MXPA95001794A - Ctla4 molecules and il-4 link molecules and uses of mis - Google Patents

Abstract

The present invention relates to the CTLA4 receptor as a ligand for the B7 antigen. The complete amino acid sequence encoding the human CTLA4 receptor gene is provided. Methods are provided for expressing CTLA4 as an immunoglobulin fusion protein, for preparing hybrid CTLA4 fusion and for using soluble fusion proteins, their fragments and derivatives, including monoclonal antibodies reactive with B7 and CTLA4 to regulate the interactions of T cells and the immune responses mediated by such interaction

Description

"MOLECULES CTLA4 AND MOLECULES OF LINK IL-4 AND USES OF THEM" Inventors: PETER S. LINSLEY, North American, domiciled at 2430 9th Avenue West, Seattle, Washington 98119, E.U.A .; JEFFREY A. LEDBETTER, North American, domiciled at 306 NW 113th Place, Seattle, Washington 98117, E.U.A .; NITIN DAMLE, North American, domiciled at 865 Ridge Road, Monmount Junction, New Jersey 08852, E.U.A .; WILLIAM BRADY, North American, domiciled at 618 219th Place SW, Bothell, Washington 98021, E.U.A. and PHILIP M. WALLACE, British, domiciled at 3020 64th Avenue SW, #D, Seattle, Washington 98116, E.U.A.

Causaire: BRISTOL-MYERS SQUIBB COMPANY, Delaware State Corporation, E.U.A. domiciled at 345 Park Avenue, New York, New York 10154, E.U.A.

FIELD OF THE INVENTION In this application, several publications are referred to. The descriptions of these publications in their entireties are incorporated herein by reference in this application, to fully describe the state of the art to which this invention pertains. The present invention relates to the expression of the CTLA4 hybrid fusion proteins, the CTLA4 receptor gene, identification of the interaction between the CTLA4 receptor and the cells expressing the B7 antigen, and methods for regulating cellular interactions involving the CTLA4 receptor and the B7 antigen.

BACKGROUND OF THE INVENTION The hallmark of a vertebrate's immune system is the ability to discriminate from "self" (from oneself) to "non-self" (strange). This property has led to the evolution of a system that requires multiple signals to achieve optimal immune activation (Janeway, Cold Spring Harbor Symp. Quant. Biol. 54: 1-14 (1989)). The interactions of B-cells T cells are essential for the immune response. The concentrations of many cohesive molecules found in T cells and B cells increase during an immune response (Springer et al., (1987), supra; Shaw and Shimuzu, Current Opinion in Immunoloqy, Eds. Kindt and Long, 1:92 -97 (1988)); and Hemler Immunology Today 9: 109-113 (1988)). The increased concentrations of these molecules may help explain why activated B cells are more effective in stimulating the proliferation of antigen-specific T cells than resting B cells (Kaiuchi et al., J. Immunol., 131: 109- 114 (1983), Kreiger et al., J. Immunol., 135: 2937-2945 (1985), McKenzie, J. Immunol., 141: 2907-2911 (1988), and Hawrylowicz and Unanue, J. Immunol., 141: 4083. -4088 (1988)). The generation of an immune response of T lymphocytes ("T cells") is a complex process involving cell-cell interactions (Springer et al., A. Rev. Immunol., 5: 223-252 (1987)), particularly between T cells and accessory cells such as B cells and production of soluble immune mediators (cytokines or lymphokines) (Dinarello and Mier, New Enql., Jour. Med 317: 940-945 (1987)). This response is regulated by several T-cell surface receptors, including the T-cell receptor complex (Weiss et al., Ann. Rev. Immunol. 4: 593-619 (1986) and other "accessory" surface molecules. "(Springer et al., (1987) supra) Many of these accessory molecules occur naturally in cell surface differentiation (CD) antigens defined by the reactivity of monoclonal antibodies on the surface of cells (McMichael, Ed., Leukocyte Tvping III, Oxford Univ. Press, Oxford, NY (1987).) Antigen-independent intercellular interactions involving lymphocyte accessory molecules are essential for an immune response (Springer et al., (1987), For example, the binding of the T-cell associated protein, CD2, to its LFA-3 ligand, a widely expressed glycoprotein (reviewed in Shaw and Shimuzu, supra), is important for optimizing T cell activation is of the antigen (Moingeon et al., Nature 339: 314 (1988)). An important adhesion system involves the binding of the LFA-1 glycoprotein found in lymphocytes, macrophages and granulocytes (Springer et al., (1987), supra.; Shaw and Shimuzu (1988), supra) to their ICAM-1 ligands (Makgoba et al., Nature 331: 86-88 (1988)) and ICAM-2 (Staunton et al., Nature 339: 61-64 (1989) ). The accessory molecules of the CD8 and CD4 T cells reinforce the adhesion of the T cell by interaction with MHC class I molecules (Norment et al., Nature 336: 79-81 (1988)) and class II (Doyle and Strominger, Nature 330: 256-259 (1987)), respectively. "Local receptors" are important for the control of lymphocyte migration (Stoolman, Cell 56: 907-910 (1989)).

The VLA glycoproteins are integrins, which appear to mediate the functions of lymphocytes that require adhesion to components of the extracellular matrix (Hemler, supra). The adhesion systems CD2 / LFA-3, LFA-l / ICAM-1 and ICAM-2; and VLA are distributed in a wide variety of cell types (Springer et al., (1987), supra; Shaw and Shimuzu, (1988) supra and Hemler, (1988), supra). Numerous in vitro studies have shown that cytokines are involved in the generation of alloreactive effector cells. For example, membrane bound IL-4 and soluble IL-4 receptor are administered separately to mice and shown to increase the lymphoproliferative response (William C. Fanslow et al. "Regulation of Alloreactivity in vivo by IL-4. and the soluble IL-4 receptor "J. Immunol., 147: 535-540 (1991)). Specifically, administration of IL-4 to BALB / c mice resulted in a slight increase in the lymphoproliferative response. In contrast, the soluble IL-4 receptor suppressed this response to allogeneic cells in a dose-dependent manner. In addition, a neutralizing antibody against IL-4 and another against the soluble IL-4 receptor were effective inhibitors of the lymphoproliferative response. It was proposed many years ago that activation of B lymphocytes requires two signals (Bretscher and Cohn, Science 169: 1042-1049 (1970)) and it is now believed that all lymphocytes require two signals for optimal activation, a specific signal from antigen or clonal signal, as well as a second signal, a non-specific antigen signal (Janeway, supra). Freeman et al. (J. Immunol., 143 (8): 2714-2722 (1989)) isolated and sequenced a cDNA clone encoding an antigen for the activation of B cells recognized by mAb B7 (Freeman et al., J. Immunol 138: 3260 (1987)). COS cells transfected with this cDNA have been shown to stain by both mAb B7 and mAb BB-1 (Clark et al., Human Immunol., 16: 100-113 (1986); Yokochi et al., J. Immunol. 823 (1981)); Freeman et al., (1989) supra; and Freedman et al., (1987), supra)). In addition, the expression of this antigen has been detected on the cells of other lineages, such as monocytes (Freeman et al., Supra). The signals required for the antigenic response of the T (T- ^) helper cells are provided by the cells presenting the antigen (APC). The first signal is initiated by the interaction of the T cell receptor complex (Weiss, J. Clin Invest 86: 1015 (1990)) with the antigen presented in the context of the class II molecules of the histocompatibility complex. major (MHC) on APC (Alien, Immunol. Today 8: 270 (1987)). This antigen-specific signal is not sufficient to generate a complete response and in the absence of a second signal, can actually lead to clonal inactivation or anergy (Schwartz, Science 248: 1349 (1990)). The requirement for a second "costimulatory" signal provided by the MHC has been demonstrated in many experimental systems (Schwartz, supra, Weaver and Unanue, Immunol. Today 11:49 (1990)). The molecular nature of this second signal or signals is not fully understood, although it is clear that in some cases both soluble molecules such as interleukin (IL) -l (Weaver and Unanue, supra) and the membrane receptors involved in the adhesion intercellular (Springer, Nature 346: 425 (1990)) can provide costimulatory signals. The CD28 antigen, a homodimeric glycoprotein of the immunoglobulin superfamily (Aruffo and Seed, Proc. Nati, Acad. Sci. 84: 8573-8577 (1987)), is an accessory molecule found in most mature human T cells. (Damle et al., J, Immunol., 131: 2296-2300 (1983)). Current evidence suggests that this molecule operates on an alternate T cell activation pathway other than that initiated by the T cell receptor complex (June et al., Mol.Cell. Biol. 7: 4472-4481 (1987) ). Monoclonal antibodies (mAbs) reactive with the CD28 antigen can increase the responses of the T cells initiated by various polyclonal stimuli (reviewed by June et al., Supra). These stimulatory effects may result from the production of the cytokine induced by mAb (Thompson et al., Proc. Nati. Acad. Sci 86: 1333-1337 (1989); and Lindsten et al., Science 244: 339-343 (1989)) as a consequence of the stabilization of the increased mRNA (Lindsten et al., (1989), supra). The anti-CD28 mAbs can also have inhibitory effects, that is, they can block the reactions of autologous mixed lymphocytes (Damle et al., Proc.Nat.Acid.Sci.78: 5096-6001 (1981)) and the activation of the clones. of antigen-specific T cells (Lesslauer et al., Eur. J. Immunol., 16: 1289-1296 (1986)). Studies have shown that CD28 is a counter-receptor for the B cell activation antigen, B7 / BB-1 (Linsley et al., Proc. Nati, Acad. Sci. USA 87: 5031-5035 (1990)). For convenience, the B7 / BB-1 antigen is referred to hereafter as the "B7 antigen". B7 ligands are also members of the imunoglobulin superfamily but have, in contrast to CD28 and CTLA4, two Ig domains in their extracellular region, a domain similar to the N-terminal variable domain (V) followed by a domain similar to the domain constant (C). An important non-specific costimulatory signal is delivered to the T cells, when there are at least two members of the homologous B7 family found in the APCs, B7-1 (also called B7 or CD80) and B7-2, both of which can provide costimulatory signals to the T cells either by means of CD28 or CTLA4. Co-stimulation by means of CD28 or CTLA4 is essential for the activation of T cells since a soluble Ig fusion protein of CTLA4 (CTLA4-Ig) was successfully used to block the activation events of T cells in vitro and in vivo . Failure to deliver this second signal can lead to clonal inactivation or T-cell anergy. Interactions between the CD28 and B7 antigen have been characterized using genetic fusions of the extracellular portions of the B7 antigen and the CD28 receptor and the immunoglobulin ( Ig) Cgammal (heavy chains of the constant region) (Linsley et al., J. Exp. Med. 173: 721-730 (1991)). The immobilized B7Ig fusion protein, as well as B7 CHO positive cells, have been shown to co-stimulate T cell proliferation. The stimulation of T cells with B7 CHO positive cells also specifically stimulates increased concentrations of the transcribed fragments for IL. -2. Additional studies have shown that the anti-CD28 mAb inhibited the production of IL-2 induced in certain T cell leukemia cell lines by cell interactions with a B-cell leukemia line (Kohno et al., Cell.Immunol. -1-10 (1990)). CD28 has a domain similar to the individual extracellular variable (V) region (Aruffo and Seed, supra). A homologous molecule, CTLA4 has been identified by differential screening of a cDNA library of murine T-cytolytic cells (Brunet et al., Nature 328: 267-270 (1987)). Transcribed fragments of the CTLA4 molecule have been found in populations of T cells that have cytotoxic activity, suggesting that CTLA4 must function in the cytolytic response (Brunet et al., Supra; and Brunet et al., Immunol., Rev. 103- 21-36 (1988)). Researchers have reported the cloning and mapping of a gene for the human counterpart of CTLA4 (Dariavach et al., Eur. J.

Immunol. 18: 1901-1905 (1988)) to the same chromosomal region (2q33-34) than CD28 (Lafage-Pochitaloff et al., Immunoqenetics 31: 198-201 (1990)). An Ig fusion of CTLA4 binds to B7-1 with «20 times greater avidity than a corresponding Ig fusion of CD28. The comparison of the sequence between this DNA of human CTLA4 and that codifies for the CD28 proteins, reveals significant homology of the sequence, with the highest degree of homology in the juxtamembrane and cytoplasmic regions (Brunet et al., 1988, supra, - Dariavach et al., 1988, supra). The high degree of homology between CD28 and CTLA4, together with the co-localization of their genes, raises questions as to whether these molecules are also functionally related. However, since the protein product of CTLA4 has not yet been expressed successfully, these questions remain unanswered. The expression of the soluble derivatives of the cell surface glycoproteins in the immunoglobulin gene superfamily has been achieved for CD4, the receptor for HIV-1 and CD28 and B7 receptors, using hybrid fusion molecules consisting of DNA encoding the amino acids corresponding to the portions of the extracellular domain of the CD4 receptor fused to the antibody domains (gammal immunoglobulin (Capon et al., Nature 337: 525-531 (1989) (CD4) and Linsley et al., J. Exp. Med., Supra (CD28 and B7).) There is a need for molecules which can identify in vitro B7-positive B cells, ie B cells activated for leukocyte typing and for classification. In addition, there is a need for molecules which were used to prevent the rejection of organ transplants and to inhibit the symptoms associated with lupus erythematosus and other autoimmune diseases. In the past, most therapies were based on paninmunosuppressive drugs, such as cyclosporin A or monoclonal antibodies (MAbs) for CD3 to prevent organ transplants or inhibit lupus symptoms. Unfortunately, these drugs must often be taken during the individual's life, diminish the entire immune system and frequently produce secondary diseases such as increased frequency of infections and cancer.

BRIEF DESCRIPTION OF THE INVENTION Accordingly, the present invention provides the complete and correct DNA sequence encoding the amino acid sequence corresponding to the CTLA4 receptor protein and identifying the B7 antigen (e.g. B7-1 and B7-2 antigens) as a natural ligand for the CTLA4 receptor. The invention also provides a method for expressing DNA as a protein product of the immunoglobulin CTLA4 (Ig) fusion. Modes of the invention include the CTLA4Ig fusion protein and hybrid fusion proteins including the CD28 / CTLA4Ig fusion proteins (which is also referred to herein as the CTLA4 / CD28Ig fusion protein). They also provide methods for using the CTLA4 fusion protein, the B7Ig fusion protein, the hybrid fusion proteins and fragments and / or their derivatives, such as monoclonal antibodies reactive with CTLA4 and the B7 antigen to regulate cellular interactions and immune responses. .

The human CTLA receptor protein of the invention is encoded by 187 amino acids and includes a newly identified N-linked glycosylation site. The CTLA4Ig fusion protein of the invention binds to the B7 antigen expressed on the activated B cells and the cells of other lineages, a ligand for the CD28 receptor on the T cells. The CTLA4Ig binds to the B7 antigen with significantly higher affinity than B7 which binds to the CD28 receiver. The CTLA4Ig construct has a first amino acid sequence corresponding to the extracellular domain of the CTLA4 receptor fused to a second amino acid sequence corresponding to the Cgammal domain of human Ig. The first amino acid sequence contains amino acid residues from about position 1 to about position 125 of the amino acid sequence corresponding to the extracellular domain of CTLA4 linked to a second amino acid sequence containing the amino acid residues corresponding to the hinge regions, CH2 and CH3 of human IgCgammal. The fusion protein is preferably produced in dimeric form. Soluble CTLA4Ig is a potent in vitro inhibitor of T and B lymphocyte responses. Soluble CTLA4 and its hybrid fusion proteins, for example soluble hybrid fusion proteins, such as CD28 fusion proteins, are also contemplated in the invention. / CTLA4Ig. The extracellular domain of CTLA4 is an example of a soluble CTLA4 molecule. Alternatively, a molecule having the extracellular domain of CTLA4 bound to a tag peptide is another example of a soluble CTLA4 molecule. As an example of a soluble hybrid fusion protein, the present invention provides the CD28 / CTLA4Ig fusion proteins, which have a first amino acid sequence corresponding to the extracellular domain fragments of CD28 linked to a second amino acid sequence corresponding to fragments of the extracellular domain of CTLA4Ig and a third amino acid sequence corresponding to the hinge, CH2 and CH3 regions of human IgCgammal. One embodiment of the hybrid fusion proteins is a CD28 / CTLA4Ig fusion construct having a first amino acid sequence containing the amino acid residues from about position 1 to about position 94 of the amino acid sequence corresponding to the extracellular domain of CD28 , linked to a second amino acid sequence containing the amino acid residues from about position 94 to about position 125 of the amino acid sequence corresponding to the extracellular domain of CTLA4, attached to a third amino acid sequence containing amino acid residues corresponding to the hinge regions CH2 and CH3 of the human IgCgammal. Other embodiments of the hybrid fusion proteins of the invention are described in Tables I and II and Example 7. A method for regulating the interactions of T cells with other cells, inhibiting the interaction of cells, is also included in the invention. T CTLA4 -positive with B7 positive cells, reacting T cells with ligands for the CTLA4 receptor. Ligands include the B7Ig fusion protein, a monoclonal antibody reactive with the CTLA4 receptor and fragments of the antibody. The invention also provides a method for regulating the interactions of T cells with B7 positive cells using a ligand for the B7 antigen. Such a ligand is the soluble CTLA4 fusion protein, for example, the CTLA4Ig fusion protein of the invention, its fragments or derivatives, the soluble CD28 / CTLA4 hybrid fusion protein, for example the hybrid fusion protein CD28 / CTLA4Ig or an antibody monoclonal reagent with the B7 antigen. The invention further includes a method for the treatment of diseases of the immune system mediated by the interactions of T cells with B7 positive cells by administering a ligand reactive with B7 antigen to regulate the interactions of T cells with B7 positive cells. The ligand is the CTLA4Ig fusion protein or the hybrid of the CD28 / CTLA4Ig fusion protein or a monoclonal antibody reactive with the B7 antigen. A monoclonal antibody reactive with the soluble CTLA4 fusion protein and a monoclonal antibody reactive with the soluble CD28 / CTLA4 fusion protein are described for use in the regulation of cellular interactions. Also described is a novel Chinese Hamster Ovary cell line that stably expresses the CTLA4Ig fusion protein. In addition, the present invention provides a method for blocking the B7 interaction to regulate the immune response. This method comprises contacting the lymphocytes with a B7 binding molecule and an IL4 binding molecule. Additionally, the present invention provides a method for regulating an immune response, which comprises contacting positive B7 lymphocytes with a B7 binding molecule and an IL4 binding molecule. As well, the invention provides a method for inhibiting the rejection of tissue transplantation by an individual, the individual is a recipient of the transplanted tissue. This method comprises administering to the individual a B7 binding molecule and an IL4 binding molecule.

