CZ20011021A3 - Method of modulating 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

Technical field

The invention relates to compositions and methods for treating hemostatic disorders using agents that promote hemostasis and agents that inhibit the co-stimulatory signal in a T cell. These compositions and methods are capable of treating hemostatic disorders using foreign therapeutic proteins while attenuating the immune response to the therapeutic proteins.

and.

BACKGROUND OF THE INVENTION

One of the primary limitations of therapeutic therapy using biological proteins is the body's immune response, the presence of foreign substances in the body. This in response to the immune response is particularly problematic when foreign substances have to be repeatedly administered in order to optimize their efficacy. One example of such a situation is the repeated administration of agents suitable for the treatment of haemostatic disorders, such as the condition

disease due to lack of factor

VIII (for example, classical haemophilia by factor deficiency)

Classical hemophilia,

IX, and von Willebrand's disease) or which is also chromosome X haemophilia which is known as haemophilia B.

And, there is one

Von Willebrand's disease is the most common bleeding that occurs up to individuals. Hemophilia B, which is a disease, (Harrison's et al., Eds.

One factor occurs

Principles of

13 ^th Edition.

VIII is a disorder associated with 10,000 men.

hereditary disorder of 800 to 1000 also known as Christmas of one

International Medicine, Isselbacher about u

100 ALIGN!

000 men

1994. McGraw-Hill, N.Y., N.Y.).

a single chain protein of the molecular which circulates in complex with von

Willebrand Factor (VWF). Factor VIII is an important mass of 265,000, the regulatory protein in the thrombin coagulation cascade increases blood. After activation of the activated factor, the activation rate

IX (factor IXa), factor X by which ultimately leads to the formation of platelet glycoproteins.

fibrin. Molecule

VWF is an adhesive carrier for platelets by the same factor VIII in the plasma of the vessel walls. VWF is a subunit of approximately 230,000.

clots which plays a central role in agglutination Serves as and allows blood interactions to create more likely the molecular weight of each subunit is

VWF is synthesized in endothelial cells and megakaryocytes. a single chain molecular weight proenzyme is converted to active protease (IXa) by factor-VIIa. Activated Factor X.

Factor

000, factor

IX is which

The Xla or tissue activating complex of the proteins mentioned may result in the proteins. When factor IX and activated factor VIII then

Repeated administration of foreign to immune responses to cells to foreign proteins must be presented by cells containing antigen (APC) to resting T (1987) J. Exp. Copper.

and Schwartz, R.

recipient of the T response two signals to lymphocytes

165, 302-319. Mueller, D.

3701-3709). The first signal transduces recognition

L., et al. (1990) J. Immunol. 144, responses, with (TCR) presented by a stimulation complex that delivers specificity through a foreign antigen receptor in the context of the major immune

T cells et al., 1996.

histocompatibility (MHC) peptide. The second signal, referred to as metals, induces T cell proliferation and functionality (Lenschow Annu. Rev. Immunol. 14: 233). Co-stimulation is neither antigen specific nor limited to one or more different

MHC and is thought to be expressed by APC cells (Jenkins, 3324-3330, Linsley, P.

721-730, Gimmi, C. D., molecules on the surface

M.

K., et al., 1988

J.

Immunol. 140,

WITH.

et al., 1991, J.

Exp.

Acad.

Copper. 173, et al.,

Why. Nati.

Clin.

88, 6575-6579; Young, J.

Invest. 90, 229-237, Koulova L.,

Sci. USA.

W., et al., 1992 J.

1991 J. Exp.

Copper. 173, 759-762, Reiser, H., et al., 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, Μ. I., et al., 1989 J. Exp. Copper. 169, 503, Armitage, R.J., et al., 1992 Nature 357, 80-82, Liu, Y., et al., 1992 J. Exp. Copper. 175, 437-445).

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

The B7-2 receptor appears to be more essential during the primary immune response, while the B7-1 receptor, which is positively regulated later during the immune response, may be important in prolonging primary T cell responses or costimulatory secondary T cell responses (Bluestone, 1995). Immunity 2: 555).

B7-1 and B7-2 receptors are receptors for two ligands expressed on T cells. One ligand to which B7-1 and B7-2 binds is CD28 and is constitutively expressed on resting T cells and enhances expression upon activation. Following signaling through the T cell receptor, ligation of CD28 and transduction of the costimulatory signal induces T cell proliferation and IL-2 secretion (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, 11, 211-6, Harding, F.A., et al., 1992, Nature. 356, 607-609). The second ligand, called CTLA4 (CD152), is homologous to CD28, but does not express on resting T cells and occurs after T cell activation (Brunet, J. F. et al., 1987, Nature 328: 267-270). CTLA4 appears to be critical in the negative regulation of T cell responses (Waterhouse et al., 1995, Science 270: 985). CTLA4 blockade has been found to remove inhibitory signals, while aggregation of CTLA4 provides inhibitory signals, while blocking CTLA4 provides inhibitory signals. Which attenuates T cell response (Allison and Krummel. 1995. Science 270: 932). B7 molecules show a higher affinity for CTLA4 compared to CD28 (Linsley, PS, et al., 1991, J. Exp. Med. 174, 561-569) and B7-1 and B7-2 receptors were found to bind to different regions of the CTLA4 molecule and exhibit different kinetics of binding to CTLA4 (Linsley et al., 1994. Immunity. 1: 793).

