MXPA99004296A - Identification of unique binding interactions between certain antibodies and the human b7.1 and b7.2 co-stimulatory antigens - Google Patents

Abstract

The present invention relates to the identification of antibodies which are specific to human B7.1 antigen (CD80) and which are capable of inhibiting the binding of B7.1 to a CD28 receptor and which are not capable of inhibiting the binding of B7.1 to a CTLA-4 receptor. Two of these antibodies, 16C10 and 7C10, significantly inhibit the production of IL-2, in spite of the existence of a second activating ligand B7.2 (CD86). Blocking of the primary activation signal between CD28 and B7.1 (CD80) with these antibodies while allowing the unimpaired or coincident interaction of CTLA-4 and B7.1 and/or B7.2 represents a combined antagonistic effect on positive co-stimulation with an agonistic effect on negative signalling. These antibodies may be used as specific immunosuppressants, e.g., for the treatment of autoimmune diseases and to prevent organ transplant rejection.

Description

IDENTIFICATION OF UNIQUE LINK INTERACTIONS BETWEEN CERTAIN ANTIBODIES AND THE ANTIGENS CO-- STIMULATORS B7.1 AND B7.2 HUMANS FIELD OF THE INVENTION The present invention relates to the identification and use of monoclonal antibodies that are specific for the B7.1 (CD80) antigens. More specifically, the present invention relates to the identification and use of monoclonal antibodies or primatized forms thereof which are capable of inhibiting the binding of the human antigen B7.1 to a CD28 receptor and which are not capable of inhibiting the binding of B7.1 to a CTLA-4 receiver. Thus, the invention relates to the identification and use of monoclonal antibodies and primatized forms thereof which recognize specific sites in the B7.1 antigen that are unique to the CTLA-4 receptor link. The invention further relates to monoclonal antibodies or primatized forms thereof which recognize specific sites in the human B7.1 antigen and are capable of inhibiting the production of IL-2. Ref. 30250 Also, the present invention relates to pharmaceutical compositions containing monoclonal or primate-specific antibodies to humans B7.1 and its use as immunosuppressants by modulating the pathway B7: CD28, for example, for the treatment of autoimmune diseases and the prevention of organ rejection.

BACKGROUND OF THE INVENTION The clinical interface between immunology, hematology and oncology has long been appreciated. Many conditions treated by hematologists or oncologists have either an autoimmune or immunodeficient component to their pathophysiology that has led to the widespread adoption of immunosuppressive drugs by hematologists, while oncologists have sought immunological adjuvants that could improve endogenous immunity to tumors To date, these interventions have generally consisted of non-specific modes of immunosuppression and immune stimulation. In addition to the limited efficacy of these interventions, toxicities secondary to their non-specificity have also limited their complete success. Therefore, alternative strategies have been sought. The elucidation of the functional role of a rapidly increasing number of surface molecules of cells has greatly contributed to the integration of immunology with clinical hematology and oncology. Nearly 200 cell surface antigens have been identified in the cells of the immune and hematopoietic systems (Schlossman SF, Boumsell L, Gilks JM, Harlan T, Kishimoto, C Morimoto C, Ritz J., Shaw S, Silverstein RL, Springer TA , Tedder TF, Todd RF: CD antigens (1993), Blood 83: 879, 1994). These antigens represent both the most widely distributed and restricted lineage molecules involved in a variety of processes, including cell recognition, recognition, adhesion, induction and maintenance of proliferation, cytokine secretion, effector function, and even the death of the cells. The recognition of the functional attributes of these molecules has led to new attempts to manipulate the immune response. Although the molecules involved in cell adhesion and the specific recognition of the antigen have previously been evaluated as targets for immunological therapeutic intervention, recent attention has focused on a subgroup of surface molecules of molecules called co-stimulators (Bretscher P: "The t o-signal model of lymphocyte activation t enty-one years later. " Immunol. Today 13:73, (1992): Jenkins MK, Johnson JG: "Molecules involved in T-cell co-stimulation." Curr Opin Immunol 5: 351, (1993); Geppert T, Davis L. Gur H. Wacholtz M. Lipsky Q: "Accessory cell sign involved in T-cell activation." Immunol Rev 117: 5, (1990); Weaver CT, Unanue ER: "The co-sti ulatory function of antigen-presenting cells." Immunol Today 11:49, (1990); Stennam RM, Young J: "You call arising from antigen-presenting cells." Curr Opin Immunol 3: 361, (1991)). Co-stimulatory molecules do not initiate but allow the generation and amplification of antigen-specific T cell responses and effector function (Bretscher P: "The two-signal model of lymphocyte activation twenty-one years later." Immunol. Today 13:73, (1992): Jenkins MK, Johnson JG: "Molecules involved in T-cell co-stimulation." Curr Opin Immunol 5: 351, (1993); Geppert T, Davis L. Gur H. Wacholtz M. Lipsky Q: "Accessory cell sign involved in T-cell. Activation." Immunol Rev 117: 5, (1990); Weaver CT, Unanue ER: "The co-stimulatory function of antigen-presenting cells." Immunol Today 11:49, (1990); Stennam RM, Young JW: "You sign out arising from antigen-presenting cells." Curr Opin Immunol 3: 361, (1991); June CH, Bluestone JA, Linsley PS, Thompson CD: "Role of the CD28 receiver in T-cell activation." Immunol Today 15: 321, (1994)). Recently, a specific co-stimulatory pathway called B7: CD28 has been studied by different research groups due to its significant role in the activation of B and T cells (June CH, Bluestone JA, Linsley PS, Thompson CD: "Role of the CD28 receptor in T-cell activation. "Immunol Today 15: 321, (1994); June CH, Ledbetter JA:" The Role of the CD28 receptor during T-cell responses to antigen. "Annu Rev Immunol 11: 191, ( 1993); Schwartz RH: "Co-stimulation of T lymphocytes: The role of CD28, CTLA-4, and B7 / BB1 in interleukin-2 production and i munotherapy." Cell 71: 1065-1068, (1992); Jenkins MK , Taylor PS, Norton SD, Urdahl KB: "CD28 delivers a costimulatory signal involved in antigen-specific IL-2 production by human T cells." Journal of Immunology 147: 2461-2466 (1991)). Since this ligand: receptor pathway was discovered four years ago, a large body of evidence has accumulated suggesting that the interactions of B7: CD28 represents one of the critical junctions in the determination of immune reactivity against anergy (June CH, Bluestone JA, Linsley PS, Thompson CD: "Role of the CD28 receiver in T-cell activation. "Immunol Today : 321, (1994); June CH, Ledbetter JA: "The role of the CD28 receptor during T-cell responses to antigen." Annu Rev Immunol 11: 191, (1993); Schwartz RH: "" Co-stimulation of T lymphocytes: The role of CD28, CTLA-4, and B7 / BB1 in interleukin-2 production and im unotherapy. "Cell 71: 1065-1068, (1992); Cohen J:" Mounting a targeted strike on unwanted immune responses "(news; comment) Science 257: 751, (1992): Cohen J: New prstein steals the show as co -stimulator 'of T cells "(news; comment). Science 262: 844, (1993)). In particular, the role of human B7 antigens, that is, the human antigens B7.1 (CD80) and B7.2 (CD86), has been reported to play a co-stimulating role in the activation of T cells. , for example, Gimmi CD, Freeman, GJ, Gribben JG, Sugita K, Freedman AS, Morimoto C, Nadler LM: "B-cell surface antigen B7 provides a costimulatory signal that induces T cells to proliferate and secrete interleukin 2." Proc. Nati Acad. Sci. (USA) 88: 6575-6579 (1991). 1. The co-stimulatory role of B7.1 and B7.2 in the activation of T cells The elaboration of a successful immune response depends on a series of specific interactions between a T cell and an antigen representing the cell. Although the essential first step in this process depends on the binding of the antigen to the T cell receptor, in the context of the MHC class II molecule (Lane, PJL, FM McConnell, GL Schieven, EA Clark, and JA Ledbetter, (1990) , "The Roie of Class II Molecules in Human B Cell Activation." Journal of Immunology, 144: 3684-3692), this interaction alone is not sufficient to induce all the events necessary for a sustained response to a given antigen (Schwartz, RH (1990), "A Cell Culture Model for T Lymphocyte Clonal Anergy. "Science, 248: 1349; Jenkins, MK (1992)," The Role of Cell Division in the Induction of Clonal Anergy. "Immunology Today, 13:69; Azuma, M., M. Cayabyab, D. Buck, JH Phillips , and LL Lanier, (1992), "Involvement of CD28 in MHC-unrestrictred Cytotropicity Mediated by a Human Natural Killer Leukemia Cell Line." The Journal of Immunology, 149: 1115-1123; Azuma, M., M. Cayabyab, D Buck, JH Phillips, and LL Lanier, (1992), "CD28 Interaction with B7 Costimulates Primary Allogeneic Proliferative Responses and Cytotoxicity Mediated by Small Resting T Lymphocytes." J. Exp. Med., 175: 353-360); S.D. Norton, L. Zuckerman, K.B. Urdahl, R. Shefner, J. Miller, and M.K. Jenkins, (1992), "The CD28 Ligand, B7," Enhances IL-2 Production by Providing a Costimulatory Signal to T Cells. "The Journal of Immunology, 149: 1557-1561; RH Schwartz, (1992)," Costimulation of T Lymphocytes: The Role of CD28, CTLA-4, and B7 / BB1 in Interleukin-2 Production and Immunotherapy. "Cell 71: 1065-1068.) The implication of other co-stimulatory molecules is necessary (Norton SD, L. Zuckerman , KB Urdahl, R. Shefner, J. Miller, and MK Jenkins, (1992), "The CD28.Ligand, B7, Enhances IL-2 Production by Providing a Costimulatory Signal to T Cells." The Journal of Immunology, 149: 1557-1561). "The ho odimers CD28 and CTLA-4 expressed on T cells" (June, CH, JA Ledbetter, PS Linsley and Thompson, (1990), "Role of the CD28 Receptor in T-cell Activation." I munology Today 11: 211-216; Linsley, P.S., W. Brady, M. Urnes, L.S. Grosmaire, N.K. Damle, and J.A. Ledbetter, (1991), "CTLA-4 is a Second Receptor for the B Cell Activation Antigen B7. "J. Exp. Med., 174: 561), together with B7.1 (CD28) and B7.2 (CD86) expressed in the antigen-presenting cells are the principal pairs of the co-stimulatory molecules necessary for a sustained immune response (Azuma, M., H. Yssel, JH Phillips, H.

Spits, and L.L. Lanier, (1993), "Functional Expression of B7 / BB1 on Activated T Lymphocytes." J.

Exp. Med., 177: 845-850; Freeman, G.J., A.S. Freedman, J.M. Segil, G. Lee, J.F. Whitman, and LM. Nadler, (1989), "B7, A New Member of the Ig Superfamily with Unique Expression on Activated and Neoplastic B Cells. "The Journal of Immunology, 143: 2714-2722: Hathcock, KS, G. Laslo, HB Dickler, J. Bradshaw, P. Linsley, and RJ Hodes, (1993)," Identification of and Alternative CTLA-4 Ligand Costimulatory for T Cell Activation. "Science, 262: 905-911; Hart, DNJ, GC Starling, VL Calder, and NS Fernando, (1993). "B7 / BB-1 is a Leucocyte Differentiation Antigen on Human Dendritic Cells Induced by Activation. ". Im unology, 79: 616-620.) It can be shown in vitro that the absence of these co-stimulatory signals leads to a pathway of aborted T-cell activation and the development of non-response specific antigens or to the anergy (See, for example Harding, FA, JG McArthur, JA Gross, D.M. Raulet, and J.P. Allison, (1992), "CD28 Mediated Signaling Co-simulates Murine T Cells and Prevents Induction of Anergy in T Cell Clones. "Nature, 356: 607-609; Gimmi, C.D., G.J. Freeman, J.G. Gribben, G. Gray, and L.M. Nadler, (1993); "Human T-Cell Clonal Anergy is Induced by Antigen Presentation in the Absence of B7 Costimulation. "Proc. Nati, Acad. Sci., 90: 6586-6590; Tan, P., C. Anasefti, JA Hansen, J. Melrose, M. Brunvand, J. Bradshaw, JA Ledbetter, and PS Linsley, (1993) , "Induction of Alloantigen-specific Hyporesponsiveness in Human T Lymphocytes by Blocking Interaction of CD28 with Its Natural Ligand B7 / BB1." J. Exp. Med., 177: 165-173.) Achieving a tolerance in vi vo constitutes a mechanism for the immunosuppression of a viable therapy for the rejection of organ transplantation and for the treatment of autoimmune diseases This has been achieved in experimental models following the administration of CTLA-4Ig (Lenschow, DJ., Y. Zeng, RJ Thistlethwaite, A. Montag, W. Brady, MG Gibson, PS Linsley, and JA Bluestone, (1992), "Long-Term Survival of Xenogeneic Pancreatic Islet Grafts Induced by CTLA-4lg." Science, 257: 789-795. B7.1 and B7.2 can be linked to either CD28 or CTLA-4, although B7.1 binds to CD28 with a Kd of 200 Nm and to CTLA-4 with an affinity 20 times highest (Linsley, P.S., E.A. Clark, and J.A.