The present invention further provides a method for inhibiting graft-versus-host disease in an individual, which comprises administering to the individual a B7 linker molecule and an IL4 linker molecule.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagrammatic representation of the CTLA4Ig fusion constructs as described in Example 2, infra. Figure 2 is a photograph of a gel obtained from the chromatographic purification of SDS-PAGE of CTLA4Ig as described in Example 2, infra. Figure 3 depicts the complete amino acid sequence encoding the human CTLA4 receptor (SEQ ID NOS: 13 and 14) fused to the oncostatin M signal peptide (position -25 to -1) and including the N-glycosylation site - newly identified unit (position 109-111), as described in Example 3, infra. Figure 4 represents the results of the analysis FACSR of the binding of the B7Ig fusion protein to COS cells transfected with CD28- and CTLA4 as described in Example 4, infra. Figure 5 depicts the results of FACSR analysis of purified CTLA4Ig binding on CHO cells positive for B7 antigen (B7 +) and on a lymphoblastoid cell line (PM LCL) as described in Example 4, infra. Figure 6 is a graph illustrating the competition binding assays of B7Ig labeled with 125j to immobilized cTLA4Ig as described in Example 4, infra. Figure 7 is a graph showing the results of the Scatchard analysis of 125 I-labeled B7Ig binding to immobilized CTLA4Ig as described in Example 4, infra. Figure 8 is a photograph of a gel of SDS-PAGE chromatography of the immunoprecipitation analysis of CHO positive cells to B7 and PM LCL cells labeled on the surface with 125? as described in Example 4, infra. Figure 9 is a graph depicting the effects on the proliferation of CTLA4Ig T cells as measured by the incorporation of thymidine- [- ^ H] as described in Example 4, infra. Figure 10 is a bar graph illustrating the effects of CTLA4Ig on immunoglobulin secretion induced by helper T cells (^) by human B cells as determined by the enzyme immunoassay (ELISA) as described in FIG. Example 4, infra.

Figures HA, 11B, and 11C are line graphs showing the survival of human pancreatic islet xenografts. Figures 12A, 12B, 12C and 12D are photographs of histopathological sections of human islets transplanted under the BIO mouse kidney capsule. Figure 13 is an online graph showing the prolongation of islet survival grafted with MAb to human B7. Figure 14 is an online graph showing the induction of donor-specific non-response for islet graft antigens by CTLA4Ig. Figure 15 is an online graph showing serum titre titers of the antibody from mice injected with sheep blood red cells (SRBC)., MAb L6 and rat Ig, mAb L6 and anti-IL4, CTLA4Ig and rat Ig, CTLA4Ig and anti-IL4. The X axis measures the antibody titer in the serum. The Y axis measures time in days.

The filled square represents the mice injected with SRBC on day 0 and day 46. The empty box represents the mice injected with SRBC on day 46. The filled circle represents the mice injected with mAb L6 and the rat immunoglobulin. The empty circle represents the mice injected with mAb L6 and the anti-IL4 antibody. The full triangle represents the mice represented with CTLA4Ig and the rat immunoglobulin. The open triangle represents mice injected with CTLA4Ig and the anti-IL4 antibody. Figure 16 is an online graph showing the serum titer levels of the antibody of mice injected with KLH, L6 mAb and rat Ig, L6 and anti-IL4 mAb, CTLA4Ig and rat Ig, CTLA4Ig and anti-IL4. The X axis measures the antibody titer in the serum. The Y axis measures time in days. The filled square represents the mice injected with hemocyanin herring (KLH) on day 46. The full circle represents the mice injected with mAb L6 and the rat immunoglobulin. The open circle represents the mice injected with mAb L6 and the anti-IL4 antibody. The closed triangle represents mice injected with CTI-A4Ig and rat immunoglobulin. The empty triangle represents the mice injected with CTLA4Ig and the anti-IL antibody. Figure 17 is a graph showing the isolation of the sequence of members of the CD28 and CTLA4 family. The sequences of human (H), mouse (M), rat (R) and chicken (Ch) CD28 are aligned with human and mouse CTLA4. The residues are numbered from the N-terminus of the mature protein with the signal peptides and the transmembrane domains underlined and the regions similar to CDR being noted. The dark shaded areas underline the complete preservation of the residues, while the light shaded areas underline the amino acid substitutions conserved in all members of the family. Figure 18 is a line graph showing mutants CTLA4Ig and CD28Ig that bind to B7-1. Figure 19 is a schematic map of the hybrid fusion proteins CTLA4 / CD28Ig. The empty areas represent the sequence of CD28; the filled areas represent the sequence of CTLA4; the cross-hatched areas represent the start of the Fc IgG (also refer to Table I). Figures 20A / B. An online graph shows that the CTLA4 / CD28Ig hybrid fusion proteins bind with a lot of avidity to CHO B7-1 cells. Figure 21. A molecular model of the extracellular domain similar to domain v of monomeric CTLA4Ig.

DETAILED DESCRIPTION OF THE INVENTION DEFINITION As used in this application, the following words or phrases have the specified meanings. As used herein the "B7-blocking interaction" means that it interferes with the binding of the B7 antigen to its ligands such as CD28 and / or CTLA4 thereby obstructing the interaction of T cells and B cells. used herein, a "B7 binding molecule" means any molecule, which will bind to the B7 antigen. As used herein, an "IL4 binding molecule" means any molecule which will recognize and bind to IL4. As used herein, a "CTLA4 mutant" means a molecule having amino acids which are similar to the amino acid sequence of the extracellular domain of CTLA4, such that the molecule recognizes and binds to the B7 antigen. As used herein a "CD28 mutant" means a molecule having amino acids which are similar to the amino acid sequence of the extracellular domain of CD28, such that the molecule recognizes and binds to the B7 antigen. As used herein a "hybrid CTLA4 / CD28 fusion protein" is a molecule that has at least portions of the extracellular domains of both CTLA4 and CD28, in such a way that the molecule recognizes and binds to the B7 antigen.

In order that the invention described herein may be more fully understood, the following description is established. This invention is related to the expression isolation of the human CTLA4 receptor found on the surfaces of the T cells, which bind to the B7 antigen expressed on the activated B cells and the cells of other lineages and to the expression of the fusion protein products soluble of the CTLA4 receptor gene. The invention also provides methods for the use of the expressed CTLA4 receptor to regulate cellular interactions, including the interactions of T cells with B7 positive cells. In a preferred embodiment, the complete and correct DNA sequence encoding the amino acid sequence corresponding to the human CTLA4 receptor protein of the invention is cloned using PCR. The cDNA containing the predicted, complete coding sequence of CTLA4 is assembled from two amplified PCR fragments of H38 RNA and inserted into the expression vector, CDM8 as described in detail in the Examples, infra. The isolates are transfected into COS cells and tested for binding to B7 Ig, a soluble fusion protein having an amino acid sequence corresponding to the extracellular domain of B7 and a region of human immunoglobulin (Ig) Cgammal, as described by Linsler, et al., J. Exp. Med. 173: 721-730 (1991). The DNA sequence of an isolate, designated OMCTLA4, is determined and found to correspond exactly to the predicted human CTLA4 sequence, fused at the N-terminus to the oncostatin M signal peptide. The CTLA4 receptor is encoded by 187 amino acids (exclusive of signal peptide and stop codons) and includes a newly identified N-linked glycosylation site at the amino acid positions 109-111 (see Figure 3, infra). The CTLA4 receptor is expressed using the oncostatin M signal peptide. In another preferred embodiment, the soluble forms of the CTLA4 receptor gene product protein (CTLA4Ig) are prepared using the fusion proteins having a first amino acid sequence corresponding to the domain extracellular CTLA4 and a second amino acid sequence corresponding to the human IgCgamal domain. Cloning and expression plasmids (CDM8 and 7TLN) are constructed to contain the cDNAs encoding portions of the amino acid sequence corresponding to the human CTLA4 receptor based on the cDNA sequences described herein, wherein the cDNA which encodes a first sequence of amino acids corresponding to a fragment of the extracellular domain of the CTLA4 receptor gene binds to the DNA encoding a second amino acid sequence corresponding to an IgC region, which allows the expression of the CTLA4 receptor gene by alternating the solubility of the expressed CTLA4 protein. In this way, the soluble CTLA4Ig fusion protein is encoded by a first amino acid sequence containing the amino acid residues from about position 1 to about position 125 of the amino acid sequence corresponding to the extracellular domain of CTLA4 attached to a second sequence of amino acids containing the amino acid residues corresponding to the hinge, CH2 and CH3 regions of human IgCgammal. The fusion protein is preferably produced in the dimeric form. The construct is transfected into COS or CHO cells and the CTLA4Ig is purified and identified as a dimer. In accordance with the practice of this invention, CTLA4Ig and the hybrid CTL4 / CD28 fusion protein can have amino acid substitutions in the amino acid sequence corresponding to the external domain of CTLA4 to produce molecules, which could retain the functional property of CTLA4 , namely, the molecule having such substitutions will still bind to the B7 antigen. These amino acid substitutions include, but are not necessarily limited to, amino acid substitutions known in the art as "conservative". For example, it is a well-established principle of protein chemistry that certain amino acid substitutions, called "conservative amino acid substitutions", can often be made in a protein without altering the conformation or function of the protein. Such changes include the substitution of either isoleucine (I), valine (V) and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) by glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) by threonine (T) and vice versa. Other substitutions may also be considered conservative, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) can often be interchangeable, such as alanine and valine (V). Methionine (M), which is relatively hydrophobic, can often be exchanged with leucine and isoleucine and sometimes with valine. The lysine (K) and arginine (R) are frequently interchangeable in locations in which the significant characteristic of the amino acid residue is its charge and the pK different from these two amino acid residues is not important. Still other changes can be considered "conservative" in particular environments. In fact, using the methodologies described herein, mutants of the B7 linker molecule are produced. A mutant comprises (1) a sequence starting with the amino acid at position 1 and ending with the amino acid at position 95 of the CD28 receptor protein; (2) a sequence starting with the amino acid at position 95 and ending with the amino acid at position 125 of the extracellular domain of CTLA4; and (3) a sequence corresponding to the human IgGgammal domain. The second mutant comprises (1) a sequence starting with the amino acid at position 1 and ending with the amino acid at position 95 of the CD28 receptor protein; (2) a sequence starting with the amino acid at position 95 and ending with the amino acid at position 120 of the extracellular domain of CTLA4; and (3) a sequence corresponding to the human IgCgammal domain. The present invention provides a method for blocking the interaction of B7 to regulate the immune response, which comprises contacting the lymphocytes with a B7 binding molecule and an IL4 binding molecule. Lymphocytes can be B7-positive lymphocytes. In addition, the present invention provides a method for regulating an immune response, which comprises contacting B7-positive lymphocytes with a B7-binding molecule and an IL-4 binding molecule. The immune response may be a response of B cells that result in the inhibition of antibody production. Additionally, the immune response may be a response of T cells that result in the inhibition of cell-mediated immunity. In addition, the immune response may be an inhibition of lymphocyte proliferation. Also, the present invention provides a method for inhibiting rejection of tissue transplantation by an individual, the individual is a recipient of the transplanted tissue. This method can comprise administering to the individual a B7-linker molecule and an IL4-linker molecule. The invention further provides a method for inhibiting graft-versus-host disease in an individual, which comprises administering to the individual a B7-binding molecule and an IL-4 binding molecule. In accordance with the practice of this invention, the B7-linker molecule can be a CTLA4Ig fusion protein. For example, the CTLA4Ig fusion protein can be a fusion protein, tending a first amino acid sequence containing amino acid residues from about position 1 to about position 125 of the amino acid sequence corresponding to the extracellular domain of CTLA4 and a second amino acid sequence containing the amino acid residues corresponding to the hinge, CH2 and CH3 regions of the human immunoglobulin Cgammal. Alternatively, the B7 linker molecule can be a soluble CD28 / CTLA4 hybrid fusion protein. For example, the hybrid CD28 / CTLA4Ig fusion protein can be a hybrid fusion protein having a first amino acid sequence that corresponds to a portion of the extracellular domain of the CD28 receptor fused to a second amino acid sequence corresponding to a portion of the domain extracellular CTLA4 receptor and a third amino acid sequence corresponding to the hinge, CH2 and CH3 regions of the human immunoglobulin Cgammal. In addition, the IL4 binding molecule can be a monoclonal antibody, which specifically recognizes and binds IL4. Alternatively, the IL4 binding molecule is a soluble IL4 receptor, which recognizes and binds to IL4 (Fanslow et al., 1991). The DNA encoding the amino acid sequence corresponding to the CTLA4Ig fusion protein has been deposited with the American Type Culture Collection (ATCC) in Rockville, Maryland, under the stipulations of the Budapest Treaty on May 31, 1991 and the ATCC accession number: 68629 has been agreed. The present invention provides the first protein product of the transcribed fragments of CTLA4 in the form of a protein of soluble fusion. The CTLA4Ig protein forms a disulfide bond dimer having two subunits, each of which has an M of about 50,000, indicating that the native CTLA4 probably exists on the surface of the T cells as a homodimer bound to the disulfide. The B7 antigen has been shown to be a ligand for the CD28 receptor on T cells (Linsley et al., Proc. Nati, Acad. Sci. USA, supra). The CTLA4 receptor molecule appears functionally and structurally related to the CD28 receptor; both are receptors for the activation antigen of B cells, B7, although CTLA4 seems to have greater affinity for B7, among the higher lymphoid adhesion systems already reported. However, CTLA4Ig is shown to bind more strongly to positive B7 (B7 +) cell lines than CD28Ig. Other experiments show that CTLA4 is a higher affinity receptor for the B7 antigen than the CD28 receptor. Additionally, CTLA4Ig is shown to bind to an individual protein on lymphoblastoid cells, which is similar in size to the B7 antigen. CTLA4Ig inhibited the proliferation of T cells and inhibited the production of IgM induced by T? _. In another preferred embodiment, hybrid fusion proteins are constructed which have amino acid sequences corresponding to fragments of different receptor proteins. For example, amino acid sequences corresponding to fragments selected from the extracellular domains of CD28 and CTLA4 are joined to form soluble hybrid CD28 / CTLA4 fusion proteins, for example the CD28 / CTLA4Ig fusion protein. This protein is obtained so that it has a first amino acid sequence that contains the amino acid residues that correspond to a fragment of the extracellular domain of CD28 linked to a second amino acid sequence that corresponds to a fragment of the extracellular domain of CTLA4Ig and to a third amino acid sequence which correspond to the hinge, CH2 and CH3 regions of the human IgGgammal. One embodiment of the hybrid fusion proteins is a CD28 / CTLA4Ig fusion construct having a first amino acid sequence containing amino acid residues from about position 1 to about position 94 of the amino acid sequence, which corresponds to the extracellular domain of CD28 , linked to a second amino acid sequence containing the amino acid residues from about position 94 to about position 125 of the amino acid sequence corresponding to the extracellular domain of CTLA4, joined by a third amino acid sequence corresponding to the hinge regions, CH2 and CH3 of human IgCgammal. Techniques for cloning and expressing the DNA sequences encoding the amino acid sequences corresponding to the CTLA4 receptor protein, soluble fusion proteins and hybrid fusion proteins, eg oligonucleotide synthesis, PCR, cell transformation , vector construction, expression systems and the like are well established in the art, and most technicians are familiar with the standard resource materials for specific conditions and procedures. However, the following paragraphs are provided for convenience and in other modifications when necessary and may serve as a guide.

Cloning and Expression of the Coding Sequences for Fusion Protein and Receptors The fusion protein constructs corresponding to CD28IgCgammal and B7IgCgammal to characterize the CTLA4Ig of the present invention and to prepare hybrid CD28 / CTLA4 fusion proteins, are prepared as described by Linsley et al., J. Exp. Med. 173: 721-730 (1991), incorporated herein by reference. Alternatively, cDNA clones can be prepared from RNA obtained from cells expressing B7 antigen and CD28 receptor based on knowledge of the published sequences for those proteins (Aruffo and Seed, and Freeman, supra) using standard procedures. CTLA4Ig fusions consisting of the DNA encoding the amino acid sequences corresponding to the extracellular domain of CTLA4 and the hinge, CH2 and CH3 regions of human IgCgammal were constructed by ligation of the fragments by PCR. The cDNA encoding the amino acid sequences is amplified using the polymerase chain reaction ("PCR") technique (U.S. Patent Nos. 4,683,195 and 4,683,202 to Mullis et al., And Mullís &Faloona, Methods Enzvmol 154: 335-350 (1987)). The CTLA4Ig fusion polypeptides are obtained from the DNA encoding the amino acid sequences containing the amino acid residues from about position 1 to about position 125 of the amino acid sequence corresponding to the extracellular domain of CTLA4 and the DNA encoding the amino acid sequences corresponding to the hinge, CH2 and CH3 regions of IgCgammal.

Because the expression of the CTLA4 receptor protein in human lymphoid cells had not previously been reported, it was necessary to locate a source of CTLA4 mRNA. The cDNA is selected by PCR made from the total cellular RNA of several human leukemia cell lines, using, as primers, oligonucleotides of the published sequence of the CTLA4 gene (Dariavach et al., Supra). From the cDNA tested, H38 cells (a leukemia line associated with HTLV II) provided the best performance of the products by PCR having the expected size. Since a signal peptide for CTLA4 was not identified in the CTLA4 gene, the N-terminus of the predicted CTLA4 sequence was fused to the oncostatin M signal peptide (Malik et al., Molec., And Cell. Biol. : 2847 (1989)) in two steps using the oligonucleotides as described in the examples, infra. The product of the PCR reaction was ligated with the / cDNA encoding the amino acid sequences corresponding to the hinge, CH2 and CH3 regions of the IgCgammal in an expression vector, such as CDM8 or 7TLN. To obtain the DNA encoding full-length human CTLA4, a cDNA encoding the transmembrane and cytoplasmic domains of CTLA4 is obtained by PCR of H38 cells and linked with a CTLA4Ig fragment, obtained as described in FIG. above, which encodes the oncostatin M signal peptide fused to N-terminus of CTLA4, using oligonucleotide primers as described in the Examples, infra. The PCR fragments are ligated into plasmid CDM8, resulting in an expression plasmid that codes for the full length of the CTLA4 gene and designated 0MCTLA4. For the construction of the DNA encoding the amino acid sequence corresponding to the hybrid fusion proteins, the DNA encoding the amino acids corresponding to the extracellular domain portions of a receptor gene binds to the DNA encoding the corresponding amino acids. to the portions of the extracellular domain of another receptor gene and to the DNA encoding the amino acid sequences corresponding to the hinge, CH2 and CH3 regions of the human IgCgammal using procedures as described above for the B7Ig, CD28Ig and CTLA4Ig. Thus, for example, DNA encoding amino acid residues from about position 1 to about position 94 of the amino acid sequence corresponding to the extracellular domain of the CD28 receptor, bind to DNA encoding amino acid residues from about position 94 to about position 125 of the amino acid sequence corresponding to the extracellular domain of the CTLA4 receptor and to the DNA encoding the amino acid sequences corresponding to the hinge, CH2 and CH3 regions of human IgGgammal. To produce large amounts of the cloned DNA, the vectors containing the DNA encoding the fusion constructs of the invention are transformed into suitable host cells, such as the bacterial cell line of E. coli strain MC106l / p3 (Invitrogen Corp ., San Diego, CA) using standard procedures and colonies are selected for the appropriate plasmids. The clones containing the DNA encoding the fusion constructs obtained as described above are then transfected into host cells suitable for expression. Depending on the host cell used, the transfection is performed using standard techniques appropriate for such cells. For example, transfection in mammalian cells is performed using the transfection measured with DEAE-dextran, co-precipitation with CaP04, lipofection, electroporation or fusion of protoplasts and other methods known in the art including: fusion of lysozyme or fusion of the erythrocytes, seeding with debris, direct incorporation, osmotic shock or sucrose, direct microinjection, indirect microinjection such as techniques mediated by erythrocytes and / or subjecting host cells to electric currents. The above list of transfection techniques is not considered exhaustive, since there is no doubt that other procedures are developed to introduce genetic information into cells. Expression in a culture of eukaryotic host cells derived from multicellular organisms is preferred (Tissue Cultures, Academic Press, Cruz and Patterson, Eds. (1973)). These systems have the additional advantage of the ability to bind introns and thus be used directly to express the genomic fragments. Useful host cell lines include Chinese hamster ovary (CHO) cells, monkey kidney cells (COS), VERA cells and HeLa cells. In the present invention, cell lines that stably express the fusion constructs are preferred. Expression vectors for such cells commonly include promoter and control sequences compatible with mammalian cells such as, for example, CMV promoters (CDM8 vector) and avian sarcoma virus.