10 to 25% of patients with haemophilia develop an immune response to factor VIII. These patients develop inhibitors, usually IgG antibodies, that neutralize factor VIII activity and thus prevent effective treatment. Two types of inhibitors have been identified. Patients with a high response with type I inhibitors exhibit an anamnestic response to factor VIII, which leads to an increase in antibody titer to factor VIII. Patients with a low response type II inhibitor exhibit a low antibody titer that does not increase with factor VIII administration. The current strategy of blunting the antibody response in these patients has had little success. The development of antibodies to the replaced proteins is a critical problem that must be addressed if gene therapy is to be successful in the treatment of hemophilia and other deficient diseases (Conelly S., et al., Blood 88: 3846, 1996, Kuna SH et al., Blood 91: 784 (1988).

SUMMARY OF THE INVENTION

The invention provides compositions and methods that allow the administration of a therapeutic protein to treat disorders while reducing the development and / or progression of an immune response to the therapeutic protein.

The invention provides compositions comprising a first agent that promotes hemostasis and a second agent that inhibits the costimulatory signal in a T cell.

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

In one embodiment, the first agent is Factor VIII. In another embodiment, the first agent is a B-region deleted Factor VIII variant. In one embodiment, the first agent is Factor IX. In another embodiment of the invention, the first agent is Von Willebrand factor.

In one embodiment of the invention, the second agent is a soluble form of the co-stimulatory molecule. In a preferred embodiment of the invention, the second agent is a soluble form of CTLA4. In another preferred embodiment of the invention, the second agent is a soluble form of the B7-1 receptor, a soluble form of the B7-2 receptor, or a combination thereof. In a more preferred embodiment of the invention, the second agent is CTLA4Ig. In another preferred embodiment of the invention the second agent is B7-1Ig or B7-2Ig. In another preferred embodiment, the second agent is a soluble form of CD40 or CD40L.

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

The invention further provides methods of treating a hemostatic disorder in a subject comprising administering to the subject a composition of the invention such that the hemostatic disorder is treated.

In one embodiment of the invention, the subject exhibits a substantial titer of antibodies that bind to the first agent. In another embodiment, the subject does not exhibit a substantial titer of antibodies that bind to the first agent.

In one embodiment, the methods comprise administering a composition comprising an agent that inhibits a co-stimulatory signal in a T cell.

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

· * * * * * * * * «· · · · · · · · · · · · ·

The invention further provides methods of treating a hemostatic disorder in a subject comprising administering to the subject a first agent that promotes hemostasis and a second agent that inhibits a costimulatory signal in a T cell by treating the hemostatic disorder.

Another embodiment of the invention provides methods for treating a hemostatic disorder in a subject, comprising administering to the subject a first agent that promotes hemostasis and a second agent that inhibits a co-stimulatory signal in a T cell such that immunological tolerance to the first agent occurs while treating the hemostatic disorder. .

In one embodiment of the invention, the first agent is Factor VIII. In another embodiment, the first agent is a B-region deleted Factor VIII variant. In another embodiment, the first agent is Factor IX. In another embodiment of the invention, the first agent is von Willebrand factor.

In one embodiment of the invention, the second agent is a soluble form of the agent that introduces a costimulatory signal into a T cell. In a preferred embodiment of the invention, the second agent is a soluble form of CTLA4. In a more preferred embodiment of the invention, the agent is CTLA4Ig. In another preferred embodiment of the invention, the second agent is a soluble form of the B7-1 receptor, a soluble form of the B7-2 receptor, or a combination thereof. In another preferred embodiment of the invention the second agent is B7-1Ig or B7-2Ig.

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

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

In one embodiment, the subject exhibits a substantial titer of antibodies that bind to the first agent.

The invention represents hemostatic disorders by allowing administration to treat the disorder, the progression of an immune response to significant that provides therapeutic whilst treating progress with the means and protein, reducing the development of the therapeutic protein.

methods, wherein and / or

I. Definitions

The term "hemostatic disorder" includes disorders that lead to abnormal bleeding and / or thrombosis. Normal hemostasis limits blood loss by a series of interactions between components of blood vessel walls, platelets and plasma proteins. For example, hemostatic disorders occur due to failure of platelet aggregation and / or fibrin clot formation, which may lead to an inappropriate response to a disease or trauma, such as uncontrolled bleeding. Such disorders can be detected, for example, by determining bleeding, partial thromboplastin time (PTT), prothrombin time (PT), thrombin time (TT), or quantitative determination of fibrinogen using methods well known in the art. Examples of haemostatic disorders include haemophilia A, B and von Willebrand's disease.

The term "hemostasis promoting agent" includes a protein or polypeptide that is absent or insufficient in a subject and which, when administered to the subject, ameliorates or treats a hemostatic disorder. Preferred agents that promote hemostasis include coagglutination factors such as factor VIII, factor IX, VWF, and analogs thereof.

The term "B7 family or" B7 molecules include costimulatory molecules that share amino acid sequence identity with B7 polypeptides, for example, B7-1, B7-2 polypeptides. 4 4 4 4 4 4 4 44 444 444 or B7-3 (recognized by antibody) In addition, the B7 family molecules share a common function, such as the ability to bind to a B7 family ligand (e.g., one or more of CD28, CTLA4 or ICOS) and the ability to co-stimulate T cell activation.

