MXPA99008621A - Immunotherapeutic ctla-4 binding peptides - Google Patents

Abstract

The present invention relates to methods and products for immunotherapy resulting in the stimulation of T-cell proliferation. The products of the invention are peptides that bind to CTLA-4 and co-stimulate the proliferation of T-cells by inhibiting the binding of B7 to CTLA-4. Pharmaceutical compositions including such peptides are also provided. The invention further provides in vitro and in vivo therapeutic methods employing the peptides of the invention.

Description

PEPTIDES OF UNION TO CTLA-4, IMMUNOTERAPEUTICOS FIELD OF THE INVENTION The present invention relates generally to the field of immunology and specifically to peptides that bind to CTLA-4 and stimulate the proliferation of T cells. BACKGROUND OF THE INVENTION The complex process of activation and proliferation of T cells, it is based on various interactions such as antigen presentation, cell-cell contact and soluble immune mediators, eg cytokines or lymphokines. Many of these interactions are mediated in T cells through the surface receptors. The T helper cells, for example, require for their activation both the presentation of an antigen by an antigen-presenting cell (APC) in association with the major histocompatibility complex (MHC), as a secondary signal. The secondary signal may be a soluble factor or may involve an interaction with another set of receptors on the surface of the T cells. The presentation of the antigen in the absence of the secondary signal, however, is not sufficient to activate the T helper cells . The CTLA-4 / CD28 / B7 system is a group of REF .: 31287 proteins involved in the regulation of T cell proliferation through its secondary signaling pathway. The proliferative response of T cells is controlled by the interaction of the family of B7 proteins, which are expressed on the surface of the CPA, with CTLA-4 (antigen of cytotoxic T lymphocytes # 4) and CD28. The B7 family of proteins is composed of structurally related glycoproteins, which include the B7-1, B7-2 and B7-3 proteins (Galea-Laure et al., Cancer Gene Therapy, vol.3, p.202-213 (1996 ), Boussiotis et al., Proc. Nat. Acad. Sci. USA, v. 90, p.11059-11063 (1993)). The different B7 proteins appear to have different expression patterns on the surface of the antigen-presenting cells. For example, B7-2 is constitutively expressed on the surface of monocytes, whereas B7-1 does not, although the expression of B7-1 is induced in these cells when said cells are stimulated with interferon gamma (IFN- ?) The different expression patterns may indicate a different function for each member of the B7 family. It is believed that B7 proteins are involved in events that are related to the stimulation of an immune response by their ability to interact with surface receptors of several immune cells. For example, it is believed that B7 plays a role in increasing T-cell proliferation and cytokine production through its interaction with the CD28 receptor. CD28, which is a homodimeric glycoprotein, has two 44-kd subunits linked by disulfide sources, is found in 95% of CD4 cells and in 50% of CD8 cells. Studies carried out using monoclonal antibodies reactive with DC28, have shown that CD28 participates in a secondary signal pathway in the activation of T cell proliferation. It has been found that antibodies that block the interaction of CD28 with its ligand, inhibit T-cell proliferation in vitro, resulting in antigen-specific T cell anergy (Harding et al., Nature, v. 356, p.607 (1991)). Recently, a T cell surface receptor protein, CTLA-4 protein, has been identified which has approximately 20% sequence homology with the CD28 receptor. Although CTLA-4 is not endogenously expressed on the surface of T cells, its expression is induced when CD28 interacts with B7 on the surface of a CPA. Once CTLA-4 is expressed on the surface of the T cell, it is able to interact with B7.

Several groups of researchers hypothesize that CTLA-4 and CD28 could have opposite effects on a T cell and that CTLA-4 and CD28 could compete for binding with B7 (Krummel et al., International Immunology, v. 8, p. 519-523 (1995); Galea-Lauri et al. , Cancer Gene Therapy, v. 3, p. 202-213 (1996)). When an antigen is presented to a T cell by a CPA and B7 interacts with CD28 on the surface of the T cell, a secondary signal is created that stimulates the T cell to proliferate. However, when B7 interacts with CTLA-4-4, the secondary signal is not created. It is not yet clear whether the interaction of CTLA-4 with B7 initiates an inhibitory signaling pathway to prevent the cell from proliferating, or whether the interaction of CTLA-4 with B7 simply acts to reduce the amount of B7 available to bind with the receiver CD28. In any case, it appears that CTLA-4-4, CD28 and B7, each of them, plays an important role in the intricate regulation of T cell proliferation. BRIEF DESCRIPTION OF THE INVENTION The present invention relates to peptides that they bind with CTLA-4 and they co-stimulate the proliferation of T cells. The peptides of the present invention, which include monoclonal antibodies, functionally active antibody fragments and functionally active polypeptides, specifically interact with human CTLA-4 and prevent the interaction of the B7 with human CTLA-4. These peptides have particular utility as pharmaceuticals for the immunotherapy of disorders sensitive to T-cell proliferation, due to their ability to co-stimulate the proliferation of T cells. The peptides of the present invention are particularly effective for the immunotherapy of disorders sensitive to T cells. the proliferation of T cells, when administered in combination with conventional therapeutic agents used for the treatment of such disorders. For example, a tumor, which is a disorder sensitive to T cell proliferation, is conventionally treated with a chemotherapeutic agent that works by killing rapidly dividing cells. The peptides of the present invention, when administered in conjunction with a chemotherapeutic agent, enhance the tumoricidal effect of the chemotherapeutic agent by stimulating the proliferation of T cells to increase the immunological rejection of the tumor cells. A major advantage of the peptides of the present invention derives from the fact that they interact specifically with human CTLA-4. Because the peptides of the present invention specifically interact with human CTLA-4, they can be used in vivo in humans to co-stimulate T cell proliferation. In vivo enhancement of T cell proliferation is desirable as an aid in the treatment of numerous disorders sensitive to the proliferation of T cells of the immune system, such as diseases resulting from immunodeficiency, as well as disorders involving unwanted cell invasion or growth, such as the invasion of the body by foreign microorganisms or the growth of tumors. In particular, the present invention provides a composition of a peptide that selectively binds to human CTLA-4 and co-stimulates T cell proliferation. In one embodiment, the peptide has a CDR3 region binding to CTLA-4. The CDR3 binding region to CTLA-4 can be a CDR3A3.4H2 or a functional variant thereof of a monoclonal antibody produced by the hybridoma A3.4H2, deposited in the American Type Culture Collection (ATCC) with the access number HB-12319. In another embodiment, the CDR3 binding region to CTLA-4 is a CDR3A3.6BIO OR a functional variant thereof of a monoclonal antibody produced by the hybridoma A3.6.B10, deposited with the ATCC with accession number HB- 12318 The peptide can be an intact soluble monoclonal antibody. In accordance with one embodiment of the present invention, the peptide is a soluble monoclonal antibody intact? A3, 4H2 produced by the hybridoma cell line deposited with the ATCC with accession number HB-12319 or an intact antibody having the characteristics of binding of the deposited monoclonal antibody. In another embodiment, the peptide is a soluble monoclonal antibody intact? A3.6Bio produced by the hybridoma cell line deposited with the ATCC with accession number HB-12318 or an intact antibody having the binding characteristics of the deposited monoclonal antibody. In accordance with yet another embodiment of the present invention, the peptide is a humanized monoclonal antibody. The peptide may also be a functionally active monoclonal antibody fragment or a functionally active polypeptide. In one embodiment, the peptide is a monoclonal antibody fragment that is selected from the group consisting of an F (ab ') 2 fragment, an Fd fragment and a Fab fragment. In another embodiment, the peptide has a light chain CDR2 region that is selected from the group consisting of a CDR2A3.4H2 OR a functional variant thereof of a monoclonal antibody produced by hybridoma A3.4H2 deposited with the ATCC with the number of access HB-12319 and a CDR2A3.6B10 or a functional variant thereof of a monoclonal antibody produced by the hybridoma A3.6B10 deposited in the ATCC with the accession number HB-12318. According to another embodiment of the present invention, the peptide has a light chain CDR1 region that is selected from the group consisting of a CDR1A3.4H2 OR a functional variant thereof of a monoclonal antibody produced by the hybridoma A3.4H2, deposited in the ATCC with the accession number HB-12319 and a CDR1A3.6B10 or a functional variant thereof of a monoclonal antibody produced by the hybridoma A3.6B10, deposited in the ATCC with the accession number HB-12318. In accordance with another aspect of the present invention, there is provided a pharmaceutical composition for the treatment of a disorder responsive to human T cell proliferation. The pharmaceutical composition includes an effective amount for the treatment of the human T cell proliferation-sensitive disorder of a peptide that selectively binds to human CTLA-4 and which co-stimulates the proliferation of T cells, and a pharmaceutically acceptable carrier. In one embodiment, the peptide has a CDR3 binding to CTLA-4. In accordance with one embodiment of the present invention, the CDR3 binding region to CTLA-4 is a CDR3A3.4H2 OR a functional variant thereof of a monoclonal antibody produced by the hybridoma A3.4H2, deposited with the ATCC with the number of access HB-12319. In another embodiment, the CDR3 binding region to CTLA-4 is a CDR3A3.6BIO OR a functional variant thereof of a monoclonal antibody produced by the hybridoma A3.6B10, deposited with the ATCC with the accession number