>Perrin et al. Immunol Res 14:189-99 (1995</text></nplcit>). EAE is an autoimmune disorder, induced by Th1 cells directed against myelin antigens that provides an <i>in vivo</i> model for studying the role of B7-mediated costimulation in the induction of a pathological immune response. Using a soluble fusion protein ligand for the B7 receptors, as well as monoclonal antibodies specific for either CD80 or CD86, Perrin et al. demonstrated that B7 costimulation plays a prominent role in determining clinical disease outcome in EAE.

The interaction between B7 and CD28 is one of several co-stimulatory signaling pathways that appear to be sufficient to trigger the maturation and proliferation of antigen specific T-cells. Lack of co-stimulation, and the concomitant inadequacy of IL-2 production, prevent subsequent proliferation of the T cell and induce a state of non-reactivity termed "anergy". A variety of viruses and tumors may block T cell activation and proliferation, leading to insufficient activity or non-reactivity of the host's immune system to the infected or transformed cells. Among a number of possible T-cell disturbances, anergy may be at least partly responsible for the failure of the host to clear the pathogenic or tumorgenic cells.

The use of the B7 protein to mediate anti-tumor immunity has been described in <nplcit id="ncit0002" npl-type="s"><text>Chen et al. Cell 71:1093-1102 (1992</text></nplcit>) and <nplcit id="ncit0003" npl-type="s"><text>Townsend and Allison Science 259:368 (1993</text></nplcit>).<nplcit id="ncit0004" npl-type="s"><text> Schwartz Cell 71:1065 (1992</text></nplcit>) reviews the role of CD28, CTLA-4, and B7 in IL-2 production and immunotherapy. <nplcit id="ncit0005" npl-type="s"><text>Harding et al. Nature 356:607-609 (1994</text></nplcit>) demonstrates that CD28 mediated signaling co-stimulates murine T cells and prevents the induction of anergy in T cell clones. <i>See also</i><patcit id="pcit0002" dnum="US5434131A"><text>U.S. Patent Nos. 5,434,131</text></patcit>, <patcit id="pcit0003" dnum="US5770197A"><text>5,770,197</text></patcit>, and <patcit id="pcit0004" dnum="US5773253A"><text>5,773,253</text></patcit>, and International Patent Application Nos. <patcit id="pcit0005" dnum="WO9300431A"><text>WO 93/00431</text></patcit>, <patcit id="pcit0006" dnum="WO9501994A"><text>WO 95/01994</text></patcit>, <patcit id="pcit0007" dnum="WO9503408A"><text>WO 95/03408</text></patcit>, <patcit id="pcit0008" dnum="WO9524217A"><text>WO 95/24217</text></patcit>, and <patcit id="pcit0009" dnum="WO9533770A"><text>WO 95/33770</text></patcit>.

From the foregoing, it was clear that T-cells required two types of signals from the antigen presenting cell (APC) for activation and subsequent differentiation to effector function. First, there is an antigen specific signal generated by interactions between the TCR on the T-cell and MHC molecules presenting peptides on the APC. Second, there is an antigen-independent signal that is mediated by the interaction of CD28 with members of the B7 family (B7-1 (CD80) or B7-2 (CD86)). Exactly where CTLA-4 fit into the milieu of immune responsiveness was initially evasive. Murine CTLA-4 was first identified and cloned by <nplcit id="ncit0006" npl-type="s"><text>Brunet et al. Nature 328:267-270 (1987</text></nplcit>), as part of a quest for molecules that are preferentially expressed on cytotoxic T lymphocytes. Human CTLA-4 was identified and cloned shortly thereafter by <nplcit id="ncit0007" npl-type="s"><text>Dariavach et al. Eur. J. Immunol. 18:1901-1905 (1988</text></nplcit>). The murine and human CTLA-4 molecules possess approximately 76% overall sequence homology and approach complete sequence identity in their cytoplasmic domains (<nplcit id="ncit0008" npl-type="s"><text>Dariavach et al. Eur. J. Immunol. 18:1901-1905 (1988</text></nplcit>)). CTLA-4 is a member of the immunoglobulin (Ig) superfamily of proteins. The Ig superfamily is a group of proteins that share key structural features of either a variable (V) or constant (C) domain of Ig molecules. Members of the Ig superfamily include, but are not limited to, the immunoglobulins themselves, major histocompatibility complex (MHC) class molecules (i.e., MHC class I and II), and TCR molecules.

In 1991, <nplcit id="ncit0009" npl-type="s"><text>Linsley et al. J. Exp. Med. 174:561-569 (1991</text></nplcit>), proposed that CTLA-4 was a second receptor for B7. Similarly, <nplcit id="ncit0010" npl-type="s"><text>Harper et al. J Immunol 147:1037-44 (1991</text></nplcit>) demonstrated that the CTLA-4 and CD28 molecules are closely related in both mouse and human as to sequence, message expression, gene structure, and chromosomal location. <i>See also</i><nplcit id="ncit0011" npl-type="s"><text>Balzano et al. Int J Cancer Suppl 7:28-32 (1992</text></nplcit>). Further evidence of this role arose through functional studies. For example, <nplcit id="ncit0012" npl-type="s"><text>Lenschow et al. Science 257:789-792 (1992</text></nplcit>) demonstrated that CTLA-4-Ig induced long term survival of pancreatic islet grafts. <nplcit id="ncit0013" npl-type="s"><text>Freeman et al. Science 262:907-909 (1993</text></nplcit>) examined the role of CTLA-4 in B7 deficient mice. Examination of the ligands for CTLA-4 are described in <nplcit id="ncit0014" npl-type="s"><text>Lenschow et al. P.N.A.S. 90:11054-11058 (1993</text></nplcit>). <nplcit id="ncit0015" npl-type="s"><text>Linsley et al. Science 257:792-795 (1992</text></nplcit>) describes immunosuppression <i>in vivo</i> by a soluble form of CTLA-4. <nplcit id="ncit0016" npl-type="s"><text>Linsley et al. J Exp Med 176:1595-604 (1992</text></nplcit>) prepared antibodies that bound CTLA-4 and that were not cross-reactive with CD28 and concluded that CTLA-4 is coexpressed with CD28 on activated T lymphocytes and cooperatively regulates T cell adhesion and activation by B7.<nplcit id="ncit0017" npl-type="s"><text> Kuchroo et al. Cell 80:707-18 (1995</text></nplcit>) demonstrated that the B7-1 and B7-2 costimulatory molecules differentially activated the Th1/Th2 developmental pathways.<nplcit id="ncit0018" npl-type="s"><text> Yi-qun et al. Int Immunol 8:37-44 (1996</text></nplcit>) demonstrated that there are differential requirements for co-stimulatory signals from B7 family members by resting versus recently activated memory T cells towards soluble recall antigens. <i>See also</i> de <nplcit id="ncit0019" npl-type="s"><text>Boer et al. Eur J Immunol 23:3120-5 (1993</text></nplcit>).

Several groups proposed alternative or distinct receptor/ligand interactions for CTLA-4 as compared to CD28 and even proposed a third B-7 complex that was recognized by a BB1 antibody. <i>See</i>, <i>for example</i>, <nplcit id="ncit0020" npl-type="s"><text>Hathcock et al. Science 262:905-7 (1993</text></nplcit>), <nplcit id="ncit0021" npl-type="s"><text>Freeman et al. Science 262:907-9 (1993</text></nplcit>), <nplcit id="ncit0022" npl-type="s"><text>Freeman et al. J Exp Med 178:2185-92 (1993</text></nplcit>),<nplcit id="ncit0023" npl-type="s"><text> Lenschow et al. Proc Natl Acad Sci U S A 90:11054-8 (1993</text></nplcit>), <nplcit id="ncit0024" npl-type="s"><text>Razi-Wolf et al. Proc Natl Acad Sci U S A 90:11182-6 (1993</text></nplcit>), and <nplcit id="ncit0025" npl-type="s"><text>Boussiotis et al. Proc Natl Acad Sci U S A 90:11059-63 (1993</text></nplcit>). <i>But</i>, <i>see</i>, <nplcit id="ncit0026" npl-type="s"><text>Freeman et al. J Immunol 161:2708-15 (1998</text></nplcit>) who discuss finding that BB1 antibody binds a molecule that is identical to the cell surface form of CD74 and, therefore, the BB1 mAb binds to a protein distinct from B7-1, and this epitope is also present on the B7-1 protein. Thus, this observation required the field to reconsider studies using BB1 mAb in the analysis of CD80 expression and function.

Beginning in 1993 and culminating in 1995, investigators began to further delineate the role of CTLA-4 in T-cell stimulation. First, through the use of monoclonal antibodies against CTLA-4, <nplcit id="ncit0027" npl-type="s"><text>Walunas et al. Immunity 1:405-13 (1994</text></nplcit>) provided evidence that CTLA-4 can function as a negative regulator of T cell activation. Thereafter, <nplcit id="ncit0028" npl-type="s"><text>Waterhouse et al. Science 270:985-988 (1995</text></nplcit>) demonstrated that mice deficient for CTLA-4 accumulated T cell blasts with up-regulated activation markers in their lymph nodes and spleens. The blast cells also infiltrated liver, heart, lung, and pancreas tissue, and amounts of serum immunoglobulin were elevated and their T cells proliferated spontaneously and strongly when stimulated through the T cell receptor, however, they were sensitive to cell death induced by cross-linking of the Fas receptor and by gamma irradiation. Waterhouse et al. concluded that CTLA-4 acts as a negative regulator of T cell activation and is vital for the control of lymphocyte homeostasis. In a comment in the same issue, <nplcit id="ncit0029" npl-type="s"><text>Allison and Krummel Science 270:932-933 (1995</text></nplcit>), discussed the work of Waterhouse et al. as demonstrative that CTLA-4 acts to down regulate T-cell responsiveness or has an inhibitory signaling role in T-cell activation and development. <nplcit id="ncit0030" npl-type="s"><text>Tivol et al. Immunity 3:541-7 (1995</text></nplcit>) also generated CTLA-4-deficient mice and demonstrated that such mice rapidly develop lymphoproliferative disease with multiorgan lymphocytic infiltration and tissue destruction, with particularly severe myocarditis and pancreatitis. They concluded that CTLA-4 plays a key role in down-regulating T cell activation and maintaining immunologic homeostasis. Also, <nplcit id="ncit0031" npl-type="s"><text>Krummel and Allison J Exp Med 182:459-65 (1995</text></nplcit>) further clarified that CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation. They generated an antibody to CTLA-4 and investigated the effects of its binding to CTLA-4 in a system using highly purified T cells. In their report, they showed that the presence of low levels of B7-2 on freshly explanted T cells can partially inhibit T cell proliferation, and this inhibition was mediated by interactions with CTLA-4. Cross-linking of CTLA-4 together with the TCR and CD28 strongly inhibits proliferation and IL-2 secretion by T cells. Finally, the results showed that CD28 and CTLA-4 deliver opposing signals that appear to be integrated by the T cell in determining the response to antigen. Thus, they concluded that the outcome of T cell antigen receptor stimulation is regulated by CD28 costimulatory signals, as well as inhibitory signals derived from CTLA-4. <i>See also</i><nplcit id="ncit0032" npl-type="s"><text> Krummel et al. Int Immunol 8:519-23 (1996</text></nplcit>) and <patcit id="pcit0010" dnum="US5811097A"><text>U.S. Patent No. 5,811,097</text></patcit> and International Patent Application No. <patcit id="pcit0011" dnum="WO9720574A"><text>WO 97/20574</text></patcit>.

A variety of additional experiments have been conducted further elucidating the above function of CTLA-4. For example,<nplcit id="ncit0033" npl-type="s"><text> Walunas et al. J Exp Med 183:2541-50 (1996</text></nplcit>), through the use of anti-CTLA-4 antibodies, suggested that CTLA-4 signaling does not regulate cell survival or responsiveness to IL-2, but does inhibit CD28-dependent IL-2 production. Also, <nplcit id="ncit0034" npl-type="s"><text>Perrin et al. J Immunol 157:1333-6 (1996</text></nplcit>), demonstrated that anti-CTLA-4 antibodies in experimental allergic encephalomyelitis (EAE), exacerbated the disease and enhanced mortality. Disease exacerbation was associated with enhanced production of the encephalitogenic cytokines TNF-alpha, IFN-gamma and IL-2. Thus, they concluded that CTLA-4 regulates the intensity of the autoimmune response in EAE, attenuating inflammatory cytokine production and clinical disease manifestations. <i>See also</i><nplcit id="ncit0035" npl-type="s"><text>Hurwitz et al. J Neuroimmunol 73:57-62 (1997</text></nplcit>) and <nplcit id="ncit0036" npl-type="s"><text>Cepero et al. J Exp Med 188:199-204 (1998</text></nplcit>) (an anti-CTLA-4 hairpin ribozyme that specifically abrogates CTLA-4 expression after gene transfer into a murine T-cell model).

In addition,<nplcit id="ncit0037" npl-type="s"><text> Blair et al. J Immunol 160:12-5 (1998</text></nplcit>) assessed the functional effects of a panel of CTLA-4 monoclonal antibodies (mAbs) on resting human CD4+ T cells. Their results demonstrated that some CTLA-4 mAbs could inhibit proliferative responses of resting CD4+ cells and cell cycle transition from G0 to G1. The inhibitory effects of CTLA-4 were evident within 4 h, at a time when cell surface CTLA-4 expression remained undetectable. Other CTLA-4 mAbs, however, had no detectable inhibitory effects, indicating that binding of mAbs to CTLA-4 alone was not sufficient to mediate down-regulation of T cell responses. Interestingly, while IL-2 production was shut off, inhibitory anti-CTLA-4 mAbs permitted induction and expression of the cell survival gene bcl-X(L). Consistent with this observation, cells remained viable and apoptosis was not detected after CTLA-4 ligation.

In connection with anergy,<nplcit id="ncit0038" npl-type="s"><text> Perez et al. Immunity 6:411-7 (1997</text></nplcit>) demonstrated that the induction of T cell anergy was prevented by blocking CTLA-4 and concluded that the outcome of antigen recognition by T cells is determined by the interaction of CD28 or CTLA-4 on the T cells with B7 molecules. Also,<nplcit id="ncit0039" npl-type="s"><text> Van Parijs et al..J Exp Med 186:1119-28 (1997</text></nplcit>) examined the role of interleukin 12 and costimulators in T cell anergy <i>in vivo</i> and found that through inhibiting CTLA-4 engagement during anergy induction, T cell proliferation was blocked, and full Th1 differentiation was not promoted. However, T cells exposed to tolerogenic antigen in the presence of both IL-12 and anti-CTLA-4 antibody were not anergized, and behaved identically to T cells which have encountered immunogenic antigen. These results suggested that two processes contribute to the induction of anergy <i>in vivo</i>: CTLA-4 engagement, which leads to a block in the ability of T cells to proliferate, and the absence of a prototypic inflammatory cytokine, IL-12, which prevents the differentiation of T cells into Th1 effector cells. The combination of IL-12 and anti-CTLA-4 antibody was sufficient to convert a normally tolerogenic stimulus to an immunogenic one.

In connection with infections, <nplcit id="ncit0040" npl-type="s"><text>McCoy et al. J Exp Med 186:183-7 (1997</text></nplcit>) demonstrated that anti-CTLA-4 antibodies greatly enhanced and accelerated the T cell immune response to <i>Nippostrongylus brasiliensis</i>, resulting in a profound reduction in adult worm numbers and early termination of parasite egg production. <i>See also</i><nplcit id="ncit0041" npl-type="s"><text>Murphy et al. J. Immunol. 161:4153-4160 (1998</text></nplcit>) <i>(Leishmania donovani).</i>

In connection with cancer, <nplcit id="ncit0042" npl-type="s"><text>Kwon et al. PNAS USA 94:8099-103 (1997</text></nplcit>) established a syngeneic murine prostate cancer model and examined two distinct manipulations intended to elicit an antiprostate cancer response through enhanced T cell costimulation: (i) provision of direct costimulation by prostate cancer cells transduced to express the B7.1 ligand and (ii) <i>in vivo</i> antibody-mediated blockade of T cell CTLA-4, which prevents T cell down-regulation. It was demonstrated that <i>in vivo</i> antibody-mediated blockade of CTLA-4 enhanced antiprostate cancer immune responses. Also, <nplcit id="ncit0043" npl-type="s"><text>Yang et al. Cancer Res 57:4036-41 (1997</text></nplcit>) investigated whether the blockade of the CTLA-4 function leads to enhancement of antitumor T cell responses at various stages of tumor growth. Based on <i>in vitro</i> and <i>in vivo</i> results they found that CTLA-4 blockade in tumor-bearing individuals enhanced the capacity to generate antitumor T-cell responses, but the expression of such an enhancing effect was restricted to early stages of tumor growth in their model. Further, <nplcit id="ncit0044" npl-type="s"><text>Hurwitz et al. Proc Natl Acad Sci U S A 95:10067-71 (1998</text></nplcit>) investigated the generation of a T cell-mediated antitumor response depends on T cell receptor engagement by major histocompatibility complex/antigen as well as CD28 ligation by B7. Certain tumors, such as the SM1 mammary carcinoma, were refractory to anti-CTLA-4 immunotherapy. Thus, through use of a combination of CTLA-4 blockade and a vaccine consisting of granulocyte-macrophage colony-stimulating factor-expressing SM1 cells, regression of parental SM1 tumors was observed, despite the ineffectiveness of either treatment alone. This combination therapy resulted in long-lasting immunity to SM1 and depended on both CD4(+) and CD8(+) T cells. The findings suggested that CTLA-4 blockade acts at the level of a host-derived antigen-presenting cell.

In connection with diabetes, <nplcit id="ncit0045" npl-type="s"><text>Luhder et al. J Exp Med 187:427-32 (1998</text></nplcit>) injected an anti-CTLA-4 mAb into a TCR transgenic mouse model of diabetes at different stages of disease. They found that engagement of CTLA-4 at the time when potentially diabetogenic T cells are first activated is a pivotal event; if engagement is permitted, invasion of the islets occurs, but remains quite innocuous for months. If not, insulitis is much more aggressive, and diabetes quickly ensues.

In connection with vaccine immunization, <nplcit id="ncit0046" npl-type="s"><text>Horspool et al. J Immunol 160:2706-14 (1998</text></nplcit>) found that intact anti-CTLA-4 mAb but not Fab fragments suppressed the primary humoral response to pCIA/beta gal without affecting recall responses, indicating CTLA-4 activation inhibited Ab production but not T cell priming. Blockade of the ligands for CD28 and CTLA-4, CD80 (B7-1) and CD86 (B7-2), revealed distinct and nonoverlapping function. Blockade of CD80 at initial immunization completely abrogated primary and secondary Ab responses, whereas blockade of CD86 suppressed primary but not secondary responses. Simultaneous blockade of CD80 + CD86 was less effective at suppressing Ab responses than either alone. Enhancement of costimulation via coinjection of B7-expressing plasmids augmented CTL responses but not Ab responses, and without evidence of Th1 to Th2 skewing. These findings suggest complex and distinct roles for CD28, CTLA-4, CD80, and CD86 in T cell costimulation following nucleic acid vaccination.

In connection with allograft rejection, <nplcit id="ncit0047" npl-type="s"><text>Markees et al. J Clin Invest 101:2446-55 (1998</text></nplcit>) found in a mouse model of skin allograft rejection that acceptance initially depended on the presence of IFN-gamma, CTLA-4, and CD4(+) T cells. Addition of anti-CTLA-4 or anti-IFN-gamma mAb to the protocol was associated with prompt graft rejection, whereas anti-IL-4 mAb had no effect.

In connection with the role of CTLA-4 in relation to CD28, <nplcit id="ncit0048" npl-type="s"><text>Fallarino et al. J Exp Med 188:205-10 (1998</text></nplcit>) generated TCR transgenic/recombinase activating gene 2-deficient/CD28-wild-type or CD28-deficient mice which were immunized with an antigen-expressing tumor. Primed T cells from both types of mice produced cytokines and proliferated in response to stimulator cells lacking B7 expression. However, whereas the response of CD28+/+ T cells was augmented by costimulation with B7-1, the response of the CD28-/- T cells was strongly inhibited. This inhibition was reversed by monoclonal antibody against B7-1 or CTLA-4. Thus, CTLA-4 can potently inhibit T cell activation in the absence of CD28, indicating that antagonism of a TCR-mediated signal is sufficient to explain the inhibitory effect of CTLA-4. Also, <nplcit id="ncit0049" npl-type="s"><text>Lin et al. J Exp Med 188:199-204 (1998</text></nplcit>) studied rejection of heart allografts in CD28-deficient mice. H-2(q) hearts were transplanted into allogeneic wild-type or CD28-deficient mice (H-2(b)). Graft rejection was delayed in CD28-deficient compared with wild-type mice. Treatment of wild-type recipients with CTLA-4-immunoglobulin (Ig), or with anti-B7-1 plus anti-B7-2 mAbs significantly prolonged allograft survival. In contrast, treatment of CD28-deficient mice with CTLA-4-Ig, anti-B7-1 plus anti-B7-2 mAbs, or a blocking anti-CTLA-4 mAb induced acceleration of allograft rejection. This increased rate of graft rejection was associated with more severe mononuclear cell infiltration and enhanced levels of IFN-gamma and IL-6 transcripts in donor hearts of untreated wild-type and CTLA-4-Ig- or anti-CTLA-4 mAb-treated CD28-deficient mice. Thus, the negative regulatory role of CTLA-4 extends beyond its potential ability to prevent CD28 activation through ligand competition. Even in the absence of CD28, CTLA-4 plays an inhibitory role in the regulation of allograft rejection.

Also, further characterization of the expression of CTLA-4 has been investigated. For example, <nplcit id="ncit0050" npl-type="s"><text>Alegre et al. J Immunol 157:4762-70 (1996</text></nplcit>) proposed that surface CTLA-4 is rapidly internalized, which may explain the low levels of expression generally detected on the cell surface. They concluded that both CD28 and IL-2 play important roles in the up-regulation of CTLA-4 expression. In addition, the cell surface accumulation of CTLA-4 appeared to be primarily regulated by its rapid endocytosis. Also, <nplcit id="ncit0051" npl-type="s"><text>Castan et al. Immunology 90:265-71 (1997</text></nplcit>) based on <i>in situ</i> immunohistological analyses of the expression of CTLA-4, suggested that germinal center T cells, which were CTLA-4 positive, could be important to immune regulation.

Accordingly, in view of the broad and pivotal role that CTLA-4 appears to possess in immune responsiveness, it would be desirable to generate antibodies to CTLA-4 that can be utilized effectively in immunotherapy. Moreover, it would be desirable to generate antibodies against CTLA-4 that can be utilized in chronic diseases in which repeat administrations of the antibodies are required.