The B7 polypeptides are capable of providing costimulation for activated T cells, thereby inducing T cell proliferation and / or cytokine secretion, or inhibiting co-stimulation of T cells, for example when they occur in soluble form. Representatives of the B7 family include B7-1, B7-2 and soluble fragments or derivatives thereof. In one embodiment of the invention, B7 family members bind to CTLA4, CD28, ICOS, and / or other ligands on immune cells and have the ability to inhibit or induce co-stimulation of immune cells.

The term "agent that inhibits a co-stimulatory signal in a T cell includes agents that inhibit a signal generated by the interaction of a co-stimulatory molecule on an antigen-containing cell (APC), for example, a B7 family molecule and its receptor on a T cell. Co-stimulatory molecules on APCs (e.g., members of the B7 family) and their analogous ligands on T cells (e.g., CTLA4, CD28, and ICOS) are collectively referred to herein as co-stimulatory molecules. An agent that inhibits the co-stimulatory signal may act either extracellularly to inhibit the interaction between the co-stimulatory molecules, thereby blocking the production of intracellular signals, or may act intracellularly, and thus inhibit the co-stimulatory signals on the signal transduction pathway. Examples of agents are described below and include, for example, soluble forms of costimulatory molecules and antibodies that bind to costimulatory molecules.

The term "immune response suppression" includes the reduction of an immune response (e.g., suppression, inhibition or inhibition) in a patient who does not exhibit an existing immune response or shorten the length and duration of an existing immune response. The term "immune response" includes any type of immune response that is initiated by or on the basis of co-stimulatory signals. 999 99999 depends, for example, on a cellular or subject's humoral response, which may occur in a subject in response to a foreign antigen. In one embodiment, the immune response is an antibody response to an antigen that promotes hemostasis (e.g.

VII, VWF or factor IX). The term "immunotolerization" involves the induction of antigen-specific tolerance, which can be measured using a method known in the art, for example by measuring a secondary immune response (e.g., a cellular or humoral response) to an antigen.

II. Agents that promote hemostasis

In one embodiment, the agent that promotes hemostasis is Factor VIII. The term "factor VIII" includes proteins exhibiting procoagulant activity characteristic of factor VIII. In one embodiment, the Factor VIII proteins are naturally occurring Factor VIII proteins. Such proteins may be purified from the blood or administered as a blood product or as an enriched blood product. In one embodiment of the invention, high purity factor VIII can be produced by adsorption and elution of factor from a blood product on an antibody column with monoclonal antibodies. Alternatively, such naturally occurring proteins may be recombinantly prepared using nucleic acid molecules, with naturally occurring nucleic acid molecules being preferred. For example, in one embodiment of the invention, factor VIII proteins are prepared by expressing a factor VIII nucleic acid molecule in a cell using a method well known in the art to produce the factor VIII protein.

VIII. The nucleotide sequence (and corresponding amino acid sequence) of human factor VIII is known (see, for example, Toole et al., Nature 1984, 312: 5992, or GenBank accession numbers X01179, K01740).

In one embodiment of the invention, the haemostasis promoting agent is a non-naturally occurring factor VIII, such as, for example, a non-naturally occurring factor VIII, e.g. A mutant form of factor VIII that retains the therapeutic function of factor VIII, such as hemostasis-promoting activity. For example, DNA sequences capable of hybridizing to DNA encoding human Factor VIII under conditions that precede hybridization with genes other than Factor VIII genes (e.g., conditions that correspond to a temperature of 65 ° C in 5x concentrated SSC (1x SSC = 150 mM NaCl / 0, 15 M sodium citrate) or homologous DNA sequences that retain sequence identity along regions of a nucleic acid molecule that encode protein domains that are important in the function of Factor VIII can be used to produce Factor VIII proteins within the scope of the invention. For example, U.S. Patent Nos. 5,744,446, 5,663,060, 5,583,209, 5,661,008, 5,422,260 and 5,707,832 are cited.

In another embodiment of the invention, the haemostasis promoting agent is a Factor VIII protein in which at least one region (e.g., a non-essential region) of the protein is deleted. For example, in one embodiment, the Factor VIII protein is a modified Factor VIII protein in which one or more amino acids have been deleted or substituted between 90 Kd and 69 Kd cleavage sites with respect to native Factor VIII, as described in more detail in US Patent No. 4,868,112.

In another embodiment of the invention, the haemostasis promoting agent is a Factor VIII analogue which comprises a deletion of one or more amino acids between the 50/40 and 73 kD cleavage site, which can be obtained by methods analogous to those described in US Patent No. 5,940,549. In a preferred embodiment of the invention, the Factor VIII analog retains some or all of the amino acid sequence region between the 80 kD and 73 kD cleavage sites. In one embodiment of the invention, part or all of this region is replaced by a corresponding acidic region immediately adjacent to the 50/40 cleavage site. In another embodiment of the invention, Factor VIII proteins are analogs (with or without deletions as described above) as described in International Application PCT / US87 / 01299 where there were spanning sites

740, 776, 1313, 1648, for example, to one or more arginine cleavage residues at positions 226, 336, 562, to confer proteolytic cleavage of multiple amino acids by different known directed or 1721 resistance, e.g. standard site mutagenesis.