HB-12318. The peptide can be an intact soluble monoclonal antibody. According to one embodiment of the present invention, the peptide is a soluble monoclonal antibody intact? A3.4H2 produced by the hybridoma cell line deposited with the ATCC with accession number HB-12319 or an intact antibody having the characteristics of binding of the deposited monoclonal antibody. In another embodiment, the peptide is a soluble monoclonal antibody intact? A3.6Bio produced by the hybridoma cell line deposited with the ATCC with accession number HB-12318 or an intact antibody having the binding characteristics of the deposited monoclonal antibody. In accordance with yet another embodiment of the present invention, the peptide is a humanized monoclonal antibody. The peptide may also be a functionally active monoclonal antibody fragment or a functionally active polypeptide. In a modality, the peptide is a monoclonal antibody fragment that is selected from the group consisting of an F (ab ') 2 fragment / an Fd fragment and a Fab fragment. In another embodiment, the peptide has a light chain CDR2 region that is selected from the group consisting of a CDR2A3. H2 or a functional variant thereof of a monoclonal antibody produced by the hybridoma A3.4H2, deposited in the ATCC with the accession number HB-12319 and a CDR2.A3.6BIO or a functional variant thereof of a monoclonal antibody produced by the hybridoma A3.6B10, deposited in the ATCC with accession number HB-12318. According to another embodiment of the present invention, the peptide has a light chain CDR1 region which is selected from the group consisting of the group consisting of a CDR1A3.4H2O or a functional variant thereof of a monoclonal antibody produced by the hybridoma. A3.4H2, deposited in the ATCC with the accession number HB-12319 and a CDR1A3.6BIO OR a functional variant thereof of a monoclonal antibody produced by the hybridoma A3.6B10, deposited in the ATCC with the access number HB -12318. The present invention further provides a method for stimulating the proliferation of in situ T cells. The method includes the step of adding a peptide that binds to human CTLA-4, to a population of cells that includes T cells and antigen presenting cells, to co-stimulate the proliferation of T cells. In one embodiment, the peptide has a CDR3 region binding to CTLA-4. In accordance with another aspect of the present invention, there is provided a method for the treatment of a disorder responsive to T cell proliferation. The method involves the step of administering to a subject suffering from a disorder responsive to T cell proliferation. , a peptide that binds to human CTLA-4 in an amount effective to increase T cell proliferation. In one embodiment, the peptide has a CDR3 region binding to CTLA-4. In another embodiment, the disorder responsive to T cell proliferation is a tumor. In another embodiment, the disorder responsive to T cell proliferation is an immunodeficiency disease. The present invention also provides antibody producing hybridomas deposited in the ATCC with accession numbers HB-12318 and HB-12319. These hybridomas produce antibodies having the CDR3 region that specifically interact with CTLA-4 and co-stimulate T cell proliferation. In accordance with another aspect of the present invention, a nucleic acid molecule is provided. In one embodiment, the nucleic acid molecule is the nucleic acid sequence of the antibodies produced by the deposited hybridomas. Each of the deposited hybridomas makes possible the production of the nucleic acid molecules of the present invention, because it is within the routine of those skilled in the art to isolate and sequence the DNA of an established cell line. According to another embodiment, the nucleic acid molecule is selected from the group consisting of intact monoclonal antibodies that selectively bind to human CTLA-4, humanized antibodies that selectively bind to human CTLA-4, fragments of antibodies that are selectively bind to human CTLA-4 and CDR regions that selectively bind to human CTLA-4. DETAILED DESCRIPTION OF THE INVENTION The present invention involves the finding that specific peptides that bind to human CTLA-4 in T cells and inhibit B7 molecules from binding to CTLA-4-4, co-stimulate cell proliferation T. By stimulating T-cell proliferation, the peptides of the present invention are useful for enhancing an immune response in vivo, which would be very useful for the treatment of numerous disorders that are related to immune function. The peptides can be used on their own as primary therapy, or in combination with other therapeutic agents as adjuvant therapy to increase the therapeutic benefits of other medical treatments. The peptides of the present invention are typically useful when it is desirable to co-stimulate the proliferation of T cells. As is well known in the art, the complementarity determining regions (CDR) of an antibody, are the portions of the antibody that are largely responsible for the specificity thereof. CDRs interact directly with the antigen epitope (see, in general, Clark, 1986).; Roitt, 1991). In both the heavy chain and light chain variable regions of IgG immunoglobulins, there are four framework regions (from FR1 to FR4) separated, respectively, by three complementarity determining regions (from CDR1 to CDR3). The framework regions (FR) maintain the tertiary structure of the paratope, which is the portion of the antibody that is involved in the interaction with the antigen. The CDRs and in particular the CDR3 regions and more particularly the heavy chain CDR3 contribute to the specificity of the antibody. Because these CDR regions and in particular the CDR3 region confer antigen specificity on the antibody, these regions can be incorporated into other antibodies or peptides to confer identical antigen specificity on that antibody or peptide. The present invention encompasses peptides that include a CTLA-4 binding region that specifically binds to human CTLA-4 and prevents it from interacting with B7. Optionally, the CTLA-4 binding region is a CDR3 binding region to CTLA-4. CTLA-4 is a surface receptor protein of T cells. The term WB7"as used herein, is a family of structurally related glycoproteins that include the B7-1, B7-2 and B7-3 proteins. The term "CTLA-4 binding CDR3 region" as used herein, is a CDR3 peptide sequence derived from the monoclonal antibodies produced by the hybridomas deposited with the ATCC under accession numbers HB-12318 and HB-12319 Hybridoma cell lines producing antibodies (A3-4H2 and A3-6B10) were deposited by the Applicants at the ATCC on March 21, 1997. The hybridoma A3.4H2 produces the monoclonal antibody 1A3.4H2 which has binding specificity by CTLA-4 Monoclonal antibody A3.4H2 includes the CDR3A3.4H2 region within its sequence As used herein, the term "CD 3A3.4H2" includes the CDR3 region of the monoclonal antibody A3.4H2- The hybridoma A3. 6B10 produces the antibody monoclone A3.6Bio that has binding specificity for CTLA-4. The 3.6Bio monoclonal antibody includes the CDR3A3.6B10 region within its sequence. As used herein, the term "CDR3A3.6BIO" includes the CDR3 region of the monoclonal antibody.A3.6Bio-Both the monoclonal antibodyA3.4H2 and the monoclonal antibodyA3.6Bi? / Bind specifically to CTLA-4 and they prevent it from interacting with B7. The term "CTLA-4 binding CDR3 region" refers to the peptide sequences CDR3A3.4H2 and CDR3.R3.6BIO 'In one embodiment of the present invention, the peptides of the present invention include functional variants of the CDR3 region of union to CTLA-4. The term "functional variant" as used herein, is a peptide having the sequence of the CDR3.A3.4H2 or CDR3A3.6BIO regions with conservative substitutions therein. As used herein, the term "conservative substitution" refers to an amino acid substitution that does not alter the relative charge or size characteristics of the peptide in which the amino acid substitution was made. Conservative amino acid substitutions include substitutions made between amino acids with the following groups: (1) M, K, L, V; (2) F, Y, W; (3) K, R, H; (4) A, G; (5) S, T; (6) Q, N; and (7) E, D. Such substitutions can be made by a variety of methods known to those skilled in the art. For example, amino acid substitutions can be performed by PCR (polymerase chain reaction, Polymerase Chain Reaction, PCR), site-directed mutagenesis according to the Kunkel method (Kunkel, Proc. Nat Acad. Sci. USA 82 : 488-492, 1985), or by chemical synthesis of a gene coding for the CDR3 region. These and other methods for altering the peptide of the CDR3 region are known to those skilled in the art and can be found in references compiling such methods, for example Sa brook or Ausubel, already referred to above. The equivalent variants in activity or functionality of the CDR3 region of binding to CTLA-4 can be tested by the binding and activity assays described in more detail below. For brevity purposes, the term "hybridoma deposited in the ATCC" is used in the present description to refer to both hybridomas deposited with the ATCC on March 21, 1997. The term "deposited monoclonal antibody" is used herein to mean to both monoclonal antibodies (monoclonal antibody A3.4H2 or monoclonal antibodyA3.6Bio) produced by the hybridomas deposited in the ATCC. For purposes of definition, in the appended claims each of the hybridomas and monoclonal antibodies is specifically described. As used herein, the term "peptide" includes monoclonal antibodies, functionally active antibody fragments and functionally active polypeptides. The peptides of the present invention are isolated peptides. As used herein, with respect to peptides, the term "isolated peptides" means that the peptides are substantially pure and are substantially free of other substances with which they could be found in nature or in vivo systems, up to a practical and appropriate degree for its intended use. In particular, the peptides are sufficiently pure and sufficiently free of other biological constituents of their host cells to be useful, for example, in the production of pharmaceutical preparations or in sequencing. Because an isolated peptide of the present invention can be mixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the peptide can comprise only a small percentage by weight of the preparation. However, the peptide is substantially pure because it has been substantially separated from the substances with which it could be associated in living systems.

