MXPA01002898A - Methods of downmodulating the immune response to therapeutic proteins. - Google Patents

Abstract

Compositions and methods for treating a hemostatic disorder using agents which promote hemostasis and agents which inhibit a costimulatory signal in a T cell are provided. The instant compositions and methods enable the treatment of hemostatic disorders using foreign therapeutic proteins, while downmodulating immune responses to the therapeutic proteins.

Description

"METHODS OF DESCENDING MODULATION OF THE IMMUNE RESPONSE TO THERAPEUTIC PROTEINS" GOVERNMENT FUNDS This invention is supported, in part, by the NIH Concession, HL 36099 granted to the American Red Cross. Therefore, the government can, have certain rights in this invention.

BACKGROUND OF THE INVENTION One of the primary limitations of therapeutic treatment using biological proteins is the immune response generated by the body in response to the presence of foreign substances in the body. This immune response is especially problematic when the foreign substances must be administered repeatedly in order to be effective optimally. An example of this situation is the repeated administration of agents for the treatment of haemostatic disorders, such as factor VIII deficiency diseases (eg, classical hemophilia A and von Illebrand disease) or factor IX deficiency, also known as hemophilia B Classical hemophilia, hemophilia A, is a disorder linked by X that affects 1 in 10,000 males. Von Willebrand disease is the most common inherited bleeding disorder, which occurs in as many as 1 in 800 to 1000 individuals. Hemophilia B, also known as Christmas sickness, occurs at least 1 in 100,000 males (Harrison's Principies of Infernal Medicine, Isselbacher et al., Editors, Thirteenth Edition 1994. McGraw-Hill N.Y., N.Y.). Factor VIII is a single chain 265 kD protein that circulates in a complex with von Willebrand factor (VWF). Factor VIII is an important regulatory protein in the blood coagulation cascade. After activation by thrombin, it accelerates the rate of activation of factor X by activated factor IX (factor IXa), eventually leading to the formation of a fibrin clot. The VWF molecule is an adhesive glycoprotein that plays a central role in the agglutination of platelets. It serves as a carrier for factor VIII in plasma and facilitates the interactions of the plate-wall of the container. VWF is composed of multiple subunits, probably identical each of approximately 230 kD. VWF is synthesized in endothelial cells and megakaryocytes. Factor IX is a single chain proenzyme of 55 kD that is converted to an active protease (IXa) by factor Xla or by the factor Vlla factor complex. Activated factor IX and activated factor VIII then activate factor X. Repeated administration of foreign proteins can lead to an immune response to those proteins in a receptor. In the response of the T cell to foreign proteins, two signals must be provided by antigen-presenting cells (APCs) to the resting T lymphocytes (M. Jenkins, and R. Schwartz, (1987) J. Exp. Med. 165 , 302-319; DL Mueller, et al. (1990) J. Immunol., 144, 3701-3709). The first signal, which confers specificity to the immune response, is transduced through the T cell receptor (TCR) following the recognition of the foreign antigenic peptide presented in the context of the main histocompatibility complex.

(MHC). The second signal, called co-stimulation, induces T cells to proliferate and become functional (Lenschow et al., 1996. Annu. Rev. Immunol. 14: 233). The co-stimulation is neither specific to the antigen nor restricted MHC and is believed to be provided by one or more other cell surface molecules expressed by APCs (MK Jenkins, et al 1988 J. Immunol 140, 3324-3330; PS Linsley , and others 1991 J. Exp. Med. 173, 721-730; CD Gimmi, et al., 1991 Proc Na tl. Acad. Sci. USA 88, 6575-6579; JW Young, et al. 1992 J. Clin.

- - Jnvest. 9_0, 229-237; L. Koulova and others 1991 J. Exp. Med. 173, 759-762; H. Reiser, and others 1992 Proc. Na tl. Acad. Sci. USES. 89, 271-275; G.A. van-Seventer et al. (1900) J. Immunol. L44, 4579-4586; J.M. LaSalle, 1991 J. Immunol. 147, 774-80; MY. Dustin, et al. 1989 J. Exp. Med. 169, 503; R.J. Armitage, et al. 1992 Nature 3_57, 80-82; Y. Liu, and others 1992 J. Exp. Med. 175, 437-445). The CD80 (B7-1) and CD86 (B7-2) proteins, expressed in APCs, are critical co-stimulatory molecules (Freeman et al., 1991. J. Exp. Med. 174: 625; Freeman et al. 1989 J. Immunol. : 2714; Azuma et al., 1993 Na ture 366: 76; Freeman et al. 1993. Sci en 262: 909). B7-2 seems to become more significant during primary immune responses, whereas B7-1, which is upregulated later in the course of an immune response, may be important for prolonging primary T cell responses or secondary T cell responses co-stimulants (Bluestone, 1995, Immuni ty, 2: 555). B7-1 and B7-2 are the counter-receptors for two coordinating groups expressed in T lymphocytes. A coordinating group to which B7-1 and B7-2, CD28 are linked, is constitutively expressed in rest and increase T cells. in expression after activation. After signaling through the T cell receptor, the binding of CD28 and transduction of a costimulatory signal induces the T cell to proliferate and secrete IL-2 (PS Linsley et al., 1991 J. Exp. Med. 173, 721-730: CD Gimmi, and others 1991 Proc. Natl. Acad. Sci. USA 88, 6575-6579; CH June, and others 1990 Immunol. Today 11, 211-6; FA Harding, and others 1992 Na ture 356, 607-609). The second coordinating group, called CTLA4 (CD152) is homologous to CD28 but is not expressed in resting T cells and appears following the activation of the T cell (JF Brunet, and others 1987 Na ture 3 ^ 8, 267-270) . CTLA4 appears to be critical in the negative regulation of T cell responses (Waterhouse et al. 1995. Sci ence 270: 985). CTLA4 blockade has been found to remove inhibitory signals, whereas CTLA4 aggregation has been found to provide inhibitory signals that downregulate T cell responses (Allison and Krummel 1995, Science 270: 932). B7 molecules have a higher affinity for CTLA4 than for CD28 (PS Lensley, et al., 1991 J. Exp. Med. 174, 561-569) and B7-1 and B7-2 have been found to bind to different regions of the CTLA4 molecule and have different kinetics of CTLA4 binding (Lensley et al 1994. Immuni ty 1: 793). Between 10 percent and 25 percent of patients with hemophilia develop an immune response to - - Factor VIII. These patients develop inhibitors, usually IgG antibodies, that neutralize the activity of factor VIII and, therefore, prevent effective therapy. Two types of inhibitors have been identified. High response patients with type I inhibitors have anamnestic response to factor VIII which results in an increased concentration of antibodies to factor VIII. The patients with the lowest response with the type II inhibitor have a low antibody concentration that is not increased by the administration of factor VIII. Current strategies to mitigate the antibody response in these patients have been only marginally satisfactory. In addition, the development of antibodies to replaced proteins is a critical problem that needs to be resolved if gene therapy is to be successful in the treatment of hemophilia and other deficiency diseases (S Connelly et al., Blood 88: 3846, 1996; SH Kuna and others, Blood 91: 784, 1998).

COMPENDIUM OF THE INVENTION The present invention provides, inter alia, compositions and methods that allow the administration of a therapeutic protein to treat a disorder while - - which reduces the development and / or progress of an immune response to the therapeutic protein. In one aspect, the invention relates to compositions comprising a first agent that promotes haemostasis and a second agent that inhibits a co-stimulatory signal in a T cell. In one embodiment, the subject subject compositions further comprise a pharmaceutically acceptable carrier. In one modality, the first agent is the factor VIII. In another embodiment, the first agent is a deleted variant of domain B of factor VIII. In one embodiment, the first agent is factor IX. In another modality, the first agent is the Von Willebrand factor. In one embodiment, the second agent is a soluble form of a costimulatory molecule. In a preferred embodiment, the second agent is a soluble form of CTLA4. In another preferred embodiment, the second agent is a soluble form of B7-1, a soluble form of B7-2, or a combination of a soluble form of B7-1 and a soluble form of B7-2. In a preferred embodiment, the second agent is CTLA4Ig. In another preferred embodiment, the second agent is B7-lIg or B7-2Ig. In another still preferred embodiment, the second agent is a soluble form of CD40 or CD40L.

In another embodiment of the second agent is an antibody that binds to a costimulatory molecule. In a preferred embodiment, the second agent is selected from the? Group which consists of an anti-B7-l antibody, an anti-B7-2 antibody, and a combination of an anti-B7-l antibody and an anti-B7 antibody. -B7-2. In one embodiment, the antibody is a non-activating form of the anti-CD28 antibody. The invention further relates to methods of treating a hemostatic disorder in a subject comprising administering to a subject the present compositions in such a manner that a hemostatic disorder is treated. In one embodiment, the subject has a significant concentration of antibodies that bind to the first agent. In another embodiment, the subject does not have a significant concentration of antibodies that bind to the first agent. In one embodiment, the methods comprise administering a composition consisting of an agent that inhibits a co-stimulatory signal in a T cell. In one embodiment, the hemostatic disorder is selected from the group consisting of hemophilia A, hemophilia B, and the disease. of von Willebrand. In another aspect, the invention relates to methods for treating a hemostatic disorder in a subject - which comprises administering to the subject the first agent that promotes haemostasis and a second agent that inhibits a co-stimulatory signal in a T cell, such that a hemostatic disorder is treated. In another aspect, the invention relates to methods for treating a hemostatic disorder in a subject comprising administering to the subject a first agent that promotes haemostasis and a second agent that inhibits a co-stimulatory signal in a T cell in such a way, that immunotolerance occurs to the first agent, thus treating a hemostatic disorder. In one embodiment, the first agent is factor VIII. In other embodiments, the first agent is a deleted variant of domain B of factor VIII. In another embodiment, the first agent is factor IX. In another modality, the first agent is a Von Willebrand factor. In one embodiment, the second agent is a soluble form of an agent that delivers a costimulatory signal to a T cell. In a preferred embodiment, the agent is a soluble form of CTLA4. In an especially preferred embodiment, the agent is CTLA4Ig. In another preferred embodiment, the agent is a soluble form of B7-1, a soluble form of B7-2 or a combination of both B7-1 and B7-2. In another modality especially - - preferred, the agent is B7-lIg, B7-2Ig, or a combination of both B7-lIg and B7-2Ig. In one embodiment, the second agent is an antibody that binds to a costimulatory molecule. In another embodiment, the second agent is selected from the group consisting of an anti-B7-l antibody, an ar-ti-B7-2 antibody, and a combination of an anti-B7-1 antibody and an anti-B7-1 antibody. -B7-2. In another embodiment, the antibody is a non-activating form of an anti-CD28 antibody. In one embodiment, the hemostatic disorder is selected from the group consisting of hemophilia A, hemophilia B, and von Willebrand's disease. In one embodiment, the subject has a significant concentration of antibodies that bind to the first agent.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the experimental design used for Example 1 to test the inhibition of primary antibody responses to factor VIII. Figure 2 shows that even when the mice did not receive CTLA4Ig they had high concentrations of the antibody starting as early as day 20 (Gl), the mice that received CTLA4Ig do not develop antibodies until day 82 (G-2 and G-3) . Figure 3 illustrates the experimental design used for Example 2 to test the inhibition of secondary antibody responses to factor VIII. Figure 4 shows that animals that did not receive CTLA4Ig had high concentrations of anti-factor VIII (Gl) antibodies, whereas mice that received CTLA4Ig (G-2), with the exception of 1 mouse, did not develop a response immune secondary to factor VIII. Figure 5 shows the effect of mCTLA4Ig on the formation of anti-factor VIII antibody. Figure 6 shows the effect of repeated administration of mCTLA4Ig on the formation of anti-factor VIII antibody. Figure 7 shows the effect of the simultaneous administration of mCTLA4Ig and factor VIII. Figure 8 shows the effect of mCTLA4Ig on the immune response secondary to factor VIII. Figure 9 shows the role of B7-1 and B7-2 in the anti-factor VIII antibody response. Figure 10 shows the response of the T cell to factor VIII for mice with hemophilia A / B7-1 - / - and hemophilia A / B7-2 - / -.