Agents that promote the haemostasis of hybrid factor VIII, which include factor VIII and a portion of the non-human factor VIII protein). Such also includes a protein of human factor VIII species of the species (e.g., proteins may be in the art, such as

744,446, 5,663,060, and 5 porcine preparations are described from another hybrid using methods known in U.S. Pat.

583

209.

Furthermore, these documents US No. 5,693 facts

499, 5,681 describe i

746, 5,663,060, in U.S. Pat

583 209,

455 031.

563,045, 5,560,951 and 5

In another embodiment, the agent is

IX includes factor IX cell isolated

IX may be factor IX. The term "factor that promotes hemostasis, isolated by line and recombinantly from culture is purified from blood or enriched media or from plasma transformed with the produced factor IX host cells. The factor can be applied as a blood product

In one embodiment of the invention, the highly purified factor is produced by adsorption and elution of the factor from the blood on a column also containing the product.

IX can purify the product monoclonal

639 857, 5 patent 849. In other occurring proteins, the proteins may, preferably, be acids. IN

457 181 and 5 286 blood antibodies.

documents

Methods

U.S. Pat.

For example, Factor IX proteins are prepared by expressing a Factor IX encoding nucleic acid molecule in a cell using methods known in the art to obtain Factor protein.

IX Examples of genetic constructs for Factor IX expression are described in U.S. Patent Nos. 5,650,503 and 4,994,371.

The nucleotide sequence and amino acid sequence of factor IX is known in the art (see, for example, Yoshitake et al., 1985. Biochemistry 24: 3726 or GenBank accession numbers K02402, A07407, A01819 or X54500).

For example, in other embodiments, Factor IX includes proteins described in U.S. Patent Nos. 4,994,371, 5,171,569, 5,679,639, and 5,714,583.

In addition to naturally occurring forms of Factor IX, the term Factor IX also includes non-naturally occurring forms, such as mutant forms of Factor IX, which retain the therapeutic functions of Factor IX, such as hemostasis-promoting properties. For example, DNA sequences capable of hybridizing to DNA encoding human Factor IX under conditions that prevent hybridization with non-Factor IX genes (e.g., equivalent to 65 ° C in a 5x concentrated SSC solution (wherein the 1x concentrated SSC solution comprises 150 mM NaCl) In addition, DNA sequences that retain sequence identity along regions of a nucleic acid molecule that encode protein domains that are important in the function of Factor IX may be used to produce the Factor IX proteins of the invention.

Factor VIII or factor IX proteins may also be obtained in a commercial manner. For example, concentrated forms of factor VIII are available, for example, as Immunate® (Immuno), Beriate® (Behring), monoclonal antibody-purified forms of factor VIII are available, for example, as OctanativM® (Pharmacia), Hemofil M® (Baxtar) and Monoclate-P® (Armor) and recombinant forms of factor VIII are available, for example, as Recombinate® (Baxter) and Kogenate® (Bayer). Also available is a recombinant form with deleted Factor B region VIII, rVIIISQ® (Pharmacia and Upjohn, Stockholm). Factor IX can be obtained, for example, as Nanotiv® (Kabi Pharmacia) or

Immunine® (Immuno), monoclonal antibody-purified factor IX is also available as Mononine® (Armor). Recombinant factor IX is also available, for example, as BeneFIX® (Genetics Institute).

VWF is a large multimeric plasma protein composed of subunits of a single glycoprotein. The VWF subunits are linked together by disulfide bridges. VWF circulates in plasma as a • multimer within the dimer range to multimers containing more than 50 subunits. The dimers contain two linked subunits, probably at their C-terminus, with flexible rod-shaped regions and are believed to be protomers of multimerization. Protomers are linked through large, probably N-terminal globular regions to form multimers. It turns out that VWF is produced as a glycosylated precursor with a molecular weight of 260,000, which is subsequently processed and sulfated. After dimerization and multimerization and proteolytic cleavage, the mature protein has a molecular weight of about 225,000. VWF produced in a recombinant manner. The nucleotide and amino acid sequence of VWF is known in the art (Sadler et al., 1986, Cold Spring Harbor Symposium in Qunatitative Biology 51: 515 or GenBank accession number L15333 or K03028). This is further described in EP 0197592 B1.

In addition to naturally occurring forms of VWF, the term "VWF" also includes non-naturally occurring forms, such as mutant forms of VWF, that retain therapeutic properties, such as those promoting VWF hemostasis. For example, DNA sequences capable of hybridizing to DNA encoding human VWF can be used to produce VWF proteins of the invention under conditions that prevent hybridization with non-VWF genes (e.g., conditions that correspond to a temperature of 65 ° C at 5 x concentrated SSC solution (1 x concentrated SSC solution contains 150 mM NaCl / 0.15 M citrate)

4 < 4 &gt; 44 4444 &gt; 444 44 444 44 444 sodium), and DNA sequences that retain sequence identity across regions of the nucleic acid molecule that encode protein regions, which are important for VWF function.

In one embodiment of the invention, the agents that promote hemostasis are from mammals. In a preferred embodiment of the invention, the agents that promote hemostasis are derived from pigs. In another preferred embodiment of the invention, the agents that promote hemostasis are human. In another embodiment of the invention, the agents that promote hemostasis are hybrid molecules.