According to one embodiment, the peptide of the present invention is an intact soluble anti-CTiA-4 monoclonal antibody in isolated form or in a pharmaceutical preparation. An intact soluble monoclonal antibody, as is well known in the art, is an assembly of polypeptide chains linked by disulfide bridges. Two major polypeptide chains, referred to as the light chain and the heavy chain, make up the major structural classes (isotypes) of the antibodies. Both heavy and light chains are further divided into sub-regions, referred to as variable regions and constant regions. As used herein, the term "monoclonal antibody" refers to a homogeneous population of immunoglobulins that specifically bind to an epitope (i.e. antigenic determinant) of CTLA-4. The peptide of the present invention in one embodiment, for example, is the deposited monoclonal antibody. The preparation and use of the deposited monoclonal antibody is described more fully in the appended Examples. In another embodiment, the peptide of the present invention is an intact antibody that has the binding characteristics of the deposited monoclonal antibody. An antibody that has the binding characteristics of the deposited monoclonal antibody is one that binds to CTLA-4 and that inhibits CTLA-4 from its interaction with B7. A person skilled in the art will readily be able to identify antibodies having the binding characteristics of the deposited monoclonal antibody using selection and binding assays, which are presented in more detail below. In a set of embodiments, the peptide useful according to the methods of the present invention is an intact humanized anti-CTLA-4 monoclonal antibody in isolated form or in a pharmaceutical preparation. The following Examples of methods for the preparation of humanized monoclonal antibodies that interact with CTLA-4 and co-stimulate T cell activation are exemplary and are provided for illustrative purposes only. The term "humanized monoclonal antibody" as used herein, is a human monoclonal antibody or a functionally active fragment thereof that has human constant regions and a CDR3 region binding to CTLA-4, derived from a mammal of a species different to the human being. Humanized monoclonal antibodies can be prepared by any method known in the art. Humanized monoclonal antibodies, for example, can be constructed by replacing the non-CDR regions of an antibody of a non-human mammal with similar regions of human antibodies, while retaining the epitope specificity of the original antibody. For example, non-human CDRs and optionally some framework regions can be covalently linked to human VF and / or Fc / pFc 'regions to produce a functional antibody. There are entities in the United States that commercially synthesize humanized antibodies from specific murine antibody regions, such as Protein Design Labs (Mountain Vie, California, USA). European Patent Application 0239400, the entire contents of which are incorporated herein by reference, provides exemplary teaching of the production and use of humanized monoclonal antibodies in which at least the CDR portion of a murine antibody (or of some other mammal) non-human) is included in the humanized antibody. In brief the following methods are useful for constructing a humanized CDR monoclonal antibody including at least a portion of a murine CDR. A first replicable expression vector is prepared which includes a suitable promoter operably linked to a DNA sequence encoding at least one variable domain of a heavy or light chain of an Ig and the variable domain comprising the framework regions of a human antibody and a CDR region of a murine antibody. Optionally, a second replicable expression vector is prepared which includes a suitable promoter operably linked to a DNA sequence encoding at least the variable domain of a light or heavy chain of a complementary human Ig, respectively. Then a cell line is transformed with the vectors. Preferably, the cell line is an immortalized mammalian cell line of lymphoid origin, such as a myeloma, hybridoma, trioma or quadroma cell line, or is a normal lymphoid line that has been immortalized by transformation with a virus. The transformed cell line is then cultured under conditions known to those skilled in the art, to produce the humanized antibody. As set out in European Patent Application 0239400, several techniques are known in this field to create the particular antibody domains that are to be inserted into the replicable vector (preferred vectors and recombinant techniques are described in more detail below) . For example, the DNA sequence encoding the domain can be prepared by oligonucleotide synthesis. Alternatively, a synthetic gene lacking the CDR regions in which four framework regions are fused together with suitable restriction sites at the junctions, such as synthetic double-stranded filaments or restricted subcloned CDR cartridges with sticky ends, is could link at the junctions of the framework regions. Another method involves the preparation of the DNA sequence encoding the variable CDR-containing domain by site-directed oligonucleotide mutagenesis. Each of these methods is known in the art. Therefore, those skilled in the art could construct humanized antibodies containing a murine CDR region without destroying the specificity of the antibody for its epitope. In preferred embodiments, the humanized antibodies of the present invention are humanized monoclonal antibodies that include at least the CDR3 region of CTLA-4 binding of the deposited monoclonal antibody. As noted above, such humanized antibodies can be produced in which some or all of the FR regions of the deposited monoclonal antibody have been replaced by homologous human FR regions. In addition, Fe portions can be replaced to produce IgA or IgM, as well as IgG antibodies carrying all or a portion of the CDR regions of the deposited monoclonal antibody. Of particular importance is the inclusion of the CDR3 binding region to CTLA-4 of the deposited monoclonal antibody and, to a lesser extent, the other CDR portions and framework regions of the deposited monoclonal antibody. Such humanized antibodies will have particular clinical utility because they will specifically recognize human CTLA-4, but will not induce any immune response in humans against the proper antibodies. In a more preferred embodiment, a murine CDR is grafted into the framework region of a human antibody to prepare the "humanized antibody". See e.g., L. Riechmann et al. , Nature 332, 323 (1988); M. S. Neuberger et al. , Nature 314, 268 (1985) and European Patent EP A 0 239 400 (published September 30, 1987). In one embodiment of the present invention, the peptide that contains a CTLA-4 binding region is a functionally active antibody fragment. Significantly, as is known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope. (see, in general, Clark, W. R. (1986) The Experimental Foundations of Modern Immunology, Wiley & Sons, Inc., New York Roitt, I. (1991) Essential Immunology, 7th edition, Blackwell Scientific Publications, Oxford). The pFc 'and Fe regions of the antibody, for example, are effectors of the complement cascade, but do not participate in binding with the antigen. An antibody from which the pFc 'regions have been enzymatically detached, or which has been produced without the pFc' region, then designated as fragment (ab ') 2 / retains both binding sites with the antigen of an intact antibody. An isolated fragment of F (abf) 2 is referred to as a divalent monoclonal fragment due to its two binding sites with the antigen. Similarly, an antibody from which the Fe region has been enzymatically removed, or which has been produced without the Fe region, designated as Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Further widening, the Fab fragments consist of a light chain of covalently bound antibody and a portion of the heavy chain of the antibody denoted as Fd (heavy chain variable region). The Fd fragments are the main determinant of the specificity of the antibody (a single Fd fragment can be associated with up to ten different light chains, without altering the specificity of the antibody) and the Fd fragments retain the ability to bind with their epitope when isolated. The terms Fab, Fe, pFc ', F (ab') 2 and Fv are used with their immunological meanings [Klein, Immunology (John Wiley, New York, N. Y. 1982); Clark, W.

R. (1986) The Experimental Foundations of Modern Immunology (Wiley &Sons, Inc., New York); Roitt, I. (1991) Essential Immunology, 7th edition, (Blackwell Scientific Publications, Oxford)]. As used herein, the term "functionally active antibody fragment" means a fragment of an antibody molecule that includes a CTLA-4 binding region of the present invention, which retains the functionality of stimulating the T cells of an intact antibody that has the same specificity than that of deposited monoclonal antibodies. Such fragments are also known in the art and are used regularly both in vi tro and in vivo. In particular, known functionally active antibody fragments include, but are not limited to, F (ab) 2 / Fab, Fv and Fd fragments of antibodies. These fragments, which lack the Fe fragment of the intact antibody, are cleared more rapidly from the bloodstream and may have fewer non-specific binding to tissues than the intact antibodies (Wahl et al., J. Nucí, Med., 24: 316-325). (1983)). For example, single chain antibodies can be constructed in accordance with the methods described in US Patent No. 4,946,778 to Ladner et al. Such single chain antibodies include the variable regions of the light and heavy chains linked by a flexible linking portion. Methods for obtaining an antibody with a single domain ("Fd") which comprises a single isolated variable heavy chain domain have also been reported (see for example Ward et al., Nature, 341: 644-646 (1989), describing a screening method for identifying an antibody heavy chain variable region (antibody with a single VH domain) with sufficient affinity for its target epitope to bind to it in isolation. Methods for preparing fragments are known and described in the art. Recombinant Fv based on sequences of light chain variable regions and known heavy chains of antibodies, eg, Moore et al., US Patent No. 4,462,334 Other references describing the use and generation of antibody fragments, include, for example, fragments Fab (Tijssen, Practice and Theory of Enzyme Immunoassays (Elsevieer, Amsterdam, 1985)), Fv fragments (Hochman et al., Biochemistry 12: 1130 (1973); Sharon et al. , Biochemistry 15: 1591 (1976); Ehrilch et al. , U.S. Patent No. 4,355,023) and portions of antibody molecules (Audilore-Hargreaves, U.S. Patent No. 4,470,925). Thus, those skilled in the art could construct fragments of antibodies from several portions of intact antibodies, without destroying the specificity of the antibodies for the CTLA-4 epitope. The functionally active antibody fragments also encompass the "humanized antibody fragments". As would be recognized by a person skilled in the art, such fragments could be prepared by a traditional enzymatic digestion of intact humanized antibodies. However, if intact antibodies are not susceptible to such degradation, due to the nature of the construction involved, the constructions can be prepared with immunoglobulin fragments used as raw materials; or, if recombinant techniques are used, the DNA sequences themselves can be conditioned to encode the desired "fragment" which, when expressed, can be combined in vivo or in vitro by chemical or biological means, to prepare the immunoglobulin fragment. intact final desired. Smaller antibody fragments and small binding polypeptides having binding specificity for CTLA-4 are also encompassed within the peptides of the present invention. Several routine assays can be used to easily identify such peptides. Selection tests to identify the peptides of the present invention, for example, are carried out using phage methods such as those described in Hart, et al. , J. Biol. Chem., 259: 12468 (1994). Hart et al. , report a filamentous phage library to identify new peptide ligands for mammalian cell receptors. In general, phage libraries that use, for example, phages M13 or fd, are prepared using conventional methods such as those described in the above references. The library inserts contain from 4 to 80 amino acid residues. The inserts optionally represent a completely degenerate or deviated arrangement of peptides. Ligands are obtained that selectively bind to CTLA-4 by selecting those phages that express on their surface a ligand that binds to CTLA-4. Then, these phages are subjected to several reselection cycles to identify the phages expressing the ligand peptide that have the most useful binding characteristics. Typically, phages that exhibit the best binding characteristics (eg, the highest affinity) are further characterized by nucleic acid analysis, to identify the particular amino acid sequences of the peptides used on the phage surface and the optimal length of the phage. peptide expressed to achieve an optimal binding with CTLA-4. Alternatively, such peptide ligands can be selected from peptide combining libraries containing one or more amino acids. Such libraries can be further processed to contain synthetic non-peptide portions that are less subject to