Compositions and use thereof for downmodulating the immune response to therapeutic proteins

One of the major limitations of therapeutic treatment using biological proteins is the immune response the body produces to the production of foreign substances in the body. This immune response is particularly problematic if foreign substances have to be reintroduced for optimal efficacy.

One example of this situation is the reintroduction of agents for the treatment of hemostatic disorders such as factor VIII deficiency diseases (e.g., classical haemophilia A and von Willebrand's disease) or factor IX deficiency, also known as haemophilia B. Classical haemophilia (haemophilia A) is an X-related disorder that affects 1 in 10,000 men. Von Wilbright's disease is the most severe hereditary bleeding disorder that occurs in 1 in 800 to 1,000 individuals. Haemophilia B, also known as Christmas disease, occurs in about 1 in every 100,000 men (Harrison's Principles of Internal Medicine. Isselbacher et al., Eds. 13^th Edition. 1994. McGraw-Hill NY, NY).

Factor VIII is a 265 kD single-stranded protein that circulates in complex with von Vilebrand Factor (VWF). Factor VIII is an important regulatory protein in the blood coagulation cascade. After thrombin activation, it increases the rate of factor X activation by activated factor IX (IXa), eventually leading to the formation of a fibrin clot. The VWF molecule is an adhesive glycoprotein that plays a central role in platelet agglutination. It plays the role of a factor VIII carrier in plasma and facilitates the interaction of platelets with vessel walls. VWF is composed of many, probably uniform, subunits of approximately 230 kD. VWF is synthesized by endothelial cells and megakaryocytes. Factor IX is a single-stranded 55 kD pro-enzyme which is converted by factor Xla or tissue factor-Vlla complex into active protease (IXa). Activated factor IX or activated factor VIII then activates factor X.

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Repeated administration of foreign proteins may elicit an immune response to these proteins in the recipient. In the case of T cells responding to foreign proteins, antigen presenting cells (APCs) must give two signals to resting T lymphocytes (Jenkins, M. and Schvvartz, R. (1987) J. Exp. Med. 165, 302-319; Mueller, DL. , et al (1990) J. Immunol. 144, 37013709). The first signal that confers specificity to the immune response is transmitted through the T cell receptor (TCR) following recognition of a foreign antigenic peptide presented under conditions of the large histocompatibility complex (MHC). The second signal, called costimulatory, induces T cell proliferation and conversion to activity (Lenschow et al., 1996. Annu. Rev. Immunol. 14: 233). Costimulation is neither antigen specific nor restricted to MHC and is thought to be caused by one or more individual cell surface molecules expressed by APCs (Jenkins, MK, et al. 1988 J. Immunol. 140: 3324-3330; Linsley, PC). , et al., 1991 J. Exp. Med. 173. 721-730; Gimmi, CD, et al., 1991 Proc. Natl. Acad. Sci. USA 88, 6575-6579; Young, JW, et al., 1992 J. Dyn. Invest. 90, 229-237; Koulova, L., et al., 1991 J. Exp. Med. 173, 759-762; Reiser, H., et al., 1992 Proc. Acad. Sci. USA. 89, 271-275; vanSeventer, GA, et al. (1990) J. Immunol. 144, 4579-4586; LaSalle, JM, et al., 1991 J. Immunol. 147, 774-80; Dustin, MI, et al., 1989 J. Exp. Med. 169. 503; Armitage, RJ, et al. 1992 Nature 357, 80-82; Liu, Y., et al. 1992 J. Exp. Med. 175. 437-445).

CD80 (B7-1) and CD86 (B7-2) proteins expressed on APCs are critical costimulatory molecules (Freeman et al., 1991. J. Exp. Med. 174: 625; Freeman et al., 1989 J. Immunol. 143 : 2714; Azuma et al. 1993 Nature 366: 76; Freeman et al. 1993 Science 262: 909).

It turns out that B7-2 is more important in the primary immune response, and B7-1, which is activated later in the immune response, may be important in the continuation of primary T cell responses or costimulation of the secondary T cell responses (Bluestone. 1995, Immunity. 2: 555 ).

B7-1 and B7-2 are counter receptors for two ligands expressed on T lymphocytes. One ligand to which B7-1 and B7-2 bind (CD28) is constitutively expressed on resting T cells and its activation is increased upon activation. After signaling through the T cell receptor, CD28 binding and costimulatory signal transduction induce T

L 4920 B cell proliferation and IL-2 release (Linsley, PS, et al. 1991 J. Exp. Med. 173. 721-730; Gimmi, CD, et al. 1991, Proc. Natl. Acad. Sci. USA. 88, 65756579; June, CH, et al. 1990 Immunol. Today 11, 211-6; Harding, FA, et al. 1992 Nature 356, 607-609). The second ligand, called CTLA4 (CD152), is homologous to CD28, but is not expressed by T cells in the dormancy state and appears after T cell activation (Brunet, J. F., et al., Nature 328. 267-270). CTLA4 appears to be crucial in the negative regulation of T cell responses (Waterhouse et al. 1995. Science 270: 985). Blocking CTLA4 has been found to eliminate inhibition signals, whereas CTLA4 aggregation delivers inhibitory signals that suppress T cell responses (Allison and KruVnmel. 1995. Science 270: 932). B7 molecules have a higher affinity for CTLA4 than CD28 (Linsley, PS, 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 their binding kinetics to CTLA4 are also different (Linsley et al., 1994. Immunity. 1: 793).

About 10-25% of patients with haemophilia develop an immune response to factor VIII. These patients develop inhibitors, usually IgG antibodies, which neutralize factor VIII activity and thus inhibit effective treatment. Two types of inhibitors were identified. Highly reactive patients with type I inhibitors have an anamnestic response to factor VIII, which produces an increased titer of factor VIII antibodies. Patients with a low response to type II inhibitors have a low antibody titer that does not increase with factor VIII administration. Current strategies to reduce antibody response in these patients have been only partially successful. In addition, the development of antibodies against substituted proteins is a critical problem that requires a solution if gene therapy is to be expected to be successful in the treatment of haemophilia and other deficiency-related diseases (Cornelly S. et al., Blood 88: 3846, 1996; Kuna SH. et al., Blood 91: 784, 1998).

Summary of the Invention

Among other things, the present invention provides compositions that allow the use of a therapeutic protein for the treatment of a disorder by reducing the onset and / or progression of an immune response to the therapeutic protein.

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In one aspect, the present invention relates to compositions comprising a first agent that promotes hemostasis and a second agent that inhibits a costimulatory signal in a T cell.

In one embodiment, the target composition further comprises a pharmaceutically acceptable carrier.

In one embodiment, the first agent is factor VIII. In another embodiment, the first agent is factor VIII, with the B domain removed. In one embodiment, the first agent is factor IX. In another embodiment, the first agent is the von Willebrand factor.

In one embodiment, the second agent is a soluble form of a costimulatory molecule. In a more preferred embodiment, the second agent is a soluble form of CTLA4. In another preferred embodiment, the second agent is soluble form B7-1, soluble form B7-2, or a combination of soluble form B7-1 and soluble form B7-2. In a more preferred embodiment, the second agent is CTLA4lg. In another highly preferred embodiment, the second agent is B7-1 Ig or B7-2lg. In still another preferred embodiment, the second agent is a CD40 or CD-40L soluble form.

In another embodiment, the second agent is an antibody that binds to a costimulatory molecule. In a preferred embodiment, the second agent is selected from the group consisting of anti-B7-1 antibody, anti-B7-2 antibody, and a combination of anti-B7-1 and anti-B7-2 antibodies. In one embodiment, the antibody is a non-activating form of an anti-CD28 antibody.

The invention also relates to the use of a rapidly prepared composition for treating a subject having a hemostatic disorder.

In one embodiment, the subject has a high titer of antibody that binds to the first agent. In another embodiment, the subject has no high titer of antibodies that bind to the first agent.

In one embodiment, a composition comprising an agent that inhibits a costimulatory signal in a T cell is administered for this purpose.

In one embodiment, the hemostatic disorder is selected from the group consisting of hemophilia A, hemophilia B, and von Willebrand's disease.

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In another aspect, the present invention relates to the use of a first agent that promotes hemostasis and a second agent that inhibits costimulatory signaling in a T cell for treating a subject's hemostatic disorder.

In another aspect, the present invention relates to administering to a subject a first agent which promotes hemostasis and a second agent which inhibits costimulatory signaling in a T cell, thereby providing immunotolerance to the first agent and thereby treating a hemostatic disorder.

In one embodiment, the first agent is factor VIII. In another embodiment, the first agent is a variant of Factor VIII with the B domain removed. In another embodiment, the first agent is factor IX. In another embodiment, the first agent is the von Willebrand factor.

In one embodiment, the second agent is in a soluble form of an agent that delivers a costimulatory signal to a T cell. In a more preferred embodiment, the agent is a soluble form of CTLA4. In another more preferred embodiment, the agent is CTLA4lg. In another preferred embodiment, the agent is soluble Form B7-1, soluble Form B7-2, or a combination of B7-1 and B7-2. In another preferred embodiment, the agent is B7-1 Ig, B7-2lg, or a combination of B7-1 Ig and B7-2lg.

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 anti-B7-1 antibody, antiB7-2 antibody, and a combination of anti-B7-1 and anti-B7-2 antibodies. 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 high titer of antibody that binds to the first agent.

Brief description of the drawings

Figure 1 illustrates the experimental design used in Example 1 for inhibiting the primary antibody response to factor VIII.

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Figure 2 shows that mice that did not receive CTLA4lg have high antibody titers starting at day 20 (G-1), while mice that received CTLA4lg have no antibody until day 82 (G-2 and G-3).

Figure 3 shows the experimental design used in Example 2, which investigates the inhibition of the secondary antibody response to factor VIII.

Figure 4 shows that animals that did not receive CTLA4lg have high titers of anti-factor VIII antibody (G-1), whereas mice that received CTLA4lg (G-2) did not develop a secondary immune response to factor VIII except 1 mouse. .

Figure 5 shows the effect of mCTLA4-Ig on the formation of anti-Factor VIII antibodies.

Figure 6 shows the effect of repeated administration of mCTLA4-Ig on the formation of anti-Factor VIII antibodies

Figure 7 shows the effect of simultaneous administration of mCTLA4-Ig and factor VIII.

Figure 8 shows the effect of mCTLA4-Ig on the secondary immune response to factor VIII.

Figure 9 shows the role of B7-1 and B7-2 in the formation of an antibody response to factor VIII.

Fig. 10 shows T cell response to factor VIII Α / Β7-Γ^/'' mice with haemophilia and A / B7-2 '' 'haemophilia.

Detailed description

The present invention represents an important advance in the treatment of a hemostatic disorder by providing compositions which allow the use of a therapeutic protein for the treatment of such a disorder by reducing the onset and / or progression of the immune response to this therapeutic protein.

Prior to the following description of the invention, some of the terms used in the description, examples, and appended definition have been assembled herein.

/. Definitions

As used herein, the term "hemostatic disorder" includes disorders characterized by abnormal bleeding and / or thrombosis. Normal hemostasis limits blood loss through a series of interactions between blood vessel wall components, platelets and plasma proteins. Hemostatic disorders

L 4920 B occurs, for example, due to a decrease in platelet aggregation and / or inability to form a fibrin clot, which may provide inappropriate responses to disease or trauma e.g. uncontrolled bleeding Such diseases can be detected e.g. determination of bleeding time partial thromboplastin time (PTT), prothrombin time (PT), thrombin time (TT), or quantification of fibrinogen using well-known methods. Examples of hemostatic disorders are haemophilia A, haemophilia B, and von Wilbright's disease.

As used herein, the term "haemostatic promoting agent" includes a protein or peptide that is deficient or absent in the subject and which, when administered to the subject, ameliorates or ameliorates the treatment of a hemostatic disorder. Between t

the most suitable agents that promote hemostasis are coagulation factors such as factor VIII, factor IX, VWF, and analogues thereof.

As used herein, the term "B7 family" or "B7 molecules" includes costimulatory molecules that have amino acid sequence identity to B7 polypeptides, e.g. with B7-1, B7-2, or B7-3 (recognized by the BB-1 antibody). In addition, the B7 family of molecules share a common function e.g. the ability to bind to a B7 family ligand (e.g., one or more of CD28, CTLA4, or ICOS) and the ability to costimulate T cell activation.