III. Immunomodulating agents

In one embodiment, the agent that inhibits the costimulatory signal in a T cell is a naturally occurring form of a co-stimulatory molecule. Naturally occurring forms of costimulatory molecules can be purified from cells or can be recombinantly produced using methods known in the art. For example, costimulatory proteins can be prepared by expressing a nucleic acid molecule encoding a co-stimulatory molecule in a cell such that a co-stimulatory molecule is produced. The nucleotide sequences of the co-stimulatory molecules are known in the art and can be found in the literature or in a database such as GenBank. For example, B7-2 (Freeman et al., 1993 Science. 262: 909 or GenBank accession number P42081 or A48754), B7-1 (Freeman et al., J. Exp. Med. 1991. 174: 625 or GenBank , accession number P33681 or A45803), CTLA4 (Ginsberg et al., 1985. Science 228: 1401 or GenBank, accession number P16410 or 291929) and CD28 (Aruffo and Seed. Proc. Natl. Acad. Sci. 84: 8573 or GenBank). , accession number 180091), ICOS (Hutloff et al., 1999, Nature. 397: 263, WO 98/38216) and related sequences.

In addition to naturally occurring forms of co-stimulatory molecules, the term "co-stimulatory molecules" also includes non-naturally occurring forms, for example, mutant forms of co-stimulatory molecules that retain the function of a co-stimulatory molecule, such as the ability to bind to the corresponding receptor. For example, DNA sequences capable of hybridizing to DNA encoding a B7 molecule, a CTLA4 molecule, a CD28 molecule, or an ICOS molecule under conditions that prevent hybridization with genes that are not genes of the co-stimulatory molecules (e.g., conditions equivalent to 65 ° C at 5 x concentrated) SSC solution (wherein 1x concentrated SSC solution contains 150 mM NaCl / 0.15 M sodium citrate) are co-stimulatory molecules of the invention. Alternatively, DNA sequences that retain sequence identity along regions of a nucleic acid molecule that encode protein regions that are important in the function of co-stimulatory molecules, e.g., binding to other co-stimulatory molecules, can be used to produce co-stimulatory proteins. which can be used as agents that inhibit the co-stimulatory signal in a T cell. It is preferred that the non-naturally occurring co-stimulatory molecules exhibit substantial amino acid identity (e.g., greater than 70%, preferably greater than 80%, and in particular greater than 90-95%) with the naturally occurring amino acid sequence of the extracellular region costimulatory molecule.

To determine the amino acid residues of the co-stimulatory molecule that are likely to be important in binding the costimulatory molecule to its receptor, amino acid sequences containing the extracellular regions of co-stimulatory molecules of various species, such as mice and human, can be aligned and recorded conserved (i.e. identical) residues. This can be done, for example, using any standard program such as MegAlign (DNA STAR). Such conserved or identical residues are likely to be necessary for proper binding of the co-stimulatory molecules to their receptors and are unlikely to be subject to alteration.

Specific residues of co-stimulatory molecules that are important in binding have also been determined. For example, • • 999999 9 9 »· 9 9 9 9 999 • 9 · 999 9 99 • 9999 9 · 9 · 9 • · 9 99999 ♦ · ♦ ··· ♦♦ 999 99999

CD28, which is critical in the interaction with the B7-1 and B7-2 receptors, was determined using site-directed mutagenesis, CD28 monoclonal antibody epitope mapping, receptor-based adhesion assays, and direct binding of Ig-fusion proteins to cell surface receptors. It has been found that the proline-rich sequence band of CD28, MYPPPY, is critical for the function of said protein (Truneh et al., 1996. Mol. Immunol. 33: 321). Regions of the B7-1 receptor molecule that are important in mediating functional interaction with CD28 and CTLA4 have been identified by mutation. Two hydrophobic residues in the B7-1 receptor-like domain, including the Y87 residue, which is conserved in all B7-1 and B7-2 molecules cloned into different species, have been found to be critical (Fargeas et al., 1995. J Exp. Med. 182: 667). Using this or a similar method, amino acid residues of the extracellular regions of co-stimulatory molecules can be determined that are critical and therefore not subject to change.

The co-stimulatory molecules can be expressed in soluble form or can be used as immunogens to generate antibodies. Such soluble costimulatory molecules or

antibodies can be used as reagents, which further inhibit in the text. ko- stimulation signal in T cell, as described A. Reagents that act extracellularly and inhibit ko- stimulating signal in T cell

1. Soluble forms of co-stimulatory molecules

In one embodiment, the agent that blocks the costimulatory signal in a cell is a soluble form of a T cell co-stimulatory molecule (e.g., CTLA4, CD28, and / or ICOS) that is capable of blocking the co-stimulation signal transduction in a T cell.

In one embodiment, an agent that blocks a co-stimulatory signal in a T cell, a soluble form of CTLA4. DNA sequences encoding the human and murine CTLA4 protein are known in the art (Dariavich, et al., (1988) Eur. J. Immunol. 18 (12), 1901-1905, Brunet, J. F.

et al., (1987) Nature 328: 267-270, Brunet, J. F. et al. (1988) Immunol. roar. 103: 21-36 and Ereeman, G. J. et al., (1992) J. Immunol. 149, 3795-3801. In certain embodiments, the soluble CTLA4 protein comprises the entire CTLA4 protein. In preferred embodiments of the invention, the soluble CTLA4 protein comprises an extracellular region of the CTLA4 protein. For example, a soluble recombinant form of the extracellular region of CTLA4 was expressed in yeast (Gerstmayer et al., 1997. FEBS Lett. 407: 63). In other embodiments, the soluble CTLA4 proteins comprise at least a portion of the extracellular region of the CTLA4 protein that retains the ability to bind to the B7-1 and / or B7-2 receptor.