Ledbetter, (1990), "T-Cell Antigen CD28 Mediates Adhesion with B Cells by Interacting with Activation Antigen B7 / BB-1. "Proc. Nati. Acad. Sci., 87: 5031-5035;" Linsley et al, (1993), "The Role of CD28 receptor during T cell responses to antigen," Annu. Rev. Immunol., 11: 191-192; Linsley et al, (1993), "CD28 Engagement by B7 / BB-1 Induces Transient Down-Regulation of CD28 Synthesis and Prolonged Unresponsiveness to CD28 Signaling," The Journal of Immunology, 150: 3151-3169). B7.1 is expressed in activated B cells and monocytes induced by interferon, but does not remain in B cells (Freeman, GJ, GS Gray, Gimmi CD, Lomarrd DB, LJ Zhou, M. White, JD Fingeroth, JG Gribben, and LM.Nadler, (1991). "Structure, Expression and T Cell Costimulatory Activity of the Murine Homologue of the Human B Lymphocyte Activation Antigen B7," J. Exp. Med., 174: 625-631) . On the other hand, B7.2 is constitutively expressed at very low levels in the remaining monocytes, drendritic cells and B cells, and its expression is enhanced in activated T cells, NK cells and lymphocytes.

B (Azuma, M. D. Ito, H. Yagita, K. Okuura, J.H. Phillips, L.L. Lanier, and C. Somoza, 1993, "B70 Antigen is a Second League for CTLA-4 and CD28, " Nature, 366: 76-79). Although B7.1 and B7.2 can be expressed in the same cell type, their expression in B cells occurs with different kinetics (Lenschow, D.J., G.H. Su, L.A. Zuckerman, N. Nabavi, C.L. Jellis, G.S. Gray, J. Miller, and J.A. Bluestone, (1993), "Expression and Functional Significance of an Additional Ligand for CTLA-4, "Proc. Nati. Acad.

Sci., USA, 90: 11054-11058; Boussiotis, V.A. , G.J. Freeman, J.G. Gribben, J. Daley, G. Gray, and L.M. Nadler, (1993), "Activated Human B Lymphocytes Express Three CTLA-4 Counter-receptors that Co-stimulate T-Cell Activation." Proc. Nati Acad. Sci., USA, 90: 11059-11063). Further analysis of RNA levels has shown that B7.2 mRNA is constitutively expressed, whereas B7.1 mRNA is detected 4 hours after activation and with low initial levels of B7.1 protein. detectable until 24 hours after stimulation (Boussiotis, VA, GJ Freeman, J.G. Gribben, J. Daley, G. Gray, and L.M.

Nadler, (1993), "Activated Human B Lymphocytes Express Three CTLA-4 Counter-receptors that C0-stimulate T-Cell Activation, "Proc. Nati. Acad. Sci., USA, 90: 11059-11063.) The CTLA-4 / CD28 counter-receptors, therefore, They can be expressed at various times after the activation of B cells. More recently, it has been suggested that the second CTLA-4 co-receptor associated with T cells apparently functions as a negative modulator to overexpress and prevent a pathway of the immune system. (Krummel M, Allison J: "CD28 and CTLA-4 have opposing effects on the response of T cells to sti ulation." J. Exp. Med. 182: 459-466 (1995) &. • CTLA-4 receptor plays a critical role in the down-regulation of the immune response, as shown in mice that were given CTLA-4, treated mice born without the ability to express the CTLA-4 gene died within 3-4 weeks of a severe lymphoproliferative disease (Tivol EA, Borriello G, Schweitzer AN, Lynch WP, Bluestone JA, Sharpe AH: "Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4." Immunity 3: 541-547 (1995)). It is thought that CTLA-4 works through signaling mechanisms linked to the induction of apoptosis (Gribben JG, Freeman GJ, Boussiotis VA, Rennert P, Jellis CL, Greenfield E, Barber M, Restivo Jr. VA, Ke X, Gray GS, Nadler LM: "CTLA-4 mediates antigen specific apoptosis of human T cells." Proc.

Nati Acad. Sci. (USA) 92: 811-815 (1995)), driven via an undefined ligand link to specific sites in the receptor. It has been shown in vitro that blocking B7.1 / B7.2 dependent on the co-stimulatory signals in various ways leads to an aborted path of activation of T cells and the development of non-responses to the specific antigen (Lederman S, Chess L, Yellin MJ: "Murine monoclonal antibody (5c8) recognizes human glycoprotein on the surface of T-lymphocytes, compositions containing same." U.S. Patent No. 5,474,771 (December 12, 1995); Linsley PS, Ledbetter JA, Damle NK, Brady W: "Chimeric CTLA4 receptor and methods for its use." U.S. Patent No. 5,434,131 (July 18, 1995), Harding, 1992, Gimmi CD, Freeman GJ, Bribben JG, Gray G, Nadler LM: "Human T-cell clonal anergy is induced by antigen presentation in the absence of B7 costimulation. "Proc. Nati. Acad. Sci. (USA) 90: 6586-6590 (1993); Tan P, Anasetti C, Hansen JA, Melrose J, Brunvand M, Bradshaw J, Ledbetter JA, Linsley PS: "Induction of allogeneic-specific hyporesponsiveness in human T lymphocytes by blocking interaction of CD28 with its natural ligand B7 / BB1." J. Exp. Med. 177: 165-173 (1993)). The achievement of an in vivo tolerance, anergy, or decrease in antigen-specific T cells would constitute a mechanism for immunosuppression and a viable therapy for the rejection of organ transplantation or an adequate treatment for autoimmune diseases. The differential temporal expression of B7.1 and B7.2 suggests that the interaction of these two molecules with CTLA-4 and / or CD28 provide distinct but related signals to the T cells CLaSalle, J.M., P.J. Tolentino, G.J. Freeman, L.M. Nadler, and D.A. Hafler, (1992), "CD28 and T Cell Antigen Receptor Signal Transduction Coordinately Reg late Interleukin 2 Gene Expression In Response to Superantigen Stimulation," J ^ Exp. Med., 176: 177-186; Vandenberghe, P., G.J. Freeman, L.M. Nadler, M.C. Fletcher, M. Kamoun, L.A. Turka, J.A. Ledbetter, C.B. Thompson, and C.H. June, (1992), "Antibody and B7 / BBl-Mediated Ligation of the CD28 Receptor Induces Tyrosine Phosphorylation in Human T Cells, The Journal of Experimental Medicine, 175: 951-960.) The exact signaling functions of CTLA-4 and CD28 on T cells are currently unknown (Janeway, C.A., Jr. and K. Bottomly, (1994), "Signs and Signs for Lymphocyte Responses, "Cell, 76.275285) However, it is possible that a set of receptors could provide the initial stimulus for the activation of the T cells, and the second set, a sustained signal to allow an additional elaboration of the path or path and so that clonal expansion (Linsley, PS, JL Greene, P. Tan, J. Bradshaw, JA Ledbetter, C. Anasetti, 'and NK Damle, (1992), "Coexpression and Functional Cooperation of CTLA-4 and CD28 on Activated T Lymphocytes, "J. Exp. Med., 176: 1595-1604.) The present data support the hypothesis of two signals proposed by Jenkins and Schwartz (Schwartz, RH, (1990), "A Cell Culture Model for Clonal Lymphocyte T Anergy, "Science, 248: 1349; Jenkins, MK, (1992)," The Role of Cell Division in the Induction of Clonal Anergy, "Immunology Today, 13:69) that both a TCR signal and a co-stimulatory signal are necessary for the expansion of T cells, lymphokine secretion and the full development of effector function (Greenan, V. and G. Kroemer, (1993), "Multiple Ways to Cellular Immune Tolerance," Immunology Today, 14: 573). The failure of the supply of the second signal results in the inability of the T cells to secrete IL-2 and returns to the cell not responsive to the antigen. Structurally, both B7.1 and B7.2 contain V domains and C-like domains of the extracellular immunoglobulin superfamily, a hydrophobic transmembrane region and a cytoplasmic tail (Freeman, _ GJ, JG Gribben, VA Boussiotis, JW Ng, V Restivo, Jr., LA Lombard, GS Gray, and LM Nadler, (1993), "Cloning of B7.2: A CTLA-4 Counter-receptor that Co-stimulates Human T Cell Proliferation," Science, 262: 909) . Both B7.1 and B7.2 are widely glycosylated. B7.1 is a glycoprotein of 44-54 kD comprised of an extracellular domain of 223 amino acids, a transmembrane domain of 23 amino acids, and a cytoplasmic tail of 61 amino acids. B7.1 contains 3 potential protein kinase phosphorylation sites. (Azuma, M., H. Yssel, JH Phillips, H. Spits, and LL Lanier, (1993), "Functional Expression of B7 / BB1 on Activated T Lymphocytes," J. Exp. Med., 177: 845-850. ). B7.2 is a membrane glycoprotein of 306 amino acids. It consists of an extracellular region of 220 amino acids, a hydrophobic transmembrane domain of 23 amino acids and a cytoplasmic tail of 60 amino acids (Freeman, G.J., ACE. Freedman, J.M. Segil, G. Lee, J.F. Whitman, and LM. Nadler, (1989), "B7, A New Member of the Ig Superfamily with Unique Expression on Activated and Neoplastic B Cells, "The Journal of Immunology, 143: 2714-2722). Although both B7.1 and B7.2 genes are located in the same chromosomal region (Freeman, G.J., "D.B. Lombard, C.D. Gimmi, S.A. Brod, L. Lee, J.C. Laning, D.A. Hafler, M.E. Dorf, G.S. Gray, H.

Reiser, C.H. June, C.B. Thompson, and L.M. Nadler, (1992), "CTLA-4 and CD28 MRNA are Coexpressed in Most T Cells After Activation, "The Journal of Immunology, 149: 3795-3801; Schwartz, R.H., (1992)," Costi ulation of T Lymphocytes: The Role of CD28, CTLA-4, and B7 / BB1"in Selvakumar, A., B.K. Mohanraj, R.L. Eddy, T.B. Shows, P.C. White, C. Perrin, and B. Dupont, (1992), "Genomic Organization and Chromosomal Location of the Human Gene Encoding the B-Lymphocyte Activation Antigen B7," Immunogenetics, 36: 175-181), these antigens do not share a high level of homology. The complete homology between B7.1 and B7.2 is j of 26% and between murine B7.1 and human B7.2 is 27% (Azuma, M., H. Yssel, JH Phillips, H. Spits, and LL Lanier, (1993), "Functional Expression of B7 / BB1 on Activated T Lymphocytes, "J. Exp. Med., 177. -845-850; Freeman, G.J., A.S. Freedman, J.M.