(ASV) (vector pLN). Other commonly used early and late promoters include those from Simian Virus 40 (SV 40) (Fiers, et al., Nature 273: 113 (1973)) or other viral promoters such as those derived from polyoma, Adenovirus 2 and papilloma virus. bovine. The controllable promoter, hMTII (Karin, et al., Nature 299: 797-802 (1982)) may also be used. The general aspects of the transformations of the mammalian host cell system have been described by Axel (U.S. Patent No. 4,399,216 issued August 16, 1983). Now it seems that the "better" regions are important in optimizing expression. These are, generally in sequences found upstream or downstream of the promoter region in the uncoded DNA regions. The origins of replication can be obtained, if needed, from viral sources. However, integration into the chromosome is a common mechanism for DNA replication in eukaryotes. Although preferred host cells for the expression of the fusion constructs include eukaryotic cells such as COS or CHO cells, other eukaryotic microorganisms can be used as hosts. Laboratory strains of Saccharomyces cerevisiae, Baker's yeast, are more commonly used although other strains such as Schizosaccharomyces pombe can be used. Using the vectors, for example, the 2 μ origin of Broach replication, Meth. Enz. 101: 307 (1983) or other compatible yeast replication origins (eg, Stinchcomb et al., Nature 282: 39 (1979)); Tschempe et al., Gene 10: 157 (1980); and Clarke et al., Meth. Enz. 101: 300 (1983)) can be used. The control sequences for the yeast vectors include promoters for the synthesis of glycolytic enzymes (Hess et al., J. Adv. Enzvme Reg. 7: 149 (1968); Holland et al., Biochemistry 17: 4900 (1978)). Additional promoters known in the art include the CMV promoter provided in the CDM8 vector (Toyama and Okayama, FEBS 268: 217-221 (1990); the promoter for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol.

Chem. 255: 2073 (1980)) and those for other glycolytic enzymes. Other promoters which have the additional advantage of transcription controlled by growth conditions are the promoter regions for alcohol dehydrogenase 2, isocitochrome C, acid phosphatase, the degrading enzymes associated with nitrogen metabolism and the enzymes responsible for the use of maltose and galactose. It is also believed that the terminator sequences are advantageous at the 3 'end of the coding sequences. Such terminators are found in the. 3 'untranslated region following the coding sequences in genes derived from yeast. Alternatively, prokaryotic cells can be used as hosts for expression. The prokaryotes are most frequently represented by the various strains of E. coli; however, other microbial strains can also be used. Commonly used prokaryotic control sequences which are defined herein, includes promoters for transcription initiation, optionally with an operator, together with ribosome binding site sequences, include commonly used promoters such as beta promoter systems -lactamase (penicillinase) and lactose (lac), the tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res. 8: 4057 (1989)) and the PL promoter derived from lambda and the ribosome binding site of the N gene (Shimatake et al., Nature 292: 128 (1981)). The sequence of nucleotides encoding the CD28Ig and CTLA4Ig proteins and the hybrid fusion proteins such as CD28 / CTLA4Ig can be expressed in a variety of systems as set forth in the following. The cDNA can be cut by the appropriate restriction enzymes and ligated into prokaryotic or eukaryotic expression vectors suitable for such expression. Because the CD28 and CTLA4 receptor proteins naturally occur as dimers, it is believed that the successful expression of these proteins requires an expression system, which allows these proteins to be formed as dimers. Truncated versions of these proteins (ie, formed by the introduction of a stop codon in the sequence in an upward position of the transmembrane region of the protein) do not appear to be expressed. The expression of the CD28 and CTLA4 receptors as fusion proteins allows the formation of the dimer of these proteins. In this way, expression of the CTLA4 protein as a fusion product is preferred in the present invention. A stable CHO line of the invention, designated Chinese Hamster Ovarian Cell Line CTLA4Ig-24, is preferred for the expression of CTLA4Ig and has been deposited with the ATCC under the terms of the Budapest Treaty on May 31, 1991 and agreed accession number of ATCC 10762. Expression of the CTLA4 receptor of the invention is accomplished by transfecting a cell line such as COS cells and detecting expression by binding of transfected CTLA4 cells to a ligand for the CTLA4 receptor, by example by testing the binding of the cells to the B7Ig fusion protein. The sequences of the resulting constructs are confirmed by DNA sequencing using known methods, for example as described by Sanger et al., Proc. Nati Acad. Sci. USA 74: 5463 (1977), further as described by Messing et al., Nucleic Acids Res. 9: 309 (1981) or by the method of Maxam et al., Methods Enzvmol. 65: 499 (1980)).

Recovery of Protein Products As observed in the above, the CD28 and CTLA4 receptor genes are not stably expressed as mature proteins using the direct expression of the DNA encoding the truncated protein. To allow the formation of the homodimer, the DNA encoding the amino acid sequence corresponding to the extracellular domains of CD28 and CTLA4 and including the codons for a signal sequence, such as that of Oncostatin M in the cells, capable of processing is fused to the DNA encoding the amino acid sequence, which corresponds to the Fc domain of a naturally dimeric protein. Purification of these fusion protein products after secretion of the cells is facilitated using antibodies reactive with the anti-immunoglobulin portion of the fusion proteins. When secreted into the medium, the product of the fusion protein is recovered using standard protein purification techniques, for example by application to protein A columns.

USE The protein and / or CTLA4Ig fusion fragments of the fusion protein can be used to react with B7 positive cells, such as B cells to regulate immune responses mediated by the interactions of T cells with antigen-positive cells. B7 or in vitro for the typing of leukocytes, to define the mature stages of B cells and / or diseases associated with B cells (Yokochi et al., J. Immuno 128 (2): 823). Leukocyte surface is made by immunofluorescence technology or immunoenzymatic methods, but other detection means are possible.Soluble CTLA4 proteins and CTLA4 / CD28 hybrid fusion proteins, and / or fragments and derivatives of these proteins, can also be used for react with B7 positive cells, including B cells, to regulate immune responses mediated by B-cell responses dependent on the B cells T. The term "fragment" as used herein, meant a portion of the amino acid sequence that codes for the protein referred to as "CTLA4". A fragment of the soluble CTLA4 protein that can be used is a polypeptide having an amino acid sequence that corresponds to some portion of the amino acid sequence corresponding to the CTLA4 receptor used to obtain the soluble CTLA4 protein, as described herein. The B7 antigen expressed on activated B cells and cells of other lineages and the CD28 receptor expressed on T cells can be directly linked together and this interaction can mediate cell-cell interaction. Such interactions directly trigger the CD28 activation pathway to T cells, leading to cytokine production, T cell proliferation and B cell differentiation in cells that produce immunoglobulins. Activation of the B cells that occurs can cause increased expression of the B7 antigen and in addition the stimulation of CD28, leading to a state of chronic inflammation such as in autoimmune diseases, allograft rejection, graft versus host disease or reactions chronic allergies. The blocking or inhibition of this reaction can be carried out to prevent the production of the cytokine of the T cells and in this way avoid or reverse the inflammatory reactions. Soluble CTLA4, for example CTLA4Ig is shown herein to be a potent inhibitor of in vitro lymphocyte functions that require the interaction of T and B cells. This indicates the importance of the interactions between the B7 antigen and its counterreceptors, CTLA4 and / or CD28. The cytoplasmic domains of murine and human CTLA4 are similar (Dariavach et al., Supra, 1988), suggesting that this region has important functional properties. The cytoplasmic domains of CD28 and CTLA4 also share homology. CTLA4 is a more potent in vitro inhibitor of lymphocyte responses than either anti-BBl or anti-CD28 mAbs. CTLA4Ig has no direct stimulatory effects on T cell proliferation to counteract its inhibitory effects. Thus, the CTLA4Ig fusion protein may function as a better inhibitor in vivo than the anti-CD28 monoclonal antibodies. The immunosuppressive effects of CTLA4Ig in vitro suggest its use in therapy for the treatment of autoimmune disorders involving the activation of abnormal T cells or the production of abnormal Ig. The CTLA4Ig fusion protein is expected to exhibit inhibitory properties in vivo. In this way, it is expected that CTLA4Ig will act to inhibit T cells in a manner similar to the effects observed for the anti-CD28 antibody, under similar conditions in vivo. Under conditions where T cell / B cell interactions are occurring as a result of contact between T cells and B cells, the CTLA4Ig binding introduced to react with B7 antigen-positive cells, e.g. B cells, may interfere, that is, they inhibit the interactions of T cells / B cells that result in the regulation of immune responses. Because of this uniquely inhibitory effect, CTLA4Ig is expected to be useful in vivo as an inhibitor of T cell activity, on non-specific inhibitors such as cyclosporin and glycosteroids. In one embodiment, the CTLA4Ig fusion protein or the hybrid CTLA4 / CD28Ig proteins can be introduced into a suitable pharmaceutical carrier in vivo, ie, administered in a human subject for the treatment of pathological conditions such as diseases of the immune system or cancer. Introduction of the fusion protein in vivo is expected to result in interference with T cell interactions with other cells, such as B cells, as a result of the binding of the ligand to B7 positive cells. The prevention of normal T cell interactions can result in decreased activity of T cells, for example, decreased T cell proliferation. In addition, administration of the fusion protein in vivo is expected to result in the regulation of in vivo levels of cytokines, including, but not limited to, interleukins, for example interleukin ("IL") - 2, IL-3 , IL-4, IL-6, IL-8, growth factors including tumor growth factor ("TGF"), a factor that stimulates the formation of colonies ("CSF"), interferons ("IFNs") and tumor necrosis factor ("TNF") to promote the desired effects in an individual.

For example, when the fusion protein is introduced in vivo, it can block the production of cytokines, which contribute to malignant growth, for example of tumor cells. The fusion protein can also block the proliferation of virus-dependent activation of T cells, such as the virus that causes AIDS, HTLV1. Under some circumstances, as seen in the above, the effect of the administration of the CTLA4Ig fusion protein or its fragments in vivo is inhibitory, resulting from the blocking by the fusion protein of CTLA4 and CD28 activation resulting from the contact of T cells / B cells. For example, the CTLA4Ig protein can block the proliferation of T cells. The introduction of the CTLA4Ig fusion protein in vivo will produce effects in both immune responses mediated by T and B cells. The fusion protein also it can be administered to an individual in combination with the introduction of cytokines or other therapeutic reagents. In a further embodiment of the invention, other reagents, including derivatives reactive with the CTLA4Ig fusion protein or the CTLA4 receptor are used to regulate the interactions of T cells. For example, antibodies and / or antibody fragments reactive with the CTLA4 receptor. they can be selected to identify those capable of inhibiting the binding of the CTLA4Ig fusion protein to the B7 antigen. Antibodies or antibody fragments such as Fab or F (ab ') 2 / fragments can then be used to react with T cells, for example to inhibit the proliferation of T cells. Monoclonal antibodies reactive with the CTLA4 receptor can produced by hybridomas prepared using known procedures, such as those introduced by Kohler and Milstein (Kohler and Milstein, Nature, 256: 495-97 (1975)) and their modifications, to regulate cellular interactions. These techniques involve the use of an animal which was primed to produce a particular antibody. The animal can be primed by injection of an immunogen (e.g. B7Ig fusion protein, CTLA4Ig fusion protein or CD28 / CTLA4Ig hybrid fusion protein or other of its soluble, functional forms) to produce the desired immune response, is say the production of the antibodies from the primed animal. A primed animal is also one which is expressing a disease. Lymphocytes derived from the lymph nodes, spleens or peripheral blood of sick, primed animals can be used to investigate a particular antibody. The chromosomes of the lymphocytes that code for the desired immunoglobulins are immortalized by fusing the lymphocytes with myeloma cells, generally in the presence of a fusion agent such as polyethylene glycol (PEG). Any of many myeloma cell lines can be used as a fusion partner according to standard techniques; for example, P3-NSl / l-Ag4-l, P3-x63-Ag8.653, Sp2 / 0-Agl4, or myeloma lines HL1-653. These lines of. Myeloma are available from the ATCC, Rockville, Maryland. The resulting cells, which include the desired hybridomas, are grown in a selective medium such as HAT medium, in which the original unmyelinated myeloma or the lymphocyte cells eventually die. Only the hybridoma cells survive and can be grown under limiting dilution conditions to obtain isolated clones. The supernatants of the hybridomas are selected for the presence of the desired specificity, for example by immunoassay techniques using the CTLA4Ig protein that has been used for immunization. Positive clones can then be subcloned under limiting dilution conditions and the monoclonal antibody produced can be isolated. Various conventional methods can be used for the isolation and purification of monoclonal antibodies, to obtain them free of other contaminating proteins. Methods commonly used to purify monoclonal antibodies include ammonium sulfate precipitation, ion exchange chromatography and affinity chromatography (Zola et al., In Monoclonal Hybridoma Antibodies: Techniques and Applications, Hurell (ed.) Pp. 51-52 (CRC Press, 1982)). Hybridomas produced according to these methods can be propagated in vitro or in vivo (in ascitic fluids) using techniques known in the art (Fink et al., Prog. Clin. Pathol., 9: 121-33 (1984), Fig. 6-1 to p.123). Generally, the individual cell line can be propagated in vitro, for example, in laboratory culture vessels and the culture medium containing high concentrations of an individual specific monoclonal antibody, can be harvested by decanting, filtration or centrifugation. In addition, fragments of those antibodies that contain the active binding region, reactive with the extracellular domain of the CTLA4 receptor, such as Fab fragments, F (ab ') 2 and Fv can be produced. Such fragments can be produced using techniques well established in the art (for example Rousseaux et al., in Methods Enzymol., 121: 663-69, Academic Press (1986)).

The anti-B7 monoclonal antibodies prepared as described above can be used to bind B7 antigen to inhibit the interactions of CD28 positive T cells or CTLA4 positive T cells with B7 positive cells. The anti-CTLA4 monoclonal antibodies can be used to bind to the CTLA4 receptor to inhibit the interaction of CTLA4 positive T cells with other cells. In another embodiment, the CTLA4Ig fusion protein can be used to identify additional compounds capable of regulating the interaction between CTLA4 and the B7 antigen. Such compounds can include naturally occurring small molecules, which can be used to react with B cells and / or T cells. For example, fermentation broths can be tested for the ability to inhibit CTLA4 / B7 interactions . In addition, derivatives of the CTLA4Ig fusion protein as described above, can be used to regulate the proliferation of T cells. For example, fragments or derivatives can be used to block the proliferation of T cells in the disease. graft against the host (GVH), which accompanies the transplantation of allogenic bone marrow. The proliferation pathway of CD28-mediated T cells is resistant to cyclosporin, in contrast to the proliferation activated by the CD3 / TÍ cell receptor complex (June et al., 1987, supra). Cyclosporin is relatively ineffective as a treatment for GVH disease (Storb, Blood 68: 119-125 (1986)). GVH disease is believed to be mediated by T lymphocytes which express the CD28 antigen (Storb and Thomas, Immunol Rev. 88: 215-238 (1985)). In this way, the CTLA4Ig fusion protein may be useful alone or in combination with immunosuppressants such as cyclosporin, to block the proliferation of T cells in GVH disease. The regulation of the interactions of CTLA4-positive T cells with B7 positive cells, including B cells, by the methods of the invention can be used in this way to treat pathological conditions such as autoimmunity, transplantation, infectious diseases and neoplasia. The B7 linker molecules and the IL4 linker molecules described herein, can be in a variety of dosage forms which include, but are not limited to, liquid solutions or suspensions, tablets, pills, powders, suppositories, polymeric microcapsules. or microvesicles, liposomes and injectable solutions or that can be administered by infusion. The preferred form depends on the mode of administration and the therapeutic application.

The most effective form of administration and the dosage regimen for the molecules of the present invention, depends on the severity and of course the disease, the patient's health and response to treatment and the judgment of the treating physician. Therefore, the doses of the molecules must be titrated for the individual patient. The interrelationship of the dose for animals of various sizes and species and humans based on mg / m2 of the surface area is described by Freireich, E. J. et al. (Quantitative Comparison of Toxicity of Anticancer Agents in Mouse, Rat, Hamster, Dog, Monkey and Man. Cancer Chemother, Rep., 50, No. 4, 219-244, May 1966). Adjustments in the dosage regimen can be made to optimize the growth inhibitory response. Doses can be divided and administered on an area basis or the dose can be reduced proportionally depending on the situation. For example, various divided doses may be administered daily or the dose may be reduced proportionally as indicated by the specific therapeutic situation. In accordance with the practice of the invention, an effective amount for the treatment of an individual may be between about 0.1 and about 10 mg / kg of the individual's body weight. Also, the effective amount may be an amount between about 1 and about 10 mg / kg of the individual's body weight. Advantages of the Invention: The subject invention overcomes the problems associated with current therapies directed to prevent tissue rejection or organ transplantation. In contrast to current therapies, the present invention affects only the immune responses deviated by the B7 interactions. For example, the present invention affects the transplantation of antigen-specific T cells, thereby inducing specific and donor-specific tolerance of the antigen. The binding of CD28 by its ligand, B7 / BB1 (B7), during the coupling of the T cell, is critical for signaling of the appropriate T cell in some systems (MK Jenkins, PS Taylor Norton SD, KB Urdahl, J. Imminol 147: 2461 (1991); C.H. June, J.A. Ledbetter, P.S. Linsley, C.B. Thompson, Immunol. Today 11: 211 (1990); H. Reiser, G.J. Freeman, Z. Razi-Wolf, C.D. Gimmi, B. Benacerraf, L.M. Nadler, Proc. Nati Acad. Sci. U.S.A. 8.9: 271 (1992); N.K. Damie, K. Klussman, P. S. Linsley, A. Aruffo, J. Immunol. 148: 1985 (1992)). When the interaction of CD28 with its ligand is blocked, the antigen-specific T cells are inappropriately induced in an anergy state of the antigen-specific T cells (M.K. Jenkins, P.S. Taylor, S. D.