In one embodiment of the invention, the soluble CTLA4 protein or portion thereof is a fusion protein comprising at least a portion of CTLA4 that binds to B7-1 and / or B7-2 and at least a portion of the second non-CTLA4 protein. In preferred embodiments of the invention, the fusion protein comprises an extracellular region of CTLA4 that is fused at the amino terminus with a signal peptide, for example from oncostatin M (see, for example, WO 93/00431).

In a particularly preferred embodiment of the invention, the soluble form of CTLA4 is a fusion protein comprising an extracellular region of CTLA4 fused to a portion of an immunoglobulin molecule. Such a CTLA4Ig fusion protein can be prepared using methods well known in the art (Linsley 1994, Perspectives in Drug Discovery and Design 2: 221, Linsley WO 93/00431 and US Patent No. 5,770,197).

In one embodiment, the agent that blocks the costimulatory signal in a T cell is a soluble form of a cell-stimulating molecule comprising an antigen (e.g., B7 family molecules such as B7-1 receptor, B7-2, and / or ICOS ligand). For example, in one embodiment of the invention, the soluble form of the co-stimulatory molecule comprises a soluble form of the B7-1 receptor or a soluble form of the B7-2 receptor or a combination of said soluble forms. DNA sequences encoding the B7 proteins are known in the art and are, for example, B7-2 (Freeman et al., 1993, 44, 44).

4 4 · • 4444 44 4 4 4 4 «

4444 «

Science 262:

909 or GenBank accession number P42081 or

A48754), B7-1 or GenBank embodiments (Freeman et al., J. Exp. Med. 1991. 174: 625 accession number P33681 or A45803). In certain embodiments, the soluble B7 protein comprises the entire B7 protein. In preferred embodiments, the soluble B7 protein comprises an extracellular region of the B7 protein. For example, the soluble recombinant form of the extracellular region of CTLA4 was expressed in yeast (Gerstmayer et al., 1997. FEBS Lett.

In another embodiment, at least an embodiment of the invention retains a portion of the extracellular soluble B7 protein of the B7 region, on CTLA4 and / or CD28.

contain which ability to bind

In one embodiment, the portion is a fusion protein comprising at least a portion of B7 that binds to CD28 and / or CTLA4 and at least a portion of a second protein that is not B7. In preferred embodiments of the invention, the B7 fusion protein comprises an extracellular region of B7 that forms a fusion at the N-terminus of soluble B7 protein or its with a signal peptide, for example from oncostatin M (described, for example, in WO 93/00431).

In a particularly preferred embodiment of the invention, the soluble form of B7 is a fusion protein comprising an extracellular region of B7 fused to a portion of an immunoglobulin molecule. the B7Ig protein can be prepared for use in the art (Linsley et al., 1994, Perspectives in and Design 2: 221, Linsley WO 93/00431, U.S. Patent No. 5,580,756).

Such a fusion method known Drug Discovery

US 5,770,197 and blocks binds to binds

2. Antibodies that bind to co-stimulatory molecules

In certain embodiments, the agent that co-stimulates the signal in a T cell is an antibody that co-stimulates the molecule. In preparing antibodies to the co-stimulatory molecules, a co-stimulatory protein, a portion of the co-stimulatory protein (e.g., a peptide derived from the co-stimulatory protein), or a fusion protein that includes all or part of the amino acid sequence of a co Stimulating molecules to generate polyclonal serum or monoclonal antibodies against the protein and / or peptide using standard methods. The term "antibodies" includes whole antibodies as well as fragments thereof. Antibody fragments (e.g., Fab 'fragments, F (ab') 2 fragments, or single chain antibodies) can be prepared using methods well known in the art. The term antibodies also include chimeric and humanized antibodies.

For example, a mammal (e.g., a mouse, hamster, or rabbit) can be immunized with an immunogenic form of a co-stimulatory protein or peptide that elicits an antibody response in a mammal. For example, the immunogen may be a recombinant protein co-stimulatory molecule or a fragment thereof, a synthetic peptide fragment, or a cell that expresses a bone-stimulating molecule on its surface. For example, the cell may be a cell comprising an antigen or a T cell or a cell transfected with a nucleic acid encoding a co-stimulatory molecule such that the co-stimulatory molecule is expressed on the cell surface. The host cells transfected to express the peptides may be any prokaryotic or eukaryotic cells. For example, a peptide that exhibits co-stimulatory molecule activity may be expressed in bacterial cells such as E. coli, insect cells (baculoviruses), yeast or mammalian cells such as Chinese hamster ovary (CHO) cells and NSO cells . Other host cells and expression vectors are described in Goeddel, (1990) or are well known in the art. Examples of vectors suitable for expression in S. cerivisae yeast include pYepSec1 (Baldari. Et al., (1987) Embo J. 6: 229-234), pMFα (Kurjan and Herskowitz, (1982) Cell 30: 933-943), pJRY88 (Schutz et al., (1987) Gene 54: 113-123) and pYES2 (Invitrogen Corporation, San Diego, CA). Baculovirus vectors available for expression of proteins in cultured insect cells (SF 9 cells) include a series of pAc (Smith et al., (1983) Mol. Cell Biol. 3: 2156-2165) and pVL