Segil, G. Lee, J.F. Whitman, and LM. Nadler, (1989), "B7, A New Member of the Ig Superfamily with Unique Expression on Activated and Neoplastic B Cells, "The Journal of Immunology, 143: 2714-2722). Although the alignment of the sequences of human B7.1 and human B7.2 and murine B7.1 show few stretches of homology length, it is known that all three molecules bind to human CTLA-4 and CD28. Thus, there is a most probable common region, or a very close homologous region shared by the three molecules that can be either contiguous or conformational. This r-tion can constitute the binding site of molecules B7.1 and B7.2 to their counter-receptors. Antibodies raised against these epitopes could potentially inhibit the interaction of B7 with its counter receptor in T cells. In addition, antibodies that cross-react with this region in both B7.1 and B7.2 molecules would have potentially practical advantages over targeted antibodies. against B7.1 or B7.2 separately. 2. Blockade of the B7 / CD28 interaction Blocking the B7 / CD28 interaction offers the possibility of inducing a specific immunosuppression, with the potential to generate specific therapeutic effects of long-term antigens.

Antibodies or agents that temporarily prevent this interaction may be useful; they may be useful, specific agents and clinically safe immunosuppressants, with the potential to generate specific therapeutic effects of long-term antigens. Antibodies to either B7.1 or B7.2 have been shown to block the activation of T cells, as measured by the inhibition of IL-2 production in vi tro (DeBoer, M., P. Parren, J Dove, F. Ossendorp, G. van der Horst, and J. Reeder, (1992), "Functional Characterization of a Novel Anti-B7 Monoclonal Antibody," Eur. Journal of Immunology, 22: 3071-3075; Azuma, M ., H. Yssel, JH Phillips, H. Spits, and LL Lanier, (1993), "Functional Expression of B7 / BB1 on Activated T Lymphocytes," J. Exp. Med., 177: 845-850). However, it has been shown that different antibodies vary in their immunosuppressive potency, which reflects either their affinity or epitope specificity. One possible explanation for this may lie in the ability of some antibodies to block only the binding of B7 to CD28, while promoting apoptosis or some other form of negative signaling through the CTLA-4 receptor in cells.

T activated. Some antibodies to B7.1 or B7.2 can, in fact, hide the activity of CTLA-4 by cross-reaction with the binding domain of CTLA-4. The fusion protein of CTLA-4Ig and the anti-CD28 Fabs have been shown to have similar effects in the down-regulation of IL-2 production. In vivo administration of some CTLA-4Ig soluble fusion protein has been shown to suppress T cell-dependent antibody responses in mice (Linsley, PS, JL Greene, P. Tan, J. Bradshaw, JA Ledbetter, C. Anasetti, and NK Damle, (1992), "Coexpression and Functional Cooperation of CTLA-4 and CD28 on Activated T Lymphocytes," J. Exp Med., 176:. 1595-1604; Lin, H., builing SF, Linsley PS , RO Wei, Thompson CD, and Turka LA, (1993), "Long-term Acceptance of Major Histocompatibility Complex Mismatched Cardiac Allografts Induced by CTLA-4-Ig Plus Donor Specific Transfusion," J. Exp Med., 178:. 1801 )) and, in addition, larger doses were also able to suppress the responses to a second immunization, demonstrating the feasibility of this approach for the treatment of autoimmune diseases mediated by antibodies. In addition, CTLA-4Ig was able to prevent rejection of pancreatic islet cells in mice by directly inhibiting the interaction of T cells and cells displaying B7.1 / B7.2 antigens (Lenschow, DJ, GH Su, 17.A. Zuckerman, N. Nabavi, CL Jellis, GS Gray, J. Miller, and JA Bluestone, (1993), "Expression and Functional Significance of an Additional Ligand for CTLA-4," Proc. Nati. Acad. Sci., USA, 90: 11054-11058). In this case, a specific long-term tolerance of the donor was achieved. 3. Recombinant Phage Sample Technology for Antibody Selection Up to this date, monoclonal antibodies that cross-react with both B7.1 and B7.2 have not been reported. In addition, no monoclonal antibodies have been reported that are "specific to B7.1 or B7.2 and that also recognize specific sites in antigens that are restricted for the binding of the coactivation CD28 receptor, or alternatively, no antibodies have been reported. monoclonal that are specific to B7.1 or B7.2 and which recognize specific sites on the antigens which are exclusive of CTLA-4 binding receptor. as discussed above, such antibodies would potentially desirable as immunosuppressants significantly. technology sample of the phage is the beginning to replace the traditional methods to isolate the antibodies generated during the immune response, because a much larger percentage of the immune repertoire can be assessed than is possible using traditional methods. This is partly due to the inefficiency of the PEG fusion, the chromosomal instability and the large number of tissue cultures and the selection associated with the production of heterohybridomas. The phage display technology, in contrast, relies on molecular techniques for a potential capture of the full repertoire of immunoglobulin genes associated with the response to a given antigen. This technique is described by Barbas et al., Proc. Nati Acad. Sci., USA, 88, 7978-7982, (1991). Essentially, the immunoglobulin heavy chain genes are amplified with PCR and cloned into a vector containing the gene encoding a minor coat protein of the M13 filamentous phage in such a way that a heavy chain fusion protein is created. The heavy chain fusion protein is incorporated into the M13 phage particle together with light chain genes as they are assembled. Each recombinant phage contains, within its gene, the genes for a different antibody Fab molecule which is shown on its surface. Within these libraries, an excess of different antibodies can be cloned and displayed. The phage library is screened in the microliter wells coated with antigen, the non-specific phage is washed, and the phage binding to the antigen is eluted. The genome of the antigen-specific clones are isolated and the gene III is cut, so that the antibody can be expressed in the soluble Fab form for further characterization. Once the unique Fab is selected as a potential therapeutic candidate, it can be easily converted to a complete antibody. One expression system previously described for converting Fab sequences to whole antibodies is the mammalian expression vector of IDEC NEOSPLA. This vector contains either genes from the human gamma 1 or gamma 4 constant region. CHO cells are transfected with NEOSPLA vectors and after amplification this vector system has reported that very high expression levels can be achieved (>; 30 pg / cell / day). 4. Primatized Antibodies Another highly efficient means to generate recombinant antibodies is described by Newman, (1992), Biotechnology, 10, 1455-1460. More particularly, this technique results in the generation of primatized antibodies containing monkey variable domains and constant human sequences. This reference is incorporated here as a reference in its entirety. In addition, this technique is also described in commonly assigned U.S. Patent Applications No. 08 / 379,072, filed January 25, 1995, which is a continuation of US Application Serial No. 07 / 912,292, filed on October 10, 1995. of July 1992, which is a continuation in part of the American Application Serial No. 07 / 856,281, filed March 23, 1992, which is finally a continuation in part of the US Application Serial No. 07 / 735,064, presented on July 25, 1991. The North American Application Serial No. 08 / 379,072 and the prior application thereto are hereby incorporated by reference in their entirety. This technique modifies antibodies that are not antigenically rejected after administration in humans. This technique relies on the immunization of cynomolgus monkeys with human antigens or receptors. This technique was developed to create high affinity monoclonal antibodies directed to human cell surface antigens. The identification of macaque antibodies to B7.1 and B7.2 by selection of phage sample libraries or monkey heterohybridomas obtained g the B lymphocytes of immunized monkeys B7.1 and / or B7.2 also is disclosed in commonly assigned US Patent Application No. 08 / 487,550, filed June 7, 1995, incorporated by reference in its entirety herein. More specifically, Application No. 08 / 487,550 provides four monoclonal antibodies 7B6, 16C10, 7C10 and 20C9 that inhibit the pathway or pathway B7: CD28 and therefore function as effective immunosuppressants. Antibodies generated in the manner described by these co-assigned applications have previously been reported as an effector function in humans, which have reduced immunogenicity and have a long serum half-life. The technology rests on the fact that even though cynomolgus monkeys are phylogenetically similar to humans, they still recognize many human proteins as foreign and therefore generate an immune response. In addition, because cynomolgus monkeys are phylogenetically very close to humans, the antibodies generated in these monkeys have been found to have a high degree of amino acid homology to those produced in humans. In fact, after forming the sequence of the light chain and heavy chain variable region of the macaque immunoglobulin, it was found that the sequence of each gene family was 85-98% homologous to its human counterpart (Newman et al, (1992), id ..). The first antibody generated in this manner, an anti-CD4 antibody, was 91-92% homologous to the consensus sequence of the human immunoglobulin structure regions. Newman et al, Biotechnology, 10: 1458-1460, (1992). Monoclonal antibodies specific to human B7 antigens have been previously described in the literature. For example, Weyl et al, Hum. Immunol. , 31 (4), 271-276, (1991) describes the mapping of epitopes of human monoclonal antibodies against HLA-B-27 g natural and mutated antigenic variants. Also Toubert et al, Clin. Exp. Im unol., 82 (1), 16-20, (1990) describes the epitope mapping of a monoclonal antibody HLA-B27 that also reacts with a bacterial outer membrane protein of 35-KD. Also, Valle et al, Immunol. , 69 (4), 531-535, (1990) describes a monoclonal antibody of the IgG1 subclass that recognizes the B7 antigen expressed on activated B cells and T cells transformed with HTLV-1. further, Toubert et al, J. Immunol., 141 (7), 2503-9, (1988) describes the mapping of epitopes of antigens HLA-B27 and HLA-B7 using intradomain recombinants constructed by making the hybrid genes between those two alleles in E. coli. The high expression of the B7 antigen has correlated to autoimmune diseases by some researchers. For example, Ionesco-Tirgoviste et al, Med. Interre, 24 (1), 11-17, (1986) reports an increased expression of B7 antigen in insulin-dependent diabetes type 1. Also, the implication of the expression of B7 antigen in dermal drendritic cells obtained from patients with psoriasis has been reported. (Nestle et al, J. Clin. Invest., 94 (1), 202-209, (1994)). In addition, inhibition of alloreactive CTL with anti-HLA-B7 using soluble HLA-B7 purified by affinity has been reported in the literature.

(Zavazava et al, Transplantation, 51 (4), 838-42, (1991)). In addition, the use of the soluble ligand of the B7 receptor, CTLA-4Ig blocks B7 activity (See, for example, Lenschow et al, Science, 257, 789, 7955 (1992)) in animal models and a B7 fusion protein. 1-Ig capable of inhibiting B7 has been reported. Evidence is provided in this description for the identification of monoclonal antibodies that recognize specific sites in the B7.1 antigen that are restricted to the CD28 receptor binding. In addition, we present here evidence for the identification of antibodies that recognize sites in the B7.1 antigen that are unique to the CTLA-4 receptor binding. Thus, evidence is presented here to support the existence of unique antigen binding sites in co-stimulatory antigens B7.1 (CD80) human. The claimed sites are identified by the antibodies PRIMATIZED® anti-B7.1 and evidence is presented confirming that the binding to an interaction site in the B7.1 antigen which is restricted to the receptor binding CD28 co-activation.

BRIEF DESCRIPTION AND OBJECTIVES OF THE INVENTION An object of the invention is to identify new antibodies that are specific to the human B7.1 antigen. More specifically, it is an object of the invention to identify antibodies that are specific to the human B7.1 antigen and that are also capable of inhibiting the binding of B7.1 to the CD28 receptor. It is also an object of the invention to identify antibodies that are specific to the B7.1 antigen and that are not capable of inhibiting the binding of B7.1 to a CTLA-4 receptor. Thus, it is an object of this invention to identify antigens that recognize specific sites in the B7.1 antigen, where the recognized sites are restricted to the CD28 receptor link and that are unique to the receptor link.

CTLA-4. It is a further object of the invention to identify antibodies that are specific to the human B7.1 antigen and that can not recognize the B7.2 antigen. It is another objective of the invention to identify monoclonal antibodies and primatized forms thereof which recognize the specific sites in the human antigen B7.1 and which inhibit the production of IL-2 and the proliferation of T cells and that function as effective immunosuppressants. More specifically, it is an object of this invention to identify antibodies that are specific to B7.1 and that are capable of inhibiting the production of IL-2. It is another object of the invention to provide monoclonal antibodies and primatized forms thereof that inhibit antigen-driven responses in spleen donor cell cultures, eg, antigen-specific IgG responses, IL-2 production and proliferation. of cells. It is another specific object of the invention to identify the particular monoclonal antibodies specific to the human B7.1 antigens and the primatized forms thereof which have the advantageous properties, for example, affinity, immunosuppressive activity, which are useful as therapeutic products. More specifically, these antibodies and the primatized forms thereof are to be used, for example, as immunosuppressants, that is, to block the immune responses triggered by the antigen, to treat autoimmune diseases, such as psoriasis, rheumatoid arthritis, systemic erythematosus ( SLE), type 1 diabetes mellitus, idiopathic thrombocytopenia purpura (ITP), allergy, biliary inflammatory conditions, and to avoid organ rejection. It is another object of the invention to provide pharmaceutical compositions containing one or more monoclonal antibodies specific to the B7.1 antigen or primatized forms thereof, and a pharmaceutically acceptable carrier or excipient. These compositions will be used, for example, as immunosuppressants to treat autoimmune conditions, for example, idiopathic thrombocytopenia purpura (ITP) and systemic lupus erythematosus (SLE), to block the immune responses triggered by the antigen, and to prevent rejection. of organs in transplant recipient organisms.It is another object of the invention to provide novel methods of therapy by administering therapeutically effective amounts of one or more or primatized monoclonal antibodies that specifically bind to the human B7.1 antigen.

