LV12768A - Methods of downmodulating the immune response to therapeutic proteins - Google Patents

Abstract

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

Description

LV 12768

METHODS OF DOWNMODULATING THE EMMUNE RESPONSE TO

THERAPEUTIC PROTEĪNS

Government Fundlng

This invention was supported, in part, by NIH grant HL 36099 awarded to the American Red Cross. The government may, therefore, have certain rights in this invention.

Background of the Invention

One of the primary limitations of therapeutic treatment using biologicai proteīns is the imraune response which the body generates in response to the presence of foreign substances in the body. This immune response is especially problematic when foreign substances must be repeatedly administered in order to be opdmaIly effective.

One example of such a situation is the repeated administration of aģents for the treatment of hemostatic disorders, such as the factor VIII deficiency diseases (e.g., classic hemophilia A and von Willebrand's disease) or factor IX deficiency, also known as hemophilia B. Classic hemophilia, hemophilia A, is an X-linked disorder which afFects 1 in 10,000 males. Von Willebrand's disease is the most common inherited bleeding disorder, occurring in as many as 1 in 800 to 1000 individuāls. Hemophilia B, also knovm as Christmas disease, occurs in roughly 1 in 100,000 males (Harrison's Principles of Internai Medicine. Isselbacher et al., eds. 13th Edition. 1994. McGraw-Hill Ν.Υ., Ν.Υ.).

Factor Vili is a 265 kD singlc chain protein which circulates in a complex with von Willebrand factor (VWF). Factor VIII is an important regulaiory protein in the blood coagulation Cascade. After activarion by tbrombin, it accelerates the rāte of factor X activation by activated factor IX (factor EXa), eventually leading to the formation of the fibrin clot The VWF molecule is an adhesive glycoprotein that plays a Central role in platelet agglutination. It serves as a carrier for factor VIII in plasma and facilitales platelet-vcssel wall interactions. VWF is made up of multiple, probably identical, subunits each of about 230 kD. VWF is synthesized in endothelial celis and megakaryocytes. Factor IX is a single-chain 55 kD procnzyme which is converted to an -2- active protease (IXa) by factor XIa or by tissue factor-VIIa complex. Activated factor IX and activated factor VIII then activate factor X.

Repeated administration of foreign proteīns can lead to an immune response to those proteīns in a recipient. In the T celi response to foreign proteīns two signāls must be provided by antigen-presenting celis (APCs) to resting T lymphocytes (Jenkins, M. and Schvvartz, R. (1987) J. Exp. Med ]65,302-319; Mueller, D.L., et al. (1990) J. Immunol. 144,3701-3709). The first signal, which confers specificity to the immune response, is transduced via the T celi receptor (TCR) following recognition of foreign antigenic peptide presented in the context of the major histocompatibility complex (MHC). The second signal, termed costimulation, inducēs T celis to proliferate and become functional (Lenschovv et al. 1996. Annu. Rev. Immunol. 14:233). Costimulation is neither antigen-specific, nor MHC restricted and is thought to be provided by one or more distinct celi surface molecules expressed by APCs (Jenkins, M.K., et al. 1988 J. Immunol. 140, 3324-3330; Linsley, P.S., et al. 1991 J. Exp. Med 173, 721-730; Gimmi, C.D., et al., 1991 Proc. Nati. Acad Sci. USA. 88, 6575-6579; Young, J.W., et al. 1992 J. Clin. Invest. 90,229-237; Koulova, L., et al. 1991 J. Exp. Med 173, 759-762; Reiser, H., etal. 1992 Proc. Nati. Acad Sci. USA. 89,271-275; van-Seventer, G_A., et al. (1990) J. Immunol. 144,4579-4586; LaSalle, J.M., et al., 1991 J. Immunol. 147,774-80; Dustin, M.I., et al., 1989 J. Exp. Med 169,503; Armitage, R.J., et al. 1992 Nature 357, 80-82; Liu, Y., et al. 1992 J. Exp. Med 175,437-445).

The CD80 (B7-1) and CD86 (B7-2) proteīns, expressed on APCs, are critical costimulatory molecules (Freeman et al. 1991. J. Exp. Med. 174:625; Freeman et al. 1989 J. Immunol. 143:2714; Aruma et al. 1993 Nature 366:76; Freeman et al. 1993. Science 262:909). B7-2 appears to be more signifieant during primary immune responses, while B7-1, which is upregulated later in the course of an immune response, may be important in prolonging primary T celi responses or costimulating secondary T celi responses (Bluestone. 1995. Immunity. 2:555). B7-1 and B7-2 are the 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 celis and inereases in expression after activation. After signaling through the T celi receptor, ligation of CD28 and transduetion of a costimulatory signal -3- LV 12768 induces T celis to proliferate and secrete IL-2 (Linsley, P.S., et al. 1991 J. Exp. Med. 173,721-730; Gimmi, C.D., et al. 1991 Proc. Nati. Acad Sci. USA. 88, 6575-6579; June, C.H., et al. 1990 Immunol. Today. 1_1,211-6; Harding, F.A., et al. 1992 Nature. 356,607-609.). The second ligand, tenned CTLA4 (CD152) is homologous to CD28 but is not «cpressed on resting T celis and appears following T celi activation (Brūnēt, J.F., et al., 1987 Nature 328,267-270). CTLA4 appears to be critical in negative regulation of T celi responses (Waterhouse et al. 1995. Science 270:985). Blockade of CTLA4 has been found to remove inhibitory signāls, while aggregation of CTLA4 has been found to provide inhibitory signāls that downregulate T celi responses (Allison and Krununel. 1995. Science 270:932). The B7 molecules have a higher af5nity for CTLA4 than for CD28 (Linsley, P.S., et al., 1991J. Exp. Med 174,561-569) and B7-1 and B7-2 have been found to bind to distinct reģions of the CTLA4 molecule and have different Iemeties of binding to CTLA4 (Linsley et al. 1994. Immunity. 1:793).

Between 10 and 25 percent of patients with hemophilia develop an immune response to Factor VIII. These patients develop inhibitors, usually IgG antibodies, which neutralize factor VIII activity and, thus, prevent effective therapy. Two types of inhibitors have been identified. High responder patients with type I inhibitors have an anam nestie response to factor VIII vvhich results in an inereased titer of antibodies to factor Vm. Low responder patients with type II inhibitor have a low antibody titer which is not inereased by administration of factor VHI. Current strategies to blunt the antibody response in these patients have only been marginally successful. Moreover, the development of antibodies to replaced proteīns is a critical problem that needs to be solved if gene therapy is to be successfiil in the treatment of hemophilias and other deficiency diseases (Connelly S et al, Blood 88:3846,1996; Kuņa S-H et al, Blood 91:784,1998).

Snmmary of the Invention

The present invention pro vides, inter alia, compositions and methods which allow the administration of a therapeutic protein to treat a disorder while reducing the development and/or progression of an immune response to the therapeutic protein. -4-

In one aspect, the invention pertains to compositions comprising a first aģent which promotes hemostasis and a second aģent which inhibits a costimulatory signal in a T celi.

In one embodiment the subject compositions further comprise a 5 phannaceutically acceptable carrier.

In one embodiment, the first aģent is factor VIII. In another embodiment, the first aģent is a B*domain deleted variant of factor VIII. In one embodiment, the first aģent is factor EX. In another embodiment, the first aģent is Von Willebrand factor.

In one embodiment, the second aģent is a soluble form of a costimulatory 10 molecule. In a preferred embodiment, the second aģent is a soluble form of CTLA4. In another preferred embodiment, the second aģent is a soluble form of B7-1, a soluble form of B7-2, or a combination of a soluble form of B7-1 and a soluble form of B7-2. In a more preferred embodiment, the second aģent is CTLA4Ig. In another more preferred embodiment, the second aģent is B7-lIg or B7-2Ig. In yet another preferred 15 embodiment, the second aģent is a soluble form of CD40 or CD40L.

In another embodiment,. the second aģent is an antibody which binds to a costimulatory molecule. In a preferred embodiment, the second aģent is selected from the group consisting of an anti-B7-l antibody, anti-B7-2 antibody, and a combination of an anti-B7-l and an anti-B7-2 antibody. In one embodiment, the antibody is a non-20 activating form of an anti-CD28 antibody.

The invention further pertains to mejhods of treating a hemostatic disorder in a subject comprising administering to a subject the instant compositions such that a hemostatic disorder is treated.

In one embodiment, the subject has a significant titer of antibodies which bind to 25 the first aģent In another embodiment the subject does not have a significant titer of antibodies which bind to the first aģent

In one embodiment the methods comprise administering a composition comprising an aģent which inhibits a costimulalory signal in a T celi.

In one embodiment the hemostatic disorder is selected from the group consisting 30 of hemophilia A, hemophilia B, and von Willebrand's disease. -5- LV 12768

In another aspect, the invention pertains to methods of treating a hemostatic disorder in a subject comprising administering to the subject a first aģent which promotes hemostasis and a second aģent which inhibits a costīmulatory signal in a T celi, such that a hemostatic disorder is treated.

In another aspect, the invention pertains to methods of treating a hemostatic disorder in a subject comprising administering to the subject a first aģent which promotes hemostasis and a second aģent which inhibits a cosfimulatoiy signal in a T celi, such that immunotolerance to the first aģent occurs thereby treating a hemostatic disorder.

In one embodiment, the first aģent is factor VIII. In another embodiment, the first aģent is a B-domai.n deleted variant of factor VIII. In another embodiment, the first aģent is factor IX. In another embodiment, the first aģent is Von Willebrand factor.

In one embodiment, the second aģent is a soluble fotm of an aģent which delivers a costimulatory signal to a T celL In a preferred embodiment, the aģent is a soluble form of CTLA4. In a more preferred embodiment, the aģent is CTLA4Ig. In another preferred embodiment, the aģent is a soluble form of B7-1, a soluble form of B7-2 or a combinaiion of both B7-1 and B7-2. In another more preferred embodiment, the aģent is B7-lIg, B7-2Ig, or a combinaiion of both B7-lIg and B7-2Ig.

In one embodiment, the second aģent is an antibody which binds to a costimulatory molecule. In another embodiment, the second aģent is selected from the group consisting of an anti-B7-l antibody, an anti-B7-2 antibody, and a combinaiion of an anti-B7-l and an anti-B7-2 antibody. In another embodiment, the antibody is a non-activating form of an anti-CD28 antibody.

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

In one embodiment, the subject has a significant titer of antibodies which bind to the first aģent

Brief Description of the Drawings

Figurē 1 illustrates the experimental design used for £xample 1 to tēst the inhibition of primary antibody responses to factor VHI. -6-

Figure 2 shows that while mice that did not receive CTLA4Ig had high titers of antibody beginning as early as day 20 (G-l), mice that received CTLA4Ig did not develop antibodies until day 82 (G-2 and G-3). 5 Figurē 3 illustrates the experimental design used for Example 2 to tēst the inhibition of secondary antibody responses to factor Vili.

Figurē 4 shows that animals that did not receive CTLA4Ig had high titers of anti-factor VIH antibodies (G-l), while the mice that received CTLA4Ig, (G-2),with the 10 exception of 1 mouse, did not develop a secondary immune response to factor VIII.

Figurē 5 shows the effect of mCTLA4-Ig on anti-factor VIII antibody formation.

Figurē 6 shows the effect of repeated administration of mCTLA4-Ig on anti-factor 15 Vin antibody formation.

Figurē 7 shows the effect of simultaneous administration of mCTLA4-Ig and factor Vm. 20 Figurē 8 shows the effect of mCTLA4-Ig on the secondary immune response to

factor VEL

Figurē 9 shows the role of B7- 1 and B7-2 in the anti-factor VHI antibody response. 25

Figurē 10 shows the T celi response to factor VIII for hemophilia A/B7-lv' and hemophilia A/B7-2V" mice.

Detailed Description

The present invention represents an important advance in the treatment of hemostadc disorders by providing compositions and methods which allow 30 -7- LV 12768 administration of a therapeutic protein to treat a disorder while reducing the development and/or progression of an immune response to the therapeutic protein.

Before further description of the invention, certain terms employed in the specification, examples and appended claims are, for convenience, collected here. 5 1. Definitions

As used herein, the language "hemostatic disorder" includes disorders which result in abnonnai bleeding and/or thrombosis. Normai hemostasis limits blood loss by a series of interactions between components of blood vessel vvalls, platelets, and plasma io proteīns. Hemostatic disorders occur, for example, due to a failure in platelet aggregation and/or fibrin clot formation which can result in inappropriate responses to disease or trauma, e.g., uncontrolled bleeding. Such disorders can be detected, e.g., by determining bleeding time, partial thromboplastin time (PTT), prothrombin time (PT), thrombin time (TT), or by a quantitative fibrinogen determination using methods well 15 known in the art Exemplary hemostatic disorders include hemophilia A, hemophilia B, and von Willebrand's disease.

As used herein, the language "aģent which promotes hemostasis" includes a protein or polypepdde which is deficient or deleted in a subject and which, when administered to the subject, ameliorates or treats a hemostatic disorder. Preferred aģents 20 which promote hemotstasis include coagulation factors such as Factor vm, Factor IX, VWF and analogs thereof.