DETAILED DESCRIPTION The present invention represents an important advance in the treatment of hemostatic disorders by providing compositions and methods that allow the administration of a therapeutic protein to treat a disorder while reducing the development and / or progress of an immune response to the therapeutic protein. Before further describing the invention, certain terms used in the specification, the examples and the appended claims are listed here for reasons of convenience. I. Definitions As used herein, the term "hemostatic disorder" includes disorders that result in abnormal bleeding and / or thrombosis. Normal hemostasis limits blood loss through a series of interactions between the components of blood vessel walls, platelets, and plasma proteins. Haemostatic disorders occur, for example, due to a failure in platelet aggregation and / or the formation of fibrin clot which can result in inappropriate responses to the disease or trauma, e.g., uncontrolled bleeding. These disorders can be detected, eg by determining the bleeding time, the partial thromboplastin time (PTT), the prothrombin time (PT), the thrombin time (TT), or by a quantitative fibrinogen determination using well-known methods in the raimo. Exemplary hemostatic disorders include hemophilia A, hemophilia B, and von Willebrand diseases. As used herein, the term "haemostasis promoting agent" includes a protein or polypeptide that is deficient or suppressed in a subject and that, when administered to the subject, alleviates or treats a hemostatic disorder. Preferred agents that promote homostasis include coagulation factors such as Factor VIII, Factor IX, VWF and analogs thereof. The term "B7 family" or "B7 molecules" as used herein includes costimulatory molecules that share the identity of the amino acid sequence with B7 polypeptides, eg, with B7-1, B7-2 or B7-3 (recognized by the BB-1 antibody). In addition, the B ~ 'family of molecules share a common function (eg, the ability to bind to a coordinating group of the B7 family (eg, one or more of CD28, CTLA4 or ICOS) and the ability to co-stimulate the activation of T cell. B7 polypeptides are able to provide co-stimulation to activated T cells to thereby induce T cell proliferation and / or cytokine secretion or to inhibit T-cell co-stimulation, eg, when present in T cells. Soluble form The members of the B7 family include B7-1, B7-2 and the fragments or soluble derivatives thereof In one embodiment, members of the B7 family join CTLA4, CD28, ICOS, and / or other coordinating groups or immune cells and have the ability to inhibit or induce the co-stimulation of immune cells As used herein, the term "agent that inhibits a costimulatory signal in a T cell" includes agents that inhibit a signal generated by the immune cell. interaction of a co-stimulatory molecule in a cell presenting an antigen (APC), eg, a molecule of the B7 family and its counter receptor in a T cell. The costimulatory molecules in APCs (eg, member of the B7 family) and its known coordinating groups in T cells (eg, CTLA4, CD28 and ICOS) are collectively referred to herein as costimulatory molecules. An agent that inhibits a costimulatory signal can act either extracellularly to inhibit the interaction between the costimulatory molecules, thereby blocking the production of intracellular signals, or it can act intracellularly to inhibit the costimulatory signals in a signal transduction pathway. Exemplary agents are described in further detail and include, for example, soluble forms of costimulatory molecules and antibodies that bind to costimulatory molecules. As used herein, the phrase "downward modulation of the immune response" includes reduction in an immune response (eg, suppression, quenching or inhibition) in a patient who does not have an existing immune response or reduction in length and magnitude of an existing immune response. The term "immune response" includes any type of immune response that is initiated by or dependent on costimulatory signals, e.g., a cellular or humoral response, which may occur in a subject in response to a foreign antigen. In one embodiment, the immune response is an antibody response to an agent that promotes hemostasis (e.g., Factor VII, VWF or Factor IX). The term "immunotolerance" includes the induction of antigen-specific tolerance that can be measured using techniques that are known in the art, e.g., by measuring secondary immune responses (e.g., cellular or humoral responses) to an antigen. II. Agents That Promote Haemostasis In one modality, the agent that promotes haemostasis is factor VIII. The term "Factor VIII", as used herein, includes proteins that exhibit procoagulant activity characteristic of factor VIII.

In one embodiment of the invention, the factor VIII proteins are the factor VIII proteins that occur naturally. These proteins can be purified from the blood or they can be administered as a blood product or an enriched blood product. In one embodiment, highly purified factor VIII can be produced by adsorbing and eluting the factor of a blood product on a monoclonal antibody column. Alternatively, these naturally occurring proteins can be made recombinantly using nucleic acid molecules, preferably naturally occurring nucleic acid molecules. For example, in one embodiment, factor VIII proteins are made by expressing a nucleic acid molecule encoding factor VIII in a cell, using techniques known in the art, such that the factor VIII protein is produced. The nucleotide sequence (and the corresponding amino acid sequence) of human factor VIII is already known in the art. (See e.g. Toóle et al., Nature 1984. 312: 5992; or GenBank Accession Nos. X01179; K01740). In another embodiment, the agent promoting haemostasis is a Factor VIII that occurs unnaturally, e.g., the mutant form of factor VIII that retains therapeutic function, e.g., the activity that promotes hemostasis of factor VIII. For example, DNA sequences capable of hybridizing to DNA encoding human factor VIII under conditions that prevent hybridization to non-factor VIII genes, (eg, under equivalent conditions at 65 ° C in 5 X SSC (1 X SSC = 150 mM NaCl / 0.15 M Na citrate) or the homologous DNA sequences that retain the identity of the sequence through the regions of the nucleic acid molecule encoding the protein domains that are important in the function of the Factor VIII can be used to produce factor VIII proteins within the scope of the invention. As examples, the contents of U.S. Patent Nos. 5,744,446; 5,663,060; 5,583,209; 5,661,008; 5,422,260; and 5,707,832 are expressly incorporated herein by reference. In another embodiment, an agent that promotes haemostasis is a factor VIII protein in which at least one domain (e.g., a non-essential domain) of the protein will be deleted. For example, in one embodiment, the factor VIII protein is a modified factor VIII protein wherein one or more amino acids will be deleted or substituted between the 90 Kd and 69 Kc dissociation sites with respect to native factor VIII, as described in greater detail in U.S. Patent Number 4,868,112, the content of which is incorporated herein by reference.

In another embodiment, the agent promoting haemostasis is an analogue of factor VIII that contains a deletion (s) of one or more amino acids between the 50/40 dissociation site and the 73 kD dissociation site that can be produced by methods analogous to those disclosed in U.S. Patent Number 4,868,112, the content of which is incorporated herein by reference. In a preferred embodiment, a factor VIII analogue retains part or all of the acidic amino acid region between the dissociation sites of 80 kD and 73 kD. In other embodiments, part or all of this region is replaced with the corresponding acidic region immediately adjacent to the 50/40 dissociation site. In yet other embodiments, the factor VIII proteins are analogous (with or without deletions as mentioned above) in such a manner that they are disclosed in International Application PCT / US87 / 01299 (the contents of which are incorporated herein by reference). incorporated herein by reference), eg, wherein one or more of the arginine residues spanning the dissociation sites at positions 226, 336, 562, 740, 776, 1313, 1648 or 1721 have become resistant to proteolytic dissociation, eg, by replenishing one or more of the amino acids with different amino acids by mutagenesis of the techniques using cDNAs known in the art, eg, site-directed mutagenesis.

Agents that promote haemostasis also include hybrid factor VIII proteins that includes a portion of a human factor VIII protein and a portion of a protein in non-human factor VIII of another species (e.g., porcine factor VIII). These hybrid proteins can be made using techniques that are known in the art, e.g., as shown in U.S. Patent Nos. 5,744,446; 5,663,060; and 5,583,209. The contents of U.S. Patent Nos. 5,693,499, 5,681,746; 5,663,060; 5,583,209; 5,563,045; 5,460,951; and 5,455,031 are also expressly incorporated herein by this reference. In another modality, the agent that promotes haemostasis is factor IX. As used herein, the term "factor IX" includes, but is not limited to, factor IX isolated from plasma, lines of transformed cells, and factor IX recombinantly produced from the culture medium of the host cell. The factor IX can be purified from the blood or it can be administered as a blood product or an enriched blood product. In one embodiment, highly purified factor IX can be produced by adsorbing and eluting the factor of a blood product on a monoclonal antibody column. Exemplary methods of purification are those disclosed in U.S. Patent Nos. 5,639,857; 5,457,181 and 5,286,849. Alternatively, these naturally occurring proteins can be made recombinantly using nucleic acid molecules preferably nucleic acid molecules that occur naturally. For example, in certain embodiments, factor IX proteins are made by expressing a nucleic acid molecule encoding factor IX in a cell, using techniques known in the art, such that the Factor IX protein is produced. Exemplary genetic constructs for expressing factor IX can be found in U.S. Patent Nos. 5,650,503 and 4,994,371. The nucleotide sequence and the amino acid sequence of factor IX are known in the art, (see, e.g., Yoshitake et al. 1985. Biochemi stry 24: 3726 or GenBank Accession Nos. K02402; A07407; A01819; or X54500). In other embodiments, Factor IX includes, for example, the proteins described in U.S. Patent Nos. 4,994,371; 5,171,569; 5,679,639; 5,621,039; and 5,714,583, all expositions of which are each incorporated herein by reference. In addition to the naturally occurring forms of factor IX, the term factor IX also includes forms that occur unnaturally, eg, mutant forms of factor IX that retain therapeutic properties, eg, properties that promote hemostasis of factor IX . For example, DNA sequences capable of hybridizing to human factor IX that encode DNA under conditions that prevent hybridization to non-factor IX genes (eg, under equivalent conditions at 65 ° C in 5 X SSC (1 X SSC = 150 mM NaCl / 0.15 M Na citrate)). In addition, DNA sequences that retain the identity of the sequence through the regions of the nucleic acid molecule encoding the protein domains that are important in factor IX function can be used to produce factor IX proteins within the scope of the invention. Factor VIII or factor IX proteins can also be purchased commercially. For example, concentrated forms of factor VIII are available, e.g., Immunate® (Immuno). Beriate® (Behring); purified forms of the factor VIII monoclonal antibody are available, e.g., Octanativ-M® (Pharmacia), Hemofil M® (Baxter) and Monoclate-P® (Armor); and recombinant forms of factor VIII are also available, e.g., Recombinate® (Baxter) and Kogenate® (Bayer). A deleted form of the recombinant B domain of factor VIII, r-VIII SQ® (Pharmacia and Upjohn, Stockholm) is also available. Factor IX can be purchased, e.g., as Nanotiv® (Kabi Pharmacia) or Immunine® (Immuno); the purified factor IX of the monoclonal antibody is also available as Mononine® (Armor). Recombinant factor IX is also available, e.g., as BeneFIX® (Institute of Genetics). VWF is a large multimeric plasma protein composed of simple glycoprotein subunits. The subunits of VWF are joined together by disulfide bonds. In VWF plasma circulates as multimers, ranging from dimers to multimers of more than 50 subunits. The dimers consist of two subunits linked, probably by their C terms, by flexible "rod-shaped" domains and are presumed to be protomers in naultimerization. The promoters bind through probably large N-terminal globular domains to form the multimers. VWF appears to be produced as a 260 kD glycosylated precursor which is subsequently processed and sulfated. After dimerization and multimerization and proteolytic cleavage, the mature protein is approximately 225 kD. VWF has been produced recombinantly. The nucleotide and amino acid sequence of VWF is known in the art. (see e.g., Sadler et al., 1986. Cold Spring Harbor Symposium in Quna ti ta ti ve Bi olgy 51: 515 or GenBank Accession Nos. L15333 or K03028). The contents of EP 0197592 Bl are incorporated herein by reference.

In addition to the naturally occurring forms of the VWF factor, the term "VWF factor also includes forms that occur unnaturally, eg, mutant forms of the VWF factor that retain therapeutic properties, eg, properties that promote haemostasis. VWF factor For example, DNA sequences capable of hybridizing to human VWF factor DNA coding under conditions that prevent hybridization in non-VWF factor genes under equivalent conditions at 65 ° C in 5 X SSC (1 X SSC = 150 mM NaCl / 0.15 M Na citrate).) In addition, DNA sequences that retain sequence identity through the regions of the nucleic acid molecule that encode the protein domains that are important in factor function VWF, can be used to produce VWF factor proteins within the scope of the invention In one embodiment, the agents that promote haemostasis are of mammalian origin. The agents that promote haemostasis are of porcine origin. In still another especially preferred embodiment, the agents that promote haemostasis are of human origin. In another embodiment, the agent that promotes haemostasis is made up of hybrid molecules. III. Immunomodulatory agents.