Such therapeutic methods are useful for the treatment of treatable conditions by inhibiting the B7: CD28 pathway, for example, autoimmune conditions such as idiopathic thrombocytopenia purpura (ITP), systemic lupus erythematosus (SLE), type 1 diabetes mellitus, psoriasis, rheumatoid arthritis. , multiple sclerosis, aplastic anemia, as well as to avoid rejection in subjects subjected to transplantation. It is still another object of the invention to provide transfectants, for example, CHO cells, which express at least the heavy and light variable domains of monoclonal antibodies specific to the human B7.1 antigen.

Definitions The following terms are defined so that the invention can be understood more clearly.

Reducing antibodies - an antibody that kills activated B cells or other cells that present antigens.

Non-reducing antibodies - an antibody that blocks the co-stimulatory action of B7 and T cells that activate the ligands CD28 and CTLA-4. Thus, it energizes but does not eliminate the cells that present antigens.

Primatized Antibody - a recombinant antibody that has been engineered to contain the variable heavy and light domains of a monkey antibody, in particular, a cynomolgus monkey antibody, and which contains human constant domain sequences, preferably the gamma constant domain 1 or gamma 4 of human immunoglobulin (or PE variant). The preparation of such antibodies is described in Newman et al., (1992), "Primatization of Recombinant Antibodies for Immunotherapy of Human Diseases: A Macaque / Human Chimeric Antibody Against Human CDH, Biotechnology, 10: 1458-1460; also in the application commonly assigned patent 08/379, 072 both incorporated herein by reference in their entirety.These antibodies have been reported to show a high degree of homology to human antibodies, ie, 85-98%, show human effector functions, have immunogenicity reduced, and may exhibit high affinity to human antigens.

B7 antigens - B7 antigens in this application include, for example, human B7, B7.1 and B7.2 antigens. These antigens bind to CD28 and / or CTLA-4. These antigens have a co-stimulatory role in the activation of T cells. Also, these B7 antigens contain all extracellular immunoglobulin superfamily V and C-like domains, a region of hydrophobic transmembrane and a cytoplasmic tail. (See, Freeman, et al., Science, 262: 909, (1993)), and are heavily glycosylated.

Anti-B7 antibodies - antibodies, preferably monkey monoclonal antibodies or primatized forms thereof, which bind specifically to human B7 antigens, eg, B7.1 and / or B7.2 antigens with sufficient affinity to block the B7: CD28 interaction and thus induce immunosuppression.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the pMS vector used to select recombinant immunoglobulin libraries produced against B7 shown on the surface of the filamentous phage containing primers based on the immunoglobulin sequences. Figure 2 shows the NEOSPEA expression vector .. used to express the primatized antibodies of the subject specific to the human B7.1 antigen. Figure 3a shows the amino acid and nucleic acid sequence of a primatized form of the light chain of 7C10. Figure 3b shows the amino acid and nucleic acid sequence of a primatized form of the 7C10 heavy chain. Figure 4a shows the amino acid and nucleic acid sequence of a primatized form of the light chain of 7B6. Figure 4b shows the amino acid and nucleic acid sequence of a primatized form of the heavy chain of 7B6.

Figure 5a shows the amino acid and nucleic acid sequence of a primed light chain of 16C10. Figure 5b shows the amino acid and nucleic acid sequence of a primed heavy chain of 16C10. Figure 6 shows the inability of P16C10 to block the binding of CTLA-4Ig-Biotin to CHO cells transfected with B7.1. Xa. Figure 7 shows the inability of CTLA-4Ig to block the binding of CTLA-4Ig-Biotin to CHO cells transfected with B7.1. Figure 8 shows that BB-1 completely blocks the binding of CTLA-4Ig-Biotin to CHO cells transfected with B7.1 and further shows the inability of BB-1 to significantly affect the binding of Pl6C10-Biotin to CHO cells transfected with B7.1. Figure 9 shows that CTLA-4Ig-Biotin is effectively blocked by all inhibitors B7.1 except for P16C10. Figure 10 shows the inability of P16C10 to block the binding of the CD28 / B7-llg interaction. The data shown are averages of the values obtained from four separate experiments.

DETAILED DESCRIPTION OF THE INVENTION As described above, the present invention relates to the identification of monoclonal antibodies or primatized forms thereof which are specific to the human B7.1 antigen and which are capable of inhibiting B7.1 binding to a CD28 receptor and which are not capable of inhibiting the binding of B7.1 to a CTLA-4 receptor. Blocking the primary activation site between CD28 and B7.1 (CD80) with the identified antibodies while allowing the combined antagonist effect in positive co-stimulation with an agonist effect in negative signaling will be a useful therapeutic approach to intervening in the forms of relapses of autoimmune diseases. The functional activity of the identified antibodies is defined by the blocking of the production of the cytokine IL-2 stimulating the T cells. The identified antibodies have demonstrated the ability to block the production of IL-2 in excess of 50%, despite of the existence of a second acting B7.2 ligand, suggesting an alternative mechanism of action that exists which is not typical of the observed effects of other anti-B7.1 antibodies defined in the literature. The manufacture of novel monkey monoclonal antibodies that specifically bind to human antigen B7.1 and / or human antigen B7.2, as well as primatized derived antibodies is disclosed in co-pending co-pending US Application Serial No. 08 / 487,550, and as stated here. These antibodies possess a high affinity to human B7.1 and / or B7.2 and therefore can be used as immunosuppressants that inhibit the pathway or B7: CD86 pathway. The preparation of monoclonal antibodies of the monkey will preferably be carried out by the selection of the phage sample libraries or by the preparation of monkey heterohybridomas using B lymphocytes obtained from monkeys immunized with B7. (for example, human B7.1 and / or human B7.2). As explained, the first method for generating anti-B7 antibodies involves the recombinant phage display technology. This technique was described in a general manner before. Essentially, this will comprise the synthesis of recombinant immunoglobulin libraries against the B7 antigens shown on the surface of the filamentous phage and the selection of the phage that secretes antibodies having high antigen affinity.

B7.1 and / or B7.2. As noted above, the antibodies will preferably be selected so that they bind to both human B7.1 and B7.2. To effect such methodology, the present inventors have created a unique library for monkeys libraries that reduces the possibility of recombination and improves stability. This vector, PMS, is described in detail below, and is shown in Figure 1. Essentially, to adopt the phage sample for use with macaque libraries, this vector contains primers specific for monkey immunoglobulin genes that amplify PCR. These primers are based on the macaque sequences obtained while the primatized technologies and the human sequences containing the databases are developed. Suitable primers are described in the commonly assigned patent 08/379, 072 incorporated herein by reference. The second method involves the immunization of monkeys, ie, macaques, against the human B7 antigen, preferably against the human B7.1 and B7.2 antigen. The inherent advantage of the macaques for - the generation of monoclonal antibodies was discussed above. In particular, such monkeys, ie, cynomolgus monkeys, can be immunized against human antigens or receptors. In addition, the resulting antibodies can be used to make primatized antibodies according to the methodology of Newman et al, Biotechnology, 10, 1455-1460, (1992), and Newman et al, commonly assigned US patent application Serial No. 08 / 379,072, filed January 25, 1995, which is hereby incorporated by reference in its entirety. The significant advantage of the antibodies obtained from the cynomolgus monkeys is that these monkeys recognize many human proteins as foreign and thus provide the formation of antibodies, some with high affinity to the desired human antigens, for example, cell receptors. and human surface proteins. In addition, because they are phylogenetically close to humans, the resulting antibodies exhibit a high degree of homology of the acids to those produced in humans. As noted above, after sequencing the genes of the light and heavy variable region of the macaque immunoglobulin, it was found that the sequence of each gene family was 85-88% 85-88% homologous to its human counterpart ( Newman et al, (1992), Id ..). Essentially, cynomolgus macaque monkeys are administered with human B7 antigens, ie, human B7.1 antigens and / or human B7.2 antigens, B cells are isolated from them, for example, lymph node biopsies are taken from the animals, and the B lymphocytes are fused with heteromyeloma cells KH6 / B5 (mouse x human) using polyethylene glycol (PEG). Antibodies that secrete heterohybridomas bind to human B7 antigens, for example, human B7.1 antigen and / or human B7.2, are then identified. Antibodies that bind to both B7.1 and B7.2 are desirable because such antibodies can potentially be used for inhibition of B7.1 and B7.2, as well as B7 with their counter-receptors, ie, CTLA-4 and CD28 human. Antibodies against these epitopes can inhibit the interaction of both human B7.1 and human B7.2 with their counter-receptors on T cells. This may provide potential synergistic effects. However, antibodies that bind only to one of the human B7 antigens, B7.1 antigen or B7.2 antigen, are also highly desirable because of the co-involvement of these molecules in the activation of T cells, secretion of lymphokine (IL-2) clonal expansion, and the response to antigen. Since both human B7.1 and B7.2 bind to CTLA-4 and CD28, it is likely that there is at least one homologous or common region (perhaps an epitope or shared conformational epitopes) to which macaque antibodies can potentially be generated. "The invention described involves the use of an animal that is primed to produce a particular antibody.Animals that are useful for such a process include, but are not limited to the following: mice, rats, guinea pigs, hamsters, monkeys, pigs , Goats, and Rabbits A preferred means of generating human antibodies using SCID mice is described in the co-pending US patent application Serial No. 08 / 488,376.The present inventors chose to immunize macaques against human B7.1 antigens. using recombinant soluble B7.1 antigen produced by CHO cells and purified by affinity chromatography using an affinity column of L307.4-sepharose.However, the particular source of human B7 antigen.However, the particular source of B7 antigen human, human B7.1 antigen or B7.2 antigen, is not critical, considering that it is of sufficient purity to result in a specific antibody to the B7 antigen administered particularly and potentially to other B7 antigens. The genes of the human B7 antigens, antigen Human B7.1 (also called CD80) and human B7.2 antigen (also called CD86) have been cloned and sequenced, and therefore can be easily manufactured by recombinant methods. Preferably, the human B7 antigen administered, the human B7.1 antigen and / or the human B7.2 antigen will be administered in soluble form, for example, by expression of a B7, B7.1 or B7.2 gene having its own transmembrane and cytoplasmic domains eliminated, thus leaving only the extracellular portion, that is, the domains of the V superfamily and similar to C. (See, for example, Gru et al., Huía. Immunol., 40 (3), p 228-234, 1994, which shows the expression of a soluble form of human B7, which is incorporated herein by reference in its entirety).