enzymatic degradation compared to their naturally occurring counterparts. Additionally, small polypeptides including those containing the CDR3 region of CTLA-4 binding can be synthesized or produced by recombinant means to produce the peptide of the present invention. Such methods are known to those skilled in the art. Peptides can be synthesized, for example, using automatic peptide synthesizers which are commercially available. The peptides can be produced by recombinant techniques incorporating the DNA expressing the peptide into an expression vector and transforming cells with the expression vector to produce the peptide. The sequence of the CDR regions, for use in the synthesis of the peptides of the present invention, can be determined by methods known in the art. The variable region of the heavy chain is a peptide that generally varies from 100 to 150 amino acids in length. The variable region of the heavy chain is a peptide that generally varies from 80 to 130 amino acids in length. The CDR sequences within the variable regions of the heavy and light chains that include only about 3 to 25 amino acid sequences can easily be sequenced by a person skilled in the art. The peptides can even be synthesized by commercial sources such as Scripps protein and Nucleic Acids Core Sequencing Facility (La Jolla, California, USA). To determine whether a peptide binds to CTLA-4, any known binding assay can be used. For example, the peptide can be immobilized on a surface and then contacted with labeled CTLA-4. The amount of CTLA-4 that interacts with the peptide or the amount that does not bind to the peptide can subsequently be quantified to determine whether the peptide binds to CTLA-. A surface having the monoclonal antibody deposited immobilized therein can serve as a positive control. The selection of the peptides of the present invention can also be carried out using a competition assay. If the peptide to be tested competes with the deposited monoclonal antibody, as demonstrated by a decrease in the binding of the deposited monoclonal antibody, then it is likely that the peptide and the deposited monoclonal antibody bind to the same or a closely related epitope. . Still another way of determining whether the peptide has the specificity of the deposited monoclonal antibody of the present invention, is to pre-incubate the monoclonal antibody deposited with CTLA-4 with which it is normally reactive, and then add the peptide being tested to determine whether said Peptide is inhibited in its ability to bind to CTLA-4. If the peptide being tested is inhibited, then there is a possibility that it has the same epitope or a functional equivalent and the same specificity as the deposited monoclonal antibody. Using routine procedures known to those skilled in the art, it can be determined whether a peptide that binds to CTLA-4 is useful in accordance with the present invention, determining whether the peptide blocks the binding of CTLA-4 to B7 and co-stimulates the proliferation of T cells in an in vi tro assay. A peptide that co-stimulates the proliferation of T cells, is one that when added to a T cell results in a secondary signal of stimulation of T cell proliferation or the production of soluble immune mediators by the T cell, when the T cell is exposed to a primary signal, such as that caused by an antigen in the CMH context on the surface of a CPA. An example of a T cell proliferation assay is described in Walunas et al. , J. Exp. Med., Vol. 183, p. 2541-2550 (1996). Briefly, lymph node cells are isolated and enriched in T cells by passing them through a nylon wool column and the purity of T cells is assessed by flow cytometry using an anti-CDR3 monoclonal antibody. T cells are seeded at a density of 2 x 10 cells / well in the presence of 1 x B6 splenocytes free of erythrocytes, syngenically irradiated. Once the cells are prepared, the test conditions are established. A first condition of the assay includes incubating the mixture of T cells with 0.1 mg / ml of anti-CDR3 and 1.0 mg / ml of anti-CD28 for 72 hours at 37 ° C. The second condition of the assay includes incubating the mixture of T cells with 0.1 mg / ml of anti-CDR3 alone for 72 hours at 37 ° C. Each of the test conditions is mixed with control Ig (50 mg / ml) or with anti-CTLA-4 (50 mg / ml) (such as the deposited monoclonal antibody), or with the test peptide of the present invention (50 mg / ml). During the last 16 hours of incubation, the mixture is pressed with 1 mCi / well [H] thymidine. The samples are then counted in a scintillation counter and the proliferation of the cells is measured as a function of the [H] thymidine incorporated in the cells. Other assays will be apparent to those skilled in the art once having read the present disclosure, such assays being useful in determining whether a peptide that binds to CTLA-4 also co-stimulates the activation of T cells. By using the monoclonal antibody deposited from In the present invention, it is now possible to produce anti-idiotypic antibodies that can be used to select other antibodies to identify whether the antibody has the same binding specificity as the deposited monoclonal antibody of the present invention. In addition, such anti-idiotypic antibodies can be used for active immunization (Herlyn, et al., Science, 232: 100, 1986). Such anti-idiotypic antibodies can be produced using the known hybridoma techniques (Kohler and Milstein, Nature, 256: 495, 1975). An anti-idiotypic antibody is an antibody that recognizes unique determinants present in the deposited monoclonal antibody. These determinants are located in the hypervariable region of the antibody. It is this region that binds to a given epitope and, therefore, is responsible for the specificity of the antibody. An anti-idiotypic antibody can be prepared by immunizing an animal with the deposited monoclonal antibody. The immunized animal will recognize and respond to the idiotypic determinants of the deposited immunizing monoclonal antibody and will produce an antibody against these idiotypic determinants. By using the anti-idiotypic antibodies of the immunized animal, which are specific for the deposited monoclonal antibody of the present invention, it is possible to identify other clones with the same idiotype as the deposited monoclonal antibody used for immunization. The idiotypic identity between monoclonal antibodies from two cell lines demonstrates that the two monoclonal antibodies are the same with respect to their recognition of the same epitope determinant. Thus, using anti-idiotypic antibodies it is possible to identify other hybridomas that express monoclonal antibodies that have the same epitope specificity. It is also possible to use anti-idiotypic antibody technology to produce monoclonal antibodies that mimic an epitope. For example, an anti-idiotypic monoclonal antibody prepared against a first monoclonal antibody will have a binding domain in the hypervariable region which is the image of the epitope to which the first monoclonal antibody binds. Thus, the anti-idiotypic monoclonal antibody can be used for immunization, since the binding domain of the anti-idiotypic monoclonal antibody effectively acts as an antigen. Each of the compositions described above includes a peptide having a CTLA-4 binding region. As described above, the variable region of an antibody that includes the CDR3 region is responsible for the specificity of the antibody. The sequences responsible for the specificity of the deposited monoclonal antibody can be easily determined by a person skilled in the art, so that the peptides according to the present invention can be prepared using recombinant DNA technology. There are entities in the United States that perform this work commercially, such as Thomas Jefferson University and the Scripps Protein and Nucleic Acids Core Sequencing Facility (La Jolla, California, USA). For example, the variable region cDNA can be prepared by a polymerase catalyzed chain reaction from the deposited hybridoma RNA using degenerate or non-degenerate primers (derived from the amino acid sequence). The cDNA can be subcloned to produce sufficient quantities of double-stranded DNA for sequences by reactions or conventional sequencing equipment. These procedures are established in more detail in the appended examples. Once the nucleic acid sequences of the Fd region of the heavy chain and of the variable domains of the light chain of the deposited monoclonal antibody CTLA-4 have been determined, a person skilled in the art will be able to produce nucleic acids coding for this antibody or which code for the various antibody fragments, humanized antibodies or polypeptides such as those described above. It is contemplated that such nucleic acids will be operably linked with other nucleic acids, forming a recombinant vector for cloning or for the expression of the peptides of the present invention. The present invention includes any recombinant vector containing the coding sequences, or part thereof, either for transformation of prokaryotic or eukaryotic cells, for transfection or for gene therapy. Such vectors can be prepared using conventional molecular biology techniques known to those skilled in the art and will comprise DNA sequences coding for the CDR3 region and additional variable sequences that contribute to the specificity of the antibodies or parts thereof, as well as other non-specific peptide sequences and a suitable promoter, either with (Whittle et al., Protein Eng., 1: 499, 1987; and Burton et al., Science, 266: 1024-1027, 1994), or, without (Marasco et al., Proc. Nati, Acad. Sci. (USA), 90: 7889, 1993; and Duan et al. , Proc. Nati, Acad. Sci. (USA), 91: 5075-5079, 1994) a signal sequence for export or secretion. Such vectors can be transformed or transfected into prokaryotic cells (Huse et al., Science, 246: 1275, 1989, Ward et al., Nature, 341: 644-646, 1989, Marks et al., J. Mol. Biol. , 222: 581, 1991; and Barbas et al., Proc. Nati, Acad. Sci. (USA), 88: 7978, 991) or in eukaryotic cells (Whittle et al., 1987; and Burton et al., 1994). ) or can be used for gene therapy (Marasco et al., 1993; and Duan et al., 1994) by conventional techniques known to those skilled in the art. As used herein, the term "vector" can be any of a number of nucleic acids in which a desired sequence can be inserted by restriction and ligation, to be transported between different genetic environments or for expression in a host cell. Vectors are typically made up of DNA, although RNA vectors are also available. Vectors include, but are not limited to, plasmids and phagemids. A cloning vector is one that is capable of replicating in a host cell and which is further characterized by one or more restriction sites of endonucleases in which the vector can be cut in a certain way and in which a vector can be ligated. DNA sequence in such a way that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence can occur many times as the plasmid increases its number of copies within the host bacterium or only once for each host before the host reproduces by mitosis. In the case of phage, replication can occur actively during a lytic phase or passively during a lysogenic phase. An expression vector is one in which a desired DNA sequence can be inserted by restriction or ligation, such that it is operably linked to regulatory sequences and can be expressed as an RNA transcript. The vectors may additionally contain one or more marker sequences suitable for use in the identification of cells that have or have not been transformed or transfected with the vector. Markers include, for example, gene coding proteins that increase or decrease their resistance or sensitivity to antibiotics or other compounds, genes that code for enzymes whose activities are detectable by standard assays known in the art (eg, β-galactosidase or alkaline phosphatase) and genes that visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques. Preferred vectors are those capable of autonomous replication and of expressing structural gene products present in the DNA segments to which they are operably linked. The expression vectors of the present invention include regulatory sequences operably linked to a nucleotide sequence that codes for one of the peptides of the present invention. As used herein, the term "regulatory sequences" means nucleotide sequences that are necessary for, or that lead to, the transcription of a nucleotide sequence that encodes a desired polypeptide and / or that are necessary for, or that lead, the translation of the resulting transcript to obtain the desired polypeptide. Regulatory sequences include, but are not limited to, 5 'sequences such as operators, promoters and ribosome binding sequences; and 3 'sequences such as polyadenylation signals. The vectors of the present invention also optionally include 5 'leader sequences or signal sequences, 5' or 3 'sequences that code for fusion products to aid in the purification of proteins and various markers that aid in the identification or selection of transformants. . The selection and design of the appropriate vector is within the capabilities and discretion of those skilled in the art. the subsequent purification of the peptides can be carried out by any of a variety of standard means known in the art.