In one embodiment, an agent that inhibits a costimulatory signal in a T cell is a naturally occurring form of a costimulatory molecule. The naturally occurring forms of the costimulatory molecules can be purified from the cells or can be produced recombinantly using techniques known in the art. For example, co-stimulatory proteins can be made by expressing a nucleic acid molecule encoding a co-stimulatory molecule in a cell in such a way that a co-stimulatory molecule is produced. The nucleotide sequence of the costimulatory molecules are known in the art and can be found in the literature or in a database such as GenBank. See, for example, B7-2 (Freeman et al., 1993 Science, 262: 909 or GenBank Accession Numbers P42081 or A48754); B7-1 (Freeman et al., J. Exp. Med. 1991. 174: 625 or GenBank Accession Numbers P33681 or A45803; CTLA4 (See, eg, Ginsberg et al. 1985. Sci ence 228: 1401; GenBank Accession P16410 or 29929), and CD28 (Aruffo and Seed, Proc.Nal.l Acad.Sci.84: 8573 or GenBank Accession No. 180091), ICOS (Hutloff et al. 1999, Nature, 397: 263; WO 98/38216), and related sequences In addition to the forms of co-stimulatory molecules that occur naturally, the term "co-stimulatory molecule" also includes forms that occur unnaturally, eg, mutant forms of costimulatory molecules that retain function. of a costimulatory molecule, eg, the ability to bind to the known counter-receptor, eg, DNA sequences capable of hybridizing to DNA encoding a B7 molecule, a CTLA4 molecule, a CD28, or an ICOS molecule under conditions that prevent Hybridization in non-co-stimulatory molecule genes (eg, low equivalent conditions at 65 ° C in 5 X SSC (1 X SSC = 150 mM NaCl / 0.15 M Na citrate) are co-stimulatory molecules within the scope of the invention. Alternatively, DNA sequences that retain the identity of the sequence through the regions of the nucleic acid molecule encoding the protein domains that are important in the function of the costimulatory molecule, eg, joining other costimulatory molecules, are they can be used to produce costimulatory proteins that can be used as agents that inhibit a costimulatory signal in a T cell. Preferably, costimulatory molecules that occur unnaturally have significant amino acid identity (eg, greater than 70 percent, preferably higher of 80 percent, and especially preferred greater than 90 to 95 percent) with an amino acid sequence that occurs naturally from an extracellular domain of costimulatory molecule. To determine the amino acid residues of a costimulatory molecule that has a tendency to be important in the binding of a co-stimulatory molecule to its counter receptor, the amino acid sequences comprising the extracellular domains of the costimulatory molecules of different species, e.g., from mouse and from humans, may be named and aligned (conserved) residues (e.g., identical). This can be carried out, for example, using any normal alignment program, such as MegAlign (DNA STAR). These conserved or identical residues tend to be necessary for the proper binding of the co-stimulatory molecules in their receptors and, thus, do not tend to be subject to alteration. The specific residues of the costimulatory molecules that are important in the binding have also been determined. For example, the portion of CD28 that is critical to the interaction with B7-1 and B7-2 has been determined using site-directed mutagenesis, epitope map preparation of monoclonal antibody CD28, receptor-based adhesion assays, and binding Direct Ig-fusion proteins to receptors on the cell surface. A stretch of the prolific sequence in CD28, MYPPPY, has been found to be critical as the function of that protein (Truneh et al., 1996. Mol Immun I. 33: 321). Also, the regions of the B7-1 molecule that are important for mediating the functional interaction with CD28 and CTLA4 have been identified by mutation. Two hydrophobic residues in the V-like domain of B7-1, including the Y87 residue, which is conserved in all cloned B7-1 and B7-2 molecules of several species, were found to be critical (Fargeas et al., 1995. J. Exp. Med. 182: 667). Using these, or similar techniques can determine the amino acid residues of the extracellular domains of the costimulatory molecules that are critical and, therefore, not subject to alteration. The costimulatory molecules can be expressed in soluble form or used as immunogens to make antibodies. These soluble coestimulatory molecules or antibodies are useful as agents that inhibit a costimulatory signal in a T cell, as described herein in greater detail. A. Agents that Act Extracellularly to Inhibit a Coestimulatory Signal in a Cell T 1. Sun-form forms of co-stimulatory molecules In one embodiment, the agent that blocks a co-stimulatory signal in a T cell is a soluble form of a - T-cell costimulatory molecule (eg, CTLA4, CD28, and / or ICOS) that is capable of blocking the transduction of a costimulatory signal in a T cell. In one embodiment, the agent that blocks a costimulatory signal in a T cell is a soluble form of CTLA4. DNA sequences encoding the murine human CTLA4 protein are known in the art, see, e.g., by Dariavich, et al. (1988) Eur. J. Immunol. 18 (12), 1901-1905; J.F. Brunet, et al. (1987) supra; J.F. Brunet et al. (1988) Immunol. Rev. 103: 21-36; and G.J. Freeman et al. (1992) J. Immunol 14_9, 3795-3801. In certain embodiments, the soluble CTLA4 protein comprises the entire CTLA4 protein. In preferred embodiments, a soluble CTLA4 protein comprises the extracellular domain of a CTLA4 protein. For example, a soluble recombinant form of the extracellular domain of CTLA4 has been expressed in yeast (Gerstmayer et al. 1997. FEBS Let t 407: 63). In other embodiments, the soluble CTLA4 proteins comprise at least a portion of the extracellular domain of the CTLA4 protein that retains the ability to liquefy B7-1 and / or B7-2. In one embodiment, the soluble CTLA4 protein or a portion thereof is a fusion protein comprising at least a portion of CTLA4 that binds to B7-1 and / or B7-2 and at least a portion of a second non-CTLA4 protein. In preferred embodiments, the CTLA4 fusion protein comprises an extracellular CTLA4 domain that is fused at the amino terminus to a signal peptide, e.g., of on :: ostatin M (see e.g., WO93 / 00431). In a particularly preferred embodiment, a soluble form of CTLA4 is a fusion protein comprising the extracellular domain of CTLA4 fused to a portion of an immunoglobulin molecule. This CTLA4Ig fusion protein can be made using methods known in the art (see, e.g., Linsley 1994. Perspectives in Drug Discovery and Design 2: 221, Linsley WO 93/00431 and U.S. Patent No. 5,770,197). In one embodiment, the agent that blocks a costimulatory signal in a T cell is a soluble form of an antigen that has a cell costimulatory molecule (eg, a molecule of the B7 family, such as B7-1, B7-2 and / or an ICOS coordinating group For example, in one embodiment, a soluble form of a costimulatory molecule includes a soluble form of B7-1 or a soluble form of B7-2 or a combination of a soluble form of B7-1 and a form Soluble of B7-2 DNA sequences encoding B7 proteins are known in the art, see, eg, B7-2 (Freeman et al., 1993 Science 262: 909 or GenBank accession number P42081 or A48754); B7-1 (Freeman et al., J. Exp. Med. 1991. 174: 625 or GenBank Accession Numbers P33681 or A45803) In certain embodiments, the soluble B7 protein comprises an entire B7 protein. Soluble B7 protein comprises the extracellular domain of a B7 protein.For example, a recombinant form Soluble content of the extracellular domain of CTLA4 has been expressed in yeast (Gerstmayer et al. 1997. FEBS Let t. 407: 63). In other embodiments, the soluble B7 proteins comprise at least a portion of the extracellular domain of the B7 protein that retains the ability to bind to CTLA4 and / or CD28. In one embodiment, the soluble B7 protein or a portion thereof is a fusion protein comprising at least a portion of B7 that binds to CD28 and / or CTLA4 and at least a portion of a second protein that is not B. In preferred embodiments, the B7 fusion protein comprises an extracellular B7 domain that is fused at the amino terminus to a peptide from the signal, e.g., from oncostatin M (see, e.g., WO93 / 00431). In a more particularly preferred embodiment, a soluble form of B7 is a fusion protein comprising the extracellular domain of B7 fused to a portion of an immunoglobulin molecule. This fusion protein, a B7Ig, can be made using methods known in the art (see, e.g., Linsley 1994. Perspecti ves in Drug Design and Design 2: 221; Linsley Number WO 93/00431, U.S. Patent Number 5,770,197, and U.S. Patent Number 5,580,756). 2. Antibodies that bind to co-mouscular molecules. In certain embodiments, the agent that blocks a costimulatory signal in a T cell is an antibody that is urged to a costimulatory molecule. In making the antibodies that bind to the costimulatory molecules, a co-stimulatory protein, a portion of a costimulatory protein, (eg, a peptide derived from a costimulatory protein), or a fusion protein that includes all or a portion of a co-stimulatory sequence. The amino acid of a co-stimulatory molecule can be used to generate anti-protein antisera and / or anti-peptide polyclonal or monoclonal antibodies using normal methods. The term "antibody" as used herein is meant to mean including whole antibodies as well as fragments thereof. Antibody fragments (e.g., Fab1 fragments, F (ab ') 2' fragments, single chain antibodies) can be made using methods well known in the art. The term "antibody" also includes chimeric and humanized antibodies. For example, a mammal, (e.g., a mouse, marmooa or rabbit) can be humanized with an immunogenic form of the co-stimulatory protein or peptide and which emits an antibody response in the mammal. The immunogen, for example, can be a recombinant co-stimulatory molecule protein or fragment thereof, a synthetic peptide fragment or a cell expressing a co-stimulatory molecule on its surface. The cell, for example, can be a cell that presents the antigen or a T cell, or a cell transfected with a nucleic acid that encodes a costimulatory molecule in such a way that the costimulatory molecule is expressed on the surface of the cell. The host cells transferred to express the peptides can be any prokaryotic or eukaryotic cells. For example, a peptide having co-stimulatory molecule activity can be expressed in bacterial cells such as E. coli, insect cells (baculovirus), yeast, or mammalian cells such as cells of the ovary of China marmot (CHO) and NSO cells. Other suitable host cells and expression vectors can be found in Goeddel, (1990) supra or are known to those skilled in the art. Examples of vectors for expression in yeast S. ceri vi sae include pYepSecl (Baldri et al., (1987) Embo J. 6: 229-234), pMFa (Kurjan and I-erskowitz, (1982) Cell 30: 993-943), pJRY88 (Schultz et al. (1987) Gene 54: 113-123), and pYES2 (Invitrogen Corporation, of San Diego, CA). Baculovirus vectors available for expression of proteins in cultured insect cells (SF 9 cells) include the pAc series (Smith et al., (1983) Mol Cell Bi ol. 3: 2156-2165) and the pVL series (V.A. Lucklow, and M.D. Summers, (1989) Virol ogy 170: 31-39). Usually, COS cells (Y. Gluzman, (1981) Cell 23_: 175-182) are used together with such vectors as pCDMd (B. Seed, (1987) Nature 329: 840) for expression and transient amplification / in mammalian cells, while CHO cells ( dhfr- Chínese Hamster Ovary) are used with vectors such as pMT2PC (Kaufman et al. (1987), EMBO J. 6: 187-195) for amplification / stable expression in mammalian cells. A preferred cell line for the production of the recombinant protein is the NSO myeloma cell line which can be obtained from ECACC (catalog number 85110503) and which is described in the article by G. Galfre and C. Milstein, ((1981) Methods in Enzymology 73_ (13): 3-46; and Prepare ti on of Monoclonal Antibodies: Strategies and Procedures, Academic Press, N.Y., N.Y.). The DNA vector can be introduced into mammalian cells through conventional techniques such as co-precipitation of calcium phosphate or calcium chloride. Transfection, lipofectin or electroporation mediated with DEAE-dextran. Appropriate methods for transforming host cells can be found in the Sambrook article and others. (Molecular Cloni ng; A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks. When used in mammalian cells, their function of controlling the expression vector is often provided by viral material. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and most frequently, Simian Virus 40. Peptides having an activity of a co-stimulatory molecule expressed in mammalian cells or otherwise can be purified from according to standard procedures of the art including ammonium sulfate precipitation, fractionation column chromatography (eg, ion exchange, gel filtration, electrophoresis, affinity chromatography, etc.) and finally crystallization (see generally "Enzyme"). Purification and Related Techniques ", Methcds in Enzymol ogy, 22: 233-577 (1971)). It will be appreciated by those skilled in the art that it is within their skill to generate antibodies towards human co-stimulatory molecules following normal techniques. The antibodies can be either polyclonal or monoclonal antibodies, or antigen binding fragments of these antibodies. They are antibodies of specific importance for use in therapeutic applications that inhibit the binding of a costimulatory molecule with its natural coordinating group (s) on the surface of immune cells, thereby inhibiting the co-stimulation of the immune cell. Antibodies of the preferred anti-co-stimulatory molecule are those capable of inhibiting or downregulating the immune responses mediated by the T cell by binding B7-2 or B7-1 on the surface of B lymphocytes and preventing interaction with CTLA4 and / or CD28. Other preferred anti-co-stimulatory molecule antibodies are those which, in combination with a second antibody that binds to another costimulatory molecule, result in increased inhibition of co-stimulation of a T cell when compared to the first antibody alone, eg, a combination of anti-B7-ly and anti-B7-2 antibodies. A. The Immunogen. The term "immunogen" is used herein to describe a composition containing a peptide having an activity of a costimulatory molecule as an active ingredient used for the preparation of antibodies against a costimulatory molecule. When a peptide having a co-stimulatory molecule activity is used to induce antibodies it will be understood that the peptide can be used alone, or linked to a carrier as a conjugate, or as a peptide polymer. To generate the appropriate anti-co-stimulatory molecule antibodies, the immunogen must contain an effective immunogenic amount of a peptide having a co-stimulatory molecule activity typically as a conjugate linked to a carrier. The effective amount of the peptide per unit dose depends, among other things, on the species of animal inoculated, the body weight of the animal and the immunization regime selected as is well known in the art. The preparation of the immunogen will typically contain peptide concentrations of about 10 micrograms up to about 500 milligrams per immunization dose, preferably from about 50 micrograms to about 50 milligrams per dose. An immunization preparation may also include a portion of the diluent adjuvant. Adjuvants such as complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) and alum are materials well known in the art, and can be obtained commercially from various sources or sources. Those skilled in the art will appreciate that, instead of using naturally occurring forms of the costimulatory molecule for immunization, synthetic peptides can alternatively be used towards which antibodies can be provided for use in this invention. Both soluble and membrane-bound co-stimulatory molecule or peptide fragments are suitable for use as an immunogen and can also be isolated by immunoaffinity purification as well. A purified form of a co-stimulatory molecule protein such as that which can be isolated as described above which is known in the art, in case it can be used directly as an immunogen, or alternatively, it can be linked to an appropriate carrier protein by conventional techniques, including a chemical coupling means as well as genetic engineering using the cloned gene of the costimulatory molecule. The peptide or protein that is selected for immunization can be modified to increase its immunogenicity. For example, techniques for conferring immunogency in a peptide include conjugation to carriers or other techniques well known in the art. Any peptide selected for immunization can also be synthesized. In certain embodiments, these peptides can be synthesized as branched polypeptides, to enhance immune responses, as is well known in the art (see e.g., Peptides, Edited by Bernd Gutte Academic Press 1995, pages 456-493).