The term ”B7 family” or “B7 molecules” as used herein includes costimulatory molecules that share amino acid sequence identity with B7 polypeptides, e.g., with B7-1, B7-2, or B7-3 (recognized by the antibody BB-1). In addition, the B7 family of 25 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 celi activation. B7 polypeptides are capable of providing costimuladon to acdvated T celis to thereby inducē T celi proliferadon and/or cytokine secretion or of inhibiting costimuladon of T celis, e.g., when present in soluble form. B7 family members include 30 B7-1, B7-2, and soluble fragments or derivatives thereof. In one embodiment, B7 family members bind to CTLA4, CD28, ICOS, and/or other ligands on immune celis and have the ability to inhibit or inducē costimuladon of immune celis. -8-

As used herein, the Ianguage "aģent which inhibits a costmmlatory signai in a T celi" includes aģents which inhibit a signai generated by the interaction of a costimulatory molecule on an antigen presenting celi (APC), e.g., a B7 family molecule and its counter receptor on a T celi. Costimulatory molecules on APCs (e.g., B7 family 5 members) and their cognate ligands on T celis (e.g., CTLA4, CD28, and ICOS) are herein collectiveIy referred to as costimulatory molecules. An aģent which inhibits a costimulatory signai can act either extracellularly to inhibit the interaction between costimulatory molecules, thus blocking the production of intiacellular signāls, or can act intracellularly to inhibit costimulatory signāls in a signai transduction pathway. 10 Exemplary aģents are described in further detail herein and include, for example, soluble fonns of costimultory molecules and antibodies which bind to costimulatory molecules.

As used herein, the phrase "downmodulation of the immune response" includes reduction in an immune response (e.g., suppression, dampening, or inhibition) in a patient that does not have an existing immune response or reduction in the length and 15 magnitude of an existing immune response. The term "immune response" includes any type of immune response which is initiated by or dependent upon costimulatory signāls, e.g., a cellular or a 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 aģent which promotes hemostasis (e.g., Factor VII, VWF, or Factor IX). The term 20 “immunotolerization” includes the induction of antigen specific tolerance which can be measured using techniques that are known in the art, e.g., by measuring secondary immune rcsponses (e.g., cellular or humoral responses) to an antigen. II. Aģents Which Promote Hemostasis 25 In one embodiment, the aģent which promotes hemostasis is factor VIII. The term "Factor VHT, as used herein, includes proteīns exhibiting procoagulant activity characteristic of factor VTH. In one embodiment of the invention, factor VIII proteīns are naturally occurring factor VIII proteīns. Such proteīns can be purified from blood or . can be administered as a blood product or an enriched blood product Inone 30 embodiment, highly purified factor VIII can be produced by adsorbing and eluting the factor from a blood product on a monoclonal antibody column. Altematively, such naturally occurring proteīns can be made recombinantly using nucleic acid molecules, -9- LV 12768 preferably naturally occumng nucleic acid molecules. For example, in one embodiment, factor Vili proteīns are made by expressing a nucleic acid molecule encoding factor VIII in a celi, using techniques known in the art, such that factor VIII protein is produced.

The nucleotide sequence (and the corresponding amino acid sequence) of human factor Vili is known in the art. (See e.g., Toole et al. Nature 1984. 312:5992; or GenBank AccessionNos. Χ01179; K01740).

In another embodiment, the aģent which promotes hemostasis is a non-naturally occumng Factor VTII, e.g., mutant form of factor VIII which rētains the therapeutic function, e.g., hemostasis promoting activity, of factor VTII. For example, DNA sequences capable of hybridizing to DNA encoding human factor VIII under conditions that avoid hybridization to non-factor VIII genes, (e.g., under conditions equivalent to 65°C in 5 X SSC (1 X SSC = 150 mM NaCl/ 0.15 M Na citrate)) or homologous DNA sequences which retain sequence identity over reģions of the nucleic acid molecule which encode protein domains which are important in factor VIII function can be used to producē factor VIII proteīns within the scope of the invention. As examples, the contents of U.S. Patents 5,744,446; 5,663,060; 5,583,209; 5,661,008; 5,422,260; and 5,707,832 are expressly incorporated herein by reference.

In another embodiment, an aģent which promotes hemostasis is a factor VIII protein in which at least one domain (e.g., a nonessential domain) of the protein has been deleted. For example, in one embodiment, a factor VIII protein is a modified factor Vm protein in which one or more amino acids have been deleted or substituted between the 90 Kd and 69 Kd cleavage sites with respect to native factor Vm, as described in greater detail in United States Patent 4,868,112, the contents of which are incorporated herein by reference.

In another embodiment, the aģent which promotes hemostasis is a factor Vm analog containing a deletion(s) of one or more amino acids between the 50/40 cleavage site and the 73 kD cleavage site which may be produced by methods analogous to those disclosed in United States Patent 4,868,112, the contents of which are hereby incorporated by this reference. In a preferred embodiment, a factor Vm analog rētains part or ali of the acidic amino acid region between the 80 kD and the 73 kD cleavage sites. In other embodiments part or ali of this region is replaced with the corresponding acidic region immediately adjacent to the 50/40 cleavage site. In stili other - 10- embodiments, factor VIII proteīns are analogs (with or vvithout deletions as mentioned above) such as are disciosed in International Application PCT/US87/01299 (the contents of vvhich are incorporated herein by reference), e.g. wherein one or more of the cleavage sites spanning arginine residues at positions 226,336,562,740,776, 1313, 1648 or 1721 5 have been rendered resistant to proteolytic cleavage, e.g., by replacement of one or more amino acids with different amino acids by mutagenesis of the cDNA using techniques knovvn in the art, e.g., Standard site directed mutagenesis. Aģents which promote hemostasis also include hybrid factor VIII proteīns which include a portion of a human factor VIII protein and a portion of a non-human factor 10 Vm protein from another species (e.g., porcine factor VIII). Such hybrid proteīns can be made using techniques which are knovvn in the art, e.g., as shovvn in U.S. Patents 5,744,446; 5,663,060; and 5,583,209.

The contents of U.S. Patents 5,693,499; 5,681,746; 5,663,060; 5,583,209; 5,563,045; 5,460,951; and 5,455,031 are also expressly incorporated herein by this 15 reference.

In another embodiment, the aģent vvhich promotes hemostasis is factor IX. As used herein, the term "factor EX" includes, but is not limited to, factor IX isolated from plasma, transformed celi lines, and recombinantly produced factor IX isolated from host celi culture medium. Factor IX can be purifred from blood or can be administered as a 20 blood product or an enriched blood product In one embodiment, highly purifred factor IX can be produced by adsorbing and eluting the factor from a blood product on a monoclonal antibody column. Exemplary methods of purificarion are also disciosed in U.S. Patents 5,639,857; 5,457,181, and 5,286,849. Altematively, such naturally occuning proteīns can be made recombinantly using nucleic acid molecules, preferably 25 naturally occurring nucleic acid molecules. For example, in certain embodiments, factor IX proteīns are made by expressing a nucleic acid molecule encoding factor IX in a celi, using techniques known in the art, such that Factor IX protein is produced. Exemplary genetic constructs for expressing factor EX can be found in U.S. Patents 5,650,503 and 4,994,371. 30 The nucleotide sequence and amino acid sequence of factor IX is knovvn in the art (See, e.g., Yoshitake et al. 1985. Biochemistry 24:3726 or GenBank Accession Nos. K02402; A07407; A01819; or Χ54500). - 11 - LV 12768

In other embodiments, factor IX includes, for example, the proteīns described in United States Patents 4,994,371; 5,171,569; 5,679,639; 5,621,039; and 5,714,583, the entire disclosures of which are each incorporated herein by reference.

In addition to naturally occurring forms of factor IX, the term factor IX also 5 includes non-naturally occurring forms, e.g., mutant forms of factor IX which retain the therapeutic, e.g., hemostasis promoting properties of factor IX. For example, DNA sequences capable of hybridizing to ONA encoding human factor IX under conditions that avoid hybridization to non-factor IX genes, (e.g., under conditions equivalent to 65°C in 5 X SSC (1 X SSC = 150 mM NaCl/ 0.15 M Na citrale)). In addition, DNA 10 sequence$ which retain sequence identity over reģions of the nucleic acid molecule which encode protein domains which are important in factor IX function can be used to producē factor IX proteīns within the scope of the invention.

Factor Vīli or factor IX proteīns can also be purchased commercially. For example, concentrated forms of factor VIII are available, e.g., Immunate® (Immuno), 15 Beriate® (Behring); monoclonal antibody purified forms of factor VIII are available e.g., Octanativ-M® (Pharmacia), Hemofil M® (Baxter), and Monoclate-P® (Armour); and recombinant forms of factor Vm are also available, e.g., Recombinate® (Baxter) and Kogenate® (Bayer). A recombinant B-domain-deleted form of factor VIII, r-VIII SQ® (Pharmacia and Upjohn, Stockholm) is also available. Factor IX can be purchased, 20 e.g., as Nanotiv® (Kabi Pharmacia) or Immunine® (Immuno); monoclonal antibody purified factor IX is also available as Mononine® (Armour). Recombinant factor IX is also available, e.g., as BeneFDC® (Genetics Institute). VWF is a large multimeric plasma protein, composed of single glycoprotein subunits. The subunits of VWF are linked together by disulfide bonds. In plasma VWF 25 circulates as multimers, ranging from dimers to multimers of more than 50 subunits.

Dimers consist of two subunits joined, probably at their C-termini, by flexible "rod-shapcd" domains and are presumed to be the protomers in multimerization. The protomers are linked through large, probably N-tenninal, globular domains to form multimers. VWF appears to be produced as a 260 kD glycosylated precursor that is 30 subsequendy processed and sulfated. After dimerization and multimerization and proteolytic cleavage, the mature protein is about 225kD. VWF has been produced recombinantly. The nucleotide and amino acid sequence of VWF is known in the art. -12- (See e.g., Sadler et al. 1986. ColdSpring Harbor Symposium irt Qunatitative Biology 51:515 or GenBank Accession Nos. L15333 or K03028). The contents of EP 0197592 B1 are incorporaled herein by reference.

In addition to naturally occuiring foims of factor VWF, the term “factor VWF 5 also includes non-naturally occurring fonns, e.g., mutant fonns of factor VWF which retain the therapeutic properties, e.g., hemostasis promoting properties of factor VWF. For example, DNA sequences capable of hybridizmg to DNA encoding human faaor VWF under conditions that avoid hybridization to non-factor VWF genes, (e.g., under conditions equivalent to 65°C in 5 X SSC (IX SSC =150 mM NaCl/ 0.15 M Na 10 citrate)). In addition, DNA sequences which retain sequence identity over reģions of the nucleic acid molecule which encode protein domains which are important in factor VWF function can be used to producē factor VWF proteins vvitbin the scope of the invention.

In one embodiment, the aģents which promote hemostasis are mammalian in origin. In a preferred embodiment, the aģents which promote hemostasis are porcine in 15 origin. In yet another more preferred embodiment, the aģents for which promote hemostasis are human in origin. In another embodiment the aģent which promotes hemostasis are hybrid moiecules. 111. Immurtomodulatory Aģents 20 In one embodiment, an aģent which inhibits a costimulatory signal in a T celi is a naturally occurring form of a costimulatory molecule. Naturally occurring fonns of costimulatory moiecules can be purified from celis or can be recombinantly produced using techniques known in the art For example, costimulatory proteins can be made by expressing a nucleic acid molecule encoding a costimulatory molecule in a celi such that 25 a costimulatoiy molecule is produced. Nucleotide sequences of costimulatory moiecules are known in the art and can be found in the literature or on a database such as GenBank. See, for exampie, B7-2 (Freeman et al. 1993 Science. 262:909 or GenBank Accession numbers P42081 or A48754); B7-1 (Freeman et al. J. Exp. Med. 1991.174:625 or GenBank Accession numbers P33681 or A45803; CTLA4 (See e.g., Ginsberg et al. 30 1985. Science. 228:1401; or GenBank Accession numbers P16410 or 291929); and CD28 (Aruffo and Seed. Proc Nati. Acad. Sci. 84:8573 or GenBank Accession number -13- LV 12768 180091), ICOS (HutlofFet al. 1999. Nature. 397:263; WO 98/38216), and related sequences.

In addition to naturally occurring fonus of costimulatory molecules, the term "costimulatory molecule" also includes non-naturally occurring fonns, e.g., mutant foims of costimulatory molecules which retain the function of a costimulatory molecule, e.g., the ability to bind to cognate counter receptor. For example, DNA sequences capable of hybridizing to DNA encoding a B7 molecule, a CTLA4 molecule, a CD28, or an ICOS molecule under conditions that avoid hybridīzation to non-costimulatory molecule genes, (e.g., under conditions equivalent to 65°C in 5 X SSC (1 X SSC = 150 mM NaCl/ 0.15 M Na citrate)) are costimulatoiy molecules within the scope of the invention. Altematively, DNA sequences which retain sequence identity over reģions of the nucleic acid molecule which encode protein domains which are important in costimulatory molecule function, e.g., binding to other costimultory molecules, can be used to producē costimulatory proteīns which can be used as aģents which inhibit a costimulatory signal in a T celi. Preferably, nonnaturally occurring costimulatory molecules have significant (e.g., greater than 70%, preferably greater than 80%, and more preferably greater than 90-95%) amino acid identity with a naturally occurring amino acid sequence of a costiinulatory molecule extracellular domain.

To determine amino acid residues of a costimulatory molecule which are likely to be important in the binding of a costimulatory molecule to its counter receptor, amino acid sequences comprising the extracellular domains of costimulatory molecules of different species, e.g., mouse and human, can be aligned and conserved (e.g., identical) residues noted. This can be done, for example, using any Standard alignment program, such as MegAlign (DNA STAR). Such conserved or identical residues are likely to be necessary for proper binding of costimulator^ molecules to their receptors and are, thus, not likely to be amenable to alteration.

Specific residues of costimultory molecules which are important in binding have also been determined. For example, the portion of CD28 which is critical for interaction with B7-1 and B7-2 has been determined using site diiected mutagenesis, CD28 monoclonal antibody epitope mapping, receptor based adhesion assays, and direct binding of Ig-fusion proteīns to celi surface receptors. A stretch of proline rich sequence in CD28, ΜΥΡΡΡΥ, has been found to be critical to the function of that protein (Truneh -14- et al. 1996. Mol. Immunol. 33:321). Likewise, the reģions ofthe B7-1 molecule which are important in mediating the functional interaction with CD28 and CTLA4 have been identified by mutation. Two hydrophobic residues in the V-like domain of B7-1, including the Υ87 residue, which is conserved in ali B7-1 and B7-2 molecules cloned 5 from various species, were found to be critical (Fargeas et al. 1995. J. Exp. Med 182:667). Using these, or similar, techniques amino acid residues of the extracellular domains of costimulatory molecules which are critical and, therefore, not amenable to alteration can be determined.