• • 9 ···· 99 99 • * • • • 99 • • 9 • 99 • • • • 9 • • 9 9 • • • • • 9 • • 9 999 ·· ·· « ·· 99

(Lucklow, V.A. and Summers, M. D., (1989) Virology 170: 3139). Typically, COS cells (Gluzman, Y. (1981) Cell 23: 175182) are used in conjunction with vectors such as pCDM8 (Seed, B., (1987) Nature 329: 840) for transient amplification / expression in mammalian cells, while CHO cells (Chinese hamster ovary cells dhfr -) are used with vectors such as pMT2PC (Kaufman et al., (1987), EMBO J., 6: 187-195), for stable amplification / expression in mammalian cells. A preferred cell line for recombinant protein production is the NSO myeloma cell line, available from 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, NY, NY). The vector DNA may be introduced into mammalian cells by conventional methods such as co-precipitation with sodium phosphate or calcium chloride, DEAE-dextran mediated transfection, use of lipofectin, or electroporation. Suitable methods for transforming host cells are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press (1989) and other laboratory guidelines. When used in mammals, expression vectors of control function are often comprised of viral material. Commonly used promoters were obtained from polyoma, adenovirus 2, cytomegalovirus, and most commonly from SV40 virus.

Peptides showing the activity of a co-stimulatory molecule expressed in mammalian cells or by other means may be purified according to a standard procedure that includes ammonium sulfate precipitation, fractionation column chromatography (e.g., interchange, gel filtration, electrophoresis, affinity chromatography, etc.) and crystallization (Enzyme)

Purification and Related

Techniques,

Methods in Enzymology,

22: 233-577 (1971)].

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ··· ··· ♦ · »· · · ·

It is within the ability of one of skill in the art to generate antibodies against human co-stimulatory molecules according to standard methods. The antibodies may be either polyclonal or monoclonal or fragments of such antibodies that bind to the antigen. Of particular interest for use in therapeutic application are antibodies that inhibit the binding of the co-stimulatory molecule to its natural ligand on the surface of the immune cell, thereby inhibiting co-stimulation of the immune cell. Preferred antibodies to the co-stimulatory molecule are antibodies capable of inhibiting or inhibiting T cell-mediated immune responses by binding B7-2 or B7-1 receptors to the surface of B cells and preventing interaction with CTLA4 and / or CD28. Other preferred antibodies against the co-stimulatory molecules are those which, in combination with a second antibody that binds to another co-stimulatory molecule, result in an enhanced inhibition of T cell co-stimulation compared to the first antibody alone. For example, a combination of anti-B7-1 and B7-2 antibodies.

A. Immunogen

The term "immunogen" refers to a composition comprising as an active ingredient a peptide having the activity of a co-stimulatory molecule, which is used in the preparation of antibodies against a costimulatory molecule. When a peptide exhibiting the activity of a co-stimulatory molecule is used in the induction of antibodies, it is clear that the peptide can be used alone or as a peptide polymer.

In order to generate suitable antibodies against the co-stimulatory molecule, the immunogen should contain an effective immunogenic amount of a peptide that exhibits the activity of the co-stimulatory molecule and is typically conjugated to a carrier. The effective amount of the peptide per unit dose depends, inter alia, on the species of inoculated animal, the animal's body weight, and the immunization regimen selected, as is known in the art. The immunogenic composition will typically contain a peptide concentration in the range of 10 micrograms to 500 milligrams per immunization dose, with about 50 micrograms to about 50 milligrams per dose being preferred. The immunizing composition may also include an adjuvant as part of a diluent. Ajuvants such as complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) and alum are adjuvants that are well known in the art and are available from several sources.

One skilled in the art will appreciate that instead of using naturally occurring forms of the co-stimulatory molecule in immunization,

synthetic peptides can be used to produce the desired antibodies.

Co-stimulatory molecules or peptide fragments, both soluble and membrane bound, can be used as the immunogen and can also be isolated by immunoaffinity purification. The purified form of the co-stimulatory protein molecule, the isolation of which is described above, can also be used directly as an immunogen, or alternatively can be coupled to a suitable protein carrier using a suitable method, which may be chemically coupled or genetically engineered using cloning gene of the co-stimulatory molecule.

The peptide or protein selected for immunization may be engineered to enhance its immunogenicity. For example, methods for conferring immunogenicity on a peptide include conjugation to a carrier or other methods well known in the art. It is further possible to synthesize any peptide that has been selected for immunization. In certain embodiments, such peptides may be synthesized as branched polypeptides to enhance immune responses as known in the art (see, for example, Peptides, Edited by Bernd Gutte Academic Press 1995. pp. 456-493).

The purified protein of the co-stimulatory molecule may also be covalently or non-covalently treated with a non-proteinaceous material such as lipids or carbohydrates to increase immunogenicity or solubility. In another case purified • · · · · ·

the protein of the co-stimulatory molecule may be associated with or incorporated into a viral particle, a replicating virus or other microorganism to enhance immunogenicity. For example, the protein of the co-stimulatory molecule may be chemically coupled to a viral particle or microorganism, or an immunogenic portion thereof.