Norton, K. B. Urdahl, J. Immunol. 147: 2461 (1991); F. A.

Harding, J. G. McArthur, J. A. Gross, D. H. Raulet, J. P.

Allison, Nature 356: 607 (1992)). The CTLA4Ig fusion proteins bind to human and murine B7 (with 20 times higher affinity than CD28), block the binding of CD28 to B7, inhibit T cell activation and induce non-response of T cells in vi tro (FA Harding, JG McArthur, JA Gross, DH RAulet, J. P. Allison, Nature 356: 607 (1992); P. S. Linsley et al., J. Exp. Med. 174: 561 (1991). In addition, the present invention would be useful to obtain the expression of a soluble protein product of the hitherto non-expressing CTLA4 gene and to identify a natural ligand for CTLA4 that is involved in the functional responses of T cells. The soluble protein product is would then use to regulate the responses of T cells in vivo to treat pathological conditions. The following examples are presented to illustrate the present invention and to assist one skilled in the art in the preparation and use thereof.

The examples are not intended in any way to limit the scope of the invention.

EXAMPLE 1 Preparation of the B7Iq and CD28Ig Fusion Proteins The B7Ig and CD28Ig receptor-immunoglobulin C gamma fusion proteins (IgCgamma) are prepared as described by Linsley et al., In J. Exp. Med. 173: 721-730 (1991), incorporated herein by reference. In summary, the DNA encoding the amino acid sequences corresponding to the respective receptor protein (e.g. B7) bind to the DNA encoding the amino acid sequences corresponding to the hinge, CH2 'and CH3 regions of the IgCgammal human This is done as follows. Polymerase Chain Reaction (PCR). For PCR, the DNA fragments are amplified using primer pairs as described in the following for each fusion protein. The PCR reactions (0.1 ml of final volume) are run in Taq polymerase buffer (Stratagene, La Jolla, CA), which contains 20 pmol of each of dNTP; 50-100 pmoles of the indicated primers; template (1 ng of the plasmid or cDNA synthesized of = 1 μg of the total RNA using the random hexamer primer, as described by Kawasaki in PCR Protocols, Academic Press, pp. 21-27 (1990), incorporated herein by reference); and the Taq polymerase (Stratagene). The reactions are run in a thermal cycle former (Perkin Elmer Corp., Norwalk, CT) for 16-30 cycles (a typical cycle consists of steps of 1 minute at 94 ° C, 1-2 minutes at 50 ° C and 1 -3 minutes at 72 ° C). Construction of the Plasmid. Expression plasmids containing the cDNA encoding CD28, as described by Aruffo and Seed, Proc. Nati Acad. Sci. USA 84: 8573 (1987)), were provided by Drs. Aruffo and Seed (Mass General Hospital, Boston, MA). Plasmids containing the cDNA encoding CD5, as described by Aruffo, Cell 61: 1303 (1990)), were provided by Dr. Aruffo. Plasmids containing the cDNA encoding B7, as described by Freeman et al., J. Immunol. 143: 2714 (1989)), were provided by Dr. Freeman (Dana Farber Cancer Institute, Boston, MA). For initial attempts at the expression of the soluble forms of CD28 and B7, the constructs (OMCD28 and OMB7) are prepared as described by Linsley et al., J. Ex. Med., Supra, in which the stop codons are introduced upstream of the transmembrane domains and the native signal peptides are replaced with the oncostatin M signal peptide (Malik et al., Mol. Cell Biol. 9: 2847 (1989)). These are formed using synthetic oligonucleotides for reconstruction (OMCD28) or as primers (0MB7) for PCR. OMCD28 is a modified CD28 cDNA for more efficient expression by replacing the signal peptide with the analogous region of Oncostatin M. The fusion constructs CD28Ig and B7Ig are made in two parts. The 5 'portions are formed using OMCD28 and 0MB7 as templates and the oligonucleotide CTAGCCACTGAAGCTTCACCATGGGTGTACTGCTCACAC (SEQ ID NO: 1), (encoding the sequence of amino acids corresponding to the oncostatin signal peptide M) as a forward primer and either TGGCATGGGCTCCTGATCAGGCTTAGAAGGTCCGGGAAA (SEQ ID NO: 2), or, TTTGGGCTCCTGATCAGGAAAATGCTCTTGCTTGGTTGT (SEQ ID NO: 2): 3), as the inverse primers, respectively.

The products of the PCR reactions are split with the restriction endonuclease (Hind III and Bell) as the sites introduced into the PCR primers and gel purified. The 3 'portion of the fusion constructs corresponding to the IgCgammal human sequences is made by reverse transcriptase (avian myeloblastosis virus, Life Sciences Associates, Bayport, NY) -CRP reaction using the RNA of a line of myeloma cells that produces the human-mouse chimeric L6 mAb (proproced by Dr. P. Fell and M. Gayle, Bristol-Myers Squibb Company, Pharmaceutical Research Institute, Seattle, WA) as the template. The oligonucleotide AAGCAAGAGCATTTTCCTGATCAGGAGCCCAAATCTTCTGACAAAACTCACACATCCCCAC CGTCCCCAGCACCTGAACTCCTG (SEQ ID NO: 4) is used as the forward primer and CTTCGACCAGTCTAGAAGCATCCTCGTGCGACCGCGAGAGC (SEQ ID NO: 5) as the reverse primer. The products of the reaction are split with Bell and Xbal and gel-purified. The final constructs are assembled by ligating the split HindIII / Bcll fragments containing the CD28 or B7 sequences together with the unfolded Bcll / Xbal fragment containing the IgCgammal sequences in CDM8 unfolded with HindIII / Xbal. The ligation products are transformed into MC1061 / p3 E. coli cells and the colonies are selected for the appropriate plasmids. The sequences of the resulting constructs are confirmed by DNA sequencing. The construct encoding B7 contained in the DNA encoding the amino acids corresponding to the amino acid residues is from about position 1 to about position 215 of the extracellular domain of B7. The construct encoding CD28 containing the DNA encoding amino acids corresponding to amino acid residues from about position 1 to about position 134 of the extracellular domain of CD28. CD5Ig is constructed identically using CATTGCACAGTCAAGCTTCCATGCCCATGGGTTCTCTGGCCACCTTG (SEQ ID NO: 6) as the forward primer and ATCCACAGTGCAGTGATCATTTGGATCCTGGCATGTGAC (SEQ ID NO: 7) as the reverse primer. The PCR product was the restriction endonuclease digested and ligated with the IgCgammal fragment as described above. The resulting construct (CD5Ig) encoded for a mature protein having an amino acid sequence containing the amino acid residues from position 1 to position 347 of the sequence corresponding to CD5, two amino acids introduced by the construction method (amino acids DQ) , followed by the DNA that codes for the amino acids that correspond to the hinge region of IgCgammal. Cell Culture and Transfections. The COS (monkey kidney cells) are transfected with the expression plasmids, which express CD28 and B7 using a modification of the Seed and Aruffo protocol (Proc.Nat.Acid.Sci.84: 3365 (1987)), incorporated for reference in the present. The cells are seeded at 106 by 10 cm in diameter of the culture dish, 18-24 h before transfection. The plasmid DNA is added (approximately 15 μg / disc) in a volume of 5 ml of DMEM without serum, containing 0.1 mM chloroquine and 600 μg / ml Dextran DEAE and the cells are incubated for 3-3.5 h at 37 ° C . The transfected cells are briefly treated (approximately 2 minutes) with 10% dimethyl sulfoxide PBS and incubated at 37 ° C for 16-24 h in DMEM containing 10% FCS. At 24 h after transfection, the culture medium is removed and replaced with DMEM without serum (6 ml / disc). Incubation is continued for 3 days at 37 ° C at which time the spent medium is collected and medium is added without freshly prepared serum. After an additional 3 days at 37 ° C, the spent medium is collected again and the cells are discarded. CHO cells expressing CD28, CD5 or B7 are isolated as described by Linsley et al., (1991) supra, as follows: In summary, stable transfectants expressing CD28, CD5, or B7 were isolated following cotransfection of Chinese Hamster ovary cells deficient in dihydrofolate reductase (dhfr-CHO) with a mixture of the appropriate expression plasmid and the selectable marker, pSV2dhfr (Linsley et al., Proc. Nati, Acad. Sci. USA 87: 5031 (1990)), incorporated herein by reference. The transfectants are then grown in increasing concentrations of methotrexate at a final consideration of 1 μM and maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 0.2 mM proline and 1 μM methotrexate. CHO lines expressing high levels of CD28 (CD28 + CHO) or B7 (B7 + CHO) are isolated by multiple rounds of fluorescence activated cell sorting (FACSR) after indirect immunostaining with mAbs 9.3 or BB-1. CHO cells amplified negative for surface expression of CD28 or B7 (dhfr + CHO) were also isolated by FACSR from populations transfected with CD28.

Immunostaining and FACSE Analysis. Transfected CHO or COS cells or activated T cells are analyzed by indirect immunostaining. Before staining, the CHO cells are removed from their culture vessels by incubation in PBS containing 10 mM EDTA. The cells are first incubated with the murine mAbs 9.3 (Hansen et al., Immunogenetics 10: 247 (1980)) or BB-1 (Yokochi et al., J. Immunol. 128: 823 (1981)), or with Ig fusion (all alO μg / ml in DMEM containing 10% FCS) for 1-2 h at 4 ° C. The cells are then washed and incubated for an additional 0.5-2 h at 4 ° C with a second-stage FITC-conjugated reagent (Ig is goat anti-mouse serum for murine mAbs or Ig Cgamma of anti-goat serum. human for fusion proteins (Tago, Inc., Burlingame, CA)). Fluorescence is analyzed in a FACS IVR cell sorter (Becton Dickinson and Co., Mountain View, CA) equipped with a logarithmic amplifier for four decades. Purification of Ig Fusion Proteins. The first, second and third collections of serum-free, spent culture media from the transfected COS cells are used as sources for the purification of the Ig fusion proteins. After removal of the cell debris by low speed centrifugation, the medium is applied to a column (approximately 200-400 ml packed medium / ml bed volume) of the immobilized protein A (Repligen Corp. Cambridge, MA ) balanced with 0.05 M sodium citrate, pH8.0. After the application of the medium, the column was washed with 1 M postasium phosphate, pH 8, and the bound pretein is eluted with 0.05 M sodium citrate pH3. The fractions are immediately collected and neutralized by the addition of 1/10 volumes of 2 M Tris, pH 8. The fractions containing the peak of absorbent material A280 are pooled and dialysed against PBS before being used. The extinction coefficients of 2.4 and 2.8 ml / mg for CD28Ig and B7Ig, respectively, are determined by the amino acid analysis of the known absorbance solutions. Recovery of the binding activities of purified CD28Ig and B7Ig was almost quantitative as classified by FACSR analysis after indirect fluorescence staining of CHO B7 + and CD28 + cells.

EXAMPLE 2 Preparation of the CTLA4Ig Fusion Protein A soluble genetic fusion that codes for CTLA4Ig between the extracellular domain of CTLA4 and a domain IgCgammal is constructed in a manner similar to that described above for the CD28Ig construct. The extracellular domain of the CTLA4 gene is cloned by PCR using synthetic oligonucleotides corresponding to the published sequence (Dariavach et al., Eur. Journ.Immunol.18: 1901-1905 (1988)). Because a signal peptide for CTLA4 was not identified in the CTLA4 gene, the N-terminus of the predicted CTLA4 sequence is fused to the oncostatin M signal peptide (Malik et al., Mol, and Cell. Biol. : 2847 (1989)) in two steps using overlapping oligonucleotides. For the first step, the oligonucleotide CTCAGTCTGGTCCTTGCACTCCTGTTTCCAAGCATGGCGAGCATGGCAATGCACGTGGCCC AGCC (SEQ ID NO: 8) (which codes for the terminal 15 amino acids C of the signal peptide oncostatin M fused to the 7 N-terminal amino acids of CTLA4) is used as the forward primer and TTTGGGCTCCTGATCAGAATCTGGGCACGGTTG (SEQ ID NO: 9) (coding for amino acid residues 119-125 of the amino acid sequence encoding the CTLA4 receptor and containing a Bel I restriction enzyme site) as the reverse primer. The template for this step was the cDNA synthesized from 1 μg of the total RNA of H38 cells (line of T-cell leukemic cells infected with HTLV II provided by Drs. Salahudin and Gallo, NCT, Bethesda, MD). A portion of the PCR product from the first step is re-amplified using an overlapping forward primer, which codes for the N-terminal portion of the oncostatin M signal peptide and contains a restriction endonuclease site.2 CTAGCCACTGAAGCTTCACCAATGGGTGTACTGCTCACACAGAGGACGCTGCTCAGTCTGG TCCTTGCACTC (SEQ ID NO: 10) and the same reverse primer. The product of the PCR reaction is digested with Hind III and Bel I and ligated together with the cleaved ANDc fragment with Bel l / Xba I encoding the amino acid sequence corresponding to the hinge, CH2 and CH3 regions of IgCgammal in the expression vector unfolded with Hind III / Xba I CDM8 or the expression vector DLN unfolded with Hind Ill / Xba I (provided by Dr. Aruffo). A map of the resulting CTLA4Ig fusion construct is shown in Figure 1. The sequences shown in this figure show the junctions between CTLA4 (uppercase letters, unshaded regions) and the SP signal peptide, oncostatin M (shaded dark regions). and the hinge, H, of IgCgammal (dotted regions). The amino acid in parentheses is introduced during construction. Asterisks (*) indicate the mutations of cysteine to serine introduced into the hinge region of IgCgamma. The domain similar to domain V of the immunoglobulin superfamily present in CTLA4 is indicated as the • CH2 and CH3 domains of IgCgammal. Expression plasmids, CDM8 containing CTLA4Ig are transfected into COS cells using transfection with DEAE / dextran by modification (Linsley et al., 1991, supra) of the protocol described by Seed and Aruffo, 1987, supra. The expression plasmid constructs (DLN or CDM8) containing the cDNA encoding the amino acid sequence of CTLA4Ig are transfected by lipofection using standard procedures in dhfr "CHO lines to obtain new cell lines that stably express CTLA4Ig. coding for the amino acid sequence corresponding to CTLA4Ig has been deposited with the ATCC under the Budapest Treaty on May 31, 1991, and has been registered with ATCC under accession number 68629. A preferred stable transfectant, expressing CTLA4Ig , designated Chinese Hamster Ovary Cell Line, CTLA4Ig-24, is prepared by selecting the B7 cell line CHO positive for B7 binding activity in the medium using immunostaining.The transfectant is maintained in DMEM supplemented with 10% serum Fetal bovine (FBS), 0.2 mM proline and 1 μM methotrexate The CTLA4Ig-24 CHO cell line has been deposited with the ATCC under the Treaty of Budapes t on May 31, 1991 and has been registered with accession number ATCC 10762. CTLA4Ig is purified by protein A chromatography of conditioned supernatants without serum (Figure 2). CTLA4Ig concentrations are determined by assuming an extinction coefficient at 280 nm of 1.6 (determined experimentally by the amino acid analysis of a known absorbance solution). The molecular weight standards (lines 1 and 3, Figure 2) and samples (1 μg) of CTLAIg (lines 2 and 4) are subjected to SDS-PAGE (gradient of 4-12% acrylamide) under non-reducing conditions (- SME, lines 1 and 2) or reducing conditions (+ ßMe, lines 3 and 4). The proteins are visualized by staining with Coomassie Brilliant Blue. Under nonreducing conditions, CTLA4Ig migrates as a Mr species of approximately 100,000 and under reducing conditions as a Mr species of approximately 50,000 (Figure 2). Because the disulfides of the IgC gamma hinge are removed during construction, CTLA4Ig, like CD28Ig, is a dimer supposedly bound by means of a native disulfide bond.

EXAMPLE 3 CTLA4 receiver To reconstruct the DNA encoding, for the amino acid sequence corresponding to the full length of the human CTLA4 gene, the cDNA encoding the amino acids corresponding to a fragment of the transmembrane and the cytoplasmic domains of CTLA4, is cloned by PCR and then it binds to the cDNA encoding the amino acids corresponding to a CTLA4Ig fragment corresponding to the oncostatin M signal peptide fused to the N-terminus of CTLA4. The procedures for PCR and cell culture and transfections were as described above in Example 1 using COS cells and transfection of DEAE-dextran. Since the expression of the receptor protein CTLA4 in human lymphoid cells had not previously been reported, it was necessary to locate a source of CTLA4 mRNA. The PCR of the inverse cDNA transcribed from the total cellular RNA of the H38 cells as observed in the above, it is used for cloning by PCR. For this purpose, the oligonucleotide GCAATGCACGTGGCCCAGCCTGCTGTGGTAGTG (SEQ ID NO: 11), (coding for the first 11 amino acids in the predicted coding sequence) is used as a forward primer and TGATGTAACATGTCTAGATCAATTGATGGGAATAAAATAAGGCTG (SEQ ID NO: 12) ) (homologous for the last 8 amino acids in CTLA4 and containing an Xba I site) as the reverse primer. Again the template was a cDNA synthesized from 1 μg of RNA from H38 cells. The products of the PCR reaction are cleaved with the restriction endonucleases Neo I and Xba I and the resulting 316 bp product is gel purified. A Hind IIl / Nco I fragment of 340 bp of the CTLAIg fusion described above was also gel purified and both restriction fragments are ligated into CDM8 split with Hind Ill / Cba I to form OMCTLA. The resulting construct corresponded to the full length of CTLA4 (IDENTIFICATION SEQUENCES NOS: 13 and 14) and the oncostatin signal peptide M. The construct is shown in Figure 3 and was designated OMCTLA4. The sequence for CTLA4 shown in Figure 3 differs from the predicted human CTLA4 DNA sequence (Dariavach et al., Supra) by a change in a base in such a way that the alanine previously reported at the amino acid position 111 of the sequence of amino acids shown, codes for a threonine. This threonine is part of a newly identified N-linked glycosylation site that may be important for the successful expression of the fusion protein. The ligation products are transformed into MC106l / p3 E. coli cells and the colonies are selected for the appropriate plasmids. The sequences of the resulting constructs are confirmed by the analysis of the DNA sequence.