<u>BRIEF DESCRIPTION OF THE DRAWING FIGURES</u>

<ul id="ul0001" list-style="none" compact="compact"><li><figref idref="f0001 f0002 f0003 f0004 f0005 f0006 f0007 f0008 f0009 f0010 f0011 f0012 f0013 f0014 f0015 f0016">Figure 1</figref> provides a series of nucleotide and an amino acid sequences of heavy chain and kappa light chain immunoglobulin molecules in accordance with the invention: 4.1.1 (<figref idref="f0001 f0002">Figure 1A</figref>), 4.8.1 (<figref idref="f0003 f0004">Figure 1B</figref>), 4.14.3 (<figref idref="f0005">Figure 1C</figref>), 6.1.1 (<figref idref="f0006 f0007">Figure 1D</figref>), 3.1.1 (<figref idref="f0008">Figure 1E</figref>), 4.10.2 (<figref idref="f0009">Figure 1F</figref>), 2.1.3 (<figref idref="f0010">Figure 1G</figref>), 4.13.1 (<figref idref="f0011">Figure 1H</figref>), 11.2.1 (<figref idref="f0012">Figure 1I</figref>), 11.6.1 (<figref idref="f0013">Figure 1J</figref>), 11.7.1 (<figref idref="f0014">Figure 1K</figref>), 12.3.1.1 (<figref idref="f0015">Figure 1L</figref>), and 12.9.1.1 (<figref idref="f0016">Figure 1M</figref>).</li><li><figref idref="f0017 f0018 f0019">Figure 2</figref> provides a sequence alignment between the predicted heavy chain amino acid sequences from the clones 4.1.1, 4.8.1, 4.14.3, 6.1.1, 3.1.1, 4.10.2, 4.13.1, 11.6.1, 11.7.1, 12.3.1.1, and 12.9.1.1 and the germline DP-50 (3-33) amino acid sequence. Differences between the DP-50 germline sequence and that of the sequence in the clones are indicated in bold. The Figure also shows the positions of the CDR1, CDR2, and CDR3 sequences of the antibodies as shaded.</li><li><figref idref="f0020">Figure 3</figref> provides a sequence alignment between the predicted heavy chain amino acid sequence of the clone 2.1.3 and the germline DP-65 (4-31) amino acid sequence. Differences between the DP-65 germline sequence and that of the sequence in the clone are indicated in bold. The Figure also shows the positions of the CDR1, CDR2, and CDR3 sequences of the antibody as underlined.</li><li><figref idref="f0021">Figure 4</figref> provides a sequence alignment between the predicted kappa light chain amino acid sequence of the clones 4.1.1, 4.8.1, 4.14.3, 6.1.1, 4.10.2, and 4.13.1 and the germline A27 amino acid sequence. Differences between the A27 germline sequence and that of the sequence in the clone are indicated in bold. The Figure also shows the positions of the CDR1, CDR2, and CDR3 sequences of the antibody as underlined. Apparent deletions in the CDR1s of clones 4.8.1, 4.14.3, and 6.1.1 are indicated with "0s".</li><li><figref idref="f0022">Figure 5</figref> provides a sequence alignment between the predicted kappa light chain amino acid sequence of the clones 3.1.1, 11.2.1, 11.6.1, and 11.7.1 and the germline 012 amino acid sequence. Differences between the 012 germline sequence and that of the sequence in the clone are indicated in bold. The Figure also shows the positions of the CDR1, CDR2, and CDR3 sequences of the antibody as underlined.</li><li><figref idref="f0023">Figure 6</figref> provides a sequence alignment between the predicted kappa light chain amino acid sequence of the clone 2.1.3 and the germline A10/A26 amino acid sequence. Differences between the A10/A26 germline sequence and that of the sequence in the clone are indicated in bold. The Figure also shows the positions of the CDR1, CDR2, and CDR3 sequences of the antibody as underlined.</li><li><figref idref="f0024">Figure 7</figref> provides a sequence alignment between the predicted kappa light chain amino acid sequence of the clone 12.3.1 and the germline A17 amino acid sequence. Differences between the A17 germline sequence and that of the sequence in the clone are indicated in bold. The Figure also shows the positions of the CDR1, CDR2, and CDR3 sequences of the antibody as underlined.</li><li><figref idref="f0025">Figure 8</figref> provides a sequence alignment between the predicted kappa light chain amino acid sequence of the clone 12.9.1 and the germline A3/A19 amino acid sequence. Differences between the A3/A19 germline sequence and that of the sequence in the clone are indicated in bold. The Figure also shows the positions of the CDR1, CDR2, and CDR3 sequences of the antibody as underlined.</li><li><figref idref="f0026">Figure 9</figref> provides a summary of N-terminal amino acid sequences generated through direct protein sequencing of the heavy and light chains of the antibodies.</li><li><figref idref="f0027 f0028">Figure 10</figref> provides certain additional characterizing information about certain of the antibodies in accordance with the invention. In <figref idref="f0027">Figure 10A</figref>, data related to clones 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.14.3, and 6.1.1 is summarized. Data related to concentration, isoelectric focusing (IEF), SDS-PAGE, size exclusion chromatography, liquid chromatography/mass spectroscopy (LCMS), mass spectroscopy (MALDI), light chain N-terminal sequences is provided. Additional detailed information related to IEF is provided in <figref idref="f0028">Figure 10B</figref>; related to SDS-PAGE is provided in 10C; and SEC of the 4.1.1 antibody in 10D.</li><li><figref idref="f0029">Figure 11</figref> shows the expression of B7-1 and B7-2 on Raji cells using anti-CD80-PE and anti-CD86-PE mAbs.</li><li><figref idref="f0030">Figure 12</figref> shows the concentration dependent enhancement of IL-2 production in the T cell blast/Raji assay induced by anti-CTLA-4 blocking antibodies (BNI3, 4.1.1, 4.8.1, and 6.1.1).</li><li><figref idref="f0031">Figure 13</figref> shows the concentration dependent enhancement of IFN-γ production in the T cell blast/Raji assay induced by anti-CTLA-4 blocking antibodies (BNI3, 4.1.1, 4.8.1, and 6.1.1)(same donor T cells).</li><li><figref idref="f0032">Figure 14</figref> shows the mean enhancement of IL-2 production in T cells from 6 donors induced by anti-CTLA-4 blocking antibodies in the T cell blast/Raji assay.</li><li><figref idref="f0033">Figure 15</figref> shows the mean enhancement of IFN-γ production in T cells from 6 donors induced by anti-CTLA-4 blocking antibodies in the T cell blast/Raji assay.</li><li><figref idref="f0034">Figure 16</figref> shows the enhancement of IL-2 production in hPBMC from 5 donors induced by anti-CTLA-4 blocking mAbs as measured at 72 hours after stimulation with SEA.</li><li><figref idref="f0035">Figure 17</figref> shows the enhancement of IL-2 production in whole blood from 3 donors induced by anti-CTLA-4 blocking mAbs as measured at 72 and 96 hours after stimulation with SEA.</li><li><figref idref="f0036">Figure 18</figref> shows the inhibition of tumor growth with an anti-murine CTLA-4 antibody in a murine fibrosarcoma tumor model.</li><li><figref idref="f0037">Figure 19</figref> shows enhancement of IL-2 production induced by anti-CTLA4 antibodies (4.1.1 and 11.2.1) of the invention in a 72 hour T blast/Raji and Superantigen (whole blood and peripheral blood mononuclear cells from 6 donors) assays.</li><li><figref idref="f0038">Figure 20</figref> shows dose dependent enhancement of IL-2 production induced by anti-CTLA4 antibodies (4.1.1 and 11.2.1) of the invention in a 72 hour T blast/Raji assay.</li><li><figref idref="f0039">Figure 21</figref> shows dose dependent enhancement of IL-2 production induced by anti-CTLA4 antibodies (4.1.1 and 11.2.1) of the invention in a 72 hour Superantigen whole blood assay stimulated with 100 ng/ml superantigen.</li><li><figref idref="f0040 f0041 f0042 f0043 f0044 f0045 f0046 f0047 f0048">Figure 22</figref> provides a series of additional nucleotide and amino acid sequences of the following anti-CTLA-4 antibody chains: full length 4.1.1 heavy chain (cDNA 22(a), genomic 22(b), and amino acid 22(c)), full length aglycosylated 4.1.1 heavy chain (cDNA 22(d) and amino acid 22(e)), 4.1.1 light chain (cDNA 22(f) and amino acid 22(g)), full length 4.8.1 heavy chain (cDNA 22(h) and amino acid 22(i)), 4.8.1 light chain (cDNA 22(j) and amino acid 22(k)), full length 6.1.1 heavy chain (cDNA 22(l) and amino acid 22(m)), 6.1.1 light chain (cDNA 22(n) and amino acid 22(o)), full length 11.2.1 heavy chain (cDNA 22(p) and amino acid 22(q)), and 11.2.1 light chain (cDNA 22 (r) and amino acid 22(s)).</li></ul>

<u>SUMMARY OF THE INVENTION</u>

In accordance with a first aspect of the present invention, there is provided an antibody that is capable of binding CTLA-4, comprising a heavy chain variable region amino acid sequence that comprises a contiguous amino acid sequence from within an FR1 sequence through an FR3 sequence that is encoded by a human V_H3-33 family gene and that comprises at least one of the amino acid substitutions in the CDR1 sequences, CDR2 sequences, or framework sequences shown in <figref idref="f0017 f0018 f0019">Figure 2</figref>. In a preferred embodiment, the amino acid sequence comprises a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, and SEQ ID NO:70. In another preferred embodiment, the antibody further comprises a light chain variable region amino acid sequence comprising a sequence selected from the group consisting of a sequence comprising SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, and SEQ ID NO:71.

In accordance with a second aspect of the present invention, there is provided an antibody comprising a heavy chain amino acid sequence comprising SEQ ID NO:1 and a light chain variable amino acid sequence comprising SEQ ID NO:14.

In accordance with a third aspect of the present invention, there is provided an antibody comprising a heavy chain amino acid sequence comprising SEQ ID NO:2 and a light chain variable amino acid sequence comprising SEQ ID NO:15.

In accordance with a fourth aspect of the present invention, there is provided an antibody comprising a heavy chain amino acid sequence comprising SEQ ID NO:4 and a light chain variable amino acid sequence comprising SEQ ID NO:17.

In accordance with a fifth aspect of the present invention, there is provided an isolated human monoclonal antibody that is capable of binding to CTLA-4. In a preferred embodiment, antibody is capable of competing for binding with CTLA-4 with an antibody selected from the group consisting of 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.2.1, 11.6.1, 11.7.1, 12.3.1.1, and 12.9.1.1. In another preferred embodiment, the antibody possesses a substantially similar binding specificity to CTLA-4 as an antibody selected from the group consisting of 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.2.1, 11.6.1, 11.7.1, 12.3.1.1, and 12.9.1.1. In another preferred embodiment, the antibody is selected from the group consisting of 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.2.1, 11.6.1, 11.7.1, 12.3.1.1, and 12.9.1.1. In another preferred embodiment, the antibody is not cross reactive with CTLA-4 from lower mammalian species, preferably the lower mammalian species comprises mouse, rat, and rabbit and more preferably mouse and rat. In another preferred embodiment, the antibody is cross reactive with CTLA-4 from primates, preferably the primates comprise cynomolgous and rhesus monkeys. In another preferred embodiment, the antibody possesses a selectivity for CTLA-4 over CD28, B7-2, CD44, and hIgG1 of greater than about 100:1 and preferably about 500:1 or greater. In another preferred embodiment, the binding affinity of the antibody is about 10^-9 M or greater and preferably about 10^-10 M or greater. In another preferred embodiment, the antibody inhibits binding between CTLA-4 and B7-2 with an IC₅₀ of lower than about 100 nM and preferably lower than about 0.38 nM. In another preferred embodiment, the antibody inhibits binding between CTLA-4 and B7-1 with an IC₅₀ of lower than about 100 nM or greater and preferably lower than about 0.50 nM. In another preferred embodiment, the antibody enhances IL-2 production in a T cell blast/Raji assay by about 500 pg/ml or greater and preferably by about 3846 pg/ml or greater. In another preferred embodiment, the antibody enhances IFN-γ production in a T cell blast/Raji assay by about 500 pg/ml or greater and preferably by about 1233 pg/ml or greater. In another preferred embodiment, the antibody enhances IL-2 production in a hPBMC or whole blood superantigen assay by about 500 pg/ml or greater. In another preferred embodiment, the antibody enhances IL-2 production in a hPBMC or whole blood superantigen assay by about 500 pg/ml or preferably 1500 pg/ml or greater or by greater than about 30% or preferably 50% relative to control.

In accordance with a sixth aspect of the present invention, there is provided a humanized antibody that possesses a substantially similar binding specificity to CTLA-4 as an antibody selected from the group consisting of 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.2.1, 11.6.1, 11.7.1, 12.3.1.1, and 12.9.1.1. In a preferred embodiment, the antibody is not cross reactive with CTLA-4 from lower mammalian species, preferably the lower mammalian species comprises mouse, rat, and rabbit and preferably mouse and rat. In another preferred embodiment, the antibody is cross reactive with CTLA-4 from primates, preferably the primates comprise cynomolgous and rhesus monkeys. In another preferred embodiment, the antibody possesses a selectivity for CTLA-4 over CD28, B7-2, CD44, and hIgG1 of greater than about 100:1 and preferably about 500:1 or greater. In another preferred embodiment, the binding affinity of the antibody is about 10^-9 M or greater and preferably about 10^-10 M or greater. In another preferred embodiment, the antibody inhibits binding between CTLA-4 and B7-2 with an IC₅₀ of lower than about 100 nM and preferably lower than about 0.38 nM. In another preferred embodiment, the antibody inhibits binding between CTLA-4 and B7-1 with an IC₅₀ of lower than about 100 nM or greater and preferably lower than about 0.50 nM. In another preferred embodiment, the antibody enhances IL-2 production in a T cell blast/Raji assay by about 500 pg/ml or greater and preferably by about 3846 pg/ml or greater. In another preferred embodiment, the antibody enhances IFN-γ production in a T cell blast/Raji assay by about 500 pg/ml or greater and preferably by about 1233 pg/ml or greater. In another preferred embodiment, the antibody induces IL-2 production in a hPBMC or whole blood superantigen assay by about 500 pg/ml or greater. In another preferred embodiment, the antibody enhances IL-2 production in a hPBMC or whole blood superantigen assay by about 500 pg/ml or preferably 1500 pg/ml or greater or by greater than about 30% or preferably 50% relative to control.

In accordance with a seventh aspect of the present invention, there is provided an antibody that binds to CTLA-4, comprising a heavy chain amino acid sequence comprising human FR1, FR2, and FR3 sequences encoded by a human V_H 3-33 gene family operably linked in frame with a CDR1, a CDR2, and a CDR3 sequence, the CDR1, CDR2, and CDR3 sequences being independently selected from the CDR1, CDR2, and CDR3 sequences illustrated in <figref idref="f0017 f0018 f0019">Figure 2</figref>. In a preferred embodiment, the antibody of Claim 32, further comprising any of the somatic mutations to the FR1, FR2, and FR3 sequences as illustrated in <figref idref="f0017 f0018 f0019">Figure 2</figref>.

In accordance with an eighth aspect of the present invention, there is provided an antibody that binds to CTLA-4, comprising a heavy chain amino acid sequence comprising human FR1, FR2, and FR3 sequences encoded by a human V_H 3-33 gene family operably linked in frame with a CDR1, a CDR2, and a CDR3 sequence, which antibody has the following properties: a binding affinity for CTLA-4 of about 10^-9 or greater; inhibits binding between CTLA-4 and B7-1 with an IC₅₀ of about 100 nM or lower; inhibits binding between CTLA-4 and B7-2 with an IC₅₀ of about 100 nM or lower; and enhances cytokine production in an assay of human T cells by 500 pg/ml or greater.

In accordance with a ninth aspect of the present invention, there is provided an antibody that binds to CTLA-4, comprising a heavy chain amino acid sequence comprising FR1, FR2, and FR3 sequences operably linked in frame with a CDR1, a CDR2, and a CDR3 sequence independently selected from the CDR1, CDR2, and CDR3 sequences illustrated in <figref idref="f0017 f0018 f0019">Figures 2</figref> and <figref idref="f0020">3</figref>, which antibody has the following properties: a binding affinity for CTLA-4 of about 10^-9 or greater; inhibits binding between CTLA-4 and B7-1 with an IC₅₀ of about 100 nM or lower; inhibits binding between CTLA-4 and B7-2 with an IC₅₀ of about 100 nM or lower; and enhances cytokine production in an assay of human T cells by 500 pg/ml or greater.

In accordance with a tenth aspect of the present invention, there is provided a cell culture system for assaying T cell stimulation, comprising a culture of human T cell blasts co-cultured with a Raji cell line. In a preferred embodiment, the T cell blasts are washed prior to culture with the Raji cell line.

In accordance with an eleventh aspect of the present invention, there is provided an assay for measuring T cell stimulation, comprising: providing a culture of human T cell blasts and a Raji cell line; contacting the culture with an agent; and measuring cytokine production by the culture.

In accordance with an twelfth aspect of the present invention, there is provided a functional assay for screening a moiety for T cell stimulatory function, comprising: providing a culture of human T cell blasts and a Raji cell line; contacting the culture with the moiety; and assessing cytokine production by the culture.

In accordance with a thirteenth aspect of the present invention, there is provided a T cell stimulatory assay for CTLA-4 inhibitory function, comprising contacting a culture comprising human T cell blasts and a Raji cell line with an agent and assessing cytokine production by the culture.

In accordance with a fourteenth aspect of the present invention, there is provided a method for screening an agent for T cell stimulatory activity, comprising: contacting the agent with a cell culture comprising human T cell blasts and a Raji cell line; and assessing cytokine production by the culture.

In each of the tenth through the fourteenth aspects of the present invention, in a preferred embodiment, the T cell blasts are washed prior to culture with the Raji cell line. In another preferred embodiment, the cytokine is IL-2 or IFN-γ. In a preferred embodiment, cytokine production is measured in supernatant isolated from the culture. In a preferred embodiment, the agent is an antibody and preferably binds to CTLA-4.

In accordance with a fifteenth aspect of the present invention, there is provided an assay for measuring T cell stimulation, comprising: providing a population of human peripheral blood mononuclear cells or human whole blood stimulated with staphylococcus enterotoxin A; contacting the culture with an agent; and measuring cytokine production by the cell population.

In accordance with a sixteenth aspect of the present invention, there is provided a functional assay for screening a moiety for T cell stimulatory function, comprising: providing a population of human peripheral blood mononuclear cells or human whole blood stimulated with staphylococcus enterotoxin A; contacting the culture with the moiety; and assessing cytokine production by the cell population.

In accordance with a seventeenth aspect of the present invention, there is provided a T cell stimulatory assay for CTLA-4 inhibitory function, comprising contacting a population of human peripheral blood mononuclear cells or human whole blood stimulated with staphylococcus enterotoxin A with an agent and assessing cytokine production by the cell population.

In accordance with an eighteenth aspect of the present invention, there is provided a method for screening an agent for T cell stimulatory activity, comprising: contacting the agent with a population of human peripheral blood mononuclear cells or human whole blood stimulated with staphylococcus enterotoxin A; and assessing cytokine production by the cell population.

In each of the fifteenth through the eighteenth aspects of the present invention, in a preferred embodiment, the cytokine is IL-2. In another preferred embodiment, cytokine production is measured in supernatant isolated from the culture. In a preferred embodiment, the agent is an antibody and preferably binds to CTLA-4.

<u>DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS</u>

In accordance with the present invention, there are provided fully human monoclonal antibodies against human CTLA-4. Nucleotide sequences encoding and amino acid sequences comprising heavy and light chain immunoglobulin molecules, particularly sequences corresponding to a contiguous heavy and light chain sequences from FR1 and CDR1 through CDR3 and FR4, are provided. Further provided are antibodies having similar binding properties and antibodies (or other antagonists) having similar functionality as antibodies disclosed herein. Hybridomas expressing such immunoglobulin molecules and monoclonal antibodies are also provided.

<i><u>Definitions</u></i>

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. <i>See e</i>.<i>g</i>., <nplcit id="ncit0052" npl-type="b"><text>Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989</text></nplcit>)), which is incorporated herein by reference. The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings: The term "isolated polynucleotide" as used herein shall mean a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the "isolated polynucleotide" (1) is not associated with all or a portion of a polynucleotide in which the "isolated polynucleotide" is found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence.

The term "isolated protein" referred to herein means a protein of cDNA, recombinant RNA, or synthetic origin or some combination thereof, which by virtue of its origin, or source of derivation, the "isolated protein" (1) is not associated with proteins found in nature, (2) is free of other proteins from the same source, e.g. free of murine proteins, (3) is expressed by a cell from a different species, or (4) does not occur in nature.

The term "polypeptide" as used herein as a generic term to refer to native protein, fragments, or analogs of a polypeptide sequence. Hence, native protein, fragments, and analogs are species of the polypeptide genus. Preferred polypeptides in accordance with the invention comprise the human heavy chain immunoglobulin molecules and the human kappa light chain immunoglobulin molecules represented in <figref idref="f0001 f0002 f0003 f0004 f0005 f0006 f0007 f0008 f0009 f0010 f0011 f0012 f0013 f0014 f0015 f0016">Figure 1</figref>, as well as antibody molecules formed by combinations comprising the heavy chain immunoglobulin molecules with light chain immunoglobulin molecules, such as the kappa light chain immunoglobulin molecules, and vice versa, as well as fragments and analogs thereof.

The term "naturally-occurring" as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory or otherwise is naturally-occurring.

The term "operably linked" as used herein refers to positions of components so described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

The term "control sequence" as used herein refers to polynucleotide sequences which are necessary to effect the expression and processing of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term "control sequences" is intended to include, at a minimum, all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

The term "polynucleotide" as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.

The term "oligonucleotide" referred to herein includes naturally occurring, and modified nucleotides linked together by naturally occurring, and non-naturally occurring oligonucleotide linkages. Oligonucleotides are a polynucleotide subset generally comprising a length of 200 bases or fewer. Preferably oligonucleotides are 10 to 60 bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides are usually single stranded, e.g. for probes; although oligonucleotides may be double stranded, e.g. for use in the construction of a gene mutant. Oligonucleotides of the invention can be either sense or antisense oligonucleotides.

The term "naturally occurring nucleotides" referred to herein includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" referred to herein includes nucleotides with modified or substituted sugar groups and the like. The term "oligonucleotide linkages" referred to herein includes oligonucleotides linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, phosphoroamidate, and the like. <i>See e</i>.<i>g</i>., <nplcit id="ncit0053" npl-type="s"><text>LaPlanche et al. Nucl. Acids Res. 14:9081 (1986</text></nplcit>); <nplcit id="ncit0054" npl-type="s"><text>Stec et al. J. Am. Chem. Soc. 106:6077 (1984</text></nplcit>); <nplcit id="ncit0055" npl-type="s"><text>Stein et al. Nucl. Acids Res. 16:3209 (1988</text></nplcit>); <nplcit id="ncit0056" npl-type="s"><text>Zon et al. Anti-Cancer Drug Design 6:539 (1991</text></nplcit>);<nplcit id="ncit0057" npl-type="b"><text> Zon et al. Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991</text></nplcit>)); <patcit id="pcit0012" dnum="US5151510A"><text>Stec et al. U.S. Patent No. 5,151,510</text></patcit>; <nplcit id="ncit0058" npl-type="s"><text>Uhlmann and Peyman Chemical Reviews 90:543 (1990</text></nplcit>), the disclosures of which are hereby incorporated by reference. An oligonucleotide can include a label for detection, if desired.

The term "selectively hybridize" referred to herein means to detectably and specifically bind. Polynucleotides, oligonucleotides and fragments thereof in accordance with the invention selectively hybridize to nucleic acid strands under hybridization and wash conditions that minimize appreciable amounts of detectable binding to nonspecific nucleic acids. High stringency conditions can be used to achieve selective hybridization conditions as known in the art and discussed herein. Generally, the nucleic acid sequence homology between the polynucleotides, oligonucleotides, and fragments of the invention and a nucleic acid sequence of interest will be at least 80%, and more typically with preferably increasing homologies of at least 85%, 90%, 95%, 99%, and 100%. Two amino acid sequences are homologous if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less are preferred with 2 or less being more preferred. Alternatively and preferably, two protein sequences (or polypeptide sequences derived from them of at least 30 amino acids in length) are homologous, as this term is used herein, if they have an alignment score of at more than 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater. See <nplcit id="ncit0059" npl-type="b"><text>Dayhoff, M.O., in Atlas of Protein Sequence and Structure, pp. 101-110 (Volume 5, National Biomedical Research Foundation (1972</text></nplcit>)) and Supplement 2 to this volume, pp. 1-10. The two sequences or parts thereof are more preferably homologous if their amino acids are greater than or equal to 50% identical when optimally aligned using the ALIGN program. The term "corresponds to" is used herein to mean that a polynucleotide sequence is homologous (i.e., is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence. In contradistinction, the term "complementary to" is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. For illustration, the nucleotide sequence "TATAC" corresponds to a reference sequence "TATAC" and is complementary to a reference sequence "GTATA".

The following terms are used to describe the sequence relationships between two or more polynucleotide or amino acid sequences: "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity", and "substantial identity". A "reference sequence" is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing or may comprise a complete cDNA or gene sequence. Generally, a reference sequence is at least 18 nucleotides or 6 amino acids in length, frequently at least 24 nucleotides or 8 amino acids in length, and often at least 48 nucleotides or 16 amino acids in length. Since two polynucleotides or amino acid sequences may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide or amino acid sequence) that is similar between the two molecules, and (2) may further comprise a sequence that is divergent between the two polynucleotides or amino acid sequences, sequence comparisons between two (or more) molecules are typically performed by comparing sequences of the two molecules over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window", as used herein, refers to a conceptual segment of at least 18 contiguous nucleotide positions or 6 amino acids wherein a polynucleotide sequence or amino acid sequence may be compared to a reference sequence of at least 18 contiguous nucleotides or 6 amino acid sequences and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions, deletions, substitutions, and the like (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of <nplcit id="ncit0060" npl-type="s"><text>Smith and Waterman Adv. Appl. Math. 2:482 (1981</text></nplcit>), by the homology alignment algorithm of<nplcit id="ncit0061" npl-type="s"><text> Needleman and Wunsch J. Mol. Biol. 48:443 (1970</text></nplcit>), by the search for similarity method of <nplcit id="ncit0062" npl-type="s"><text>Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85:2444 (1988</text></nplcit>), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, (Genetics Computer Group, 575 Science Dr., Madison, Wis.), Geneworks, or MacVector software packages), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected.

The term "sequence identity" means that two polynucleotide or amino acid sequences are identical (i.e., on a nucleotide-by-nucleotide or residue-by-residue basis) over the comparison window. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) or residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The terms "substantial identity" as used herein denotes a characteristic of a polynucleotide or amino acid sequence, wherein the polynucleotide or amino acid comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 18 nucleotide (6 amino acid) positions, frequently over a window of at least 24-48 nucleotide (8-16 amino acid) positions, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the comparison window. The reference sequence may be a subset of a larger sequence.

As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. <i>See</i><nplcit id="ncit0063" npl-type="b"><text>Immunology - A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991</text></nplcit>)), which is incorporated herein by reference. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the lefthand direction is the amino terminal direction and the righthand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

Similarly, unless specified otherwise, the lefthand end of single-stranded polynucleotide sequences is the 5' end; the lefthand direction of double-stranded polynucleotide sequences is referred to as the 5' direction. The direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA and which are 5' to the 5' end of the RNA transcript are referred to as "upstream sequences"; sequence regions on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the RNA transcript are referred to as "downstream sequences".

As applied to polypeptides, the term "substantial identity" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity, and most preferably at least 99 percent sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic-aspartic, and asparagine-glutamine.