B7 polypeptides may provide costimulation to activated T cells and thereby induce T cell proliferation and / or cytokine secretion or may inhibit T cell costimulation e.g. when in soluble form. B7 family members include B7-1, B7-2, and soluble fragments or derivatives thereof. In one embodiment, members of the B7 family bind to CTLA4, CD28, ICOS and / or other ligands on immune cells and can inhibit or induce costimulation 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 interaction of a costimulatory molecule on an antigen presenting cell (APC), e.g. B7 family molecule, with its counter-receptor on the T cell. Costimulatory molecules on APCs (e.g., members of the B7 family) and related ligands on T cells (e.g., CTLA4, CD28, and ICOS) are collectively referred to herein as costimulatory molecules. The agent which inhibits the costimulatory signal may act either extracellularly. by inhibiting the interaction between costimulatory molecules and thereby blocking intracellular signals

L 4920 B or may act intracellularly by inhibiting costimulatory signals in the signaling pathway. Examples of agents are detailed below and include, for example, soluble forms of costimulatory molecules and antibodies that bind to costimulatory molecules.

As used herein, the expression "suppressive modulation of the immune response" includes reducing (e.g., suppressing or inhibiting) an immune response in a patient without an existing immune response, or decreasing the length and magnitude of an existing immune response. The term "immune response" includes any type of immune response that is initiated or dependent on costimulatory signals e.g. a cellular or humoral response that can 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 antigenic induction of specific tolerance which can be measured by methods known in the art, e.g. by measuring a secondary immune response (eg, a cellular or humoral response) to an antibody.

//. Agents that promote hemostasis

In one embodiment, the agent that induces hemostasis is factor VIII. As used herein, the term "factor VIII" includes proteins that exhibit factor VIII-specific procoagulant activity. In one embodiment of the present invention, the Factor VIII type proteins are natural Factor VIII proteins. Such proteins may be isolated from the blood or may be administered as a blood product or as an enriched blood product. In one embodiment, highly purified factor VIII can be obtained by adsorbing and eluting this factor from a blood product onto a monoclonal antibody column. Alternatively, such natural proteins may be produced recombinantly using nucleic acid molecules, preferably natural nucleic acid molecules. For example, in one embodiment, the Factor VIII protein is produced by expressing a Factor VIII-encoding nucleic acid molecule in a cell according to methods known in the art to provide a Factor VIII protein. The human factor VIII nucleotide sequence (and the corresponding amino acid sequence)

L 4920 Sequence B) is known. (See, e.g., Toole et al. Nature 1984, 312: 5992; or GenBank Accession Nos. 1701179; K01740).

In another embodiment, the haemostasis-promoting agent is non-natural factor VIII, e.g. A factor VIII mutant that retains a therapeutic function, ie, factor VIII haemostasis-promoting activity. For example, DNA sequences that can hybridize to human factor VIII-encoding DNA under conditions that prevent hybridization with non-factor VIII genes (e.g. conditions equivalent to 65 ° C at 5 x SSC (1 X SSC = 150 mM NaCl / 0). , 15 M Na citrate), or homologous DNA sequences that retain sequence identity in the portions of the nucleic acid molecule that encode protein regions important for Factor VIII function can be used in the present invention to produce Factor VIII. U.S. Patent 5,744,446; 5,663,060; 5,583,209; 5,661,008; 5,422,260 and 5,707,832.

In another embodiment, the haemostasis-promoting agent is a factor VIII-type protein in which at least one portion (e.g., a minor portion) is deleted. For example, in one embodiment, the Factor VIII protein is a modified Factor VIII protein in which one or more amino acids have been removed or are substituted between 90 kD and 69 kD cleavage sites relative to native Factor VIII; it is described in more detail in United States Patent 4,868,112, which is incorporated herein by reference.

In another embodiment, the haemostasis-promoting agent is a factor VIII analog having one or more amino acid deficiencies between a 50/40 cleavage site and a 73 kD cleavage site, which may be prepared by methods analogous to those described in United States Patent 4,868,112 (incorporated herein by reference). In a preferred embodiment, the factor VIII analog retains some or all of the acidic amino acid regions between the 80 kD and 73 kD cleavage sites. In another embodiment, part or all of this region is replaced by the corresponding acidic region immediately adjacent to the 50/40 cleavage site. In still other embodiments, the Factor VIII proteins are analogs (with or without the deletions mentioned above) such as those described in International Application PCT / US87 / 01299 (which is hereby incorporated by reference), e.g. wherein one or more cleavage sites comprising arginine residues at positions 226, 336, 562, 740, 776, 1313, 1648 or 1721 have been made resistant to proteolytic cleavage, e.g. replacing

L 4920 B, one or more amino acids by other amino acids using cDNA mutagenesis, known methods, e.g. by standard site-directed mutagenesis.

Agents that promote haemostasis also include hybrid Factor VIII proteins, which consist partly of human factor VIII and some non-human factor VIII of another species (e.g., porcine factor VIII). Such hybrid proteins may be prepared using known techniques, e.g. U.S. Patent 5,744,446; 5,663,060 and 5,583,209.

U.S. Patent 5,693,499; 5,681,746; 5,663,060; 5,583,209; 5,563,045; 5,460,951 and 5,455,031 are also appended as references.

In another embodiment, the agent that induces hemostasis is factor IX. As used herein, the term "factor IX" includes, but is not limited to, plasma-derived, transformed cell line factor IX and recombinantly derived factor IX isolated from host cell culture medium. Factor IX can be secreted from the blood or administered as a blood product or as an enriched blood product. In one embodiment, highly purified factor IX can be obtained by adsorbing and eluting this factor from a blood product onto a monoclonal antibody column. Examples of isolation methods are also described in U.S. Patent 5,639,857; 5,457,181 and 5,286,849. Alternatively, such natural proteins may be produced recombinantly using nucleic acid molecules, preferably natural nucleic acid molecules. For example, in one embodiment, the Factor IX protein is produced by expressing a factor IX-encoding nucleic acid molecule in a cell according to techniques known in the art to provide a Factor IX protein. Examples of genetic constructs for factor IX can be found in U.S. Patents 5,650,503 and 4,994,371.

The nucleotide sequence of factor IX is known. (See, e.g., Yoshitake et al. 1985. Biochemistry 24: 3726 or GenBank Accession Nos. K022402; A07407; A01819 or Χ54500).

In other embodiments, factor IX includes, for example, United States Patents 4,994,371; 5,171,569; 5,679,639; 5,621,039; and 5,714,583 (the disclosures of which are hereby incorporated by reference).

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In addition to the natural forms of factor IX, the term "factor IX" also includes non-natural forms, e.g. Factor IX mutants that retain therapeutic function, ie, factor IX promoting haemostasis. For example, DNA sequences that can hybridize to human factor IX-encoding DNA under conditions that prevent hybridization with non-factor IX genes (e.g. conditions equivalent to 65 ° C at 5 x SSC (1 X SSC = 150 mM NaCl / 0). , 15 M Na citrate), or DNA sequences that retain sequence identity in the portions of the nucleic acid molecule that encode protein regions important for Factor IX function can be used in the present invention to produce Factor IX.

Factor VIII or factor IX proteins can also be obtained from commercial suppliers. For example, concentrated forms of factor VIII can be obtained, e.g. Immunate® (Immuno, Beriate® (Behring); purified forms of Factor VIII monoclonal antibody are available, e.g., Oktanativ-M® (Pharmacia), Hemofil® M® (Baxter), and Monoclate-P® (Armor); Factor VIII forms such as Recombinate® (Baxter) and Kogenate® (Bayer). Also available is a recombinant form VIII with deleted B-part, r-VIII SQ® (Pharmacia and Upjohn, Stockholm). Factor IX can be purchased, e.g., as Nanotiv® (Kabi Pharmacia) or Immunine® (Immuno); it is also possible to obtain a purified Factor IX monoclonal antibody as Mononine® (Armor). Recombinant factor IX can also be obtained, e.g. as BeneFIX® (Genetics Institute).

VWF is a large multimeric plasma protein composed of individual subunits. VWF subunits are linked to each other by disulfide bonds. In plasma, VWF circulates as multimers, ranging from dimers to more than 50 subunits. The dimers consist of two subunits joined probably at the C-terminus by flexible rod-shaped moieties and are considered multimerization promoters. These promoters are linked in large, probably N-terminal globular moieties, to form multimers. It turns out that VWF is made as a 260 kD glycosylated precursor, which is then processed and sulfated. After dimerization, multimerization and proteolytic cleavage, the mature protein is approximately 225 kD. VWF was obtained recombinantly. The nucleotide and amino acid sequences of VWF are known. (See, e.g., Sadler et al. 1986. Cold Spring Harbor Symposium in

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Quantitative Biology 51: 515 or GenBank Accession Nos. L15333 or K033028). EP 0197592 B1 is hereby incorporated by reference.

In addition to the natural form of VWF, the term "VWF factor" also includes non-natural forms, e.g. Mutants of the VEF factor that retain the therapeutic properties of χ · ', i.e., haemostasis promoting VWF factor. For example, DNA sequences capable of hybridizing to human VWF-encoding DNA conditions that prevent hybridization with non-VWF factor genes (e.g. conditions equivalent to 65 ° C at 5 x SSC (1 X SSC = 150 mM NaCl / 0) , 15 M Na citrate), or DNA sequences that retain sequence identity in parts of the nucleic acid molecule that encode protein regions important for VWF factor function can be used in the present invention to produce VWF factor.

In one embodiment, the agents that promote hemostasis are of mammalian origin. In a more preferred embodiment, the agents that induce hemostasis are of porcine origin. In yet another preferred embodiment, the agents that promote hemostasis are of human origin. In yet another embodiment, the agents that promote hemostasis are hybrid molecules.

III. Immunomodulatory agents

In one embodiment, the agent which inhibits a costimulatory signal in a T cell is a natural form of a costimulatory molecule. Natural forms of costimulatory molecules may be isolated from cells or may be obtained by recombinant techniques using known techniques. For example, costimulatory proteins may be made by expressing a nucleic acid molecule encoding a costimulatory molecule in a cell to produce a costimulatory molecule. The nucleotide sequences of costimulatory molecules are known and may be found in literature or databases such as GenBank. See, for example, B7-2 (Freeman et al. 1993 Science, 262: 909 or GenBank Accession Nos. P42081 or A48754); B7-1 (Freeman et al., J. Exp. Med. 1991, 174: 625 or GenBank Accession Nos. P33681 or A45803); CTLA 4 (see, e.g., Ginsberg et al. 1985. Science, 228: 1401; or

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GenBank Accession Nos. P16410 or 291929); and CD28 (Aruffo and Seed. Proc. Natl. 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 natural forms of costimulatory molecules, the term "costimulatory molecules" also includes non-natural forms, e.g. mutants of costimulatory molecules, which deplete the function of the costimulatory molecule, ie the ability to bind to a cognate counter-receptor. For example, DNA sequences that can hybridize to DNA encoding a B7 molecule, a CTLA4 molecule, a CD28, or an ICOS molecule, which prevent hybridization to genes from a non-costimulatory molecule (e.g. under conditions equivalent to 65 ° C in 5 x SSC (1 X SSC = 150 mM NaCl / 0.15 M Na Citrate) are costimulatory molecules according to the present invention. Alternatively, DNA sequences that retain sequence identity within regions of the nucleic acid molecule that encode for the function of the costimulatory molecule, e.g. important areas of the protein for binding to other costimulatory molecules can also be used as agents that inhibit the costimulatory signal in a T cell. Preferably, the non-natural costimulatory molecules have high (e.g., greater than 70%, preferably greater than 80%, and more preferably greater than 90-95%) amino acid identity to the natural amino acid sequence of the extracellular portion of the costimulatory molecule.

In order to determine the amino acid sequences of a costimulatory molecule that may be important in binding the costimulatory molecule to its counter-receptor, various species, e.g. amino acid sequences comprising extracellular portions of mouse and human costimulatory molecules, and labeled retained (e.g., identical) residues. This can be done, for example, using standard alignment programs such as MegAlign (DNA STAR). It is likely that such retained or identical residues are necessary for the proper binding of the costimulatory molecule to its receptors and thus should not be altered.