Costimulatory molecules can be expressed in soluble form or used as 10 immunogens to make antibodies. Such soluble costimulatory molecules or antibodies are useful as aģents which inhibit a costimulatory signal in a T celi as described in further detail herein. A. Aģents Which Act Extracellularly To Inhibit A Costimulatory Signal InAT Celi 15 1. Soluble forms of costimulatory molecules

In one embodiment, the aģent which blocks a costimulatory signal in a T celi is a soluble form of a T celi costimulatory molecule (e.g., CTLA4, CD28, and/or ICOS) which is capable of blocking the transduction of a costimulatory signal in a T celi.

In one embodiment, the aģent which blocks a costimulatoiy signal in a T celi is a 20 soluble form of CTLA4. DNA sequences encoding the human and murine CTLA4 protein are known in the art, see e.g., Dariavich, et al. (1988) Eur. J. Immunol. 18(12), 1901-1905; Bnmet, J.F., et al. (1987) supra; Brūnēt, JJ7. et al. (1988) Immunol. Rev. 103:21-36; and Frecman, GJ., et al. (1992) J. Immunol. 149,3795-3801. In certain embodiments, the soluble CTLA4 protein comprises the entire CTLA4 protein. In 25 preferred embodiments, a soluble CTLA4 protein comprises the extracellular domain of a CTLA4 protein. For example, a soluble, recombinant form of the extracellular domain of CTLA4 has been expressed in yeast (Gerstmayer et al. 1997. FEBSLett. 407:63). In other embodiments, the soluble CTLA4 proteins comprise at least a portion of the extracellular domain of CTLA4 protein which rētains the ability to bind to B7-1 and/or 30 B7-2. - 15- LV 12768

In one embodiment the soluble CTLA4 protein or portion thereof is a fusion protein comprising at least a portion of CTLA4 which binds to B 7-1 and/or B7-2 and at least a portion of a second non-CTLA4 protein. In preferred embodiments, the CTLA4 fusion protein comprises a CTLA4 extracellular domain which is fused at the amino terminus to a signal peptide, e.g., from oncostatin M (see e.g., W093/00431).

In a particularly preferred embodiment, a soluble form of CTLA4 is a fusion protein comprising the extracellular domain of CTLA4 fused to a portion of an immunoglobulin molecule. Such a fusion protein, CTLA4Ig, can be made using methods known in the art (see e.g., Linsley 1994. Perspectives in Drug Discovery and Design 2:221; Linsley WO 93/00431 and U.S. Patent 5,770,197).

In one embodiment, the aģent which blocks a costimulatory signal in a T celi is a soluble form an antigen presenting celi costimulatory molecule (e.g., a B7 family molecule, such as B7-1, B7-2 and/or an ICOS ligand. For example, in one embodiment, a soluble form of a costimuiatory molecule includes a soluble form of B7-1 or a soluble form of B7-2 or a combination of a soluble form of B7-1 and a soluble form of B7-2. DNA sequences encoding B7 proteīns are known in the art, see e.g., B7-2 (Freeman et al. 1993 Science. 262:909 or GenBank Accession numbers P42081 or A48754); B7-1 (Freeman et al. J. Exp. Med. 1991.174:625 or GenBank Accession numbers P33681 or A45803. In cērtam embodiments, the soluble B7 protein comprises an entire B7 protein. In preferred embodiments, a soluble B7 protein comprises the extracellular domain of a B7 protein. For example, a soluble, recombinant form of the extracellular domain of CTLA4 has been expressed in yeast (Gerstmayer et al. 1997. FEBS Leti. 407:63). In other embodiments, the soluble B7 proteīns comprise at least a portion of the extracellular domain of B7 protein which rētains the 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 which binds to CD28 and/or CTLA4 and at least a portion of a second non-B7 protein. In preferred embodiments, the B7 fusion protein comprises a B7 extracellular domain which is fused at the amino terminus to a signal peptide, e.g., from oncostatin M (see e.g., W093/00431). -16-

In a particularly more preferred embodiment, a soluble fonn of B7 is a fusion protein comprising the extracellular domain of B7 fiised to a portion of an immunoglobulin molecule. Such a fusion protein, a B7Ig, can be made using methods known in the art (see e.g., Linsley 1994. Perspectives in Drug Discovery and Design 5 2:221; Linsley WO 93/00431, U.S. Patent 5,770,197, and U.S. Patent 5,580,756). 2. Antibodies which bind to costimulatory molecules

In certain embodiments, the aģent which blocks a costimulatory signal in a T celi is an antibody which binds to a costimulatory molecule. In making antibodies which 10 bind to costimulatory molecules, a costimulatory protein, a portion of a costimulatory protein, (e.g., a peptide derived frora a costimulatory protein), or fusion protein which includes ali or a portion of an amino acid sequence of a costimulatory molecule can be used to generate anti-protein and/or anti-peptide polydonal antisera or monoclonal antibodies using Standard methods. The term Mantibody” as used herein is meant to 15 include whoIe antibodies as well as fragments thereof. Fragments of antibodies (e.g., Fab' fragments, F(ab')2 fragments, or single chain antibodies) can be made using methods well known in the art. The term antibody also includes chimeric and humanized antibodies.

For example, a mammai, (e.g., a mouse, hamster, or rabbit) can be immunized 20 with an immunogenic fonn of the costimulatory protein or peptide which elicits an antibody response in the mammai. The immunogen can be, for example, a recombinant costimulatory molecule protein, or fragment thereof, a synthetic peptide fragment or a celi that expresses a costimulatory molectile on its surface. The celi can be, for example, an antigen presenting celi, or a T celi, or a celi transfected with a nucleic acid encoding a 25 costimulatory molecule such that the costimulatory molecule is expressed on the celi surface. Host celis transfected to express peptides can be any procaryotic or eucaryotic celi. For example, a peptide having costimulatory molecule activity can be expressed in bacterial celis such as E. coli, insect celis (baculovirus), yeast, or mammalian celis such as Chinese hamster ovary celis (CHO) and NS0 celis. Other suitable host celis and 30 expression vectors may be found in Goeddel, (1990) supra or are known to those skilled in the art Examples of vectors for expression in yeast S. cerivisae include pYepSecl (Baldari. ct ak, (1987) Embo J. 6:229-234), pMFa (Kuijan and Herskowitz, (1982) Celi -17- LV 12768 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, CA). Baculovirus vectors available for expression of proteīns in cultured insect celis (SF 9 celis) include the pAc series (Smith et al·, (1983) Mol. Celi Biol. 3:2156-2165) and the pVL series (Lucklow, V.A., and Summers, M.D., (1989) 5 Virology 170:31-39). Generally, COS celis (Gluzman, Y., (1981) Celi 23:175-182) are used in conjunction with such vectors as pCDM8 (Seed, B., (1987) Nature 329:840) for transient amplification/expression in mammalian celis, while CHO (dhfr* Chinese Hamster Ovary) celis are used with vectors such as pMT2PC (Kaufinan et al· (1987), EMBO J. 6:187-195) for stable amplification/exprcssion in mammalian celis. A 10 prefeired celi line for production of recombinant protein is the NSO myeloma celi line available from the ECACC (catalog #85110503) and described in Galfre,G. and Milstein, C. ((1981) Methods in Enzymology 73( 13):3-46; and Preparation of Monoclonal Antibodies: Strategies and Procedures, Academic Press, Ν.Υ., Ν.Υ). Vector DNA can be introduced into mammalian celis via conventional techniques such 15 as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofectin, or electroporation. Suitable methods for transforming host celis can be found in Sambrook et al· (Molecular Cloning: A Labora.tory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks. When used in mammalian celis, the expression vector's control functions are often 20 provided by virai material. For example, commonly used promoters are derived firom polyoma, Adenovirus 2, cytomegalovirus and most frequently, Simian Vīrus 40.

Peptides having an activity of a costhnulalory molecule expressed in mammalian celis or othenvise can be purified according to Standard procedures of the art, including ammonium sulfate precipitaiion, fractionation column chromatography (e.g. ion 25 exchange, gel filtration, electrophoresis, affinity chromatography, etc.) and ultimately, crystallization (see generally, "Enzyme Purification and Related Techniques", Methods in Enzymology, 22:233-577 (1971)).

It will be appreciated by those skilled in the art that it is within their skill to generate antibodies to human costimulatory molecules by following Standard techniques. 30 Antibodies may either be polycIonal or monoclonal antibodies, or antigen binding fragments of such antibodies. Of particular significance for use in therapeutic applications are antibodies that inhibit binding of a costimulalory molecule with its - 18- natural ligand(s) on the surface of immune celis, thereby inhibiting coslimulation of the immune celi. Prefeired anti-costimuIatory molecule antibodies are those capable of inhibiting or downregulating T celi mediated immune responses by binding B7-2 or B7-1 on the surface of B lymphocytes and preventing interaction with CTLA4 and/or CD28. 5 Other preferred anti-costimulatory molecule antibodies are those which, in combination with a second antibody which binds to another costimulatory molecule, results in increased inhibition of costimulation of a T celi when compared to the first antibody alone, e.g., a combination of anti-B7-l and anti-B7-2 antibodies. A. The Immunogen. The tenn "immunogen" is used herein to describe a 10 composition containing a peptide having an activity of a costimulatory molecule as an active ingredient used for the preparation of antibodies against a costimulatory molecule. When a peptide having a costimulatory molecule activity is used to inducē antibodies it is to be understood that the peptide can be used alone, or linked to a carrier as a conjugate, or as a peptide polymer. 15 To generate suitable anti-costimulatory molecule antibodies, the immunogen should contain an effective, immunogenic amount of a peptide having a costimulatory molecule activity, typically as a conjugate linked to a carrier. The effective amount of peptide per unit dose depends, among other things, on the species of animal inoculated, the body weight of the animal and the chosen immunization regimen as is well known in 20 the art The immunogen preparation will typically contain peptide concentrations of about 10 micrograms to about 500 milligrams per immunization dose, preferably about 50 micrograms to about 50 milligrams per dose. An immunization preparation can also include an adjuvant as part of the diluent Adjuvants such as complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) and alum are materiāls well known in the 25 art, and are available commercially from several sources.

Those skilled in the art will appreciate that, instead of using naturally occurring forms of a costimulatory molecule for immunization, synthetic peptides can aiternatively be employed towards which antibodies can be raised for use this invention. Both soluble . and membrane bound costimulatory molecule or peptide fragments are suitable for use 30 as an immunogen and can also be isolated by immunoaffinity purification as well. A purified form of a costimulatory molecule protein, such as may be isolated as described above or as known in the art, can itseif be directly used as an immunogen, or -19- LV 12768 altematively, can be linked to a suitable carrier protein by conventional techniques, including by Chemical coupling means as well as by genetic engineering using a cloned gene of the a costimulatory molectiie.

The peptide or protein chosen for immunization can be modified to inciease its 5 immunogenicity. For example, techniques for conferring immunogenicity on a peptide include conjugation to carriers or other techniques well known in the art. Any peptide chosen for immunization can also be synthesized. In certain embodiments, such peptides can be synthesized as bnnched polypeptides, to enhance immune responses, as is knovvn in the art (see, e.g., Peptides. Edited by Bemd Gutte Academic Press 1995. pp. 10 456-493).

The purified costimulatory molecule protein can also be covalently or noncovalently modified with non-proteinaceous materiāls such as lipids or carbohydrates to enhance immunogenecity or solubility. Altematively, a purified costimulatory molecule protein can be coupled vvith or incorporated into a virai particle, l S a replicating virus, or other microorganism in order to enhance immunogenicity. The costimulatory molecule protein may be, for example, chemically attached to the virai particle or microorganism or an immunogenic portion thereof.

In an illustrative embodiment, a purified costimulatory molecule protein, or a peptide fragment having a costimulatory molecule activity (e.g., produccd by limited 20 proteolysis or recombinant DNA techniques) is conjugated to a cairier which is immunogenic in animals. Preferred carriers include proteins such as albumin, serum proteins (e.g., globulins and lipoproteins), and polyamino acids. Examples of useful proteins include bovine serum albumin, rabbit serum albumin, thyroglobulin, keyhole limpet hemocyanin, egg ovalbumin and bovine gamma-globulins. Synthetic polyamino 25 acids such as polylysine or polyarginine are also useful carriers. With respect to the covalent attachment of a costimulatory molecule protein or peptide fragments to a suitable immunogenic carrier, there are a number of Chemical cross-linking aģents that are known to those skilled in the art Preferred cross-linking aģents are heterobifunctional cross-linkers, which can be used to link proteins in a stepwise 30 manner. A wide variety of heterobifunctional cross-linkers are known in the art, including succinimidyl 4-(N-maleimidomethyl) cyclohexane- 1 -carboxylatc (SMCC), m-Maleimidobenzoyl-N- hydroxysuccinimide ester (MBS); N-succinimidyl (4- -20- iodoacetyl) aminobenzoate (SIAB), succinimidyl 4-(p-maleimidophenyl) butyrate (SMPB), l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC); 4-succinimidyloxycarbonyl- a-methyl-a-(2-pyridyldithio)-tolune (SMPT), N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), succinimidyl 6-[3-(2-pyridyIdithio) propionate] 5 hexanoate (LC-SPDP).