As discussed herein, minor variations in the amino acid sequences of antibodies or immunoglobulin molecules are contemplated as being encompassed by the present invention, providing that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99%. In particular, conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into families: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3) nonpolar=alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. More preferred families are: serine and threonine are aliphatic-hydroxy family; asparagine and glutamine are an amide-containing family; alanine, valine, leucine and isoleucine are an aliphatic family; and phenylalanine, tryptophan, and tyrosine are an aromatic family. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the binding or properties of the resulting molecule, especially if the replacement does not involve an amino acid within a framework site. Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those of ordinary skill in the art. Preferred amino- and carboxy-termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and/or amino acid sequence data to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function. Methods to identify protein sequences that fold into a known three-dimensional structure are known.<nplcit id="ncit0064" npl-type="s"><text> Bowie et al. Science 253:164 (1991</text></nplcit>). Thus, the foregoing examples demonstrate that those of skill in the art can recognize sequence motifs and structural conformations that may be used to define structural and functional domains in accordance with the invention.

Preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (4) confer or modify other physicochemical or functional properties of such analogs. Analogs can include various muteins of a sequence other than the naturally-occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally-occurring sequence (preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts. A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in <nplcit id="ncit0065" npl-type="b"><text>Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984</text></nplcit>)); <nplcit id="ncit0066" npl-type="b"><text>Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991</text></nplcit>)); and <nplcit id="ncit0067" npl-type="s"><text>Thornton et at. Nature 354:105 (1991</text></nplcit>), which are each incorporated herein by reference.

The term "polypeptide fragment" as used herein refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the naturally-occurring sequence deduced, for example, from a full-length cDNA sequence. Fragments typically are at least 5, 6, 8 or 10 amino acids long, preferably at least 14 amino acids long, more preferably at least 20 amino acids long, usually at least 50 amino acids long, and even more preferably at least 70 amino acids long. The term "analog" as used herein refers to polypeptides which are comprised of a segment of at least 25 amino acids that has substantial identity to a portion of a deduced amino acid sequence and which has at least one of the following properties: (1) specific binding to CTLA-4, under suitable binding conditions, (2) ability to block CTLA-4 binding with its receptors, or (3) ability to inhibit CTLA-4 expressing cell growth <i>in vitro</i> or <i>in vivo.</i> Typically, polypeptide analogs comprise a conservative amino acid substitution (or addition or deletion) with respect to the naturally-occurring sequence. Analogs typically are at least 20 amino acids long, preferably at least 50 amino acids long or longer, and can often be as long as a full-length naturally-occurring polypeptide.

Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drus with properties analogous to those of the template peptide. These types of non-peptide compound are termed "peptide mimetics" or "peptidomimetics". <nplcit id="ncit0068" npl-type="s"><text>Fauchere, J. Adv. Drug Res. 15:29 (1986</text></nplcit>); <nplcit id="ncit0069" npl-type="s"><text>Veber and Freidinger TINS p.392 (1985</text></nplcit>); and <nplcit id="ncit0070" npl-type="s"><text>Evans et al. J. Med. Chem. 30:1229 (1987</text></nplcit>), which are incorporated herein by reference. Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biochemical property or pharmacological activity), such as human antibody, but have one or more peptide linkages optionally replaced by a linkage selected from the group consisting of: --CH₂NH--, --CH₂S--, - CH₂-CH₂--, --CH=CH--(cis and trans), --COCH₂--, --CH(OH)CH₂--, and - CH₂SO--, by methods well known in the art. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) may be used to generate more stable peptides. In addition, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (<nplcit id="ncit0071" npl-type="s"><text>Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992</text></nplcit>), incorporated herein by reference); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.

"Antibody" or "antibody peptide(s)" refer to an intact antibody, or a binding fragment thereof that competes with the intact antibody for specific binding. Binding fragments are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab', F(ab')₂, Fv, and single-chain antibodies. An antibody other than a "bispecific" or "bifunctional" antibody is understood to have each of its binding sites identical. An antibody substantially inhibits adhesion of a receptor to a counterreceptor when an excess of antibody reduces the quantity of receptor bound to counterreceptor by at least about 20%, 40%, 60% or 80%, and more usually greater than about 85% (as measured in an <i>in vitro</i> competitive binding assay).

The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. An antibody is said to specifically bind an antigen when the dissociation constant is ≤1 µM, preferably ≤ 100 nM and most preferably ≤ 10 nM.

The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.

As used herein, the terms "label" or "labeled" refers to incorporation of a detectable marker, e.g., by incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). In certain situations, the label or marker can also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (<i>e</i>.<i>g</i>., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (<i>e</i>.<i>g</i>., FITC, rhodamine, lanthanide phosphors), enzymatic labels (<i>e</i>.<i>g</i>., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

The term "pharmaceutical agent or drug" as used herein refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient. Other chemistry terms herein are used according to conventional usage in the art, as exemplified by <nplcit id="ncit0072" npl-type="b"><text>The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985</text></nplcit>)), incorporated herein by reference).

The term "antineoplastic agent" is used herein to refer to agents that have the functional property of inhibiting a development or progression of a neoplasm in a human, particularly a malignant (cancerous) lesion, such as a carcinoma, sarcoma, lymphoma, or leukemia. Inhibition of metastasis is frequently a property of antineoplastic agents.

As used herein, "substantially pure" means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, more preferably more than about 85%, 90%, 95%, and 99%. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.

The term patient includes human and veterinary subjects.

<i><u>Antibody Structure</u></i>

The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. <i>See generally</i>, <nplcit id="ncit0073" npl-type="b"><text>Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989</text></nplcit>)) (incorporated by reference in its entirety for all purposes). The variable regions of each light/heavy chain pair form the antibody binding site.

Thus, an intact IgG antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are the same.

The chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is in accordance with the definitions of <nplcit id="ncit0074" npl-type="b"><text>Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991</text></nplcit>)), or <nplcit id="ncit0075" npl-type="s"><text>Chothia &amp; Lesk J. Mol. Biol. 196:901-917 (1987</text></nplcit>); <nplcit id="ncit0076" npl-type="s"><text>Chothia et al. Nature 342:878-883 (1989</text></nplcit>).

A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. <i>See, e.g.,</i><nplcit id="ncit0077" npl-type="s"><text> Songsivilai &amp; Lachmann Clin. Exp. Immunol. 79: 315-321 (1990</text></nplcit>), <nplcit id="ncit0078" npl-type="s"><text>Kostelny et al. J. Immunol. 148:1547-1553 (1992</text></nplcit>). In addition, bispecific antibodies may be formed as "diabodies" (<nplcit id="ncit0079" npl-type="s"><text>Holliger et al. "'Diabodies': small bivalent and bispecific antibody fragments" PNAS USA 90:6444-6448 (1993</text></nplcit>)) or "Janusins" (<nplcit id="ncit0080" npl-type="s"><text>Traunecker et al. "Bispecific single chain molecules (Janusins) target cytotoxic lymphocytes on HIV infected cells" EMBO J 10:3655-3659 (1991</text></nplcit>) and <nplcit id="ncit0081" npl-type="s"><text>Traunecker et al. "Janusin: new molecular design for bispecific reagents" Int J Cancer Suppl 7:51-52 (1992</text></nplcit>)). Production of bispecific antibodies can be a relatively labor intensive process compared with production of conventional antibodies and yields and degree of purity are generally lower for bispecific antibodies. Bispecific antibodies do not exist in the form of fragments having a single binding site (e.g., Fab, Fab', and Fv).

<i><u>Human Antibodies and Humanization of Antibodies</u></i>

Human antibodies avoid certain of the problems associated with antibodies that possess murine or rat variable and/or constant regions. The presence of such murine or rat derived proteins can lead to the rapid clearance of the antibodies or can lead to the generation of an immune response against the antibody by a patient. In order to avoid the utilization of murine or rat derived antibodies, it has been postulated that one can develop humanized antibodies or generate fully human antibodies through the introduction of human antibody function into a rodent so that the rodent would produce antibodies having fully human sequences.

<u>Human Antibodies</u>

The ability to clone and reconstruct megabase-sized human loci in YACs and to introduce them into the mouse germline provides a powerful approach to elucidating the functional components of very large or crudely mapped loci as well as generating useful models of human disease. Furthermore, the utilization of such technology for substitution of mouse loci with their human equivalents could provide unique insights into the expression and regulation of human gene products during development, their communication with other systems, and their involvement in disease induction and progression.

An important practical application of such a strategy is the "humanization" of the mouse humoral immune system. Introduction of human immunoglobulin (Ig) loci into mice in which the endogenous Ig genes have been inactivated offers the opportunity to study the mechanisms underlying programmed expression and assembly of antibodies as well as their role in B-cell development. Furthermore, such a strategy could provide an ideal source for production of fully human monoclonal antibodies (Mabs) an important milestone towards fulfilling the promise of antibody therapy in human disease. Fully human antibodies are expected to minimize the immunogenic and allergic responses intrinsic to mouse or mouse-derivatized Mabs and thus to increase the efficacy and safety of the administered antibodies. The use of fully human antibodies can be expected to provide a substantial advantage in the treatment of chronic and recurring human diseases, such as inflammation, autoimmunity, and cancer, which require repeated antibody administrations.

One approach towards this goal was to engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci in anticipation that such mice would produce a large repertoire of human antibodies in the absence of mouse antibodies. Large human Ig fragments would preserve the large variable gene diversity as well as the proper regulation of antibody production and expression. By exploiting the mouse machinery for antibody diversification and selection and the lack of immunological tolerance to human proteins, the reproduced human antibody repertoire in these mouse strains should yield high affinity antibodies against any antigen of interest, including human antigens. Using the hybridoma technology, antigen-specific human Mabs with the desired specificity could be readily produced and selected.

This general strategy was demonstrated in connection with our generation of the first XenoMouse™ strains as published in 1994. <i>See</i><nplcit id="ncit0082" npl-type="s"><text>Green et al. Nature Genetics 7:13-21 (1994</text></nplcit>). The XenoMouse™ strains were engineered with yeast artificial chromosomes (YACs) containing 245 kb and 190 kb-sized germline configuration fragments of the human heavy chain locus and kappa light chain locus, respectively, which contained core variable and constant region sequences. <i>Id</i>. The human Ig containing YACs proved to be compatible with the mouse system for both rearrangement and expression of antibodies and were capable of substituting for the inactivated mouse Ig genes. This was demonstrated by their ability to induce B-cell development, to produce an adult-like human repertoire of fully human antibodies, and to generate antigen-specific human Mabs. These results also suggested that introduction of larger portions of the human Ig loci containing greater numbers of V genes, additional regulatory elements, and human Ig constant regions might recapitulate substantially the full repertoire that is characteristic of the human humoral response to infection and immunization. The work of Green et al. was recently extended to the introduction of greater than approximately 80% of the human antibody repertoire through introduction of megabase sized, germline configuration YAC fragments of the human heavy chain loci and kappa light chain loci, respectively, to produce XenoMouse™ mice. <i>See</i><nplcit id="ncit0083" npl-type="s"><text>Mendez et al. Nature Genetics 15:146-156 (1997</text></nplcit>), <nplcit id="ncit0084" npl-type="s"><text>Green and Jakobovits J. Exp. Med. 188:483-495 (1998</text></nplcit>), and <patcit id="pcit0013" dnum="US75962096A"><text>U.S. Patent Application Serial No. 08/759,620, filed December 3, 1996</text></patcit>, the disclosures of which are hereby incorporated by reference.

Such approach is further discussed and delineated in <patcit id="pcit0014" dnum="US46600890A"><text>U.S. Patent Application Serial Nos. 07/466,008, filed January 12, 1990</text></patcit>, <patcit id="pcit0015" dnum="US07610515B"><text>07/610,515, filed November 8, 1990</text></patcit>, <patcit id="pcit0016" dnum="US07919297B"><text>07/919,297, filed July 24, 1992</text></patcit>, <patcit id="pcit0017" dnum="US07922649B"><text>07/922,649, filed July 30, 1992</text></patcit>, filed <patcit id="pcit0018" dnum="US08031801B"><text>08/031,801, filed March 15,1993</text></patcit>, <patcit id="pcit0019" dnum="US08112848B"><text>08/112,848, filed August 27, 1993</text></patcit>, <patcit id="pcit0020" dnum="US08234145B"><text>08/234,145, filed April 28, 1994</text></patcit>, <patcit id="pcit0021" dnum="US08376279B"><text>08/376,279, filed January 20, 1995</text></patcit>, <patcit id="pcit0022" dnum="US08430938B"><text>08/430, 938, April 27, 1995</text></patcit>, <patcit id="pcit0023" dnum="US08464584B"><text>08/464,584, filed June 5, 1995</text></patcit>, <patcit id="pcit0024" dnum="US08464582B"><text>08/464,582, filed June 5, 1995</text></patcit>, <patcit id="pcit0025" dnum="US08463191B"><text>08/463,191, filed June 5, 1995</text></patcit>, <patcit id="pcit0026" dnum="US08462837B"><text>08/462,837, filed June 5, 1995</text></patcit>, <patcit id="pcit0027" dnum="US08486853B"><text>08/486,853, filed June 5, 1995</text></patcit>, <patcit id="pcit0028" dnum="US08486857B"><text>08/486,857, filed June 5, 1995</text></patcit>, <patcit id="pcit0029" dnum="US08486859B"><text>08/486,859, filed June 5, 1995</text></patcit>, <patcit id="pcit0030" dnum="US08462513B"><text>08/462,513, filed June 5, 1995</text></patcit>, <patcit id="pcit0031" dnum="US08724752B"><text>08/724,752, filed October 2, 1996</text></patcit>, and <patcit id="pcit0032" dnum="US08759620B"><text>08/759,620, filed December 3, 1996</text></patcit>. <i>See also</i><nplcit id="ncit0085" npl-type="s"><text>Mendez et al. Nature Genetics 15:146-156 (1997</text></nplcit>) and <nplcit id="ncit0086" npl-type="s"><text>Green and Jakobovits J. Exp. Med. 188:483-495 (1998</text></nplcit>). <i>See also</i> European Patent No., <patcit id="pcit0033" dnum="EP0463151B1"><text>EP 0 463 151 B1</text></patcit>, grant published June 12, 1996, International Patent Application No., <patcit id="pcit0034" dnum="WO9402602A"><text>WO 94/02602, published February 3, 1994</text></patcit>, International Patent Application No., <patcit id="pcit0035" dnum="WO9634096A"><text>WO 96/34096, published October 31, 1996</text></patcit>, and <patcit id="pcit0036" dnum="WO9824893A"><text>WO 98/24893, published June 11, 1998</text></patcit>. The disclosures of each of the above-cited patents, applications, and references are hereby incorporated by reference in their entirety.

In an alternative approach, others, including GenPharm International, Inc., have utilized a "minilocus" approach. In the minilocus approach, an exogenous Ig locus is mimicked through the inclusion of pieces (individual genes) from the Ig locus. Thus, one or more V_H genes, one or more D_H genes, one or more J_H genes, a mu constant region, and a second constant region (preferably a gamma constant region) are formed into a construct for insertion into an animal. This approach is described in <patcit id="pcit0037" dnum="US5545807A"><text>U.S. Patent No. 5,545,807 to Surani et al.</text></patcit> and <patcit id="pcit0038" dnum="US5545806A"><text>U.S. Patent Nos. 5,545,806</text></patcit>, <patcit id="pcit0039" dnum="US5625825A"><text>5,625,825</text></patcit>, <patcit id="pcit0040" dnum="US5625126A"><text>5,625,126</text></patcit>, <patcit id="pcit0041" dnum="US5633425A"><text>5,633,425</text></patcit>, <patcit id="pcit0042" dnum="US5661016A"><text>5,661,016</text></patcit>, <patcit id="pcit0043" dnum="US5770429A"><text>5,770,429</text></patcit>, <patcit id="pcit0044" dnum="US5789650A"><text>5,789,650</text></patcit>, and <patcit id="pcit0045" dnum="US5814318A"><text>5,814,318 each to Lonberg and Kay</text></patcit>, <patcit id="pcit0046" dnum="US5591669A"><text>U.S. Patent No. 5,591,669 to Krimpenfort and Berns</text></patcit>, <patcit id="pcit0047" dnum="US5612205A"><text>U.S. Patent Nos. 5,612,205</text></patcit>, <patcit id="pcit0048" dnum="US5721367A"><text>5,721,367</text></patcit>, <patcit id="pcit0049" dnum="US5789215A"><text>5,789,215 to Berns et al.</text></patcit>, and <patcit id="pcit0050" dnum="US5643763A"><text>U.S. Patent No. 5,643,763 to Choi and Dunn</text></patcit>, and GenPharm International <patcit id="pcit0051" dnum="US57474890A"><text>U.S. Patent Application Serial Nos. 07/574,748, filed August 29, 1990</text></patcit>, <patcit id="pcit0052" dnum="US07575962B"><text>07/575,962, filed August 31, 1990</text></patcit>, <patcit id="pcit0053" dnum="US07810279B"><text>07/810,279, filed December 17, 1991</text></patcit>, <patcit id="pcit0054" dnum="US07853408B"><text>07/853,408, filed March 18, 1992</text></patcit>, <patcit id="pcit0055" dnum="US07904068B"><text>07/904,068, filed June 23, 1992</text></patcit>, <patcit id="pcit0056" dnum="US07990860B"><text>07/990,860, filed December 16, 1992</text></patcit>, <patcit id="pcit0057" dnum="US08053131B"><text>08/053,131, filed April 26, 1993</text></patcit>, <patcit id="pcit0058" dnum="US08096762B"><text>08/096,762, filed July 22, 1993</text></patcit>, <patcit id="pcit0059" dnum="US08155301B"><text>08/155,301, filed November 18, 1993</text></patcit>, <patcit id="pcit0060" dnum="US08161739B"><text>08/161,739, filed December 3, 1993</text></patcit>, <patcit id="pcit0061" dnum="US08165699B"><text>08/165,699, filed December 10, 1993</text></patcit>, <patcit id="pcit0062" dnum="US08209741B"><text>08/209,741, filed March 9, 1994</text></patcit>, the disclosures of which are hereby incorporated by reference. <i>See also</i> European Patent No. <patcit id="pcit0063" dnum="EP0546073B1"><text>0 546 073 B1</text></patcit>, International Patent Application Nos. <patcit id="pcit0064" dnum="WO9203918A"><text>WO 92/03918</text></patcit>, <patcit id="pcit0065" dnum="WO9222645A"><text>WO 92/22645</text></patcit>, <patcit id="pcit0066" dnum="WO9222647A"><text>WO 92/22647</text></patcit>, <patcit id="pcit0067" dnum="WO9222670A"><text>WO 92/22670</text></patcit>, <patcit id="pcit0068" dnum="WO9312227A"><text>WO 93/12227</text></patcit>, <patcit id="pcit0069" dnum="WO9400569A"><text>WO 94/00569</text></patcit>, <patcit id="pcit0070" dnum="WO9425585A"><text>WO 94/25585</text></patcit>, <patcit id="pcit0071" dnum="WO9614436A"><text>WO 96/14436</text></patcit>, <patcit id="pcit0072" dnum="WO9713852A"><text>WO 97/13852</text></patcit>, and <patcit id="pcit0073" dnum="WO9824884A"><text>WO 98/24884</text></patcit>, the disclosures of which are hereby incorporated by reference in their entirety. <i>See further</i> Taylor et al., 1992, Chen et al., 1993, Tuaillon et al., 1993, Choi et al., 1993, Lonberg et al., (1994), Taylor et al., (1994), and Tuaillon et al., (1995), Fishwild et al., (1996), the disclosures of which are hereby incorporated by reference in their entirety.

The inventors of Surani et al., cited above and assigned to the Medical Research Counsel (the "MRC"), produced a transgenic mouse possessing an Ig locus through use of the minilocus approach. The inventors on the GenPharm International work, cited above, Lonberg and Kay, following the lead of the present inventors, proposed inactivation of the endogenous mouse Ig locus coupled with substantial duplication of the Surani et al. work.

An advantage of the minilocus approach is the rapidity with which constructs including portions of the Ig locus can be generated and introduced into animals. Commensurately, however, a significant disadvantage of the minilocus approach is that, in theory, insufficient diversity is introduced through the inclusion of small numbers of V, D, and J genes. Indeed, the published work appears to support this concern. B-cell development and antibody production of animals produced through use of the minilocus approach appear stunted. Therefore, research surrounding the present invention has consistently been directed towards the introduction of large portions of the Ig locus in order to achieve greater diversity and in an effort to reconstitute the immune repertoire of the animals.

Human anti-mouse antibody (HAMA) responses have led the industry to prepare chimeric or otherwise humanized antibodies. While chimeric antibodies have a human constant region and a murine variable region, it is expected that certain human anti-chimeric antibody (HACA) responses will be observed, particularly in chronic or multi-dose utilizations of the antibody. Thus, it would be desirable to provide fully human antibodies against CTLA-4 in order to vitiate concerns and/or effects of HAMA or HACA response.

<u>Humanization and Display Technologies</u>

As was discussed above in connection with human antibody generation, there are advantages to producing antibodies with reduced immunogenicity. To a degree, this can be accomplished in connection with techniques of humanization and display techniques using appropriate libraries. It will be appreciated that murine antibodies or antibodies from other species can be humanized or primatized using techniques well known in the art. <i>See e.g.,</i><nplcit id="ncit0087" npl-type="s"><text>Winter and Harris Immunol Today 14:43-46 (1993</text></nplcit>) and <nplcit id="ncit0088" npl-type="s"><text>Wright et al. Crit. Reviews in Immunol. 12125-168 (1992</text></nplcit>). The antibody of interest may be engineered by recombinant DNA techniques to substitute the CH1, CH2, CH3, hinge domains, and/or the framework domain with the corresponding human sequence (<i>see</i><patcit id="pcit0074" dnum="WO9202190A"><text>WO 92/02190</text></patcit> and <patcit id="pcit0075" dnum="US5530101A"><text>U.S. Patent Nos. 5,530,101</text></patcit>, <patcit id="pcit0076" dnum="US5585089A"><text>5,585,089</text></patcit>, <patcit id="pcit0077" dnum="US5693761A"><text>5,693,761</text></patcit>, <patcit id="pcit0078" dnum="US5693792A"><text>5,693,792</text></patcit>, <patcit id="pcit0079" dnum="US5714350A"><text>5,714,350</text></patcit>, and <patcit id="pcit0080" dnum="US5777085A"><text>5,777,085</text></patcit>). Also, the use of Ig cDNA for construction of chimeric immunoglobulin genes is known in the art (<nplcit id="ncit0089" npl-type="s"><text>Liu et al. P.N.A.S. 84:3439 (1987</text></nplcit>) and <nplcit id="ncit0090" npl-type="s"><text>J.Immunol.139:3521 (1987</text></nplcit>)). mRNA is isolated from a hybridoma or other cell producing the antibody and used to produce cDNA. The cDNA of interest may be amplified by the polymerase chain reaction using specific primers (<patcit id="pcit0081" dnum="US4683195A"><text>U.S. Pat. Nos. 4,683,195</text></patcit> and <patcit id="pcit0082" dnum="US4683202A"><text>4,683,202</text></patcit>). Alternatively, a library is made and screened to isolate the sequence of interest. The DNA sequence encoding the variable region of the antibody is then fused to human constant region sequences. The sequences of human constant regions genes may be found in <nplcit id="ncit0091" npl-type="s"><text>Kabat et al. (1991) Sequences of Proteins of Immunological Interest, N.I.H. publication no. 91-3242</text></nplcit>. Human C region genes are readily available from known clones. The choice of isotype will be guided by the desired effector functions, such as complement fixation, or activity in antibody-dependent cellular cytotoxicity. Preferred isotypes are IgG1, IgG2, IgG3 and IgG4. Particularly preferred isotypes for antibodies of the invention are IgG2 and IgG4. Either of the human light chain constant regions, kappa or lambda, may be used. The chimeric, humanized antibody is then expressed by conventional methods.

Antibody fragments, such as Fv, F(ab')₂ and Fab may be prepared by cleavage of the intact protein, e.g. by protease or chemical cleavage. Alternatively, a truncated gene is designed. For example, a chimeric gene encoding a portion of the F(ab')₂ fragment would include DNA sequences encoding the CHI domain and hinge region of the H chain, followed by a translational stop codon to yield the truncated molecule.

In one approach, consensus sequences encoding the heavy and light chain J regions may be used to design oligonucleotides for use as primers to introduce useful restriction sites into the J region for subsequent linkage of V region segments to human C region segments. C region cDNA can be modified by site directed mutagenesis to place a restriction site at the analogous position in the human sequence.

Expression vectors include plasmids, retroviruses, cosmids, YACs, EBV derived episomes, and the like. A convenient vector is one that encodes a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that any VH or VL sequence can be easily inserted and expressed. In such vectors, splicing usually occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C region, and also at the splice regions that occur within the human CH exons. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding regions. The resulting chimeric antibody may be joined to any strong promoter, including retroviral LTRs, e.g. SV-40 early promoter, (<nplcit id="ncit0092" npl-type="s"><text>Okayama et al. Mol. Cell. Bio. 3:280 (1983</text></nplcit>)), Rous sarcoma virus LTR (<nplcit id="ncit0093" npl-type="s"><text>Gorman et al. P.N.A.S. 79:6777 (1982</text></nplcit>)), and moloney murine leukemia virus LTR (<nplcit id="ncit0094" npl-type="s"><text>Grosschedl et al. Cell 41:885 (1985</text></nplcit>)); native 1g promoters, etc.