Specific residues of costimulatory molecules that are important in linkage formation have also been identified. For example, the CD28 region, which is critical for interaction with B7-1 and B7-2, has been identified by targeted mutagenesis, CD28 mAb epitope mapping, receptor-based adhesion assays, and direct Ig-fusion protein

L 4920 B binding to surface receptors. The proline-rich sequence insert of CD28, MYPPPY, has been found to be critical for the function of this protein (Trunch et al., 1996, Mol. Immunol. 33: 321). Similarly, regions of the B7-1 molecule that are critical for functional interaction with CD28 and CTLA4 have been identified using the mutation. Two hydrophobic residues, including the Y87 residue in the V-shaped portion of B7-1, have been found to be of major importance, retaining in all B7-1 and B7-2 molecules cloned from various species (Fargeas et al., 1995, J. Exp. Med. 182: 667). Using these or similar techniques, amino acid sequences of extracellular portions of costimulatory molecules can be identified, which are very important and therefore need not be altered.

Costimulatory molecules may be expressed in soluble form or used as immunogens to produce antibodies. Such soluble costimulatory molecules or antibodies are suitable as agents that inhibit costimulatory signaling in a T cell as detailed below.

A. Agents that inhibit costimulatory signaling in a T cell by extracellular route.

I. Soluble forms of costimulatory molecules

In one embodiment, the agent that blocks a costimulatory signal in a T cell is a soluble form of a T cell costimulatory molecule (e.g., CTLA4, CD28, and / or ICOS) that can block the transmission of a costimulatory signal in a T cell.

In one embodiment, the agent that blocks the costimulatory signal in a T cell is a soluble form of CTLA4. DNA sequences encoding human and mouse CTLA4 protein are known; see e.g., Dariavich, et al. (1988) Eur. J. Immunol. 18 (12), 1901-1905; Brunei, JF, et al. (1987) supra; Brunet, JF et al. (1988) Immunol. Rev. 103: 21-36; and Freeman, GJ et al. (1992) J. Immunol. 149. 3795–3801. In some embodiments, the soluble CTLA4 protein comprises the entire CTLA4 protein. In a more preferred embodiment, the soluble CTLA4 protein comprises an extracellular portion of the CTLA4 protein. For example, a recombinant form of the soluble CTLA4 extracellular portion was expressed in yeast (Gerstmayer et al. 1997. FEBS Lett. 407: 63). In other implementations

In variants of L 4920 B, the soluble CTLA4 protein comprises at least a portion of the extracellular portion of the CTLA4 protein that retains its ability to bind to B7-1 and / or B7-2.

In one embodiment, the soluble CTLA4 protein or portion thereof is a fusion protein comprising at least a portion of CTLA4 that binds B7-1 and / or B7-2 and at least a portion of a second non-CTLA4 protein. In a more preferred embodiment, the CTLA4 fusion protein comprises an extracellular portion of CTLA4 fused at the amino terminus to a signal peptide, e.g. from oncostatin M (see, e.g., WO093 / 00431).

In a particularly preferred embodiment, the soluble form of CTLA4 is a fusion protein comprising an extracellular portion of CTLA4 fused to a portion of an immunoglobulin molecule. Such a fusion protein, CTLA4lg, can be made using known techniques (see, e.g., Linsley 1994. Perspectives in Drug Discovery and Design 2: 221; Linsley WO 93/00431 and US Patent 5,770,197).

In one embodiment, the agent that blocks a costimulatory signal in a T cell is a soluble form of an antigen presenting cell costimulatory molecule (e.g., B7 family molecules such as B7-1, B7-2 and / or ICOS ligand). For example, in one embodiment, the soluble form of the costimulatory molecule comprises a soluble form of B7-1 or a soluble form of B7-1 or a combination of a soluble form of B7-1 and a soluble form of B7-2. DNA sequences encoding B7 proteins are also known; see e.g., B7-2 (Freeman et al. 1993, Science, 262: 909 or GenBank Accession Nos. P42081 or A48754); B7-1 (Freeman et al., J. Exp. Med. 1991. 174: 625 or GenBank Accession Nos. P33681 or A45803). In some embodiments, the soluble B7 protein comprises the entire B7 protein. In preferred embodiments, the soluble B7 protein comprises an extracellular portion of the B7 protein. For example, a recombinant form of the soluble extracellular portion of CTLA4 was expressed in yeast (Gerstmayer et al. 1997). FEBS Lett. 407: 63). In other embodiments, the soluble B7 protein comprises at least a portion of the extracellular portion of the B7 protein that retains its ability to bind to CTLA4 and / or CD28.

In one embodiment, the soluble B7 protein or 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 non-B7 protein. In a more preferred embodiment B7

The L 4920 B fusion protein comprises an extracellular portion of B7 fused at the amino terminus to a signal peptide, e.g. from oncostatin M (see, e.g., WO093 / 00431).

In a particularly preferred embodiment, soluble form B7 is a fusion protein comprising an extracellular portion of B7 fused to a portion of an immunoglobulin molecule. Such a fusion protein, B7lg, can be made using known techniques (see, e.g., Linsley 1994. Perspectives in Drug Discovery and Design 2: 221; Linsley WO 93/00431 and US Patent 5,770,197 and US Patent 5,580,756).

2. Antibodies that bind to costimulatory molecules

In some embodiments, the agent that blocks a costimulatory signal in a T cell is an antibody that binds to a costimulatory molecule. In the production of antibodies that bind to costimulatory molecules, the costimulatory protein, part of the costimulatory protein (e.g. peptide costimulatory protein derivative) or a fusion protein containing the whole or part of the amino acid sequence of the costimulatory molecule may be used to generate anti-protein and / or anti-peptide polyclonal antisera or monoclonal antibodies using standard techniques. As used herein, the term "antibody" is intended to include whole antibodies or fragments thereof. Antibody fragments (e.g. Fab 'fragments, F (ab')₂ fragments or single-chain antibodies) can be made using well-known techniques. The term "antibody" also encompasses chimeric and humanized antibodies.

For example, a mammal (e.g., mouse, hamster, or rabbit) may be immunized with an immunogenic form of a costimulatory protein or peptide that elicits an antibody response in a mammal. The immunogen may be, for example, a molecule or fragment of a recombinant costimulatory protein, a fragment of a synthetic peptide, or a cell that expresses a costimulatory molecule on its surface. The cell may be, for example, an antigen presenting cell, or a T cell, or a cell transfected with a nucleic acid encoding a costimulatory molecule such that the costimulatory molecule is expressed on the cell surface. Host cells transfected to express peptides may be any prokaryotic or eukaryotic cell. For example, having the activity of a costimulatory molecule

L 4920 B peptide can be expressed in bacterial cells such as E. coli, insect cells (baculovirus), yeast or mammalian cells such as Chinese hamster ovary (CHO) cells and UFOs. Other suitable host cells and expression vectors may be found in Goeddel (1990) supra or known to those skilled in the art. Examples of vector expression in yeast S. cervisae include pYepSecl (Baldari et al., (1987) Embo J. 6: 229-234), pMFa (Kurjan and Herskovvitz, (1982) Cell 30: 933-943), pJRY88 (Schultz et al., (1987) Gene 54: 113-123), and pYES2 (Invitrogen Corporation, San Diego). CA). Baculovirus vectors suitable for protein expression in cultured insect cells (SF 9 cells) include the pAc series (Smith et al., (1983) Mol. Cell Biol. 3: 2156-2165) and the pVL series (Lucklovv, VA, and Summers, MD, (1989) Virology 170: 31-39). In general, COS cells (Gluzman, Y., (1981) Cell 23: 175-182) were used in combination with vectors such as pCDM8 (Seed, B., (1987) Nature 329: 840) for transient amplification / expression in mammalian cells. while CHO (decoded Chinese Hamster Ovary) cells were used in combination with vectors such as pMT2PC (Kaufman et al. (1987) EMBO J. 6: 187-195) for stable amplification / expression in mammalian cells. The preferred cell line for obtaining the recombinant protein is the UFO myeloma cell line available from ECACC (Cat. No. 85110603); it is described by Galfre, G. and Milstein, C. ((1981) Methods in Enzymology 73 (13): 3-46: and Preparation of Monoclonal Antibodies: Strategies and Procedures, Academic Press, NY, NY). The vector DNA may be introduced into mammalian cells by conventional techniques such as calcium phosphate or calcium chloride conidation, DEAE-dextran-mediated transfection, lipofectin or electroporation. Suitable methods for transforming host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2^nd Edition, Cold Spring Harbor Laboratory Press (1989) and other laboratory textbooks. In mammalian cells, control functions of the expression vector are often transferred using viral material. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and most commonly, Simian virus 40.

Peptides expressed in mammalian cells having costimulatory molecule activity may be isolated by standard procedures known in the art.

L 4920 B including ammonium sulfate precipitation, column chromatography fractionation (eg ion exchange, gel filtration electrophoresis, affinity chromatography, etc.) and finally crystallization (see mainly Enzyme Purification and Related Techniques, Methods in Enzyme Biology, 22: 233-577 (1971).

It will be appreciated by those skilled in the art that generating antibodies to human costimulatory molecules according to standard techniques is a matter of skill. The antibodies may be either polyclonal or monoclonal antibodies, or antigen-binding fragments of such antibodies. Of particular interest for therapeutic use are antibodies which inhibit the costimulatory binding of the molecule to its natural ligand (s) on the surface of immune cells, thereby inhibiting the costimulation of the immune cell. Preferred antibodies to the costimulatory molecule are those that can inhibit or inhibit inverse T cell-mediated immune responses by binding B7-2 or B7-1 to the surface of B lymphocytes and preventing interaction with CTLA4 and / or CD28. Other preferred antibodies to a costimulatory molecule are those which, in combination with a second antibody that binds to another costimulatory molecule, exhibit enhanced inhibition of T cell costimulation compared to one of the first antibody, e.g. combination of anti-B7-1 and anti-B7-2 antibodies.

A. Immunogen

The term "immunogen" is used herein to describe a composition comprising, as an active ingredient, a costimulatory molecule activity peptide used to produce antibodies against a costimulatory molecule. When a costimulatory molecule activity peptide is used to induce antibodies, it should be understood that this peptide may be used alone, conjugated to the carrier, or as a peptide polymer.

In order to generate suitable antibodies against costimulatory molecules, the immunogen must contain an effective immunogenic amount of a peptide having costimulatory activity of the molecule, usually as a conjugate attached to a carrier. The effective amount per unit dose of peptide will depend, among other things, on the species of animal being inoculated, the body weight of that animal and the choice of

L 4920 B immunization regimen as is well known in the art. The immunogenic preparation should generally contain from about 10 micrograms to about 500 milligrams, preferably from about 50 micrograms to about 50 milligrams of peptide per immunization dose. The immunization preparation may also contain an adjuvant as part of the diluent. Adjuvants such as complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) and alum are well known in the art and can be purchased from several sources.

Those skilled in the art will appreciate that instead of using natural costimulatory molecule forms for immunization, synthetic peptides may otherwise be used to elicit suitable antibodies for use in the present invention. Fragments of both soluble and membrane-bound costimulatory molecules or peptides are also suitable for use as immunogens and can also be isolated by immunoaffinity purification. The purified protein form of the costimulatory molecule, such as isolated as described above or known form, may be used directly as an immunogen or may otherwise be coupled to a suitable protein carrier according to known techniques, including chemical copolymerization, and genetic engineering using a cloned gene of a costimulatory molecule.

The peptide or protein selected for immunization may be modified to enhance their immunogenicity. For example, methods for conferring immunogenicity on a peptide include attachment to carriers or other well known techniques. Any peptide selected for immunization may also be synthesized. In some embodiments, such peptides may be synthesized as branched polypeptides to enhance the immune response known to those skilled in the art (see, e.g., peptides. Edited by Bemd Gutte Academic Press 1995, p. 456-493).

The purified costimulatory molecule protein may also be covalently or non-covalently modified with non-protein substances such as lipids or carbohydrates to enhance immunogenicity or solubility. Alternatively, the purified costimulatory molecule protein may be copolymerized or inserted into a viral particle (replication virus) or other microorganism to increase immunogenicity.

L 4920 B

For example, a protein of a costimulatory molecule may be chemically attached to a viral particle or a microorganism or an immunogenic part thereof.