In may also be desirable to simply immunize with whole celis which express a costimulatory molecule protein on their surface. Various celi lines can be used as immunogens to generate monoclonal antibodies to a costimulatory molecule antigen, including, but not limited to activated B celis. For example, splenic B celis can be 10 obtained from a subject and activated with anti-immunoglobulin. Altematively, a B celi Iine can be used, provided that a costimulatory molecule is expressed on the celi surface, such as the Rāji celi line (B celi Buikett's lymphoma, see e.g., Freeman, G.J. et al. (1993) Science 262:909-911) or the JY B lymphoblastoid celi line (see e.g., Azuma, M. et al. (1993) Nature 366:76-79). Whole celis that can be used as immunogens to 15 producē costimulatory molecule specific antibodies also include recombinant transfectants. For example, COS and CHO celis can be reconstituted by transfection with a costimulatory molecule cDNA, such as described by Knudson et al. (1993, PNAS 90:4003-4007); Travemor et al. (1993, Immunogenitics 37:474-477); Dougherty et al. (1991, JExp Med 174:1-5); and Aruffo et al. (1990, Celi 61:1303-1313), to producē 20 intact costimulatory molecule on the celi surface. These transfectant celis can then be used as immunogen to producē anti-costimulatory molecule antibodies of preselected specificity. Other examples of transfectant celis are knovvn, particularly eukaryotic celis able to glycosylate the costimulatoiy molecule protein, but any procedure that works to express transfected costimulatory molecule genes on the celi surface could be used to 25 producē the whole celi immunogen. B. Polyclonal Anti-Costimulatory Molecule Antibodies.

Polycolonal antibodies to a purified costimulatory molecule protein or peptide having a costimulatory molecule activity can generally be raised in animals by multiple 30 subcutaneous (sc) or intraperitoneal (ip) injections of a costimulatory molecule immunogen, such as the extracellular domain of a costimulatory molecule protein, and an adjuvant For example, as described above, it may be useful to conjugate a -21 - LV 12768 costimulatory molecule (including fragments containing particular eptitope(s) of interest) to a protein that is immunogenīc in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin.

The route and schedule of the host animal or cultured antibody-producing celis 5 therefrom can generally make use of established and conventional techniques for antibody stimulation and production. In an illustrative embodiment, animals are typically immunized against the immunogenic costimulatory molecule conjugates or derivatives by combining about lpg to lmg of conjugate with Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the 10 animals are boosted with 1/5 to 1/10 the original amount of conjugate in Freund's complete adjuvant (or other suitable adjuvant) by subcutaneous injection at multiple sites. Seven to 14 days later, the animals are bled and the serum is assayed for anti-costimulatory molecule titer. Animals are boosted until the titer plateaus. Preferably, the animal is boosted with the conjugate of the same costimulatory molecule protein, but 15 conjugated to a different protein and/or through a different cross-linking aģent Conjugates also can be made in recombinant celi culture as protein fusions. Also, aggregating aģents such as alum can be used to enhance the immune response.

Such mammai-prnduced populations of antibody molecules are referred to as ”poiycional" because the population comprises antibodies with differing 20 immunospecificities and affinities for a costimulatory molecule. The antibody molecules are then collected from the mammai and isolated by well known techniques such as, for example, by using DEAE Sephadex to obtain the IgG fraction. To enhance the specificity of the antibody, the antibodies may be purified by immunoa£ELnity chromatography using solid phase-affixed immunogen. The antibody is contacted vvith 25 the solid phase-afSxed immunogen for a period of time sufficient for the immunogen to immunoreact vvith the antibody molecules to form a solid phase-af5xed immunocomplex. The bound antibodies are separated from the complex by Standard techniques. 30 C. Monoclonal Anti-Costimulatory Molecule Antibodies. Thetenn "monoclonal antibody" or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding -22- site capable of iirununoreacting with a particular epitope of a costimulatory molecule. A monoclonai antibody composition thus typically displays a single binding afSnity for a particular costimulatory molecule protein with which it immunoreacts. Preferably, the monoclonai antibody used in the subject method is further characterized as 5 immunoreacting with a costimulatory molecule derived from humāns.

Monoclonai antibodies useful in the compositions and methods of the invention are directed to an epitope of a costimulatory molecule antigen, such that complex formation between the antibody and the costimulatory molecule antigen inhibits interaction of the costimulatory molecule with its natūrai ligand(s) on the surface of 10 immune celis, thereby inhibiting costimulation of a T celi through the costimulatory molecule-ligand interaction. A monoclonai antibody to an epitope of a costimulatory molecule can be prepared by using a technique which provides for the production of antibody molecules by continuous celi lines in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975, Nature 15 256:495-497), and the more recent human B celi hybridoma technique (Kozbor et al. (1983) Immunol Today 4:72), EBV-hybridoma technique (Cole et al. (1985),

Monoclonai Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96), and trioma techniques. Other methods which can effectively yield monoclonai antibodies useful in the present invention include phage display techniques (Marks et al. (1992) JBiol Chem 20 16007-16010).

In one embodiment, the antibody preparation applied in the subject method is a monoclonai antibody produced by a hybridoma celi line. Hybridoma fusion techniques 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) JBiol Chem 255:4980-25 83; Yeh et al. (1976) PNAS 76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75).

Thus, the monoclonai antibody compositions of the present invention can be produced by the following method, which comprises the steps of: (a) Immunizing an animal with a cosdmulatory molecule. The immunization is typically accomplished by administering a costimulatory molecule immunogen to an 30 immunologically competent mammai in an immunologically effective amount, i.e., an amount sufficient to producē an immune response. Preferably, the mammai is a rodent such as a rabbit, rat or mouse. The mammai is then maintained for a time peri od -23- LV 12768 sufīicient for the mammai to producē celis secreting antibody molecules that immunoreact with the costimulatory molecule immunogen. Such immunoreaction is detected by screening the antibody molecules so produced for immunoreactivity with a preparation of the immunogen protein. Optionally, it may be desired to screen the 5 antibody molecules with a preparation of the protein in the form in which it is to be detected by the antibody molecules in an assay, e.g., a membrane-associated form of a costimulatory molecule. These screening methods are well known to those of skili in the art (b) A suspension of antibody-producing celis removed from each immunized 10 mammai secreting the desired antibody is then prepared. After a sufficient time, the mouse is sacrificed and somatic antibody-producing lymphocytes are obtained. Antibody-producing celis may be derived from the lymph nodēs, spleens and peripheral blood of primed animals. Spleen celis are preferred, and can be mechanically separated into individual celis in a physiologically tolerable medium using methods well known in IS the art. Mouse lymphocytes give a higher percentage of stable fusions with the mouse myelomas described below. Rat, rabbit and frog somatic celis can also be used. The spleen celi chromosomes encoding desired immunoglobulins are immortalized by fusing the spleen celis with myeloma celis, generally in the presence of a fusing aģent such as polyethylene glycol (PEG). Any of a number of myeloma celi lines may be used as a 20 fusion partner according to Standard techniques; for example, the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/0-AgI4 myeloma lines. These myeloma lines are available from the American Type Culture Collection (ATCC), Rockville, Md.

The resulting celis, which include the desired hybridomas, are then grown in a selective medium, such as HAT medium, in which unfiised parental myeloma or 25 lymphocyte celis eventually die. Only the hybridoma celis survive and can be grovvn under limiting dilution conditions to obtain isolated clones. The supematants of the hybridomas are screened for the presence of antibody of the desired specificity, e.g., by immunoassay techniques using the antigen that has been used for immunization.

Positive clones can then be subcloned under limiting dilution conditions and the 30 monoclonal antibody produced can be isolated. Various conventional methods exist for isolation and purifrcation of the monoclonal antibodies so as to free them from other proteīns and other contaminants. Commonly used methods for purifymg monoclonal -24- antibodies include ammonium sulfate precipitation, ion excbange chromatography, and affinity chromatography (see, e.g., Zola et al. in Monoclonal Hybridoma Andbodics: Technigues And Applications, Hurell (ed.) pp. 51-52 (CRC Press 1982)). Hybridomas produced according to these methods can be propagated in vitro or in vivo (in ascites 5 fluid) using techniques known inthe art

Generally, the individual celi line may be propagated in vitro, for example in laboratory culture vessels, and the culture medium containing high concentrations of a single specific monoclonal antibody can be harvested by decantation, filtration or centrifugation. Altematively, the yield of monoclonal antibody can be enhanced by 10 injecting a sample of the hybridoma into a histocompatible animal of the type used to provide the somatic and myeloma celis for the original fusion. Tumors secreting the specific monoclonal antibody produced by the fused celi hybrid develop in the injected animal. The body fluids of the animal, such as ascites fluid or serum, provide monoclonal antibodies in high concentrations. When human hybridomas or EBV-15 hybridomas are used, it is necessary to avoid rejection of the xenograft injected into animals such as mice. Immunodeficient or nude mice may be used or the hybridoma may be passaged first into irradiated nude mice as a solid subcutaneous tumor, cultured in vitro and then injected intraperitoneally into pristane primed, irradiated nude mice which develop ascites tumors secreting large amounts of specific human monoclonal 20 antibodies.

Media and animals usefiil for the preparation of these compositions are both well known inthe art and commercially available and include synthetic culture media, inbred mice and the like. An exemplary synthetic medium is Dulbecco's minimal essential medium (DMEM; Dulbecco et al. (1959) Virol 8:396) supplemented with 4.5 gm/1 25 glucose, 20 mM glutamine, and 20% fetal caf serum. An exemplary inbred mouse strain is the Balb/c. D. Humanized Anti-Costimulatory Molecule Antibodies. When antibodies produced in non-human subjects are used therapeutically in humāns, they are 30 recognized to varying degrees as foreign and an immunc response may be generated in the patient One approach for minimizing or eliminating this problem, which is preferable to general immunosuppression, is to producē chimeric antibody derivatives, -25- LV 12768 i.e., antibody molecules that combine a non-human animai variabie region and a human constant region. Such antibodies are the equivalents of the monoclonal and polyclonal antibodies described above, but may be less immunogenic when administered to humāns, and therefore more likely to be tolerated by the patient S Chimeric mouse-human monoclonal antibodies (i.e., chimeric antibodies) reactive with a costimulatory molecule can be produced, for example, by techniques recently developed for the production of chimeric antibodies. Humanized antibodies may be produced, for instance, by replacing an immunogenic portion of an antibody with a corresponding, but non-immunogenic portion. Accordingly, genes encoding the 10 constant reģions of the murine (or other species) anti-costimulatory molecule antibody molecule are substituted with genes encoding human constant reģions. (Robinson et al., International Patent Publication PCT/US86/02269; Akira, et aL, European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Moirison et al., European Patent Application 173,494; Neuberger et al., PCT Application WO 15 86/01533; Cabilly 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. Nati Ccmcer Inst. 80:1553-1559). General reviews of "humanized" chimeric 20 antibodies are provided by Monison, S. L. (1985) Science 229:1202-1207 and by Oi et al. (1986) BioTechniques 4:214. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode ali or part of an immunoglobulin variabie region from at least one of a heavy or light chain. Sources of such nucleic acid are well known to those skilled in the art and, for example, may be obtained from an 25 anti-costimulatory molecule antibody producing hybridoma. The chimeric cDNA can then be cloned into an appropriate expression vector.

Suitable "humanized" antibodies can be altematively produced by CDR or CEA substitution (The Winter U.S. Patent 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol 30 141:4053-4060). -26- E. Combinatorial Anti-Costiraulatory Molecule Antibodies. Both monoclonal and polyclonal antibody compositions of the invention can also be produced by other methods well knovvn to those skilled in the art of recombinant DNA technology. An altemative method, referred to as the "combinatorial antibody display" s method, has been developed to identify and isolate antibody fragments having a particular antigen specificity, and can be utilized to producē monoclonal anti-costimulatory molecule antibodies, as well as a polyclonal anti-costimulatory molecule population (Sastry et ai. (1989) PNAS 86:5728; Huse et al. (1989) Science 246:1275; and Orlandi et al. (1989) PNAS 86:3833). After immunizing an animal with a costimulatory 10 molecule immunogen as described above, the antibody repertoire of the resulting B-cell pool is cloned. Methods are generally knovvn for directly obtaining the DNA sequence of the variable reģions of a diverse population of immunoglobulin molecules by using a mixture of oligomer primers and PCR. For instance, mixed oligonucleotide primers corresponding to the 5' leader (signal peptide) sequences and/or framework 1 (FR1) 15 sequences, as well as primer to a conserved 3' constant region primer can be used for PCR amplification of the heavy and light chain variable reģions from a number of murine antibodies (Larrick et al. (1991) Biotechniques 11:152-156). A similar strategy can also been used to amplify human heavy and light chain variable reģions from human antibodies (Larrick et al. (1991) Methods: Companion to Methods in Enzymology 2:106-20 110). The ability to clone human immunoglobulin V-genes takes on special significance in light of advancements in creating human antibody repertoires in transgenic arti mals (see, for example, Bruggeman et al. (1993) Year Immunol 7:33-40; Tuaillon et al. (1993) PNAS 90:3720-3724; Bruggeman et al. (1991) EurJ Immunol 21:1323-1326; and Wood et al. PCT publication WO 91/00906). 25 In an illustrative embodiment, RNA is isolated from activated B celis of, for example, peripheral blood celis, bone marrow, or spleen prepararions, using Standard protocols (e.g., U.S. PatentNo. 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 primers specific for the constant region 30 of the heavy chain(s) and each of the κ and λ light chains, as well as primers for the signal sequence. Using variable region PCR primers, the variable reģions of both heavy and light chains are amplified, each alone or in combinantion, and ligated into -27- LV 12768 appropriate vectors for further manipulation in generating the display packages. Oligonucleotide primers usefiil in amplification protocols may be unique or degenerate or incorporate inosine at degenerate positions. Restriction endonuclease recognition sequences may also be incorporated into the primers to allow for the cloning of the 5 amplified fragment into a vector in a predetermined reading frame for expression.