Further, human antibodies or antibodies from other species can be generated through display-type technologies, including, without limitation, phage display, retroviral display, ribosomal display, and other techniques, using techniques well known in the art and the resulting molecules can be subjected to additional maturation, such as affinity maturation, as such techniques are well known in the art. Wright and Harris, <i>supra</i>., <nplcit id="ncit0095" npl-type="s"><text>Hanes and Plucthau PNAS USA 94:4937-4942 (1997</text></nplcit>) (ribosomal display), <nplcit id="ncit0096" npl-type="s"><text>Parmley and Smith Gene 73:305-318 (1988</text></nplcit>) (phage display),<nplcit id="ncit0097" npl-type="s"><text> Scott TIBS 17:241-245 (1992</text></nplcit>), <nplcit id="ncit0098" npl-type="s"><text>Cwirla et al. PNAS USA 87:6378-6382 (1990</text></nplcit>), <nplcit id="ncit0099" npl-type="s"><text>Russel et al. Nucl. Acids Research 21:1081-1085 (1993</text></nplcit>), <nplcit id="ncit0100" npl-type="s"><text>Hoganboom et al. Immunol. Reviews 130:43-68 (1992</text></nplcit>), <nplcit id="ncit0101" npl-type="s"><text>Chiswell and McCafferty TIBTECH 10:80-84 (1992</text></nplcit>), and <patcit id="pcit0083" dnum="US5733743A"><text>U.S. Patent No. 5,733,743</text></patcit>. If display technologies are utilized to produce antibodies that are not human, such antibodies can be humanized as described above.

Using these techniques, antibodies can be generated to CTLA-4 expressing cells, CTLA-4 itself, forms of CTLA-4, epitopes or peptides thereof, and expression libraries thereto (<i>see e.g.</i><patcit id="pcit0084" dnum="US5703057A"><text>U.S. Patent No. 5,703,057</text></patcit>) which can thereafter be screened as described above for the activities described above.

<u>Additional Criteria for Antibody Therapeutics</u>

As will be appreciated, it is generally not desirable to kill CTLA-4 expressing cells. Rather, one generally desires to simply inhibit CTLA-4 binding with its ligands to mitigate T cell down regulation. One of the major mechanisms through which antibodies kill cells is through fixation of complement and participation in CDC. The constant region of an antibody plays an important role in connection with an antibody's ability to fix complement and participate in CDC. Thus, generally one selects the isotype of an antibody to either provide the ability of complement fixation, or not. In the case of the present invention, generally, as mentioned above, it is generally not preferred to utilize an antibody that kills the cells. There are a number of isotypes of antibodies that are capable of complement fixation and CDC, including, without limitation, the following: murine IgM, murine IgG2a, murine IgG2b, murine IgG3, human IgM, human IgG1, and human IgG3. Those isotypes that do not include, without limitation, human IgG2 and human IgG4.

It will be appreciated that antibodies that are generated need not initially possess a particular desired isotype but, rather, the antibody as generated can possess any isotype and the antibody can be isotype switched thereafter using conventional techniques that are well known in the art. Such techniques include the use of direct recombinant techniques (<i>see e</i>.<i>g</i>., <patcit id="pcit0085" dnum="US4816397A"><text>U.S. Patent No. 4,816,397</text></patcit>), cell-cell fusion techniques (<i>see e</i>.<i>g</i>., <patcit id="pcit0086" dnum="US73063996A"><text>U.S. Patent Application No. 08/730,639, filed October 11, 1996</text></patcit>), among others.

In the cell-cell fusion technique, a myeloma or other cell line is prepared that possesses a heavy chain with any desired isotype and another myeloma or other cell line is prepared that possesses the light chain. Such cells can, thereafter, be fused and a cell line expressing an intact antibody can be isolated.

By way of example, the majority of the CTLA-4 antibodies discussed herein are human anti-CTLA-4 IgG2 antibody. Since such antibodies possess desired binding to the CTLA-4 molecule, any one of such antibodies can be readily isotype switched to generate a human IgG4 isotype, for example, while still possessing the same variable region (which defines the antibody's specificity and some of its affinity).

Accordingly, as antibody candidates are generated that meet desired "structural" attributes as discussed above, they can generally be provided with at least certain additional "functional" attributes that are desired through isotype switching.

<i><u>Design and Generation of Other Therapeutics</u></i>

In accordance with the present invention and based on the activity of the antibodies that are produced and characterized herein with respect to CTLA-4, the design of other therapeutic modalities including other antibodies, other antagonists, or chemical moieties other than antibodies is facilitated. Such modalities include, without limitation, antibodies having similar binding activity or functionality, advanced antibody therapeutics, such as bispecific antibodies, immunotoxins, and radiolabeled therapeutics, generation of peptide therapeutics, gene therapies, particularly intrabodies, antisense therapeutics, and small molecules. Furthermore, as discussed above, the effector function of the antibodies of the invention may be changed by isotype switching to an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM for various therapeutic uses.

In connection with the generation of advanced antibody therapeutics, where complement fixation is a desirable attribute, it may be possible to sidestep the dependence on complement for cell killing through the use of bispecifics, immunotoxins, or radiolabels, for example.

In connection with bispecific antibodies, bispecific antibodies can be generated that comprise (i) two antibodies one with a specificity to CTLA-4 and another to a second molecule that are conjugated together, (ii) a single antibody that has one chain specific to CTLA-4 and a second chain specific to a second molecule, or (iii) a single chain antibody that has specificity to CTLA-4 and the other molecule. Such bispecific antibodies can be generated using techniques that are well known for example, in connection with (i) and (ii) <i>see e</i>.<i>g</i>., <nplcit id="ncit0102" npl-type="s"><text>Fanger et al. Immunol Methods 4:72-81 (1994</text></nplcit>) and Wright and Harris, <i>supra.</i> and in connection with (iii) <i>see e</i>.<i>g</i>., <nplcit id="ncit0103" npl-type="s"><text>Traunecker et al. Int. J. Cancer (Suppl.) 7:51-52 (1992</text></nplcit>).

In addition, "Kappabodies" (<nplcit id="ncit0104" npl-type="s"><text>Ill et al. "Design and construction of a hybrid immunoglobulin domain with properties of both heavy and light chain variable regions" Protein Eng 10:949-57 (1997</text></nplcit>)), "Minibodies" (<nplcit id="ncit0105" npl-type="s"><text>Martin et al. "The affinity-selection of a minibody polypeptide inhibitor of human interleukin-6" EMBO J 13:5303-9 (1994</text></nplcit>)), "Diabodies" (<nplcit id="ncit0106" npl-type="s"><text>Holliger et al. "'Diabodies': small bivalent and bispecific antibody fragments" PNAS USA 90:6444-6448 (1993</text></nplcit>)), or "Janusins" (<nplcit id="ncit0107" npl-type="s"><text>Traunecker et al. "Bispecific single chain molecules (Janusins) target cytotoxic lymphocytes on HIV infected cells" EMBO J 10:3655-3659 (1991</text></nplcit>) and <nplcit id="ncit0108" npl-type="s"><text>Traunecker et al. "Janusin: new molecular design for bispecific reagents" Int J Cancer Suppl 7:51-52 (1992</text></nplcit>)) may also be prepared.

In connection with immunotoxins, antibodies can be modified to act as immunotoxins utilizing techniques that are well known in the art. <i>See e</i>.<i>g</i>., <nplcit id="ncit0109" npl-type="s"><text>Vitetta Immunol Today 14:252 (1993</text></nplcit>). <i>See also</i><patcit id="pcit0087" dnum="US5194594A"><text>U.S. Patent No. 5,194,594</text></patcit>. In connection with the preparation of radiolabeled antibodies, such modified antibodies can also be readily prepared utilizing techniques that are well known in the art. <i>See e.g.</i>, <nplcit id="ncit0110" npl-type="b"><text>Junghans et al. in Cancer Chemotherapy and Biotherapy 655-686 (2d edition, Chafner and Longo, eds., Lippincott Raven (1996</text></nplcit>)). <i>See also</i><patcit id="pcit0088" dnum="US4681581A"><text>U.S. Patent Nos. 4,681,581</text></patcit>, <patcit id="pcit0089" dnum="US4735210A"><text>4,735,210</text></patcit>, <patcit id="pcit0090" dnum="US5101827A"><text>5,101,827</text></patcit>, <patcit id="pcit0091" dnum="US5102990A"><text>5,102,990</text></patcit> (<patcit id="pcit0092" dnum="USRE35500E"><text>RE 35,500</text></patcit>), <patcit id="pcit0093" dnum="US5648471A"><text>5,648,471</text></patcit>, and <patcit id="pcit0094" dnum="US5697902A"><text>5,697,902</text></patcit>. Each of immunotoxins and radiolabeled molecules would be likely to kill cells expressing CTLA-4, and particularly those cells in which the antibodies of the invention are effective.

In connection with the generation of therapeutic peptides, through the utilization of structural information related to CTLA-4 and antibodies thereto, such as the antibodies of the invention (as discussed below in connection with small molecules) or screening of peptide libraries, therapeutic peptides can be generated that are directed against CTLA-4. Design and screening of peptide therapeutics is discussed in connection with <nplcit id="ncit0111" npl-type="s"><text>Houghten et al. Biotechniques 13:412-421 (1992</text></nplcit>), <nplcit id="ncit0112" npl-type="s"><text>Houghten PNAS USA 82:5131-5135 (1985</text></nplcit>), <nplcit id="ncit0113" npl-type="s"><text>Pinalla et al. Biotechniques 13:901-905 (1992</text></nplcit>), <nplcit id="ncit0114" npl-type="s"><text>Blake and Litzi-Davis BioConjugate Chem. 3:510-513 (1992</text></nplcit>). Immunotoxins and radiolabeled molecules can also be prepared, and in a similar manner, in connection with peptidic moieties as discussed above in connection with antibodies.

Important information related to the binding of an antibody to an antigen can be gleaned through phage display experimentation. Such experiments are generally accomplished through panning a phage library expressing random peptides for binding with the antibodies of the invention to determine if peptides can be isolated that bind. If successful, certain epitope information can be gleaned from the peptides that bind.

In general, phage libraries expressing random peptides can be purchased from New England Biolabs (7-mer and 12-mer libraries, Ph.D.-7 Peptide 7-mer Library Kit and Ph.D.-12 Peptide 12-mer Library Kit, respectively) based on a bacteriophage M13 system. The 7-mer library represents a diversity of approximately 2.0 x 10⁹ independent clones, which represents most, if not all, of the 20⁷ = 1.28 x 10⁹ possible 7-mer sequences. The 12-mer library contains approximately 1.9 x 10⁹ independent clones and represents only a very small sampling of the potential sequence space of 20¹² = 4.1 x 10¹⁵ 12-mer sequences. Each of 7-mer and 12-mer libraries are panned or screened in accordance with the manufacturer's recommendations in which plates were coated with an antibody to capture the appropriate antibody (a goat anti-human IgG Fc for an IgG antibody for example) followed by washing. Bound phage are eluted with 0.2 M glycine-HCl, pH 2.2. After 3 rounds of selection/amplification at constant stringency (0.5% Tween), through use of DNA sequencing, one can characterize clones from the libraries that are reactive with one or more of the antibodies. Reactivity of the peptides can be determined by ELISA. For an additional discussion of epitope analysis of peptides <i>see also</i><nplcit id="ncit0115" npl-type="s"><text>Scott, J.K. and Smith, G.P. Science 249:386-390 (1990</text></nplcit>); <nplcit id="ncit0116" npl-type="s"><text>Cwirla et al. PNAS USA 87:6378-6382 (1990</text></nplcit>); <nplcit id="ncit0117" npl-type="s"><text>Felici et al. J. Mol. Biol 222:301-310 (1991</text></nplcit>), and<nplcit id="ncit0118" npl-type="s"><text> Kuwabara et al. Nature Biotechnology 15:74-78 (1997</text></nplcit>).

The design of gene and/or antisense therapeutics through conventional techniques is also facilitated through the present invention. Such modalities can be utilized for modulating the function of CTLA-4. In connection therewith the antibodies of the present invention facilitate design and use of functional assays related thereto. A design and strategy for antisense therapeutics is discussed in detail in International Patent Application No. <patcit id="pcit0095" dnum="WO9429444A"><text>WO 94/29444</text></patcit>. Design and strategies for gene therapy are well known. However, in particular, the use of gene therapeutic techniques involving intrabodies could prove to be particularly advantageous. <i>See e.g.</i>, <nplcit id="ncit0119" npl-type="s"><text>Chen et al. Human Gene Therapy 5:595-601 (1994</text></nplcit>) and <nplcit id="ncit0120" npl-type="s"><text>Marasco Gene Therapy 4:11-15 (1997</text></nplcit>). General design of and considerations related to gene therapeutics is also discussed in International Patent Application No. <patcit id="pcit0096" dnum="WO9738137A"><text>WO 97/38137</text></patcit>. Genetic materials encoding an antibody of the invention (such as the 4.1.1, 4.8.1, or 6.1.1, or others) may be included in a suitable expression system (whether viral, attenuated viral, non-viral, naked, or otherwise) and administered to a host for <i>in vivo</i> generation of the antibody in the host.

Small molecule therapeutics can also be envisioned in accordance with the present invention. Drugs can be designed to modulate the activity of CTLA-4 based upon the present invention. Knowledge gleaned from the structure of the CTLA-4 molecule and its interactions with other molecules in accordance with the present invention, such as the antibodies of the invention, CD28, B7, B7-1, B7-2, and others can be utilized to rationally design additional therapeutic modalities. In this regard, rational drug design techniques such as X-ray crystallography, computer-aided (or assisted) molecular modeling (CAMM), quantitative or qualitative structure-activity relationship (QSAR), and similar technologies can be utilized to focus drug discovery efforts. Rational design allows prediction of protein or synthetic structures which can interact with the molecule or specific forms thereof which can be used to modify or modulate the activity of CTLA-4. Such structures can be synthesized chemically or expressed in biological systems. This approach has been reviewed in <nplcit id="ncit0121" npl-type="b"><text>Capsey et al. Genetically Engineered Human Therapeutic Drugs (Stockton Press, NY (1988</text></nplcit>)). Indeed, the rational design of molecules (either peptides, peptidomimetics, small molecules, or the like) based upon known, or delineated, structure-activity relationships with other molecules (such as antibodies in accordance with the invention) has become generally routine. <i>See, e.g.</i>, <nplcit id="ncit0122" npl-type="s"><text>Fry et al. "Specific, irreversible inactivation of the epidermal growth factor receptor and erbB2, by a new class of tyrosine kinase inhibitor" Proc Natl Acad Sci U S A 95:12022-7 (1998</text></nplcit>); <nplcit id="ncit0123" npl-type="s"><text>Hoffman et al. "A model of Cdc25 phosphatase catalytic domain and Cdk-interaction surface based on the presence of a rhodanese homology domain" J Mol Biol 282:195-208 (1998</text></nplcit>); <nplcit id="ncit0124" npl-type="s"><text>Ginalski et al. "Modelling of active forms of protein kinases: p38-a case study" Acta Biochim Pol 44:557-64 (1997</text></nplcit>); <nplcit id="ncit0125" npl-type="s"><text>Jouko et al. "Identification of csk tyrosine phosphorylation sites and a tyrosine residue important for kinase domain structure" Biochem J 322:927-35 (1997</text></nplcit>); <nplcit id="ncit0126" npl-type="s"><text>Singh et al. "Structure-based design of a potent, selective, and irreversible inhibitor of the catalytic domain of the erbB receptor subfamily of protein tyrosine kinases" J Med Chem 40:1130-5 (1997</text></nplcit>); <nplcit id="ncit0127" npl-type="s"><text>Mandel et al. "ABGEN: a knowledge-based automated approach for antibody structure modeling" Nat Biotechnol 14:323-8 (1996</text></nplcit>); <nplcit id="ncit0128" npl-type="s"><text>Monfardini et al. "Rational design, analysis, and potential utility of GM-CSF antagonists" Proc Assoc Am Physicians 108:420-31 (1996</text></nplcit>); <nplcit id="ncit0129" npl-type="s"><text>Furet et al. "Modelling study of protein kinase inhibitors: binding mode of staurosporine and origin of the selectivity of CGP 52411" J Comput Aided Mol Des 9:465-72 (1995</text></nplcit>).

Further, combinatorial libraries can be designed and sythesized and used in screening programs, such as high throughput screening efforts.

<i><u>Therapeutic Administration and Formulations</u></i>

It will be appreciated that administration of therapeutic entities in accordance with the invention will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: <nplcit id="ncit0130" npl-type="b"><text>Remington's Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, PA (1975</text></nplcit>)), particularly Chapter 87 by Blaug, Seymour, therein. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as Lipofectin™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be appropriate in treatments and therapies in accordance with the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. <i>See also</i><nplcit id="ncit0131" npl-type="s"><text>Powell et al. "Compendium of excipients for parenteral formulations" PDA J Pharm Sci Technol. 52:238-311 (1998</text></nplcit>) and the citations therein for additional information related to excipients and carriers well known to pharmaceutical chemists.

<i><u>Preparation of Antibodies</u></i>

Antibodies in accordance with the invention are preferably prepared through the utilization of a transgenic mouse that has a substantial portion of the human antibody producing genome inserted but that is rendered deficient in the production of endogenous, murine, antibodies. Such mice, then, are capable of producing human immunoglobulin molecules and antibodies and are deficient in the production of murine immunoglobulin molecules and antibodies. Technologies utilized for achieving the same are disclosed in the patents, applications, and references disclosed in the Background, herein. In particular, however, a preferred embodiment of transgenic production of mice and antibodies therefrom is disclosed in <patcit id="pcit0097" dnum="US75962096A"><text>U.S. Patent Application Serial No. 08/759,620, filed December 3, 1996</text></patcit>, the disclosure of which is hereby incorporated by reference. <i>See also</i><nplcit id="ncit0132" npl-type="s"><text>Mendez et al. Nature Genetics 15:146-156 (1997</text></nplcit>), the disclosure of which is hereby incorporated by reference.

Through use of such technology, we have produced fully human monoclonal antibodies to a variety of antigens. Essentially, we immunize XenoMouse™ lines of mice with an antigen of interest, recover lymphatic cells (such as B-cells) from the mice that express antibodies, fuse such recovered cells with a myeloid-type cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. We utilized these techniques in accordance with the present invention for the preparation of antibodies specific to CTLA-4. Herein, we describe the production of multiple hybridoma cell lines that produce antibodies specific to CTLA-4. Further, we provide a characterization of the antibodies produced by such cell lines, including nucleotide and amino acid sequence analyses of the heavy and light chains of such antibodies.

The antibodies derived from hybridoma cell lines discussed herein are designated 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.2.1, 11.6.1, 11.7.1, 12.3.1.1, and 12.9.1.1. Each of the antibodies produced by the aforementioned cell lines are either fully human IgG2 or IgG4 heavy chains with human kappa light chains. In general, antibodies in accordance with the invention possess very high affinities, typically possessing Kd's of from about 10^-9 through about 10^-11 M, when measured by either solid phase or solution phase.

As will be appreciated, antibodies in accordance with the present invention can be expressed in cell lines other than hybridoma cell lines. Sequences encoding the cDNAs or genomic clones for the particular antibodies can be used for transformation of a suitable mammalian or nonmammalian host cells. Transformation can be by any known method for introducing polynucleotides into a host cell, including, for example packaging the polynucleotide in a virus (or into a viral vector) and transducing a host cell with the virus (or vector) or by transfection procedures known in the art, as exemplified by <patcit id="pcit0098" dnum="US4399216A"><text>U.S. Patent Nos. 4,399,216</text></patcit>,<patcit id="pcit0099" dnum="US4912040A"><text> 4,912,040</text></patcit>, <patcit id="pcit0100" dnum="US4740461A"><text>4,740,461</text></patcit>, and <patcit id="pcit0101" dnum="US4959455A"><text>4,959,455</text></patcit> (which patents are hereby incorporated herein by reference). The transformation procedure used depends upon the host to be transformed. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, particle bombardment, encapsulation of the polynucleotide(s) in liposomes, peptide conjugates, dendrimers, and direct microinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, NSO₀, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and a number of other cell lines. Non-mammalian cells including but not limited to bacterial, yeast, insect, and plants can also be used to express recombinant antibodies. Site directed mutagenesis of the antibody CH2 domain to eliminate glycosylation may be preferred in order to prevent changes in either the immunogenicity, pharmacokinetic, and/or effector functions resulting from non-human glycosylation. The expression methods are selected by determining which system generates the highest expression levels and produce antibodies with constitutive CTLA-4 binding properties.

Further, expression of antibodies of the invention (or other moieties therefrom) from production cell lines can be enhanced using a number of known techniques. For example, the glutamine sythetase and DHFR gene expression systems are common approaches for enhancing expression under certain conditions. High expressing cell clones can be identified using conventional techniques, such as limited dilution cloning and Microdrop technology. The GS system is discussed in whole or part in connection with European Patent Nos.<patcit id="pcit0102" dnum="EP0216846A"><text> 0 216 846</text></patcit>, <patcit id="pcit0103" dnum="EP0256055A"><text>0 256 055</text></patcit>, and <patcit id="pcit0104" dnum="EP0323997A"><text>0 323 997</text></patcit> and European Patent Application No. <patcit id="pcit0105" dnum="EP89303964A"><text>89303964.4</text></patcit>.

Antibodies of the invention can also be produced transgenically through the generation of a mammal or plant that is transgenic for the immunoglobulin heavy and light chain sequences of interest and production of the antibody in a recoverable form therefrom. In connection with the transgenic production in mammals, antibodies can be produced in, and recovered from, the milk of goats, cows, or other mammals. <i>See, e.g.</i>, <patcit id="pcit0106" dnum="US5827690A"><text>U.S. Patent Nos. 5,827,690</text></patcit>, <patcit id="pcit0107" dnum="US5756687A"><text>5,756,687</text></patcit>, <patcit id="pcit0108" dnum="US5750172A"><text>5,750,172</text></patcit>, and <patcit id="pcit0109" dnum="US5741957A"><text>5,741,957</text></patcit>.

Antibodies in accordance with the present invention have been analyzed structurally and functionally. In connection with the structures of the antibodies, amino acid sequences of the heavy and kappa light chains have been predicted based on cDNA sequences obtained through RT-PCR of the hybridomas. <i>See</i> Examples 3 and 4 and <figref idref="f0016 f0017 f0018 f0019 f0020 f0021 f0022 f0023 f0024 f0025">Figures 1-8</figref>. N-terminal sequencing of the antibodies was also conducted in confirmation of the results discussed in Examples 3 and 4. <i>See</i> Example 5 and <figref idref="f0026">Figure 9</figref>. Kinetic analyses of the antibodies were conducted to determine affinities. <i>See</i> Example 2. Antibodies in accordance with the invention (and particularly the 4.1.1, 4.8.1, and 6.1.1 antibodies of the invention) have high affinities (4.1.1:1.63 X 10¹⁰ 1/M; 4.8.1:3.54 X 10¹⁰ 1/M; and 6.1.1:7.2 X 10⁹ 1/M). Further, antibodies were analyzed by isoelectric focusing (IEF), reducing gel electrophoresis (SDS-PAGE), size exclusion chromatography, liquid chromatography/mass spectroscopy, and mass spectroscopy and antibody production by the hybridomas was assessed. <i>See</i> Example 6 and <figref idref="f0027 f0028">Figure 10</figref>.

In connection with functional analysis of antibodies in accordance with the present invention, such antibodies proved to be potent inhibitors of CTLA-4 and its binding to its ligands of the B7 family of molecules. For example, antibodies in accordance with the present invention were demonstrated to block CTLA-4 binding to either B7-1 or B7-2. <i>See</i> Example 7. Indeed, many of the antibodies in accordance with the invention possess nanomolar and subnanomolar IC₅₀s with respect to inhibiting CTLA-4 binding to B7-1 and B7-2. Further, antibodies of the invention possess excellent selectivity for CTLA-4 as compared to CD28, CD44, B7-2, or hIgG1. <i>See</i> Example 8. Selectivity is a ratio that reflects the degree of preferential binding of a molecule with a first agent as compared to the molecules binding with a second, and optionally other molecules. Herein, selectivity refers to the degree of preferential binding of an antibody of the invention to CTLA-4 as compared to the antibody's binding to other molecules such as CD28, CD44, B7-2, or hIgG1. Selectivity values of antibodies of the invention greater than 500:1 are common. Antibodies of the invention have also been demonstrated to induce or enhance expression of certain cytokines (such as IL-2 and IFN-γ) by cultured T cells in a T cell blast model. <i>See</i> Examples 9 and 10 and <figref idref="f0030 f0031 f0032 f0033 f0034 f0035">Figures 12-17</figref>. Further, it is expected that antibodies of the invention will inhibit the growth of tumors in appropriate <i>in vivo</i> tumor models. The design of which models are discussed in Example 11 and 12.

The results demonstrated in accordance with the present invention indicate that antibodies of the present invention possess certain qualities that may make the present antibodies more efficacious than current therapeutic antibodies against CTLA-4.