- - The macaques will be immunized with the antigen B7, B7.1 and / or B7.2, preferably a soluble form thereof, under conditions that result in the production of specific antibodies thereto. Preferably, the soluble human antigen B7, B7.1 or B7.2 will be administered in combination with an adjuvant, eg Freund's Complete Adjuvant (CFA), Alu, Saponin, or other known adjuvants, as well as combinations thereof. In general, this will require a repeated immunization, for example, by repeated injection for several months. For example, administration of the soluble B7.1 antigen was effected as an adjuvant, to generate the immunizations, over a period of 3 to 4 months, which results in the production of antibodies contained in the serum that bind to the B7 antigen. 1 human After the immunization B cells are collected, for example, by biopsies of the lymph node taken from the immunized animals and from the B lymphocytes fused with the heteromyeloma cells KH6 / B5 (mouse x human) using polyethylene glycol. Methods for the preparation of such heteromyelomas are known and can be found in US Patent Application Serial No. 08 / 379,072 by Newman et al, filed January 25, 1995, and incorporated herein by reference. The heterohybridomas that secrete antibodies that bind to human B7, B7.1 and / or B7.2 are thus identified. This can be done by known techniques. For example, it can be determined by ELISA or radioimmunoassay using enzymes or B7, B7.1 and / or B7.2 antigens labeled with radio nucleotides. Cell lines that secrete antibodies having the desired specificity to human antigens B7, B7.1 and / or B7.2 can then be subcloned to a monoclonality. In the present invention, the inventors selected antibodies purified for their ability to bind to those coated with soluble B7.1 antigens in an ELISA assay, B cells positive with antigens, and CHO transfectomes expressing the human B7.1 antigen or its cellular surface. In addition, the antibodies are selected for their ability to block B cell / T cell interactions as measured by IL-2 production and resumed by tritiated thymidine in a mixed lymphocyte reaction (MLR), with the binding - -B7 which is detected using soluble B7.1 radiolabelled with 125I (SB7.1). Also, antibodies affinity purified from the macaques were tested for their reactivity against the CHO transients that are expressed in the B7.1 / Ig fusion proteins, and against the CHO cells that produce the human B7.2 antigen.

These results indicated that the immune serum of B7.1 was linked to transfectomas B7.2. The binding of the antibodies to the B7.2 antigen can be confirmed using soluble B7.2-Ig reagents. As discussed in the examples, this can be done by producing and purifying B7.2-Ig from CHO transfectants in sufficient quantities to prepare an affinity column B7.2-Ig-sepharose. These antibodies cross-reacting with B7.2 will bind to the B7.2-Ig-sepharose column. Cell lines that express antibodies that bind specifically to the human B7 antigen, the human B7.1 antigen and / or the human B7.2 antigen are then used to clone variable domain sequences for the manufacture of primatized antibodies essentially as described in Newman et al, (1992), Id. and Newman et al. , - US Patent Serial No. 379,072, filed January 25, 1995, both incorporated herein by reference. Essentially, this generates the extraction of RNA, the conversion to cDNA, and the amplification thereof by the PCR technique using the specific Ig primers. Suitable primers are described in Newman et al, 1992, Id. And in the American Patent Serial No. 379,072. (See, in particular, Figure 1 of the US Patent Serial No. 379,072). The cloned monkey variable genes are then inserted into an expression vector containing genes from constant regions of heavy and light chains of humans. Preferably, this is effected using an expression vector owned by IDEC, Inc., referred to as NEOSPLA. This vector is shown in Figure 2 and contains the cytomegalovirus promoter / enhancer, the mouse beta globin major promoter, the SV40 origin of replication, the polyadenylation sequence of bovine growth hormone, exon 1 and exon 2 of the neomycin phosphotransferase, the human immunoglobulin kappa or lambda constant region, the dihydrofolate reductase gene, the human immunoglobulin gamma 1 or gamma 4 constant region and the leader-9 sequence. It has been found that this vector results in a very high level of expression of the primatized antibodies after incorporation of the variable region genes of the monkey, transfection in the CHO cells, followed by sectioning in the medium containing G418 and the amplification of methotrexate. For example, this expression system has been previously described and results in primatized antibodies having a high affinity (Kd = 10"10 M) against CD4 and other surface receptors of human cells. the same affinity, specificity and functional activity as the original monkey antibody This vector system is described substantially in US Patent No. 379,072, incorporated herein by reference as well as from the American Serial No. 08 / 149,099, filed on November 3, 1993, also incorporated herein by reference in its entirety.This system provides high levels of expression, i.e., > 30 pg / cell / day.As discussed below, the inventors of the present invention have selected four Monoclonal antibodies of candidate monkeys that bind specifically to the B7.1 antigen. These monkey monoclonal antibodies refer to such as 7B6, 16C10, 7C10 and 20C9. As discussed in more detail above, these antibodies were evaluated for their ability to block B cell / T cell interactions as measured by IL-2 production and retention of tritiated thymidine in a mixed lymphocyte reaction for T-cell binding experiments for binding, peripheral blood lymphocytes from the human skin lining were cultured for 3-6 days in the presence of the PHA stimulator. Link B7 was identified by a radioassay using B7.1 so-luble radiolabelled with 125I. The observed results indicated that all these antibodies bind to B7.1 antigen with high affinity and effectively blocked the interactions of B cells / T cells as evidenced by reduced IL-2 production and reduced proliferation of lymphocyte cultures. mixed The properties of these monoclonal antibodies specific to monkeys are summarized below: 1. The Scatchard analysis showed that the apparent affinity constants (Kd) for - the monkey antibody bonds to the B7-Ig coated plates were approximately: a: 7C10: 6.2 x 10"9 M b: 16C10: 8.1 x 10 ~ 9 M c: 7B6: 10.7 x 10" 9 M d: 20C9: 16.8 x 10"9 M The antibodies were tested in vitro in a reaction assay of Mixed lymphocytes (MLR) The MLR showed that all 4 anti-B7.1 antibodies inhibit the production of IL-2 to different degrees as shown by the following IC50 values: a: 7B6: 5.0 μg / M b: 16C10: <0.1 μg / M c: 20C9: 2.0 μg / M d: 7C10: 5.0 μg / M The anti-B7.1 monkey antibodies were tested to verify their ability to bind to B7 in lymphocytes of human peripheral blood ( PBL) The FACS analysis showed that all 4 monkey antibodies generated a positive response., 7B6, 7C10 and 20C9 were tested for their - - link to Clq by FACS analysis. The results showed that the Ig of monkey 7C10 had a strong binding to human Clq after incubation with cells transfected with CHO of B7.1. The 16C10 was positive while the antibodies of monkey 20C9 and 7B6 were negative. 5. To select an animal model for the tox pathway studies, monkey antibodies were tested with blood from animals of different species. It was determined that anti-B7.1 monkey antibodies that cross-reacted with humans were the most convenient and corresponded to chimpanzees. Based on these properties, it would be apparent that the three monkey monoclonal antibodies possess the most advantageous properties, 16C10, 7C10 and 20C9, with 16C10 and 7C10 being better than 20C9. Using the techniques described above, and in commonly assigned U.S. Patent Application Serial No. 08 / 379,072, the present inventors have cloned the variable domains of 7C10, 7B6 and 16C10, and provide the amino acid and nucleic acid sequences of the - - primatized forms of the light chain of 7C10, the heavy chain of 7C10, the light chain of 7B6, the heavy chain of 7B6, the light chain of 16C10 and the heavy chain of 16C10. These amino acid and nucleic acid sequences can be found in the Figures3a and 3b, 4a and 4b, and 5a and 5b. The amino acid and DNA sequence for the human gamma 1, and gamma 4 constant domains have been found in the US Patent Application No. 08 / 379,072. "As discussed above, these primatized antibodies are preferably expressed using the NEOSPLA expression vector shown in Figure 2 which is substantially described in commonly assigned US Patent Nos. 08 / 379,072 and 08 / 149,099, both applications incorporated herein. As previously noted, the primatized antibodies of the invention will preferably contain either the gamma 1 or gamma 4 constant regions of human immunoglobulin, preferably with human gamma 4 mutated at two positions to create gamma 4 PE. It contains two mutations, a glutamic acid in the CH2 region introduced to eliminate the residual FCR bond, and a substitution of proline in the hidden region, in order to improve the stability of the interaction of the disulfide bond in the heavy chain. , Alegre et al, J. Immunol., 148, 3461-3468, (1992), and Angel et al, Mol.Iromunol., 30, 105-158, ( 1993), both of which are incorporated herein for reference). Whether the primatized antibodies of the invention contain the constant regions gamma 1, gamma 4 or gamma 4 PE, this depends significantly on the particular target condition. Preferably, the reducing and non-reducing antibodies IgG1 and IgG4 are created and tested against specific objective conditions. Given the described linkage and functional properties of the monoclonal antibodies of the monkey of the invention, these anti-B7.1 monoclonal antibodies and the primatized forms thereof should be well suited as therapeutic agents to block the B7: CD28 interaction thus providing immunosuppression. In particular, given its high affinity to the B7.1 antigen and its ability to block B cell / T cell interactions as measured by IL-2 production and tritiated thymidine uptake in the culture of mixed lymphocytes as well as its ability to inhibit - indeed the responses triggered by the antigen in donor spleen cell cultures as shown by reduced antigen-specific IgG responses, IL-2 production and cell proliferation, these antibodies Monoclonal monkeys and primatized forms thereof should function as effective immunosuppressants that modulate the B7: CD28 route. This is significant for the treatment of many conditions in which immunosuppression is therapeutically desirable, for example, in autoimmune conditions, to inhibit undesirable anti-IgG specific responses, and also to avoid organ rejection and graft-versus-host disease. Essentially, the antibodies of the invention will be useful in the treatment of any condition wherein the deletion of the B7: CD28 pathway is therapeutically desirable. The key therapeutic indications for anti-B7.1 antibodies of the invention include, by way of example, autoimmune conditions such as idiopathic thrombocytopenia purpura (ITP), systemic lupus erythematosus (SLE), type 1 diabetes mellitus, multiple sclerosis, aplastic anemia , psoriasis, - - allergy, inflammatory biliary disease and rheumatoid arthritis. Other significant therapeutic indications of the anti-B7.1 antibodies of the invention is to avoid graft-versus-host disease (GVHD) during bone marrow transplantation and organ transplantation (BMT). The antibodies of the invention can be used to induce host tolerance to specific antigens of the donor and thereby facilitate grafting and reduce the incidence of graft rejection. It has been shown in murine models of allogeneic cardiac transplantation that intravenous administration of CTLA4-Ig can result in immunosuppression or even induction of tolerance to alloantigens.

(Lin et al, J. Exp. Med. 179: 1801, 1993; Torka et al, Proc. Nati Acad. Sci., USA, 89: 11102, 1992). It is expected that the primed anti-B7.1 antibodies of the invention will exhibit similar or greater activity. The antibodies produced in the manner described above, or by equivalent techniques, can be purified by a combination of affinity and size exclusion chromatography for characterization in functional biological assays. These assays include affinity-binding determination and specificity as well as effector function associated with the associated isotype, e.g., ADCC, or complementary fixation. Such antibodies can be used as passive or active therapeutic agents against a number of human conditions, including B cell lymphoma, diseases infections including viral conditions such as HIV / AIDS, autoimmune and inflammatory conditions, and transplantation. The antibodies can be used either in their native form or as part of an antibody / chelate, antibody / drug or antibody / toxin complex. Additionally, whole antibodies or fragments of the antibodies (Fab2, Fab, Fv) can be used as imaging reagents or as potential or uninogenic vaccines in active immunotherapy for the generation of anti-idiotypic responses. The amount of the antibody useful to produce a therapeutic effect can be determined by standard techniques well known to those of ordinary skill in the art. Antibodies will generally be provided by standard techniques within a pharmaceutically acceptable buffer, and may be administered by any desired route. Due to the efficiency of the antibodies claimed herein and their tolerance for humans it is possible to administer these antibodies respectively to combat various conditions or conditions of suffering within a human. The anti-B7.1 antibodies (or fragments thereof) of this invention are useful for inducing immunosuppression, i.e. inducing a suppression of an immune system of an animal or a human. This invention therefore relates to a method for immunosuppression induced prophylactically or therapeutically in a human or other animal in need thereof by administering an effective amount of such antibody of this invention to a human or other animal. The ability of the compounds of this invention to induce immunosuppression has been demonstrated in standard tests used for this purpose, for example, a mixed lymphocyte reaction test or a test that measures the inhibition of T cell proliferation, as measured by the Thymidine intake. The fact that the antibodies of this invention have utility in the Indian-induced immunosuppression that should be useful in the treatment or prevention of resistance or rejection of transplanted organs or tissues (eg, kidney, heart, lung, bone marrow, skin, cornea, etc.); the treatment or prevention of autoimmune, inflammatory, proliferative, and cutaneous manifestations of immunologically medicated conditions (for example, rheumatoid arthritis, lupus erythematosus, lupus erythematosus systemic, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, diabetes type 1, uveitis, nephrotic syndrome, psoriasis, atopic dermatitis, contact dermatitis and also eczematous dermatitis, seborrheic dermatitis, lichen planus, Pemplugus, bullous pemphigus, epidermolysis bullosa, urticaria, angioedema, vasculitis, erythema, cutaneous eosinophilia, Alopecia areata, etc. ), the treatment of reversible obstructive airways, intestinal inflammations and allergies (for example, inflammatory biliary disease, Coeliac disease, proctitis, eosinophilic gastroenteritis, mastocytosis, Crohn's disease and ulcerative colitis), food-related allergies (for example, migraine, rhinitis and eczema), and other types of allergies.