In an illustrative embodiment, the purified costimulatory molecule protein or peptide fragment having the activity of the costimulatory molecule (e.g., obtained by limited proteolysis or recombinant DNA techniques) is coupled to a carrier that is immunogenic to animals. Preferred carriers include proteins such as albumin, serum proteins (e.g., globulins and lipoproteins), and polyamino acids. Examples of suitable proteins include bovine serum albumin, rabbit serum albumin, thyroglobulin, snail lymph hemocyanin, egg ovalbumin and bovine gamma-globulins. Synthetic polyamino acids such as polylysine or polyarginine are also suitable as carriers. Costimulatory molecules for attaching protein or peptide moieties to a suitable immunogenic carrier contain many chemical cross-linking agents known to those skilled in the art. Preferred cross-linking agents are heterobifunctional cross-linkers that can be used in a stepwise manner to attach proteins. A wide variety of heterobifunctional cross-linkers are known, including succinimidyl 4- (Nmaleimidomethyl) cyclohexane-1-carboxylate (SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-succinimidyl (4-iodacetyl) aminobenzoimide (SIAB), p-maleimidophenyl) butyrate (SMPB), 1-ethyl-3- (3dimethylaminopropyl) carbodiimide hydrochloride (EDC), 4-succinimidoyloxycarbonyl-α-methyl-α- (2-pyridyldithio) -toluene (SMPT), N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP), succinimidyl 6- [3- (2-pyridyldithio) propionate hexanoate (LC-SPDP).

It may also be desirable to simply immunize whole cells expressing the protein of the costimulatory molecule on its surface. A variety of cell lines, including but not limited to activated B cells, can be used as immunogens to generate monoclonal antibodies against the costimulatory molecule antigen. For example, spleen B cells can be obtained from a subject and activated with anti-immunoglobulin. Alternatively, if a costimulatory molecule is expressed on the cell surface, a B cell line such as Raji may be used.

L 4920 B cell line (B cell Burkett's lymphoma, see e.g. Freeman, GJ et al. (1993) Science 262: 909-911) or JY B lymphoblastoid cell line (see e.g. Azuma, M. et al. (1993) Nature 366: 76-79). Whole cells that can be used as immunogens to produce antibodies specific for the costimulatory molecule also include recombinant transfectants. For example, COS and CHO cells can be reconstructed by transfecting the cDNA of a costimulatory molecule such as that described by Knudson et al. (1993, PNAS 90: 40034007); Travernor et al. (1993, Immunogenetics 37: 474-477); Dougherty et al. (1991, J. Exp. Med. 174: 1-5); and Aruffo et al. (1990, Cell 61: 1303-1313), yielding an intact costimulatory molecule on the cell surface. These transfectant cells can then be used as an immunogen to generate antibodies of pre-selected specificity to the costimulatory molecule. Other examples of transfectant cells are known, in particular eukaryotic cells, which are capable of glycosylating costimulatory cell protein to produce any whole cell-type immunogen, and any method that allows expression of transfected costimulatory molecule genes on the cell surface can be used.

B. Polyclonal antibodies against costimulatory molecules

Polyclonal antibodies to purified costimulatory molecule protein or peptide-containing costimulatory molecule activity can be mainly induced in animals by subcutaneous (ip) or intraperitoneal (SC) or intraperitoneal injection of an immunogen of the costimulatory molecule, such as the costimulatory molecule protein. For example, as described above, it may be useful to conjugate a costimulatory molecule (including certain epitope-containing fragments of interest) to a protein that is immunogenic to the species to be immunized, e.g. snail lymph hemocyanin, serum albumin.

Usually, established routine procedures for stimulating and producing antibodies can be used to immunize a host animal or its cultured antibody-producing cell for a method and mode of immunization. In an illustrative embodiment, the animals are generally immunized against conjugates or derivatives of an immunogenic costimulatory molecule by mixing about 1 pg to 1 mg of the conjugate with Freund's complete adjuvant and injecting this solution with

L 4920 B skin in many places. After one month, the animals are reintroduced 1/5 to 1/10 of the initial amount of Freund's complete adjuvant conjugate (or other suitable adjuvant) by subcutaneous injection at multiple sites. After 7-14 days, the animals are bled and the titre of the anticostimulatory molecule is determined. Animals are exposed to the immunogen until the titer is switched to plateau. Preferably, the animals are conjugated to a protein of the same costimulatory molecule but conjugated to a different protein and / or via a different crosslinking agent. Conjugates can also be made in recombinant cell culture as fusion proteins. Aggregating agents, such as, for example, alum, may also be used to enhance the immune response.

Such mammalian populations of antibody molecules are called "polyclonal" because populations include antibodies with different immunospecificities and affinities to the costimulatory molecule. The antibody molecules are then taken from mammals and isolated by well known methods such as using DEAE to obtain the Sephadex IgG fraction. To increase antibody specificity, antibodies can be purified by immunoaffinity chromatography using solid phase-attached immunogen. The antibody is contacted with the solid-phase immunogen for a period of time required to react with the antibody molecule to form a solid-phase immuno-complex. . Bound antibodies are isolated from the complex using standard procedures.

C. Monoclonal antibodies against costimulatory molecules

As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a population of antibody molecules that have only one antigen-binding site capable of immunoreacting with a particular epitope of a costimulatory molecule. Thus, a monoclonal antibody composition typically has only one binding affinity for a particular costimulatory molecule protein with which it is immunoreactive. Preferably, the monoclonal antibody used in the method of the invention is further characterized as immunoreactive with a costimulatory molecule derived from a human.

L 4920 B

Monoclonal antibodies suitable for the compositions and methods of the present invention are directed to an epitope on a costimulatory molecule antigen such that the formation of a complex between the antibody and the costimulatory molecule antigen inhibits the interaction of the costimulatory molecule with its natural ligand (s) on immune cell surfaces; thereby inhibiting T cell costimulation through the costimulatory molecule-ligand interaction. The monoclonal antibody against the epitope of the costimulatory molecule can be produced using techniques that allow the production of antibody molecules using continuous cell lines in culture. Such methodologies include, but are not limited to, hybridoma methodology, first described by Kohler and Milstein (1975, Nature 256: 495-497), and subsequently described by human B cell hybridoma methodology (Kozbor et al. (1983) Immunol. Today 4:72). , EBV Hybridoma Methods (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Ine. 77-96) and triom methodologies. Other methods that can efficiently produce monoclonal antibodies suitable for the present invention include phage display techniques (Marks et al. (1992) J. Biol. Chem. 16007-16010).

In one embodiment, the antibody preparation of the invention is a monoclonal antibody produced by a hybridoma cell line. Hybridoma fusion methodologies were first introduced by Kohler and Milstein (Kohler et al., Nature (1975) 256: 495-97; Brown et al. (1981) J. Immunol. 127: 539-46; Brown et al. (1980) J. Biol. 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 way, this. The monoclonal antibody compositions of the invention may be prepared by the following process, which comprises the steps of:

(a) Immunization of an animal with a costimulatory molecule. Immunization is generally accomplished by administering an immunologically effective amount, i.e., an amount sufficient to elicit an immune response, to an immunologically susceptible mammal of an immunogen of a costimulatory molecule. Preferably, the mammal is a rodent, such as a rabbit, rat or mouse. The mammal is then maintained for a period of time to produce in the mammalian cells expressing antibody molecules that immunoreact with the immunogen of the costimulatory molecule. Such immunoreaction is determined by screening the antibody molecules so produced for immunoreactivity with the immunogenic protein preparation. Other

In the case of L 4920 B, it may be desirable to screen for antibody molecules with a protein preparation in a form where it must be recognized by the antibody molecule in the assay, e.g. costimulatory molecules in membrane-bound form. Such selection techniques are well known to those skilled in the art.

(b) Subsequently, a suspension of antibody-producing cells taken from each immunized mammal expressing the desired antibody is prepared. After some time, the mice are sacrificed and somatic antibody-producing lymphocytes are obtained. Antibody-producing cells can be obtained from the spleen of lymph nodes and from peripheral blood of primed animals. Spleen cells are most suitable and can be mechanically disaggregated into individual cells in physiologically acceptable media using well known techniques. Mouse lymphocytes give a higher percentage of stable fusions with the myeloma myeloma described below. Rat, rabbit and frog somatic cells may also be used. Cellular chromosomes encoding the desired immunoglobulins are immortalized by fusing spleen cells with myeloma cells, usually in the presence of a fusion agent such as polyethylene glycol (PEG). As a fusion partner, any of a number of myeloma cell lines can be used for fusion according to known procedures; for example, the P3NS1 / 1-Ag4-1, P3-x63-Ag8.653, or Sp2 / O-g14 myeloma cell lines. These myeloma lines are available from the American Type Culture Collection (ATCC), Rockville, Md.

The resulting cells containing the desired hybridoma are then grown in a selective medium, such as HAT medium, in which non-lysed parent myeloma or lymphocyte cells eventually die. Only hybridoma cells survive and can be grown under limiting dilution to produce isolated clones. Selection of hybridoma supernatants is performed for the presence of antibodies of the desired specificity e.g. by immunoassay methodologies using the antigen used for immunization. Positive clones can then be cloned under limiting dilution conditions and the produced monoclonal antibody can be isolated. There are various commonly accepted methods for isolating and purifying monoclonal antibodies, which allow them to be distinguished from other proteins and other impurities. Methods commonly used to purify monoclonal antibodies include ammonium sulfate precipitation, ion exchange chromatography, and affinity chromatography (see, e.g., Zola et al.

L 4920 B

Monoclonal Hybridoma Antibodies: Techniques and Applications, Hurell (ed) pp. 51-52 (CRC Press 1982). The hybridomas obtained by these methods may be propagated in vitro or in vivo (ascites fluid) using known techniques.

Typically, individual cell lines may be propagated in vitro, for example in laboratory culture vessels, and growth media containing a high concentration of a single specific monoclonal antibody may be processed by decantation, filtration or centrifugation. Alternatively, the yield of the monoclonal antibody may be increased by injecting the hybridoma sample into a histosatable animal of the type used for the initial somatic and myeloma cell fusion. This animal develops tumors that express a specific monoclonal antibody produced by a fusion cell hybrid. Animal body fluids, such as ascites fluid or serum, produce high levels of monoclonal antibodies. If human hybridomas or EBV-hybridomas are used, the rejection of foreign bodies injected into animals such as mice must be avoided. Immunodeficient or nude mice may be used, or the hybridoma may be transplanted primarily into irradiated nude mice as solid subcutaneous tumors grown in vitro, followed by intraperitoneal pristane-irradiated nude mice expressing high levels of anti-human monoclonal .

Suitable media and animals for making such compositions are well known to those skilled in the art, are commercially available, and include synthetic culture media, inbred mice, and the like. An example of a synthetic medium is Dulbecco's Minimum Master Medium (DMEM; Dulbecco et al. (1959) Virol. 8: 396) supplemented with 4.5 gm / L glucose, 20 mM glutamine and 20% fetal calf serum. An example of an inbred mouse strain is Balb / c.

D. Humanized antibodies to costimulatory molecules

When human antibodies are used to treat human beings, they are recognized to varying degrees as foreign bodies and can induce an immune response in the patient. One strategy to reduce or eliminate this problem, which is most appropriate for general immunosuppression, is the production of chimeric antibody derivatives,

L 4920 B in which animal-non-human variable areas and human constant areas are combined. Such antibodies are the equivalents of the monoclonal and polyclonal antibodies described above, but may be less immunogenic when administered to humans and are therefore likely to be better transmitted by the patient.

Chimeric mouse-human monoclonal antibodies (i.e., chimeric antibodies) that react with a costimulatory molecule may be made, for example, using recently developed protocols for the production of chimeric antibodies. Humanized antibodies can be produced, for example, by replacing the immunogenic portion of the antibody with a corresponding but non-immunogenic portion. For this purpose, the genes encoding the constant regions of the mouse (or other species) antibody to the costimulatory molecule are replaced by the genes encoding the human constant region. (Robinson et al., International Patent Publication PCT / US86 / 02269; Akira et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al. PCT Application WO 86/01533; Cabillity et al., European Patent Application 125,023; Better et al. (1988 Science 240: 1041-1043); Liu et al. (1987) PNAS 84: 3439-3443; Liu et al. (1987) J. Immunol. 139: 3521-3526; Sun et al. (1987 PNAS 84: 214-218; Nishimura et al. (1987) Canc. Res. 47: 999-1005; Wood et al. (1985) Nature 314: 446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80: 1553-1559). Major reviews of humanized chimeric antibodies have been provided by Morrison, SL (1985) Science 229: 1202-1207 and Oi et al. (1986) BioTechniques 4: 214. Such methods include the isolation, manipulation and expression of nucleic acid sequences encoding all or part of the variable region of at least one heavy or light chain of an immunoglobulin. Sources of such nucleic acids are well known to those skilled in the art and can be derived, for example, from hybridomas producing antibodies to costimulatory molecules. The chimeric cDNA can then be cloned into the appropriate expression vector.