In particular, the 4.1.1, 4.8.1, and 6.1.1 antibodies of the invention possess highly desirable properties. Their structural characteristics, functions, or activities provide criteria that facilitate the design or selection of additional antibodies or other molecules as discussed above. Such criteria include one or more of the following: <ul id="ul0002" list-style="none" compact="compact"><li>Ability to compete for binding to CTLA-4 with one or more of the antibodies of the invention;</li><li>Similar binding specificity to CTLA-4 as one or more of the antibodies of the invention;</li><li>A binding affinity for CTLA-4 of about 10^-9 M or greater and preferably of about 10^-10M or greater;</li><li>Does not cross react with lower mammalian CTLA-4, including, preferably, mouse, rat, or rabbit and preferably mouse or rat CTLA-4;</li><li>Cross reacts with primate CTLA-4, including, preferably, cynomolgous and rhesus CTLA-4;</li><li>A selectivity for CTLA-4 over CD28, B7-2, CD44, or hIgG1 of at least about 100:1 or greater and preferably of about 300, 400, or 500:1 or greater;</li><li>An IC₅₀ in blocking CTLA-4 binding to B7-2 of about 100 nM or lower and preferably 5, 4, 3, 2, 1, 0.5, or 0.38 nM or lower;</li><li>An IC₅₀ in blocking CTLA-4 binding to B7-1 of about of about 100 nM or lower and preferably 5, 4, 3, 2, 1, 0.5, or 0.50 nM or lower;</li><li>An enhancement of cytokine production in one or more <i>in vitro</i> assays, for example: <ul id="ul0003" list-style="none" compact="compact"><li>An enhancement of IL-2 production in a T cell blast/Raji assay of about 500 pg/ml or greater and preferably 750, 1000, 1500,2000, 3000, or 3846 pg/ml or greater,</li><li>An enhancement of IFN-γ production in a T cell blast/Raji assay of about 500 pg/ml or greater and preferably 750, 1000, or 1233 pg/ml or greater; or</li><li>An enhancement of IL-2 production in a hPBMC or whole blood superantigen assay of about 500 pg/ml or greater and preferably 750, 1000, 1200, or 1511 pg/ml or greater. Expressed another way, it is desirable that IL-2 production is enhanced by about 30, 35, 40, 45, 50 percent or more relative to control in the assay.</li></ul></li></ul>

It is expected that antibodies (or molecules designed or synthesized therefrom) having one or more of these properties will possess similar efficacy to the antibodies described in the present invention.

The desirable functional properties discussed above can often result from binding to and inhibition of CTLA4 by a molecule (i.e., antibody, antibody fragment, peptide, or small molecule) in a similar manner as an antibody of the invention (i.e., binding to the same or similar epitope of the CTLA4 molecule). The molecule may either be administered directly (i.e., direct administration to a patient of such molecules). Or, alternatively, the molecule may be "administered" indirectly (i.e., a peptide or the like that produces an immune response in a patient (similar to a vaccine) wherein the immune response includes the generation of antibodies that bind to the same or similar epitope or an antibody or fragment that is produced <i>in situ</i> after administration of genetic materials that encode such antibodies or fragments thereof which bind to the same or similar epitope). Thus, it will be appreciated that the epitope on CTLA4 to which antibodies of the invention bind to can be useful in connection with the preparation and/or design of therapeutics in accordance with the invention. In drug design, negative information is often useful as well (i.e., the fact that an antibody which binds to CTLA4 does not appear to bind to an epitope that acts as an inhibitor of CTLA4 is useful). Thus, the epitope to which antibodies of the invention bind that do not lead to the desired functionality can also be very useful. Accordingly, also contemplated in accordance with the present invention are molecules (and particularly antibodies) that <u>bind to the same or similar epitopes</u> as antibodies of the invention.

In addition to the fact that antibodies of the invention and the epitopes to which they bind are contemplated in accordance with the invention, we have conducted some preliminary epitope mapping studies of certain antibodies in accordance with the invention and particularly the 4.1.1 and the 11.2.1 antibodies of the invention.

As a first step, we conducted BIAcore competition studies to generate a rough map of binding as between certain antibodies of the invention in connection with their ability to compete for binding to CTLA4. To this end, CTLA4 was bound to a BIAcore chip and a first antibody, under saturating conditions, was bound thereto and competition of subsequent secondary antibodies binding to CTLA4 was measured. This technique enabled generation of a rough map in to which families of antibodies can be classified.

Through this process, we determined that the certain antibodies in accordance with the invention could be categorized as falling into the following epitopic categories: <tables id="tabl0001" num="0001"><table frame="all"><tgroup cols="3"><colspec colnum="1" colname="col1" colwidth="19mm" /><colspec colnum="2" colname="col2" colwidth="21mm" /><colspec colnum="3" colname="col3" colwidth="127mm" /><thead><row><entry align="center" valign="top">Category</entry><entry align="center" valign="top">Antibodies</entry><entry align="center" valign="top">Competition for CTLA4 Binding</entry></row></thead><tbody><row><entry morerows="1" align="center">A</entry><entry align="center">BO1M*</entry><entry morerows="1">Freely cross-compete with one another; cross-compete with category B; some cross-competition with category D</entry></row><row><entry align="center">BO2M**</entry></row><row><entry morerows="1" align="center">B</entry><entry align="center">4.1.1</entry><entry morerows="1">Freely cross-compete with one another; cross-compete with category A, C and D.</entry></row><row><entry align="center">4.13.1</entry></row><row><entry morerows="5" align="center">C</entry><entry align="center">6.1.1</entry><entry morerows="5">Freely cross-compete with one another; cross-compete with category B and category D</entry></row><row><entry align="center">3.1.1</entry></row><row><entry align="center">4.8.1</entry></row><row><entry align="center">11.2.1</entry></row><row><entry align="center">11.6.1</entry></row><row><entry align="center">11.7.1</entry></row><row><entry align="center">D</entry><entry align="center">4.14.3</entry><entry>Cross-compete with category C and B; some cross-competition with category A</entry></row><row><entry morerows="1" align="center">E</entry><entry align="center">4.9.1</entry><entry morerows="1">BNI3 blocks 4.9.1 binding to CTLA4 but not the reverse</entry></row><row><entry align="center">BNI3***</entry></row></tbody></tgroup><tgroup cols="3" rowsep="0"><colspec colnum="1" colname="col1" colwidth="19mm" /><colspec colnum="2" colname="col2" colwidth="21mm" /><colspec colnum="3" colname="col3" colwidth="127mm" /><tbody><row><entry namest="col1" nameend="col3" align="justify">(*) (**) Available from Biostride. (***) Available from Pharmingen.</entry></row></tbody></tgroup></table></tables>

As a next step, we endeavored to determine if the antibodies of the invention recognized a linear epitope on CTLA4 under reducing and non-reducing conditions on Western blots. We observed that none of the 4.1.1, 3.1.1, 11.7.1, 11.6.1, or 11.2.1 antibodies of the invention appeared to recognize a reduced form of CTLA4 on Western blot. Accordingly, it appeared likely that the epitope to which each of these antibodies bound was not a linear epitope but more likely was a conformational epitope the structure of which may have been abrogated under reducing conditions.

Therefore, we sought to determine whether we could learn about residues within the CTLA4 molecule that are important for binding of antibodies of the invention. One manner that we utilized was to conduct kinetic assessments of off-rates as between human CTLA4 and two highly conserved primate CTLA4 molecules (cynomologous and marmoset CTLA4). BIAcore studies demonstrated that the 4.1.1 antibody of the invention bound to human, cynomologous, and marmoset CTLA4 at the same rate. However, with respect to off-rates (affinity), the 4.1.1 antibody had the highest affinity (slowest off-rate) for human, a faster off-rate with cynomologous, and a much faster off-rate for marmoset. The 11.2.1 antibody of the invention, on the other hand, binds to human, cynomologous, and marmoset CTLA4 at the about the same rate and has about the same relative off-rate for each of the three. This information further indicates that the 4.1.1 and 11.2.1 antibodies of the invention bind to different epitopes on CTLA4.

To further study the epitope to which the category B and C antibodies of the invention bind, we conducted certain site directed mutagenesis studies. Marmoset CTLA4 possesses two important changes at residues 105 and 106 relative to human CTLA4. Such differences are a leucine to methionine change at residue 105 and a glycine to serine change at residue 106. Accordingly, we mutated cDNA encoding human CTLA4 to encode a mutated CTLA4 having the L105M and G106S changes. The homologue replacement mutant CTLA4 did not effect binding of a B7.2-IgG1 fusion protein. Further, binding with the 11.2.1 antibody of the invention was not effected. However, such molecule was significantly inhibited in its ability to bind with the 4.1.1 antibody of the invention (similar to marmoset). Next, we mutated a cDNA encoding marmoset CTLA4 to create a mutant marmoset CTLA4 having a S106G change. Such change resulted in restoration of stable binding between the 4.1.1 antibody and the marmoset CTLA4 mutant. In addition, we mutated a cDNA encoding marmoset CTLA4 to create a mutant marmoset CTLA4 having a M105L change. Such change partially restored binding between the 4.1.1 antibody and the mutant CTLA4.

Each of the category B through D antibodies of the invention appear to possess similar functional properties and appear to have the potential to act as strong anti-CTLA4 therapeutic agents. Further, each of the molecules certain cross-competition in their binding for CTLA4. However, as will be observed from the above discussion, each of the molecules in the different categories appear to bind to separate conformational epitopes on CTLA4.

From the foregoing, it will be appreciated that the epitope information discussed above indicates that antibodies (or other molecules, as discussed above) that cross-compete with antibodies of the invention will likely have certain therapeutic potential in accordance with the present invention. Further, it is expected that antibodies (or other molecules, as discussed above) that cross-compete with antibodies of the invention (i.e., cross-compete with category B, C and/or D antibodies) will likely have certain additional therapeutic potential in accordance with the present invention. Additionally, it is expected that antibodies (or other molecules, as discussed above) that cross-compete with antibodies of the invention (i.e., cross-compete with category B, C and/or D antibodies) and that (i) are not reduced in their binding to marmoset CTLA4 (similar to the 11.2.1 antibody) or (ii) are reduced in their binding to marmoset CTLA4 (similar to the 4.1.1 antibody) will likely have certain additional therapeutic potential in accordance with the present invention. Antibodies (or other molecules, as discussed above) that compete with categories A and E may also have certain therapeutic potential.

<u>EXAMPLES</u>

The following examples, including the experiments conducted and results achieved are provided for illustrative purposes only and are not to be construed as limiting upon the present invention.

<u>EXAMPLE 1</u>

<u>Generation of Anti-CTLA-4-Antibodv Producing Hybridomas</u>

Antibodies of the invention were prepared, selected, and assayed in accordance with the present Example.

<b><i><u>Antigen Preparation</u></i>:</b> Three distinct immunogens were prepared for immunization of the XenoMouse™ mice: (i) a CTLA-4-IgG fusion protein, (ii) a CTLA-4 peptide, and (iii) 300.19 murine lymphoma cells transfected with a mutant of CTLA-4 (Y201V) that is constitutively expressed on the cell surface.

(i)

<u>CTLA-4-IgG1 Fusion Protein:</u>

Expression Vector Construction:

The cDNA encoding the mature extracellular domain of CTLA-4 was PCR amplified from human thymus cDNA library (Clontech) using primers designed to published sequence (<nplcit id="ncit0133" npl-type="s"><text>Eur. J Immunol 18:1901-1905 (1988</text></nplcit>)). The fragment was directionally subcloned into pSR5, a Sindbis virus expression plasmid (InVitrogen), between the human oncostatin M signal peptide and human IgG gamma 1 (IgG1) CH1/CH2/CH3 domains. The fusion protein does not contain a hinge domain but contains cysteine 120 in the extracellular domain of CTLA-4 to form a covalent dimer. The resulting vector was called CTLA-4-IgG1/pSR5. The complete CTLA-4-IgG1 cDNA in the vector was sequence confirmed in both strands. The amino acid sequence the CTLA4-Ig protein is shown below. The mature extracellular domain for CD44 was PCR amplified from human lymphocyte library (Clontech) and subcloned into pSinRep5 to generate a control protein with the identical IgG1 tail. <ul id="ul0004" list-style="none" compact="compact"><li>OM-CTLA4-IgG1 Fusion Protein: <img file="EP3553085A1_D0001.tif" /></li><li>Underlined: signal peptide Bold: CTLA4 extracellular domain</li></ul>

The cDNAs for mature extracellular domain of CD28 were PCR amplified from human lymphocyte library (Clontech) and then subcloned into pCDM8 (<nplcit id="ncit0134" npl-type="s"><text>J. Immunol. 151: 5261-71 (1993</text></nplcit>)) to produce a human IgG1 fusion protein containing both thrombin cleavage and hinge regions. Marmoset, Cynomologous, and Rhesus CTLA4 were cloned from mRNA isolated from PHA stimulated PBMCs using standard techniques of degenerate PCR. Sequencing demonstrated that rhesus and cynomologous amino acid sequence were identical with three differences from mature human CTLA4 extracellular domain (S13N, I17T and L105M). Marmoset demonstrated ten amino acid differences from the mature human CTLA4 extracellular domain (V21A, V33I, A41T, A51G, 541, S71F, Q75K, T88M, L105M and G106S). Site directed mutagenesis was used to make single point mutations of all amino acids different in marmoset CTLA4 to map amino acids important for interation of the antibodies with human CTLA4-IgG. Mutations of human and marmoset CTLA-IgG for epitope mapping were generated by matchmaker site-directed mutagenesis (Promega). The IgG fusion proteins were produced by transient transfection of Cos7 cells and purified using standard Protein A techniques. Mutant CTLA4-IgG proteins were evaluated for binding to antibodies by immunoblotting and using BIAcore analyses.

Recombinant Protein Expression/Purification:

Recombinant sindbis virus was generated by electroporating (Gibco) Baby Hamster Kidney cells with SP6 <i>in vitro</i> transcribed CTLA-4-IgG1/pSR5 mRNA and DH-26S helper mRNA as described by InVitrogen. Forty eight hours later recombinant virus was harvested and titered for optimal protein expression in Chinese hamster ovary cells (CHO-K1). CHO-K1 cells were cultured in suspension in DMEM/F12 (Gibco) containing 10% heat-inactivated fetal bovine serum (Gibco), non-essential amino acids (Gibco), 4mM glutamine (Gibco), penicillin/streptomycin (Gibco), 10mM Hepes pH 7.5 (Gibco). To produce CTLA-4-IgG, the CHO-K1 cells were resuspended at 1x10⁷ cells/ml in DMEM/F12 and incubated with sindbis virus for one hour at room temperature. Cells were then diluted to 1x10⁶/ml in DMEM/F12 containing 1% fetal bovine serum depleted of bovine IgG using protein A sepharose (Pharmacia), non-essential amino acids, 4mM glutamine, 12.5mM Hepes pH 7.5, and penicillin/streptomycin. Forty eight hours post-infection cells were pelleted and conditioned media was harvested and supplemented with complete protease inhibitor tablets (Boehringer Mannheim), pH adjusted to 7.5, and filtered 0.2µ (Nalgene). FPLC (Pharmacia) was used to affinity purify the fusion protein using a 5ml protein A HiTrap column (Pharmacia) at a 10ml/min flow rate. The column was washed with 30 bed volumes of PBS and eluted with 0.1M glycine/HCl pH 2.8 at 1ml/min. Fractions (1ml) were immediately neutralized to pH 7.5 with Tris pH 9. The fractions containing CTLA-4-IgG1 were identified by SDS-PAGE and then concentrated using centriplus 50 (Amicon) before applying to sepharose 200 column (Pharmacia) at 1ml/min using PBS as the solvent. Fractions containing CTLA-4-IgG1 were pooled, sterile filtered 0.2µ (Millipore), aliquoted and frozen at -80°C. CD44-IgG1 was expressed and purified using the same methods. CD28-IgG was purified from conditioned media from transiently transfected Cos7 cells.

Characterization CTLA-4-IgG1

:

The purified CTLA-4-IgG1 migrated as a single band on SDS-PAGE using colloidal coomassie staining (Novex). Under non-reducing conditions CTLA-4-IgG1 was a dimer (100kDa), that reduced to a 50kDa monomer when treated with 50mM DTT. Amino acid sequencing of the purified CTLA-4-IgG1 in solution confirmed the N-terminus of CTLA-4 (MHVAQPAVVLAS), and that the oncostatin-M signal peptide was cleaved from the mature fusion protein . The CTLA-4-IgG1 bound to immobilized B7.1-IgG in a concentration dependent manner and the binding was blocked by a hamster-anti-human anti-CTLA-4 antibody (BNI3: PharMingen). The sterile CTLA-4-IgG was endotoxin free and quantitated by OD280 using 1.4 as the extinction coefficient. The yield of purified CTLA-4-IgG ranged between 0.5-3mgs/liter of CHO-K1 cells.

(ii)

<u>CTLA-4 Peptide:</u>

The following CTLA-4 peptide was prepared as described below: <img file="EP3553085A1_D0002.tif" />

Abbreviations/Materials:

NMP, N-Methylpyirolidinone; TFE, 2,2,2-Trifluoroethanol; DCM, Dichloromethane; FMOC, Fluorenyl Methoxycarbonyl. All reagents were supplied by Perkin Elmer, with the following exceptions: TFE, Aldrich Chemical, FMOC-PAL-PEG resin, Perseptive Biosystems. Fmoc-Arg(PMC)-OH, FMOC-Asn(Trt)-OH, FMOC-Asp(tBu)-OH, FMOC-Cys(Trt)-OH, FMOC-Glu(tBu)-OH, FMOC-Gln(Trt)-OH, FMOC-His(Boc)-OH, FMOC-Lys(BOC)-OH, FMOC-Ser(tBu)-OH, FMOC-Thr(tBu)-OH and FMOC-Tyr(tBu)-OH were used for those amino acids requiring side chain protecting groups

Peptide Synthesis:

Peptide synthesis was performed on a Perkin-Elmer 431A, retrofitted with feedback monitoring via UV absorbance at 301nm (Perkin-Elmer Model 759A detector). The peptide sequence was assembled on a FMOC-PAL-PEG resin using conditional double coupling cycles. Forced double couplings were performed at cycles 10,11,18,19,20 and 28 through 33. The resin was washed with a 50% mixture of DCM and TFE at the completion of each acylation cycle, followed by capping of unreacted amino groups with acetic anhydride in NMP. Resin was removed from the reactor after completing cycle 49 and the remainder continued to completion. Peptide cleavage from the resin was performed using Reagent K (<nplcit id="ncit0135" npl-type="s"><text>King et al. International Journal of Protein and Peptide Research 36:255-266 (1990</text></nplcit>)) for 6 hours on 415mg of resin affording 186mg crude CTLA-4 peptide.

Peptide Characterization:

25mg aliquots of the crude CTLA-4 peptide were dissolved in 5ml 6M Guanidine HCl/100mM K₂PO₃ at pH6.4 and eluted over a Pharmacia Hi Load Superdex 75 16/60 column (16mm x 600mm, 120ml bed volume) with 2M Guanidine.HCl / 100mM K₂PO₃ at pH6.4 at 2 ml / min for 180 minutes collecting 5 ml fractions. The fractions were analyzed by loading 1.7µl of fractions onto a NuPAGE Laemeli gel running with MES running buffer and visualizing via Daichii silver stain protocol. Those fractions exhibiting a molecular weight of 12 KDa, as judged versus molecular weight standards, were pooled together and stored at 4°C. The combined fractions were analyzed by UV and gel electrophoresis. Amino acid sequencing was performed by absorbing a 100 microliter sample in a ProSorb cartridge (absorbed onto a PVDF membrane) and washing to remove the buffer salts. Sequencing was performed on an Applied Biosystems 420. The expected N-terminal sequence (M H V A Q P A V V L A) was observed. Immunoblotting demonstrated that the peptide was recognized by the BNI3 anti-human CTLA-4 (PharMingen). To desalt, an aliquot containing 648µg of material was placed in 3500 Da MWCO dialysis tubing and dialyzed against 0.1% TFA / H2O at 4°C for 9 days with stirring. The entire contents of the dialysis bag was lyophilyzed to a powder.

(iii)

<u>300.19 cells transfected with CTLA-4 (Y201V)</u>

The full length CTLA-4 cDNA was PCR amplified from human thymus cDNA library (Stratagene) and subcloned into pIRESneo (Clontech). A mutation of CTLA-4 that results in constitutive cell surface expression was introduced using MatchMaker Mutagenesis System (Promega). Mutation of tyrosine, Y201 to valine inhibits binding of the adaptin protein AP50 that is responsible for the rapid internalization of CTLA-4 (<nplcit id="ncit0136" npl-type="s"><text>Chuang et al. J. Immunol. 159:144-151 (1997</text></nplcit>)). Mycoplasma-free 300.19 murine lymphoma cells were cultured in RPMI-1640 containing 10% fetal calf serum, non-essential amino acids, penicillin/streptomycin, 2mM glutamine, 12.5mM Hepes pH 7.5, and 25uM beta-mercaptoethanol. Cells were electroporated (3x10⁶/0.4ml serum free RPMI) in a 1ml chamber with 20ug CTLA-4-Y201V/pIRESneo using 200V/1180uF (Gibco CellPorator). Cells were rested for 10 minutes and then 8mls of prewarmed complete RPMI media. At 48 hours cells were diluted to 0.5 x10⁶/ml in complete RPMI media containing 1mg/ml G418 (Gibco). Resistant cells were expanded and shown to express CTLA-4 on the cell surface using the BNI3 antibody conjugated with phycoerythrin (PharMingen). High level expressing cells were isolated by sterile sorting.

<b><i><u>Immunization and hybridoma generation:</u></i></b> XenoMouse mice (8 to 10 weeks old) were immunized (i) subcutaneously at the base of tails with 1x10⁷ 300.19 cells that were transfected to express CTLA-4 as described above, resuspended in phosphate buffered saline (PBS) with complete Freund's adjuvant, or (ii) subcutaneously at the base of tail with (a) 10 µg the CTLA-4 fusion protein or (b) 10 µg CTLA-4 peptide, emulsified with complete Freund's adjuvant. In each case, the dose was repeated three or four times in incomplete Freund's adjuvant. Four days before fusion, the mice received a final injection of the immunogen or cells in PBS. Spleen and/or lymph node lymphocytes from immunized mice were fused with the [murine non-secretory myeloma P3 cell line] and were subjected to HAT selection as previously described (<nplcit id="ncit0137" npl-type="s"><text>Galfre, G. and Milstein, C., "Preparation of monoclonal antibodies: strategies and procedures." Methods Enzymol. 73:3-46 (1981</text></nplcit>)). A large panel of hybridomas all secreting CTLA-4 specific human IgG₂κ or IgG₄κ (as detected below) antibodies were recovered.

<b><i><u>ELISA assay</u>:</i></b> ELISA for determination of antigen-specific antibodies in mouse serum and in hybridoma supernatants was carried out as described (<nplcit id="ncit0138" npl-type="s"><text>Coligan et al., Unit 2.1, "Enzyme-linked immunosorbent assays," in Current protocols in immunology (1994</text></nplcit>)) using CTLA-4-Ig fusion protein to capture the antibodies. For animals that are immunized with the CTLA-4-Ig fusion protein, we additionally screen for non-specific reactivity against the human Ig portion of the fusion protein. This is accomplished using ELISA plates coated with human IgG1 as a negative control for specificity.

In a preferred ELISA assay, the following techniques are used: ELISA plates are coated with 100 µl/well of the antigen in plate coating buffer (0.1 M Carbonate Buffer, pH 9.6 and NaHCO₃ (MW 84) 8.4g/L). Plates are then incubated at 4°C overnight. After incubation, coating buffer is removed and the plate is blocked with 200 µl/well blocking buffer (0.5% BSA, 0.1% Tween 20, 0.01% Thimerosal in 1x PBS) and incubated at room temperature for 1 hour. Alternatively, the plates are stored in refrigerator with blocking buffer and plate sealers. Blocking buffer is removed and 50 µl/well of hybridoma supernatant, serum or other hybridoma supernatant (positive control) and HAT media or blocking buffer (negative control) is added. The plates are incubated at room temperature for 2 hours. After incubation, the plate is washed with washing buffer (1x PBS). The detecting antibody (i.e., mouse anti-human IgG2-HRP (SB, #9070-05) for IgG2 antibodies or mouse anti-human IgG4-HRP (SB #9200-05) for IgG4 antibodies) is added at 100µl/well (mouse anti-human IgG2-HRP @ 1:2000 or mouse anti-human IgG4-HRP @ 1:1000 (each diluted in blocking buffer)). The plates are incubated at room temperature for 1 hour and then washed with washing buffer. Thereafter, 100 µl/well of freshly prepared developing solution (10 ml Substrate buffer, 5 mg OPD (o-phenylenediamine, Sigma Cat No. P-7288), and 10 µl 30% H₂O₂ (Sigma)) is added to the wells. The plates are allowed to develop 10-20 minutes, until negative control wells barely start to show color. Thereafter, 100 µl/well of stop solution (2 M H₂SO₄) is added and the plates are read on an ELISA plate reader at wavelength 490 nm.

<i><u>Determination of affinity constants of fully human Mabs by BIA core:</u></i> Affinity measurement of purified human monoclonal antibodies, Fab fragments, or hybridoma supernatants by plasmon resonance was carried out using the BIAcore 2000 instrument, using general procedures outlined by the manufacturers.