EXAMPLE 4 Characterization of CTLA4Ig To characterize the CTLA4Ig constructs, several CD28Ig, B7Ig, and CD5Ig isolates are prepared as described above and transfected into COS cells as described in Examples 2 and 3 and tested by FACSR analysis to the B7Ig linkage. In addition to the constructs mentioned in the above, CDM8 plasmids containing cDNAs encoding CD7 are also used as described by Aruffo and Seed, (EMBO Journal 6: 3313-3316 (1987)), incorporated herein by reference. reference. mAb. Murine monoclonal antibodies (mAbs) 9.3 (anti-CD28) and G19-4 (anti-CD3), G3-7 (anti-CD7), BB-1 (anti-B7 antigen) and rat mAb 187.1 (anti-mouse K chain) have been previously described (Ledbetter et al., Proc. Nati.

Acad. Sci. 84: 1384-1388 (1987); Ledbetter et al., Blood 75: 1531 (1990); Yokochi et al., Supra) and purified from ascitic fluids before use. The hybridoma that produces the OKT8 mAb is obtained from ATCC, Rockville, MD and the mAb is also purified from ascites before being used. The 4G9 mAb (anti-CD19) was provided by Dr. E. Engleman, Stanford University, Palo Alto, CA). The purified human-mouse chimeric L16 mAb, (which has the Fc portion of human Cgammal) was presented by Dr. P. Fell and M. Gayle (Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA). Immunostaining and FACSS Analysis. Before staining, COS or CHO cells are removed from their culture vessels by incubation in PBS containing 10 mM EDTA. The cells are first incubated with the mAbs or Ig fusion proteins at 10 μg / ml in DMEM containing 10% FBS for 1-2 hours at 4 ° C. The cells are then washed and incubated for an additional 0.5-2 hours at 4 ° C with goat anti-mouse conjugated immunoglobulin-FITC or goat anti-human IgG gamma conjugated-FITC serum (both from Tago, Burlingame, CA). When both mAbs bind and the Ig fusion proteins are measured in the same experiment, the reagents of the second stage anti-mouse and anti-human FITC conjugates are mixed together before being used. Fluorescence in a total of 10,000 cells is then analyzed by FACSR. Separation and Stimulation of Peripheral Blood Lymphocytes. Peripheral blood lymphocytes (PBL) are isolated by centrifugation through the Lymphocyte Separation Medium (Litton Bionetics, Kensington, MD). The alloreactive cells are isolated by stimulation of PBL in a reaction of primary mixed lymphocytes (MLR). The PBL are cultured at 106 / ml irradiated (5000 rad) T51 LCL. Lymphoblastoid cell lines transformed with EBV (LCL), PM (Bristol-Myers Squibb Co.) and T51 (Bristol-Myers Squibb Co.) Are maintained in RPMI supplemented with 10% FBS. After 6 days, the alloreactive "blasto" cells are cryopreserved. Secondary MLR is carried out by culturing blasts to the frozen reagents together with freshly irradiated T51 LCL in the presence and absence of mAbs and Ig fusion proteins. Cells are grown in 96 well flat bottom plates (4 x 104 blasts to the reagents and 1 x 10 4 irradiated T51 LCL cells / well in a volume of 0.2 ml) in RMPI containing 10% FBS. The cell proliferation of the quadrupled cultures is measured by the incorporation of [3 H] -thymidine during the last 6 hours of a culture of 2-3 days. PHA-activated T cells are prepared by culturing the PBL with 1 μg / ml PHA (Wellcome, Charlotte, NC) for five days, and one day in medium lacking PHA. Viable cells are harvested by sedimentation through the Lymphocyte Separation Medium before use. The cells are stimulated with mAb or CHO cells or transfected for 4-6 hours at 37 ° C, harvested by centrifugation and used to prepare the RNA. CD4 + T cells are isolated from PBL by separating PBL from healthy donors on T and non-T cells using the sheep erythrocyte rosette formation technique and also separating T cells by extension on CD4 + cells as described by Damle et al., J_¡_ Immunol. 139: 1501 (1987), incorporated for reference herein. B cells are also purified from peripheral blood by separation as described by Wysocki and Sato, Proc. Nati Acad. Sci. 75: 2844 (1978), incorporated herein by reference, using the anti-CD19 mAb 4G9. To measure the production of Ig induced with T- ^, 10 ^ CD4 + cells are mixed with 10d CD19 + B cells in 1 ml of RPMI containing 10% FBS. After cultivation for 6 days at 37 ° C, the production of human IgM is measured in the culture supernatants using the solid phase ELISA as described by Volkman et al. , Proc. Nati Acad. Sci. USA, 78: 2528 (1981) incorporated herein by reference. Briefly, 96 well flat bottom microtiter plate ELISA plates (Corning, Corning, NY) are coated with 200 μl / well of sodium carbonate buffer (ph 9.6) containing 10 μg / ml affinity of goat antibody purified anti IgG or human IgM (Tago, Burlingame, CA), incubated overnight at 4 ° C and then washed with PBS and the wells are further blocked with 2% BSA in PBS (BSA-PBS). The samples to be analyzed are added at an appropriate dilution to these wells and incubated with 200 μl / well of a 1: 1000 dilution of horseradish peroxidase (HRP) - F (ab ') 2 conjugate fraction of affinity antibody -purified goat anti IgG or human IgM (Tago). Then the plates are washed and 100 μl / well of an o-phenylenediamine solution (Sigma Chemical Co., St. Louis, MO) (0.6 mg / ml in phosphate-citrate buffer with pH 5.5 and 0.045% hydrogen peroxide). The color development is stopped with 2 N sulfuric acid. The absorbance at 490 nm is measured with an automated ELISA reader plate. The test and control samples are run in triplicate and the absorbance values are compared with those obtained with standard, known IgG or IgM, run simultaneously with the supernatants to generate the standard curve, the use of which quantifies Ig concentrations in the culture supernatant. The data are expressed as ng / ml of Ig ± SEM, either triplicate or quadruplicate cultures. Immunoprecipitation Analysis and SDS PAGE. Cells are labeled on the surface with 125j and subjected to immunoprecipitation analysis. Briefly, PHA-activated T cells are labeled on the surface with 125j using lactoperoxidase and H2O2 as described by Vitetta et al., J. Exp. Med. 134: 242 (1971), incorporated for reference herein. SDS-PAGE chromatography is performed on gels in linear acrylamide gradients with 5% acrylamide row gels. The gels are stained with Coomassie Blue, fade and photographed and dried and exposed to an X-ray film (Kodak X7AR-5). Link Testing B7Ig is labeled with 125j at a specific activity of approximately 2 x 10 >; cpm / pmoles. Ninety-six well plastic plates are coated for 16-24 hours with a solution containing CTLA4Ig (0.5 μg in a volume of 0.06 ml of 10 mM Tris, pH 8). The wells are blocked with the binding buffer (DMEM containing BES 50 mM (Sigma Chemical Co.), pH 6.8, 0.1% BAS, and 10% FCS) before the addition of a solution (0.09 ml) containing 12 ^ I-B7Ig (approximately 5 x 105 cpm) in the presence or absence of a competitor). After incubation for 2-3 hours, at 23 ° C, the wells are washed once with binding buffer and four times with PBS. The bound radioactivity is solubilized by the addition of 0.5N NaOH and quantified by gamma counting. Link to B7Ig. The functional activity of the 0MCTLA4 construct encoding the complete, human CTLA4 DNA gene is shown in the experiment shown in the Figure. The COS cells are transfected with the expression plasmids CD7, OMCD28 and OMCTLA4 as described above. Forty-eight hours after transfection, cells are harvested and incubated only with medium (no addition) or with mAbs 9.3, B7Ig, CD5Ig or G3-7. The cells are then washed and the binding is detected by a mixture of the reagents of the second step of the goat anti-mouse Ig-FITC conjugate and the goat anti-human-FITC conjugate. The transfected cells are tested for the expression of the appropriate cell surface markers by indirect immunostaining and the fluorescence is measured using the FACSR analysis as described above. As shown in Figure 4, the mAb 9.3 bound to the COS cells transfected with CD28, but not to the transfected CTLA4 cells. In contrast, the B7Ig fusion protein (but without the control CD5Ig fusion protein) bound to the transfected CD28 and CTLA4 cells. COS cells transfected with CD7 did not bind mAb 9.3 or any of the fusion proteins. This indicates that CD28 and CTLA4 bind to the activation antigen of B cells, B7. In addition, mAb 9.3 does not bind detectably to CTLA4. Link CTLA4Ig in Positive CHO Cells to B7. To further characterize the binding of CTLA4Ig and B7, the binding activity of purified CTLA4Ig in CHO B7 + cells and in a lymphoblastoid cell line (PM LCL) is measured in the experiment shown in Figure 5. Transfected, amplified CHO cell lines and PM LCL are incubated only with medium (no addition) or an equivalent concentration of the proteins containing the human IgCgammal. (10 μg / ml) of CD5Ig, CD28Ig or CTLA4Ig. The linkage is detected by FACSR after the addition of the reagents of the second step of the goat anti-human Ig-FITC conjugate. A total of 10,000 stained cells are analyzed by FACSR. As shown in Figure 5, CD28Ig binds to CHO B7 + cells, but not to PM LCL, a cell line which expresses relatively low concentrations of B7 antigen (Linsley et al., Supra, 1990). CTLA4Ig binds more strongly to both cell lines than CD28Ig, suggesting that it binds with higher affinity. Neither CD28Ig nor CTLA4Ig bind to CD28 + CHO cells. Linkage affinity of CTLA4Ig and B7Ig. The apparent affinity of the interaction between CTLA4Ig and B7Ig is then measured using a solid phase competition binding assay. Ninety-six well plastic discs are coated with CTLA4Ig, as described above. The B7Ig is radiolabelled with 125j (5 x] _Q5 cpm, 2 x 106 cpm / pmoles), and added at a concentration of 4 nM in the presence of the indicated concentrations (Figure 6) of mAb L6, mAb 9.3, mAb BB-1 or unlabeled chimeric B7Ig. The radioactivity bound to the plate is determined and expressed as a percentage of the radioactivity bound to the treated wells without the competitor (28,300 cpm). Each point represents the mean of the determinations in duplicate; the replicas usually vary from the mean by μ 20%. The concentrations are calculated based on a Mr of 75,000 per link site for the mAbs and 51,000 per link site for B7Ig.

As shown in Figure 6, only the mAb 'BB-1 and the unlabeled B7Ig compete significantly for binding with 1 5j_B7jg (semi-maximal effects at approximately 22 nM and approximately 175 nM, respectively). Neither the L6 mAb nor the chimeric mAb 9.3 compete effectively at the tested concentrations. In other experiments, the concentrations of mAb 9.3 used, were sufficient to inhibit the binding of 125 I-B7Ig to immobilized CD28Ig or to CD28 expressed on the cell surface by > 90% When the competition data of Figure 6 are plotted on a Scatchard plot, a dissociation constant K ^ of about 12 nM is calculated for the binding of 125 I-B7 for immobilized CTLA4Ig (Figure 7). This value is approximately 20 times less than the K¿ determined previously of the bond between 125I-B7Ig and CD28 (approximately 200 nM) (Linsley et al, (1991), supra) indicating that CTLA4 is a higher affinity receptor for the B7 antigen than the CD28 receiver. To identify the molecule or molecules on the lymphoblastoid cells, which bind to CTLA4Ig (Figure 7), cells labeled on the surface with 125j are subjected to immunoprecipitation analysis (Figure 8). CHO B7 + and PM LCL cells were labeled on the surface with 125? and they are extracted with a nonionic detergent solution as described above. Aliquots of extracts containing about 1.5 x 10 ^ cpm in a volume of 0.1 ml are subjected to immunoprecipitation analysis as described above without addition or 2 μg of each of CD28Ig, CTLA4Ig or CD5Ig. The washed immunoprecipitates are analyzed by SDS-PAGE (gradient of acrylamide of 10-20%) under reducing conditions. The gel is then dried and subjected to autoradiography. The left panel of Figure 8 shows an autoradiogram obtained after 1 day of exposure. The right panel of Figure 8 shows an autoradiogram of the same gel after a 10-day exposure. The autoradiogram in the panel of Figure 8 is also exposed for 10 days. The positions of the standard molecular weight are also indicated in this figure. As shown in Figure 8, a radiolabeled protein of diffuse migration (Mr of about 50,000-75,000, center to about 60,000) was immunoprecipitated by CTLA4Ig, but not with CD28Ig or CD5Ig. This molecule co-migrates with B7 immunoprecipitated from CHO B7 + cells by CTLA4Ig and much more weakly by CD28Ig. These findings indicate that CTLA4Ig binds to an individual protein on lymphoblastoid cells, which is similar in size to the B7 antigen.

Inhibition of In Vitro Immune Responses by CTLA4Ig Inhibition of Proliferation. Previous studies have shown that the anti-CD28 mAb, mAb 9.3 and the anti-B7 mAb, the BB-1 mAb, inhibit the proliferation of Tj- specific alloantigen. as well as the secretion of immunoglobulin by B cells displaying the alloantigen (Damle, et al., Proc.Nat.Acid.Sci.78: 5096 (1981); Lesslauer et al., Eur. J. Immunol. 16: 1289 (1986)). Because CTLA4 is a high affinity receptor for the B7 antigen as demonstrated herein, soluble CTLA4Ig is tested for its ability to inhibit these responses. The effects of CTLA4Ig on T-cell proliferation are examined in the experiment shown in Figure 9. Blasts from the reaction of primary mixed lymphocytes (MLR) are stimulated with irradiated T51 lymphoblastoid (LC) cells in the absence or presence of concentrations of Fab fragments of murine mAb 9.3, or the Cgamma fusion proteins of immunoglobulin B7Ig, CD28Ig or CTLA4Ig. Proliferation is measured by the incorporation of [3 H] thymidine after 4 days and is expressed as the percentage of incorporation by untreated culture (21.00.0 cpm). Figure 9 shows the average of determinations in quadruplicate (SEM = 10%).

As shown in Figure 9, CTLA4Ig inhibited the MLR reaction in a dose dependent manner by a maximum of > 90% with a maximum response of 1/2 to approximately 30 ng / ml (approximately 0.8 nM). The Fab fragment of mAb 9.3, which had previously shown to be a more potent inhibitor of MLR than the whole mAb 9.3 (Damle et al., J. Immunol.140: 1753-1761 (1988)), also inhibited MLR, but at higher concentrations (approximately 800 ng / ml or approximately 30 nM for 1/2 of the maximum response). B7Ig and CD28Ig do not significantly inhibit MLR even at higher concentrations. In another experiment, the addition of B7Ig together with CTLA4Ig partially exceeded the inhibition of MLR by CTLA4Ig, indicating that the inhibition was specifically due to interactions with the B7 antigen. Inhibition of Immunoglobulin Secretion. The CTLA4Ig effects on the immunoglobulin secretion induced by helper T cells (T ^) were also examined (Figure 10). The CD4 + T cells are mixed with halogistic CD19 + B cells in the presence or absence of the immunoglobulin molecules indicated as described above. The murine mAbs OKT8, 9.3 and BB-1 are added at 20 μg / ml and the Ig fusion proteins at 10 μg / ml. After 6 'days of culture, the concentrations of human IgM (SEM <5%) in the culture supernatants are determined by the enzyme immunoassay (ELISA) as described above. The production of IgM by B cells cultured in the absence of CD4 + T cells was 11 ng / ml. As shown in Figure 10, CD4 + T cells stimulated IgM production by allogeneic CD19 + B cells (in the absence of CD4 + T cells, IgM concentrations were reduced by 93%). MAbs 9.3 and BB-1 significantly inhibit IgM production induced by T ^ (63% and 65% inhibition, respectively). CTLA4Ig was even more effective as an inhibitor (89% inhibition) than these mAbs were. Inhibition by the control Ig molecules, the mAb OKT8 and CD5Ig, was much lower (= 30% inhibition). None of these molecules significantly inhibits Ig production measured in the presence of Staphylococcal aureus enterotoxin B. Similar results were obtained with CD4 + T cells and B cells derived from other donors. These results indicate that the inhibition by CTLA4Ig is specific. The above data also demonstrate that CTLA4 and CD28 receptors are functionally as well as structurally related. Like CD28, CTLA4 is also a receptor for the B cell activation antigen, B7. The CTLA4Ig bound to B7-125I with a constant affinity, K ^, of approximately 12 nM, a value 20 times greater than the affinity between CD28 and B7Ig (approximately 200 nM). In this way, CTLA4 and CD28 can be believed to be high and low affinity receptors, respectively, for the same ligand, the B7 antigen. The apparent affinity between CD28 and B7 is similar to the affinity reported for the binding of soluble alloantigen to the receptor of the T cells of a hybridoma of the cells Murine T (approximately 100 nM; Schnek et al., Cell 56:47 (1989)), and it is higher affinity than the interactions between CD2 and LFA3 (Recny et al., J. Biol. Chem. 265: 8542 (1990)) or CD4 and the MHC molecules of class II (Clayton et al. ., Nature 339: 548 (1989)). The apparent affinity constant, K1, between CTLA4 and B7 is even greater and compares favorably with the higher affinity mAbs (K¿ of 2-10,000 nM; Alzari et al., Ann.Rev. Immuno., 6: 555 ( 1988)). The K¿ between CTLA4 and B7 is similar to or greater than the Kd values of the integrin receptors and their ligands (10-2000 nM, Hautanen et al., J. Biol. Chem. 264: 1437-1442 (1989); et al., Blood 61: 140-148 (1983), Thiagarajan and Kelley, J. Biol. Chem.263: 035-3038 (1988)). The affinity of interaction between CTLA4 and B7 in this way is among the highest reported for lymphoid adhesion systems. These results demonstrate the first expression of a functional protein product of the transcribed fragments of CTLA4. The CTLA4Ig, a fusion construct containing the extracellular domain of CTLA4 fused to an IgCgamma 1 domain forms a disulfide-linked dimer of a Mr of approximately 50,000 subunits (Figure 1). Because the interchain disulfides can not be predicted to form in the Ig production of this fusion, it seems likely that the CTLA4 cysteines are involved in the formation of the disulfide bond. The analogous CD28Ig fusion protein (Linsley et al, supra, 1991) also contains the interchain chain disulfide bond (s). These results suggest that the CTLA4 receptor, such as CD28 (Hansen et al., Immunogenetics 10: 247-260 (1980)) exists on the surface of T cells as a homodimer bound to the disulfide. Although CD28 and CTLA4 are highly homologous proteins they are immunologically distinct, because the anti-CD28 mAb, mAb 9.3, does not recognize CTLA4 (Figures 4 and 5). It is not known if CTLA4 can activate T cells by a signal path analogous to CD28. The cytoplasmic domains of murine and human CTLA4 are identical (Dariavach et al., Supra, 1988), suggesting that this region has important functional properties. The cytoplasmic domains of CD28 and CTLA4 also share homology, although it is not clear if this is not enough to impart signal properties similar to the two molecules. CTLA4Ig is a potent inhibitor of in vitro lymphocyte functions that require the collaboration of T cells and B cells (Figures 9 and 10). These findings, together with previous studies, indicate the fundamental importance of interactions between the B7 antigen and its counter-receptors CD28 and / or CTLA4, in regulating the responses of T and B lymphocytes. CTLA4Ig should be a useful reagent for future investigations in the role of these interactions during immune responses. CTLA4Ig is a more potent inhibitor of lymphocyte responses in vitro than either mAb BB-1 or mAb 9.3 (Figures 9 and 10). The higher potency of CTLA4Ig over mAb BB-1 is probably due more to the difference in affinities for B7 between these molecules (Figure 6). CTLA4Ig is also more potent than mAb 9.3, probably unlike mAb, which also has no direct stimulatory effects on T cell proliferation.