A preferred vector for the selection of peptides, but not necessarily preferred for the mass production of the peptides of the present invention, is a recombinant DNA molecule that contains a nucleotide sequence that codes for, and is capable of, expressing a fusion polypeptide containing, in the direction of the amino terminus towards the carboxyl terminus, (1) a prokaryotic secretion signal domain, (2) a polypeptide of the present invention and, optionally, (3) a fusion protein domain . The vector includes DNA regulatory sequences for expressing the fusion polypeptide, preferably prokaryotic regulatory sequences. Such vectors can be constructed by those skilled in the art and have been described by Smith et al. (Science, 228: 1315-1317, 1985), Clalkson et al. (Nat? Re, 352: 624-628, 1991); Kang et ai. (in "Methods: A Companion to Methods in Enzymology: Vol. 2"; R. A. Lerner and D. R. Burton, Academic Press, N. Y., pp. 111-118, 1991); Barbas et al. (Proc. Nati, Acad. Sci. (USA), 88: 7978-7982, 1991); Roberts et al. (Proc. Nati, Acad. Sci. (USA) 89: 2429-2433, 1992). A fusion polypeptide may be useful for the purification of the peptides of the present invention. The fusion domain, for example, can include a poly-His tail, which allows purification on Ni + columns or the maltose binding protein of the commercially available pMAL vector (New England BioLabs, Beverly, MA). A fusion domain currently preferred, but not necessarily necessary, is a filamentous phage membrane anchor. This domain is particularly useful for selecting phage libraries of monoclonal antibodies, but may be less useful for mass production of antibodies. The filamentous phage membrane anchor is preferably a domain of the cpIII or cpVIII coat protein, capable of associating with the matrix of a filamentous phage, thereby incorporating the fusion polypeptide on the phage surface, to make possible the solid binding of the phage to specific antigens or epitopes and, therefore, allow the enrichment and selection of specific antibodies or fragments encoded by the phagemid vector. The secretion signal is a leader peptide domain of a protein that targets the host cell's protein membrane, such as the periplasmic membrane of gram-negative bacteria. A preferred secretion signal for E. coli is a pelB secretion signal. The predicted sequences of amino acid residues of the secretion signal domain of two pelB gene producer variants of Erwinia carotova are described in Lei, et al. . { Nature, 381: 543-546, 1988). The leader sequence of the pelB protein has previously been used as a secretion signal for fusion proteins (Better et al., Science, 240: 1041-1043, 1988, Sastry et al., Proc. Nati. Acad. Sci (USA) , 86: 5728-5732, 1989, and Mullinax, et al., Proc. Nati, Acad. Sci. (USA), 87: 8095-8099, 1990). The amino acid residue sequences for other E. coli secretion signal polypeptide domains useful in the present invention can be found in Oliver, Neidhard, FC (ed), Escherichia coli and Salmonella Typhimurium, American Society for Microbiology, Washington , DC, 1: 56-69 (1987). To achieve high levels of gene expression in E. coli, it is necessary to use not only strong promoters to generate large amounts of mRNA, but also ribosome binding sites to ensure that the mRNA is efficiently translated. In E. coli, the ribosome binding site includes an initiation codon (AUG) and a sequence of 3 to 9 nucleotides of localized length from 3 to 11 nucleotides upstream of the initiation codon (Shine et al., Nature, 254: 34, 1975). Sequence AGGAGGU, which is called the Shine-Dalgarno sequence (SD), is complementary to the 3 'end of the 16S rRNA of E. coli. The binding of the ribosome to the mRNA and the sequence at the 3 'end of the mRNA can be affected by several factors: (i) The degree of complementarity between the sequence Fd and 3 'end of the 16S rRNA. (ii) The separation and possibly the DNA sequence that lies between the SD sequence and the AUG (Roberts et al., Proc. Nati. Acad. Sci. (USA), 76: 760, 1979a; Roberts et al., Proc. Nati Acad. Sci. (USA), 76: 5596, 1979b; Guarente et al., Science, 209: 1428, 1980; and Guarente et al., Cell, 20: 543, 1980). Optimization is achieved by measuring the level of expression of genes in plasmids in which this separation is systematically altered. The comparison of different mRNAs shows that there are statistically preferred sequences from the position -20 to position +13 (where A of the AUG is in position 0) (Gold et al., Annu Rev. Microbiol., 35: 365, 1981). The leader sequences have been shown to drastically influence translation (Roberts et al., 1979a, b, supra). (iii) The nucleotide sequence after the AUG, which affects ribosome binding (Tainiguchi et al., J. Mol. Biol., 118: 533, 1978). The 3 'regulatory sequences define at least one termination or stop codon in the framework, being operably linked to the heterologous fusion polypeptide. In preferred embodiments with a prokaryotic expression host, the vector used includes a prokaryotic origin of replication or replicon, ie, a DNA sequence that has the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in the cell prokaryotic host, such as a bacterial host cell, transformed therewith. Such origins of replication are known in the art. The preferred origins of replication are those that are efficient in the host organism. A preferred host cell is E. coli. To be used as a vector in E. coli, a preferred origin of replication is ColEl found in pBR322 and a variety of other common plasmids. Also preferred is the pl5A origin of replication found in pACYC and its derivatives. The ColEl and pl5A replicons have been used extensively in molecular biology, are available in a variety of plasmids and are described in Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989). In addition, those modalities that include a prokaryotic replicon, preferably also include a gene whose expression confers a selective advantage, such as drug resistance, to a host bacterium transformed therewith. The typical bacterial resistance genes to drugs are those that confer resistance to ampicillin, tetracycline, neomycin / kanamycin or chloramphenicol. Vectors typically also contain convenient restriction sites for the insertion of translatable DNA sequences. Examples of vectors are the plasmids pUC18 and pUC19 and derived vectors such as pcDNAII, available from Invitrogen (San Diego, CA, USA). When the peptide of the present invention is an antibody that includes both heavy chain and light chain sequences, these sequences can be encoded in separate vectors or, more conveniently, can be expressed by a single vector. The heavy chain and the light chain, after translation or after secretion, can form the heterodimeric structure of the natural antibody molecules. Such a heterodimeric antibody may or may not be stabilized by disulfide bridges between the heavy and light chains. A vector for the expression of heterodimeric antibodies such as the intact antibodies of the present invention or the F (ab ') 2 antibody fragments., Fab or Fv of the present invention, is a recombinant DNA molecule adapted to receive and express a first and second translatable DNA sequences. That is, a DNA expression vector for expressing a heterodimeric antibody provides a system for independently cloning (inserting) the two translatable DNA sequences into two separate cartridges present in the vector, to form two separate cistrons for the expression of the first and second polypeptides of a heterodimeric antibody. The expression vector of DNA to express two cistrons is called dicistronic expression vector. Preferably, the vector comprises a first cartridge that includes upstream and downstream DNA regulatory sequences operably linked through a sequence of nucleotides adapted for directional attachment to a DNA insert. The upstream translatable sequence preferably codes for the secretion signal such as that described above. The cartridge includes DNA regulatory sequences for expressing the first antibody polypeptide that is produced when a translatable DNA sequence insert (DNA insert) is directionally inserted into the cartridge through the sequence of nucleotides adapted for directional linkage. The dicistronic expression vector also contains a second cartridge for expressing the second antibody polypeptide. The second cartridge includes a second translatable DNA sequence that preferably codes for a secretion signal such as that described above, operably linked at its 3 'end through a sequence of nucleotides adapted for directional attachment to a DNA sequence downstream of the vector that typically defines at least one stop codon in the cartridge reading frame. The second translatable DNA sequence is operably linked at its 5 'end to DNA regulatory sequences that form the 5' elements. The second cartridge is capable, after inserting a translatable DNA sequence (DNA insert), of expressing the second fusion polypeptide comprising a secretion signal with a polypeptide encoded by the DNA insert. The peptides of the present invention can also, of course, be produced by eukaryotic cells such as CHO cells, human hybridomas, immortalized B-lymphoblastoid cells and the like. In this case, a vector is constructed in which the eukaryotic regulatory sequences are operably linked to the nucleotide sequences encoding the peptide. The design and selection of an appropriate eukaryotic vector is within the capabilities and discretion of those skilled in the art. The subsequent purification of the peptides can be carried out by any of a variety of standard methods known in the art. In another embodiment, the present invention provides host cells, both prokaryotic and eukaryotic, transformed or transfected with, and therefore including, the vectors of the present invention. As used herein with respect to nucleic acids, the term "isolated" means: (i) amplified in vi tro, for example by a polymerase chain reaction (PCR); (ii) produced recombinantly by cloning; (iii) purified, for example by gel separation and separation; or (iv) synthesized, for example, by chemical synthesis. An isolated nucleic acid is one that is easily manipulated by recombinant DNA techniques known in the art. Thus, a nucleotide sequence contained in a vector of which 5 'and 3' restriction sites are known or for which primer sequences have been described for a polymerase chain reaction (PCR), is considered isolated; but a nucleic acid sequence existing in its native state in its natural host is not considered isolated. An isolated nucleic acid can be substantially purified, but does not need to be. For example, a nucleic acid that is isolated within a cloning or expression vector is not pure, since it can comprise only a small percentage of the material of the cell in which it resides. However, such nucleic acid is isolated according to the term used in the present invention, because it is easily manipulated by standard techniques known to those skilled in the art. As used herein, a coding sequence is said and the regulatory sequences are "operably linked" when they are covalently linked in such a way that the expression or transcription of the coding sequence is placed under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably linked if the induction of a promoter in the 5 'regulatory sequences results in the treinscription of the coding sequence and if the of the junction between the two non-(1) DNA sequences results in the introduction of a frame change mutation, (2) interferes