- - Someone with skills in the technique would be able, through routine experimentation, to determine that a non-toxic, effective amount of antibodies would be for the purpose of inducing immunosuppression. Generally, however, an effective dose in the range of about 0.05 to 100 milligrams per kilogram of body weight per day. The antibodies (or fragments thereof) of this invention should also be useful for the treatment of tumors in a mammal. More specifically, they should be useful in reducing the size of tumors, inhibiting the growth of tumors and / or prolonging the survival time of animals that have tumors. Accordingly, the invention also relates to a method of treating tumors in a human or other animal by administering to an animal or human of a non-toxic, effective amount of an antibody. One skilled in the art will be able, by routine experimentation, to determine what non-toxic, effective amount of the anti-B7 antibody would be necessary for the purpose of the treatment of carcinogenic tumors. Generally, however, an effective dose is expected to range from -about 0.05 to 100 milligrams per kilogram of body weight per day. The antibodies of this invention can be administered to a human or other animal according to the aforementioned methods of treatment in an amount sufficient to produce such an effect to a therapeutic or prophylactic degree. Such antibodies of the invention can be administered to a human or other animal in a conventional dosage form prepared by combining the antibody of the invention with a conventional pharmaceutically acceptable carrier or diluent, according to known techniques. It will be recognized by one of skill in the art that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of the active ingredient with which it is to be combined, the route of administration and other well-known variables. The route of administration of the antibody (or fragment thereof) of the invention can be oral, parenteral, by inhalation or topical. The term parenteral as used includes intravenous, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. The intramuscular subcutaneous and intramuscular forms of parenteral administration are generally preferred. Daily parenteral and oral dosage regimens for the compounds employed in the invention to induce prophylactic or therapeutical immunosuppression, or for the therapeutic treatment of carcinogenic tumors will generally be in the range of about 0.05 to 100, but preferably about 0.5 to 10, milligrams per kilogram of body weight per day. The antibodies of the invention can also be administered by inhalation. By "inhalation" is indicated intranasal administration or oral inhalation. Appropriate dosage forms for such administration, such as an aerosol formulation or a metered dose inhaler, can be prepared by conventional techniques. The preferred dosage amount of a compound of the invention to be employed is generally within the range of about 10 to 100 milligrams. The antibodies of the invention can also be administered topically. By topical administration is indicated non-systemic administration and include the application of a compound - of the antibody (or fragment thereof) of the invention externally to the epidermis, to the oral cavity and instillation of such antibody in the ears, eyes and nose, and where it does not significantly enter blood pressure. By systemic administration oral, intravenous, intraperitoneal and intramuscular administration is indicated. The amount of an antibody required for the therapeutic or prophylactic effect will, of course, be varying within the chosen antibody, the nature and severity of the condition to be treated and the treatment through which the animal passes, and is finally subject to the discretion of the doctor. A suitable topical dose of an antibody of the invention will generally be in the range of about 1 to 100 milligrams per kilogram of body weight per day.

Formulations Although it is possible for an antibody or fragment thereof to be administered alone, it is preferable that it be present as a pharmaceutical formulation. The active ingredient may comprise for topical administration, from 01001% to 10% w / w, for example from 1% to 2% by weight of the formulation, although it may comprise as much as 10% w / w but - preferably not in excess of 5% w / w and more preferably from 0.1% to 1% w / w of the formulation. Topical formulations of the present invention comprise an active ingredient together with one or more acceptable carrier (s) therefor and optionally any other therapeutic ingredient (s). He (the) carrier (s) must (be) "acceptable" in the sense of being compatible with other "ingredients of the formulation and not harmful to the containers thereof Formulations suitable for administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site in where the treatment is required, such as liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eyes, ears or nose Drops according to the present invention may comprise sterile aqueous or oily solutions or suspensions and can be prepared by dissolving the active ingredient in a suitable aqueous solution of a bactericidal and / or fungicidal agent and / or any other suitable preservative, and preferably including an active-surface agent.The resulting solution can then be clarified by filtration, transferred to an appropriate container that is sealed and sterilized by an autoclave or keeping at 90 ° -100 ° C for half an hour. Alternatively, the solution can be sterilized by filtration and can be transferred to the container by aseptic technique.

Examples of suitable bactericidal and fungicidal agents for inclusion in the drops are nitrate or phenylmercuric acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate. (0.01%). Suitable solvents for the preparation of an oily solution include a glycerol, dilute alcohol and propylene glycol. Lotions according to the present invention include those suitable for application to the skin or eyes. An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those for the preparation of drops. Lotions or liniments for application to the skin may also include an agent or a blotter and skin cooler, such as an alcohol or acetone, and / or a humectant such as -glycerol or an oil such as castor oil or arachis oil. The creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient for external application. They can be made by mixing the active ingredient in the form of finely divided powder, alone or in solution or in suspension with an aqueous or non-aqueous fluid, with the help of suitable machinery, with a greasy or non-greasy base. The base may comprise hydrocarbons such as liquid, soft or hard paraffins, glycerol, beeswax, metallic soap; a mucilage; an oil of natural origin such as oil of almonds, corn, araquis, castor or olive; wool fats or their derivatives or a fatty acid such as stearic or oleic acid together with an alcohol such as a propylene glycol or macrogols. The formulation can incorporate any suitable surface active agent such as an anionic, cationic or nonionic surface agent such as sorbitan esters or polyoxyethylene derivatives thereof. Suspending agents such as natural gums, cellulose derivatives, or inorganic materials such as silicas, silicaceas, and other ingredients such as lanolin, may also be included.

- The anti-B7.1 antibodies of the invention or fragments thereof can also be administered in combination with other portions that modulate the B7: CD28 route. Such portions include, by way of example, cytokines such as IL-7 and IL-10, CTLA4-Ig, soluble CTLA-4 and anti-CD28 antibodies and fragments thereof. Also, the antibodies of the invention can be administered in combination with other immunosuppressants. Such immunosuppressants include small molecules such as cyclosporin (CSA) and FK506; monoclonal antibodies such as anti-tumor necrosis factor, (anti-TNFa), anti-CD54, anti-CDII, anti-CDlla, and anti-IL-1; and, other soluble receptors such as rTNFa and rIL-1. It will be recognized by one skilled in the art that the optimum amount and spacing of the individual dosages of an antibody or fragment thereof of the invention will be determined by the nature and severity of the condition to be treated, the shape, the route and place of administration, and the particular animal to be treated, and that such optimal points can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimum course of treatment is - ie, the number of doses of an antibody or fragment thereof of the invention given per day for a defined number of days, can be identified by those skilled in the art using a conventional course of treatment determination tests. Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its full extent. The following formulations are, therefore, constructed as merely illustrative embodiments and not as a limitation of the scope of the present invention in any way.

Capsule Composition A pharmaceutical composition of this invention in the form of a capsule is prepared by filling a standard two-piece hard gelatin capsule with 50 mg of an antibody or fragment thereof of the invention, in powder form, 100 mg of lactose, 32 mg of talc and 8 mg of magnesium stearate.

- - Injectable Parenteral Composition A pharmaceutical composition of this invention in a form suitable for administration by injection is prepared by stirring 1.5% by weight of an antibody or fragment thereof of the invention in 10% by volume of propylene glycol and water. The solution is sterilized by filtration.

Ointment Composition ~ The antibody or fragment thereof of the invention 1.0 g. Soft white paraffin at 100.0 g. The antibody or fragment thereof of the invention is dispersed in a small volume of the vehicle to produce a smooth, homogeneous product.

Then collapsible metal tubes are filled with the dispersion.

Topical Cream Composition Antibody or fragment thereof of the invention 1.0 g. Polawax GP 200 20.0 g. Anhydrous Lanolin 2.0 g White Bee Wax 2.5 g. Methyl hydroxybenzoate 0.1 g. - 0 - Distilled water up to 100.0 g. The polawax, beeswax and lanolin are heated together at 60 ° C. A solution of methyl hydroxybenzoate is added and homogenization is achieved using high speed agitation. The temperature is then allowed to fall to 50 ° C. The antibody or fragment thereof of the invention is then added and dispersed completely, and the composition is allowed to cool with low speed agitation.

Topical Lotion Composition Antibody or fragment thereof of the invention 1.0 g. Sorbitan monolaurate 0.6 g. Polysorbate 20 0.6 g. Cetostearyl Alcohol 1.2 g. Glycerin 6.0 g. Methyl Hydroxybenzoate 0.2 g. Purified water B.P. at 100-00 mi. (B.P. British Pharmacopoeia) Methyl hydroxybenzoate and glycerin are dissolved in 70 ml of water at 75 ° C. The sorbitan monolaurate, the polysorbate 20 and the cetostearyl alcohol are melted together at 75 ° C and added to the aqueous solution. The resulting emulsion is homogenized, allowed to cool with continuous stirring and the antibody or fragment thereof of the invention is added as a suspension in the remaining water. The complete suspension is stirred until homogenized.

Composition of Eye Drops Antibody or fragment thereof of the invention 0.5 g: Methyl Hydroxybenzoate 0.01 g. Propyl Hydroxybenzoate 0.04 g. Purified water B.P. up to 100-00 mi. The methyl and propyl hydroxybenzoates are dissolved in 70 ml of purified water at 75 ° C and the resulting solution is allowed to cool. The antibody or fragment thereof of the invention is then added, and the solution is sterilized by filtration through a membrane filter (0.022 μm pore size), and aseptically packed into the appropriate sterile containers.

- -Composition for Administration by Inhalation For an aerosol container with a capacity of 15-20 ml: mix 10 mg of an antibody or fragment thereof of the invention with 0.2-0.5% of a lubricating agent, such as polysorbate 85 or oleic acid, and such a mixture is dispersed in a propellant, such as freon, preferably in a combination of (1,2-dichlorotetrafluoroethane) and difluorochloro-methane and placed in a suitable aerosol container adapted for either intranasal or oral inhalation administration.

Composition for Inhalation Administration For an aerosol container with a capacity of 15-20 ml: dissolve 10 mg of an antibody or fragment thereof of the invention in ethanol (6-8 ml), add 0.1-0.2% of a lubricating agent, such as polysorbate 85 or oleic acid; and dispersed such as in a propellant, such as freon, preferably in combination of (1,2-dichlorotetra-fluoroethane) and difluorochloromethane, and placed in an appropriate aerosol container adapted for either administration for nasal or oral administration.

- The antibodies and pharmaceutical compositions of the invention are used particularly for parenteral administration, ie, subcutaneous, intramuscular or intravenous administration. Compositions for parenteral administration will commonly comprise a solution of an antibody or fragment thereof of the invention or a mixture thereof dissolved in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be employed, for example, water, buffered water, 0.4% saline, 0.3% glycine, and the like. These solutions are sterile and usually free of particulate material. These solutions can be sterilized by conventional well known or sterilization techniques. The compositions may contain pharmaceutically acceptable excipients as required up to approximate physiological conditions such as pH adjustment and buffering agents, etc. The concentration of the antibody or fragment thereof of the invention in such a pharmaceutical formulation can vary widely, ie, from less than about 0.5%, usually or at least about 1% or as much as 15 or 20% by weight, and will be selected primarily in base to fluid volumes, viscosities, etc., according to the particular mode of administration selected. Thus, a pharmaceutical composition of the invention for intramuscular injection could be prepared containing 1 MI sterile buffered water, and 50 mg of an antibody or fragment thereof of the invention. Similarly, a pharmaceutical composition of the invention for intravenous infusion could be made containing 250 ml of sterile Ringer's solution and 150 mg of an antibody or fragment thereof of the invention. The real methods for the preparation of compositions "parenterally administrable are well known or will be apparent to those skilled in the art, and are described in greater detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pennsylvania, incorporated herein by reference The antibodies (or fragments thereof) of the invention can be lyophilized for storage and can be reconstituted in a suitable carrier before use.This technique has been shown to be effective with conventional immunoglobulins and known reconstitution and lyophilization techniques can be employed. in the technique.