(June et al., Immunoloqy Today 11: 211 (1989)) for its inhibitory effects. The immunosuppressive effects of CTLA4ig in vitro suggest that future research is warranted on possible therapeutic effects of this molecule, for the treatment of autoimmune disorders involving aberrant activation of T cells or Ig-production. As will be apparent by those with skill in the technique to which the invention pertains, the present invention may be contemplated in ways different from those specifically described in the foregoing without departing from the spirit or essential features of the invention. The particular embodiments of the invention described in the foregoing are, therefore, considered as illustrative and not as restrictive. The scope of the present invention is as set forth in the appended claims rather than limited to the examples contained in the foregoing description.

EXAMPLE 5 Female BALB / c and C57BL / 6 (H-26) mice 6 to 8 weeks old are obtained from The Jackson Laboratory (Bar Harbor, ME). The human pancreatic islet cells are purified after digestion with collagenase as described (C. Ricordi et al, Transplantation 52.:519 (1991), AG Tzakis et al., Lancet 336: 402 (1990), C. Ricordi, PE Lacy, EH Finke, BJ Olack, DW Scharp, Diabetes 37: 413 (1988)). B6 or BIO mice treated with streptozocin (175 mg per kilogram of body weight) 3 to 5 days before transplant and have fasting plasma glucose levels greater than 280 mg / dL (with most with above 300 mg / ml), were used as recipients.

Each animal received approximately 800 freshly prepared human islets 150 μm in diameter below the left renal capsule (D. Faustman and C. Coe, Science 252: 1700 (1991); YJ Zeng et al., Transplantation 53: 277 (1992)). ). The treatment started immediately after the transplant. Control animals are treated with PBS (solid lines) or L6 (dotted lines) at 50 μg every third day after 14 days immediately after transplantation (Figure HA). Islet transplants are considered rejected when glucose levels were greater than 250 mg / dl for three consecutive days. Animals treated with PBS (n = 14) and L6 (n = 8) have average graft survival of 5.6 and 6.4 days, respectively. The animals are treated with 10 μg of CTLA4Ig for 14 consecutive days immediately after transplantation (n = 7) (Figure 11B). Three of seven animals maintained their grafts during > 80 days The remaining four animals had an average survival of 12.75 days. Animals are treated with 50 μg of CTLA4Ig every third day for 14 days immediately after human islet transplantation (Figure 11C). All animals (n = 12) treated with this dose maintained the grafts throughout the analysis (Figure 11C). The selection mice are nephrectomized on days 21 and 29 after transplantation to evaluate graft function (Figure 11C). Histology was performed on the kidneys transplanted with human islet cells (Figures 12A, 12B, 12C, 12D). The cuts are analyzed in a blind analysis. Hematoxylin and eosin staining of a control human islet grafted to the mouse 29 days after transplantation showed a massive lymphocyte infiltration (Figure 12A). The same tissue, stained for insulin, did not show detectable insulin production (Figure 12B). Histological examination of mouse tissue treated with CTLA4Ig 21 days after transplantation showed intact islets under the renal capsule with very few lymphocytes infiltrating the transplanted tissue (Figure 12C). The tissue is stained with hematoxylin and eosin. The same tissue of the mouse treated with CTLA4Ig, stained for insulin, showed the production of insulin by the grafted islets (Figure 12D). Similar results are observed in the grafted tissue examined at later time points. The upper, middle and lower arrows identify the renal capsule, islet transplant and renal parenchyma, respectively. The histopathology analysis in all tissues is fixed in 10% buffered formalin and processed and the 5 μm sections are stained with either hematoxylin and eosin or for insulin with the avidin-biotin-peroxidase method (SM Hsu, L Raine, H. Fanger, J. Histochem, Cytochem, 29: 577 (1981)). The amplification was x 122. In Figure 13, animals treated with streptozotocin are transplanted as described in the above for Figure 11. Mice are treated either with PBS (dotted lines) or with mAb for human B7 (lines continuous) at a dose of 50 μg every third day for 14 days (Figure 13). The control animals (treated with PBS) (n = 3) have an average graft survival of 3.5 days, while the animals treated with anti-B7 (n = 5) maintain the grafts from 9 a >50 days (Figure 13). In Figure 14, transplanted mice, treated with normal CTLA4Ig glycemics, (dotted lines) are nephrectomized on day 44 after transplantation and immediately re-transplanted with either 1000 islets of the first donor part (dotted lines, full circles ) or 1000 islets of the second part (dotted lines, empty circles) below the remainder of the renal capsule. These islets, frozen at the time of the first transplant, are thawed and cultured for 3 days before the transplant to ensure the island's function. B10 mice that have been treated with streptozotocin and had fasting glucose levels greater than 280 mg / dl are used as controls (solid lines) (Figure 14). Without treatment after the transplant. The control animals rejected the first part (solid lines, empty circles) and the second part (solid lines open circles) and islet grafts on day 4 after transplantation (Figure 14). The retransplantation of mice treated with CTLA4Ig with the islets of the second part have an average graft survival of 4.5 days, whereas the animals retransplanted with the islets of the first donor part maintained the grafts while they are analyzed (> 80 days) (Figure 14).

CTLA4Ig significantly prolongs the survival of the human islet graft in mice in a specific form of the donor, thereby providing an approach for immunosuppression C57BL / 6 (B6) or C57BL / 10 (BIO) mice are treated with streptozotocin to eliminate the function of the pancreatic B-islet valley of mouse. The diabetic animals were grafted under the renal capsule and the treatment is started immediately after the surgery. The survival of islet grafts is verified by the analysis of blood glucose concentrations.

Transplanted control animals, treated either with phosphate buffered saline (PBS) (n = 14) or L6 (chimeric MAb and human IgGl, n = 8), had a graft survival by means of 5.6 and 6.4 days, respectively (Figure HA). In contrast, islet rejection was delayed in animals treated with CTLA4Ig (10 μg per day for 14 days), with four of the seven animals showing moderately prolonged average graft survival (12.75 days), while all three remaining animals maintained normal glucose levels during > 80 days (Figure 11B). This eventual increase in glucose concentration may be a result of islet wear because no evidence of active cellular rejection was observed. In the three mice that maintained the long-term islet grafts, the transient increase in glucose concentrations around day 21 after transplantation may have represented a self-limiting rejection episode, consistent with the pharmacokinetics of CTLA4Ig clearance after therapy (PS Linsley et al., Science 257: 792 (1992)). In the subsequent experiments, the dose of CTLA4Ig was increased to 50 μg per animal every third day for approximately 14 days. This treatment resulted in 100% of the animals that maintain the function of the normal island throughout the experiment without signs of a rejection crisis (Figure 11C). To confirm that the insulin production originated from the transplanted islets and not from the native mouse pancreas, the animals were nephrectomized on days 21 and 29 to remove the islet grafts (Figure 11C). In these animals, glucose concentrations increased above 350 mg / dl at 24 hours, which indicated that the islet xenograft was responsible for maintaining normal glucose levels. It seems that blocking the CD28-B7 interaction inhibits the rejection of the xenogenic islet graft. The effects of the treatment with the soluble receptor, namely the CTLAIg fusion protein, were not a result of Fc binding (L6 does not effect graft rejection) or general effects on the function of T cells or B cells in vivo . Historical analyzes of the islet xenograft of the control (treated with PBS) and mice treated with CTLA4Ig were performed (Figures 12A, 12B, 12C, 12D). The islet tissue of the control animal demonstrated evidence of immune rejection with a marked lymphocytic infiltrate in the graft and few remaining islets (Figure 12A).

Immunohistochemical staining showed that insulin-positive cells were only rarely present and somatostatin-positive cells were not present (Figure 12B). In contrast, the transplantation tissue of mice treated with CTLA4Ig was left devoid of any lymphocytic infiltrate (Figure 12C). The grafts were intact, with many visible islets. further, the B cells observed in the tissue of the human islet produced human insulin (Figure 12D) and somatostatin. The human CTLA4Ig used in this study reacts with murine and human B7. An advantage of the xenogeneic transplant model is the availability of a MAb for human B7 that does not react with mouse B7 (T. Yokochi, R.D. Holly, E.A. Clark, J. Immunol.128: 823 (1982)). In this way, the role of cells presenting the antigen carrying the human B7 (APC) could be examined directly. Mice are transplanted as described and then treated with 50 μg of human B7 MAb every third day for 14 days after transplantation. This treatment prolonged graft survival in the treated mice (from 9 a >50 days) compared to that for the control mice (Figure 13). The anti-B7 MAb is unable to block rejection as effectively as CTLA4Ig.

CTLA4Ig therapy resulted in acceptance of the graft in most of the mice. However, animals can not be tolerant. The transient immunosuppression can lead to the acceptance of the permanent islet graft, due to graft adaptation (the loss of immunogenicity as a result of the loss of APC function) (L. Hao, Y. Wang, RG Gilí, KJ Lafferty, J. Immunol, 139: 4022 (1987), KJ Lafferty, SJ Prowse, M. Simeonovic, Annu, Rev. Immunol., 1: 143 (1983)). To differentiate between these possibilities, mice treated with nephrectomized xenografted CTLA4Ig were selected (day 40) and retransplanted under the rest of the renal capsule with the original donor islands (first part) or the human islets of the unrelated second part (Figure 1). 14). Control animals treated with streptozotocin who have never received the graft from an islet are also transplanted with the islets of the first or second parts. There is no treatment after the transplant is performed. The control animals rejected the islets of the first and second part on day 4. The animals treated with CTLA4Ig that received the islets of the second part rejected these islets on day 5, while the animals receiving the donor islets of the first part maintained their graft during > 80 days (Figure 14).

These results suggest that treatment with CTLA4Ig resulted in the lack of specific sensitivity of the prolonged donor for the xenogenic islets. The ability of the murine immune response to distinguish the differences between the donors of the human islet will also support the direct recognition of the polymorphic MHC products expressed in the cells of the human islets.

EXAMPLE 6 Female BALB / c (H-2d) and C57BL / 6 (H-2d) mice, 6 to 8 weeks old, are obtained from The Jackson Laboratory (Bar Harbor, ME). Monoclonal antibody 11B11 is a murine IL-4 rat IgGl (Ohara, J., and WE Paul, 1985, Production of a monoclonal antibody to and molecular characterization of B-cell stimulatory factor-1., Nature 315: 333) ( Verax (Lebanon, NH)). BALB / c mice (five per group) are immunized intravenously with 108 SRBC alone or together with 200 μg of the chimeric L6 mAb or the human CTLA4Ig fusion protein. The indicated groups are treated 2 hours before the injection of the SRBC by intraperitoneal injection 2 ml of either the rat immunoglobulin or the murine anti-IL-4 rat mAb 11B11 at 5 mg / ml. Treatment with chimeric mAb L6 or CTLA4Ig was repeated daily for an additional 4 days. All animals are given intravenous injections of the SRBC (Figure 15) or KLH (Figure 16) on day 46. Specifically, Figure 15, the full circle represents the mice that were only given SRBC on the day 0 and day 46. The empty circle represents the mice administered with SRBC only on day 46. The remaining mice depicted in Figure 15 were further administered with SRBC on day 46. In contrast, in Figure 16, mice were administered with a different immunogen, KLH, only on day 46. The serum concentrations of the mice measuring the antibodies directed against SRBC or KLH are determined by ELISA as described (Linsley et al., Science 1992). The antibody titers in serum are calculated as the dilution giving an A450 of five times the base. The values of the antibody titer in serum of Figure 15 is determined from the pooled sera of five mice per group, while the values of the antibody titer in serum of Figure 16 represent average titers of five individual sera. The arrows indicate an injection of SRBC or KLH on day 46. Figures 15 and 16 show that the immunological response in mice injected together with CTLA4Ig and anti-IL4 (empty triangle) is suppressed in a specific form of the antigen. Figure 15 shows that there is no increase in serum antibody titer (i.e., no primary or secondary immunological response) in mice injected together with CTLA4Ig and anti-IL4 and injected with SRBC on day 0 and day 46. The combination of CTLA4Ig and anti-IL4 suppresses a primary and secondary immune response and induces lasting immunological insensitivity to SRBC. Additionally, Figure 15 shows that there is no primary immunological response in the mice injected together with CTLA4Ig and the Ig of the control rat (Cappel, organonteckinka, Palo Alto, CA). However, these mice have a secondary immune response after injection with SRBC on day 46 (full triangle, Figure 15). Figure 16 shows that the administration of CTLA4Ig and anti-IL4 followed by a different immunogen, KLH at day 46 in mice does not suppress a primary immune response to KLH in mice. In contrast, these mice exhibited a primary immune response for KLH (empty triangle, Figure 16). In this way, mice treated with CTLA4Ig and anti-IL4 had a very specific immune response depending on the antigen administered.

EXAMPLE 7 For site-specific and homologous mutagenesis, regions have been identified in CTLA4Ig, which are required for their high binding avidity for B7-1. The following is a description of how to prepare hybrid fusion proteins CTLA4 / CD28 soluble which bind to B7.

MATERIALS AND METHODS Monoclonal antibodies (mAbs). the murine mAb specific for CTLA4 are prepared and characterized as previously described (Linsley, et al., J. Ex. Med., (1992) 176: 1595-1604). Antibody 9.3 (anti-CD28) has been previously described ((Hansen et al., Immunogenetics 10: 247-260 (1980)). Cell culture The preparation of stably transfected B7-1 CHO positive cells is previously described (Linsley et al., in J. Exp. Med. 173: 721-730 (1991); PS Linsley et al., J. Exp. Med. 174: 561 (1991)). The cells are maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 0.2 mM proline and 1 μM methotrexate. The COS cells are grown in DMEM supplemented with 10% FBS. CTLA4Ig is prepared in CHO cells as previously described (Example 2). Plasmids for expression of the mutant if uncle CTLA4Ig and CD28Ig. Site-directed mutagenesis is performed on a vector that codes for the soluble chimeric form of CTLA4 (CTLA4Ig) in which the extracellular domain of CTLA4 is genetically fused to the hinge and constant regions of the heavy chain of human IgG (Example 2).

The CTLA4Ig site-directed mutants are prepared by coding the desired mutation in the overlapping oligonucleotide primers and generating the mutants by PCR (Ho et al., 1989, supra.) Using the CTLA4Ig plasmid construct as a template. Six mutants are prepared which code for substitutions for alanine in the highly conserved hexapeptide 98MYPPPY103 which forms part of the domain similar to CDR3-course (Figures 17 and 22) (Ho et al., 1989, supra.).

These mutants are described in Table II. In addition, two mutants coding for residues P103A and Y104A are also prepared by the same methods (MYPPAY and MYPPPA, respectively) of the hexapeptide 99MYPPPY104 CD28Ig, using CD28Ig as a template.

These mutants are also described in Table II. The primers required for the PCR reactions but not for the introduction of the mutations, included (1) a CDM8 forward primer (CDM8FP) encoding a sequence upstream of the HindIII restriction site at the 5 'end of the CDM8 filled region and (2) a reverse primer (CDM8RP) that codes for a complementary sequence downstream of the Xbal site at the 3 'end of the CDM8 filled region. These primers encoded for the following sequences: CDM8FP: 5 '-AATACGACTCACTATAGG CDM8RP: 5' -CACCACACTGTATTAACC The conditions for PCR consisted of 6 minutes at 94 ° C followed by 25 cycles of 1 minute at 94 ° C, 2 minutes at 55 ° C and 3 minutes at 72 ° C. The Taq polymerase and the reaction conditions were used as suggested by the supplier (Perkin Elmer, Cetus, Emeryville, CA). The PCR products are digested with HindIII and Xbal and ligated to the CDM8 expression vector cut with HindIII / Xbal. To confirm that the desired mutations have been inserted and to verify the absence of the secondary mutations, the mutant CTLA4Ig fusion protein (an example of a soluble CTLA4 mutant fusion protein is sequenced by the dideoxy chain termination / extension reaction by The Sequenase reagents used in accordance with the recommendations of the manufacturers (United States Biochemical Corp., Cleveland, OH).

The plasmids are transfected into COS cells (Aruffo et al., Cell 61: 1303 (1990)) and the conditioned media is used as a source for the resulting Ig mutant fusion proteins. Hybrid expression plasmids CTLA4 / CD28Ig. The CTLA4 / CD28Ig hybrid plasmids coding for the HS2, HS4, HS4-A, HS4-B and HS5 constructs (Figure 19 and Table I) are screened by PCR using the overlapping oligonucleotide primers designed to introduce the sequences CTLA4 on CD28Ig, while at the same time, the equivalent region of CD28 is deleted. The same primers of the forward and reverse PCR of CDM8 described in the above were also used. The following is a list of hybrid CTLA4 / CD28 fusion proteins which were prepared.

DESIGNATION OF THE WORK FRAMEWORK MODIFICATIONS HS1 CTLA4 1-24 FROM CD28 97-125 FROM CD28 HS2 CD28 1-22 FROM CTLA4 96-125 FROM CTLA4 HS3 CTLA4 96-125 FROM CD28 HS4 CD28 96-123 FROM CTLA4 HS4A CD28 96-113 FROM CTLA4 HS4B CD28 114-123 OF CTLA4 HS5 CD28 25-32 OF CTLA4 HS6 CTLA4 25-32 OF CD28 HS7 CD28 96-123 OF CTLA4 25-32 OF CTLA4 HS8 CD28 25-32 OF CTLA4 96-113 OF CTLA4 HS9 CD28 25-32 OF CTLA4 114-123 OF CTLA4 HS10 CD28 96-123 OF CTLA4 51-58 OF CTLA4 HS11 CD28 25-32 OF CTLA4 51-58 OF CTLA4 96-123 OF CTLA4 HS12 CD28 51-58 OF CTLA4 96-113 OF CTLA4 HS13 CD28 25-32 OF CTLA4 51-58 OF CTLA4 96-113 OF CTLA4 HS14 CD28 51-58 OF CTLA4 Each cDNA construct is genetically linked to the cDNA encoding the hinge and constant regions of a human IgG1 to prepare the soluble chimeras. An HS6 hybrid is prepared in a manner similar to that described above, except that the CDR1-like region in CTLA4Ig is replaced with the equivalent region of CD281g. The HS7, HS8 and HS9 constructs are prepared by replacing a base pair of ~ 350 of a HindIII / Hpal 5 'fragment of HS4, HS4-A, and HS4-B, respectively, with the equivalent cDNA fragment digested in a similar manner to starting from HS5, thus introducing the CDLA-like loop of CTLA4 in those hybrids that already contain the CDR3-like region of CTLA4. The HS10-HS13 constructs are homologous mutants in the domain, which are prepared by introducing the CDR2-like loop of CTLA4Ig into previously constructed homologous mutants. This is done by the overlap of mutagenesis by PCR, whereby the primers are designed to introduce CDR2-like sequences of CTLA4 into homologous templates, while at the same time removing the CDR2-like region of CD28 equivalent to the molecule. Accordingly, HS4 served as a template for preparing HS10; HS7 served as a template to prepare HSll; HS4-A served as a template to prepare HS12; and HS8 served as a template for preparing HS13 (Figure 19 and Table I). The CDM8 primers described in the above were also used in these constructions.