with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interferes with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably linked to a coding sequence if the promoter region were capable of transcribing the DNA sequence such that the resulting transcript could be translated into the desired protein or polypeptide. The precise nature of the regulatory sequences necessary for gene expression may vary between species or cell types, but in general will include, as necessary, 5 'non-transcription and non-translational sequences involved with the initiation of transcription and translation, respectively, such as a TATA box, a capping sequence, a CAAT sequence and the like. Especially, such non-transcriptional regulatory sequences will include a promoter region that includes a promoter sequence for the transcriptional control of the operably linked gene. Regulatory sequences may also include enhancer sequences or upstream activating sequences, as desired. The peptides can also be used immunotherapeutically for disorders sensitive to the proliferation of T cells in humans. The term "immunotherapeutically" or "immunotherapy", as used herein, in conjunction with the monoclonal antibodies of the present invention, denotes both prophylactic administration and therapeutic administration. Thus, the peptides can be administered to high-risk subjects in order to decrease the likelihood and / or the severity of a disease sensitive to T-cell proliferation, such as a tumor, or it can be administered to subjects who already have Evidence of tumors. In one aspect, the present invention encompasses a method for stimulating T cell proliferation in si tu. The method includes the step of adding a peptide of the present invention to a population of cells that includes T cells and antigen-presenting cells. As used in this, the term "T cells" refers to T cells expressing CTLA-4 and CD28 on the surface thereof and the term "antigen-presenting cells" refers to any immune cell that expresses B7 and that presents an antigen on the CMH context on the surface. When a peptide of the present invention is added to a population of T cells and antigen-presenting cells, the peptide interacts with CTLA-4, preventing it from interacting with B7 on the surface of the antigen presenting cell. Then, the antigen presenting cell is free to interact with the T cell to generate a primary and secondary signal that results in the proliferation of T cells. By definition, the word "in situ" encompasses and includes the terms ^ in vivo " , "ex-vivo" and "in vi tro." The compositions of the present invention are useful for many in vi tro purposes, For example, the compositions of the present invention are useful for screening compounds that inhibit T cell proliferation. Such a screening assay can be performed in vitro by preparing cell proliferation assays including a peptide of the present invention and a cell population that includes T cells and antigen presenting cells, Potential inhibitors of T cell proliferation can be added to the mix and the effect on proliferation can be measured Other in vi tro uses, such as research purposes, are also known for the technicians in the matter. The ex-vivo uses will also be easily identified by the technicians in the matter. Ex vivo uses include, for example, the stimulation of the proliferation of T cells that have been removed from a human subject and that are subsequently returned to the body of the human subject. The ex-vivo uses are useful when other ex vivo processes are being performed in a subject and when it is desirable to separate the T cells from the other components of the blood prior to manipulation. The present invention also includes a method for the treatment of a disorder responsive to T cell proliferation. The method includes the step of administering a peptide of the present invention to a subject suffering from a disorder responsive to T cell proliferation, in an amount effective to increase the proliferation of T cells. As used herein, the term "T cell proliferation sensitive disorder" is any disorder associated with the adverse physiological consequences in which an enhancement of function of the immune cells, characterized by an increase in the proliferation of T cells, results in an improvement of the adverse physiological consequences. Disorders sensitive to T cell proliferation include disorders of the immune system, such as immunodeficiency, as well as disorders involving undesirable cell invasion by microorganisms or the growth of undesirable cells such as tumors. The peptide of the present invention is a secondary costimulatory signal that requires a primary co-stimulatory event to initiate the proliferation of T cells. The primary co-stimulatory event may be the interaction of a T cell with an antigen in the context of MHC. When a T cell has been exposed to an antigen in the context of MHC, it is not necessary to add a primary costimulatory signal when the secondary costimulatory signal is added in order to stimulate cell proliferation. For example when the peptide of the present invention is administered to a human subject infected with a particular antigen or expressing a particular antigen in a tumor cell, such that the antigen is incorporated and expressed on the surface of the circulating CPAs, it is not necessary to administer a primary costimulatory signal in order to stimulate the proliferation of T cells. The antigen displayed on the surface of circulating CPAs of the subject, it works as a primary costimulatory signal. When the antigen is not expressed on the surface of circulating CPAs, a primary costimulatory signal can be administered in conjunction with the secondary costimulatory signal (the peptide of the present invention). For example, an antigen may not be expressed on the surface of circulating CPAs of a subject when said subject has not been exposed to a particular infectious agent. It may be desirable to stimulate the proliferation of T cells in such a subject when the subject may be exposed to the infectious agent in the future. The stimulation of the proliferation of T cells under such conditions intensifies the immunity of the subject against the particular infectious agent when said subject is exposed to the agent in the future. The compositions of the present invention are administered in therapeutically effective amounts. As used herein, the term "effective amount" of the peptide of the present invention is a dose that is sufficient to inhibit B7 binding to CTLA-4 to a degree at which the proliferation of the B7 is co-stimulated. T-cells. Stimulation of T-cell proliferation is sufficient to produce the desired effect in which the symptoms associated with the disorder responsive to T-cell proliferation improve or decrease. Preferably an effective amount of the peptide is an amount effective to promote proliferation of T cells in vivo. In general, a therapeutically effective amount may vary according to the age, condition and sex of the patient, as well as the degree of the patient's disease, and may be determined by a person skilled in the art. The dosage can be adjusted by the individual doctor in case of any complication. A therapeutically effective amount will typically range from about 0.01 to about 500 mg / kg, typically from about 0.1 to about 200 mg / kg and often from about 0.2 to about 20 mg / kg, in one or more daily administrations, over or several days (depending on the course or mode of administration and the factors described above). A person skilled in the art can determine what is an effective amount of a peptide by selecting the ability thereof to inhibit the activation of T cell proliferation in an in vi tro assay. The effectiveness of the peptide can be defined in terms of the ability thereof to inhibit the proliferation of T cells. An exemplary assay for measuring the ability of a putative peptide of the present invention to inhibit T cell proliferation is provided in Examples and described above. The exemplary assay is predictive of the ability of a peptide to co-stimulate T cell proliferation in vivo and, therefore, can be used to screen peptides for therapeutic applications. Proliferation assays measure the ability of a peptide to co-stimulate T cell proliferation. In accordance with the methods of the present invention, the peptide can be administered in a pharmaceutically acceptable composition. In general, pharmaceutically acceptable carriers for monoclonal antibodies, antibody fragments and peptides are known to those skilled in the art. As used herein, the term "pharmaceutically acceptable carrier" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients, ie, the ability of the peptide to co-stimulate the activation of T cells. Pharmaceutically acceptable carriers include diluents, fillers, salts, buffer solutions, stabilizers, solubilizers and other materials that are known in the art. Exemplary pharmaceutically acceptable carriers for particular peptides are described in U.S. Patent No. 5,211,657. The peptides of the present invention can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, deposited implants, inhalers and injections; and the normal forms of administration are oral, parenteral or surgical. The present invention also encompasses pharmaceutical compositions that are formulated for local administration, such as implants. In accordance with the methods of the present invention, the peptides can be administered by injection, by gradual infusion over time or by any other medically acceptable method. The administration, for example, can be intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous or transdermal. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable organic esters such as ethyl oleate. Aqueous vehicles include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and media with regulated pH. Parenteral vehicles include sodium chloride solution, dextrose Ringer's solution, dextrose and sodium chloride solution, lactated Ringer's solution or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on dextrose Ringer's solution), and the like. Preservatives and other additives such as, for example, antimicrobial agents, antioxidants, chelants and inert gases and the like may also be present. Those skilled in the art will readily be able to determine the various parameters for preparing these alternative pharmaceutical compositions, without resorting to further experimentation.

The methods of the present invention also encompass the step of administering the peptides of the present invention in conjunction with conventional therapies for the treatment of the disorder responsive to underlying T-cell proliferation. For example, the method of the present invention can be practiced simultaneously with conventional treatment. Particular conventional treatment depends on the course and nature of the disorder responsive to T cell proliferation. When for example the disorder sensitive to T cell proliferation is, a tumor, a conventional mode of treatment is chemotherapy. The peptides of the present invention can be administered in conjunction with chemotherapy in the treatment of tumors, in order to intensify the tumoricidal effects. The following Examples are provided to illustrate specific cases of the practice of the present invention and it should not be considered that the present invention is limited to these Examples. As will be apparent to those skilled in the art, the present invention will find applications in a variety of compositions and methods.