The protein of the purified costimulatory molecule can also be modified covalently or non-covalently with non-protein materials such as lipids or carbohydrates to improve immunogenicity or solubility. Alternatively, a purified co-stimulatory molecule protein can be coupled with or incorporated into a viral particle, a duplicating virus, or other microorganism in order to improve immunogenicity. The protein of the costimulatory molecule for example can be chemically fixed to the viral particle or to the microorganism or to an immunogenic portion thereof. In an exemplary embodiment, a purified co-stimulatory molecule protein, or a peptide fragment having co-stimulatory molecule activity (e.g., produced by limited proteolysis or recombinant DNA techniques) is conjugated to a carrier that is immunogenic in animals. Preferred carriers include proteins such as albumin, whey proteins (e.g., globulins and lipoproteins), and polyamino acids. Examples of useful proteins include bovine serum albumin, rabbit serum albumin, thyroglobulin, limpet hemocyanin, egg ovalbumin and bovine gamma-globulins. Synthetic polyamino acids such as polylysine or polyarginine are also useful carriers. With respect to the covalent attachment of a co-stimulatory molecule protein or fragments of peptide to an appropriate immunogenic carrier, there are a number of chemical crosslinking elements that are already known to those skilled in the art. The preferred crosslinking agents are the heterobifunctional crosslinkers, which can be used to link proteins in a stepwise manner. A wide variety of heterobifunctional crosslinkers are known in the art including 4- (N-maleimidomethyl) cyclohexane-1-succinyl idyl carboxylate (SMCC), m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), 4- (p-maleimidophenyl) succinimidyl butyrate (SMPB), l-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC); 4-succinimidyloxycarbonyl-α-methyl-α- (2-pyridyldithio) -toluene (SMPT), N-succinimidyl 3- (2-pyridyldithio) propionate (SMPT), 6- [3- (2-pyridyldithio) propionate] hexanoate of succinimidyl (LC-SPDP). It may also be desirable to simply immunize with whole cells expressing a co-stimulatory molecule protein on their surface. Several cell lines can be used as immunogens to generate monoclonal antibodies to a co-stimulatory molecule antigen including, but not limited to, activated B cells. For example, the splenic B cells of a subject can be obtained and activated with anti-immunoglobulin. Alternatively, a B-cell line can be used, as long as a co-stimulatory molecule is expressed on the surface of the cell such as the Raji cell line (Burkett cell B lymphoma, see, eg, GJ Freeman, et al. (1993) Science 262: 909-911) or the lymphoblastoid cell line JY B (see, eg, M. Azuma, et al. (1993) Na ture 366: 76-79). Whole cells that can be used as immunogens to produce antibodies specific to the costimulatory molecule also include recombinant transfectants. For example, COS and CHO cells can be reconstituted by transfection with the co-stimulatory molecule cDNA, as described by Knudson et al. (1993, PNAS 90: 4003-4007); Travernor et al. (1993, Immunogeni tics 37: 474-477); Dougherty et al. (1991, J. Fxp Med 174: 1-5); and Aruffo et al. (1990, Cell 61: 1303-1313), to produce an intact costimulatory molecule on the surface of the cell. These transfectant cells can then be used as an immunogen to produce antibodies of the anti-co-stimulatory molecule of preselected specificity. Other examples of transfectant cells are known, particularly eukaryotic cells capable of glycosylating the co-stimulatory molecule protein, but any procedure that - It works to express the genes of the co-stimulatory molecule transfected on the surface of the cell could be used to produce the immunogen of the enter cell. B. Anti-Coestimulatory Molecule Antibodies Polyclonal Polyclonal antibodies to a protein or peptide from a costimulatory purified molecule that have a costimulatory molecule activity can usually be used in animals by subcutaneous injections (se) or multiple intraperitoneal (ip) immunostimulators of a costimulatory molecule, such as the extracellular domain of a co-stimulatory molecule protein and an adjuvant.

For example, as described above, it may be useful to conjugate a costimulatory molecule (including fragments containing specific epitope (s) of interest) to a protein that is immunogenic in the species to be immunized, eg, serum albumin. of limpet hemocyanin. The route and project of the host animal or cells that produce the cultured antibody therefore can generally use established and conventional techniques for stimulation and production of the antibody. In an illustrative embodiment, the animals were typically immunized against the conjugates or derivatives of the immunogenic costimulatory molecule or by combining about 1 microgram to 1 milligram of the conjugate with Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals were boosted with 1/5 to 1/10 of the original amount of conjugate in Freund's complete adjuvant (or other appropriate adjuvant) by subcutaneous injection at multiple sites. Seven to 14 days later, the animals are bled and the serum is assayed for concentration of the anti-co-stimulatory molecule. The animals are reinforced up to the concentration plateau. Preferably, the animal is reinforced with the conjugate of the same protein of the costimulatory molecule, but is conjugated to a different protein and / or through a different cross-linking agent. Conjugates can also be made in a recombinant cell culture as protein fusions. Also, aggregation agents such as alum can be used to improve the immune response. These populations produced by mammals of the antibody molecules are referred to as "polyclonal" because the population comprises antibodies with different immunospecificities and affinities for a costimulatory molecule. The antibody molecules are then harvested from the mammal and isolated by well-known techniques such as, for example, using Sephadex DEAE to obtain the IgG fraction. To improve the specificity of the antibody, the antibodies can be purified by immunoaffinity chromatography using the immunogen set to the solid phase. The antibody is contacted with the immunogen bound to the solid phase for a sufficient period of time for the immunogen to react with the antibody molecules to form an immunocomplex fixed in the solid phase. The ligated antibodies are separated from the complex by standard techniques. C. Antibodies to Monoclonal Anti-Coestimulatory Molecule. The term "monoclonal antibody" or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of antigen-binding antigen reaction site with a specific epitope of a costimulatory molecule. A monoclonal antibody composition thus typically exhibits a unique binding affinity for a protein of the specific costimulatory molecule with which it reacts immunally. Preferably, the monoclonal antibody used in the method is further characterized as being immune by reacting with a co-stimulatory molecule derived from humans.

Monoclonal antibodies useful in the compositions and methods of the invention are directed to an epitope of an antigen of the costimulatory molecule, in such a way that the formation of the complex between the antibody and the co-stimulatory molecule antigen inhibits the interaction of the costimulatory molecule with its natural coordinating group (s) on the surface of the immune cells, thereby inhibiting the co-stimulation of a cell T through an interaction of co-stimulatory molecule-coordinating group. A monoclonal antibody to an epitope of a costimulatory molecule can be prepared using a technique that provides means for the production of the antibody molecules by continuous cell lines in the culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256: 495-497), and the most recent human B cell hybridoma technique (Kozbor et al. (1983) Immunol Today 4 : 72), the EBV-hybridoma technique (Cole and others (1985), Monoclonal Antibodies and Cancer Therapy, by Alan R. Liss, Inc., pages 77-96), and trioma techniques. Other methods that can be effectively rendered monoclonal antibodies useful in the present invention include bacteriophage display techniques (Marks et al. (1992) J Bi ol Chem 16007-16010).

I In one embodiment, the antibody preparation applied in the present method is a monoclonal antibody produced by a hybridoma cell line. Hybridoma fusion techniques were first introduced by Kohler and Milstein (Kohler and others Na ture (1975) 256: 495-97; Brown et al. (1981) J. Immunol 127: 539-46; Brown et al. (1980) J Bi ol Chem 255: 4980-83; Yeh et al. (1976) PNAS 76: 2927-31; and Yeh et al. (1982) Int. J. Cancer 29: 269-75). In this manner, the monoclonal antibody compositions of the present invention can be produced by the following method, comprising the steps of: (a) Immunizing an animal with a co-stimulatory molecule. Immunization is typically achieved by administering an immunogen of the costimulatory molecule to an immunologically competent mammal in an immunologically effective amount, i.e., an amount sufficient to produce an immune response. Preferably, the mammal is a rodent, for example a rabbit, rat or mouse. The mammal is then maintained for a period of time sufficient for the mammal to produce cells that secrete the antibody molecules that immunoreact with the immunogen of the costimulatory molecule. This immunoreaction is detected by the selection of the antibody molecules! produced in this manner for immunoreactivity with a preparation of the immunogen protein. Optionally, it may be desirable to select the antibody molecules with a preparation of the protein in the form in which it will be detected by the antibody molecules in an assay, e.g., a shape associated with the membrane of a costimulatory molecule. These selection methods are well known to those skilled in the art. (b) A suspension of the antibody-producing cells removed from each immunized mammal that secretes the desired antibody is then prepared. After a sufficient period of time, the mouse is sacrificed and the lymphocytes producing the somatic antibody are obtained. The antibody producing cells can be derived from lymph nodes, spleens and peripheral blood of the primed animals. Spleen cells are preferred and can be mechanically separated into individual cells in a physiologically tolerable medium using methods well known in the art. Mouse lymphocytes provide a higher percentage of stable fusions with mouse myelomas described below. Rat, rabbit and frog somatic cells can also be used. Chromosomes of the spleen cell that encode the desired immunoglobulins are immortalized by fusing the cells of the spleen with myeloma cells, generally in the presence of a fusion agent such as polyethylene glycol (PEG). Any of a number of myeloma cell lines can be used as a fusion partner in accordance with standard techniques; for example, P3-NSl / l-Ag4-l, P3-? 63-Ag8.653 or Sp2 / 0-Agl4 that are myeloma lines. These myeloma lines are available from the American Type Culture Collection (ATCC), Rockville, Md. Rejuvenating cells, which include the desired hybridomas, are then grown in a selective medium, such as HAT medium, wherein the non-melted parental myeloma or lymphocyte cells may eventually die. Only the hybridoma cells survive and can be grown under boundary dilution conditions to obtain isolated clones. The supernatants of the hybridomas are selected to determine the presence of the antibody of the desired specificity, e.g., by immunoassay techniques using the antigen that has been used for immunization. Positive clones can then be subcloned under boundary dilution conditions and the monoclonal antibody produced can be isolated. There are several conventional methods for the isolation and purification of monoclonal antibodies in order to free them from other proteins and other contaminants. Methods commonly used to purify monoclonal antibodies include ammonium sulfate precipitation, ion exchange chromatography, and affinity chromatography (see, e.g., Zola et al.

Monoclonal Hydridoma Antibodies: Techniques And Applications. Hurell (editor) pages 51-52 (CRC Press 1982)). Hybridomas produced according to these methods can be propagated in vi tro or in vivo (in the asoitis fluid) using techniques known in the art. In general, the individual cell line can be propagated in vitro, for example in laboratory culture vessels, and the culture medium containing high concentrations of a single specific monoclonal antibody can be harvested by decanting, filtration or centrifugation. Alternatively, the performance of the monoclonal antibody can be improved by injecting a sample of the hybridoma into a histocompatible animal of the type used to provide the somatic and myeloma cells for the original fusion. Tumors that secrete the specific monoclonal antibody produced by the hybrid of the molten cell develops in the injected animal. Fluids from the animal's body, such as ascites fluid or serum, provide monoclonal antibodies in high concentrations. When using human hybridomas or EBV hybridomas, it is necessary to avoid rejection of the xenograft injected into animals such as mice.

Immunodeficient or naked mice may be used or the hybridoma may be first passed to the irradiated nude mouse as a solid subcutaneous tumor, cultured in vi tro and then injected intraperitoneally in the irradiated pristane-primed nude mouse that develops large-secreting arthritis tumors. amounts of specific human monoclonal antibodies. The media and animals useful for the preparation of these compositions are well known in the art and can be obtained commercially and include mice from synthetic culture media, raised and mixed rets and the like. An exemplary synthetic medium is the minimal essential medium of Dulbecco (DMEM; Dulbecco et al. (1959) Virol 8: 396) which is supplemented with 4.5 grams per liter of glucose, 20 mM of glutamine, and 20 percent of fetal calf serum . A strain of mouse grown without extraneous breeding is the Balb / c. D. Anti-Coestimulatory Molecule Antibodies Humanized. When antibodies produced in non-human subjects are used therapeutically in humans, they are recognized up to several degrees as foreign and an immune response can be generated in the patient. One approach to minimize or eliminate this problem, which is preferable to general immunosuppression, is to produce chimeric antibody derivatives, ie, antibody molecules that combine a variable region of the non-human animal and a human constant region. These antibodies are the equivalents of the monoclonal and polyclonal antibodies described above, but they may be less immunogenic when administered to humans, and therefore are more likely to be tolerated by the patient. Monoclonal antibodies from the chimaeric-human mouse (ie, chimeric antibodies) reactive with a costimulatory molecule can be produced, for example, by newly developed techniques for the production of chimeric antibodies. Humanized antibodies can be produced, for example, by replacing an immunogenic portion of an antibody with a corresponding, but non-immunogenic, portion. Accordingly, the genes encoding the constant regions of the antibody molecule of the anti-co-stimulatory molecule murir.e (or another species) are substituted with genes that encode the human constant regions. (Robinson et al., International Patent Publication PCT / US86 / 02269; Akira., And others, European Patent Application 184,187; M. Taniguchi, European Patent Application Number 171,496; Morri.son et al., European Patent Application Number 173, 94; Neuberger et al., PCT Application Number WO 86/01533; Cabilly et al., European Patent Application Number 125,023;; Better et al. (1988 Sci en 240: 1041-1043); Liu et al. (1987) PNAS 84: 3439-3443; Liu et al. (1987) J. Immunol. 139: 3521-3526; Sun and others (1987 PNAS 84: 214-218; Nishimura et al. (1987) Canc. Res. 47: 999-1005; Wood et al. (1985) Na ture 314: 446-449; and Shaw et al. (1988) J Na tl Cancer Inst. 80: 1553-1559). General magazines of "humanized" chimeric antibodies are provided by S.L. Morrison (1985) Sci en 229: 1202-1207 and by Oi et al. (1986) Bi oTechniques 4: 214 .. Those methods include isolating, manipulating and expressing the nucleic acid sequences encoding all or part of a variable immunoglobulin region. of at least one heavy or light chain. The sources of this nucleic acid are well known to those skilled in the art and, for example, can be obtained from a hybridoma which produces the antibody of the anti-co-stimulatory molecule. The chimeric cDNA can be cloned into an appropriate expression vector. Suitable "humanized" antibodies may alternatively be produced by substitution of CDR or CEA (Winter US Patent Number 5,225,539, Jones et al. (1986) Na ture 321: 552-525, Verhoeyan et al. (1988) Sci en 239: 1534; and Beidler et al. (1988) J. Imivunol, 141-4053-4060).