The HS14 hybrid construct is prepared by replacing the CD28-like CDR2 loop with the CTLA4Ig equivalent loop (Figure 19 and Table I). Oligonucleotide primers designed to introduce these changes are used in overlap PCR mutagenesis, identical to that described for other mutants. For PCR reactions and subcloning in CDM8 are performed as described above. Again, all mutants sequence is determined by the termination / extension reaction of the dideoxy chain. The plasmids encoding each of the mutants are transfected into COS cells and the resulting soluble Ig fusion proteins are quantified in culture media and visualized by Western blotting as described in the following sections. Quantification of the Ig fusion proteins resulting in the culture media. Soluble mutant fusion proteins are quantified in an enzyme immunoassay by determining the amount of Ig present in the COS cell culture media without serum. Microtiter plates (Immulon2; Dynatech Labs., Chantilly, VA) are coated with 0.5 μg / ml goat anti-human IgG (Jackson Immunoresearch Labs., West Chester, PA) for 16-24 hours at 4 ° C. The wells are blocked for 1 h with the sample diluent (Genetic Systems, Seattle, WA), then washed with PBS containing 0.05% Tween 20 (PBS-Tw). The COS cell culture media containing the fusion proteins are added at various dilutions and incubated for 1 h at 22 ° C. The known concentrations of CTLA4Ig are also added to separate the wells in each plate for a standard curve. After washing, horseradish peroxidase (HRP) -IgG goat anti-human IgG (Tago, Burlingame, CA) diluted 1: 12,000 and incubated for 1 hour at 22 ° C. The wells are then washed and incubated with the substrate 3, 3 ', 5,5'-tetramethylbenzidine (TMB) (Genetic Systems) for 15 minutes before stopping the reaction by the addition of H 2 SO 4. The optical density is measured at double wavelengths of 450 and 630 nm in a microtiter plate reader (Genetic Systems). The concentration of the mutant Ig fusion protein is determined by comparison with a standard curve of known concentrations of CTLA4Ig. Immunoprecipitation of analysis of Western immunoblotting. The CTLA4 / CD28Ig hybrid fusion proteins present in culture media are adsorbed to protein A-Sepharose by incubation overnight at 4 ° C. The beads are washed with PBS containing 0.1% Nonidet-P40 (NP40) then the sample buffer is added to SDS PAGE and the eluted protein is loaded on an SDS polyacrylamide gel. The Western immunoblot of the protein on nitrocellulose is done by standard procedures. The nitrocellulose membranes are blocked with PBS containing 0.1% NP40 and 1% dry, fat-free milk powder. After washing in PBS-Tw, the membranes are incubated with goat anti-human IgG alkaline phosphatase-conjugated goat IgG (Boehringer Mannheim, Indianapolis, IN) diluted 1: 1,000 and incubated for Ih at 22 ° C. The immunoblots are washed and developed using standard procedures. Enzyme immunoassay of positive CHO cells to B7 The ability of CTLA4Ig mutant fusion proteins and hybrid CTLA4 / CD28Ig fusion proteins to bind B7-1 stably expressed in CHO cells is determined by an enzyme immunoassay. The round bottom tissue culture of the 96 well microtiter plates (Corning, Corning, NY) is seeded with CHO B7-1 positive cells at 103 cells / well.

Two days later, the confluent cells are fixed in 95% ethanol for 15 minutes. After washing with PBS-Tw, the mutant Ig fusion proteins are added at various concentrations and incubated for Ih at 4 ° C. After washing, HRP-conjugated Goat IgG anti-human IgG (Tago) diluted with 1: 10,000 is added and incubated for 1 h at 22 ° C. Then the wells are washed and the TMB substrate is added as above and allowed to react for 30 minutes, before stopping the reaction with H2SO4 IN. The absorbance of the wells was measured at 450 nm. Linkage assay of the mutant fusion protein, if tiodirigide CD28Ig. The mutated site-directed fusion proteins of CD28Ig are analyzed for their ability to bind to B7-1 by an indirect enzyme immunoassay. The wells of the ELISA plates are coated with a chimeric fusion protein containing the extracellular domain of human B7-1 fused to an Fc region of the Mouse IgGl at 5 μg / ml for 16 h at 4 ° C. The wells are blocked for 1 h with the sample diluent (Genetic Systems) then washed with PBS-Tw. The culture media of "COS cells containing known concentrations of the mutant fusion protein are added at various concentrations and incubated for 1 h at 22 ° C. The known concentrations of CD28Ig are also added to separate the wells in each plate. add HRP-conjugated Goat IgG anti-human IgG (Tago) diluted 1: 10,000 and incubate for lh at 22 ° C.

The TMB substrate is added and the optical density read as described for the quantification of the fusion proteins Ig in culture media. Linkage of mAb to Ig fusion proteins. The ability of anti-CD28 anti-CTLA4 and anti-CD28 mAbs 9.3 to bind hybrid CTLA4 / CD28Ig fusion proteins and mutant CTLA4Ig fusion proteins is evaluated by an enzyme immunoassay. Wells of microtiter plates (Immulon 2) are coated with 0.5 μg / ml goat anti-human IgG (Jackson) for 16-24 hours at 4 ° C. The plates are blocked for lh with the sample diluent (Genetic Systems), washed with PBS-Tw, then incubated with the Ig fusion proteins for lh at 22 ° C. After washing, the cells are incubated with the mAb at 1 μg / ml for 1 h at 22 ° C. After washing, HRP-conjugate of goat anti-mouse Ig Ig (Tago) diluted 1: 10,000 is added and incubated for 1 h at 22 ° C. The TMB substrate and the optical density measured as described above are added. Molecular model CTLA4. An approximate three-dimensional model of the extracellular domain CTLA4 is generated based on the conservation of the consensus residues of the variable cell domains of IGSF. Using such IGSF consensus residues as "anchor points" for sequence alignments, CTLA4 residues are assigned to strands A, B, C, C, C ", D, E, F, G of a variable fold Ig (Williams / Barclay, 1988 , supra.) and the connecting loop regions (Figure 22) .The CTLA4 model is constructed (InsightlI, Discover, Molecular Modeling and Mechanics Programs, respectively, Biosym Technologies, Inc., San Diego) using the variable heavy chain of HyHEL- 5 (Sheriff et al., 1987 PNAS 84: 8075-8079) as the template structure The replacements of the side chain and the conformations of the loop are approximated using conformational research (Bruccoleri et al., 1988 335: 564-568) Several versions of the model with modified assignments of some residues for ß-strands or loops are tested using the 3D profile analysis (Lüthy et al., 1992, Nature 336: 83-85) to improve the initial alignment of the sequence of the CTLA4 extracellular region with an IGSF with a variable fold.

RESULTS Construction and binding activity of mutant fusion proteins CTLA4Ig and CD28Ig. Ona alignment sequence of several CD28 and CTLA4 homologs is shown in Figure 17. In Figure 17, the human (H), mouse (M), rat (R) and chicken (Ch) sequences are aligned with human and mouse CTLA4. The residues are numbered from the N-terminus of the mature protein with the signal peptides and the transmembrane domains underlined and the analogous regions of CDR are noted. The dark shaded areas underline the complete conservation of the residues, while the underlined light shaded areas preserve the amino acid substitutions in all members of the family. The regions of sequence conservation are extended in all the extracellular domains of these proteins, with the majority of the rigorous conservation observed in the MYPPPY hexapeptide motif located in the CDR3-like loop of both CTLA4 and CD28 (Figure 17). This suggests a probable role in this region in interaction with a B7 antigen, for example B7-1 and B7-2. To test this possibility, site-directed alanine scanning mutations are introduced into this region of CTLA4Ig using the mutagenesis-directed PCR of the oligonucleotide primer, resulting in CTLA4Ig mutant fusion proteins. Similarly, two alanine mutations are introduced into the MYPPPY motif of CD28ig resulting in mutant fusion protein CD28Ig. All cDNA constructs are sequenced to confirm the desired mutations before transfection in COS cells. The concentrations of the mutant Ig fusion proteins in culture media of COS cells without serum, are determined by an Ig quantitation assay. The ability of each CTLA4Ig mutant fusion protein to bind to B7-1 expressed in CHO cells or stably transfected is then determined by an indirect cell binding immunoassay. The binding of mutant fusion proteins CD28Ig to B7-1 is evaluated by an indirect enzyme immunoassay. Each of these tests is described in Materials and Methods. The mutagenesis of each residue of the MYPPPY motif of CTLA4Ig to Ala has a profound effect on the binding to B7-1 as shown in Figure 18. Figure 18 shows that mutations in the MYPPPY motif of CTLA4Ig and CD28Ig break binding to B7-1. The mutated site-directed Ig fusion proteins are produced in transiently transfected COS cells, quantified and tested for their ability to bind to B7-1. In Figure 18, the quantifications of the fusion protein are repeated at least twice with replicate determinations. Specifically, Figure 18 shows that the CTLA4Ig mutants bind to ethanol-fixed CHO B7-1 + cells, stably transfected to confluence in ELISA tissue culture plants. Link data is expressed as the average of duplicate wells and is representative of at least two experiments.

Mutants Y99A and P101A bind to B7-1, but with considerably reduced capacity relative to wild-type CTLA4Ig. In contrast, mutants M98A, P100A, P102A and Y103A showed an almost complete loss of the binding. In addition, mutants P103A and Y104A of CD28Ig MYPPPY do not have detectable binding to B7-1 immobilized on the cells of the ELISA plates (Figure 18b). CHO cells transfected with B7-1 which are incubated with the CTLA4Ig mutant fusion protein, labeled with anti-human FITC, and analyzed, using a FACSCAN showed equivalent results. These results clearly demonstrate that a critical role for the MYPPPY motif in both CTLA4Ig and CD28Ig binds to B7-1. Characterization of the hybrid fusion proteins of CTLA4 / CD28Ig. Since the MYPPPY motif is common for both CTLA4Ig and CD28Ig, the link to B7-1 observed with CTLA4Ig and CD28Ig can not be taken into account only for the differences observed. The contribution of less conserved residues to high binding avidity B7-1 is evaluated using a series of homologous mutants. The three CD28 CDR-like regions are replaced in various combinations with the equivalent regions of the extracellular domain of CTLA4 (Figure 19 and Table I). Figure 19 is a map of mutant CTLA4 / CD28Ig fusion proteins showing% binding activity to CHO B7-1 + cells relative to CTLA4-Ig. The conserved cysteine residues (C) are shown at positions 22, 93 and 121, respectively (CTLA4 numbering). The position of the MYPPPY pattern is also displayed. The empty areas represent the sequence CD28; the filled areas represent CTLA4 sequences; the cross-hatched areas represent the start of Fc of IgG (also refer to Table I). The percentage of binding activity is determined by comparing with the binding curves (Figure 20a / b) in relation to CTLA4-Ig and the discovery of the concentration of a mutant required to give the same D.O. than the one found for CTLA4-Ig. The ratio of the mutant protein to the concentration of CTLA4-Ig to a D.O. In particular, it is expressed as the% of binding activity. At least two readings at A450 are taken from the linear part of the CTLA4-Ig link curve and the average activity link% is determined. A total of 14 hybrid cDNA constructs are prepared, the sequence determined and transfected into COS cells. The concentrations of the Ig fusion proteins in culture media without serum is determined and the electrophoretic mobility is compared by SDS-PAGE including Western blot analysis. Under reducing conditions each chimeric protein migrates with a relative molecular mass between that of CTLA4Ig (Mr-50kDa) and CD28Ig (Mr-70kDa) depending on the size of the exchanged region. Under non-reducing conditions, the proteins migrate mainly between 100-140 kDa units indicating that these fusion proteins existed as disulfide-linked dimers despite the mutagenesis of the cysteine residues in the hinge region of the Fc. Since four of the five cysteine residues conserved in CTLA4 and CD28 are thought to be involved in intrachain disulfide bonds, the dimerization of the fusion proteins is therefore very likely attributable to the fifth cysteine residue conserved at position 121 in CTLA4 (position 123 in CD28). Linkage of the hybrid fusion proteins CTLA4 / CD28Ig to B7-1. The hybrid fusion proteins are tested for their capacity for a B7-1 linkage by the same indirect cell binding immunoassay used to assay the site-specific mutant CTLA4Ig and CD28Ig fusion proteins. Under these conditions the linkage CD28Ig and B7-1 is barely detectable (Figures 20a / b). However, replacing residues 97 to 125 (the extended CDR3-like region) of CD28 with the corresponding residues of CTLA4 resulted in approximately an increase of two and a half orders of magnitude in the binding of the analog CD28Ig to B7-1 (Figure 20a / b). Figure 20a / b shows that CTLA4 / CD28Ig mutant fusion proteins are involved in the analog regions of CDR in the high binding avidity to CHO B7-1 cells. The mutants are tested as described in Figure 2. The data are expressed as the average of duplicate wells and are representative of at least three experiments. Of these curves, the% binding activity relative to CTLA4-Ig is determined as explained and shown in Figure 19. The binding to B7-1 by this construct, called HS4 (Figure 19), is approximately five times less than the wild-type CTLA4Ig. Hybrid HS2, which includes the additional N-terminal residues of CTLA4 (amino acids 1-22) does not improve the ability of the hybrid molecule to bind to B7-1 relative to HS4. The HS6 construct, which represents the sequence CTLA4Ig except that it contains the CDR1-like region of CD28 (residues 25-32), similarly bound. However, the additional inclusion of the CTL4 CDR1-like region (residues 25-32) in the HS4 construct (called HS7), further showed the enhanced linkage such that the binding affinity is approximately 44% of CTLA4Ig (FIG. 19). In contrast, inclusion of the CDR2-like region of CTLA4 (residues 51-58) in HS4 (construct HS10) does not further increase the binding (Figure 19). A similar result is found for the HSll construct, which has all three snces of the CDR-like region of CTLA4 included within CD28Ig. The HS5 hybrid containing only the CDR1 domain of CTLA4 was bound at very low levels. HS4-A of hybrid CTLA4 / CD28Ig encoded for residues 96-113 of CTLA4Ig in the CDR3-like region extended at the C-terminus; nine residues derived from CTLA4 less than HS4 (Figure 19 and Table I). HS4-A bound CHO B7-1 cells less than HS4 (Figures 19 and 20b). However, the addition of the CDR1-like loop of CTLA4 (hybrid HS8) increased the binding of B7-1 from about 2% to almost 60% of the wild type linkage. On the other hand, the addition of the CDR2-like loop of CTLA4 in HS4-A (HS12) did not increase the binding relative to HS4-A; nor the addition of all three CDR-like regions of CTLA4 (HS13, Figure 19). Another hybrid called HS4-B encoded CDR3-like region of CD28 including motif MYPPPY followed by residues 114-122 of CTLA4 (Table I and Figure 19). HS4-B and HS4-A presented similar links to B7-1. Unlike HS4-A, however, the inclusion of the CDR1-like loop of CTLA4 in HS4-B (HS9) does not improve the binding (Figure 19), suggesting that residues immediately adjacent to the MYPPPY motif of CTLA4Ig were important determinants in the high bond avidity.

Monoclonal antibody binding to the hybrid fusion proteins CTLA4 / CD28Ig. The structural integrity of each hybrid fusion protein is examined by evaluating its ability to bind to specific mAbs for CTLA4 or CD28 in an enzyme immunoassay. Specific mAbs of CTLA4, 7F8, 11D4 and 10A8 block the ligand binding (Linsley et al. (1992) supra.). These antibodies bind to each of the mutant CTLA4Ig fusion proteins, except that 11D4 which fails to bind to P100A and P102A (Table II). Since 7F8 and 10A8 bind to these mutants, the lack of 11D4 binding can probably be attributed to the mutagenesis that disrupts the epitope recognized by 11D4. In contrast, each antibody failed to bind to any of the homologous hybrid fusion proteins except that 7F8 which bound to HS6 and 11D4 binds weakly to HS8. As many of these homologous hybrid fusion proteins were, to some degree, capable of binding to B7-1, it is likely that the lack of linkage by the antibodies is due to the disruption of the conformational epitopes formed by spatially adjacent snces, but not linear The CD28-specific mAb 9.3 (Linsley et al. (1992) supra.) Failed to bind either to the CD28 site-directed mutant fusion proteins, but bound to the hybrid fusion proteins HS4, HS4-A, HS7, and HS8. With HS2, the weakest link was observed. No link was observed with the HS5 and HS6 constructs. CTLA4 model. Figure 21 shows a schematic representation of the CTLA4 model. The assignment of CTLA4 residues to CDR-like regions is shown in Figure 17. The CTLA4 model suggests the presence of an additional disulfide bond (without Ig) between the Cys49 and Cys67 residues which supports the similarity of CTLA4 and the fold Ig variable. The two possible N-linked glycosylation sites on the CTLA4 map to resolve the exposed positions of the framework regions of the ß-strand of Ig. Analysis of the 3D profile indicated that the CTLA4 snce is fully compatible with a V fold of Ig, although it is related very slowly. The Valll5 residue represents the last residue of the CTLA4Ig-like domain. The conformation of the region between Valll5 and Cysl21 close to the membrane, which is believed to form the CTLA4 homodimer is very variable in the CD28 family. The image that emerges is that of the members of the CD28 family that mainly use the residues in two of three regions similar to CDR for the B7-1 link. The MYPPPY motif represents a conserved fold to bind which seems to be increased by its C-terminal extension and which is modulated specifically by the very variable CDR1-like region. The CDR3 and CDR1-like regions are spatially contiguous in variable folds of the Ig. The CDR2-like region is spatially distinct and not, in the case of the CD28 family, it contributes significantly to the binding to B7-1.

TABLE I. Mutant binding sequences CTLA4-Ig / CD28-Ig homologous.