The V-gene library cloned from the immunization-derived antibody repertoire can be expressed by a population of display packages, preferably derived ffom filamentous phage, to form an antibody display library. Ideally, the display package comprises a system that allows the sampling of very large variegated antibody display 10 libraries, rapid sorting after each affinity separation round, and easy isolation of the antibody gene from purified display packages. In addition to commercialIy available kits for generating phage display libraries (e.g., the Phannacia Recombinant Phage Antibody System, catalog no. 27-9400-01; and the Stratagene SurfZAP™. phage display kit, catalog no. 240612), examples of methods and reaģents particularly amenable for 15 use in generating a variegated anti-costimulatory molecule antibody display library can be found in, for example, the Ladner et al. U.S. Patent No. 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 WO 92/20791; the Markland et al. International Publication No. WO 92/15679; the Breitling et al. 20 International Publication 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 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85;

Huse et al. (1989) Science 246:1275-1281; Griffihs et al. (1993) EMBOJ 12:725-734; 25 Hawkins et al. (1992) JMol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) NucAcidRes 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982.

In certain embodiments, the V region domains of heavy and light chains can be 30 expressed on the same polypeptide, joined by a flexible linker to form a single-chain Fv fragment, and the scFV gene subsequently cloned into the desired cxpression vector or phage genome. As generally described in McCa£ferty et al., Nature (1990) 348:552-554, -28- complete Vjļ and Vl domains of an antibody, joined by a flexible (Gly4-Ser)3 linker can be used to producē a single chain antibody which can render the display package separable based on antigen af5nity. Isolated scFV antibodies immunoreactive with a costimulatory molecule can subsequently be formulated into a pharmaceutical 5 prcpararion for use in the subject method. F. Hybridomas and Methods of Preparation. Hybridomas useful in the present invention are those characterized as having the capacity to producē a monoclonal antibody which will specifically immunoreact with a costimulatory molecule. As 10 described below, the hybridoma celi producing anti-costiinulatory molecule antibody can be directly implanted into the recipient animal in order to provide a constant source of antibody. The use of immuno-isolatory devices to encapsulate the hybridoma culture can prevent immunogenic response against the implanted celis, as vvell as prevent unchecked proliferation of the hybridoma celi in an immunocompromised host A 15 prefened hybridoma of the present invention is characterized as producing antibody molecules that specifically immunoreact with a costimulatory molecule expressed on the celi surfaces of activated human B celis.

Methods for generating hybridomas that producē, e.g., secrete, antibody molecules having a desired immunospecificity, i.e., having the ability to bind to a 20 particular costimulatory molecule, and/or an identifiable epitope of a costimulatoiy molecule, are well known in the art Particularly applicable is the hybridoma technology described by Niman et al. (1983) PNAS 80:4949-4953; and by Galfre et al. (1981) Metk Enzymol. 73:3-46.

In another exemplary method, transgenic mice carrying human antibody 25 repertoires can be immunized with a human costimultory molecule. Splenocytes from these immunized transgenic mice can then be used to create hybridomas that secrete human monoclonal antibodies specifically reactive with a human costimultory molecule (see, e.g., Wood et al. PCT publication WO 91/00906, Kucherlapati et al. PCT publication WO 91/10741; Lonberg et al. PCT publication WO 92/03918; Kay et al. 30 PCT publication 92/03917; Lonberg, N. et al. (1994) Nature 368:856-859; Green, L.L. et al. (1994) Nature Genet. 7:13-21; Morrison, S.L. et al. (1994) Proc. Nati. Acad. Sci. USA 81:6851-6855; Bruggeman et al. (1993) Year Immunol 7:33-40; Tuaillon et al. -29- LV 12768 (1993) PNAS 90:3720-3724; and Bruggeman et al. (1991) EurJImmmol 21:1323-1326).

The term “antibody” as used herein is intended to include fragments thereof which are also specifically reactive with a costimulatory molecule as described herein. S Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, Fiab^ fragments can be generated by treating antibody with pepsin. The resniting

Fiab^ fragment can be treated to reduce disulfide bridges to producē Fab' fragments. Antibodies made using these or other methods can be tested to determine 10 whether they inhibit a costimulatory signal in a T celi using the methods described below.

In one embodiment the aģent that inhibits a costimulatory signal in a T celi is an antibody which binds to both B7-1 and B7-2. In making such an antibody, for example, portions of the etfracellular domain which are conserved between the two costimulatory 15 molecules can be used as the immunogen. See, e.g., Metzler et al. 1997 Nat Struct.

Biol. 4:527).

In one embodiment, the aģent which inhibits a costimulatory signal in a T celi is an antibody which binds to B7-1. Such antibodies are known in the art or can be made as set forth above using a B7-1 molecule or a portion thereof as an immunogen and 20 screened using the methods set forth above or other Standard methods. Examples of B7-1 antibodies include those taught in U.S. Patent 5,747,034 and in McHugh et al. 1998. Clin. Immtmol. Immunopathol. 87:50 or Rugtveit et al. 1997. Clin Exp. Immunol 110:104.

In another embodiment, the aģent which inhibits a costimulatory signal in a T 25 celi is an antibody which binds to B7-2. Such antibodies are known in the art or can be made as set forth above using a B7-2 molecule or a portion thereof as an immunogen and screened using the methods set forth above or other Standard methods. Examples of B7-2 antibodies include those taught in Rugtveit et al. 1997. Clin Exp. Immunol 110:104. 30 In one embodiment, the aģent which blocks a costimulatory signal in a T celi is a combination of an antibody which binds to B7-1 and an antibody which binds to B7-2. -30-

In yet other embodiments, the aģent which inhibits a costimulatory signal in a T celi is an antibody which binds to CD28, but does not transduce a costimulatory signal to a T celi (e.g., an Fab fragment of an anti CD28 antibody). Such antibodies are known in the art or can be made as set forth above using a CD28 molecule or a portion thereof 5 as an immunogen and screened using the methods set forth above or other Standard methods. Examples of knovm anti-CD28 antibodies include those taught by Darling et al. 1997. Gene Ther. 4:1350.

In preferred embodiments, Fab fragments of an antibody vvhich binds to CD28 can be used. Such antibody fragments, which are unable to crosslink CD28 on the io surface of a T celi, have been found to block T celi costimulation (Walunas et al. 1994. Immunity 1:405).

In yet other embodiments, the aģent vvhich inhibits a costimulatory signal in a T celi is an antibody vvhich binds to CTLA4, which blocks a costimulatory signal in a T celi by delivering a negative signal to the T celi (i.e., vvhich is a CTLA4 agonist). For 15 example, crosslinking CTLA4 on the surface of a T celi has been shovvn to inhibit proliferation and IL-2 production (Krummel and Allison. 1996. J. Exp. Med 183:2533). Such antibodies can be made as set forth above using a CTLA4 molecule or a portion thereof as an immunogen and screened using the methods set forth above or other Standard methods. Exemplary antibodies are also taught e.g., in Vandenborre et al. 20 1998. Am. J. Pathol. 152:963.

In yet another embodiment, the aģent vvhich inhibits a costimulatory signal in a T celi is an antibody vvhich binds to ICOS, vvhich blocks a costimulatory signal in a T celi. Such antibodies can be made as set forth above using an ICOS molecule or a portion thereof as an immunogen and screened using the methods set forth above or other 25 Standard methods. IV. Aģents That Regulate The Expression Of Costimulatoty Molecules

In another embodiment, an aģent that inhibits a costunulatory signal in a T celi is an aģent that interferes with the expression of a cositmulatory molecule. For example, 30 interactions betvveen CD40 on antigen presenting celis and CD40 ligand (CD40L) on T celis have been found to be important in sustaining, enhancing, or prolonging the expression of Β7Ί or B7-2 on antigen presenting celis, resulting in enhanced -31- LV 12768 costimulation (Van Gool, et ai. 1996. Immunol. Rev. 153:47; Kļaus etal. 1994. J. Immunol. 152:5643).

In one embodiment, the aģent which blocks the expression of a costimulatory molecule, thus blocking a costimulatory signal in a T celi is a soluble fonn of CD40 or 5 CD40L. DNA sequences encoding these CD40 and CD40L are known in the art, see e.g., GenBank AccessionNos. Υ10507 or Stamenlovic et al. 1988. EMBOJ. 7:1053-1059 for CD40 or Gauchat et al. 1993. FEBS 315(3):259-266; Grāf et al. 1992. Eur. J Immunol. 22:3191-3194; Seyama 1996. Hum. Genet. 97:180-185 or GenBank Accession Nos. L07414, Χ67878, Χ96710 for CD40L. 10 In one embodiment, the aģent which blocks the expression of a costimulatory molecule, thus blocking a costimulatory signal in a T celi is a soluble form of CD40 or CD40L. In one embodiment, the soluble CD40 or CD40L protein comprises the entire protein. In preferred embodiments, a soluble CD40 or CD40L protein comprises the extracellular domain of the protein. For example, a soluble, recombinant form of the 15 extracellular domain of CD40 or CD40L protein or portion thereof can be made as a fusion protein comprising at least a portion of CD40 or CD40L such that the interaction between CD40 on an APC and CD40L on a T celi is interrupted and the delivery of a costimulatory signal to a T celi is inhibited. Such a soluble, recombinant form of a CD40 or CD40L protein comprises at least a portion of the molecule sufficient to bind to 20 its counter receptor and at least a portion of a second non-CD40 or CD40L protein. In preferred embodiments, the CD40 or CD40L fusion protein comprises a CD40 or CD40 extracellular domain vvhich is fused at the amino terminus to a signal peptide, e.g., from oncostatin M (see e.g., W093/00431).

In a particulariy preferred embodiment, a soluble form of CD40 or CD40L is a 25 fusion protein comprising the extracellular domain 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, a CD40Ig or a CD40LIg, can be made using methods known in the art (see e.g., Linsley 1994. Perspectives in Drug Discovery and Design 2:221; Linsley WO 93/00431, U.S. Palent 5,770,197, and U.S. Patent 5,580,756). 30 In addition, antibodies to CD40 ligand have.been found to synergize with aģents which inhibit a costimulatory signal in a T celi to promote grafi tolerance (Kirk et al. 1997. Proc. Nati Acad Sci. USA 94:8789; Larsen et al. 1996. Nature 381:434). -32-

Therefore, in one embodiment antibodies to CD40 or CD40L which bind to these molecules, but which do not inducē the expression of costimulatory molecules, can be used as an aģent which blocks a costimulatory signal in a T celi. V. Aģents ThatAct Intracellularly To InhibitA Costimulatory Signal

In one embodiment, the aģent which inhibits a costknulatory signal in a T celi is an aģent which acts intracellularly to inhibit such a signal. Stimulation of a T celi through the CD28 surface receptor (i.e., a costimulatory signal) leads to the production of D-3 phosphoinositides in a T celi. Therefore, in one embodiment, the production of D-3 phosphoinositides can be inhibited in a T celi to inhibit a costimulatory signal to thereby inhibit a T celi response, as measured, for example, by T celi prolifeiation and/or cytokine production. The teim "D-3 phosphoinositides" is intended to include derivatives of phosphatidylinositol that are phosphorylated at the D-3 position of the inositol ring and encompasses the compounds phosphatidylinositol(3)-monophosphate (PtdIns(3)P), phosphatidylinositol(3,4)-bisphosphate (PtdIns(3,4)P2), and phosphatidylinositol(3,4,5)-trisphosphate (Ptdlns(3,4,5)P3). D-3 phosphoinositides are generated intracellularly by the activity of a phosphatidyl-inositol 3-kinase (PI3K). PI3K is a heterodimer composed of an 85 kDa subunit that binds tyrosyl-phosphorylated proteīns via its SH2 domains and a 110 kDa catalytic subunit PDK was first identified as a lipid kina.se that phosphorylates the D-3 position of the inositol ring of phosphatidylinositol, Ptdlns (4)P, and PtdIns(4,5)P2.

Two recent studies have demonstrated that PI3K is in fact a dual-specificity kinase that possesses both lipid and serine kinase activīties (Dhand, R. et al. (1994) EMBO J. 13:522 and Carpenter, C.L. et al. (1993) Mol Celi Biol. 13:1657).