EXAMPLES Example 1: Preparation of hybridomas producing an i-CTLA-4 monoclonal antibodies 1. Immunization An intraperitoneal injection of 10 activated human T-cell clones in Dulbecco's phosphate-buffered saline was applied as an immunization, and then emulsified with an equal volume of Freund's complete adjuvant (Sigma Chemical Co., St. Louis, MO, USA) in female Balb / c mice (Jackson Laboratories, Bar Habor, ME, USA). The mice were administered two intraperitoneal booster immunizations with 10 clones of activated human T cells suspended in phosphate-regulated Dulbecco's saline solution (GIBCO, Grand Island, NY, USA) and emulsified with an equal volume of incomplete Freund's adjuvant ( Sigma Chemical Co., St. Louis, MO, USA) at intervals of 14 days after the initial immunization. 2. Selection of Mice by Flow Cytometry The presence of antibodies against surface proteins in the clones of T cells was assayed by flow cytometry. Ten days after the third and last immunization, a small amount of blood was taken by retro-orbital bleeding of each mouse and allowed to clot. Activated T cell clones were grown in tissue culture flasks and collected and washed thoroughly (3X) with Dulbecco's saline solution regulated with phosphates with 1% bovine serum albumin (1% BSA solution). A 1: 1000 dilution of each of the serum samples collected from the immunized mice (50 μl) was added to 10 clones of T cells, mixed well and incubated for 30 minutes at 4 ° C. After incubation, the cells were washed 3X with 1% BSA solution, then incubated for 30 minutes in 50 μl of an antibody detector (goat serum with mouse immunoglobulin, conjugated with fluorescein isothiocyanate; Zy ed Laboratories, San Francisco, CA, USA) diluted 1:40 in 1% BSA solution. Cells were washed 3X with 1% BSA solution, then fixed with 1% parafo-maldehyde solution (paraformaldehyde dissolved in phosphate-regulated Dulbecco's saline solution, Sigma Chemical Co., St. Louis, MO). Afterwards, the samples were analyzed in a FACSort flow cytometer (Becton-Dickinson, San José, CA, USA). The mouse sera exhibiting the highest degree of fluorescence in the T cell clones were selected for cell fusion to create the monoclonal antibodies. 3. Preparation of Hybridomas After the mouse with the best antibody titer against the T cell clones was selected, it was allowed to stand for a total of 4 weeks after the last immunization. A 7 booster with 10 T-cell clones was then applied by intraperitoneal injection in Dulbecco's phosphate-buffered saline. Four days later, the mouse was sacrificed by cervical dislocation and the spleen was removed, ground to form a cell suspension and washed with phosphate-regulated Dulbecco's saline. Then, the spleen cells were counted and mixed with SP 2/0 myeloma cells (ATCC accession number CRL8006, Rockville, MD) that were unable to secrete heavy or light chains of immunoglobulin (Kearney et al., Journal of Immunology, 1979, 123: 1548) at a ratio of 2: 1 (spleen cells / myeloma cells) and then fused using polyethylene glycol 1450 (ATCC, Rockville, MD) in accordance with the standard procedure developed by Kohler and Miltein (Nature, 1975, 256: 495) on eight tissue culture plates of 96 wells, in selective HAT medium. Between 10 and 21 days after the fusion, the hybridoma colonies were visible and were selected by flow cytometry using T cell clones, in the manner previously described. All the hybridoma colonies that gave a positive response with the T-cell clones were propagated in 24-well cultures and subcloned by limiting dilution to produce cell lines producing monoclonal antibodies. In this point, an additional selection was made with the hybridomas to identify which hybridoma produced an anti-CTLA-4 antibody. The culture media from which hybridoma cultures were harvested (supernatants) were subjected to selection by ELISA (enzyme-linked immunosorbent assay) with a panel of known T cell surface antigens, including CTLA-4. The ELISA test was performed by coating 96-well EIA plates with high protein binding (Costar, Cambridge, MA) with 50 μl / well of a 1 μg / ml solution (0.02 μg / well) of human Ig-CTLA-4 , which has the extracellular binding portion of CTLA-4 coupled to an Fe portion of an immunoglobulin (Repligen Corporation, Cambridge, MA) dissolved in phosphate-regulated Dulbecco's saline, pH 7.2 (PBS) overnight at 4 ° C. The Ig-CTLA-4 was aspirated from the plates and the plates washed well with PBS (3X). Then, the plates were blocked with 1% BSA solution for 1 hour at room temperature, to inhibit non-specific binding. After the plates were sufficiently blocked, the BSA solution was removed and 50 μl / well of hybridoma supernatant was added. The plates were incubated for 45 minutes at 37 ° C and then washed 3X with PBS. Then a detector antibody was diluted (goat serum with mouse Ig, conjugated with horseradish peroxidase); Zymed Laboratories, San Francisco, CA, USA) 1: 400 in PBS and 50 μl was added to each well for 45 minutes at 37 ° C. The plates were then washed again with PBS, followed by the addition of 50 μl / well of 1 mM ABTS in a 0.1 M sodium citrate solution, pH 4.2 (2, 2-azino-bis-3-ethylbenzthiazolin-6-acid). sulfonic, Zymed Laboratories, San Francisco, CA, USA) to which a 1: 1000 dilution of 30% hydrogen peroxide had been added, for 30 minutes at room temperature in the dark. Samples that had hybridoma supernatants containing antibodies against human CTLA-4, were highlighted in a green color, which is formed as peroxidase reacts with ABTS and hydrogen peroxide. The intensity of the green color (absorbance at 405 nm) was evaluated in a Bio-Rad microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). Two mouse monoclonal antibodies against human CTLA-4 were identified, which were identified as A3.4H2 and A3.6B10.

Example 2: Analysis of anti-CTLA-4 monoclonal antibodies Monoclonal antibodies were examined for antibody subclass using an ISOstrip Kit or Kit (Boehringer Mannheim, Indianapolis, IN, USA). 5 μl of hybridoma supernatant was diluted in PBS and drained in a test tube containing blue latex beads bound to antibodies against murine Ig. Then an isotyping strip was placed in each tube and the bead / antibody solution rises through the strip by capillary action until the solution passes a line of antibodies containing specific antibodies for the different isotypes. A blue line appeared in the area of the strip for each isotype detected in the supernatant of the hybridoma. It was found that both the A3.4H2 and the A3.6B10 antibodies are from subclass IgG2a, with kappa light chains. Subclones of the hybridomas were analyzed by ELISA and flow cytometry using a CHO cell line transfected with CTLA-4 (obtained from Dr. Gordon Freeman through Repligen Corporation, Cambridge, MA). The antibodies were also tested for their ability to block the binding of B7.1 and B7.2 proteins, which are the natural ligands for the CTLA-4 receptor, to CTLA-4. Both A3.4H2 and A3.6B10 were able to block the binding of ligand B7 (Table 1) Table 1 Example 3: Separation and sequencing of the heavy and light chains of the monoclonal antibody an i-CTLA-4 The antibody can be isolated from the hybridomas and purified by any method known in the art. At least two methods can be used to separate the heavy and light chains from the purified antibody to determine the sequence. The first method employs polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE), followed by electroblotting on a polyvinyl difluoride membrane (DFPV). Briefly, the purified antibody is subjected to gel electrophoresis with SDS after a reduction with 2-mercaptoethanol. The resolved heavy and light chains are subsequently transferred to a membrane such as an IMMOBILON® membrane (a DFPV membrane from Millipore, Bedford, Mass.) Using the Matsudaira immunoblot method [J. Biol. Chem., 261: 10035 (1987)]. The bands corresponding to the heavy and light chains, which are identified with Coomassie brilliant blue staining, can be cut from the membrane and processed for N-terminal sequencing. A second, more complicated method allows solution isolation of larger amounts of heavy and light chains. The method includes a dialysis step in which the purified antibody is dialyzed against 0.1 M Tris-HCl, 1 mM EDTA, pH 8.0, at 4 ° C and then subjected to oxidative sulfitolysis in NaS? 3 Na2S2O, essentially in the manner described by Morehead et ai. [Biochemistry 23: 2500 (1984)]. After sulfitolysis, the antibody preparation is dialyzed against freeze-dried 1 M acetic acid to dryness, reconstituted in 1 M acetic acid, and subjected to gel filtration on a 1 x 30 cm SEPHADEX G-75® column, in acid 1 M acetic acid. The purity of the heavy and light chains after this stage can be evaluated by analytical SDS-SDA and then concentrated for sequencing. N-terminal amino acid sequencing can be performed using any commercial amino acid sequencer, such as the protein-peptide sequencer from Applied Biosystems model 477A. The analysis of the isolated chains is carried out following the instructions of the sequencer manufacturer. Example: Oligonucleotide primer design and cloning of anti-CTLA-4 monoclonal antibody 1. Oligonucleotide Preparation Based on the information obtained from the above amino acid sequencing analyzes, degenerate oligonucleotide primers can be designed for use in PCR. Other non-degenerate primers can be designed based on the nucleotide sequence information obtained after a PCR amplification of the cDNA encoding the complete heavy and light chains (described below). Oligonucleotide primers are synthesized by normal methods using a commercially available sequencer, such as the Applied Biosystems Model 380B synthesizer. Alternatively, PCR amplification of light chains and fragments of IgGi Fd heavy chains can be performed using the families of variable genes of individual light and heavy chains, and primers of the constant 3 'region for IgG ?, K or 1, as described previously (Kang et al., "Methods, A Companion to Methods in Enzymology: Vol. 2", RA Lerner and DR Burton, ed. Academic Press, NY, pp. 111-118, 1991). Such primers can be obtained commercially in companies such as Operon (Alameda, CA). The primers may contain restriction enzyme sites to allow binding of Fd sequences and light chain libraries for various other recombinant uses in a phage vector. 2. PCR Amplification of Heavy and Light Chains Total cytoplasmic RNA can be isolated from the hybridoma cell lines by any method known in the art, for example by lysing the cells in a lithic buffer consisting of 10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 2 mM MgCl2 and 0.5% Nonidot P40, followed by a centrifugation to separate the nuclear and cytoplasmic fractions. The nuclear pellet is discarded and the supernatant liquid is mixed with an equal volume of a solution containing 200 mM NaCl, 10 mM Tris-HCl, pH 7.4, 20 mM ethylenediaminetetraacetate (EDTA) and 2% sodium dodecylsulfate (SDS). The mixture is subjected to extraction once with an equal volume of phenol regulated with Tris / chloroform (1: 1) and once with chloroform. After extraction, the mixture is precipitated with sodium acetate and absolute ethanol at -20 ° C. The method can be performed with commercially available kits or packages such as that sold by Stratagene, La Jolla, CA.