Depending on the expected result, the pharmaceutical composition of the invention can be administered for treatments. In the therapeutic application, the compositions are administered to a patient suffering from a condition, in an amount sufficient to cure or at least particularly to arrest the condition and its complications. In prophylactic applications, the compositions contain the present antibodies or a mixture thereof are administered to a patient who is not in a disease state to improve the patient's resistance. The single or multiple administration of the pharmaceutical compositions can be carried out with levels of dosages and standards that are to be selected by the attending physician. In any case, the pharmaceutical composition of the invention should provide an amount of the altered antibodies (or fragments thereof) of the invention sufficient to effectively treat the patient. It should also be noted that the antibodies of this invention can be used for the design and synthesis of either peptidic or non-peptidic (mimetic) compounds that would be useful therein -therapeutics as the antibody. See, for example., Saragovi et al., Science, 253, 792-795 (1991). To further illustrate the invention, the following examples are provided. These examples are not intended and are not constructed as limiting the invention.

Example 1 The recombinant immunoglobulin libraries shown on the surface of the filamentous phage were first described by McCafferty et al, Nature, 348: 552-554, 1990 and Barbas et al, Proc. Nati Acad. Sci., USA 88: 7978-7982, 1991. Using this technology, high affinity antibodies have been isolated from the immune human recombinant libraries (Barbas et al, Proc. Nati, Acad. Sci., USA 589: 10164-10168, 1992). Although the phage sample concept used was substantially similar to that described by Barbas, 1991, Id., The technique has been modified by the substitution of a single vector for monkey libraries to reduce the possibility of recombinations and improve stability. This vector, pMS, Figure 1 contains a single lac / operator promoter for efficient transcription and translation of the DNA of chain-ngera monkey, and heavy, polycistronic. This vector contains two leader sequences, omp A (Movva et al, J.

Biol. Chem., 255: 27-29, (1980), for the light chain and pei B (Lei, J. Bact., 4379-109: 4383 (1987) for the heavy chain Fd. Both leading sequences are translated in peptides of hydrophobic signals that direct the secretion of the cloned products of light and heavy chains into the periplasmic space In the oxidative environment of the periplasm, the two chains bend and the disulfide bonds are formed to create stable fragments of Fab. derived the vector structures of the bluescript phagemids (Stratagene, La Jolla, CA) and contains the genes for the beta-lactase enzyme that confers resistance to ampicillin (carbenicillin) to the bacteria that anchor the pMS DNA. The origin of the replication of the ColEl multicopy plasmids and the origin of replication of the filamentous bacteriophage were also derived from bluescript. The origin of the replication of phage fl (called the intragenic region), signals the initiation of single-stranded DNA synthesis, the initiation of capsid formation and the termination of RNA synthesis by viral enzymes. Replication and assembly of pMS DNA strands into phage particles requires viral proteins that must be provided by a helper phage. VCSM13 helper phages have been used which are particularly suitable for this, since they contain a gene that codes for kanamycin resistance. Bacteria infected with VCSM13 and pMS can be selected by adding both kanamycin and carbenicillin to the growth medium. The bacteria will eventually produce filamentous phage particles that contain either pMS or VCSM13 genomes. The packaging of the phage helper is less efficient than that of the pMS, resulting in a mixed population of phage containing recombinant pMS phage predominantly. The ends of the phage collect specific minor coat proteins at each end. Of particular interest here is gene III that is present in three of the five copies at one end of the phage. The product of gene III are the residues of 406 amino acids and are required for the infection of the phage of E. coli via the F pili. The first of the two domains of the heavy chain, the variable domain and the CH1, are fused to the terminal half with carboxy of the gene III protein. This recombinant pili protein directed by leader pei B, is secreted into the peroplasma - where it accumulates and forms disulfide bonds with the light chain before it is incorporated into the phage coat. Also, another vector contains a FLAG sequence generated by genetic engineering downstream of gene III. FLAG is a peptide of 8 amino acids expressed in the carboxy terminal of the Fd protein. We used anti-FLAG M2 monoclonal commercially available for both purification and detection of phage Fab by ELISA (Brizzard, Bio Techniques, 16 (4): 730-731, (1994)). After construction of the pMS vector its ability to produce the Fab phage linkage was tested using control antibody genes. An anti-tetanus toxoid antibody (obtained from Dr. Carlos Barbas) was cloned in pMS and transformed to XLI-blue. The cells were coinfected with VCSM13 and phages were generated which show the anti-tetanus toxoid antigen. Efficient experiments were carried out in which the phage of the anti-tetanus toxoid was combined with the phage carrying an irrelevant antibody in 1: 100,000. Three rounds of testing were performed by applying 50 μl of the mixed phage to the polystyrene walls coated with the antigen (tetanus toxoid). The non-adherent phages were washed and the adherent phages were eluted with acid. The eluted phage was used to infect a fresh aliquot of X-Ll-Blue bacteria and the helper phage was added.

After amplification overnight, the phages were prepared and again tested with plates coated with antigen. After three rounds of testing, the inventors were able to show that they had successfully enriched the anti-tetanus toxoid phages. The success of this technology also depends on the ability to prepare soluble Fabs for the characterization of the final tested or separated product. This was achieved by cutting the gene III of the pMS DNA using the restriction enzyme Nhe I followed by the re-ligation. After the III gene was cut, the FAB was no longer shown on the surface of the phage but accumulated in the periplasmic space. The Used ones were prepared from bacteria expressing soluble Fab and tested for specificity using ELISA. High levels of soluble Fab were detected. In order to adapt the phage sample technology for use with the macaque libraries, specific primers were developed for monkey immunoglobulin genes amplified with PCR. These were based on the sequences of macaques obtained while the PRIMATIZED® antibody technology was developed (see, the reference 08 / 379,072, incorporated herein by reference) and the databases containing the human sequences.

(Kabat et al, (1981), "Sequences of Proteins of Immunological Interest," U.S. Dept. of Health and Human Services, National Institute of Health). Three sets of primers were developed to cover the amplification of the macaque repertoire. The first set of primers was designed to "amplification domain of the heavy chains VH and CH1 (Fd). It consisted of a primer CH1 domain 3 'and six specific primers family 5' VH is linked to the region structure 1. the second set of primers, for amplification of the complete lambda chain, covers many lambda chain subgroups. it consists of a 3 'primer and three degenerate 5' primers that bind in the framework region 1 VL. the third set of primers was designed for amplification subgroup kappa chain. it consists of a 3 'primer and five primers structure 1 VK. Using each of these sets, PCR parameters were optimized to obtain signals sufficiently strengths of each pair of primers for sufficient material to be available for the cloning of the library. Recently combating macaque libraries were created in the vector pMS using these PCR conditions optimized. Bone marrow biopsies were taken from monkeys immunized with CD4 as the source of immunoglobulin RNA. The libraries contained approximately 106 members and had separated or specific binders in the walls coated with antigen.

Example 2 Development of Reagents B7 / CTLA-4 A number of reagents were generated for the purpose of immunizing the monkeys, develop links and functional assays in vi tro, select the heterohybridomas and separate the phage libraries. Table 1 lists each of the reagents and their intended purpose. In the case of B7.1, the RNA was extracted from the SB cells and converted to cDNA using reverse transcriptase. The first strand of cDNA was amplified with PCR using specific B7.1 primers and cloned into mammalian expression vectors NEOSPLA from IDEC. The CHO cells were transfected with NEOSPLA DNA of B7.1 and the clones expressing the associated membrane - with B7.1, were identified. The fusion protein of B7.1 was similarly generated, except that the B7.1 gene amplified with PCR was cloned into the NEOSPLA cassette vector containing the human CH2 and CH3 immunoglobulin genes. The CHO cells were transformed with NEOSPLA B7.1 / Ig DNA and the stable clones that secreted the B7.1 / Ig fusion protein were amplified. In general, reagents B7.2 and CTLA4 were generated in the same manner, except that for "B7.2 RNA was isolated from human spleen cells that had been stimulated for 124 hours with anti-Ig and IL- 4, and for CTLA4 builders, the source of the gene were T cells activated with PHA.

- - Table 1 The availability of these reagents, together with the monoclonal antibodies to B7.1 (L3074) (Becton Dickinson, 1994) and B7.2 (Fun-1 (Engel et al, Blood, 84, 1402-1407, (1994) and antiserum of goat and purified rabbit, were developed specifically to detect Fab monkey fragments, which facilitates the identification of antibodies having the desired properties.

Example 3 Generation of a Phage Sample Library The recombinant phage sample libraries were generated from monkeys immunized with B7.1 and B7.2. Biopsies of lymph nodes and bone marrow 7-12 were performed after immunization to harvest the B cells rich in RNA and plasma cells. The RNA was isolated from the lymphocytes using the method described by Chomczynski Anal. Biochem., 162 (1), 156-159, (1987). The RNA was converted to cDNA using an oligo dT primer and reverse transcriptase. The first strand of cDNA was aliquoted and amplified with PCR using the primer sets of the heavy chain and kappa, lambda Fd region described above and any of the Pfu polymerase.

(Stratagene, San Diego) or Taq polymers (Promega, Madison). The heavy chain PCR amplified products were pooled, cut with the restriction enzymes Xho VSpe I and cloned into the pMS vector. Subsequently, the light chain PCR products were pooled, cut with Sac I / Xba I restriction enzymes, and cloned to create the recombinant library. XLI-Blue E. coli was transformed with the DNA of the library and super-infected with VCSM13 to produce the antibodies that show the phage. The library was rinsed four times in polystyrene wells coated with the B7.1 or B7.2 antigen. The clones of the individual phages from each round of rinse were analyzed. The pMS vector DNA was isolated and the gene III separated. The soluble Fab fragments were generated and tested in ELISA for the binding to B7.1 and B7.2.

Example 4 Characterization of Phage Fab Fragments Fab fragments from monkey phage were characterized by their specificity and the ability to block B7.1-Ig and B7.2-Ig that bind to cells transfected with CTLA-4- Ig or CTLA-4. Phage fragments were also characterized for cross-reactivity after the first rinse for four rounds in B7 species used for immunization to select high affinity fragments. The identified Fab fragments from the four rounds of rinsing either on the surfaces coated with the B7.1 or B7.2 antigen were by infection and growth in 24-hour fermentation cultures of E coli. The fragments were purified by binding of Kodak FLAG to an anti-FLAG affinity column. Purified phage Fabs were tested for affinity by modified Scatchar analysis with direct linkage based on ELISA (Katoh et al, J. Chem. BioEng., 76: 451-454, (1993)) - using Fab anti-bodies. goat monkey or Anti-FLAG MAbs conjugated with horseradish peroxidase.

The anti-monkey Fab reagents will be absorbed against the human heavy chain constant Ig region to eliminate any cross-reactivity to the B7-Ig. The Kd values are calculated for each fragment after the measurements or the direct link to the plates coated with B7.1-Ig or B7.2-Ig.

"Example 5 Blocking the Fab Fragment of the Link Phage CTLA-4 / B7 Fab fragments that most effectively block B7-Ig binding at the lowest concentrations were selected as lead candidates. The selections are made by competition of the 125I-B7-Ig linkage to cells transfected with CTLA-4-Ig or CTLA-4. Additional selection criteria include blocking the reaction of mixed lymphocytes (MLR), as measured by the inhibition of 3H-thymidine uptake in responder cells (Azuma et al, J. Exp. Med., 177: 845-850; Azuma et al, Nature, 301: 76-79, (1993)) and direct analysis of IL-2 production using - -IL-2 assay kits. There are three or four candidates that are most effective at inhibiting MLR binding trials and CTLA-4 and are chosen to be cloned into the expression vector of the mammals described above for transfection into CHO cells and expression of chimeric monkey / human antibodies.