E. Cobinatorial Anti-co-stimulatory Molecule Antibodies. Both monoclonal and polyclonal antibody compositions of the invention can be produced by other methods well known to those skilled in the art of recombinant DNA technology. An alternative method referred to as the "combinatorial antibody display" method has been developed to identify and isolate antibody fragments that have a specific antigen specificity, and can be used to produce monoclonal anti-co-stimulatory molecule antibodies. , as well as population of polyclonal anti-co-stimulatory molecule (Sastry et al. (1989) PNAS 86: 5728; Huse et al. (1989) Science 246: 1275; and Orlandi et al. (1989) PNAS 86: 3833). After immunizing an animal with a costimulatory molecule immunogen as described above, the antibody repertoire of the resulting B cell pool is cloned. The methods are generally known to directly obtain the DNA sequence from the variable regions of a diverse population of immunoglobulin molecules using a mixture of oligomer primers and PCR. For example, mixed oligonucleotide primers corresponding to the 5 'forward sequences (signal peptide) and / or frame sequences 1 (FR1), as well as a primer to a conserved 3' constant region primer can be used. for PCR amplification of the heavy and light chain variable regions from a number of murine antibodies (Larrick et al. (1991) Biotechniques 11: 152-156). A similar strategy can also be used to amplify the human heavy and light chain variable regions of human antibodies (Larrick et al. (1991) Methods: Compani on to Methods in Enzymology 2: 106-110). The ability to clone human immunoglobulin genes acquires special importance in view of the advances in creating human antibody repertoires in transgenic animals (see, for example, Bruggeman et al. (1993) Year Immunol 7: 33-40; Tuaillon and others (1993) PNAS 90: 3720-3724; Bruggeman et al. (1991) Fur J Immunol 21: 1323-1326; and Wood et al. PCT Publication Number WO 91/00906). In an illustrative embodiment, the .RNA is isolated from activated B cells from, for example, peripheral blood cells, bone marrow, or spleen preparations, using standard protocols (eg, US Patent Number 4,683,202; Orlandi, and other PNAS (1989) 86: 3833-3837; Sastry and others PNAS (1989) 86: 5728-5732; and Huse et al. (1989) Sci en 246: 1275-1281). The cDNA is first synthesized using primers specific for the constant region of the heavy chain (s) and each of the light chains K and γ, as well as primers for the signal sequence. Using PCR primers of the variable region, the variable regions of both the heavy and light chains are amplified, either alone or in combination, and ligated into vectors suitable for further manipulation to generate presentation packages. Oligonucleotide primers useful in amplification protocols can be unique or degenerate or incorporate inosine at degenerate positions. Restriction endonuclease recognition sequences can also be incorporated into the primers to allow cloning of the amplified fragment into a vector in a predetermined reading frame for expression. The V gene library cloned from the repertoire of the antibody derived by immunization can be expressed by a population of presentation packets derived preferentially from the filamentous bacteriophage, to form an antibody display library. Ideally, the presentation package comprises a system that allows sampling of the greatly varied antibody display libraries, rapid classification after each round of affinity separation and easy isolation of the antibody gene from the purified presentation packets. In addition, of the kits commercially obtainable to generate bacteriophage display libraries (e.g., Pharmacia Recopbinant Phage Antibody System, catalog number 27-9400-01; and the bacteriogage presentation kit from Stratagene SurfZAP ™ catalog number 240612), examples of the methods and reagents particularly ready to be used to generate an antibody presentation library of varied anti-co-stimulatory molecule can be found for example in the Patent American Ladner et al. Number 5,223,409; International Publication of Kang and others Number WO 92/18619; and Dower International Publication and other WO 91/17271; Winter International Publication and other WO 92/20791; International Publication Markland et al. Number WO 92/15679; International Breitling Publication and others WO 93/01288; McCafferty International Publication and others WO 92/01047; International Publication of Garrard and others WO 92/09690; the International Publication of Ladner and others Number WO 90/02809; Fuchs et al. (1991) Bio / Technolgy 9: 1370-1372; Hay and others (1992) Hum Antibod Hybridomas 3: 81-85; Huse et al. (1989) Science 246: 1275-1281; Griffths et al. (1993) EMBO J 12: 725-734; Hawkins et al. (1992) J Mol Biol 226: 889-896; Clackson et al. (1991) Nature 352: 624-628; Gram et al. (1992) PNAS 89: 3576-3580; Garrad et al. (1991) Bi o / Technolgy 9: 1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19: 4133-4137; and Barbas et al. (1991) PNAS 88: 7978-7982. In certain embodiments, the domains of the V region of heavy and light chains can be expressed in the same polypeptide, linked by a flexible linker to form a single chain Fv fragment, and the scFV gene subsequently cloned into the expression vector desired or in the bacteriophage genome. As generally described in the article by McCafferty et al., Na ture (1990) 348: 552-554, the complete VH and VL domains of an antibody, linked by a flexible linker (Gly4 ~ Ser) 3 can be used to produce a single chain antibody that can yield the separable display package based on the affinity of the antigen. Antibodies isolated from scFV immunoreactive with a costimulatory molecule can be subsequently formulated in a pharmaceutical preparation for use in the present method. F. Hybridomas and Preparation Methods. Hybridomas useful in the present invention are those characterized as having the ability to produce a monoclonal antibody that will specifically immunoreact with a costimulatory molecule. As will be described below, the hybridoma cell that produces the antibody of the anti-co-stimulatory molecule - - it can be implanted directly into the recipient animal in order to provide a constant source of the antibody. The use of immuno-isolating devices to encapsulate the culture of the hybridoma can impede the immunogenic response against the implanted cells, as well as prevent the unproven proliferation of the hybridoma cell in an immunocompromised host. A preferred hybridoma of the present invention is characterized as producing antibody molecules that specifically immunoreact with a costimulatory molecule expressed on the cell surfaces of activated human B cells. Methods for generating hybridomas that produce, eg, secrete antibody molecules that have a desired immunospecificity, ie, have the ability to bind to a specific costimulatory molecule, and / or the identifiable epitope of a costimulatory molecule are already well known in the art. Particularly applicable is the hybridoma technology described by Niman et al. (1983) PNAS 80: 4949-4953; and by Galfree et al. (1981) Meth. Enzymol 73: 3-46. In another exemplary method, human antibody repeaters carrying transgenic mice can be immunized with a human costimulatory molecule. Splenocytes from these immunized transgenic mice can then be used to create hybridomas that secrete human monoclonal antibodies specifically reactive with a human costimulatory molecule (see, eg, from Wood et al. PCT Publication WO 91/00906, Kucherlapati et al.) PCT publication WO 91/10741; Lonberg et al PCT Publication WO 92/03918; Kay others, PCT Publication No. 92/03917; Lonberg N. et al. (1994) Na ture 368: 856-859; LL Green et al. (1994) Na ture Genet 7: 13-21; SL Morrison et al. (1994) Proc Na tl Acad. Sci. USA 81: 6851-6855; Bruggeman et al. (1993) Year Immunol 7: 33-40; Tuaillon et al. (1993) PNAS 90: 3720-3724; and Bruggeman et al. (1991) Eur J Immunol 21: 1323-1326). The term "antibody" as used herein is intended to include fragments thereof that are also specifically reactive with a costimulatory molecule as described herein. Antibodies can be fragmented using conventional techniques and fragments selected for utility in the same manner as that described above for whole antibodies. For example, F (ab ') 2 fragments can be generated by treating the antibody with pepsin. The resulting F (ab ') 2 fragment can be treated to reduce the disulfide bridges to produce Fab' fragments.

Antibodies made using these and other methods can be tested to determine if they inhibit a costimulatory signal in a T cell using the methods described below. In one embodiment, the agent that inhibits a costimulatory signal in a T cell is an antibody that binds both B7-1 and B7-2. In making this antibody, for example, portions of the extracellular domain that are conserved between two costimulatory molecules can be used as the immunogen. See, e.g., Metzler et al., 1997 Na t Struct. Bi ol. 4: 527). In one embodiment, the agent that inhibits a costimulatory signal in a T cell is an antibody that binds to B7-1. These antibodies are known in the art or can be made as indicated above by using a B7-1 molecule or a portion thereof as an immunogen and selected using the methods outlined above or other normal methods . Examples of B7-1 antibodies include those disclosed in U.S. Patent Number 5, 747,034 and in the McHugh and others article of 1998. Clin. Immunol. Immur.opa thol. 87:50 or from Rugtveit and others from 1997. Clin Exp. Immunol. 110: 104. In another embodiment, the agent that inhibits a costimulatory signal in a T cell is an antibody that binds to B7-2. These antibodies are known in the art or can be made as discussed above using a B7-2 molecule or a portion thereof as an immunogen and selected using the methods outlined above or other normal methods. Examples of B7-2 antibodies include those disclosed in the Rugtveit and other articles of 1997. Clin Exp. Immunol. 110: 104. In another embodiment, the agent that blocks a costimulatory signal in a T cell is a combination of an antibody that binds to B7-1 and an antibody that binds to B7-2. In still other embodiments, the agent that inhibits a costimulatory signal in a T cell is an antibody that binds to CD28, but does not transduce a costimulatory signal to a T cell (eg, a Fab fragment of an anti-CD28 antibody) . These antibodies are known in the art or can be made as discussed above using a CD28 molecule or a portion thereof as an immunogen and selected using the methods outlined above or other normal methods. Examples of known anti-CD28 antibodies include those disclosed by Darling et al., 1997. Gene Ther. 4: 1350 In preferred embodiments, FaB fragments of an antibody that binds to CD28 can be used. These antibody fragments that are unable to cross-link from CD28 on the surface of a T cell have been encoded that block the co-stimulus of the T cell (Waltnas et al. 1994, Immuni ty 1: 405). In still other embodiments, the agent that inhibits a costimulatory signal in a T cell is an antibody that binds to CTLA4, which blocks a costimulatory signal in a T cell by supplying a negative signal to the T cell (i.e., which is an agonist from CTLA4). For example, cross-linking of CTLA4 on the surface of a T cell has been shown to inhibit proliferation and production of 11-2 (Krummel and Allison, 1996. J. Exp. Med. 183-2533). These antibodies can be made as discussed above using a CTLA4 molecule or a portion thereof as an immunogen and selected using the methods outlined above or other normal methods. Exemplary antibodies are also disclosed e.g., in Vandenborre et al., 1998. Am. J. Pa thol. 152: 963. In still another embodiment, the agent that inhibits a costimulatory signal in a T cell is an antibody that binds to ICOS, which blocks a costimulatory signal in a T cell. These antibodies can be made as outlined above using an ICOS molecule or a portion thereof as an immunogen and selected using the methods set forth above or other normal methods. JV Agents that regulate the expression of co-stimulatory molecules In another embodiment, an agent that inhibits a co-stimulatory signal in a T cell is an agent that interferes with the expression of a costimulatory molecule. For example, the interactions between CD40 in the antigen presenting cells and the coordinating group CD40 (CD40L) in T cells has been found to be important to sustain, improve, or prolong the expression of B7-1 or B7-2 in the cells that present the antigen, resulting in enhanced co-stimulation (Van Gool, et al., 1996. Immunol Rev. 153: 47; Klaus and others 1994. J. Immunol., 152: 5643). In one embodiment, the agent that blocks the expression of a costimulatory molecule, thereby blocking a costimulatory signal in a T cell is a soluble form of CD40 or CD40L. DNA sequences encoding CD40 and CD40L are known in the art, see, eg, Accession Number of GenBank Y10507 or Stamenlovic et al. 1988. EMBO J. 7: 1053-1059 for CD40 or Gauchat et al., 1993. FEBS 315 (3): 259-266; Graf and others. 1992. Eur.

J. Immunol. 22: 3191-3194; Seyama 1996. Hum. Genet 97: 1S0-185 or Access Numbers GenBank L07414, X67878, X96710 for CD40L. In one embodiment, the agent that blocks the expression of a costimulatory molecule, thereby blocking a costimulatory signal in a T cell is a soluble form of CD40 or CD40L. In one embodiment, the soluble CD40 or CD40L protein comprises all the protein. In preferred embodiments, a soluble protein CD40 or CD40I comprises the extracellular domain of the protein. For example, a soluble recombinant form of the extracellular domain of the CD40 or CD40L protein or a portion thereof can be made as a fusion protein comprising at least a portion of CD40 or CD40L such that the interaction between CD40 in APC and CD40L in a T cell is disrupted and is inhibited by the delivery of a costimulatory signal to a T cell. This soluble, recombinant form of a CD40 or CD40L protein comprises at least a portion of the molecule sufficient to bind to its counter-receptor and at least a portion of the second protein that is not CD40 or CD40L. In preferred embodiments, the CD40 or CD40L fusion protein comprises an extracellular CD40 or CD40 domain that is fused at the amino terminus to a signal peptide, e.g., oncostatin M (see, e.g., WO93 / 00431).