The first strand of cDNA can be synthesized directly from the total cytoplasmic RNA at 37 ° C for 90 minutes, using a reverse transcriptase in a reaction buffer, such as that sold by Gibco-BRL, Grand Island, NY. Then, amplifications can be performed by polymerase chain reaction (PCR) using a Technc programmable thermal cycler or similar equipment. The PCR reaction mixture usually consists of 10 μl of the first strand of the cDNA reaction mixture, 53.5 ml of distilled H2O, 10 μl of Taq 10X polymerase reaction buffer (500 mM KC1, 100 Tris-HCl mM, pH 8.3, 15 mM MgCl2, gelatin 0. 1%), 16 μl of 1.25 mM dNTP mixture (DATP, TTP, dCTP, dGTP), 5 μl of each primer of interest (20 pmol / μl) and 5 μl of DNA polymerase. After PCR, the DNA mixtures are subjected to electrophoresis in agarose gels containing ethidium bromide. The fragments of CPR of interest can subsequently be cut from the gels and purified by electroelusion. 3. Subcloning and DNA Sequencing The gel-purified PCR fragments are digested with restriction enzymes such as Not I and Spel and then ligated to an appropriate vector such as the Bluescript plasmid vector digested with Not I / Spel. Competent cells such as E. coli strain DH5-alpha (Max Efficiency) are transformed with the ligated mixture or introduced into a phagemid library for subsequent recombination procedures. Double-stranded plasmid DNA is purified by any technique known in the art to purify the DNA, such as the Qiagen plasmid maxiprep kit or kit (Qiagen, Chatsworth, CA). Subsequently sequencing is performed on an automatic DNA sequencer (e.g., Applied Biosystems, Inc. (ABI), Foster City, CA), using a cyclic sequencing kit or kit with Taq dideoxy fluorescent terminator (ABI). The sequences derived from heavy chain Fd fragments and the light chains are subsequently aligned using a MacVector and the Genbank database (International Biotechnologies Inc., New Haven, CT). Deposits Hybridomas A3.4H2 and A3.6B10 were deposited on March 21, 1997 in the North American collection of Type Crops (being its acronym in English ATCC), with access numbers HB-12319 and HB-12318, respectively. The Assignee's transferee, Brigham and Women's Hospital, represents that the ATCC is a depositary that is responsible for the permanence of the deposit and rapid accessibility to it by the public if a patent is granted. All restrictions on the availability to the public of the material thus deposited will be irrevocably removed upon the granting of a patent. The material will be available as long as the patent application lasts, determined by the Commissioner in charge, under 37 CFR 1.14 (37 Code of Federal Regulations) and 35 U.S.C. 122 (35 Code of the United States). The deposited material will be maintained with all necessary care to keep it viable and without contamination for a period of at least five years after the most recent requirement of a sample of the deposited hybridoma and, in any case, for a period of at least thirty (30 ) years after the date of deposit or throughout the term of the patent, whichever period is the longest. The transferee of the applicants recognizes their duty to replace the deposit if the depositary is unable to provide a sample when it is required due to the conditions of the deposit. The application written here should be considered as sufficient to enable a person skilled in the art to practice the invention. The present invention is not limited in scope by the deposited cell lines, since the deposited modality is understood as a single illustration of an aspect of the present invention and any functionally equivalent cell line is within the scope of the present invention. In a similar way, the particular antibodies and peptides described herein are not to be considered as limiting the invention, as they are understood only as illustrative of particular embodiments of the present invention. Therefore, any cell line, antibody and peptide that is functionally equivalent to those described herein, is within the spirit and scope of the claims of the present invention. In fact, various modifications of the invention will be apparent to those skilled in the art in addition to those shown and described herein, after reading the description, and falling within the scope of the appended claims. All references, patents and patent publications cited in this application are incorporated by reference in their entirety. It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects to which it relates.

Claims (30)

1. CLAIMS Having described the invention as an antecedent, the content of the following claims is claimed as property: 1. A composition characterized by an isolated peptide that selectively binds to human CTLA-4 (acronym for cytotoxic T lymphocyte number 4, in English) and which co-stimulates the proliferation of T cells.
2. 2. The composition according to claim 1, characterized in that the isolated peptide has a CDR3 region binding to CTLA-4 or a functional variant thereof.
3. 3. The composition according to claim 2, characterized in that the CDR3 region of binding to CTLA-4 is a CDR3A3.4H2 of a monoclonal antibody produced by the hybridoma A3.4H2, deposited in the ATCC with accession number HB- 12319 4. The composition according to claim 2, characterized in that the CDR3 region of binding to CTLA-4 is a CDR3.R.6BIO of a monoclonal antibody produced by the hybridoma A3.6B10, deposited in the ATCC with the access number HB-12318. 5. The composition according to claim 1, characterized in that the isolated peptide is an intact soluble monoclonal antibody. 6. The composition according to claim 1, characterized in that the isolated peptide is a monoclonal antibody A3.4H2 produced by the hybridoma cell line deposited with the ATCC with the accession number HB-12319. The composition according to claim 1, characterized in that the isolated peptide is a monoclonal antibody A3.6Bio produced by the hybridoma cell line deposited with the ATCC with the accession number HB-12318. 8. The composition according to claim 1, characterized in that the isolated peptide is a humanized monoclonal antibody. The composition according to claim 1, characterized in that the isolated peptide is a monoclonal antibody fragment that is selected from the group consisting of an F (ab ') 2 fragment / an Fd fragment and a Fab fragment. The composition according to claim 1, characterized in that the isolated peptide has a light chain CDR2 region which is selected from the group consisting of a CDR2 3. H2 of a monoclonal antibody produced by the hybridoma A3.4H2, deposited in the ATCC with the accession number HB-12319 and a CDR2A3.6BIO of a monoclonal antibody produced by the hybridoma A3.6B10, deposited in the ATCC with the accession number HB-12318. The composition according to claim 1, characterized in that the isolated peptide has a light chain CDR1 region which is selected from the group consisting of a CDR1A3. H2 of a monoclonal antibody produced by the hybridoma A3.4H2, deposited in the ATCC with the accession number HB-12319 and a CDR1.R3.6BIO of a monoclonal antibody produced by the hybridoma A3.6B10, deposited in the ATCC with the access number HB-12318. 12. A hybridoma cell line deposited with the ATCC with accession number HB-12319. 13. A hybridoma cell line deposited with the ATCC with accession number HB-12318. 14. A pharmaceutical composition for the treatment of a disorder responsive to human T-cell proliferation, characterized in that it comprises: an effective amount for the treatment of the human T cell proliferation-sensitive disorder of an isolated peptide that selectively binds to the CTLA -4 and which co-stimulates the proliferation of T cells; and a pharmaceutically acceptable vehicle. 15. The composition according to claim 14, characterized in that the isolated peptide has a CDR3 region of binding to CTLA-4 or a functional variant thereof. 16. The composition according to claim 15, characterized in that the CDR3 region of binding to CTLA-4 is a CDR3A3.4H2 of a monoclonal antibody produced by the hybridoma A3.4H2, deposited in the ATCC with accession number HB- 12319 17. The composition according to claim 15, characterized in that the CDR3 binding region to CTLA-4 is a CDR3.R3.6BIO of a monoclonal antibody produced by the hybridoma A3.6B10, deposited in the ATCC with the accession number HB-12319. 18. The composition according to claim 14, characterized in that the isolated peptide is selected from the group consisting of an intact soluble monoclonal antibody and a functionally active monoclonal antibody fragment. 19. The composition according to claim 14, characterized in that the isolated peptide is a humanized antibody. 20. The composition according to claim 14, characterized in that the isolated peptide is a monoclonal antibody.R3.4H2 produced by the hybridoma cell line deposited with the ATCC with the accession number HB-12319. 21. The composition according to claim 14, characterized in that the isolated peptide is a monoclonal antibody. R3.6Bio produced by the hybridoma cell line deposited with the ATCC with the accession number HB-12318. 22. The composition according to claim 14, characterized in that the isolated peptide is a monoclonal antibody fragment that is selected from the group consisting of an F (ab ') 2 fragment, an Fd fragment and a Fab fragment. 23. The composition according to claim 14, characterized in that the isolated peptide has a light chain CDR2 region that is selected from the group consisting of a CDR2.A3.4H2 of a monoclonal antibody produced by the hybridoma A3.4H2 deposited in the ATCC with the accession number HB-12319 and a CDR2A3.6BIO of a monoclonal antibody produced by the hybridoma A3.6B10, deposited in the ATCC with the accession number HB-12318. The composition according to claim 14, characterized in that the isolated peptide has a light chain CDR1 region that is selected from the group consisting of a CDR1A3.4H2 of a monoclonal antibody produced by the hybridoma A3.4H2 deposited with the ATCC with the accession number HB-12319 and a CDR1A3.6BIO of a monoclonal antibody produced by the hybridoma A3.6B10, deposited in the ATCC with the accession number HB-12318. 25. A method for stimulating T-cell proliferation, characterized in that it comprises adding an isolated peptide that binds human CTLA-4 to a population of cells that includes T cells and antigen-presenting cells, itself. 26. The method according to claim 25, characterized in that the isolated peptide has a CDR3 region binding to CTLA-4 or a functional variant thereof. 27. A method for the treatment of a disorder responsive to T cell proliferation, characterized in that it comprises administering to an individual suffering from a disorder responsive to T cell proliferation, an isolated peptide that binds to human CTLA-4 in an effective amount for increasing the proliferation of T cells. The method according to claim 27, characterized in that the isolated peptide has a CDR3 region of binding to CTLA-4 or a functional variant thereof. 29. The method according to claim 27, characterized in that the disorder sensitive to the proliferation of T cells is a tumor. 30. The method according to claim 27, characterized in that the disorder sensitive to T cell proliferation is an immunodeficiency disease.