Alternatively, suitable "humanized" antibodies can be made using CDR or CEA substitution (The Winter US Patent 5,225,539; Jon et al. (1986) Nature 321: 552-525; Verhoeyan et al. (1988) Science 239: 1534; and Beidler et al. (1988) J. Immunol. 141: 4053-4060).

L 4920 B

E. Combinatorial antibodies against costimulatory molecules

The compositions of the present invention and monoclonal and polyclonal antibodies can also be obtained by other well-known methods of recombinant DNA technology. To identify and isolate antibody fragments of particular antigenic specificity, another method, called the combinatorial antibody demonstration method, has been developed and can be used to obtain a population of monoclonal antibodies to costimulatory molecules and polyclonal antibodies to costimulatory molecules (Sastry et al. (1989) PNAS 86: 5728; Huse et al. (1989) Science 246: 1275; and Orlandi et al. (1989) PNAS 86: 3833). After immunization of the animal with costimulatory molecules as immunogen as above, the resulting B cell pool antibody repertoire is cloned. Methods of directly obtaining DNA sequences of variable regions of immunoglobulin molecules of various populations using a mixture of oligomeric primers and PCR are generally known. For example, mixed oligonucleotide primers corresponding to 5 'leader (signal peptide) sequences and / or frame 1 (FR1) sequences, as well as a conserved 3' constant region (Larrick et al. (1991) Biotechniques 11: 152156). A similar strategy can also be used to amplify human heavy and light chain variable regions from human antibodies [Larrick et al. (1991) Methods: Companion to Methods in Enzymology 2: 106110). The ability to clone human immunoglobulin V genes is of particular importance in light of the achievements in creating a human antibody kit in transgenic animals (see, e.g., Bruggeman et al. (1993) Year Immunol. 7: 33-40; Tuaillon et al. (1993) PNAS 90: 3720 -3724; Bruggeman et al. (1991) Eur. J. Immunol. 21: 1323-1326; and Wood et al. PCT Publication WO 91/00906).

In an illustrative embodiment, RNA was isolated from activated B cells, such as peripheral blood cells, bone marrow or spleen preparations, using standard procedures (e.g., U.S. Patent No. 4,683,202; Orlandi, et al. PNAS (1989) 86: 3833-3837). Sastry et al., PNAS (1989) 86: 5728-5732; and Huse et al. (1989) Science 246: 1275-1281). First strand cDNA is synthesized using the constant region of the heavy chain (s) and each of the κ and λ light chains

L 4920 B, as well as primers for the signal sequence. Using variable region PCR primers, both heavy and light chain variable regions (individually or in combination) are amplified and incorporated into appropriate vectors for further manipulation, generating demonstration packages. Oligonucleotide primers suitable for amplification

Xmay be unique either in congenital or have inosine embedded in congenital anomalies. Restriction endonuclease recognition sequences can also be inserted into the primers so that the amplified fragment can be cloned into a vector in a predefined expression reading frame.

A cloned library of immunization-derived antibody repertoire V genes can be expressed using a population of demonstration packages, preferably derived from a filamentous phage, to form an antibody demonstration library. Ideally, the demonstration package includes a system that provides samples of a very large variety of antibody demonstration libraries, rapid sorting after each affinity separation cycle, and easy isolation of the antibody gene from the purified demonstration packages. Apart from commercial kits for generating phage demonstration libraries (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and Stratagene SurfZAP ™ Phage Demonstration Kit, Catalog No. 240612), particularly suitable for demonstrating antibodies to costimulatory molecules. examples of intermodal library generation methods and reagents can be found, for example, in The Ladner et al. U.S. Pat. 5,223,409; the Kang et al. International Publication No. WO 92/18619; the Dower et al. International Publication No. WO 91/17271; the Winter et al. International Publication No. WO 92/20791; the Markland et al. International Publication No. WO 92/15679; the Breitling et al. International Publication No. WO 93/01288; the McCafferty et al. International Publication No. WO 92/01047; the Garrard et al. International Publication No. WO 92/09690; the Ladner et al. International Publication No. WO 90/02809; Fuchs et al. (1991) Bio / Technology 9: 13701372; Hay et al. (1992) Hum. Antibod. Hybridoma 3: 81-85; Huse et al. (1989) Science 246: 1275-1281; Griffths et al. (1993) EMBO J. 12: 725-734; Havvkins et al. (1992) J. Mol. Biol. 226: 889-896; Clackson et al. (1991) Nature 352: 624628; Gram et al. (1992) PNAS 89: 3576-3580; Garrad et al. (1991)

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Bio / Technology 9: 1373-1377; Hoogenboom et al. (1991) Nuc. Acid Res. 19: 4133-4137; and Barb et al. (1991) PNAS 88: 7978-7982.

In certain embodiments, the heavy and light chain V region sites can be expressed on the same polypeptide joined by a flexible junction to form a single-stranded Fv fragment, and the scFV gene is then cloned into the desired expression vector or phage genome As generally described by McCafferty et al. Nature (1990) 348: 552554, the complete Vh and Vl portions of an antibody fused to a (Gly4-Ser) 3 junction can be used to produce a single-stranded antibody that can provide a demonstration bundle to discriminate based on antigenic affinity. The isolated immunoreactive scFV antibodies reactive with the costimulatory molecule can then be introduced into pharmaceutical formulations for use in the method of the invention.

F. Hybridomas and methods of obtaining them

Hybridomas suitable for use in the present invention are hybridomas that have the ability to produce a monoclonal antibody that will specifically immunoreact with a costimulatory molecule. As described below, hybridoma cells that produce antibodies to costimulatory molecules can be directly implanted in a recipient animal to obtain a stable source of antibodies. The use of isolation devices for encapsulating hybridoma culture can help prevent an immunogenic response against implanted cells and uncontrolled hybridoma cell proliferation in a host with suppressed immunity. Preferred hybridomas of the present invention are hybridomas that produce antibody molecules that specifically immunoreact with a costimulatory molecule expressed on activated human B cell surfaces.

Hybridomas that produce e.g. methods for generating antibody molecules having the desired immunospecificity, i.e. capable of binding to a particular costimulatory molecule and / or an identifiable epitope of the costimulatory molecule, are well known. Particularly applicable is the hybridoma technology described by Niman et al. (1983) PNAS 80: 4949-4953; and Galfre et al. (1981) Meth. Enzymol. 73: 3-46.

In another exemplary method, transgenic mice containing human antibody sets can be immunized with a human costimulatory molecule.

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Splenocytes from these immunized transgenic mice can then be used to generate hybridomas that express human monoclonal antibodies that specifically react with the human costimulatory molecule (see, e.g., Wood et al. PCT publication WO 91/00906; Kucherlapti et al. PCT publication WO 91/00906). 10741; Lonberg et al., PCT Publication WO 92/03918; Kay et al., PCT Publication 92/03917; Lonberg, N. et al. (1994) Nature 368: 856-859; Green, LL et al. (1994) Nature. Genet 7: 13-21; Morrison, SL et al. (1994) Proc. Natl. 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).

Considered ¹ that the term "antibody" as used herein includes fragments thereof that also specifically react with a costimulatory molecule as described herein. Antibodies can be fragmented using conventional techniques and selected for use in the same manner as full antibodies. For example, F (ab ')₂ fragments can be generated by exposure to the antibody with pepsin. F (ab ') received₂ fragment can be treated by reduction of disulfide bridges to produce Fab 'fragments.

Antibodies produced by these or other methods may 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 the costimulatory signal in a T cell is an antibody that binds to both B7-1 and B7-2. For example, parts of the extracellular region that are retained on both costimulatory molecules can be used to produce such an antibody as an immunogen. See also: e.g., Metzler et al. 1997 Nat. Struct. Biol. 4: 527).

In one embodiment, the agent that inhibits a costimulatory signal in a T cell is an antibody that binds to B7-1. Such antibodies are known to those skilled in the art or may be prepared as described above using the B7-1 molecule or portion thereof as an immunogen and selected by the above methods or standard methods. Examples of B7-1 antibodies include those described in U.S. Patent 5,747,034 and McHug et al. 1998, Dyn. Immunol. Immunopathol. 87:50 or Rugtveit et al. 1997, Dyn. Exp. Immunol. 110: 104.

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In another embodiment, the agent which inhibits a costimulatory signal in a T cell is an antibody that binds to B7-2. Such antibodies are known to those skilled in the art or may be prepared as described above using the B7-2 molecule or portion thereof as an immunogen and selected by the above methods or standard methods. Examples of B7-1 antibodies include the antibodies described in Rugtveitet et al. 1997, Clin. Exp. Immunol. 110: 104.

In one embodiment, the agent that blocks the costimulatory signal in a T cell is a combination of an antibody that binds B7-1 and an antibody that binds 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 transmit a costimulatory signal to a T cell (e.g., an Fab fragment of an anti-CD28 antibody). Such antibodies are known to those skilled in the art or may be prepared as described above using the CD28 molecule or portion thereof as an immunogen and selected by the above methods or standard methods. Examples of known anti-CD28 antibodies include those described in Darling et al. 1997, Gene Ther. 4: 1350.

In preferred embodiments, Fab fragments of an antibody that binds to CD28 may be used. Antibody fragments that are unable to crosslink CD28 on the surface of a T cell have been found to block T cell costimulation (Valunas et al., 1994, Immunity 1: 405).

In still other embodiments, the agent that inhibits a costimulatory signal in a T cell is a CTLA4 binding antibody that blocks a costimulatory signal in a T cell by providing a negative signal to a T cell (i.e., a CTLA4 agonist). For example, cross-linking of CTLA4 on the surface of a T cell has been shown to inhibit proliferation and IL-2 production (Krummel and Allison, 1996, J. Exp. Med. 183: 2533). Such antibodies may be prepared as described above using the CTLA4 molecule or portion thereof as an immunogen and selected by the above methods or standard methods. Examples of these antibodies include those also described by Vandenborre et al. 1998, Am. J. Pathol. 152: 963.

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In yet another embodiment, the agent that inhibits a costimulatory signal in a t cell is an ICOS antibody that blocks a costimulatory signal in a T cell. Such antibodies may be prepared as above using an ICOS molecule or portion thereof as an immunogen and selected by the above methods or standard methods.

IV. Agents that regulate the expression of costimulatory molecules

In another embodiment, the agent that inhibits a costimulatory signal in a T cell is an agent that interferes with the expression of a costimulatory molecule. For example, interactions between CD40 on antigen presenting cells and CD40 ligand (CD40L) on T cells have been found to be important in maintaining, amplifying or prolonging the expression of B7-1 or B7-2 on antigen presenting cells, and provide enhancement of costimulation (Van Gool , et al., 1996, Immunol Rev. 153: 47; Klaus et al., 1994, J. Immunol. 152: 5643).

In one embodiment, the agent that blocks costimulatory expression of a molecule thereby blocking a costimulatory signal in a T cell is a CD40 or CD40L soluble form. The DNA sequences encoding these CD40 and CD40L are known; see such as GenBank Accession Nos. Y10507 or Stamenlovic et al., 1988, EMBO J. 7: 1053-1059 for CD40 or Gauchat et al., 1993, FEBS 315 (3): 259-266; Graf et al., 1992, Eur. J. Immunol. 22: 31913194; Seyama 1996, Hum. Genet. 97: 180-185 or GenBank Accessio Nos. L07414, Χ67878, Χ96710 for CD40L.