In a particularly preferred embodiment, a soluble form of CD40 or CD40L is a fusion protein comprising the extracellular domain of CD40 or CD40L fused to a portion of an immunoglobulin molecule (eg, Chen et al. 1995. J. Immunol. 2833). This fusion protein, a CD40Ig or a CD40LIg, can be made using the methods known in the art (see e.g., the article by Linsley 1994, Perspectives in Drug Discovery and Design 2: 221; Lensley WO 93/00431, U.S. Patent Number 5,770,197 and U.S. Patent Number 5, 580, 756). In addition, antibodies to the CD40 coordinator group have been found to be synergized with agents that inhibit a costimulatory signal in a T cell to promote graft tolerance (Kirk et al. 1997. Proc. Na tl. Acad. Sci. USA 94: 8789; Larsen et al. 1996. Na ture 381-434). Therefore, in one embodiment, antibodies to CD40 or CD40L that bind to these molecules, but which do not induce the expression of co-stimulatory molecules, can be used as an agent that blocks a costimulatory signal in a TV cell. Acting Agents Intracellularly To Inhibit a Coestimulatory Signal In one embodiment, the agent that inhibits a costimulatory signal in a T cell is an agent that acts intracellularly to inhibit this signal. The stimulation of a T cell through the surface receptor CD28 (ie a costimulatory signal) leads to the production of phosphinositides D-3 in a T cell. Therefore, in one embodiment, the production of phosphoinositides D-3 can be inhibited in a T cell to inhibit a costimulatory signal to thereby inhibit a T cell response, as inhibited, for example, by proliferation of the T cell and / or cytokine production. The term "phosphoinositides D-3" is intended to include the phosphatidylinositol derivatives which are phosphorylated at the D-3 position of the inositol ring and which comprise the compounds phosphatidylinositol (3) -monophosphate (Ptdlns (3), phosphatidylinositol (3, 4) -bisphosphate (Ptdlns (3, 4) P2), and phosphatidylinositol (3, 4, 5) -trisphosphate (Ptdlns (3, 4, 5) P3). D-3 phosphoinositides are generated intracellularly by the activity of a Phosphatidyl-inositol 3-kinase (P13K) P13K is a heterodimer composed of an 85 kDa subunit that binds the phosphorylated proteins with tyrosyl through its SH2 domain and a catalytic subunit of 110 kDa.P13K was first identified as a kinase lipid that phosphorylates the D-3 position of the inositol ring of phosphatidylinositol, Ptdlns (4) P, and Ptdlns (4, 5) P2. Two recent studies have shown that P13K is indeed a double specificity kinase possessing: activities both of lipid as a serine kinase (R. Dhand, et al. (1994) EMBO J. 13: 522 and C.L. Carpenter, and others (1993) Mol. Cel Bi ol. 13: 1657). Accordingly, in one embodiment, the agent that inhibits a costimulatory signal in a T cell is an agent that inhibits the activity of a P13K. A preferred agent that inhibits P13K activity in a T cell is the wortmanin of the fungal metabolite, or analogous derivatives thereof. Wortmanin is a potent inhibitor of P13K derived from T. wortmannii (Kyowa Hakko Kohyc Co. Ltd.) or from P. fumiculosum (Sigma). Derivatives or analogs of wortmanin include compounds structurally related to wortmanin that retain the ability to inhibit P13K and T cell responses. Examples of derivatives of wortmanin analogs are disclosed in the article by D. Wiesinger and others ( 1974) Experientia 30: 135-136; A. Closse et al. (1981) J. Med. Chem. 24: 1465-1471; and M. Baggiolini, et al. (1987) Exp. Cell Res. 169: 408-418. Another inhibitor of P13K activity that can be used in bioflavenoid quercetin, or derivatives or analogues thereof. Derivatives or analogs of quercetin include compounds structurally related to quercetin which retains the ability to inhibit P13K and inhibit T cell responses. Examples derived from quercetin analogs are disclosed in C.J. Vlahos et al. (1994) J. Bi ol. Chem. 269: 5241-5284. A preferred quercetin derivative that inhibits the activity of P13K is LY294002 (2- (4-morpholinyl) -8-phenyl-4H-1-benzopyran-4-one, Lilly Indianapolis, IN) (which is described in the Vlahos article. and others, cited supra). The CD28 stimulus has also been shown to result in phosphorylation of tyrosine protein in a T cell (see, e.g., P. Vandenberghe et al. (1992) J. Exp. Med. 175: 951-960; Y. Lu et al. (1992) J. Immunol. 149: 24-29). Accordingly, in one embodiment, an agent that inhibits a costimulatory signal in a T cell inhibits tyrosine phosphorylation in the T cell. A preferred protein tyrosine kinase inhibitor is one that inhibits the protein tyrosine kinase src. In one embodiment, the protein tyrosine kinase inhibitor src is herbimycin A, or a derivative or an analog thereof. Herbricinin A derivatives and analogs include compounds that are structurally related to herbimycin A and retain the ability to inhibit the activity of protein tyrosine kinases. In another embodiment, the agent that inhibits protein tyrosine phosphorylation is a protein tyrosine phosphatase or an activator of a protein tyrosine phosphatase. By increasing the activity of tyrosine phosphatase in a T cell, the net amount of protein tyrosine phosphorylation is decreased. The protein tyrosine phosphatase may be a tyrosine phosphatase of cellular protein within the T cell, such as CD45 or Hcph. The activity of a tyrosine phosphatase on the surface of the cell in a T cell can be activated by contacting the T cell with a molecule that binds to the phosphatase and simulates its activity. For example, an antibody directed against CD45 can be used to stimulate the activity of tyrosine phosphatase in a T cell expressing CD45 on its surface. Correspondingly, in one embodiment, the agent that inhibits protein tyrosine phosphorylation within the T cell is an anti-CD45 antibody, or a fragment thereof that retains the ability to stimulate CD45 activity. Examples of antibody fragments include Fab and F (ab ') 2 fragments. Antibodies or fragments thereof can be provided in a stimulatory form, for example multimerized or immobilized, etc. In addition, the binding of CD28 has been associated with increased phospholipase C activity (see, eg, J. Nunes et al. (1993) Bi ochem J. 293: 835-842) and increased intracellular calcium levels (see, eg. , JA Ledbetter, et al. (1990) Bl ood 75: 1531-1539 and the Examples).

Correspondingly, an agent that acts intracellularly to inhibit a costimulatory signal in a T cell can act by inhibiting the activity of phospholipase C and / or by inhibiting an increase in intracellular calcium levels. For example, the tyrosine kinase inhibitor, herbimycin A also inhibits the calcium flux induced by CD28 in T cells. Serine protein and serine-threonine kinases have also been shown to be involved in the transduction trajectories of signals associated with CD28 (J.N. Siegel, et al. (1993) J. Immunol. 151: 4116-4127; S.V. Pai and others. (1994) J. Immunol. 24: 2364; Parry et al. 1997. Eur. J. Immunol. 27: 2495).

Therefore, in another embodiment of the invention, an agent that acts intracellularly to inhibit a costimulatory signal in a T cell inhibits the activity of serine kinase or serine threonine. VJ. Other Agents That Blunt Coestimulation Of The T cells Other agents that block a costimulatory signal in a T cell can be identified using normal techniques. For example, these agents can be identified by their ability to inhibit T cell proliferation and / or cytokine production. For example, a coest.Lmulo test system can be used. In this system, human CD28 + T cells are isolated, for example, by suppression of immunomagnetic counts using monoclonal antibodies directed against B cells, natural killer cells and macrophages as described above (CD, Gimmi, et al. (1993) Proc. Natl. Acad. Sci. USA 90, 6586-6590). Cells that present the antigen, eg, whole spleen cells, or purified B cells, or transfected B7-1 or B7-2 COS cells can be irradiated or treated with mitomycin-C (eg, at 25 micrograms per milliliter) for 1 hour, and then wash extensively to inhibit proliferation. T cells, 10 ^ CD28 + can be incubated with, e.g., 10 ^ -10 ^ APCs, (e.g., COS cells transfected with a B7 molecule). In this exemplary assay, a population of T cells receives a single primary activation signal (e.g., a signal from the T cell receptor), another population of T cells receives a co-stimulatory signal alone; yet another population of T cells receive both a primary activation signal and a costimulatory signal and still another population of T cells recite both a primary activation signal and a costimulatory signal in the presence of the agent to be tested due to its ability to block a co-stimulatory signal in a T cell. A primary activation signal may be provided, eg, by a submicron dose of PMA (eg, 1 ng / milliliter), a submygenic dose of mitogen, a suboptimal dose of antigen, or a dose subunit of the anti-T cell receptor antibody. Signal 2 is delivered by antigen presenting cells carrying a B7 molecule. Potential blocking agents can be tested at a concentration scale. For example, potential blocking antibodies can be used as hybridoma supernatants or as a purified antibody (e.g., at about 10 micrograms per milliliter). The proliferation of T cells can be measured by incorporation of 3 H-thymidine (1 microCi) during the last 12 to 18 hours of a 72-hour incubation. The delivery of a primary activation signal should result in some proliferation, but the T cells that receive both the primary activation signal and the costimulatory signal 2, the signals must proliferate maximally. Blocking agents are identified by their ability to reduce proliferation induced by the maximal costimulatory signal. In addition to, or as an alternative to measure the proliferation of the T cell, the production of the cytokine of the T cell can be measured using techniques that are well known in the art. For example, IL-2 and IL-4 produced in the T cell cultures can be assayed in culture supernatants harvested at 24 to 72 hours after culture initiation, using a commercially available ELISA system (R &; D, from Minneapolis, MN and BioSource, from Camarillo, CA). As noted above, blocking agents can be identified by their ability to reduce the cytokine production induced by the maximal costimulatory signal. In the case of antibodies, any of the "blocking antibodies" identified using this or another assay can be further tested to determine the costimulatory molecule to which they are linked, using techniques known in the art. For example, the ability of the blocking antibody to reduce the binding of a labeled antibody to a known coordinating group can, of course, be measured. VJJ. Additional Agents for Immune Responses of Descending Modulation In certain embodiments, the compositions and methods of the invention may comprise additional agents or the use of additional agents to enhance immunotolerance to an agent that promotes hemostasis. In one embodiment, an agent that promotes immunotolerance but does not act by inhibiting a costimulatory signal in a T cell can be added to the present compositions or administered in the present methods. For example, the anti-CD40 coordinator group (e.g., the monoclonal antibody to the human coordinator group CD40, 5C8 (Kirk et al. 1997. Proc. Natl. Acad. Sci. USA 94: 8789)) can be included in a composition. CD40 and its coordinating group based on the T cell, CD40L (CD154) play an important role in the up-regulation of B7 and in establishing the activity of the B cell (U.S. Patent Number 5,683,693, Yang et al., 1996. Science 273: 1862; Grewal et al., 1996. Sci en 273: 1864, Leterman et al., 1992. J. Exp. Med. 175: 1091, Lederman et al. 1992. J. Immunol. 149: 3817). Antibodies to the CD40 coordinator group have been found to be synergized with agents that inhibit a costimulatory signal in a T cell to promote graft tolerance (Kirk et al. 1997. Proc.Nal.l Acad.Sci. USA 94: 8789; Larsen and others 1996. Na ture 381: 434). In another embodiment, an agent that acts intracellularly to promote immunotolerance, but does not inhibit a costimulatory signal in a T cell can be used in the present compositions. For example, in one embodiment, a cyclosporin A (CSA), FK506, Rapamycin, or other agent that inhibits immune responses can be included in the present compositions or administered as part of the methods present. (See, eg, Sigal et al. 1992, Annv. Rev. Immunol.10: 519, Ruhlmann et al. 1997 or I munobiol ogy 198: 192; Shaw et al., 1996. Clin. Chem. 42: 1316) VJJJ Methods of Use of Compositions Comprising a Therapeutic Protein and an Immunotolerant Agent In one embodiment, the present compositions and / or the agents described herein are administered to subjects having a hemostatic disorder and who have been previously treated with an agent that forms the hemostasis In another embodiment, the present compositions and / or the agents described herein are administered to subjects the occulents have not been treated with an agent that promotes haemostasis. In yet another embodiment, the present compositions and / or the agents described herein are administered to a subject who has not yet developed an immune response to an agent that promotes haemostasis. In other embodiments, the compositions and / or agents present are administered to subjects having a pre-existing immune response for an agent that promotes hemostasis. Whether an object has a "pre-existing immune response" can be determined by measuring the concentration of the antibodies in the subject that reacts with the agent that promotes haemostasis using techniques that are well known in the art. If this subject has a measurable concentration of these antibodies (e.g., a statistically significant concentration) when compared to the concentrations of the control persons, a subject can be said to have a pre-existing immune response to an agent that promotes hemostasis. Alternatively, a cellular immune response to an agent that promotes haemostasis can be measured to determine whether a subject has an immune response to an agent that promotes haemostasis. These techniques are well known in the field. In one embodiment, a first agent that promotes hemostasis and a second agent that inhibits or blocks a costimulatory signal in a T cell are used to treat a hemostatic disorder. In another embodiment, compositions comprising a combination of a first agent promoting haemostasis and a second agent that inhibits a costimulatory signal in the T cell can be used to treat a hemostatic disorder. Administration of the compositions and / or agents described herein may be in any pharmacological form that includes a therapeutically active amount of an agent and a pharmaceutically acceptable carrier. The administration of a therapeutically active amount of the present agents and / or compositions is defined as an effective amount, at dosages and for periods of time necessary to achieve the treatment of hemostatic disorder in the case of people promoting hemostasis, and for achieve immunotolerance for the agent that promotes haemostasis in the case of an agent that inhibits a costimulatory signal in a T cell. A therapeutically active amount of an agent or composition may vary according to factors such as the state of the disease, the age, sex and weight of the person, and whether or not the person has already developed an immune response to an agent that promotes haemostasis. This amount can be easily determined by a person skilled in the art. The course of optimal administration of the agents and / or compositions may also vary depending on the subject to be treated. In certain modalities, a subject will require treatment with both agents during that time. In this case, it will be desirable to administer an agent that promotes haemostasis and an agent that inhibits a co-stimulatory signal in a T cell simultaneously, for example, in the form of a composition comprising both agents. In other embodiments, it will be desirable to administer the agents separately, e.g., in order to promote the stability of the agents, or to facilitate the stepwise administration of the agents. In one embodiment, the stepped administration may be desirable to achieve an optimal therapeutic effect of the agent that promotes haemostasis, while optimally inhibiting the immune response, preferably an antibody response to the agent. For example, an agent that inhibits a costimulatory signal can be administered only before the administration of an agent that promotes hemostasis, or it can be administered only for several days after administering an agent that promotes hemostasis. In one embodiment, an agent that blocks a costimulatory signal in the T cell can be chronically administered, e.g., each time the agent promoting hemostasis is administered. In another embodiment, an agent that blocks a costimulatory signal in a T cell is administered sporadically. For example, a subject may require treatment with an agent that promotes haemostasis on a regular basis, but may only require treatment with an agent that inhibits a co-stimulatory signal in a T cell, periodically. For example, treatment with an agent that promotes haemostasis may be underway, while as few as one or two treatments of the subject may be sufficient, with an agent that blocks a co-stimulatory signal in a T cell.; the additional administration of an agent that blocks a costimulatory signal may not be required.