Kinetic analysis of the antibodies was carried out using antigens immobilized onto the sensor surface at a low density. Three surfaces of the BIAcore sensorchip were immobililized with the CTLA-4-Ig fusion protein at a density ranging from approximately 390-900 using CTLA-4-Ig fusion protein at 20 or 50 µg/ml in 10 mM sodium acetate at pH 5.0 using the amine coupling kit supplied by the manufacturer (BIAcore, Inc.). The fourth surface of the BIAcore sensorchip was immobilized with IgG1 (900 RU) and was used as a negative control surface for non-specific binding. Kinetic analysis was performed at a flow rate of 25 or 50 microliters per minute and dissociation (kd or k_off) and association (ka or k_on) rates were determined using the software provided by the manufacturer (BIA evaluation 3.0) that allows for global fitting calculations.

<u>EXAMPLE 2</u>

Affinity Measurement of Anti-CTLA-4-Antibodies

In the following Table, affinity measurements for certain of the antibodies selected in this manner are provided: <tables id="tabl0002" num="0002"><table frame="all"><title><b><u>TABLE I</u></b></title><tgroup cols="6"><colspec colnum="1" colname="col1" colwidth="22mm" /><colspec colnum="2" colname="col2" colwidth="21mm" /><colspec colnum="3" colname="col3" colwidth="20mm" /><colspec colnum="4" colname="col4" colwidth="43mm" /><colspec colnum="5" colname="col5" colwidth="43mm" /><colspec colnum="6" colname="col6" colwidth="19mm" /><thead><row><entry valign="top" /><entry namest="col2" nameend="col6" align="left" valign="top"><b>Solid Phase (by BIAcore)</b></entry></row><row><entry valign="top"><b>Hybridoma</b></entry><entry valign="top"><b>On-rates K_a (M^-1 S^-1x10⁶)</b></entry><entry valign="top"><b>Off-rates K_d (S^-1x10^-4)</b></entry><entry valign="top"><b>Association Constant KA (1/M)= k_a/k_dx10¹⁰</b></entry><entry valign="top"><b>Dissociation Constant KD(M)= K_d/k_ax10^-10</b></entry><entry valign="top"><b>Surface Density [RU]</b></entry></row></thead><tbody><row rowsep="0"><entry><b>Moab01</b></entry><entry>0.68</entry><entry>1.01</entry><entry>0.67</entry><entry>1.48</entry><entry>878.7</entry></row><row rowsep="0">&lt;entry /&gt;<entry>0.70</entry><entry>4.66</entry><entry>0.15</entry><entry>6.68</entry><entry>504.5</entry></row><row rowsep="0">&lt;entry /&gt;<entry>0.77</entry><entry>6.49</entry><entry>0.19</entry><entry>8.41</entry><entry>457.2</entry></row><row>&lt;entry /&gt;<entry>0.60</entry><entry>3.08</entry><entry>0.20</entry><entry>5.11</entry><entry>397.8</entry></row><row rowsep="0"><entry><b>4.1.1</b></entry><entry>1.85</entry><entry>0.72</entry><entry>2.58</entry><entry>0.39</entry><entry>878.7</entry></row><row rowsep="0">&lt;entry /&gt;<entry>1.88</entry><entry>1.21</entry><entry>1.55</entry><entry>0.64</entry><entry>504.5</entry></row><row rowsep="0">&lt;entry /&gt;<entry>1.73</entry><entry>1.54</entry><entry>1.13</entry><entry>0.88</entry><entry>457.2</entry></row><row>&lt;entry /&gt;<entry>1.86</entry><entry>1.47</entry><entry>1.26</entry><entry>0.79</entry><entry>397.8</entry></row><row rowsep="0"><entry><b>4.8.1</b></entry><entry>0.32</entry><entry>0.07</entry><entry>4.46</entry><entry>0.22</entry><entry>878.7</entry></row><row rowsep="0">&lt;entry /&gt;<entry>0.31</entry><entry>0.23</entry><entry>1.33</entry><entry>0.75</entry><entry>504.5</entry></row><row>&lt;entry /&gt;<entry>0.28</entry><entry>0.06</entry><entry>4.82</entry><entry>0.21</entry><entry>397.8</entry></row><row rowsep="0"><entry><b>4.14.3</b></entry><entry>2.81</entry><entry>3.04</entry><entry>0.92</entry><entry>1.08</entry><entry>878.7</entry></row><row rowsep="0">&lt;entry /&gt;<entry>2.88</entry><entry>3.97</entry><entry>0.73</entry><entry>1.38</entry><entry>504.5</entry></row><row rowsep="0">&lt;entry /&gt;<entry>2.84</entry><entry>6.66</entry><entry>0.43</entry><entry>2.35</entry><entry>457.2</entry></row><row>&lt;entry /&gt;<entry>3.17</entry><entry>5.03</entry><entry>0.63</entry><entry>1.58</entry><entry>397.8</entry></row><row rowsep="0"><entry><b>6.1.1</b></entry><entry>0.43</entry><entry>0.35</entry><entry>1.21</entry><entry>0.83</entry><entry>878.7</entry></row><row rowsep="0">&lt;entry /&gt;<entry>0.46</entry><entry>0.90</entry><entry>0.51</entry><entry>1.98</entry><entry>504.5</entry></row><row rowsep="0">&lt;entry /&gt;<entry>0.31</entry><entry>0.51</entry><entry>0.61</entry><entry>1.63</entry><entry>457.2</entry></row><row>&lt;entry /&gt;<entry>0.45</entry><entry>0.79</entry><entry>0.57</entry><entry>1.76</entry><entry>397.8</entry></row><row rowsep="0"><entry><b>3.1.1</b></entry><entry>1.04</entry><entry>0.96</entry><entry>1.07</entry><entry>0.93</entry><entry>878.7</entry></row><row rowsep="0">&lt;entry /&gt;<entry>0.95</entry><entry>1.72</entry><entry>0.55</entry><entry>1.82</entry><entry>504.5</entry></row><row rowsep="0">&lt;entry /&gt;<entry>0.73</entry><entry>1.65</entry><entry>0.44</entry><entry>2.27</entry><entry>457.2</entry></row><row>&lt;entry /&gt;<entry>0.91</entry><entry>2.07</entry><entry>0.44</entry><entry>2.28</entry><entry>397.8</entry></row><row rowsep="0"><entry><b>4.9.1</b></entry><entry>1.55</entry><entry>13.80</entry><entry>0.11</entry><entry>8.94</entry><entry>878.7</entry></row><row rowsep="0">&lt;entry /&gt;<entry>1.43</entry><entry>19.00</entry><entry>0.08</entry><entry>13.20</entry><entry>504.5</entry></row><row>&lt;entry /&gt;<entry>1.35</entry><entry>20.50</entry><entry>0.07</entry><entry>15.20</entry><entry>397.8</entry></row><row rowsep="0"><entry><b>4.10.2</b></entry><entry>1.00</entry><entry>2.53</entry><entry>0.39</entry><entry>2.54</entry><entry>878.7</entry></row><row rowsep="0">&lt;entry /&gt;<entry>0.94</entry><entry>4.30</entry><entry>0.22</entry><entry>4.55</entry><entry>504.5</entry></row><row rowsep="0">&lt;entry /&gt;<entry>0.70</entry><entry>5.05</entry><entry>0.14</entry><entry>7.21</entry><entry>457.2</entry></row><row>&lt;entry /&gt;<entry>1.00</entry><entry>5.24</entry><entry>0.19</entry><entry>5.25</entry><entry>397.8</entry></row><row rowsep="0"><entry><b>2.1.3</b></entry><entry>1.24</entry><entry>9.59</entry><entry>0.13</entry><entry>7.72</entry><entry>878.7</entry></row><row rowsep="0">&lt;entry /&gt;<entry>1.17</entry><entry>13.10</entry><entry>0.09</entry><entry>11.20</entry><entry>504.5</entry></row><row>&lt;entry /&gt;<entry>1.11</entry><entry>13.00</entry><entry>0.09</entry><entry>11.70</entry><entry>397.8</entry></row><row rowsep="0"><entry><b>4.13.1</b></entry><entry>1.22</entry><entry>5.83</entry><entry>0.21</entry><entry>4.78</entry><entry>878.7</entry></row><row rowsep="0">&lt;entry /&gt;<entry>1.29</entry><entry>6.65</entry><entry>0.19</entry><entry>5.17</entry><entry>504.5</entry></row><row>&lt;entry /&gt;<entry>1.23</entry><entry>7.25</entry><entry>0.17</entry><entry>5.88</entry><entry>397.8</entry></row></tbody></tgroup></table></tables>

As will be observed, antibodies prepared in accordance with the invention possess high affinities and binding constants.

<u>EXAMPLE 3</u>

<u>Structures of Anti-CTLA-4-Antibodies Prepared in Accordance with the Invention</u>

In the following discussion, structural information related to antibodies prepared in accordance with the invention is provided.

In order to analyze structures of antibodies produced in accordance with the invention, we cloned genes encoding the heavy and light chain fragments out of the particular hybridoma. Gene cloning and sequencing was accomplished as follows: Poly(A)⁺ mRNA was isolated from approximately 2 X 10⁵ hybridoma cells derived from immunized XenoMouse mice using a Fast-Track kit (Invitrogen). The generation of random primed cDNA was followed by PCR. Human V_H or human V_κ family specific variable region primers (<nplcit id="ncit0139" npl-type="s"><text>Marks et al., "Oligonucleotide primers for polymerase chain reaction amplification of human immunoglobulin variable genes and design of family-specific oligonucleotide probes." Eur. J. Immunol 21:985-991 (1991</text></nplcit>)) or a universal human V_H primer, MG-30 (CAGGTGCAGCTGGAGCAGTCIGG) was used in conjunction with primers specific for the human Cγ2 constant region (MG-40d; 5'-GCTGAGGGAGTAGAGTCCTGAGGA-3') or Cκ constant region (hκP2; as previously described in Green et al., 1994). Sequences of human Mabs-derived heavy and kappa chain transcripts from hybridomas were obtained by direct sequencing of PCR products generated from poly(A⁺) RNA using the primers described above. PCR products were also cloned into pCRII using a TA cloning kit (Invitrogen) and both strands were sequenced using Prism dye-terminator sequencing kits and an ABI 377 sequencing machine. All sequences were analyzed by alignments to the "V BASE sequence directory" (<nplcit id="ncit0140" npl-type="s"><text>Tomlinson et al., MRC Centre for Protein Engineering, Cambridge, UK</text></nplcit>) using MacVector and Geneworks software programs.

Further, each of the antibodies 4.1.1, 4.8.1, 11.2.1, and 6.1.1 were subjected to full length DNA sequences. For such sequencing, Poly(A)⁺ mRNA was isolated from approximately 4 X 10⁶ hybridoma cells using mRNA Direct kit (Dynal). The mRNA was reverse transcribed using oligo-dT(18) and the Advantage RT/PCR kit (Clonetech). The Variable region database (V Base) was used to design amplification primers beginning at the ATG start site of the heavy chain DP50 gene (5'-TATCTAAGCTTCTAGACTCGACCGCCACC<u>ATG</u>GAGTTTGGGCTGAGC TG-3') and to the stop codon of the IgG2 constant region (5'-TTCTCTGATCAGAATTCCTATCATTTACCCGGAGACAGGGAGAGCT-3'). An optimal Kozak sequence (ACCGCCACC) was added 5' to the ATG start site. The same method was used to design a primer to the ATG start site of the kappa chain A27 gene (5'-TCTTCAAGCTTGCCCGGGCCCGCCACC<u>ATG</u>GAAACCCCAGCGCAG -3') and the stop codon of the kappa constant region (5'-TTCTTTGATCAGAATTCTCACTAACACTCTCCCCTGTTGAAGC-3'). The 012 cDNA was cloned by using a primer to the ATG start site (5'-TCTTCAAGCTTGCCCGGGCCCGCCACCATGGACATGAGGGTCCCCGC T-3) and the kappa constant region stop codon primer above. The heavy chain cDNAs were also cloned as genomic constructs by site directed mutagenesis to add an NheI site at the end of the variable J domain and subcloning an NheI-fragment containing the genomic IgG2 CH1/Hinge/CH2/CH3 regions. The point mutation to generate NheI site does not alter the amino acid sequence from germline. The primer pairs were used to amplify the cDNAs using Advantage High Fidelity PCR Kit (Clonetech). Sequence of the PCR was obtained by direct sequencing using dye-terminator sequencing kits and an ABI sequencing machine. The PCR product was cloned into pEE glutamine synthetase mammalian expression vectors (Lonza) and three clones were sequenced to confirm somatic mutations. For each clone, the sequence was verified on both strands in at least three reactions. An aglycosylated 4.1.1 antibody was generated by site directed mutagenesis of N294Q in the CH2 domain. Recombinant antibodies were produced by transient trasnfection of Cos7 cells in IgG depleted FCS and purified using standard Protein A sepharose techniques. Stable transfectants were generated by electroporation of murine NSO cells and selection in glutamine free media. Recombinant 4.1.1 with or without glycosylation exhibited identical specificity and affinity for CTLA4 in the <i>in vitro</i> ELISA and BIAcore assays.

<u>Gene Utilization Analysis</u>

The following Table sets forth the gene utilization evidenced by selected hybridoma clones of antibodies in accordance with the invention: <tables id="tabl0003" num="0003"><img file="EP3553085A1_D0003.tif" /></tables>

As will be observed, antibodies in accordance with the present invention were generated with a strong bias towards the utilization of the DP-50 heavy chain variable region. The DP-50 gene is also referred to as a V_H 3-33 family gene. Only one antibody that was selected on the basis of CTLA-4 binding and preliminary <i>in vitro</i> functional assays showed a heavy chain gene utilization other than DP-50. That clone, 2.1.3, utilizes a DP-65 heavy chain variable region and is an IgG4 isotype. The DP-65 gene is also referred to as a V_H 4-31 family gene. On the other hand, the clone, 4.9.1, which possesses a DP-47 heavy chain variable region binds to CTLA-4 but does not inhibit binding to B7-1 or B7-2. In XenoMouse mice, there are more than 30 distinct functional heavy chain variable genes with which to generate antibodies. Bias, therefore, is indicative of a preferred binding motif of the antibody-antigen interaction with respect to the combined properties of binding to the antigen and functional activity.

<u>Mutation Analysis</u>

As will be appreciated, gene utilization analysis provides only a limited overview of antibody structure. As the B-cells in XenoMouse animals stocastically generate V-D-J heavy or V-J kappa light chain transcripts, there are a number of secondary processes that occur, including, without limitation, somatic hypermutation, n-additions, and CDR3 extensions. <i>See, for example,</i><nplcit id="ncit0141" npl-type="s"><text>Mendez et al. Nature Genetics 15:146-156 (1997</text></nplcit>) and <patcit id="pcit0110" dnum="US75962096A"><text>U.S. Patent Application Serial No. 08/759,620, filed December 3, 1996</text></patcit>. Accordingly, to further examine antibody structure predicted amino acid sequences of the antibodies were generated from the cDNAs obtained from the clones. In addition, N-terminal amino acid sequences were obtained through protein sequencing.

<figref idref="f0001 f0002 f0003 f0004 f0005 f0006 f0007 f0008 f0009 f0010 f0011 f0012 f0013 f0014 f0015 f0016">Figure 1</figref> provides nucleotide and predicted amino acid sequences of the heavy and kappa light chains of the clones 4.1.1 (<figref idref="f0001 f0002">Figure 1A</figref>), 4.8.1 (<figref idref="f0003 f0004">Figure 1B</figref>), 4.14.3 (<figref idref="f0005">Figure 1C</figref>), 6.1.1 (<figref idref="f0006 f0007">Figure 1D</figref>), 3.1.1 (<figref idref="f0008">Figure 1E</figref>), 4.10.2 (<figref idref="f0009">Figure 1F</figref>), 2.1.3 (<figref idref="f0010">Figure 1G</figref>), 4.13.1 (<figref idref="f0011">Figure 1H</figref>). 11.2.1 (<figref idref="f0012">Figure 1I</figref>), 11.6.1 (<figref idref="f0013">Figure 1J</figref>), 11.7.1 (<figref idref="f0014">Figure 1K</figref>), 12.3.1.1 (<figref idref="f0015">Figure 1L</figref>), and 12.9.1.1 (<figref idref="f0016">Figure 1M</figref>). In <figref idref="f0001 f0002">Figures 1A</figref>, <figref idref="f0003 f0004">1B</figref>, and <figref idref="f0006 f0007">1D</figref>, extended sequences of the antibodies 4.1.1, 4.8.1, and 6.1.1 were obtained by full length cloning of the cDNAs as described above. In such Figures, the signal peptide sequence (or the bases encoding the same) are indicated in bold and sequences utilized for the 5' PCR reaction are underlined.

<figref idref="f0017 f0018 f0019">Figure 2</figref> provides a sequence alignment between the predicted heavy chain amino acid sequences from the clones 4.1.1, 4.8.1, 4.14.3, 6.1.1, 3.1.1, 4.10.2, 4.13.1, 11.2.1, 11.6.1, 11.7,1, 12.3.1.1, and 12.9.1.1 and the germline DP-50 (3-33) amino acid sequence. Differences between the DP-50 germline sequence and that of the sequence in the clones are indicated in bold. The Figure also shows the positions of the CDR1, CDR2, and CDR3 sequences of the antibodies as shaded.

<figref idref="f0020">Figure 3</figref> provides a sequence alignment between the predicted heavy chain amino acid sequence of the clone 2.1.3 and the germline DP-65 (4-31) amino acid sequence. Differences between the DP-65 germline sequence and that of the sequence in the clone are indicated in bold. The Figure also shows the positions of the CDR1, CDR2, and CDR3 sequences of the antibody as underlined.

<figref idref="f0021">Figure 4</figref> provides a sequence alignment between the predicted kappa light chain amino acid sequence of the clones 4.1.1, 4.8.1, 4.14.3, 6.1.1, 4.10.2, and 4.13.1 and the germline A27 amino acid sequence. Differences between the A27 germline sequence and that of the sequence in the clone are indicated in bold. The Figure also shows the positions of the CDR1, CDR2, and CDR3 sequences of the antibody as underlined. Apparent deletions in the CDR1s of clones 4.8.1, 4.14.3, and 6.1.1 are indicated with "0s".

<figref idref="f0022">Figure 5</figref> provides a sequence alignment between the predicted kappa light chain amino acid sequence of the clones 3.1.1, 11.2.1, 11.6.1, and 11.7.1 and the germline 012 amino acid sequence. Differences between the 012 germline sequence and that of the sequence in the clone are indicated in bold. The Figure also shows the positions of the CDR1, CDR2, and CDR3 sequences of the antibody as underlined.

<figref idref="f0023">Figure 6</figref> provides a sequence alignment between the predicted kappa light chain amino acid sequence of the clone 2.1.3 and the germline A10/A26 amino acid sequence. Differences between the A10/A26 germline sequence and that of the sequence in the clone are indicated in bold. The Figure also shows the positions of the CDR1, CDR2, and CDR3 sequences of the antibody as underlined.

<figref idref="f0024">Figure 7</figref> provides a sequence alignment between the predicted kappa light chain amino acid sequence of the clone 12.3.1 and the germline A17 amino acid sequence. Differences between the A17 germline sequence and that of the sequence in the clone are indicated in bold. The Figure also shows the positions of the CDR1, CDR2, and CDR3 sequences of the antibody as underlined.

<figref idref="f0025">Figure 8</figref> provides a sequence alignment between the predicted kappa light chain amino acid sequence of the clone 12.9.1 and the germline A3/A19 amino acid sequence. Differences between the A3/A19 germline sequence and that of the sequence in the clone are indicated in bold. The Figure also shows the positions of the CDR1, CDR2, and CDR3 sequences of the antibody as underlined.

<figref idref="f0040 f0041 f0042 f0043 f0044 f0045 f0046 f0047 f0048">Figure 22</figref> provides a series of additional nucleotide and amino acid sequences of the following anti-CTLA-4 antibody chains: <ul id="ul0005" list-style="none"><li><i><u>4.1.1</u>:</i><ul id="ul0006" list-style="none" compact="compact"><li>full length 4.1.1 heavy chain (cDNA 22(a), genomic 22(b), and amino acid 22(c));</li><li>full length aglycosylated 4.1.1 heavy chain (cDNA 22(d) and amino acid 22(e));</li><li>4.1.1 light chain (cDNA 22(f) and amino acid 22(g));</li></ul></li><li><i><u>4.8.1</u></i>: full length 4.8.1 heavy chain (cDNA 22(h) and amino acid 22(i)); 4.8.1 light chain (cDNA 22(j) and amino acid 22(k));</li><li><i><u>6.1.1</u></i>: <ul id="ul0007" list-style="none" compact="compact"><li>full length 6.1.1 heavy chain (cDNA 22(1) and amino acid 22(m));</li><li>6.1.1 light chain (cDNA 22(n) and amino acid 22(o));</li></ul></li><li><i><u>11.2.1</u>:</i><ul id="ul0008" list-style="none" compact="compact"><li>full length 11.2.1 heavy chain (cDNA 22(p) and amino acid 22(q)); and</li><li>11.2.1 light chain (cDNA 22 (r) and amino acid 22(s)).</li></ul></li></ul>

Signal peptide sequences are shown in bold and large text. The open reading frames in the full length 4.1.1 genomic DNA sequence (<figref idref="f0040">Fig. 22(b)</figref>) are underlined. And, the mutations introduced to make the aglycosylated 4.1.1 heavy chain and the resulting change (N294Q) are shown in doble underline and bold text (cDNA (<figref idref="f0040">Fig. 22(b)</figref> and amino acid (<figref idref="f0041">Fig. 22(c)</figref>).

<u>EXAMPLE 4</u>

<u>Analysis of Heavy and Light Chain Amino Acid Substitutions</u>

In <figref idref="f0017 f0018 f0019">Figure 2</figref>, which provides a sequence alignment between the predicted heavy chain amino acid sequences from the clones 4.1.1, 4.8.1, 4.14.3, 6.1.1, 3.1.1, 4.10.2, 4.13.1, 11.2.1, 11.6.1, 11.7.1, 12.3.1.1, and 12.9.1.1 and the germline DP-50 (3-33) amino acid sequence, an interesting pattern emerges. In addition to the fact of the bias for heavy chain DP-50 in the majority of the clones, there is relatively limited hypermutation in the antibodies relative to the germline DP-50 gene. For example, clones 3.1.1 and 11.2.1 have no mutations. Moreover, the mutations in the other clones are generally conservative changes, involving substitutions of amino acids with similar properties to the amino acids in the germline. Mutations within many of the CDR1 and CRD2 sequences are particularly conservative in nature. Three of the heavy chains represented in <figref idref="f0017 f0018 f0019">Figure 2</figref>, 4.10.2, 4.13.1, and 4.14.3, are clearly derived from a single recombination event (i.e., derive from an identical germinal center) and are nearly identical in sequence. If these three are considered as a single sequence, then, among the 10 different antibodies containing the DP50 heavy chain, in CDR1 and CDR2 there are 3 positions in which a nonpolar residue is replaced by another nonpolar residue, 12 in which a polar uncharged residue is replaced by another polar uncharged residue, and 1 in which a polar charged residue is replaced by another polar charged residue. Further, there are two positions in which two residues which are very similar structurally, glycine and alanine, are substituted for one another. The only mutations not strictly conservative involve 3 substitutions of a polar charged residue for a polar uncharged residue and one substitution of a nonpolar residue for a polar residue.

The light chains of these antibodies are derived from 5 different Vk genes. The A27 gene is the most heavily represented and is the source of 6 different light chains. Comparison of these 6 sequences reveals two noteworthy features. First, in three of them, 4.8.1, 4.14.3, and 6.1.1, contain deletions of one or two residues in CDR1, a rare event. Second, there is a strong prejudice against the germline serine at position six in CDR3 in that the serine has been replaced in every sequence. This suggests that a serine at this position is incompatible with CTLA4 binding.

It will be appreciated that many of the above-identified amino acid substitutions exist in close proximity to or within a CDR. Such substitutions would appear to bear some effect upon the binding of the antibody to the CTLA-4 molecule. Further, such substitutions could have significant effect upon the affinity of the antibodies.

<u>EXAMPLES</u>

<u>N-Terminal Amino Acid Sequence Analysis of Antibodies in Accordance with the Invention</u>

In order to further verify the composition and structure of the antibodies in accordance with the invention identified above, we sequenced certain of the antibodies using a Perkin-Elmer sequencer. Both heavy and kappa light chains of the antibodies were isolated and purified through use of preparative gel electrophoresis and electroblotting techniques and thereafter directly sequenced as described in Example 6. A majority of the heavy the heavy chain sequences were blocked on their amino terminus. Therefore, such antibodies were first treated with pyroglutamate aminopeptidase and thereafter sequenced.

The results from this experiment are shown in <figref idref="f0026">Figure 9. Figure 9</figref> also provides the molecular weight of the heavy and light chains as determined by mass spectroscopy (MALDI).

<u>EXAMPLE 6</u>

<u>Additional Characterization of Antibodies in Accordance with the Invention</u>

<figref idref="f0027 f0028">Figure 10</figref> provides certain additional characterizing information about certain of the antibodies in accordance with the invention. In the Figure, data related to clones 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.14.3, and 6.1.1 is summarized. The following data is provided: Concentration, isoelectric focusing (IEF), SDS-PAGE, size exclusion chromatography, FACS, mass spectroscopy (MALDI), and light chain N-terminal sequences.

Generally, the data was generated as follows:

Materials and Methods

<u>Protein concentration</u> was determined at 280 nm from a UV scan (200-350 nm), where 1.58 absorbance units at 280 nm equaled 1mg/ml.