MUTANT HS1 -22CKYasp27- -93ckvEV 99- -123CPSDQE- HS2 -20fvcKYS25- -94CKIelm98- -121cpdDQE- H S3 -93ckvEVM99- -123CPSDQE- HS4 -94CKIelm98- -121cpdDQE- HS5 -22CKYasp27- -30ateFRA35- -123CPSDQE- H S6 -22ceySYN27 - -30SREvrv35- -121cpdDQE- HS4-A -94CKIelm98- -ll ltqiHVKlld- -123CPSDQE- WS4-B -113? Iyvil l6- -121cpdDQE- HS7 -22CKYa-f27- -30ateFRA35- -94CKIelm98- -121cpdDQE-tfS8 -22CKYasp27 - -30ateFRA35- -94CKIelm98- -ll liqiHV l ld- -123CPSDQE- H S9 -22CKYasp27- -30aleFRA35- -U3TII vil -6- -L21cpdDQE- or I «SIO -47VCVaty53 -56gneLQV60- -94CKIelm98- -121cpdDQE- Hsn -22CKYasp27- -30ateFRA35- -47VCVaty53 -56gneLQV60- -94CKIelm98- -121cpdDQE- / - • S12 -47VCVaty53 -56gneLQV60- -94CKLelm98- -ll ltq¡HVK118- - 123CPSDQE-tfS13 -22CKYasp27- -30ateFRA35- -47VCVaty53 -56gneLQV60- -94CKIelm98- -ll ltqiHV pß- -123CPSDQE- «S14 -47VCVaty53 -56gneLQV60- -123CPSDQE- The binding sequences of the hybrid fusion proteins CTLA4-Ig / CD28-Ig. Amino acids are represented by their one-letter code with those in capital letters that are CD28-Ig residues, those in lowercase letters that are CTLA4 residues and those that are in highlighted capital letters that are residues of IgGl. The numbering is of the N-terminal mature of the respective proteins and refers to the adjacent amino acid in the table.

TABLE II. Linkage of monoclonal antibodies CTLA4 and CD28 to mutant fusion proteins CTLA4lg and CD28lg and hybrid fusion proteins CTLA4 / CD28lg. mAb anti-CTLA4 mAb an i-CD28 7F8 11D4 10A8 9.3 MUTATING FUSION PROTEIN CTLA4lq AYPPPY +++ +++ +++ MAPPPY ++ + ++ MYAPPY + - + MYPAPY +++ +++ +++ MYPPAY + ++ - + MYPPPA +++ ++++ AAPPPY + ++ +++ MUTATING FUSION PROTEIN CD28lq MYPPAY MYPPPA HYBRID FUSION PROTEINS CTLA4 / CD28lq HS1 HS2 + HS3 HS +++ HS5 HS6 HS4-A ++ HS4-B ++ HS7 +++ HS8 + +++ HS9 + HS10 HSll HS12 HS13 HS14 CTLA4Ig +++ +++ +++ CD28Ig +++ The binding of the antibody was classified from that which was observed from the wild-type protein. { +++) superior of the base (+), and non-detectable link (-).

LIST OF SEQUENCES (1. GENERAL INFORMATION (i) APPLICANTS: Linsley, Peter S. Ledbetter, Jeffrey A. Damle, Nitin K. Brady, William Wallace, Philip M. (ii) TITLE OF THE INVENTION: CTLA4 MOLECULES AND IL4 LINK MOLECULES AND USES THEREOF (iii) SEQUENCE NUMBER: 14 (iv) ADDRESS THE CORRESPONDENCE: (A) RECIPIENT: Merchant & Gould (B) STREET: 11150 Santa Monica Blvd., Suite 400 (C) CITY: Los Angeles (D) STATE: California (E) COUNTRY: E.U.A. (F) ZIP: 90025-3395 (v) READING FORM ON THE COMPUTER: (A) TYPE OF MEDIUM: soft disk (B) COMPUTER: compatible with an IBM PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.25 (vii) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) DEPOSIT DATE: (C) CLASSIFICATION: (viii) INFORMATION OF THE POWDER / AGENT: (A) NAME: Adriano, Sarah (B) REGISTRATION NUMBER: 34,470 (C) REFERENCE / FILE NUMBER: 9643 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (310) 312-9900 (B) TELEFAX: (310) 479-8340 (2) INFORMATION FOR IDENTIFICATION SEQUENCE NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) SHAPE OF THE SHEET: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 1: CTAGCCACTG AAGCTTCACC ATGGGTGTAC TGCTCACAC 39 (2) INFORMATION FOR THE IDENTIFICATION SEQUENCE NO: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) SHAPE OF THE SHEET: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 2: TGGCATGGGC TCCTGATCAG GCTTAGAAGG TCCGGGAAA 39 (2) INFORMATION FOR IDENTIFICATION SEQUENCE NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) SHAPE OF THE SHEET: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL FU FUNTE: (A) ORGANISM: Homo sapiens (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 3: TTTGGGCTCC TGATCAGGAA AATGCTCTTG CTTGGTTGT 39 (2) INFORMATION FOR IDENTIFICATION SEQUENCE NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 84 base pairs (B) TYPE: nucleic acid (C) SHAPE OF THE SHEET: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 4: AAGCAAGAGC ATTTTCCTGA TCAGGAGCCC AAATCTTCTG ACAAAACTCA CACATCCCCA 60 CCGTCCCCAG CACCTGAACT CCTG 84 (2) INFORMATION FOR IDENTIFICATION SEQUENCE NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) SHAPE OF THE SHEET: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 5: CTTCGACCAG TCTAGAAGCA TCCTCGTGCG ACCGCGAGAG C 41 (2) INFORMATION FOR IDENTIFICATION SEQUENCE NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 47 base pairs (B) TYPE: nucleic acid (C) SHAPE: simple (D) TOPOLOGY : linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO 6: CATTGCACAG TCAAGCTTCC ATGCCCATGG GTTCTCTGGC CACCTTG 47 (2) INFORMATION FOR IDENTIFICATION SEQUENCE NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) SHAPE OF THE SHEET: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 7: ATCCACAGTG CAGTGATCAT TTGGATCCTG GCATGTGAC 39 (2) INFORMATION FOR IDENTIFICATION SEQUENCE NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 65 base pairs (B) TYPE: nucleic acid (C) SHAPE OF THE SHEET: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iü) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 8: CTCAGTCTGG TCCTTGCACT CCTGTTTCCA AGCATGGCGA GCATGGCAAT GCACGTGGCC 60 CAGCC 65 (2) INFORMATION FOR IDENTIFICATION SEQUENCE NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) SHAPE OF THE SHEET: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 9: TTTGGGCTCC TGATCAGAAT CTGGGCACGG TTG 33 (2) INFORMATION FOR IDENTIFICATION SEQUENCE NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 72 base pairs (B) TYPE: nucleic acid (C) SHAPE OF THE SHEET: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 10: CTAGCCACTG AAGCTTCACC AATGGGTGTA CTGCTCACAC AGAGGACGCT GCTCAGTCTG 60 GTCCTTGCAC TC 72 (2) INFORMATION FOR IDENTIFICATION SEQUENCE NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) SHAPE OF THE SHEET: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (ix) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 11: GCAATGCACG TGGCCCAGCC TGCTGTGGTA GTG 33 (2) INFORMATION FOR IDENTIFICATION SEQUENCE NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) SHAPE OF THE SHEET: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (ix) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 12: TGATGTAACA TGTCTAGATC AATTGATGGG AATAAAATAA GGCTG 45 (2) INFORMATION FOR IDENTIFICATION SEQUENCE NO: 13: Ü) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 561 base pairs (B) TYPE: nucleic acid (C) SHAPE OF THE SHEET: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (IX) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 1,561 (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 13: GCA ATG CAC GTG GCC CAG CCT GCT GTG GTA CTG GCC AGC AGC CGA GGC 48 Wing Met His Val Wing Gln Pro Wing Val Val Leu Wing Ser Ser Arg Gly 1 5 10 15 ATC GCC AGC TTT GTG TGT GAG TAT GCA TCT CCA GGC AAA GCC ACT GAG 96 He Wing Be Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu 20 25 30 GTC CGG GTG ACA GTG CTT CGG CAG GCT GAC AGC CAG GTG ACT GAA GTC 144 Val Arg Val Thr Val Leu Arg Gln Wing Asp Ser Gln Val Thr Glu Val 35 40 45 TGT GCG GCA ACC TAC ATG ATG GGG AAT GAG TTG ACC TTC CTA GAT GAT 192 Cys Ala Wing Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp 50 55 60 TCC ATC TGC ACG GGC ACC TCC AGT GGA AAT CAA GTG AAC CTC ACT ATC 240 Ser lie Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr lie 65 70 75 80 CAG GGÁ CTG AGG GCC ATG GAC ACG GGA CTC TAC ATC TGC AAG GTG GAG 288 Gln Gly Leu Arg Wing Met Asp Thr Gly Leu Tyr He Cys Lys Val Glu 85 90 95 CTC ATG TAC CCA CCG CCA TAC TAC CTG GGC ATA GGC AAC GGA ACC CAG 336 Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly He Gly Asn Gly Thr Gln 100 105 110 ATT TAT GTA ATT GAT CCA GAA CCG TGC CCA GAT TCT GAC TTC CTC CTC 384 He Tyr Val He Asp Pro Glu Pro Cys Pro Asp As Asp Phe Leu Leu 115 120 125 TGG ATC CTT GCA GCA GTT AGT TCG GGG TTG TTT TTT TAT AGC TTT CTC 432 Trp He Leu Ala Wing Val Ser Ser Gly Leu Phe Phe Tyr Ser Phe Leu 130 135 140 CTC ACA GCT GTT TCT TTG AGC AAA ATG CTA AAG AAA AGA AGC CCT CTT 480 Leu Thr Ala Val Ser Leu Ser Lys Met Leu Lys Lys Arg Ser Pro Leu 145 150 155 160 ACA ACA GGG GTC TAT GTG AAA ATG CCC CCA ACA GAG CCA GAA TGT GAA 528 Thr Thr Gly Val Tyr Val Lys Met Pro Pro Thr Glu Pro Glu Cys Glu 165 170 175 AAG CA TTT CAG CCT TAT TTT ATT CCC ATC AAT 561 Lys Gln Phe Gln Pro Tyr Phe He Pro He Asn 180 185, ) INFORMATION FOR IDENTIFICATION SEQUENCE NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 187 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (ix) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 14: Wing Met H ± s Val Wing Gln Pro Wing Val Val Leu Wing Ser Being Arg Gly 1 S IO 15 Ha Wing Wing Phe Val Cys Glu Tyr Wing Ser Pro Gly Lys Wing Thr Glu 20 25 30 Val Arg Val T? Ir Val Leu Arg Gln Ala Asp Ser Gln Val thr Glu Val 40 45 Cys Ala Ala Thr-Tyr Met Met Gly Asn Glu Leu Thr-Phe Leu Asp Asp 50 55 60 Ser lie Cys Thr Gly Thr Ser Ser Gly? Sn Gln. Val Asn. Leu Thr lie 65 70 75 80 Gln Gly Leu Arg Wing Met Asp Thr Gly Leu Tyr lie Cys Lys Val Glu 85 90 95 Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly lie Gly Asn Gly Thr Gln 100 105 110 lie Tyr Val lie Asp Pro Glu Pro Cys Pro Asp Ser Asp Phe Leu Leu 115 120 125 Trp He Leu Wing Wing Val Ser Ser Gly Leu Phe Phe Tyr Ser Phe Leu 130 135 140 Leu Thr Wing Val Ser Leu Ser Lys Met Leu Lys Lys Arg Ser Pro Leu 145 150 155 160 Thr Thx Gly Val Tyr Val Lys Met Pro Pro Thr Glu Pro Glu Cys Glu 165 170 175 Lys Gln Phe Gln Pro Tyr Phe He Pro He Asn 180 185 It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention is the conventional one for the manufacture of the objects or products to which it refers. Having described the invention as above, the following is claimed as property:

Claims (31)

1. A CTLA4 / CD28 hybrid fusion protein reactive with the B7 antigen, characterized in that it comprises (a) a first amino acid sequence, if a fragment of the extracellular domain of CD28 is fused to a second amino acid sequence; (b) the second amino acid sequence comprises a fragment of the extracellular region of CTLA4; and (c) a third amino acid sequence of the hinge, CH2 and CH3 regions of the human immunoglobulin Cgammal.

2. The CTLA4 / CD28 hybrid fusion protein according to claim 1, characterized in that the first amino acid sequence is amino acid residues 1-24 and 97-125 of CD28 and the second amino acid sequence is amino acid residues 25-95 of CTLA4 .

3. The hybrid fusion protein CTLA4 / CD28 according to claim 1, characterized in that the first amino acid sequence is amino acid residues 23-96 of CD28 and the second amino acid sequence is amino acid residues 1-22 and 96-123 of CTLA4 .

4. The CTLA4 / CD28 hybrid fusion protein according to claim 1, characterized in that the first amino acid sequence is amino acid residues 97-125 of CD28 and the second amino acid sequence is amino acid residues 1-95 of CTLA4.

5. The CTLA4 / CD28 hybrid fusion protein according to claim 1, characterized in that the first amino acid sequence is amino acid residues 1-94 of CD28 and the second amino acid sequence is amino acid residues 96-123 of CTLA4.

6. The CTLA4 / CD28 hybrid fusion protein according to claim 1, characterized in that the first amino acid sequence is amino acid residues 1-24 and 37-125 of CD28 and the second amino acid sequence is amino acid residues 25-32 of CTLA4 .

7. The CTLA4 / CD28 hybrid fusion protein according to claim 1, characterized in that the first amino acid sequence is amino acid residues 25-32 of CD28 and the second amino acid sequence is amino acid residues 1-24 and 37-123 of CTLA4 .

8. The CTLA4 / CD28 hybrid fusion protein according to claim 1, characterized in that the first amino acid sequence is amino acid residues 1-96 and 116-125 of CD28 and the second amino acid sequence is amino acid residues 96-114 of CTLA4 .

9. The CTLA4 / CD28 hybrid fusion protein according to claim 1, characterized in that the first amino acid sequence is amino acid residues 1-115 of CD28 and the second amino acid sequence is amino acid residues 114-123 of CTLA4.

10. The CTLA4 / CD28 hybrid fusion protein according to claim 1, characterized in that the first amino acid sequence is amino acid residues 1-24 and 32-96 of CD28 and the second amino acid sequence is amino acid residues 25-32 and 96. -123 from CTLA4.

11. The CTLA4 / CD28 hybrid fusion protein according to claim 1, characterized in that the first amino acid sequence is amino acid residues 1-24, 33-96 and 116-125 of CD28 and the second amino acid sequence is amino acid residues. -32 and 96-113 of CTLA4.

12. The CTLA4 / CD28 hybrid fusion protein according to claim 1, characterized in that the first amino acid sequence is amino acid residues 1-24 and 33-115 of CD28 and the second amino acid sequence is amino acid residues 25-32 and 114. -123 from CTLA4.

13. The CTLA4 / CD28 hybrid fusion protein according to claim 1, characterized in that the first amino acid sequence is amino acid residues 1-49 and 58-96 of CD28 and the second amino acid sequence is amino acid residues 51-58 and 96 -123 from CTLA4.

14. The CTLA4 / CD28 hybrid fusion protein according to claim 1, characterized in that the first amino acid sequence is amino acid residues 1-24, 33-49 and 58-96 of CD28 and the second amino acid sequence is amino acid residues. -32, 51-58 and 96-123 of CTLA4.

15. The CTLA4 / CD28 hybrid fusion protein according to claim 1, characterized in that the first amino acid sequence is amino acid residues 1-49, 58-96 and 116-125 of CD28 and the second amino acid sequence is amino acid residues 51 -58 and 96-114 of CTLA4.

16. The CTLA4 / CD28 hybrid fusion protein according to claim 1, characterized in that the first amino acid sequence is amino acid residues 1-24, 33-49, 58-96 and 116-125 of CD28 and the second amino acid sequence are the amino acid residues 51-58 and 96-114 of CTLA4.

17. The CTLA4 / CD28 hybrid fusion protein according to claim 1, characterized in that the first amino acid sequence is amino acid residues 1-49 and 58-125 of CD28 and the second amino acid sequence is amino acid residues 51-58 of CTLA4 .

18. A mutant CTLA4 characterized in that any of the amino acid residues 98-103 which comprises the amino acid sequence MYPPY are replaced by alanine.

19. The mutant CTLA4 according to claim 18, characterized in that the 98-103 amino acid sequence comprises the amino acid sequence AYPPY, MAPPPY, MYAPPY, MYPAPY, MYPPAY, PYPPPA or AAPPPY.

20. A mutant CD28 characterized in that any of the amino acid residues 99-104 which comprises the amino acid sequence MYPPPY are replaced by alanine.

21. The CD28 mutant according to claim 20, characterized in that the amino acid residue 103 or 104 is replaced by alanine.

22. A composition characterized in that it comprises any CTLA4 / CD28 hybrid fusion protein according to any of claims 1-17 for use in the regulation of an immune response by blocking a B7 interaction with lymphocytes.

23. The composition according to claim 22, characterized in that it also comprises an IL4 binding molecule.

24. The composition according to claim 23, characterized in that the IL4 binding molecule is a monoclonal antibody, which specifically recognizes and binds IL4 or a soluble IL4 receptor, which recognizes and binds IL4.

25. The compositions according to any of claims 22-24, characterized in that the lymphocytes are B7-positive lymphocytes.

26. The compositions according to any of claims 22 to 24, characterized in that the immune response is a response of B cells that results in the inhibition of antibody production, a T cell response resulting in the inhibition of mediated immunity by cells or the immune response is an inhibition of lymphocyte proliferation.

27. The composition according to any of claims 22 to 24, characterized in that the composition is for use in inhibiting transplant rejection in an individual, the individual is a recipient of the transplanted tissue.

28. The compositions according to claims 22 to 24, characterized in that the composition is for use in inhibiting graft disease against the host.

29. The use of hybrid CTLA4 / CD28 fusion proteins to prepare a pharmaceutical composition according to any of claims 22 to 24 to regulate an immune response by blocking a B7 interaction with the lymphocytes.

30. The use of a hybrid fusion protein CTLA4 / CD28 for preparing a pharmaceutical composition according to claim 29, characterized in that the lymphocytes are B7-positive lymphocytes.

31. The use of a hybrid fusion protein CTLA4 / CD28 for preparing a pharmaceutical composition according to claim 29, characterized in that the immune response is a T cell response that results in the inhibition of antibody production, a T cell response resulting in the inhibition of the Cell-mediated immunity or the immune response is an inhibition of lymphocyte proliferation. In testimony of which I present the present in this City of Mexico, D.F., on April 12, 1995. By: BRISTOL-MYERS SQUIBB COMPANY Lie Manuel M. Soto Representative