In a preferred embodiment, an agent that blocks a costimulatory signal in a T cell is administered at appropriate intervals for at least about 6 months. In one embodiment, the present agents or compositions are administered to patients if the patient is found to have pre-existing antibodies. In another embodiment, the present agents or compositions are administered to patients not previously treated, i.e., without pre-existing antibodies. In yet another embodiment, the present agents or compositions are administered to patients who have been previously treated, but who do not have antibody concentrations against an agent that promotes hemostasis. A dosage regimen can be adjusted to provide the optimal therapeutic response for each subject without undue experimentation. For example, antibody concentrations in an agent that promotes haemostasis can be added to determine whether the subject is not developing an immune response to the agent and the dosage regimen can be adjusted accordingly. For example, if concentrations of the antibody in an agent that promotes increased hemostasis can be given more doses of an agent that blocks a costimulatory signal in a T cell.

To administer the agents or compositions present by another route than parenteral administration, it may be necessary to coat the same with, or co-administer the same with, a material to prevent its inactivation. An agent or composition of the present invention can be administered to a person in an appropriate carrier or diluent, co-administered with enzyme inhibitors or in an appropriate carrier such as liposomes. Pharmaceutically acceptable diluents include saline solutions and an aqueous stabilizer. Enzyme inhibitors include a pancreatic trypsin inhibitor, diisopropyl fluorophosphate (DEP) and trasilol. Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes (Strejan et al., (1984) J. Neuroimmunol 7:27). The active agent or composition can also be administered parenterally or intraperitoneally. The dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under regular conditions of storage and use, these preparations may contain a condom to prevent the growth of microorganisms. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where they are soluble in water) or dispersions and powders - - sterile for the extemporaneous preparation of sterile injectable solutions or a dispersion. In all cases, the agent or composition must be sterile and must be fluid to the extent that there is an easy capacity to be administered by syringe. It must be stable under manufacturing and storage conditions and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or a dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol and the like), and appropriate mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferred to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be effected by including in the composition an agent that II - retards absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active composition or agent in the required amount in an appropriate solvent with one or a combination of ingredients that are listed above, as required, followed by filtered sterilization. In general, dispersions are prepared by incorporating the active compound in a sterile vehicle containing a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze drying which yields a powder of the active ingredient (eg, agent or composition) plus any additional desired ingredient of a solution of it, previously sterile filtered. When the active agent or composition is appropriately protected, as described above, the protein can be administered orally, for example, with an inert diluent or an edible assimilable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retarding agents, and the like. The use of these media and agents for pharmaceutically active substances is well known in the art. Except as long as the conventional medium or agent is incompatible with the active compussto, the use thereof is proposed in the therapeutic compositions. Supplementary active compounds can also be incorporated into the compositions. It is especially advantageous to formulate the parenteral compositions in the unit dosage form for ease of administration and uniformity of dosage. The dosage unit form as used herein refers to physically discrete units suitable as unit dosages for the mammalian subjects to be treated; each unit contains a predetermined amount of the active compound that is calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are regulated by and directly depend on (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the inherent limitations in the mixing technique for example, to an agent or active composition for the treatment of people. As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delay agents, and the like. The use of these media and agents for pharmaceutically active substances is well known in the art. Supplementary agents can also be incorporated. The practice of the present invention will employ, unless otherwise indicated, the conventional techniques of cell bilology, cell culture, molecular biology, microbiology, recombinant DNA, and immunology, which fall within the skill of the art. These techniques are fully explained in the literature. See, for example, Genetics; Mol ecular Cl oing A Labora tory Manual, Second Edition, edited by J. Sambrook, et al. (Cold Spring Harbor Laboratory Press (1989)); Short Protocols in Molecular Biology, Third Edition, edited by F. Ausubel and others (Wiley, NY (1995)) DNA Cloning, Volumes I and II (DN Glover editor, 1985) Oligonucl eotide Synthesis (MJ Gait editor (1984)) Mullis et al. American Patent Number 4,683,195 Nucl eic Acid Hybridi za ti on (BD Mames &SJ Higgins 14 - editors (1984)); the treaty, Methods In Enzymolgy (Academic Press, Inc., N.Y.); Immunochemi cal Methods In Cell And Molecular Biolgy (Mayer and Walker, eds., Academic Press, London (1987)); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Balckwell, editors (1986)); and Miller, J. Experiments in Mol ecular Geneti cs (Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1972)). The contents of all references, publications of pending patents and published patents cited by this application are expressly incorporated herein by reference. The invention is further illustrated by the following examples, which should not be construed as additional limitation. EXAMPLES In the Examples, a model hemophilia A mouse was used to evaluate new methods for the prevention and treatment of inhibitor formation. Hemophilia A mice, generated by disruption of exon 16 of the Factor VIII gene, have no detectable Factor VIII activity in their plasma (Bi L, Nature Genetics 10: 119, 1995) and are similar in this way to patients with hemophilia A would. As expected, hemophilia A mice have in vivo signals of a coagulation path defect with fatal bleeding if the tails are cut without the use of hemostatic measures, and develop subcutaneous and intramuscular bleeding after management or temporary immobilization (J Qian, M. Borovok, L Bi, HH Kazazian, L. Hoyer Thromb Haemost 81: 940, 1999, GL Evans et al., Proc Natl Acad Sci USA 95: 5734, 1998). Intravenous infusions of 0.2 microgram, of human factor VIII, a dose equivalent on a weight basis to that provided to hemophilia A patients, resulted in a minimal antibody response or no response in these hemophilia A mice after a single injection, but repeated infusions led to high concentration of inhibitory anti-factor VIII (J Qian et al., Thromb Haemost 81: 940, 1999). In addition, a proliferative response of the T cell specific for the HIV factor was repeated three days after the first exposure to human Factor VIII, before the antibodies were detected.

Example 1. Inhibition of the primary immune response to factor VIII The experimental design of this example is shown in Figure 1. Three groups of mice were injected with pharystor VIII intravenously on day 0. A group of mice was also injected with CTLA4-Ig one day before and one day after injection of factor VIII. A second group of mice received the same CTLA4-Ig treatment (intraperitoneally) followed by daily injections of factor VIII from day 2 to day 12. The third group received no CTLA4-Ig. All the mice then received two additional injections of factor VIII on days 23 and 44. The animals were bled on days 20, 37, 58 and 82. While the mice that did not receive CTLA4Ig had high concentrations of the antibody starting so early as on day 20, mice that received CTLA4Ig did not develop anti-bodies until day 82 (Figure 2).

Examples 2. Inhibition of secondary response to factor VIII The experimental design for this example is shown in Figure 3. In this example, the mice were given multiple intravenous injections of factor VIII at two week intervals and then divided into two groups. Mice were re-injected with factor VIII on days 1, 20 and 37. One group of mice was injected with CTLA4-Ig on days -1 and day +1 relative with day 1, 20 and 37 with injections of factor VIII. Animals that did not receive CTLA4Ig had high concentrations of anti-factor VIII antibodies, whereas mice that received CTLA4Ig (with the exception of 1 mouse) did not develop a secondary immune response to factor VIII (Figure 4). The following methods and materials were used in Examples 3 to 6. Animals . The characteristics of the strain exon-16 (E-16) of mice have been reported as L Bi, and others Nature Genetics 10: 119, 1995; L Bi, and others Blood 88: 3446, 1996. E-16 male and female homozygous mice, aged 10 to 20 weeks, were used for these studies. Blood samples were obtained by bleeding from the orbital venous plexus and the serum was separated by centrifugation at 600 grams for 3 minutes. The serum samples were stored at -20 ° C until assayed. To avoid serious bleeding and death of animals, ear tags were not used to identify the mice in some experiments. Due to this reason, Figure 5 does not indicate the data in sequence for individual mice. The generation of double-knockout mice E-16 / B7-1 and B7-2 was achieved by transverse breeding of E-16 with knockout mice B7-1 and B7-2 (F Borriello et al., 1997, Immunity 6: 303 ). The homozygous E-16 / B7-1 and E-16 / B7-2 mice knocked out twice were identified by genotype determination (L Bi et al., Nature Genetics 10: 119, 1995; F. Borriello et al., Immunity. 6: 303, 1997. Reduced factor VIII activity was verified using the chromogenic bioassay Coatest (Chromogenix, Molndal, Sweden) (Bi L et al., Blood 88: 3446, 1996) Factor VIII activity was less than 1 per Hundred percent in mice deficient E-16 / B7-1 and E-16 / B7-2 Antigens Human recombinant factor VIII was obtained from Hyland Division of Baxter Healthcare Corp. (Glendale, CA). The murine CTLA4-Ig cDNA expression plasmid was prepared by binding the murine CTLA4 and extracellular domains to the main point, the CH2 and CH3 domains of IgHg2a that had undergone mutation to remove the effector functions as described in the article by Streurer et al. (Streurer, J. Immunol 155: 1165, 1995). it was left in the pED expression vector and stably transfected into the CHO cells as described previously (P Lollar et al., J Clin Invest 93: 2497, 1994). A concentrated conditioned medium was loaded onto a Sepharose Rapid Flow chromatography column of rProtein A (Amersham Pharmacia Biotech, Piscataway, NY). The column was washed with PBS of pH 7.1 and mCTLA4-Ig was eluted with 20 mM Citrate of pH 3.0. The puddle was neutralized with a Tris 1M pH of 8.0 to a final pH of 7.5 and formulated in PBS of pH 7.1 using an Amicon cell shaken with a YM30 membrane. The mCTLA4-Ig was depyrogenated using a Poros Pl chromatography column (Perceptual Biosystems) and the product was eluted from the column in a linear gradient of NaCl from 0 to 1 M NaCl in 25 mM Tris pH 7.5. MCTLA-4-Ig was then formulated in PBS of pH 7.1 using an Amicon stirred cell using a YM30 membrane. Antibody measurements. The anti-factor VIII concentration was determined by ELISA (J Qian, et al .. The development of the inhibitory antibody and the cell response to human factor VIII in hemophilia A from murine A. Thromb Haemost 81: 940, 1999). ELISA assays were carried out using microconcentration wells coated with recombinant human factor VIII, 0.8 microgram per milliliter in 0.05 mol / milliliter carbonate-bicarbonate of pH 9. The samples of the mouse plasma were then incubated in wells a 4 ° C overnight and then washed, goat anti-mouse IgG conjugated with alkaline phosphatase (Southern Biotechnology Associates Inc., Bir ingham, AL) was added for 2 hours at room temperature. After washing, P-nitrophenyl phosphate (Sigma, St. Louis, MO), 2 milligrams per milliliter in 100 millimoles per liter of glycine, 1 millimole / liter of MgCl 2, 2 millimole / liter of ZnCl 2, pH was added. of 10.4, and the absorbance read at 410 nm using an automatic microtiter plate ELISA reader. Antifactor VIII antibody concentration was calculated from a normal curve obtained using a murine anti-human murine factor VIII antibody monoclonal IgG that binds to the A2 domain (Mab 413) (P Lollar et al J Clin Invest 93: 9497, 1994) . The concentration was calculated from points that strike a linear portion in the normal curve of the test. Concentrations of the anti-factor VIII inhibitor in Bethesda Units (BU) were measured by the Bethesda Assay (Kasper CK, Thromb et Diath Hae 30: 263, 1973). Cell proliferation assay T. The spleen was used as the source of T cells for proliferation assays. Spleen cells were then cultured (5 x 10 ^ / well) in flat bottom plates of 96 wells. Various amounts of the recombinant factor VIII were added to the culture medium consisting of complete RPMI-1640 containing 0.5 percent hemophilic mouse serum. 37 kBp of 3H-thymidine / well (6.7 Ci / millimole, ICN Pharmaceuticals Irvine, CA) was added after 72 hours of cultivation at 37 ° C. The cultures were harvested 16 hours later using a 9600 Matrix (Packard, Meriden, CT). The data is expressed as the means for the triplicate wells of cpm incorporated in the insoluble DNA.

Example 3. mCTLA4-Ig blocks the induction of an anti-factor VIII response.