<u>SDS-PAGE</u> was performed using the Novex NuPAGE electrophoresis system with a 10% NuPAGE gel and MES running buffer. Samples were prepared by diluting 3:1 with 4x NuPAGE sample buffer (+/-) beta-mercaptoethanol, heated and ∼ 5 ug of protein was loaded onto the gel. The gel was then stained with Brilliant Blue R staining solution (Sigma cat.# B-6529) and molecular size estimates were made by comparing stained bands to "Perfect Protein Markers" (Novagen cat# 69149-3).

For <u>N-terminal sequencing</u>, samples were run as above on NuPAGE gels, transferred to Pro Blot immobilization membrane (Applied Biosystems) then stained with Coomassie Blue R-250. The stained protein bands were excised and subjected to sequence analysis by automated Edman degradation on an Applied Biosystems 494 Procise HT Sequencer.

<u>Isoelectric focusing (IEF)</u> was performed using Pharmacia IEF 3-9 pHast gels (cat# 17-0543-03). Samples were diluted in 10% glycerol to ∼0.8 mg/ml and 1ul was loaded onto gel and then silver stained. The pI estimates were made by comparing stained bands to broad range (pH3-10) IEF standards (Pharmacia cat # 17-0471-01)

<u>Size exclusion chromatography (SEC)</u> was carried in phosphate buffered saline (PBS) on the Pharmacia SMART system using the Superdex 75 PC 3.2/30 column. Molecular size estimates were made by comparing peak retention time to the retention times of gel

For <u>FACS studies</u>, human peripheral T cells were prepared and stimulated for 48 hours. T cells were washed once, resuspended in FACS buffer at 1x10⁶ cells/100 ul and stained for CD3 surface expression with 10 ul of anti-CD3-FITC (Immunotech, Marseille, France) for 30 minutes at room temperature. Cells were washed twice, then fixed, permeabilized (Fix and Perm, Caltag), and stained for intracellular CTLA-4 expression with 10 ul anti-CD152-PE (Pharmingen). Flow cytometry was performed using a Becton Dickinson FACSort. Quadrants were set by analysis of relevant isotype control antibodies (Caltag).

As was discussed above, anti-CTLA-4 antibodies have been demonstrated to possess certain powerful immune modulation activities. The following experiments were carried out in order to determine if antibodies in accordance with the present invention possessed such activities. In general, the experiments were designed to assess ability of the antibodies to inhibit the interaction between CTLA-4 and B7 molecules, be selective as between CTLA-4 and B7 molecules and CD28, and promote T cell cytokine production, including, but not limited to IL-2 and/or IFN-γ expression. Further, examination of cross-reactivity of antibodies of the invention with certain human tissues and CTLA-4 molecules in other species (e.g., mouse and primate) was undertaken.

<u>EXAMPLE 7</u>

<u>Competition ELISA: Inhibition of CTLA-4/B7-1 or B7-2 Interaction by Antibodies in Accordance with the Invention</u>

An <i>in vitro</i> assay was conducted to determine if antibodies in accordance with the present invention were capable of inhibiting the binding of CTLA-4 with either B7-1 or B7-2. As will be appreciated, antibodies of the invention that are capable of inhibiting the binding of CTLA-4 with B7 molecules would be expected to be candidates for immune regulation through the CTLA-4 pathway. In the assay, the following materials and methods were utilized:

Materials and Methods

3 nM B7.1-Ig(G1) or B7.2-Ig(G1) (Repligen, Inc. Needham, MA) in Dulbecco's PBS was coated on 96-well MaxiSorp plates (Nunc, Denmark, #439454) and incubated at 4°C overnight. On day 2, B7-Ig was removed and plates were blocked with 1% BSA plus 0.05% Tween-20 in D-PBS for two hours. Plates were washed 3X with wash buffer (0.05% Tween-20 in D-PBS). Antibody at appropriate test concentrations and CTLA-4-Ig(G4) (0.3 nM final cone.) (Repligen, Inc. Needham, MA) were pre-mixed for 15 minutes and then added to the B7-Ig coated plate (60 ul total volume) and incubated at RT for 1.5 hours. Plates were washed 3X and 50 µl of a 1 to 1000 dilution of HRP-labeled mouse anti-human IgG4 antibody (Zymed, San Francisco, CA, #05-3820) was added and incubated at RT for 1 hour. Plates were washed 3X and 50 µl TMB Microwell peroxidase substrate (Kirkegaard &amp; Perry, Gaithersburg, MD, #50-76-04) was added and incubated at RT for 20 minutes, and then 50 µl IN H₂SO₄ was added to the plate. Plates were read at 450 nm using a Molecular Devices plate reader (Sunnyvale, CA). All samples were tested in duplicate. Maximal signal was defined as CTLA-4-Ig binding in the absence of test antibody. Non-specific binding was defined as absorbance in the absence of CTLA-4-Ig and test antibody.

The results from the assay are provided in Table IIIA and IIIB. In Table IIIA, results are shown for a variety of antibodies in accordance with the invention. In Table IIIB, results are shown comparing the 4.1.1 antibody of the invention with the 11.2.1 antibody of the invention from a separate experiment. <tables id="tabl0004" num="0004"><table frame="all"><title><b><u>TABLE IIIA</u></b></title><tgroup cols="4"><colspec colnum="1" colname="col1" colwidth="29mm" /><colspec colnum="2" colname="col2" colwidth="16mm" /><colspec colnum="3" colname="col3" colwidth="56mm" /><colspec colnum="4" colname="col4" colwidth="56mm" /><thead><row><entry align="center" valign="top">Clone CTLA-4-Ig</entry><entry align="center" valign="top">Isotype</entry><entry align="center" valign="top">CTLA4/B7.2 Comp. ELISA IC50 (nM)</entry><entry align="center" valign="top">CTLA4/B7.1 Comp. ELISA IC50 (nM)</entry></row></thead><tbody><row><entry>CT3.1.1</entry><entry>IgG2</entry><entry>0.45 ± 0.07 (n=3)</entry><entry>0.63 ± 0.10 (n=2)</entry></row><row><entry>CT4.1.1</entry><entry>IgG2</entry><entry>0.38 ± 0.06 (n=5)</entry><entry>0.50 ± 0.05 (n=2)</entry></row><row><entry>CT4.8.1</entry><entry>IgG2</entry><entry>0.57 ± 0.03 (n=3)</entry><entry>0.17 ± 0.28 (n=2)</entry></row><row><entry>CT4.9.1</entry><entry>IgG2</entry><entry>Non-competitive (n=3)</entry><entry>non-competitive (n=2)</entry></row><row><entry>CT4.10.2</entry><entry>IgG2</entry><entry>1.50 ± 0.37 (n=3)</entry><entry>3.39 ± 0.31 (n=2)</entry></row><row><entry>CT4.13.1</entry><entry>IgG2</entry><entry>0.49 ± 0.05 (n=3)</entry><entry>0.98 ± 0.11 (n=2)</entry></row><row><entry>CT4.14.3</entry><entry>IgG2</entry><entry>0.69 ± 0.11 (n=3)</entry><entry>1.04 ± 0.15 (n=2)</entry></row><row><entry>CT6.1.1</entry><entry>IgG2</entry><entry>0.39 ± 0.06 (n=3)</entry><entry>0.67 ± 0.07 (n=2)</entry></row></tbody></tgroup></table></tables><tables id="tabl0005" num="0005"><table frame="all"><title><b><u>TABLE IIIB</u></b></title><tgroup cols="4"><colspec colnum="1" colname="col1" colwidth="29mm" /><colspec colnum="2" colname="col2" colwidth="16mm" /><colspec colnum="3" colname="col3" colwidth="56mm" /><colspec colnum="4" colname="col4" colwidth="56mm" /><thead><row><entry align="center" valign="top">Clone CTLA-4-Ig</entry><entry align="center" valign="top">Isotype</entry><entry align="center" valign="top">CTLA4/B7.2 Comp. ELISA IC50 (nM)</entry><entry align="center" valign="top">CTLA4/B7.1 Comp. ELISA IC50 (nM)</entry></row></thead><tbody><row><entry>CT4.1.1</entry><entry>IgG2</entry><entry>0.55 ± 0.08 (n=4)</entry><entry>0.87 ± 0.14 (n=2)</entry></row><row><entry>CT11.2.1</entry><entry>IgG2</entry><entry>0.56 ± 0.05 (n=4)</entry><entry>0.81 ± 0.24 (n=2)</entry></row></tbody></tgroup></table></tables>

<u>EXAMPLE 8</u>

<u>Selectivity Ratios of Antibodies of the Invention with Respect to CTLA-4 Versus Either CD28 or B7-2</u>

Another <i>in vitro</i> assay was conducted to determine the selectivity of antibodies of the invention with respect to CTLA-4 versus either CD28 or B7-2. The following materials and methods were utilized in connection with the experiments:

CTLA-4 Selectivity ELISA: Materials and Methods

A 96-well FluroNUNC plate (Nunc Cat No.475515) was platecoated with four antigens: CTLA-4/Ig, CD44/Ig, CD28/Ig, and B7.2/Ig (antigens generated in-house). The antigens were platecoated overnight at +4°C at 1µg/ml 100ul/well in 0.1M sodium bicarbonate buffer, pH 9.6. The plate was then washed with PBST (PBS + 0.1% Tween-20) three times using a NUNC plate washer. The plate was blocked with PBST+0.5%BSA at 150 µl/well. The plate was incubated at RT for 1 hour then washed with PBST three times. Next the anti-CTLA-4 antibodies of the invention were diluted in block at 1 µg/ml and were added to the plate. The plate was incubated at RT for 1 hour then washed with PBST three times. The wells that contained the antibodies of the invention were then treated with 100 µl/well anti-human IgG2-HRP (Southern Biotech Cat No.9070-05) at a 1:4000 dilution in block. Also, one row was treated with anti-human IgG (Jackson Cat No. 209-035-088) to normalize for platecoating. This antibody was diluted to 1:5000 in block and added at 100 µl/well. Also, one row was treated with anti-human CTLA-4-HRP (Pharmingen Cat No. 345815/Custom HRP conjugated) as a positive control. This antibody was used at 0.05 ug/ml diluted in block. The plate was incubated at RT for 1 hour then washed with PBST three times. LBA chemiluminescent substrate (Pierce) was added at 100 µl/well and the plate was incubated on a plateshaker for 5 min. The plate was then read using a lumi-imager for a 2 min. exposure.

IGEN CTLA-4-Ig Selectivity Binding Assay: Materials and Methods

M-450 Dynabeads (Dynal A.S, Oslo, Norway #140.02) were washed 3X with Na phosphate buffer, pH 7.4 and resuspended in Na phosphate buffer. 1.0 µg CTLA-4-Ig(G1), 1.0 µg CD28-Ig(G1) or 1.0 to 3.0 µg B7.2-Ig(G1) (Repligen, Inc. Needham, MA) were added to 100 µl of beads and incubated overnight on a rotator at 4°C. On day 2 the beads were washed 3X in 1% BSA plus 0.05% Tween-20 in Dulbecco's PBS and blocked for 30 minutes. Beads were diluted 1 to 10 with blocking buffer and 25 µl of the coated beads were added to 12x75 mm polypropylene tubes. All samples were tested in duplicate. 50 µl test antibody (1 µg/ml final concentration) or blocking buffer was added to the tubes and incubated for 30 minutes on the Origen 1.5 Analyzer carousel (IGEN International, Inc., Gaithersburg, MD) at RT, vortexing at 100 rpm. 25 µl of ruthenylated murine anti-human IgG1, IgG2 or IgG4 (Zymed, Inc. San Francisco, CA #05-3300, 05-3500 and 05-3800) (final concentration of 3 µg/ml in 100 µl total volume) was added to the tubes. Tubes were incubated for 30 minutes at RT on the carousel vortexing at 100 rpm. 200 µl of Origen assay buffer (IGEN International, Inc., Gaithersburg, MD #402-050-03) per tube was added and briefly vortexed and then the tubes were counted in the Origen Analyzer and ECL (electrochemi luminescence) units were determined for each tube. Normalization factors were determined to correct for differences in binding of fusion proteins to Dynabeads, and ECL units were corrected for nonspecific binding before calculating selectivity ratios.

The results from the assays are provided in Tables IVA and IVB. <tables id="tabl0006" num="0006"><table frame="all"><title><b><u>TABLE IVA</u></b></title><tgroup cols="7"><colspec colnum="1" colname="col1" colwidth="15mm" /><colspec colnum="2" colname="col2" colwidth="16mm" /><colspec colnum="3" colname="col3" colwidth="28mm" /><colspec colnum="4" colname="col4" colwidth="27mm" /><colspec colnum="5" colname="col5" colwidth="28mm" /><colspec colnum="6" colname="col6" colwidth="28mm" /><colspec colnum="7" colname="col7" colwidth="27mm" /><thead><row><entry align="center" valign="top">Clone</entry><entry align="center" valign="top">Isotype</entry><entry align="center" valign="top">CTLA4/CD28 ELISA</entry><entry align="center" valign="top">CTLA4/B7.2 ELISA</entry><entry align="center" valign="top">CTLA4/CD44 ELISA</entry><entry align="center" valign="top">CTLA4/CD28 IGEN</entry><entry align="center" valign="top">CTLA4/B7.2 IGEN</entry></row></thead><tbody><row rowsep="0"><entry>3.1.1</entry><entry>IgG2</entry><entry>&gt;500:1 (n=3)</entry><entry>&gt;500:1 (n=3)</entry><entry>&gt;500:1 (n=3)</entry><entry>&gt;500:1 (n=2)</entry><entry>&gt;500:1 (n=1)</entry></row><row>&lt;entry /&gt;&lt;entry /&gt;&lt;entry /&gt;&lt;entry /&gt;&lt;entry /&gt;&lt;entry /&gt;<entry>195:1 (n=1)</entry></row><row rowsep="0"><entry>4.1.1</entry><entry>IgG2</entry><entry>&gt;500:1 (n=3)</entry><entry>&gt;500:1 (n=2)</entry><entry>&gt;500:1 (n=3)</entry><entry>&gt;500:1 (n=1)</entry><entry>&gt;500:1 (n=1)</entry></row><row>&lt;entry /&gt;&lt;entry /&gt;&lt;entry /&gt;<entry>485:1 (n=1)</entry>&lt;entry /&gt;<entry>261:1 (n=1)</entry><entry>107:1 (n=1)</entry></row><row rowsep="0"><entry>4.8.1</entry><entry>IgG2</entry><entry>&gt;500:1 (n=3)</entry><entry>&gt;500:1 (n=2)</entry><entry>&gt;500:1 (n=3)</entry><entry>&gt;500:1 (n=2)</entry><entry>&gt;500:1 (n=2)</entry></row><row>&lt;entry /&gt;&lt;entry /&gt;&lt;entry /&gt;<entry>190:1 (n=1)</entry>&lt;entry /&gt;&lt;entry /&gt;&lt;entry /&gt;</row><row rowsep="0"><entry>4.9.1</entry><entry>IgG2</entry><entry>&gt;500:1 (n=2)</entry><entry>&gt;500:1 (n=2)</entry><entry>&gt;500:1 (n=3)</entry><entry>&gt;500:1 (n=1)</entry><entry>&gt;500:1 (n=1)</entry></row><row>&lt;entry /&gt;&lt;entry /&gt;<entry>244:1 (n=1)</entry><entry>33:1 (n=1)</entry>&lt;entry /&gt;&lt;entry /&gt;&lt;entry /&gt;</row><row><entry>4.10.2</entry><entry>IgG2</entry><entry>&gt;500:1 (n=3)</entry><entry>&gt;500:1 (n=3)</entry><entry>&gt;500:1 (n=3)</entry><entry>&gt;500:1 (n=1)</entry><entry>&gt;500:1 (n=1)</entry></row><row rowsep="0"><entry>4.13.1</entry><entry>IgG2</entry><entry>&gt;500:1 (n=2)</entry><entry>&gt;500:1 (n=3)</entry><entry>&gt;500:1 (n=3)</entry><entry>&gt;500:1 (n=1)</entry><entry>&gt;500:1 (n=2)</entry></row><row>&lt;entry /&gt;&lt;entry /&gt;<entry>46:1 (n=1)</entry>&lt;entry /&gt;&lt;entry /&gt;<entry>329:1 (n=1)</entry>&lt;entry /&gt;</row><row rowsep="0"><entry>4.14.3</entry><entry>IgG2</entry><entry>&gt;500:1 (n=2)</entry><entry>&gt;500:1 (n=2)</entry><entry>&gt;500:1 (n=2)</entry><entry>&gt;413:1 (n=1)</entry><entry>&gt;234:1 (n=1)</entry></row><row>&lt;entry /&gt;&lt;entry /&gt;<entry>80:1 (n=1)</entry><entry>10:1 (n=1)</entry><entry>126:1 (n=1)</entry>&lt;entry /&gt;&lt;entry /&gt;</row><row rowsep="0"><entry>6.1.1</entry><entry>IgG2</entry><entry>&gt;500:1 (n=2)</entry><entry>&gt;500:1 (n=3)</entry><entry>&gt;500:1 (n=3)</entry><entry>&gt;500:1 (n=2)</entry><entry>&gt;500:1 (n=2)</entry></row><row>&lt;entry /&gt;&lt;entry /&gt;<entry>52:1 (n=1)</entry>&lt;entry /&gt;&lt;entry /&gt;&lt;entry /&gt;&lt;entry /&gt;</row></tbody></tgroup></table></tables><tables id="tabl0007" num="0007"><table frame="all"><title><b><u>TABLE IVB</u></b></title><tgroup cols="5"><colspec colnum="1" colname="col1" colwidth="15mm" /><colspec colnum="2" colname="col2" colwidth="16mm" /><colspec colnum="3" colname="col3" colwidth="33mm" /><colspec colnum="4" colname="col4" colwidth="32mm" /><colspec colnum="5" colname="col5" colwidth="32mm" /><thead><row><entry align="center" valign="top">Clone</entry><entry align="center" valign="top">isotype</entry><entry align="center" valign="top">CTLA4/CD28 ELISA</entry><entry align="center" valign="top">CTLA4/B7.2 ELISA</entry><entry align="center" valign="top">CTLA4/hIgG ELISA</entry></row></thead><tbody><row><entry>4.1.1</entry><entry>IgG2</entry><entry>&gt;500:1 (n=3)</entry><entry>&gt;500:1 (n=2)</entry><entry>&gt;500:1 (n=3)</entry></row><row><entry>11.2.1</entry><entry>IgG2</entry><entry>&gt;500:1 (n=3)</entry><entry>&gt;500:1 (n=3)</entry><entry>&gt;500:1 (n=3)</entry></row></tbody></tgroup></table></tables>

<u>EXAMPLE 9</u>

<u>Human T-Cell Signal Model</u>

In order to further define the activity of antibodies in accordance with the invention to act as immune regulators, we developed certain T-cell assays in order to quantify the enhancement of T-cell IL-2 production upon blockade of CTLA-4 signal with the antibodies. The following materials and methods were utilized in connection with the experiments:

Materials and Methods

Freshly isolated human T cells were prepared by using Histopaque (Sigma, St. Louis, MO #A-70543) and T-kwik (Lympho-Kwik, One Lambda, Canoga Park, CA, #LK-50-T), and stimulated with PHA (1 µg/ml) (Purified Phytohemagglutinin, Murex Diagnostics Ltd. Dartford, England, #HA 16) in medium (RPMI 1640 containing L-glutamine, MEM non-essential amino acids, penicillin, streptomycin, 25 mM Hepes and 10% FBS) at a concentration of 1x10⁶ cells/ml and incubated at 37°C for 2 days. The cells were washed and diluted in medium to 2x10⁶ cells/ml. Raji cells (Burkitt lymphoma, Human ATCC No.: CCL 86 Class II American Type Culture Collection Rockville, MD) were treated with mitomycin C (Sigma St. Louis, MO, # M-4287) (25 µg/ml) for one hour at 37°C. The Raji cells were washed 4X in PBS and resuspended at 2x10⁶ cells/ml. Human T cell blasts (5x10⁵/ml), Raji cells (5x10⁵/ml) and anti-CTLA-4 antibodies or an isotyped-matched control antibody at various concentrations were added to 96-well microtiter plates and the plates were incubated at 37°C for 72 hours. Total volume per well was 200 µl. Seventy-two hours post stimulation, the plates were spun down and supernatant removed and frozen for later determination of IL-2 (Quantikine IL-2 ELISA kit, R&amp;D Systems, Minneapolis, MN, #D2050) and IFN-γ (Quantikine IFN-g ELISA kit, R&amp;D Systems). Cytokine enhancement was defined as the difference between cytokine levels in cultures containing an anti-CTLA-4 blocking mAb versus an isotype-matched control antibody. For flow cytometry experiments, Raji cells were washed 1x with FACS buffer (PBS containing 2% heat inactivated FCS, 0.025% sodium azide). Cell pellets were resuspended in FACS buffer at 1x10⁶ cells/100 µl and incubated with 10 µl of anti-CD80-PE (Becton Dickinson, San Jose, CA) or anti-CD86-PE (Pharmingen, San Diego, CA) for 30 minutes at room temperature. Cells were washed twice and resuspended in 1 ml FACS buffer. Flow cytometry was performed using a Becton Dickinson FACSort. Histogram markers were set by analysis of relevant isotype control antibodies (Caltag, Burlingame, CA).

In general, we have developed an assay that can be used for rapid determination of T-cell IL-2 upregulation. As will be appreciated, stimulation of T cells is B7 and CD28 dependent. Further, washed T blasts do not make detectable IL-2 and Raji cells do not make detectable IL-2 even when stimulated with LPS or PWM. However, in combination, the T blasts co-cultured with Raji cells can model B7, CTLA-4, and CD28 signaling events and the effects of antibodies thereon can be assessed.

<figref idref="f0029">Figure 11</figref> shows the expression of B7-1 and B7-2 on Raji cells using anti-CD80-PE and anti-CD86-PE mAbs using flow cytometry (FACs) as described in Example 6.

<figref idref="f0030">Figure 12</figref> shows the concentration dependent enhancement of IL-2 production in the T cell blast/Raji assay induced by CTLA-4 blocking antibodies (BNI3 (PharMingen) and the 4.1.1, 4.8.1, and 6.1.1 antibodies of the invention).

<figref idref="f0031">Figure 13</figref> shows the concentration dependent enhancement of IFN-γ production in the T cell blast/Raji assay induced by CTLA-4 blocking antibodies (BNI3 (PharMingen) and the 4.1.1, 4.8.1, and 6.1.1 antibodies of the invention) (same donor T cells).

<figref idref="f0032">Figure 14</figref> shows the mean enhancement of IL-2 production in T cells from 6 donors induced by CTLA-4 blocking antibodies in the T cell blast/Raji assay. It is interesting to consider that the mAb, CT4.9.1, binds to CTLA4 but does not block B7 binding. Thus, simply binding to CTLA-4 is insufficient by itself to provide a functional antibody of the invention.

<figref idref="f0033">Figure 15</figref> shows the mean enhancement of IFN-γ production in T cells from 6 donors induced by CTLA-4 blocking antibodies in the T cell blast/Raji assay.

<figref idref="f0037">Figure 19</figref> shows a comparison between the 4.1.1 and 11.2.1 antibodies of the invention at a concentration of 30µg/ml in the 72 hour T cell blast/Raji assay as described in this Example 9 and the Superantigen assay described in Example 10.

<figref idref="f0038">Figure 20</figref> shows the concentration dependent enhancement of IL-2 production in the T cell blast/Raji assay induced by the 4.1.1 and 11.2.1 CTLA4 antibodies of the invention.