EXAMPLE 6 Generation of Mono Heterohíbridomas Monohype heterohybridomas secreting monoclonal antibodies are generated from existing immunized animals whose serum gives the positive test for B7.1 and / or B7.2 Lymph node biopsies are taken from the animals positive for any or both of the antigens The method of hybridoma production is similar to the established method for the generation of monkey anti-CD4 antibodies (Newman, 1992 (Id.).) Monkeys with high titers serum will have sections of inguinal lymph nodes removed under anesthesia.The lymphocytes are washed from the tissue and fused with heteromyeloma cells KH6 / B5 (Carrol et al, J. Immunol.Meth., 89: 61-72, (1986)) using polyethylene glycol (PEG) Hybrids are selected in the HAT medium and stabilized by repeated subcloning in 96-well plates Monoclonal antibodies specific for the B7.1 antigen are selected for Reactivity cross-linked to B7.2. The anti-B7 monkey antibodies will be characterized for B7 / CTLA-4 binding block using the 125I-B7-Ig binding assay. Inhibition of MLR by taking 3 H-thymidine and direct measurements of IL-2 production are used to select three candidates. Two candidates will be carried forward in the Phase II studies and expressed in CHO cells while all functional studies are repeated. For the purpose of developing an animal model for in vivo pharmacology, anti-B7 antibodies will be tested in cells of various animal species. The establishment of an animal model will allow clinical studies to be carried out for the selected clinical indication.

Example 7 As discussed above, using the heterohbridoma methods, 4 anti-B7.1 monkey monoclonal leaders were identified: 16C10, 7B6, 7C10 and 20C9. These antibodies were characterized as follows: - The Scatchard analysis showed that the apparent affinity constants (Kd) for the monkey antibodies that bind to the B7-Ig coated plates were approximately the following: a: 7C10: 6.2 x 10"9 M b: 16C10: 8.1 x 10" 9 M c: 7B6: 10.7 x 10"9 M d: 20C9: 16.8 x 10" 9 M The antibodies were tested in vi tro in a mixed lymphocyte reaction assay (MLR ). The MLR showed that all 4 anti-B7.1 antibodies inhibit the production of IL-2 in different degrees: a: 7B6: 5.0 μg / ml b: 16C10: 0.1 μg / ml c: 20C9: 2.0 μg / ml d: 7C10: 5.0 μg / Ml Anti-B7.1 monkey antibodies were tested to verify their ability to bind to B7 in human peripheral blood lymphocytes (PBL). The FACS analysis showed that all 4 monkey antibodies gave a positive response. Mono antibodies 16C10, 7B6, 7C10 and 20C9 were tested for their binding to Clq - - by FACS analysis. The results showed that the monkey Ig 7C10 had a strong binding to Clq human after incubation with cells transfected with CHO of B7.1. 16C10 was also positive while monoclonal antibodies 20C9 and 7B6 were negative.

Example 8 Using the primatized antibody methodology incorporated as reference to US Patent Serial No. 08 / 379,072, and using the NEOSPLA vector system shown in Figure 2, the heavy and light variable domains of 7C10, 7B6 and 16C10 were cloned and the primatized forms of the. They were synthesized in CHO cells using the NEOSPLA vector system. The amino acid and nucleic acid sequences for primed heavy and light chains 7C10, heavy and light chains 7B6, and light and heavy chains of 16C10 are shown respectively in Figures 3a, 3b, 4a, 4b, 5a and 5b.

- - Example 9 Confirmation experiments of the non-crosslinked reactivity of the antibody binding sites CTLA-4 and PRIMATIZED® in B7.1. In the competitive link tests using Biotinylated CTLA-4Ig (Figure 6), primed unlabeled 16C10 (ie, P16C10) was unable to block the CTLA-4Ig binding to CHO cells transfected with B7.1. As you can see, unlabeled CTLA-4Ig and unlabelled B7.1 compete effectively under these conditions. In a second experiment using Biotinylated P16C10, the same conclusions were reached. In the experiment shown in Figure 7, the binding of P16C10-Biotin is inhibited by both P16C10 and unlabeled B7.1Ig, but not by CTLA-4Ig. Although it is reported that CTLA-4Ig will be as high as 4-10 times in affinity (Kd = 0.4 nM, Morton et al., J. Immunol. 156: 1047-1054 (1996)), there is no significant inhibition. of the P16C10 binding even at CTLA-4Ig concentrations as high as 100 fold in excess. Similar results were obtained using primed 7C10 antibody (P7C10) when substituted for P16C10 in the experiments - - (data not provided).

Example 10 Comparison of the ability of mouse antibodies L307.4 and BB-1 to bind to CHO B7 cells in the presence of CTLA-4Ig. The binding of the murine anti-B7 antibody L307.4 and BB-1 in the presence of CTLA-4Ig was studied to determine whether the binding sites of the mouse antibody "overlapped with the binding sites of CTLA-4. Competitive assays using Pl 6C10-Biotin, L307.4-Biotin, and CTLA-4Ig-Biotin were made to ensure that affinity differences will not prevent the detection of competitive binding.The results are shown in Figures 8 and 9. The results of Figure 8 confirm that previous studies of mouse BB-1 antibody do not compete with P16C10.These results also show that there is some cross-reactivity to L307.4 of about 50% .The results of Figure 8 confirm that both the BB-1 as L307.4 compete with each other and that BB-1 completely blocks the binding of CTLA-4Ig-Biotin to CHO cells transfected with B7.1 The BB-1 does not significantly affect the P16C10 binding to the cells CHO positive B7.1. The results shown in Figure 9 indicate better than 50% of the competition between CTLA-4Ig-Biotin is used in the binding experiments. The Figure 9 shows that the CTLA-4Ig-Biotin binding is effectively blocked by all B7.1 inhibitors except for P16C10, therefore P16C10 recognizes a unique binding determined in B7.1 allowing the binding of the normal ligand CTLA-4 in the generation of negative signals. Previous functional studies (data not shown) suggest a decreased ability of L307.4 to block IL-2 production in the allogeneic MLR, which correlates with the hypothesis that it may interfere with the negative signaling of CTLA-4 . It is not clear how many of the other murine antibodies reported in the literature give complete inhibition of CTLA-4 binding; however, this result may be important in defining the true functional mechanisms of specific antibodies B7.1 and B7.2. These results confirm previous studies using B7-Ig in competition with Pl6C10-Biotin to bind to CHO- -transfected cells with B7.1. The studies also confirm previous observations of non-inhibition of P16C10 by CTLA-4lg. These results strongly suggest that primate antibodies are specific for a unique B7.1 epitope independent of the CTLA-4 binding site which interacts primarily with CD28. This type of interaction would provide a benefit, since it has the ability to block the B7.1 link to the CD28 receivers while still allowing the negative signaling function of the CTLA-4 to remain uninhibited. Perceived interactions can lead to a down regulation of the activation response of total T cells regardless of the predominance of the Th1 or Th2 phenotypes. Similar results were obtained using P7C10 when it was replaced by P16C10 in the experiments (data not provided).

EXAMPLE 11 Experiment demonstrating the ability of P16C10 to bind and block the interactions of B7.1 with the CD28 receptor. An experiment to determine if the P16C10 linkage of B7.1 can block the interaction of B7.1 - with CD28 was attempted by radiolabelling B7.1lg with I, followed by the competitive binding to the non-activated, positive T-cell lymphocytes of CD28. The results shown in Figure 10 demonstrate that radiolabeled B7.1Ig binds specifically to cells T, as confirmed by inhibition with unlabeled B7.1Ig. The results also show that the CTLA-4Ig, anti-CD28 and P16C10 are all capable of blocking this interaction. The results further confirm that P16C10 blocks the binding of the CD28 / B7 interaction with an IC50 of < 1 ug / mL. The above results were obtained under conditions where CTLA-4 associated with the membrane was not expressed (Linsley et al., J. Exp. Med. 173: 721-730 (1991)) and was confirmed by blocking with the antibody anti-CD28. Similar results were obtained using P7C10 when it was replaced by P16C10 in the experiments (data not provided). It is expected that these primatized antibodies, given their probable low antigenicity and low effector function, will be well suited as therapeutic. In fact, it has recently been shown that primed 16C10 exhibits a human Clq bond.

- Those skilled in the art will recognize or be able to use no more than routine experiments equivalent to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (28)

- - CLAIMS

1. A monoclonal antibody that binds specifically to the B7.1 antigen (CD28) or the B7.2 antigen (CD86), and which antibody inhibits the binding of said B7.1 or B7.2 antigen to CD28.

2. The monoclonal antibody according to claim 1, characterized in that the antibody binds specifically to the B7.1 antigen (CD80).

3. The monoclonal antibody according to claim 2, characterized in that the antibody does not inhibit the binding of B7.1 antigen to C LA-4.

4. The monoclonal antibody according to claim 1, characterized in that the antibody binds specifically to the B7.2 antigen (CD86).

5. The monoclonal antibody according to claim 4, characterized in that the antibody does not inhibit binding of the B7.2 antigen to CTLA-4.

- -

6. The monoclonal antibody according to claim 1, characterized in that it inhibits the production of IL-2 by the T cells.

7. The monoclonal antibody according to claim 2, characterized in that it selectively inhibits the interaction of B cells and T cells via the CD28 / B7.1 pathway.

8. The monoclonal antibody according to claim 4, characterized in that it inhibits

• selectively the interaction of B cells and T cells via the CD28 / B7.2 pathway.

9. The monoclonal antibody according to claim 1, characterized in that it is able to inhibit the in vitro production of IL-2 by the T lymphocytes.

10. The monoclonal antibody according to claim 9, characterized in that the antibody is capable of inhibiting the production of IL-2 when it is added to a T lymphocyte containing the culture at a concentration of at least 10 μg / ml.

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11. - A monoclonal antibody that binds to the same epitope in B7.1 as 16C10 or 7C10, or which monoclonal antibody inhibits the interaction of 16C10 or 7C10 with B7.1.

12. - The monoclonal antibody according to claim 1, characterized in that it is a primatized antibody.

13. - The monoclonal antibody according to claim 1, characterized in that it is a chimeric or humanized human, mouse / human antibody.

14. - The monoclonal antibody according to claim 1, characterized in that B7.1 is human B7.1.

15. - The monoclonal antibody according to claim 1, characterized in that B7.2 is human B7.1.

- - 16.- A method for treating a condition involving the interactions of T cells / B cells characterized in that it comprises administering an amount of a monoclonal antibody according to claim 2 sufficient to inhibit the binding of B cells and cells T via route B7.1 / CD28.

17. - A method for treating a condition involving the interactions of T cells / B cells, characterized in that it comprises administering an amount of a monoclonal antibody according to claim 4 sufficient to inhibit the binding of B cells and T cells via the route B7.2 / CD28.

18. - The method according to claim 16, characterized in that the condition is an autoimmune condition.

19. - The method according to claim 17, characterized in that the condition is an autoimmune condition.

20. The method according to claim 16, characterized in that the condition is selected from the group consisting of idiopathic purple thrombocytopenia, systemic lupus erythematosus, diabetes mellitus type 1, rheumatoid arthritis, psoriasis, aplastic anemia, inflammatory biliary disease, allergy and multiple sclerosis .

21. The method according to claim 17, characterized in that the condition is selected from the group consisting of idiopathic purple thrombocytopenia, systemic lupus erythematosus, diabetes mellitus type 1, rheumatoid arthritis, psoriasis, aplastic anemia, inflammatory biliary disease, allergy and multiple sclerosis .

22. - The method according to claim 16, characterized in that the condition is a graft-versus-host disease.

23. - The method according to claim 17, characterized in that the condition is a graft-versus-host disease.

24. - The method according to claim 16, characterized in that the condition is selected from the group consisting of B cell lymphoma, infectious diseases and inflammatory conditions.

25. - The method according to claim 17, characterized in that the condition is selected from the group consisting of B cell lymphoma, infectious diseases and inflammatory conditions.

26. - A pharmaceutical composition suitable for the treatment of a treatable condition by inhibiting B7: CD28 binding comprising an antibody according to claim 1.

27. - The method according to claim 16, characterized in that the antibody is administered in combination with another recombinant protein or small molecule immunosuppressants.

28. - The method according to claim 17, characterized in that the antibody is administered in combination with another recombinant protein or small molecule immunosuppressants.