In one embodiment, the agent that blocks costimulatory expression of a molecule thereby blocking a costimulatory signal in a T cell is a CD40 or CD40L soluble form. In one embodiment, the soluble CD40 or CD40L protein is a complete protein. In more preferred embodiments, the soluble CD40 or CD40L protein comprises an extracellular portion of the protein. For example, the soluble recombinant form of the extracellular portion of the CD40 or CD40L protein or portion thereof may be made as a fusion protein comprising at least a portion of the CD40 or CD40L such that interactions between CD40 on the APC and CD40L on the T cell are interrupted and costimulatory signal transduction cell. Such a soluble recombinant form of the CD40 or CD40L protein comprises at least a portion of a molecule which

L 4920 B is sufficient to bind to its anti-receptor, and at least part of the second non-CD40 or CD40L protein. In preferred embodiments, the CD40 or CD40L fusion protein comprises an extracellular portion of CD40 or CD40L fused to a signal peptide at the amino terminus, e.g. from oncostatin M (see e.g. WO 93/00431).

In a particularly preferred embodiment, the soluble form of CD40 or CD40L is a fusion protein comprising an extracellular portion of CD40 or CD40L fused to a portion of an immunoglobulin molecule (e.g., Chen et al., 1995, J. Immunol. 155: 2833). Such a fusion protein, CD40lg or CD40Llg, can be made using known methods (see, e.g., Linsley, 1994, Perspectives in Discovery and Design 2: 221; Linsley WO 93/00431, U.S. Patent 5,770,197 and U.S. Patent 5,580,756).

In addition, antibodies to CD40 have been found to have a synergistic effect on agents that inhibit costimulatory signaling in a T cell by promoting foreign body tolerance (Kirk et al., 1997, Proc. Natl. Acad. Sci. USA, 94: 8789; Larsen et al. 1996, Nature 381: 434). Therefore, in one embodiment, antibodies to CD40 or CD40L that bind to these molecules but do not induce expression of costimulatory molecules can be used as an agent that blocks a costimulatory signal in a T cell.

V. Agents that Inhibit Costimulatory Signal Intracellularly

In one embodiment, the agent that inhibits a costimulatory signal in a T cell is an agent that inhibits such a signal intracellularly. Stimulation of the T cell via the CD28 surface receptor (i.e., a costimulatory signal) induces the production of D-3 phosphoinositides in the T cell. Thus, in one embodiment, the production of D-3 phosphoinositides in a T cell can be inhibited by inhibiting a costimulatory signal, thereby inhibiting a T cell response measured, for example, by T cell proliferation or cytokine production. The term "D-3 phosphoinositides" is intended to include phosphatidylinositol derivatives which are phosphorylated at the D-3 position of the inositol ring and include phosphatidylinositol (3) -monophosphates (Ptdlns (3) P), phosphatidylinositol (3,4) -bisphosphates (Ptl) 3,4) P₂) and phosphatidylinositol (3,4,5) -trisphosphate (Ptlns (3,4,5) p3).

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D-3 phosphoinositides are generated intracellularly by phosphatidylinositol 3-kinase (PI3K). PI3K is a heterodimer composed of an 85 kDa subunit that binds tyrosyl phosphorylated proteins through its SH2 moieties and a 110 kDa catalytic subunit. PI3K was first identified as a lipid kinase that phosphorylates the D-3 position of the inositol ring of phosphatidylinositol lPtdlns (4) P and Ptdlns (4,5) p2). Two recent studies have shown that PI3K is in fact a dual-specificity kinase with both lipid and serine kinase activities (Dhand, R. et al. (1994) EMBO J. 13: 522 and Carpenter, CL et al. (1993) Mol. . Cell Biol. 13: 1657).

Thus, in one embodiment, the agent that inhibits costimulatory signaling in a T cell is an agent that inhibits PI3K activity. The preferred agent that inhibits PI3K activity in the T cell is the fungal metabolite vortmanine, derivatives or analogs thereof. Vortmanin is a potent PI3K inhibitor obtained from T. wortmanii (Kyowa Hakko Kohyo Co. Ltd.) or P. fumicuiosum (Sigma). Vortmanin derivatives or analogs include structurally related Vortmanin compounds that retain the ability to inhibit PI3K and T cell responses. Examples of Vortmanin derivatives and analogs are described in Viesinger, D. et al. (1974) Experientia 30: 135136; Closse, A. et al. (1981) J. Med. Chem. 24: 1465-1471; and Baggiolini, M. et al. (1987) Exp. Cell Fri 169: 408-418. Another inhibitor of PI3K activity that may be used is bioflavenoid quercetin, derivatives or analogs thereof. Quercetin derivatives or analogs include structurally related quercetin compounds that retain the ability to inhibit PI3K and T cell responses. Examples of quercetin derivatives and analogs are described in Vlahos, CJ et al. (1994) J. Biol. Chem. 269: 5241-5284. A preferred derivative of quercetin that inhibits PI3K activity is LY294002 (2- (4-morpholinyl) -8-phenyl4H-1-benzopyran-4-one, Lilly Indianapolis, IN) (described in Vlahos et al., Supra).

Stimulation of CD28 has also been shown to yield protein tyrosine phosphorylation in a T cell (see, e.g., Vandenberghe, P. et al. (1992) J. Exp. Med. 175: 951-960: Lu, Y. et al. 1992) J. Immunol. 149: 24-29). Thus, in one embodiment, an agent that inhibits a costimulatory signal in a T cell inhibits tyrosine phosphorylation in a T cell. A preferred inhibitor of protein tyrosine kinase is an inhibitor that inhibits src

L 4920 B protein tyrosine kinase. In one embodiment of the invention, the src protein tyrosine kinase inhibitor is herbimycin A, a derivative thereof, or an analogue thereof. Derivatives or analogs of herbimycin A include structurally related herbimycin A related compounds that retain the ability to inhibit protein tyrosine kinase activity. In another embodiment, the agent which inhibits protein tyrosine phosphorylation is a protein tyrosine phosphatase, or an activator of protein tyrosine phosphatase. By increasing tyrosine phosphatase activity in a T cell, the amount of pure protein tyrosine phosphorylation is reduced. This protein tyrosine phosphatase may be a cellular protein tyrosine phosphatase T cell such as CD45 or Hcph. Cell surface tyrosine phosphatase activity on T cells can be activated by contacting the T cell with a molecule that binds to this phosphatase and stimulates its activity. For example, an antibody directed against CD45 may be used to stimulate tyrosine phosphatase activity on a T cell expressing CD45. Thus, in one embodiment, the agent that inhibits protein tyrosine phosphorylation in a T cell is an anti-CD45 antibody or fragment thereof that retains its ability to stimulate CD45 activity. Examples of antibody fragments include Fab and F (ab ') 2 fragments. The antibodies or fragments thereof may be presented in the form of a stimulant, for example multimerized or immobilized, etc.

In addition, CD28 binding has been associated with increased phospholipase C activity (see, e.g., Nuneš, J. et al. (1993) Biochem. J. 293: 835-842) and increased intracellular calcium (see, e.g., Ledbetter, JA et al (1990) Blood 75: 1531-1539 and examples). Thus, an agent that acts intracellularly by inhibiting costimulatory signaling in a T cell may act by inhibiting phospholipase C activity and / or inhibiting intracellular calcium elevation. For example, the tyrosine kinase inhibitor herbimycin A also inhibits CD28-induced calcium release in T cells.

Proteinserin and serine threonine kinases have also been shown to interact with the CD28 signaling pathways (Siegel, J.N. et al. (1993) J. Immunol. 151: 4116-4127; Pai, SV et al. (1994) J Immunol. 24: 2364; Parry et al. 1997, Eur. J. Immunol. 27: 2495). Thus, in another embodiment, an agent that acts intracellularly to inhibit a costimulatory signal in a T cell inhibits serine or serine threonine kinase activity.

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VI. Other agents that block T cell costimulation

Other agents that block the costimulatory signal in the T cell can be identified using standard techniques. For example, such agents may be identified by their ability to inhibit T cell proliferation or cytokine production. For example, a costimulation test system may be used. In such a system, human CD28⁺ T cells are isolated, for example, by harvesting immunomagnetic beads using monoclonal antibodies against B cells, natural killer cells, and macrophages as described previously (Gimmi, CD, et al. (1993) Proc. Natl. Acad. Sci. USA 90, 6586-6590). Antigen presenting cells, e.g. whole spleen cells, purified B cells, or B7-1 or B7-2 transfected COS cells may be irradiated or exposed to mitomycin-C (e.g. at 25 pg / ml) for 1 hour thereafter to wash extensively for proliferation. 10th⁵CD28 * T cells may be incubated with, e.g., 10⁵-10⁴ APC (eg COS cells transfected with B7 molecule). In this exemplary test, one population of T cells received one primary activation signal (e.g. T cell receptor signal); another T-cell population received one costimulatory signal; another T-cell population received both a primary activation signal and a costimulatory signal; The primary activation signal may be delivered, for example, using a submitogenic dose of PMA (e.g., 1 ng / ml), a submitogenic dose of mitogen, a suboptimal dose of antigen, or a submitogenic dose of an anti-T cell receptor antibody. Signal 2 is delivered using antigen presenting cells containing the B7 molecule. Potential blocking agents are tested over a range of concentrations. For example, potential blocking antibodies may be used as a hybridoma snail or as a purified antibody (e.g., about 10 µg / ml). T cell proliferation can be measured by³Of H-thymidine (1 pCi) over a minimum of 12-18 hours during a 72 hour incubation. The delivery of the primary activation signal should give some proliferation to any T cells receiving both the primary activation signal and the costimulatory signal 2,

L 4920 B should proliferate at maximum. Blocking agents are identified by their ability to reduce the maximum costimulatory signal-induced proliferation.

In addition, or as an alternative to measuring T cell proliferation, cytokine production can be measured using well-known techniques. For example, IL-2 and IL-4 produced in T cell cultures can be detected in culture supernatants collected 24-72 hours after culture initiation using a commercially available ELISA (R&D Systems, Minneapolis, MN and BioSource, Camarillo, CA). As noted above, blocking agents can be identified by their ability to reduce maximal cytokine production induced by cytosolic signal.

In the case of antibodies, any of the "blocking antibodies" identified by one or the other assay may be further tested by determining the costimulatory molecule to which they are bound according to known procedures. For example, the ability of a blocking antibody to reduce the binding of a labeled antibody to a known ligand can be measured.

VII. Other agents for suppressive modulation of immune responses

In some embodiments, the compositions and methods of the present invention may include additional agents or additional agents to enhance immunotolerance of an agent that promotes haemostasis. In one embodiment, an agent that stimulates immunotolerance but does not inhibit a costimulatory signal in a T cell may be added to these compositions. For example, an antiCD40 ligand (e.g. a monoclonal antibody against human CD40 ligand 5C8 (Kirk et al., 1997, Proc. Natl. Acad. Sci. USA 94: 8789). CD40 and its T cell based ligand, CD40L (CD154), play an important role in the positive regulation of B7 and in the determination of B cell activity (U.S. Patent 5,683,693 to Yang et al., 1996, Science 273: 1862; Grewal et al., 1996, Science 273). : 1864; Leterman et al., 1992, J. Exp. Med. 175: 1091; Lederman et al., 1992, J. Immunol. 149: 3817). Antibodies to CD40 ligand have been found to interact synergistically with agents that inhibit costimulatory signal T

L 4920 B, promoting foreign body tolerance (Kirk et al., 1997, Proc. Natl. Acad. Sci. USA 94: 8789; Larsen et al., 1996, Nature 381: 434). In another embodiment, the composition of the invention may use an agent which acts intracellularly to induce immunotolerance but does not inhibit the costimulatory signal in the T cell. For example, in one embodiment of χ · ', cyclosporin A (CSA), FK506, rapamycin, or another agent that suppresses immune responses may be included in these compositions or administered as part of these routes (See, e.g., Sigai et al., 1992 , Annv. Rev. Immunol. 10: 519; Ruhlmann et al. 1997, Immunobiology 198: 192; Shaw et al. 1996, Clin. Chem. 42: 1316).

t

VIII. Methods of using compositions comprising a therapeutic protein and an immunotolerant

In one embodiment, the compositions and / or agents of the present invention described herein are administered to subjects having a haemostatic disorder who have previously been treated with an agent that promotes hemostasis. In another embodiment, the compositions and / or agents of the present invention described herein are administered to subjects who have not yet been treated with an agent that promotes hemostasis.