The following Table IVc provides information related to mean enhancement and range of enhancement of cytokine response in the Raji and SEA assays of the invention. Each of the experiments included in the results are based on antibody at a dose of 30 µg/ml and measured at 72 hours. Numbers of donors used in the experiments as well as responses are shown. <tables id="tabl0008" num="0008"><table frame="all"><title><b><u>TABLE IVC</u></b></title><tgroup cols="8"><colspec colnum="1" colname="col1" colwidth="24mm" /><colspec colnum="2" colname="col2" colwidth="15mm" /><colspec colnum="3" colname="col3" colwidth="18mm" /><colspec colnum="4" colname="col4" colwidth="34mm" /><colspec colnum="5" colname="col5" colwidth="13mm" /><colspec colnum="6" colname="col6" colwidth="36mm" /><colspec colnum="7" colname="col7" colwidth="9mm" /><colspec colnum="8" colname="col8" colwidth="20mm" /><thead><row><entry align="center" valign="top">Assay</entry><entry align="center" valign="top">mAb</entry><entry align="center" valign="top">Cytokine</entry><entry align="center" valign="top">Mean Enhancement pg/ml</entry><entry align="center" valign="top">SEM</entry><entry align="center" valign="top">Range Enhancement pg/ml</entry><entry align="center" valign="top">n</entry><entry align="center" valign="top">Donor Response</entry></row></thead><tbody><row><entry>T cell blast/Raji</entry><entry align="center">4.1.1</entry><entry align="center">1L-2</entry><entry align="center">3329</entry><entry align="center">408</entry><entry align="center">0 to 8861</entry><entry align="center">42</entry><entry align="center">19 of 21</entry></row><row><entry>T cell blast/Raji</entry><entry align="center">4.1.1</entry><entry align="center">IFN-γ</entry><entry align="center">3630</entry><entry align="center">980</entry><entry align="center">600 to 13939</entry><entry align="center">17</entry><entry align="center">13 of 13</entry></row><row><entry>T cell blast/Raji</entry><entry align="center">11.2.1</entry><entry align="center">IL-2</entry><entry align="center">3509</entry><entry align="center">488</entry><entry align="center">369 to 6424</entry><entry align="center">18</entry><entry align="center">14 or 14</entry></row><row><entry>SEA (PBMC)</entry><entry align="center">4.1.1</entry><entry align="center">IL-2</entry><entry align="center">2800</entry><entry align="center">312</entry><entry align="center">330 to 6699</entry><entry align="center">42</entry><entry align="center">17 of 17</entry></row><row><entry>SEA (PBMC)</entry><entry align="center">11.2.1</entry><entry align="center">IL-2</entry><entry align="center">2438</entry><entry align="center">366</entry><entry align="center">147 to 8360</entry><entry align="center">25</entry><entry align="center">15 of 15</entry></row><row><entry>SEA (Whole Blood)</entry><entry align="center">4.1.1</entry><entry align="center">IL-2</entry><entry align="center">6089</entry><entry align="center">665</entry><entry align="center">-168 to 18417</entry><entry align="center">46</entry><entry align="center">15 of 17</entry></row><row><entry>SEA (Whole Blood)</entry><entry align="center">11.2.1</entry><entry align="center">IL-2</entry><entry align="center">6935</entry><entry align="center">700</entry><entry align="center">-111 to 11803</entry><entry align="center">25</entry><entry align="center">12 of 14</entry></row></tbody></tgroup></table></tables>

<u>EXAMPLE 10</u>

<u>Human T-Cell Signal Model</u>

We developed a second cellular assay in order to quantify the enhancement of T-cell IL-2 upregulation upon blockade of CTLA-4 signal with the antibodies. The following materials and methods were utilized in connection with the expeiments:

Materials and Methods

Human PBMC were prepared using Accuspin. Microtiter plates were precoated with an anti-CD3 antibody (leu4, Becton Dickinson) (60 ng/ml) and incubated for 2 hours at 37°C. hPBMC were added to the wells at 200,000 cells per well. Staphylcoccus enterotoxin A (SEA) (Sigma) was added to the wells at 100 ng/ml. Antibodies were added to the wells, usually at 30 µg/ml. Cells were then stimulated for 48, 72 or 96 hours. Plates were centrifuged at the desired time-point and supernatants were removed from the wells. Thereafter, supernatants were checked for IL-2 production using ELISA (R&amp;D Systems).

Results from these experiments are shown in <figref idref="f0034">Figures 16</figref>, <figref idref="f0035">17</figref>, and <figref idref="f0039">21</figref>. In <figref idref="f0034">Figure 16</figref>, induction of IL-2 production in hPBMC from 5 donors was measured 72 hours after stimulation. In <figref idref="f0035">Figure 17</figref>, results are shown from measurement of whole blood, analyzing the difference in induction of IL-2 production in the blood of 3 donors as measured at 72 and 96 hours after stimulation.

In <figref idref="f0039">Figure 21</figref>, the enhancement of IL-2 production in whole blood of 2 donors as measured at 72 hours after stimulation.

<u>EXAMPLE 11</u>

<u>Tumor Animal Model</u>

We have established an animal tumor model for the <i>in vivo</i> analysis of anti-murine-CTLA-4 antibodies in inhibiting tumor growth. In the model, a murine fibrosarcoma tumor is grown and the animals are treated with anti-murine-CTLA-4 antibodies. The materials and methods for establishment of the model are provided below:

Materials and Methods

Female A/J mice (6-8 weeks old) were injected subcutaneously on the dorsal side of the neck with 0.2 ml of SalN tumor cells (1x10⁶) (Baskar 1995). Anti-murine CTLA-4 or an isotype matched control antibody (PharMingen, San Diego, CA, 200 ug/animal) were injected intraperitioneally on days 0, 4, 7 and 14 following the injection of tumor cells. Tumor measurements were taken during the course of the 3-4 week experiments using a Starrett SPC <i>Plus</i> electronic caliper (Athol, MA) and tumor size was expressed as the surface area covered by tumor growth (mm²).

<figref idref="f0036">Figure 18</figref> shows the inhibition of tumor growth with an anti-murine CTLA-4 antibody in a murine fibrosarcoma tumor model. As shown in <figref idref="f0036">Figure 18</figref>, animals treated with anti-CTLA-4 had a reduction in tumor growth as compared to animals treated with an isotype control antibody. Accordingly, anti-murine CTLA4 mAbs are capable of inhibiting growth of a fibrosarcoma in a mouse tumor model.

It is expected that antibodies that are cross-reactive with murine CTLA-4 would perform similarly in the model. However, of the antibodies of the invention that have been checked for cross-reactivity, none are cross-reactive with murine CTLA-4.

<u>EXAMPLE 12</u>

<u>Tumor Animal Model</u>

In order to further investigate the activity of antibodies in accordance with the invention, a xenograft SCID mouse model was designed to test the eradication of established tumors and their derived metastases. In the model, SCID mice are provided with grafted human T cells and are implanted with patient-derived non-small cell lung cell (NSCL) or colorectal carcinoma (CC) cells. Implantation is made into the gonadal fat pads of SCID mice. The tumors are allowed to grow, and thereafter removed. The mice develop human-like tumor and liver metastases. Such a model is described in <nplcit id="ncit0142" npl-type="s"><text>Bumpers et al J. Surgical Res. 61:282-288 (1996</text></nplcit>).

It is expected that antibodies of the invention will inhibit growth of tumors formed in such mice.

<u>INCORPORATION BY REFERENCE</u>

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<u>EQUIVALENTS</u>

The foregoing description and Examples detail certain preferred embodiments of the invention and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the invention may be practiced in many ways and the invention should be construed in accordance with the appended claims and any equivalents thereof.

THE PRESENT INVENTION CAN ALSO BE CHARACTERIZED BY THE FOLLOWING ITEMS:

<ol id="ol0001"><li>1. An antibody that is capable of binding CTLA-4, comprising a heavy chain variable region amino acid sequence that comprises a contiguous amino acid sequence from within an FR1 sequence through an FR3 sequence that is encoded by a human V_H3-33 family gene and that comprises at least one of the amino acid substitutions in the CDR1 sequences, CDR2 sequences, or framework sequences shown in <figref idref="f0017 f0018 f0019">Figure 2</figref>.</li><li>2. The antibody of Item 1, wherein the amino acid sequence comprises a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, and SEQ ID NO:70.</li><li>3. The antibody of Item 1, further comprising a light chain variable region amino acid sequence comprising a sequence selected from the group consisting of a sequence comprising SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, and SEQ ID NO:71.</li><li>4. The antibody of Item 2, further comprising a light chain variable region amino acid sequence comprising a sequence selected from the group consisting of a sequence comprising SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, and SEQ ID NO:71.</li><li>5. An antibody comprising a heavy chain amino acid sequence comprising SEQ ID NO:1 and a light chain variable amino acid sequence comprising SEQ ID NO:14.</li><li>6. An antibody comprising a heavy chain amino acid sequence comprising SEQ ID NO:2 and a light chain variable amino acid sequence comprising SEQ ID NO:15.</li><li>7. An antibody comprising a heavy chain amino acid sequence comprising SEQ ID NO:4 and a light chain variable amino acid sequence comprising SEQ ID NO:17.</li><li>8. A isolated human monoclonal antibody that is capable of binding to CTLA-4.</li><li>9. The antibody of Item 8, wherein the antibody is capable of competing for binding with CTLA-4 with an antibody selected from the group consisting of 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.2.1, 11.6.1, 11.7.1, 12.3.1.1, and 12.9.1.1.</li><li>10. The antibody of Item 8, wherein the antibody possesses a substantially similar binding specificity to CTLA-4 as an antibody selected from the group consisting of 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.2.1, 11.6.1, 11.7.1, 12.3.1.1, and 12.9.1.1.</li><li>11. The antibody of Item 8, wherein the antibody is selected from the group consisting of 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.2.1, 11.6.1, 11.7.1, 12.3.1.1, and 12.9.1.1.</li><li>12. The antibody of Item 8, wherein the antibody is not cross reactive with CTLA-4 from lower mammalian species.</li><li>13. The antibody of Item 12, wherein the lower mammalian species comprises mouse, rat, and rabbit.</li><li>14. The antibody of Item 8, wherein the antibody is cross reactive with CTLA-4 from primates.</li><li>15. The antibody of Item 14, wherein the primates comprise cynomolgous and rhesus monkeys.</li><li>16. The antibody of Item 8, wherein the antibody possesses a selectivity for CTLA-4 over CD28, B7-2, CD44, and hIgG1 of greater than about 100:1.</li><li>17. The antibody of Item 16, wherein the selectivity is about 500:1 or greater.</li><li>18. The antibody of Item 8, wherein a binding affinity of the antibody is about 10^-9 M or greater.</li><li>19. The antibody of Item 18, wherein a binding affinity of the antibody is about 10^-10 M or greater.</li><li>20. The antibody of Item 8, wherein the antibody inhibits binding between CTLA-4 and B7-2 with an IC₅₀ of lower than about 100 nM.</li><li>21. The antibody of Item 20, wherein the antibody inhibits binding between CTLA-4 and B7-2 with an IC₅₀ of lower than about 0.38 nM.</li><li>22. The antibody of Item 8, wherein the antibody inhibits binding between CTLA-4 and B7-1 with an IC₅₀ of lower than about 100 nM or greater,</li><li>23. The antibody of Item 22, wherein the antibody inhibits binding between CTLA-4 and B7-1 with an IC₅₀ of lower than about 0.50 nM.</li><li>24. The antibody of Item 8, wherein the antibody enhances IL-2 production in a T cell blast/Raji assay by about 500 pg/ml or greater.</li><li>25. The antibody of Item 24, wherein the antibody enhances IL-2 production in a T cell blast/Raji assay by about 3846 pg/ml or greater.</li><li>26. The antibody of Item 8, wherein the antibody enhances IFN-γ production in a T cell blast/Raji assay by about 500 pg/ml or greater.</li><li>27. The antibody of Item 26, wherein the antibody enhances IFN-γ production in a T cell blast/Raji assay by about 1233 pg/ml or greater.</li><li>28. The antibody of Item 8, wherein the antibody induces IL-2 production in a hPBMC or whole blood superantigen assay by about 500 pg/ml or greater.</li><li>29. The antibody of Item 26, wherein the antibody induces IL-2 production in a hPBMC or whole blood superantigen assay by about 1500 pg/ml or greater</li><li>30. The antibody of Item 26, wherein the antibody induces IL-2 production in a hPBMC or whole blood superantigen assay by greater than about 30% relative to control.</li><li>31. The antibody of Item 26, wherein the antibody induces IL-2 production in a hPBMC or whole blood superantigen assay by greater than about 50% relative to control.</li><li>32. A humanized antibody that possesses a substantially similar binding specificity to CTLA-4 as an antibody selected from the group consisting of 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.13.1, 4.14.3, 6.1.1, 11.2.1, 11.6.1, 11.7.1,12.3.1.1, and 12.9.1.1.</li><li>33. The antibody of Item 32, wherein the antibody is not cross reactive with CTLA-4 from lower mammalian species.</li><li>34. The antibody of Item 33, wherein the lower mammalian species comprises mouse, rat, and rabbit.</li><li>35. The antibody of Item 32, wherein the antibody is cross reactive with CTLA-4 from primates.</li><li>36. The antibody of Item 35, wherein the primates comprise cynomolgous and rhesus monkeys.</li><li>37. The antibody of Item 32, wherein the antibody possesses a selectivity for CTLA-4 over CD28, B7-2, CD44, and hIgG1 of greater than about 100:1.</li><li>38. The antibody of Item 37, wherein the selectivity is about 500:1 or greater.</li><li>39. The antibody of Item 32, wherein a binding affinity of the antibody is about 10^-9 M or greater.</li><li>40. The antibody of Item 39, wherein a binding affinity of the antibody is about 10^-10 M or greater.</li><li>41. The antibody of Item 32, wherein the antibody inhibits binding between CTLA-4 and B7-2 with an IC₅₀ of lower than about 100 nM.</li><li>42. The antibody of Item 41, wherein the antibody inhibits binding between CTLA-4 and B7-2 with an IC₅₀ of lower than about 0.38 nM.</li><li>43. The antibody of Item 32, wherein the antibody inhibits binding between CTLA-4 and B7-1 with an IC₅₀ of lower than about 100 nM or greater.</li><li>44. The antibody of Item 43, wherein the antibody inhibits binding between CTLA-4 and B7-1 with an IC₅₀ of lower than about 0.50 nM.</li><li>45. The antibody of Item 32, wherein the antibody enhances IL-2 production in a T cell blast/Raji assay by about 500 pg/ml or greater.</li><li>46. The antibody of Item 45, wherein the antibody enhances IL-2 production in a T cell blast/Raji assay by about 3846 pg/ml or greater.</li><li>47. The antibody of Item 32, wherein the antibody enhances IFN-γ production in a T cell blast/Raji assay by about 500 pg/ml or greater.</li><li>48. The antibody of Item 47, wherein the antibody enhances IFN-γ production in a T cell blast/Raji assay by about 1233 pg/ml or greater.</li><li>49. The antibody of Item 32, wherein the antibody enhances IL-2 production in a hPBMC or whole blood superantigen assay by about 500 pg/ml or greater.</li><li>50. The antibody of Item 49, wherein the antibody enhances IL-2 production in a hPBMC or whole blood superantigen assay by about 1500 pg/ml or greater</li><li>51. The antibody of Item 32, wherein the antibody enhances IL-2 production in a hPBMC or whole blood superantigen assay by greater than about 30% relative to control.</li><li>52. The antibody of Item 50, wherein the antibody enhances IL-2 production in a hPBMC or whole blood superantigen assay by greater than about 50% relative to control.</li><li>53. An antibody that binds to CTLA-4, comprising a heavy chain amino acid sequence comprising human FR1, FR2, and FR3 sequences encoded by a human V_H 3-33 gene family operably linked in frame with a CDR1, a CDR2, and a CDR3 sequence, the CDR1, CDR2, and CDR3 sequences being independently selected from the CDR1, CDR2, and CDR3 sequences illustrated in <figref idref="f0017 f0018 f0019">Figure 2</figref>.</li><li>54. The antibody of Item 32, further comprising any of the somatic mutations to the FR1, FR2, and FR3 sequences as illustrated in <figref idref="f0017 f0018 f0019">Figure 2</figref>.</li><li>55. An antibody that binds to CTLA-4, comprising a heavy chain amino acid sequence comprising human FR1, FR2, and FR3 sequences encoded by a human V_H 3-33 gene family operably linked in frame with a CDR1, a CDR2, and a CDR3 sequence, which antibody has the following properties: <ul id="ul0014" list-style="none" compact="compact"><li>a binding affinity for CTLA-4 of about 10^-9 or greater;</li><li>inhibits binding between CTLA-4 and B7-1 with an IC₅₀ of about 100 nM or lower;</li><li>inhibits binding between CTLA-4 and B7-2 with an IC₅₀ of about 100 nM or lower; and</li><li>enhances cytokine production in an assay of human T cells by 500 pg/ml or greater.</li></ul></li><li>56. An antibody that binds to CTLA-4, comprising a heavy chain amino acid sequence comprising FR1, FR2, and FR3 sequences operably linked in frame with a CDR1, a CDR2, and a CDR3 sequence independently selected from the CDR1, CDR2, and CDR3 sequences illustrated in <figref idref="f0017 f0018 f0019">Figures 2</figref> and <figref idref="f0020">3</figref>, which antibody has the following properties: <ul id="ul0015" list-style="none" compact="compact"><li>a binding affinity for CTLA-4 of about 10^-9 or greater;</li><li>inhibits binding between CTLA-4 and B7-1 with an IC₅₀ of about 100 nM or lower;</li><li>inhibits binding between CTLA-4 and B7-2 with an IC₅₀ of about 100 nM or lower; and</li><li>enhances cytokine production in an assay of human T cells by 500 pg/ml or greater.</li></ul></li><li>57. A cell culture system for assaying T cell stimulation, comprising a culture of human T cell blasts co-cultured with a Raji cell line.</li><li>58. The cell culture system of Item 57, wherein the T cell blasts are washed prior to culture with the Raji cell line.</li><li>59. An assay for measuring T cell stimulation, comprising: <ul id="ul0016" list-style="none" compact="compact"><li>providing a culture of human T cell blasts and a Raji cell line; contacting the culture with an agent; and</li><li>measuring cytokine production by the culture.</li></ul></li><li>60. The assay of Item 59, wherein the T cell blasts are washed prior to culture with the Raji cell line.</li><li>61. The assay of Item 59, wherein the cytokine is IL-2.</li><li>62. The assay of Item 59, wherein the cytokine is IFN-γ.</li><li>63. The assay of Item 59, wherein cytokine production is measured in supernatant isolated from the culture.</li><li>64. The assay of Item 59, wherein the agent is an antibody.</li><li>65. The assay of Item 59, wherein the antibody binds to CTLA-4.</li><li>66. A functional assay for screening a moiety for T cell stimulatory function, comprising: <ul id="ul0017" list-style="none" compact="compact"><li>providing a culture of human T cell blasts and a Raji cell line;</li><li>contacting the culture with the moiety; and</li><li>assessing cytokine production by the culture.</li></ul></li><li>67. The assay of Item 66, wherein the T cell blasts are washed prior to culture with the Raji cell line.</li><li>68. The assay of Item 66, wherein the cytokine is IL-2.</li><li>69. The assay of Item 66, wherein the cytokine is IFN-γ.</li><li>70. The assay of Item 66, wherein cytokine production is assessed in supernatant isolated from the culture.</li><li>71. The assay of Item 66, wherein the moiety is an antibody.</li><li>72. The assay of Item 66, wherein the antibody binds to CTLA-4.</li><li>73. A T cell stimulatory assay for CTLA-4 inhibitory function, comprising contacting a culture comprising human T cell blasts and a Raji cell line with an agent and assessing cytokine production by the culture.</li><li>74. The assay of Item 73, wherein the T cell blasts are washed prior to culture with the Raji cell line.</li><li>75. The assay of Item 73, wherein the cytokine is IL-2.</li><li>76. The assay of Item 73, wherein the cytokine is IFN-γ.</li><li>77. The assay of Item 73, wherein cytokine production is assessed in supernatant isolated from the culture.</li><li>78. The assay of Item 73, wherein the agent is an antibody.</li><li>79. The assay of Item 73, wherein the antibody binds to CTLA-4.</li><li>80. A method for screening an agent for T cell stimulatory activity, comprising: <ul id="ul0018" list-style="none" compact="compact"><li>contacting the agent with a cell culture comprising human T cell blasts and a Raji cell line; and</li><li>assessing cytokine production by the culture.</li></ul></li><li>81. The method of Item 80, wherein the T cell blasts are washed prior to culture with the Raji cell line.</li><li>82. The method of Item 80, wherein the cytokine is IL-2.</li><li>83. The method of Item 80, wherein the cytokine is IFN-γ.</li><li>84. The method of Item ; 80, wherein cytokine production is assessed in supernatant isolated from the culture.</li><li>85. The method of Item 80, wherein the agent is an antibody that binds to CTLA-4.</li><li>86. An assay for measuring T cell stimulation, comprising: <ul id="ul0019" list-style="none" compact="compact"><li>providing a population of human peripheral blood mononuclear cells or human whole blood stimulated with staphylococcus enterotoxin A;</li><li>contacting the culture with an agent; and</li><li>measuring cytokine production by the cell population.</li></ul></li><li>87. The assay of Item 86, wherein the cytokine is IL-2.</li><li>88. The assay of Item 86, wherein cytokine production is measured in supernatant isolated from the cell population.</li><li>89. The assay of Item 86, wherein the agent is an antibody.</li><li>90. The assay of Item 86, wherein the antibody binds to CTLA-4.</li><li>91. A functional assay for screening a moiety for T cell stimulatory function, comprising: <ul id="ul0020" list-style="none" compact="compact"><li>providing a population of human peripheral blood mononuclear cells or human whole blood stimulated with staphylococcus enterotoxin A;</li><li>contacting the culture with the moiety; and</li><li>assessing cytokine production by the cell population.</li></ul></li><li>92. The assay of Item 91, wherein the cytokine is IL-2.</li><li>93. The assay of Item 91, wherein cytokine production is assessed in supernatant isolated from the cell population.</li><li>94. The assay of Item 91, wherein the moiety is an antibody .</li><li>95. The assay of Item 91, wherein the antibody binds to CTLA-4.</li><li>96. A T cell stimulatory assay for CTLA-4 inhibitory function, comprising contacting a population of human peripheral blood mononuclear cells or human whole blood stimulated with staphylococcus enterotoxin A with an agent and assessing cytokine production by the cell population.</li><li>97. The assay of Item 96, wherein the cytokine is IL-2.</li><li>98. The assay of Item 96, wherein cytokine production is assessed in supernatant isolated from the cell population.</li><li>99. The assay of Item 96, wherein the agent is an antibody.</li><li>100. The assay of Item 96, wherein the antibody binds to CTLA-4.</li><li>101. A method for screening an agent for T cell stimulatory activity, comprising: <ul id="ul0021" list-style="none" compact="compact"><li>contacting the agent with a population of human peripheral blood mononuclear cells or human whole blood stimulated with staphylococcus enterotoxin A; and</li><li>assessing cytokine production by the cell population.</li></ul></li><li>102. The method of Item 101, wherein the cytokine is IL-2.</li><li>103. The method of Item 101, wherein cytokine production is assessed in supernatant isolated from the cell population.</li><li>104. The method of Item 101, wherein the agent is an antibody that binds to CTLA-4.</li></ol><img file="EP3553085A1_D0004.tif" /><img file="EP3553085A1_D0005.tif" /><img file="EP3553085A1_D0006.tif" /><img file="EP3553085A1_D0007.tif" /><img file="EP3553085A1_D0008.tif" /><img file="EP3553085A1_D0009.tif" /><img file="EP3553085A1_D0010.tif" /><img file="EP3553085A1_D0011.tif" /><img file="EP3553085A1_D0012.tif" /><img file="EP3553085A1_D0013.tif" /><img file="EP3553085A1_D0014.tif" /><img file="EP3553085A1_D0015.tif" /><img file="EP3553085A1_D0016.tif" /><img file="EP3553085A1_D0017.tif" /><img file="EP3553085A1_D0018.tif" /><img file="EP3553085A1_D0019.tif" /><img file="EP3553085A1_D0020.tif" /><img file="EP3553085A1_D0021.tif" /><img file="EP3553085A1_D0022.tif" /><img file="EP3553085A1_D0023.tif" /><img file="EP3553085A1_D0024.tif" /><img file="EP3553085A1_D0025.tif" /><img file="EP3553085A1_D0026.tif" /><img file="EP3553085A1_D0027.tif" /><img file="EP3553085A1_D0028.tif" /><img file="EP3553085A1_D0029.tif" /><img file="EP3553085A1_D0030.tif" /><img file="EP3553085A1_D0031.tif" /><img file="EP3553085A1_D0032.tif" /><img file="EP3553085A1_D0033.tif" /><img file="EP3553085A1_D0034.tif" /><img file="EP3553085A1_D0035.tif" /><img file="EP3553085A1_D0036.tif" /><img file="EP3553085A1_D0037.tif" /><img file="EP3553085A1_D0038.tif" /><img file="EP3553085A1_D0039.tif" /><img file="EP3553085A1_D0040.tif" /><img file="EP3553085A1_D0041.tif" /><img file="EP3553085A1_D0042.tif" /><img file="EP3553085A1_D0043.tif" /><img file="EP3553085A1_D0044.tif" /><img file="EP3553085A1_D0045.tif" /><img file="EP3553085A1_D0046.tif" /><img file="EP3553085A1_D0047.tif" /><img file="EP3553085A1_D0048.tif" /><img file="EP3553085A1_D0049.tif" /><img file="EP3553085A1_D0050.tif" /><img file="EP3553085A1_D0051.tif" /><img file="EP3553085A1_D0052.tif" /><img file="EP3553085A1_D0053.tif" /><img file="EP3553085A1_D0054.tif" /><img file="EP3553085A1_D0055.tif" /><img file="EP3553085A1_D0056.tif" /><img file="EP3553085A1_D0057.tif" /><img file="EP3553085A1_D0058.tif" /><img file="EP3553085A1_D0059.tif" /><img file="EP3553085A1_D0060.tif" /><img file="EP3553085A1_D0061.tif" /><img file="EP3553085A1_D0062.tif" /><img file="EP3553085A1_D0063.tif" /><img file="EP3553085A1_D0064.tif" /><img file="EP3553085A1_D0065.tif" /><img file="EP3553085A1_D0066.tif" /><img file="EP3553085A1_D0067.tif" /><img file="EP3553085A1_D0068.tif" /><img file="EP3553085A1_D0069.tif" /><img file="EP3553085A1_D0070.tif" /><img file="EP3553085A1_D0071.tif" /><img file="EP3553085A1_D0072.tif" /><img file="EP3553085A1_D0073.tif" /><img file="EP3553085A1_D0074.tif" /><img file="EP3553085A1_D0075.tif" /><img file="EP3553085A1_D0076.tif" />