Anti-factor VIII inhibitory antibodies were induced in control mice by repeated intravenous injections of 1 microgram of recombinant human factor VIII at three week intervals. In this Example, four groups of hemophilia A mice were injected with recombinant human factor VIII on days 0, 23, 44 and 66 (1 microgram intravenously initially, and then 0.2 microgram during the second, third and fourth injections). Mice in groups G-3 and G-4 were also injected intraperitoneally with 0.2 microgram of factor VIII on days 2-12. Blood samples were obtained for the anti-factor VIII assay on days 20, 37, 58 and 82. Control groups G-1 and G-3, of open circuits, were injected with only factor VIII. Groups G-2 and G-4, solid circles, were also injected with mCTLA4-Ig (250 migrans, i.p.) on the day before the day after the first injection of factor VIII. Anti-factor VIII anti-body concentration was determined by ELISA. The anti-factor VIII assay data points indicated as < 0.16 micrograms per milliliter were similar to those for plasma samples obtained from non-immunized hemophilia A mice. The results of this experiment are shown in Figure 5 (Note that Figure 5 repeats some of the data shown in Figure 2, but adds additional data). Anti-factor VIII was detected in four of the five mice, 20 days after the first injection, and all control mice developed high concentration of anti-factor VIII after receiving two to four injections. The mean inhibitory level after four injections was 1860 Bethesda units (BU). The formation of the anti-factor VIII antibody was markedly suppressed in mice injected intraperitoneally with 250 micrograms of murine CTLA4-Ig on the day before and the day after the first injection of factor VIII (Group G-2, Figure 5) , even when no additional mCTLA4-Ig was provided with the three subsequent factor VIII injections on days 23, 44 and 66. Anti-factor VIII was not detectable in any of the mice of the G-2 group and after the first or second injections of factor VIII. Three weeks after the third injection of factor VIII, one was detected - Weak immune response in two of the six mice in the G-2 group. To investigate whether the limited duration of non-response is a result of the short lifespan of human factor VIII in these mice (4 to 5 hours in murine hemophilia A (Evans FL et al., Proc Natl Acad Sci USA 95: 5734, 1998 ), control mice treated with mCTLA-4-Ig (Groups G-3 and G-4, Figure 5) were injected intravenously with 1 microgram of factor VIII on day 0 followed by daily intraperitoneal injections of 1 microgram of the factor VIII on days 2-12 High level anti-factor VIII was present on day 20 in the control mice (Group G-3): more than 350 micrograms per milliliter by ELISA and an average inhibitory concentration of 694 BU In contrast, Group G-4 mice that were injected with mCTLA4-Ig on the day before the day after the first exposure to factor VIII did not have detectable anti-factor VIII on day 20. The anti-human antibody response -factor VIII delayed after three injections of The additional factor VIII was the same in these mice as in the animals of Group G-2. In this way, the limited persistence of factor VIII in the plasma after the CTLA4-Ig injection was not the reason for a duration - Limited lack of response in mice treated with CTLA4-Ig.

Example 4. Effects of Repeated Administration of CTLA4-Ig Because the response of delayed anti-factor VIII was detected after repeated infusions of factor VIII when mCTLA4-Ig was provided only at the time of exposure of the first factor VIII, it was determined whether mCTLA4-Ig could prevent the development of anti-factor VIII. Factor VIII if provided with each infusion of factor VIII. In that experiment (Figure 6), hemophilia A mice were simultaneously infused with both factor VIII and mCTLA4-Ig six times at three week intervals. Hemophilia A mice were injected intravenously with both 1 microgram of factor VIII and 250 micrograms of mCTLA4-Ig at 3-week intervals (solid circles) or with factor VIII only after the first injection containing both factor VIII and mCTLA4-Ig (open circles). Serum samples were obtained for the anti-factor VIII assay 4 weeks after the sixth injection of factor VIII. No antifactor VIII was detectable in many of the ten mice treated in this manner when tested four weeks after the sixth injection of factor VIII. In - - In contrast, a high concentration of anti-factor VIII was present in the serum of mice that had received only one injection of mCTLA4-Ig (at the time of the first exposure to factor VIII) followed by five injections of factor VIII alone. These mice treated with mCTLA4-Ig were then tested to determine if they would have an immune response after additional injections of factor VIII in the absence of mCTLA4-Ig. After two intravenous injections at three-week intervals, none of the five mice developed the anti-factor VIII while the low-level anti-factor VIII was detected in two of the four control mice not previously exposed to either factor VIII or mCTLA4-Ig (Figure 7). In this experiment, the hemophilia A mice treated as described for Figure 6 with six injections of both factor VIII and mCTLA4-Ig were subsequently provided with six intravenous injections of 0.2 microgram of factor VIII at time intervals of 3 weeks without mCTLA4 -Ig additional (closed circles). Control mice without prior exposures to factor VIII were immunized in parallel (open circles). Serum samples for the anti-factor VIII assay were obtained 3 weeks after the second and sixth injections. After six injections of factor VIII alone, the mean concentration of factor VIII was 93 micrograms per milliliter for mice that had previously received both factor VIII and mCTLA4-Ig, while the mean concentration was 155 micrograms per milliliter for mice. control mice. These data document the limits of the specific immune suppression of the e.ntigen following the repeated co-administration of mCTLA.4-Ig with factor VIII.

Example 5. mCTLA4-Ig suppresses the immune response secondary to factor VIII.

To determine whether mCTLA4-Ig modifies an immune response secondary to factor VIII, mCTLA4-Ig was injected at the same time that factor VIII was given to hemophilia A mice that had already developed anti-factor VIII. Initially, all hemophilia A mice had been injected three times with 0.2 microgram of factor VIII and the level of anti-factor VIII was determined by ELISA. The control mice then received three additional injections of factor VIII while the remaining mice were provided with mCTLA4-Ig at the same time they received the first three additional injections of factor VIII. Even though many mice died of bleeding complications during this experiment due to repeated injections of blood sample collections, the results were clearly different for the two groups. In this Example, all mice initially received 3 intravenous injections of 0.2 microgram of FVTII at 2 week intervals. The control mice (open circles) were then injected with factor VIII three more times and blood samples were obtained for assay (upper panel). The other mice (solid circles) were provided with mCTLA4-Ig (250 micrograms, intraperitoneally) the day before the day after the fourth injection of factor VIII (as indicated by the arrow), followed by two more injections of factor VIII alone. at intervals of 3 weeks. The number of injections of factor VIII before testing the blood sample for anti-factor VIII is indicated on the horizontal axis. An increase in the concentration of anti-factor VIII was observed after the fourth injection of factor VIII for the control mice, with the average concentration being from 16 to 230 micrograms per milliliter (Figure 8A). After the fifth injection of factor VIII, the anti-factor VIII concentrations were all more than 350 micrograms per milliliter in the four remaining control mice. In contrast, mice treated with mCTLA4-Ig in the fourth injection of factor VIII had minimal increases or no increases in anti-factor VIII (Figures 8B and C). The administration of mCTLA4-Ig inhibited this secondary immune response of factor VIII for mice that had already developed relatively high anti-factor VIII levels corresponding to concentrations of the inhibitor of 5-90 BU (J Qian and other Thromb Haemost 81: 940, 1999) (Figure 8C) as well as for mice with the minimum anti-factor VIII after three initial injections of less than 5 BU (Figure 8B).

Example 6. Determination of the role of B7-1 and B7-2 in the primary immune response to factor VIII.

The roles of B7-1 and B7-2 which are co-stimulatory coordinating groups in the antigen-presenting cells when evaluated, since it was their interaction with CD28 that was assumed to have been prevented in the experiments using mCTLA4-Ig. To do this, cross the hemophilia A mice with B7-l ~ / ~ and B7-2- / "mice (F. Borriello et al. B Immunity 6: 303, 1997; Freeman GJ et al., Science 262: 907, 1993) and mice deficient in both factor VIII and either B7-1 or B7-2 were selected by genotyping. The hemophilia A / B7-1 - / - and hemophilia A / B7-2 - / - mice were then injected intravenously with 0.2 microgram of human factor VIII at two week intervals. Serum samples for the anti-factor VIII assay were obtained 12 days after the second and sixth injections of factor VIII. After four injections, all nine hemophilia A / B7-1 - / - mice (open circles) had developed anti-factor VIII, with concentrations of more than 350 micrograms per milliliter and an average inhibitory level of 712 BU (Figure 9). ), values similar to those for otherwise normal hemophilia A mice injected with factor VIII (J. Qian, and others Thromb Haemost 81: 940, 1999). In contrast, none of the eight hemophilia mice A1B7-2 - / - (closed circuits) had detectable anti-factor VIII. Similar results were obtained in hemophilia A mice treated with anti-B7-1 / anti-B7-2 antibodies. To evaluate the T cell response of these hemophilia A mice deficient in B7-1 and B7-2, spleen cells were obtained three days after the fifth intravenous injection of factor VIII. Spleen cells placed in a reservoir of 3 mice were used to establish the proliferation data. The open and closed frames in Figure 10 are for cells of the untreated hemophilia mice A / B7-1- / ", and B7-2" / -, respectively. The open circles are from cells of hemophilia A / B7-1 - / - mice that received 5 intravenous injections of FVIII and the solid circles are for hemophilia A / Bl-2 ~ '~ mice that received 5 intravenous injections of factor VIII . The concentration of factor VIII in the cultures is indicated on the horizontal axis. The proliferative activity of the T cell determined by the incorporation of 3H-thymidine showed a dose-dependent response of factor VIII from the hemophilia / A B7-l ~ / ~ mice (Figure 10). In contrast, no response to the T cell or any level of factor VIII was detected for the spleen cells from the hemophilia A / B7-2- / mice. "Therefore, B7-2 has the main role to support a immune response to factor VIII injected intravenously, and the formation of anti-factor VIII is prevented if it is lacking.

Equivalents Those skilled in the art will recognize, or be able to make sure using no more than routine experiments, of numerous equivalents to the specific compositions and methods described herein. These equivalents are considered as falling within the scope of this invention and are protected by the following claims.

Claims (36)

1. CLAIMS: 1. A composition comprising a first agent that promotes haemostasis and a second agent that inhibits a co-stimulatory signal in a T cell. The composition of claim 1, further comprising a pharmaceutically acceptable carrier. 3. The composition of claim 1, wherein the first agent is factor VIII. The composition of claim 1, wherein the first agent is a deleted variant of domain B of factor VIII. The composition of claim 1, wherein the first agent is factor IX. 6. The composition of claim 1, wherein the first agent is a von Willebrand factor. The composition of claim 1, wherein the second agent is a soluble form of a costimulatory molecule. The composition of claim 7, wherein the second agent is a soluble form of CTLA4. The composition of claim 7, wherein the second agent is a soluble form of B7-1, a soluble form of B7-2, or a combination of the soluble form of B7-1 and the soluble form of B7-2 . 10. The composition of claim 8, wherein the second agent is CTLA4Ig. The composition of claim 9, wherein the second agent is B7-Hg or B72Ig. The composition of claim 1, wherein the second agent is a soluble form of CD40 or CD40L. The composition of claim 1, wherein the second agent is an antibody that is linked to a costimulatory molecule. The composition of claim 13, wherein the second agent is selected from the group consisting of an anti-B7-l antibody, an anti-B7-2 antibody, and a combination of an anti-B7 antibody and an antibody of B7-2. 15. The composition of claim 13, wherein the antibody is a non-activating form of an anti-CD28 antibody. 16. A method for treating a hemostatic disorder in a subject comprising administering to the subject the composition of any one of claims 1 to 15, such that a hemostatic disorder is treated. 17. The method of claim 16, wherein the subject has a pre-existing immune response to the first agent. 18. The method of claim 16, wherein the subject does not have a pre-existing immune response to the first agent. The method of claim 16, further comprising administering a composition consisting of an additional immunosuppressive agent. The composition of claim 16, wherein the hemostatic disorder is selected from the group consisting of hemophilia A, hemophilia B and von Willebrand disease. 21. A method for treating a hemostatic disorder in a subject comprising administering to the subject the first agent that promotes haemostasis and a second agent that inhibits a co-stimulatory signal in a T cell, such that a hemostatic disorder is treated. 22. A method for treating a hemostatic disorder in a subject comprising administering to the subject the first agent promoting haemostasis and a second agent that inhibits a co-stimulatory signal in a T cell, such that the immune response to the first agent is modulated descendingly to thereby treat a hemostatic disorder. 23. The method of claim 21 or 22, wherein the first agent is factor VIII. The method of claim 21 or 22, wherein the first agent is a deleted variant of domain B of factor VIII. 25. The method of claim 21 or 22, wherein the first agent is factor IX. 26. The method of claim 21 or 22, wherein the first agent is the von Willebrand factor. The method of claim 21 or 22, wherein the second agent is a soluble form of an agent that supplies a costimulatory signal to a T cell. The method of claim 27, wherein the agent is a soluble form of CTLA4. 29. The method of claim 28, wherein the agent is CTLA4Ig. 30. The method of claim 27, wherein the agent is a soluble form of B7-1, a soluble form of B7-2, or a combination of a soluble form of B7-1 and a soluble form of B7-2. 31. The method of claim 30, wherein the agent is B7-Hg, B7-2Ig, or a combination of both B7-Hg and B7-2Ig. 32. The method of claim 21 or 22, wherein the second agent is an antibody that is linked to a costimulatory molecule. The method of claim 32, wherein the second agent is selected from the group consisting of an anti-B7-l antibody, an anti-B7-2 antibody, and a combination of an anti-B7-antibody. and anti-B7-2. 34. The method of claim 32, wherein the antibody is a non-activating form of an anti-CD28 antibody. 35. The method of claim 21 or 22, wherein the hemostatic disorder is selected from the group consisting of hemophilia A, hemophilia B and von Willebrand disease. 36. The method of claim 21 or 22, wherein the subject has a significant concentration of antibodies that bind to the first agent.