In yet another embodiment, the compositions and / or agents of the present invention described herein are administered to a subject who has not yet developed an immune response to an agent that promotes hemostasis. In other embodiments, the compositions and / or agents of the invention are administered to subjects who already have an immune response to an agent that promotes hemostasis. The subject's "pre-existing immune response" can be determined by measuring the subject's titer of antibodies reacting with the haemostatic stimulating agent according to well-known procedures. If such a subject has a measurable titer of such antibodies (e.g., a statistically significant titer) compared to the titers of control subjects, it may be said that the subject has an already existing immune response to an agent that promotes hemostasis. Alternatively, the cellular immune response to an agent that promotes hemostasis may be measured by determining whether the subject has an ongoing immune response to an agent that promotes hemostasis. Such methodologies are well known to those skilled in the art.

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In one embodiment, a first agent that stimulates hemostasis and a second agent that inhibits or blocks a costimulatory signal in a T cell is used to treat a hemostatic disorder. In another embodiment, a composition comprising a combination of a first agent that promotes hemostasis and a second agent that inhibits or blocks a costimulatory signal in a T cell can be used to treat a hemostatic disorder.

The compositions and / or agents described herein may be administered in any pharmacological form which comprises a therapeutically effective amount of an agent and a pharmaceutically acceptable carrier. Use of a therapeutically effective amount of the agents and / or compositions of the present invention is defined as an amount effective at providing the doses and times required to achieve healing of a hemostatic disorder in the case of a haemostatic promoting agent and immunotolerance in the case of an agent that inhibits costimulatory signaling. The therapeutically effective amount of the agent or composition may vary depending upon factors such as the disease state, the age, sex and weight of the individual and whether or not the individual already has a developed immune response to an agent that promotes hemostasis. This amount can easily be determined by one of ordinary skill in the art.

The optimal course of administration of the agents and / or compositions may also vary with the subject being treated. In some embodiments, the subject will need to be treated with both agents simultaneously. In this case, it is desirable to use an agent which promotes hemostasis and an agent which inhibits a costimulatory signal in a T cell simultaneously, for example in the form of a composition containing both agents.

In other embodiments, it is desirable to administer the agents separately, for example to increase the stability of the agents or to facilitate the ordering of the agents. In one embodiment, sequential administration may be desirable to achieve the optimal therapeutic effect of a haemostatic stimulating agent by optimally inhibiting an immune response, preferably an antibody response to that agent. For example, an agent that inhibits a costimulatory signal may be administered alone prior to the administration of a haemostatic stimulating agent or may be administered alone for several days following the administration of a haemostatic promoting agent.

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In one embodiment, an agent that blocks a costimulatory signal in a T cell can be administered continuously, e.g. each time a haemostatic stimulating agent is administered. In another embodiment, the agent that blocks a costimulatory signal in a T cell is administered singly. For example, a subject may require regular treatment with a haemostatic stimulating agent, but treatment with an agent that inhibits a costimulatory signal in a T cell may be intermittent. For example, treatment with a haemostatic stimulating agent may be ongoing but may be sufficient with one or two subject treatment agents that block the costimulatory signal in the T cell; further introduction of an agent that blocks the costimulatory signal in the T cell may no longer be necessary. In a preferred embodiment, the agent which blocks the costimulatory signal in the T cell is administered at appropriate intervals for at least about 6 months.

In one embodiment, the agents or compositions of the present invention are administered to patients if the patient has been found to have pre-existing antibodies. In another embodiment, the agents or compositions of the present invention are administered to previously untreated patients, i.e., without pre-existing antibodies. In yet another embodiment, the agents or compositions of the present invention are for use in patients who have previously been treated but have no antibody titers against the haemostatic stimulating agent.

To achieve an optimal therapeutic response for each subject, a dosage regimen may be adapted without extensive experimentation. For example, antibody titers to the haemostatic stimulating agent may be measured to determine whether or not the subject has developed an immune response to the agent and the dosage regimen adapted accordingly. For example, if antibody titers to the haemostatic stimulating agent increase, higher doses of the agent that blocks the costimulatory signal in the T cell may be administered.

Non-parenteral administration of the agent or composition to the subject may require coating or administration with an agent that prevents their inactivation. The agent or composition of the present invention may be administered to a subject in a suitable vehicle or diluent, in combination with enzyme inhibitors, or in a suitable carrier such as liposomes. Pharmaceutically acceptable diluents include saline and aqueous buffers. Enzyme inhibitors include the pancreatic trypsin inhibitor 41

L 4920 B diisopropyl fluorophosphate (DEP) and trazilol. Liposomes include water-in-oil emulsions and conventional liposomes (Strejan et al., (1984) J. Neuroimmunol. 7:27).

The active agent or composition may also be administered parenterally or intraperitoneally. Dispersions can be made in glycerol, liquid polyethylene glycols and mixtures thereof, and oils. Under normal conditions of storage and use, substances may be added which may prevent the growth of micro-organisms.

Pharmaceutical formulations suitable for injection include sterile aqueous solutions (if water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile solutions or injectable dispersions. At all

In such cases, the agent or composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against any contamination by microorganisms such as bacteria or fungi. The carrier may be a solvent or dispersing medium containing, for example, water, ethanol, polyol (e.g., glycerol, propyl glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof. A suitable liquid may be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions and by the use of surfactants. A variety of antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like can be used to protect against the action of microorganisms. In many cases, it is desirable to include in the composition an isotonic agent, for example sugars, polyalcohols such as mannitol, sorbitol, sodium chloride. Prolonged absorption of the compositions for injection may be achieved by the addition to the composition of an agent which retains absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating the appropriate amount of the active composition or agent into an appropriate vehicle containing one or a combination of the ingredients listed above, followed by sterile filtration. Dispersions are usually prepared by incorporating the active compound into a sterile vehicle containing the main dispersion medium and other required ingredients as listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the most suitable preparation is drying

L 4920 B in vacuo and lyophilization to give a powder of the active ingredient (e.g., agent or composition) plus any desired ingredient from a previously sterile filtered solution thereof.

When the active agent or composition is suitably protected as described above, the protein may be administered orally, for example, with an inert diluent or an absorbable edible carrier. As used herein, the term "pharmaceutically acceptable carrier" means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retention agents, and the like. The use of such media and agents for pharmaceutically active substances is well known. It will be appreciated that when any conventional medium or agent is incompatible with the active compound, their use in therapeutic compositions is debatable. Additional active compounds may also be added to these compositions.

It is particularly desirable to prepare parenteral compositions in dosage unit form to facilitate administration and uniformity of dosage. As used herein, the term "unit dosage form" refers to physically discrete units suitable as unit dosage forms for a treated mammal; each unit contains a predetermined amount of the active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification of dosage unit forms of the present invention is dictated and directly dependent upon (a) the unique characteristics of the active compound and the particular effect desired and (b) the limitation of the formulation of such active agent or composition for treating individuals.

As used herein, the term "pharmaceutically acceptable carrier" means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retention agents, and the like. The use of such media and agents for pharmaceutically active substances is well known. Additional agents may also be added.

Unless otherwise stated, the practice of the present invention will employ standard techniques in cell biology, cell cultures, molecular biology, microbiology, recombinant DNA and immunology known to those skilled in the art. Such methodologies are fully explained in the literature. See also: such as Genetics; Molecular Cloning A Laboratory Manual, 2^nd Ed., Ed. by

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Sambrook, J. et al. (Cold Spring Harbor Laboratory Press (1989)); Short Protocols in Molecular Biology, 3^rd Ed., Ed. by Ausubel, F. et al. (Viiey, NY (1995)); DNA Cloning, Volumes I and II (DN Glover ed., 1985); Oligonucleotide Synthesis (MJ Gait ed. (1984)); Mullis et al. U.S. Patent No: 4,683,195; Nucleic Acid Hybridization (BU Hames & amp; SJ Higgins eds. (1984)); treatise Methods in Enzymology (Academic Press, Ine., NY); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds., Academic Press, London (1987)); Handbook of Experimental Immunology, Volumes I-IV {DM VVeir and CC Blachwell, eds. (1986)); and Miller, J. Experiments in Molecular Genetics (Cold Spring Harbor Press, Cold Spring Harbor, N¹.Y. (1972)).

The exact contents of all references cited in this application, pending patent applications and published patents are hereby incorporated by reference.

The invention is further illustrated by the following examples which should not be construed as limiting.

EXAMPLES

In the examples, a mouse model of haemophilia A was used to evaluate novel inhibitors and treatments for inhibitors. Hemophilia A mice, which are the result of purposeful disruption of plasma exon 16 of the Vili factor gene, have no detectable amount of active factor VIII (Bi L., Nature Genetics 10: 119, 1995) and are similar in this respect to patients with severe haemophilia A. As would be expected, mice with haemophilia A show signs of a coagulation cascade in vivo with lethal hemorrhage if they have their tails cut without the use of haemostatic measures and exhibit subcutaneous or intramuscular haemorrhage after support or temporary immobilization (Qian J., Borovok M Biol., Kazazian H.H., Hoyer L. Thromb. Haemost. 81: 940, 1999; Evans GL et al., Proc. Natl. Acad. Sci. USA 95: 5734, 1998).

Intravenous infusion of human villi factor 0.2 pg - dose equivalent to the dose administered to patients with haemophilia A

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- gives minimal or no antibody response to these haemophilia A mice after a single injection, but repeated infusion produces a higher titre of inhibitory anti-factor VIII (Qian J. et al. Thromb. Haemost. 81: 940, 1999). In addition, three days after the first administration of human factor VIII, a T cell proliferative response was observed before antibody detection.

example. Inhibition of the primary immune response to factor VIII

An experimental design for this example is shown in FIG. On day zero, three groups of mice were injected intravenously with factor VIII. One group of mice was also injected with CTLA4Ig the day before and the day after the Factor VIII injection. A second group of mice received the same treatment with CTLA4-Ig (intraperitoneally) after daily injections of factor VIII from day 2 to day 12. The third group did not receive CTLA4-lg at all. Blood was collected from animals on days 20, 37, 58, and 82 Mice that did not receive CTLA4-Ig had high antibody titres starting at day 20, whereas mice receiving CTLA4-Ig did not develop antibodies until day 82 (Fig.2). .

example. Inhibition of the secondary immune response to factor VIII

An experimental design for this example is shown in FIG. In this example, mice received multiple intravenous injections of factor VIII every two weeks and were then divided into two groups. Mice were re-injected on day 1, 20 and 37 of factor VIII. One group of mice was injected with CTLA4-Ig on days -1 and +1, counting from days 1, 20, and 37 when Factor VIII injections were given. Animals that did not receive CTLA4-Ig had high titers of antiVIII, and mice receiving CTLA4-Ig (except one) showed no secondary immune response to factor VIII (Fig. 4).

The following methods and materials were used in Examples 3-6:

Animals. Characteristics of hemophilic mice of exon-16 (E-16) strain were described in Bi L., et al. Nature Genetics 10: 119, 1995; Bi L., et al. Blood 88: 3446, 1996. Adult male mice and homozygous E-16 females 10 to 20 weeks of age were used in these studies. Blood samples were taken from the saphenous vein plexus and the serum was separated by centrifugation at

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600g, for 3 minutes. Serum samples were stored at -20 ° C until assay. In some experiments, ear tagging was not used to identify mice to avoid severe bleeding and death. For this reason, Fig. 5 does not provide consistent data for individual mice.

E-16 / B7-1 and B7-2 double elimination mice were obtained using E-16 crossover with B7-1 and B7-2 eliminated mice (Borriello F, et al. 1997, Immunity 6: 303). Homozygous E-16 / B7-1 and E-16 / B7-2 double elimination mice were identified by genotyping (Bi L. et al., Nature Genetics 10: 119, 1995; Borriello F., et al. Immunity 6: 303 , 1997. Reduced factor VIII activity was assayed using the Coatest chromogenic bioassay (Chromogenix, Molndal, Sweden) (Bi L. et al. Blood 88: 3446, 1996). Factor VIII activity was less than 1% in both E-16 / B7-1 and E-16 / B7-2 deficient mice.