Accordingly, in one embodiment the aģent which inhibits a costimulatory signal in a T celi is an aģent which inhibits the activity of a PI3K. A preferred aģent which inhibits PDK activity in a T celi is the fungal metabolite wortmannin, or derivatives or analogues thereof. Wortmannin is a potent PDK inhibitor derived from I wortmannii (Kyowa Hakko Kohyo Co. Ltd.) or from P. fumiculosum (Sigma). Wortmannin derivatives or analogues include compounds structurally related to vvortmannin which retain the ability to inhibit PDK and T celi responses. Examples of wortmannin derivatives and analogues are disclosed in Wiesinger, D. et al. (1974) Experientia -33- LV 12768 30:135-136; Closse, A. et ai. (1981) J. Med Chem. 24:1465-1471; and Baggiolini, M. et al. (1987) Exp. Celi Res. 169:408-418. Another inhibitor ofPI3Kactivity that canbe used is the bioflavenoid quercetin, or derivatives or analogues thereof. Quercetin derivatives or analogues include compounds structurally related to quercetin that retain 5 the ability to inhibit PI3K and inhibit T celi responses. Examples of quercetin derivatives and analogues are disclosed in Vlahos, C.J. et al. (1994) J. Biol. Chem. 269:5241-5284. A prcferred quercetin derivative which inhibits PI3K activity is LY294002 (2-(4-morpholinyl)-8-phenyl-4H-l-benzopyran-4-one, Lilly Indianapolis, IN) (described in Vlahos et al. cited supra). 10 CD28 stimulation has also been shown to result in protein tyrosine phosphorylation in a T celi (see e.g., Vandenberghe, P. et al. (1992) J. Exp. MecL 175:951-960; Lu, Y. et al. (1992) J. Immunol. 149:24-29). Accordingly, in one embodiment, an aģent which inhibits a costimulatory signal in a T celi inhibits tyrosine phosphorylation in the T celi. A preferred protein tyrosine kinase inhibitor is one which 15 inhibits src protein tyrosine kinases. In one embodiment, the src protein tyrosine kinase inhibitor is herbimycin A, or a derivative or analogue thereof. Derivatives and analogues of herbimycin A include compounds which are structurally related to herbimycin A and retain the ability to inhibit the activity of protein tyrosine kinases. In another embodiment, the aģent which inhibits protein tyrosine phosphorylation is a 20 protein tyrosine phosphatase or an activator of a protein tyrosine phosphatase. By increasing the tyrosine phosphatase activity in a T celi, the net amount of protein tyrosine phosphorylation is decreased. The protein tyrosine phosphatase can be a cellular protein tyrosine phosphatase within the T celi, such CD45 or Hcph. The activity of a celi surface tyrosine phosphatase on a T celi can be activated by contacting the T 25 celi with a molecule which binds to the phosphatse and stimulates its activity. For example, an antibody directed against CD45 can be used to stimulate tyrosine phosphatase activity in a T celi expressing CD45 on its surface. Accordingly, in one embodiment, the aģent v/hich inhibits protein tyrosine phosphoiyladon within the T celi is an anti-CD45 antibody, or a fragment thereof which rētains the ability to stimulate the 30 activity of CD45. Examples of antibody fragments include Fab and FCab^ fragments. Antibodies, or fragments thereof, can be provided in a stimulatory fonn, for example multimerized or immobilized etc. -34-

In addition, CD28 ligation has been associated with increased phospholipase C activity (see e.g., Nunes, J. et al. (1993) Biochent. J. 293:835-842) and increased intracellular calcium Ievels (see e.g. Ledbetter, J.A. et al. (1990) Blood 75:1531-1539 and the Examples). Accordingly, an aģent which acts intracellularly to inhibit a costimulatory signal in a T celi can act by inhibiting phospholipase C activity and/or inhibiting an increase in intracellular calcium Ievels. For example, the tyrosine kinase inhibitor herbimycin A also inhibits CD28-induced calcium flux in T celis.

Protein serine and serine-threonine kinases have also been shown to be involved in signal transduction pathways associated with CD28 (Siegel, J.N. et al. (1993) J. Immunol. 151:4116-4127; Pai, S.V. et al. (1994) J. Immunol. 24:2364; Parry et al. 1997. Eur. J. Immunol. 27:2495). Thus, in another embodiment of the invention, an aģent which acts intracellularly to inhibit a costimuiatory signal in a T celi inhibits serine or serine-threonine kinase activity. VI. Other Aģents That Block Costimulation OfT Celis

Other aģents which block a costimulatory signai in a T celi can be identified nsing Standard techniques. For example, such aģents can be identified by their ability to inhibit T celi proliferation and/or cytokine production. For instance, a costimulation assay system can be used. In such a system, human CD28+ T celis are isolated for example, by immunomagnetic bead depletion using monoclonal antibodies directed against B celis, natūrai killer celis and macrophages as previously described (Gimmi, C.D., et al. (1993) Proc. Nati AccuL Sci. USA 90,6586-6590). Antigen presenting celis, e.g., whole spleen celis, or purified B celis, or B7-1 or B7-2 transfected COS celis can be iiradiated or treated with mitomycin-C (e.g., at 25μg/ml) for l hour, and then extensively washed to inhibit proliferation. 10^ CD28+ T celis can be incubated with, e.g., 105- 10^ APCs, (e.g., COS celis transfected with a B7 molecule). In this exemplaiy assay, one population of the T celis receive a primary activation signal (e.g., a T celi receptor signal) alone; another population of T celis receive a costimulalory signal alone; yet another population of T celis receive both a primary activation signal and a costimulatory signal and yet another population of T celis receive both a primary activation signal and a costimulatory signal in the presence of the aģent to be tested for its ability to block a costimulatory signal in a T celi. A primaiy activation signal can be -35- LV 12768 delivered, e.g., by a submitogenic dose of PMA (e.g., lng/ml), a submitogenic dose of mitogen, a suboptimal dose of antigen, or a submitogenic dose of anti-T celi receptor antibody. Signal 2 is delivered by antigen presenting celis bearing a B7 molecule. Potential blocking aģents can be tested at a range of concentrations. For example, 5 potential blocking antibodies can be used as hybridoma supematants or as purified antibody (e.g., at about 10μg/ml). Proliferation of T celis can be measured by ^H-thymidine (lpCi) incorporation for the last 12-18 hours of a 72 hour incubation. The delivery of a primary activation signal should result in some proliferation, but T celis receiving both the primary activation signal and costimulatory signal 2, signāls should 10 proliferate maximally. Blocking aģents are identifled by their ability to reducē the maximal, costimulatory signal induced proliferation.

In addition to, or as an altemative to measuring T celi proliferation, T celi cytokine production can be measured using techniques which are well known in the art. For example, IL-2 and IL-4 produced in the T celi cultures can be assayed in culture 15 supematants collected at 24-72 hours after initiation of the culture using a commercially available ELISA (R&D Systems, Minneapolis, MN and BioSource, Camarillo, CA). As set forth above, blocking aģents can be identified by their ability to reducē the maximal, costimulatory signal induced cytokine production.

In the case of antibodies, any "blocking antibodies" identified using this or 20 another assay can be further tested to detennine the costimulatory molecule to which they bind, using techniques known in the art For example, the ability of the blocking antibody to reducē the binding of a labeled antibody to a known ligand can be measured.

Vil Additional Aģents For Downmodulating īmmune Responses 25 In certain embodiments, the compositions and methods of the invention can comprisc additional aģents or the use of additional aģents to enhance immunotolerance to an aģent which promotes hemostasis. In one embodiment, an aģent which promotes immunotolerence but does not act by inhibiting a costimulatory signal in a T celi can be added to the instant compositions or administered in the instant methods. For example, 30 anti-CD40 ligand (e.g., the monoclonal anūbody to human CD40 ligand, 5C8 (Kirk et al. 1997. Proc. Nati AcadL Sci. USA 94:8789)) can be included in a composition. CD40 and its T cell-based ligand, CD40L (CD154) play an important role in up-regulating B7 -36- and in establishing B celi activity (U.S. Patent 5,683,693, Yang et al. 1996. Science 273:1862; Grewal et al. 1996. Science 273:1864; Leterman et al. 1992. J. Exp. Med 175:1091; Ledennan et al. 1992. J. Immunol. 149:3817). Antibodies to CD40 ligand have been found to synergize with aģents which inhibit a costimulatory signal in a T celi to promote grafi tolerance (Kirk et al. 1997. Proc. Nati Acad Sci. USA 94:8789; Larsenetal. 1996. Nature 381:434). In another embodiment, an aģent that acts intracellularly to promote immunotolerization, but which does not inhibit a costimulatory signal in a T celi can be used in the subject compositions. For example, in one embodiment, cyclosporine A (CSA), FK506, Rapamycin, or another aģent which inhibits immune responses can be included in the instant compositions or administered as part of the instant methods. (See, e.g., Sigal et al. 1992. Annv. Rev. Immunol 10:519, Ruhimann et al. 1997or lmmunobiology. 198:192; Shaw et al. 1996. Clin. Chenu 42:1316) VIII. Methods ofUsing Compositions Which Comprise a Therapeutic Protein and an Immunotolerizing Aģent

In one embodiment the subject compositions and/or aģents described herein are administered to subjects who have a hemostatic disorder and have been previously treated with an aģent that promotes hemostasis. In another embodiment, the subject compositions and/or aģents described herein are administered to subjects who have not yet been treated with an aģent that promotes hemostasis.

In yet another embodiment, the subject compositions and/or aģents described herein are administered to a subject that has not yet developed an immune response to an aģent which promotes hemostasis. In other embodiments, the subject compositions and/or aģents are administered to subjects who have a preexisting immune response to an aģent which promotes hemostasis. Whether a subject has a wpreexisting immune response” can be determined by measuring the titer of antibodies in the subject that react with the aģent that promotes hemostasis using techniques that are well known in the art If such a subject has a measurable titer of such antibodies (e.g., a statistically significant titer) when compared with titers from control individuāls, a subject can be said to have a preexisting immune response to an aģent which promotes hemostasis. Altematively, a cellular immune response to an aģent that promotes hemostasis can be measured to -37- LV 12768 deteimine whether a subject has an ongoing immune response to an aģent that promotes hemostasis. Such techniques are well known in the art.

In one embodiment, a first aģent which promotes hemostasis and a second aģent which inhibits or blocks a costimulatory signal in a T celi are used to treat a hemostatic 5 disorder. In another embodiment, compositions comprising a combination of a first aģent which promotes hemostasis and a second aģent which inhibits a costimulatory signal in a T celi can be used to treat a hemostatic disorder.

Administration of the compositions and/or aģents described herein can be in any pharmacological form that includes a therapeutically active amount of an aģent and a 10 phannaceutically acceptable carrier. Administration of a therapeutically active amount of the subject aģents and/or compositions is defined as an amount effective, at dosages and for periods of time necessary to achieve treatment of the hemostatic disorder in the case of an aģent which promotes hemostasis, and to achieve immunotolerance to the aģent vvhich promotes hemostasis in the case of an aģent which inhibits a costimulatory 15 signal in a T celi. A therapeutically active amount of an aģent or composition may vaiy according to factors such as the disease state, age, sex, and weight of the individual, and whether or not the individual has already developed an immune response to an aģent which promotes hemostasis. Such an amount can be readily determined by one of ordinary skill in the art 20 The optimal course of administration of the aģents and/or compositions may also vary depending upon the subject to be treated. In certain embodiments a subject will require treatment with both aģents at one time. In that instance, it will be desirable to administer an aģent which promotes hemostasis and an aģent which inhibits a costimulatory signal in a T celi simultaneously, for example, in the form of a 25 composition comprising both aģents.

In other embodiments it will be desirable to administer the aģents separately, e.g., in order to promote stability of the aģents, or to facilitaie staggered administration of the aģents. In one embodiment, staggered administration may be desirable to achieve optimal therapeutic effect of the aģent which promotes hemostasis, while optimally 30 inhibiting the immune response, preferably an antibody response, to the aģent For example, an aģent which inhibits a costimulatoiy signal can be administered alone prior -38- to administration of an aģent which promotes hemostasis, or can be administered alone for several days after administering an aģent that promotes hemostasis.

In one embodiment, an aģent which blocks a costimulatory signal in a T celi can be administered chronically, e.g., each time the aģent which promotes hemostasis is administered. In another embodiment, an aģent which blocks a costimulatory signal in a T celi is administered sporadically. For example, a subject may require treatment with an aģent that promotes hemostasis on a regular basis, but may only require treatment with an aģent that inhibits a costimulatory signal in a T celi periodically. For example, treatment with an aģent which promotes hemostasis can be ongoing, while as few as one or two treatments of the subject with an aģent which blocks a costimulatory signal in a T celi may be sufficient; further administration of an aģent which blocks a costimulatory signal may not be required. In a preferred embodiment, an aģent that blocks a costimulatory signal in a T celi is administered at appropriate intervāls for at least about 6 months.

In one embodiment the subject aģents or compositions are administered to patients if the patient is found to have preexisting antibodies. In another embodiment, the subject aģents or compositions are administered to previously untreated patients, i.e., without preexisting antibodies. In stili another embodiment, the subject aģents or compositions are administered to patients that have been previously treated, but do not have antibody titers against an aģent which promotes hemostasis. A dosage regime may be adjusted to provide the optimum therapeutic response for each subject without undue experimeiltation. For example, antibody titers to an aģent which promotes hemostasis can be measured to determine whether or not the subject is developing an immune response to the aģent and the dosage regimen can be adjusted accordingly. For example, if antibody titers to an aģent that promotes hemostasis increase, more doses of an aģent which blocks a costimulatory signal in a T celi may be administered.

To ādminister the subject aģents or compostions by other than parenteral administration, it may be necessary to coat them with, or co-administer them with, a material to prevent its inactivation. An aģent or compostion of the present invention may be administered to an individual in an appropriate carrier or diluent, co-administer ed with enzyme inhibitors or in an appropriate carrier such as liposomes. -39- LV 12768

Pharmaceutically acceptable diluents include saline and aqueous buffer Solutions. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol. Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes (Strejan et ai., (1984) J. Neuroimmunol 7:27). 5 The active aģent or composition may also be ādmini stered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

Pharmacentical compositions suitable for injectable use include sterile aqueous 10 Solutions (where water soluble) or dispersions and sterile povvders for the extemporaneous preparation of sterile injectable Solutions or dispersion. In ali cases, the aģent 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 the contaminating action of microorganisms such as 15 bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for examplc, water, ethanol, polyol (for example, glyceroI, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of 20 surfactants. Prevention ofthe action of microorganisms can be achieved by various antibacterial and antifungal aģents, for example, parabens, chlorobutanol, phenol, asorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic aģents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be 25 brought about by including in the composition an aģent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable Solutions can be prepared by incorporating active composition or aģent in the required amount in an appropriate solvent with one or a combinadon of ingredients enumerated above, as required, followed by filtered sterilization. Generally, 30 dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basie dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable -40- solutions, the preferred methods of preparation are vacuum drying and freeze-diying which yields a powder of the active ingredient (e.g., aģent or comosition) plus any additional desired ingredient from a previously sterile-filtered solution thereof.