Antigens. Recombinant human factor VIII was obtained from Hyland Division of Baxter Healthcare Corp. (Glendale, CA).

mCTLA-4lg. The murine CTLA4-Ig cDNA expression plasmid was constructed by joining the leader and extracellular portions of murine CTLA4 to the hinge, IgHg2a CH2 and CH3 portions which were mutated to remove effector functions as described by Streurer et al. (Streurer, J. Immunol. 155: 1165, 1995). This insert was cloned into the pED expression vector and stably transfected into CHO cells as previously described (Lollar P. et al., J. Clin. Invest. 93: 2497, 1994). The concentrated conditioned medium was loaded onto an rProtein Aepharose Fast Flow Chromatographic column (Amersham Pharmacia Biotech, Piscataway, NJ). The column was washed with PBS pH 7.1 and mCTI_A4-Ig was eluted with 20 mM citrate pH 3.0. The peak fraction was neutralized with 1M Tris pH 8 to a final pH 7.5 and mixed with pBS pH 7.1 using an Amicon agitated cell with a YM30 membrane. mCTl_A4-Ig was depyrogenated using a Pore PI (Perceptive Biosystems) chromatography column and the product was eluted from the column using a linear NaCi gradient from 0 to 1 M NaCi in 25 mM Tris pH 7.5. The mCTLA4-Ig was then added to pBS pH 7.1 using an Amicon agitated cell with a YM30 membrane.

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Antibody measurements. Anti-factor VIII titer was determined by ELISA (Qian J, et al. Inhibitor antibody development and cellular response to human factor VIII in murine hemophilia A. Thromb Haemost. 81: 940, 1999). ELISAs were performed using microtiter wells coated with recombinant human factor VIII '' z '', 0.8 pg / ml 0.05 mol / ml carbonate-acid carbonate, pH 9. After maintaining mouse plasma samples in wells overnight at 4 ° C and washing, goat anti-mouse IgG (Southern Biotechnology Associates Ine., Birmingham, AL) was added and maintained for 2 hours. at room temperature. After washing, p-nitrophenyl phosphate (Sigma, St. Louis, MO), 2 mg / mL 100 mmol / L glycine, 1 mmol / L MgCl₂, 2 mmol / l ZnCl₂pH 10.4 and measuring the optical density at 410 nm with an automatic microtitre plate ELISA meter. Anti-Factor VIII antibody concentration is estimated from a standard curve obtained using a monoclonal mouse IgG anti-human Factor VIII antibody that binds to the A2 site (Mab 413) (Lollar P. et al., J. Clin. Invest. 93: 9497, 1994). ). The titre was calculated from points within the linear portion of the standard curve.

Anti-factor VIII inhibitor titers in Bethesda Units (BU) were measured using the Bethesda assay (Kasper CK Thromb et Diath Haem 30: 263, 1973).

T cell proliferation assays. The spleen was used as a T cell source for proliferation experiments. Spleen cells were cultured (5 x 10⁵/ well) in 96-well flat bottom plates. To a growth medium consisting of complete RPMI-1640 containing 0.5% hemophilic. mouse serum, various amounts of recombinant factor VIII are added. After 72 or. 37 kBp is added at 37 ° C³H-thymidine / well (6.7 Ci / mmol, ICN Pharmaceuticals Irvine, CA). After 16 hours, cultures were treated using Matrix 9600 (Packard, Meriden, CT). Data are expressed as the mean cpm of three identical wells of insoluble DNA.

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

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Anti-Factor VIII inhibitory antibodies were induced in control mice by repeated injections of 1 pg of recombinant human Factor VIII every three weeks. In this example, four groups of mice with haemophilia A were injected with recombinant human factor VIII at days 0, 23, 44, and 66 (initially 1, -pg iv, then 0.2 pg for injections 2, 3, and 4). Groups of G-3 and G-4 mice were also intraperitoneally injected with 0.2 pg Factor VIII on days 2-12. Blood samples for the anti-Factor VIII assay were taken on days 20, 37, 58 and 82. The control groups (white circles) received only factor VIII. Groups G-2 and G-4 (black circles) were also injected with mCTLA4-Ig (250 pg, ip) the day before and the day after the first factor VIII injection. Anti-Factor VIII antibody concentration was determined by ELISA. The anti-Factor VIII assay data points labeled <0.16 pg / ml were similar to those of plasma samples from unimmunized hemophilia A mice. The results of this experiment are shown in Fig. 5 (note that Fig. 5 reproduces some of the results shown in Fig. 2, but additional data are added).

Anti-Factor VIII was detected in four of the five mice 20 days after the first injection, and all control mice developed high titers of anti-VIII when given two to four injections. The average level of inhibition after four injections was 1860 Bethesda Units (BU). Anti-Factor VIII antibody formation was significantly reduced in mice injected intraperitoneally with 250 pg CTLA4-Ig mouse the day before and the day after the first Factor VIII injection (Group G-2, Figure 5), even when mCTLA4 was not administered. -lg after three consecutive doses of Factor VIII at days 23, 44 and 66. Anti-VII, no factor G was found in any of the mice in the G-2 group after the first or second injection of factor VIII. Three weeks after the third injection of Factor VIII, two of six mice had a weak immune response in the G-2 group.

To investigate whether the limited duration of absence of response is the result of the short half-life of human factor VIII in these mice (4-5 hours for murine A haemophilia) (Evans GL, et al., Proc. Natl. Acad. Sci. USA 95: 5734, 1998). , control and mCTLA4-Ig-treated mice (groups G-3 and G-4, Fig. 5) were injected intravenously with 1 pg of factor VIII for days 2-12.

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Control mice (G-3 group) had a high anti-factor VIII titer at day 20: greater than 350 µg / ml by ELISA and a mean titre of 694 BU inhibitors. In contrast, mice in the G-4 group given mCTLA4-Ig one day before and one day after the first administration of factor VIII had no detectable levels of anti-factor VIII on day 20. The delay in the anti-Factor VIII anti-body response after three additional injections of Factor VIII was the same in these mice as in G-2 animals. Thus, the limited plasma stability of factor VIII after injection of CTLA4-Ig is not a reason for the lack of response in CTLA4-Ig treated mice of limited duration.

example. Effects of repeated CTLA4-lg administration

Because of the delayed anti-Factor VIII response after repeated infusions of Factor VIII, when mCTLA4-Ig was only administered at the first administration of Factor VIII, it was determined whether mCTLA4-Ig could prevent the onset of anti-Factor VIII when given with each Factor VIII infusion. In this experiment (Figure 6), mice with haemophilia A were administered both factor VIII and mCTLA4-Ig simultaneously six times every three weeks. Mice with haemophilia A were injected intravenously with 1 µg of factor VIII and 250 µg of mCTLA4-Ig every 3 weeks (black circles), or only one factor VIII (white) after the first injection of both factor VIII and mCTLA4-Ig circles). Serum samples for the anti-Factor VIII assay were taken 4 weeks after the sixth Factor VIII injection. None of the ten mice treated in this manner were detectable with anti-Factor VIII when tested four weeks after the sixth Factor VIII injection. In contrast, mice that received only one injection of mCTLA4-Ig (at the first exposure to factor VIII) and then exposed to factor VIII alone had high serum anti-factor VIII titres.

These mCTLA4-Ig-exposed mice were then examined to determine whether they would have an immune response after additional injections of factor VIII in the absence of mCTLA4-Ig. After 2 intravenous injections every 3 weeks, none of the 5 mice developed anti-Factor VIII, whereas a small amount of anti-Factor VIII was found in two of the four control mice not previously exposed to either Factor VIII or mCTLA4-Ig (FIG. 7). In this experiment, A

L 4920 Hemophilia B mice treated as described in Figure 6 were treated with six injections of both factor VIII and mCTLA4-Ig, followed by six intravenous injections of 0.2 pg of factor VIII every three weeks without additional mCTLA4-Ig (black circles). ). Control mice were immunized in parallel without receiving factor VIII (white circles). Serum samples for the anti-Factor VIII assay were taken 3 weeks after the 2nd and 6th injections. After six injections of factor VIII alone, the mean titer of factor VIII was 93 µg / ml in mice that had previously received both factor VIII and mCTLA4-Ig, while the control titer had a mean titer of 155 µg / ml. These data represent antigen-specific immune suppression resulting from repeated administration of mCTLA4-Ig with VIII t

factor, limits?

example. mCTLA4-Ig inhibits the secondary immune response to factor VIII

To determine whether mCTLA4-Ig modifies the secondary immune response to Factor VIII, mCTLA4-Ig was injected at the same time as Factor VIII was administered to haemophiliac mice that had already developed anti-Factor VIII. Initially, all mice with haemophilia A were injected three times with 0.2 µg of factor VIII, and anti-factor VIII levels were determined by ELISA. Control mice then received three additional injections of factor VIII, and the remaining mice received mCTLA4-Ig at the same time as they received the first of three additional injections of factor VIII. Although most mice died from the complications of bleeding in this experiment, resulting from repeated injections and blood sampling, the results were clearly different between the two groups.

In this example, all mice initially received 3 intravenous injections of 0.2 µg of factor VIII every two weeks. Control mice (white circles) were then injected three more times with factor VIII and blood samples were collected for test (upper panel). Other mice (black circles) were given mCTLA4-Ig (250 µg, intraperitoneally) the day before and the day after the fourth Factor VIII injection (as shown by the arrow), followed by two additional Factor VIII injections every 3 weeks. The number of factor VIII injections prior to the blood sample assay for anti-factor VIII is shown on the horizontal axis.

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Control mice showed an increase in anti-factor VIII titer after a 4-fold injection of factor VIII, with a mean titer of 16 to 230 µg / ml (Fig. 8A). After Factor VIII injection, all anti-Factor VIII titers were greater than 350 µg / ml in the four remaining mice. In contrast, mice exposed to mCTLA4-Ig at Factor VIII injection had minimal or no increase in anti-Factor VIII levels (FIGS. 8B and C). Administration of mCTLA4-Ig inhibited this secondary immune response to factor VIII in mice that had already developed relatively high levels of anti-factor VIII corresponding to 590 BU inhibitor titers (Qian J., et al. Thromb. Haemost. 81: 940, 1999) (fig. .8C), as well as mice that had a minimal anti-factor VIII level of less than 5 BU in the three initial infections (FIG. 8B).

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

The roles of the B7-1 and B7-2 costimulatory ligands on antigen presenting cells were then evaluated, since it was assumed that their interaction with CD28 was prevented in experiments using mCTLA4-Ig. For this purpose, we cross-linked hemophilia A mice with Β7-Γ⁷'and B7-2'^zmice (Borriello F., et al., immunity 6: 303, 1997; Freeman GJ et al., Science 262: 907, 1993) and both factor VII and B7-1 or B7-2 deficient were selected by genotype analysis. mice. Then Α / Β7-Γ^ζ'haemophilia and A / B7-2'^/mice with haemophilia were injected intravenously with 0.2 µg of human factor VII every two weeks. Twelve days after the 2nd and 6th factor VII injections, serum samples were taken for anti-VII factor. After four injections for all nine Α / Β7-Γ⁷Haemophiliac mice (white circles) developed anti-factor VIII titers greater than 350 µg / ml and mean inhibition level 712 BU (FIG. 9), values ​​similar to otherwise normal haemophilia A mice. injected with factor VIII (Qian J., et al. Thromb. Haemost. 81: 940, 1999). In contrast, none of the eight AJB1-2¹mice with haemophilia (black circles) had no detectable levels of anti-factor VIII. Similar results were obtained with haemophilia A mice exposed to anti-B7-1 and anti-B7-2 antibodies.

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Spleen cells were harvested to assess the T cell response of these B7-1 and B7-2 deficient mice with haemophilia A three days after the fifth Factor VIII injection. 3 mouse spleen cells were mixed to obtain proliferation data. White and black circles fig. 10 respectively shows the unaffected Α / Β7-Γ⁷'and B7-2'^A cells of mice with haemophilia. White circles represent cells from A / B7-1 '^/'haemophilia mice who received 5 intravenous injections of FVIII and black circles A / B7-2'⁷· Haemophiliac mice who received 5 intravenous factor VIII injections. The concentration of factor VIII in the cultures is plotted on the horizontal axis. T cell proliferative activity as determined by³H-thymidine incorporation shows a dose-dependent factor A / III response in cells from mice with A / B7-1 * haemophilia (Fig. 10). In contrast, no T response was found with any amount of factor VIII in spleen cells from A / B7-2 '^z'in mice with haemophilia. Thus, B7-2 plays a key role in the maintenance of an immune response to intravenous factor VIII and, in its absence, does not produce anti-VI factor II.

Equivalents

Those skilled in the art should recognize, or can ascertain, using no more than conventional experimentation, that various equivalents of the particular compositions and methods described herein are possible. Such equivalents are considered to be within the scope of the present invention and are included in the following definitions.

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DEFINITION OF INVENTION