When the active aģent or coraposition is suitably protected, as described above, 5 the protein may be orally administered, for example, with an inert diluent or an assimilable edible carrier. As used herein "phannaceutically acceptable carrier" includes any and ali solvents, dispersion media, coatings, antibacterial and antifungal aģents, isotonic and absorption delaying aģents, and the like. The use of such media and aģents for pharmaceutically active substances is well known in the art Except insofar as any 10 conventional media or aģent is incompatible with the active compound, use thereof in the therapeutic compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

It is especially advantageous to fonnulate parenteral compositions in dosage unit form for ease of administration and unifonnity of dosage. Dosage unit form as used 15 herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetennined quantity of active compound calculated to producē the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active 20 compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active aģent or composition for the treatment of individuāls.

As used herein "pharmaceutically acceptable carrier" includes any and ali solvents, dispersion media, coatings, antibacterial and antifungal aģents, isotonic and 25 absorption delaying aģents, and the like. The use of such media and aģents for pharmaceutically active substances is well known in the art Supplementary aģents can also be incorporated.

The practice of the present invention will empIoy, unless otherwise indicated, conventional techniques of celi biology, celi culture, molecular biology, microbiology, 30 recombinant DNA, and immunology, which are within the skill of the art Such techniques are explained fully in the literature. See, for example, Genetics; Molecular ClortingA Laboratory Mannai, 2nd Ed., ed. by Sambrook, J. et ai (Cold Spring Harbor -41 - LV 12768

Laboratory Press (1989)); Short Protocols in Molecular Biology, 3rd Ed., ed. by Ausubel, F. et al. (Wiley, NY (1995)); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed. (1984)); Mullis et ai U.S. Patent No: 4,683,195; NucleicAcid Hybridization (B. D. Hames & S. J. Higgins eds. (1984)); 5 the treatise, Methods In Enzymology (Academic Press, Inc., Ν.Υ.); Immunochemical Methods In Celi And Molecular Biology (Mayer and Walker, eds., Academic Press, London (1987)); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds. (1986)); and Miller, J. Experiments in Molecular Genetics (Cold Spring Harbor Press, Cold Spring Harbor, Ν.Υ. (1972)). 10 The contents of ali references, pending patent applicadons and published patents, cited throughout this application are hereby expressly incorporated by reference.

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

15 EXAMPLES

In the Examples, a mouse modei of hemophilia A was used to evaluate new methods for the prevention and treatment of inhibitor foimation. Hemophilia A mice, generated by targeted disruption of exon 16 of Factor VIII gene, have no dctectable 20 Factor VIII activity in their plasma (Bi L, Nature Genetics 10:119,1995) and are similar in this way to padents with severe hemophilia A. As expected, hemophilia A mice have in vivo signs of a coagulation patbway defect with fatal bleeding if tails are cut without use of hemostatic measures, and they develop subcutaneous and intramuscular bleeding after handling or temporaiy immobilization (Qian J, Borovok M, Bi L, Kazazian HH, 25 Hoyer L. Thromb Haemost 81:940,1999; Evans GL et al, Proc Nati Acad Sci USA 95:5734, 1998).

Intravenous infusions of 0.2pg human factor Vm, a dose equivalent on a weight basis to that given to hemophilia A padents, resulted in minimal or no andbody response in these hemophilia A mice after a singie injection, but repeat infusions led to high dter 30 inhibitory anti-factor VIII (Qian J et al. Thromb Haemost 81:940,1999). In addition, a factor νΤΠ-specific T celi proliferative response was detected three days after first exposure to human factor VIII, before antibodies was detected. -42-

Example 1. Inhibition of the primary immune response to factor Vili

The experimental design for this example is shown in Figurē 1. Three groups of mice were injected with factor VIII intravenously on day 0. One group of mice was also 5 injected with CTLA4-Ig one day before and one day after the factor VHI injection. A second group of mice received the same CTLA4-Ig treatment (intraperitoneally) foliowed by daily factor VIII injections from day 2 to day 12. The third group did not receive any CTLA4-Ig. Ali mice then received two additional injections of factor VIII at days 23 and 44. Animals were bled at days 20,37,58, and 82. While mice that did 10 not receive CTLA4Ig had high titers of antibody beginning as early as day 20, mice that received CTLA4Ig did not develop antibodies until day 82 (Figurē 2).

Example2. Inhibition of the secondary response to factor VIII

The experimental design for this example is shown in Figurē 3. In this example 15 mice were given multiple intravenous injections of factor VIII at two week intervāls and then divided into two groups. The mice were re-injected with factor VIII at days 1,20, and 37. One group of mice was injected with CTLA4-Ig on days -1 and day +1 relaūve to the day 1,20, and 37 factor VIII injections. The animals that did not receive CTLA4Ig had high titers of anti-factor VIII antibodies, while the mice that received 20 CTLA4Ig (with the exception of 1 mouse) did not develop a secondary immune response to factor VIII (Figurē 4).

The follovving methods and materiāls were used in Examples 3-6. 25 Animals. The characteristics of the exon-16 (E-16) strain of hemophilic mice have been reported Bi L, et al. Nature Genetics 10:119,1995; Bi L, et al. Blood 88:3446, 1996. Adult male and homozygous E-16 female mice, aged 10-20 weeks, were used for these studies. Blood samples were obtained by orbītai venous plexus bleeding, and the senam was separated by centrifugation at 600 g for 3 minūtes. The serum samples were stored 30 at -20°C until assayed. To avoid severe bleeding and death of animals, ear tags were not used to identify the mice in some experiments. For this reason, Figurē 5 does not indicate sequential data for individual mice. -43- LV 12768

Generation of E-16/B7-1 and B7-2 double knockout mice was accomplished by cross breeding of E-16 with B7-1 and B7-2 knockout mice (Borriello F, et ai. 1997. Immunity 6:303). Homozygous E-16/B7-1 and E-16/B7-2 double knockout mice vvere identified by genotype determination (Bi L et al, Nature Genetics 10:119,1995; 5 Borriello F, et al. Immunity 6:303,1997. Reduced factor VIII activity was verified using the Coatest chromogenic bioassay (Chromogenix, Molndal, Sweden) (Bi L et al, Blood 88:3446,1996). The factor VIII activity was less then 1% in both E-16/B7-1 and E-16/B7-2 deficient mice. 10 Anti gēns. Recombinant human factor VIII was obtained from the Hyland Division of

Baxter Healthcare Corp. (Glendale, CA). mCTLA-4Ig. A murine CTLA4-lg cDNA expression plasmid was prepared by ligation of the leader and extracellular doraains of murine CTLA4 to the hinge, CH2 and CH3 15 domains of IgHg2a that had been mutated to remove effector fimctions as described in Streurer et al (Streurer, J Immunol 155:1165, 1995). The insert was cloned into the expression vector pED and stably transfected into CHO celis as previously described (Lollar P et al, J Clin Invest 93:2497,1994). Concentrated conditioned media was loaded onto a rProtein A Sepharose Fast Flow chromatography column (Amersham 20 Pharmacia Biotech, Piscataway, NJ). The column was washed with PBS pH 7.1 and the mCTLA4-lg eluted with 20 mM Citrate pH 3.0. The peak pool was neutralized with 1M Tris pH 8.0 to a final pH of 7.5 and formulated into PBS pH 7.1 using an Amicon stirred celi with a ΥΜ30 membrane. The mCTLA4-Ig was depyrogenated using a Poros PI (Perceptive Biosystems) chromatography column and the product eluted from the 25 column in a linear NaCl gradient from O to IMNaClin 25 mM Tris pH 7.5. The mCTLA-4-Ig was then formulated into PBS pH 7.1 using an Amicon stirred celi using a ΥΜ30 membrane. . Antibody Measurements. The anti-factor VIII titer was determined by ELISA (Qian J, et 30 al. Inhibitor antibody development and celi response to human factor VIII in murine hcmophilia A. Thromb Haemost 81:940, 1999). The ELISA assays were carricd out using microtiter wells coated vvith recombinant human factor VIII, 0.8pg/ml in 0.05 -44- mol/ml carbonate-bicarbonate, pH 9. After mouse plasma samples were incubated in the wells at 4°C ovemight and then washed, alkaline-phosphatase conjugated goat anti-mouse IgG (Southern Biotechnology Associates Inc., Birmingham, AL) was added for 2 hours at room temperature. After washing, P-nitrophenyl phosphate (Sigma, Sl 5 Louis, MO), 2mg/ml in lOOmmol/L glycine, Immol/L MgCh, 2mmol/L ZnClļ, pH 10.4, was then added and the absorbance read at 410 nm using an automated micro titer plate ELISA reader. The concentration of anti-factor VIII antibody was estimated from a Standard curve obtained using a monoclonal murine IgG anti-human factor VITI antibody that binds to the A2 domain (Mab 413) (Lollar P et al, J Clin Invest 93:9497, 1994). The 10 titer was calculated from points that fell on the linear portion of the assay Standard curve.

Anti-factor VIII inhibitor titers in Bethesda Units (BU) were measured by the Bethesda Assay (Kasper CK. Thromb et Diath Haem 30:263,1973). 15 T celi proliferation assays. The spleen was used as the source of T celis for proliferation assays. Spleen celis were then cultured (5 x 105/well) in 96 well flat bottom plates. Varying amounts of recombinant factor VIII were added to the culture medium consisting of complete RPMI-I640 containing 0.5% hemophilic mouse serum. 37 kBp of 3H-thymidine/well (6.7Ci/mmol, ICN Pharmaceuticals Irvine, CA) was added after 72 20 hours of culture at 37°C. The cultures were harvested 16 hours later using a Matrix 9600 (Packard, Meriden, CT). Data are expressed as the mean for triplicate wells of the cpm incorporated into insoluble DNA.

Example 3. mCTLA4-Ig blocks the induction of an anti-factor VIII responsc. 25 Anti-factor VIII inhibitory antibodies were induced in control mice by repeated intravenous injections of 1 pg recombinant human factor VIII at three week intervāls. In this Example, four groups of hemophilia A mice were injected with recombinant human factor VEH on days 0,23,44 and 66 (lpg i.v. initially, and then 0.2pg for the 2®1,3rf, and j. 4 injections). Mice in groups G-3 and G-4 were also injected intraperitoneally with 30 0.2pg factor VIII on days 2-12. Blood samples for anti-factor VHI assay were obtained on days 20, 37,58 and 82. Control groups G-l and G-3, open circles, were injected with only factor VIII. Groups G-2 and G-4, solid circles, were also injected with mCTLA4-Ig -45- LV 12768 (250pg, i.p.) on the day before and the day after the first factor VIII injection. The anti-factor VIII antibody concentration was determined by ELISA. Anti-factor VIII assay data points indicated as <0.16pg/ml were similar to those for plasma samples obtained from unimmunized hemophilia A mice. The results of this experiment are shown in 5 Figurē 5 (Note that Figurē 5 repeats some of the data shown in Figurē 2, but adds additional data).

Anti-factor VIII was detected in four of the five mice 20 days after the first injection, and ali control mice developed high titer anti-factor VIII after receiving two to four injections. The mean inhibitor Ievel after four injections was 1860 Bethesda units 10 (BU). Anti-factor VIII antibody formation was markedly suppressed in mice injected intraperitoneally with 250pg of murine CTLA4-lg on the day before and the day after first factor VIII injection (Group G-2, Figurē 5), even though there was no further mCTLA4-lg given with three subsequent factor VIII injections on days 23,44 and 66. Anti-factor VIII was not detectable in any Group G-2 mice after the first or second 15 factor Vīli injection. Three weeks after the third injection of factor VĪD, a weak immune response was detected in two of the six mice in the G-2 group.

To investigate if the limited duration of unresponsiveness is a result of the short half Iife of human factor VTII in these mice (4-5 hours in murine hemophilia A (Evans GL et al, Proc Nati Acad Sci USA 95:5734,1998), control and mCTLA-4-Ig treated 20 mice (Groups G-3 and G-4, Figurē 5) were injected with lpg factor VĪD intravenously on day 0 followed by daily intraperitoneal injections of Ipg factor VIII on days 2-12. High titer anti-factor VDI was present on day*20 in the control mice (Group G-3): over 350pg/ml by ELISA and a mean inhibitor titer of 694 BU. In contrast, the Group G-4 mice that were injected with mCTLA4-Ig on the day before and the day after the first 25 exposure to factor VHI had no detectable anti-factor vm on day 20. The delayed anti-factor VĪD antibody response after three additional factor VDI injections was the same in these mice as that in the Group G-2 animals. Thus, the limited persistence of factor VHI in the plasma after CTLA4-Ig injection was not the reason for a limited duration of unresponsiveness in CTLA4-Ig treated mice. 30 -46-

Example4. Effects of Repeated Administration of CTLA4-Ig

Because a delayed anti-factor VIII response was detected after repeated factor VIII infusions when mCTLA4-Ig was given only at the time of the first factor VIII exposure, it was determined if mCTLA4-Ig might prevent anti-factor VIII development 5 if given with each factor VIII infusion. In that experiment (Figurē 6), hemophilia A mice were simultaneously infused with both factor VIII and mCTLA4-Ig six times at three week intervāls. Hemophilia A mice were injected intravenously with both lpg factor VIA and 250pg mCTLA4-Ig at 3 week intervāls (solid circles) or with factor VHI alone after the first injection which contained both factor VIII and mCTLA4-Ig (open 10 circles). Serum samples for anti-factor VIII assay were obtained 4 weeks after the 6Λ factor VIII injection. There was no detectable anti-factor VHI in any of ten mice treated in this way when they were tested four weeks after the sixth factor VIII injection. In contrast, high titer anti-factor VIII was present in serum from mice that had received only one mCTLA4-Ig injection (at the time of the first exposure to factor VIII) followed 15 by five injections of factor VIII alone.

Thesc mCTLA4-lg treated mice were then tested to determine if they would have an immune response after additional factor VIII injections in the absence of mCTLA4-Ig. After two intravenous injections at three week intervāls, none of five mice developed anti-factor VĪD, wfaile low Ievel anti-factor VHI was detected in two of four 20 control mice not previously exposed to either factor VHI or mCTLA4-Ig (Figurē 7). In this experiment, hemophilia A mice treated as described for Figurē 6 with six injections of both factor VIII and mCTLA4-Ig were subsequently given six intravenous injections of 0.2pg of factor Vm at 3 week intervāls without additional mCTLA4-Ig (closed circles). Control mice with no prior factor vm exposure were immunized in parallel 25 (open circles). Serum samples for anti-factor VIII assay were obtained 3 weeks after the 2°* and 6* injections. After six injections of factor VHI alone, the mean factor VIII titer was 93pg/ml for mice that had previously received both factor VIII and mCTLA4-Ig, while the mean titer was 155pg/ml for the control mice. These dala document the limits of antigen-specific immune suppression that follow from repeated co-administration of 30 mCTLA4-Ig with factor Vm. -47- LV 12768

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

To determine if mCTLA4-lg modifies a secondary immune response to factor Vm, mCTLA4-Ig was injected at the same time that factor VIII was given to hemophilia A mice that had already developed anti-factor VIII. Initially, ali the hemophilia A mice 5 had been injected three times with 0.2pg factor VIII and the Ievel of anti-factor VIII was deteimined by ELISA. The control mice then received three additional injections of factor Vili while the remaining mice were given mCTLA4-Ig at the same time as they received the first of three additional factor VHI injections. While many mice died of bleeding complications during this experiment because of the repeated injections and 10 blood sample collections, the resnlts were clearly dififerent for the two groups.

In this Example, ali mice initially received 3 intravenous injections of 0.2μ$ FVIII at 2 week intervāls. Control mice (open circles) were then injected with factor Vm three more times and blood samples were obtained for assay (upper panei). The other mice (solid circles) were given mCTLA4-Ig (250pg, intraperitoneaUy) the day 15 before and the day after the 4* factor Vm injection (as indicated by the arrow), followed by two more injections of factor VIII alone at 3 week intervāls. The number of factor Vm injections prior to the blood sample tested for anti-factor Vm is indicated on the horizontal axis.

An increase in the anti-factor Vm titer was noted after the 4th injection of factor 20 Vm for the control mice, with the mean titer going fiom 16 to 230μς/ια1 (Figurē 8A). After the 5th factor Vm injection, anti-factor Vm titers were ali over 350pg/ml in the four remaining control mice. In contrast, mice treated with mCTLA4-Ig at the 4th factor Vm injection had minimal or no increases in anti-factor VHI (Figurē 8B and C). The administration of mCTLA4-Ig inhibited this secondary immune response to factor Vm 25 for mice that had already developed reladvely high anti-factor VHI Ievels, corresponding to inhibitor titers of 5-90 BU (Qian J, et al. Thromb Haemost 81:940,1999) (Figurē 8C) as well as for mice with minimal anti-factor Vm after the three initial injections, less than 5 BU (Figurē 8B). -48-

Example6. Determination of the role ofB7-1 and B7-2 in the primary immune response to factor VIII.

The roles of B7-1 and B7-2 co-stimuiatory ligands on antigen presenting celis were then evaluated, since it was their interaction with CD28 that was presumed to be 5 prevented in the experiments using mCTLA4-Ig. To do this we crossed hemophilia A mice with B7- lv' and B7-2V‘ mice (Borriello F, et al. B Immunity 6:303, 1997; Freeman GJ et al, Science 262:907,1993) and mice deficient in both factor VIII and either B7-1 or B7-2 were selected by genotype analysis. The hemophilia A/B7-lv‘ and hemophilia A/B7-2 m,‘ mice were then injected intravenously with 0.2pg human factor VIII at two 10 week intervāls. Serum samples for anti-factor VIII assay were obtained 12 days after the 2nd and 6* factor VIII injections. Aiter four injections, ali nine hemophilia A/B7-1 mice (open circles) had developed anti-factor VIII, with titers over 350pg/ml and a mean inhibitor Ievel of 712 BU (Figurē 9), values similar to those for otherwise normai hemophilia A mice injected with factor VIII (Qian J, et al. Thromb Haemost 81:940, 15 1999). In contrast, none of the eight hemophilia AlB7-2‘/" mice (closed circles) had detectable anti-factor VIII. Similar results were obtained in hemophilia A mice treated with anti-B7-l and anti-B7-2 antibodies.

To evaluate the T celi response of these B7-1 and B7-2 deficient hemophilia A mice, spleen celis were obtained three days aiter a fifth intravenous injection of factor 20 Vin. Pooled spleen celis firom 3 mice were used to establish the proliferation data. The open and closed squares in Figurē 10 are for celis from untreated hemophilia A/B7-lv', and B7-2V‘ mice, respectively. The open circles are for celis from hemophilia A/B7-l'/' mice that received 5 intravenous injections of FVin and the solid circles are for hemophilia A/B7-27· mice that received 5 intravenous injections of factor VIII. The 25 concentration of factor VIII in the cultures is indicated on the horizontal axis. The T celi proliferative activity detennined by 3H-thymidine incorporation showed a factor VIII dose dependent response for celis from the hemophilia/A B7-lv' mice (Figurē 10). In contrast, no T celi response was detected at any factor VIII Ievel for spleen celis from . hemophilia A/B7-2V' mice. Thus, B7-2 has the major role in suppordng an immune 30 response to factor VIII injected intravenously, and anti-factor VIII formation is prevented if it is missing. LV 12768 -49-

Eq uivalen ts

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific compositions and 5 methods described herein. Such equivalents are considered to be within the scope of this invention and are covered by the follovving claims. -50- LV 12768

What is claimed is: 1. A composition comprising a first aģent which promotes hemostasis and a second aģent which Lnhibits a costimulatory signal in a T celi. 5 2. The composition of claim I, further comprising a pharmaceutically acceptable canier. 3. The composition of claim 1, wherein the first aģent is factor VIII. 10 4. The composition of claim 1, wherein the first aģent is a B-domain deleted variant of factor VIII. 5. The composition of claim I, wherein the first aģent is factor IX. 15 6. The composition of claim 1, wherein the first aģent is Von Willebrand factor. 7. The composition of claim 1, vvherein the second aģent is a soluble form 20 of a costimulatory molecule. 8. The composition of claim 7, wherein the second aģent is a soluble fonn of CTLA4. 25 9. The composition of claim 7, vvherein the second aģent is a soluble fonn of B7-1, a soluble form of B7-2, or a combination of said soluble form of B7-1 and said soluble fonn of B7-2. 10. The composition of claim 8, vvherein the second aģent is CTLA4Ig. 30 11. The composition of claim 9, vvherein the second aģent is B7-lIg or B7-2Ig. - 51 - 12. The composition of claim 1, wherein the second aģent is a soluble form ot' CD40 or CD40L. 5 13. The composition of claim 1, wherein the second aģent is an antibody which binds to a costimulatory molecule. 14. The composition of claim 13, wherein the second aģent is selected from the group consisting of an anti-B7-1 antibodv, anti-B7-2 antibody, and a combination of an anti- 10 B7 antibody and an anti-B7-2 antibody. 15. The composition of claim 13, vvherein the antibody is a non-activating form of an anti-CD28 antibody. 15 16. The use of the composition of any of claims 1-15, for the preparation of a medicament for treatment of a hemostatic disorder in a subject. 17. The use of claim 16, vvherein the subject has a preexisting immune response to the first aģent. 20 18. The use of claim 16, vvherein the subject does not have a preexisting immune response to the first aģent. 19. The use of claim 16, a composition comprising an additional 25 immunosuppressive aģent. 20. The use of claim 16, wherein the hemostatic disorder is selected from the group consisting of hemophilia A, hemophilia B, and von Willebrand’s disease. -52 - -52 -LV 12768 21. The use of the composition for the preparation of a medicament for the treatment of a hemostatic disorder in a subject, vvherein the composition comprises a first aģent vvhich promotes hemostasis and a second aģent which inhibits a costimulatory signal in 5 a T celi, such that a hemostatic disorder is treated. 22. The use of the composition for the preparation of a medicament for the treatment of a hemostatic disorder in a subject, vvherein the composition comprises a first aģent vvhich promotes hemostasis and a second aģent vvhich inhibits a costimulatory signal in 10 a T celi, such that the immune response to the first aģent is dovvnmodulated to therebv treat a hemostatic disorder. 23. The use of claim 21 or 22, vvherein the first aģent is factor VIII. 15 24. The use of claim 21 or 22, vvherein the first aģent is a B-domain deleted variant of factor VIII. 25. The use of claim 21 or 22, vvherein the first aģent is factor IX. 20 26. The use of claim 21 or 22, vvherein the first aģent is Von Willebrand factor. 27. The use of claim 21 or 22, vvherein the second aģent is a soluble form of an aģent vvhich delivers a costimulatorv signal to a T celi. 25 28. The use of claim 27, vvherein the aģent is a soluble form of CTLA4. 29. The use of claim 28, vvherein the aģent is CTLA4Ig. 30. The use of claim 27, vvherein the aģent is a soluble form of B7-1, a soluble 30 form of B7-2, or a combination of a soluble form of B7-1 and a soluble form of B7-2. 31. The use of claim 30, vvherein the aģent is B7- llg, B7-2Ig, or a combination of both B7- llg and B7-2Ig. -53 - 32. The use of claim 21 or 22, vvherein the second aģent is an antibody which binds to a costimulatory molecule. 33. The use of claim 32, wherein the second aģent is selected from the group 5 consisting of an anti-B7-l antibody, an anti-B7-2 antibody, and a combination of an anti-B7-l and an anti-B7-2 antibody. 34. The use of claim 32, vvherein the antibody is a non-activating form of an anti-CD28 antibody. 10 35. The use of claim 21 or 22, vvherein the hemostatic disorder is selected from the group consisting of hemophilia A, hemophilia B, and von Willebrand’s disease. 36. The use of claim 21 or 22, vvherein the subject has a signifīcant titer of 15 antibodies vvhich bind to the first aģent. -1 - -1 -LV 12768

SEQUENCE LISTING 5 <110> Qian, Jiahua

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Claims (14)

A composition comprising a first agent that stimulates hemostasis and a second agent that inhibits a costimulatory signal in a T cell. A composition according to claim 1, further comprising a pharmaceutically acceptable carrier. 3. The composition of claim 1, wherein the first agent is factor VIII. The composition of claim 1, wherein the first agent is Factor VIII variant 15 with B-domain deletion. The composition of claim 1, wherein the first agent is factor IX. The composition of claim 1, wherein the first agent is von Villebrand 20 (Von VVillebrand). The composition of claim 1, wherein the second agent is a soluble form of the costimulatory molecule. The composition of claim 7, wherein the second agent is a soluble form of CTLA4. The composition of claim 7, wherein the second agent is a soluble form of B7-1, a soluble form of B7-2, or a combination of said soluble form of B7-1 and said soluble form of B7-230. The composition of claim 8, wherein the second agent is CTLA4lg. The composition of claim 7, wherein the second agent is B7-1 Ig or B7-2lg. 35 The composition of claim 1, wherein the second agent is a soluble form of CD40 or CD40L. 2. A composition according to claim 1, wherein the second agent is an antibody that binds to a costimulatory molecule. The composition of claim 13, wherein the second agent is selected from the group consisting of anti-B7-1 antibody, anti-B7-2 antibody, and anti-B7-1 antibody and anti-B7-2 antibody. The composition of claim 13, wherein the antibody is an anti-CD28 antibody inactive form. Use of a composition according to any one of claims 1 to 15 for the preparation of a medicament for the treatment of hemostatic disorders in a human. Use according to claim 16, wherein the human has a pre-existing immune response to the first agent. Use according to claim 16, wherein the human has no pre-existing immune response to the first agent. Use according to claim 16, wherein the composition comprises an additional immunosuppressive agent. Use according to claim 16, wherein the haemostatic disorder is selected from the group consisting of haemophilia A, haemophilia B, and von Villebrand disease. Use of a composition for the preparation of a medicament for the treatment of haemostatic disorders in a human, wherein the composition comprises a first haemostatic stimulating agent and a second agent that inhibits a costimulatory signal in a T cell, such as to prevent a haemostatic disorder. Use of a composition for the preparation of a medicament for the treatment of haemostatic disorders in a human, wherein the composition comprises a first haemostatic stimulating agent and a second agent that inhibits a costimulatory signal in a T cell, such as to suppress the immune response to the first agent, thereby treating the hemostatic disorder. 35 3 EN 12768 Use according to claim 21 or 22, wherein the first agent is factor VIII. Use according to claim 21 or 22, wherein the first agent is a variant of factor VIII with B-domain deletion. Use according to claim 21 or 22, wherein the first agent is factor IX. Use according to claim 21 or 22, wherein the first agent is von Villebrand factor. Use according to claim 21 or 22, wherein the second agent is a soluble form of an agent that transmits a costimulatory signal to a T cell. Use according to claim 27, wherein the agent is a soluble form of CTLA4. Use according to claim 28, wherein the agent is CTLA4lg. Use according to claim 27, wherein the agent is a soluble form of B7-1, a soluble form of B7-20, or a combination of a soluble form of B7-1 and a soluble form of B7-2. Use according to claim 30, wherein the agent is a combination of B7-1 Ig, B7-2lg or both B7-1 Ig and B7-2lg. Use according to claim 21 or 22, wherein the second agent is an antibody that binds to a costimulatory molecule. Use according to claim 32, wherein the second agent is selected from the group consisting of an anti-B7-1 antibody, an anti-B7-2 antibody, and a combination of anti-B7-1 and anti-B7-2 antibodies. Use according to claim 32, wherein the antibody is an inactive form of anti-CD28 antibody. 35. Use according to claim 21 or 22, wherein the hemostatic disorder is selected from the group consisting of haemophilia A, haemophilia B, and von Villebrand disease. 4 Use according to claim 21 or 22, wherein the human has a significant titre of antibodies that bind to the first agent.