In one embodiment of the invention, the purified costimulatory molecule protein (e.g. produced by limited proteolysis or recombinant DNA methods) is conjugated to a carrier that 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, gastropod hemocyanin, egg ovalbumin, or bovine gamaglobulins. Synthetic polyamino acids, such as polylysine or polyarginine, can also be used as carriers. With regard to the covalent attachment of the protein of the co-stimulatory molecule or peptide fragments to a suitable immunogenic carrier, there are a number of chemical cross-linking agents known in the art. Preferred cross-linking agents are heterobifunctional cross-linking agents that can be used to bind proteins sequentially. Numerous heterobifunctional cross-linking agents are known in the art, including succinimidyl 4- (N-maleimimidomethyl) cyclohexane-1-carboxylate (SMCC), mmaleinimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-succinimidyl- (4-iodoacetyl) aminobenzoate (SIAB), -4 (p-maleimidimidenyl) butyrate (SMPB), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), 4-succinimidyloxycarbonyl-α-methyl-α- (2-pyridyldithio) -toluene (SMPT), N-succinimidyl-3 - (2-pyridylthio) propionate (SPDP), succinimidyl 6- [3- (2-pyridyldithio) proponate] hexanoate (LC-SPDP).

It may also be desirable to simply immunize with whole cells that express the protein of the co-stimulatory molecule on their surface. Various cell lines may be used as immunogens to produce monoclonal antibodies against the costimulatory molecule antigen, including, but not limited to, activated B cells. For example, spleen B cells can be obtained from the subject and activated by anti-immunoglobulin. Alternatively, a B cell cell line may be used when a co-stimulatory molecule such as a Raj cell cell line is expressed on their surface (Burkett's lymphoma B cells, see, for example, Freeman, GJ et al., (1993) Science 262: 909-). 911) or JY B, which is a lymphoblastoid cell line (Azuma, M. et al., (1993) Nature 366: 76-79). Whole cells that can be used as immunogens in the production of antibodies specific for a costimulatory molecule also include recombinant transfectants. For example, COS and CHO cells may be reconstituted by transfecting cDNA of the co-stimulatory molecule as described by Knudson et al., 1993, PNAS 90: 4003-4007, Travernor et al., 1993, Immunogenitics 37: 474-477, Dougherty et al. 1991,

J. Exp. Copper. 174: 1-5) and Aruffo et al., 1990, Cell 61: 13031313) to produce an intact cell-surface co-stimulatory molecule. Said transfectants may be used as an immunogen in the production of antibodies against a co-stimulatory molecule of a preselected specificity. Other examples, particularly eukaryotic cells capable of transfectants to glycosylate the production of an immunogen, can be any method of expressing the transfected genes by co-stimulating cell surface.

stimulating molecules, however, there is known a cellular protein that acts on the molecules

B. Polyclonal antibodies against a co-stimulatory molecule

Polyclonal antibodies against a purified protein or peptide that exhibit the activity of a co-stimulatory molecule can generally be generated in animals by administering multiple subcutaneous (sc) or intraperitoneal (ip) injections of the immunogen that makes up the co-stimulatory molecule, such as 9 9 9 9 99 99 99 99 99 99 99 99 99 99 99 99 99 9 For example, the extra-cellular region of the protein of the co-stimulatory molecule, and the adjuvant. For example, as described above, it may be desirable to conjugate a co-stimulatory molecule (including fragments containing certain epitopes) with a protein that is immunogenic to the species to be immunized, for example, hemocyanin of gastropod gastropods or serum albumin.

In general, established and conventional methods of stimulating and producing antibodies can be used for methods and programs involving host animals or cultured antibody-producing cells. In one embodiment of the invention, animals are typically immunized against conjugates or derivatives of an immunogenic co-stimulatory molecule by combining about 1 µg to 1 mg of the conjugate with Freund's complete adjuvant and introducing the solution by interdermal injection at multiple sites. One month after the first injection, animals are immunized with 1/5 to 1/10 of the original amount of conjugate in Freund's complete adjuvant (or other suitable adjuvant) by subcutaneous injection at multiple sites. Seven to fourteen days later, blood samples are taken from the animals and the titer of anti-costimulatory molecules is determined in serum. Animals are immunized repeatedly until the titer value increases. It is preferred that the animals are repeatedly immunized with a conjugate of the same protein of the co-stimulatory molecule but conjugated to a different protein and / or using a different cross-linking agent. Conjugates may also be formed in recombinant cell culture as protein fusions. Aggregating agents such as alum are also used to enhance the immune response.

Such a population of antibody molecules produced by a mammal is called &quot; polyclonal because this population contains antibodies with different immunospecific and affinity for the costimulatory molecule. Antibody molecules can then be obtained from mammals and isolated by well known methods, such as using DEAE Sephadex, to obtain an IgG fraction. To enhance the specificity of the antibodies, the antibodies can be purified by immunoaffinity chromatography using a solid phase fixed immunogen. The antibodies are contacted with the solid phase-fixed immunogen for a time sufficient to allow the immunogen to immunoreact with the antibody molecules to form a solid-phase fixed immunocomplex. Bound antibodies are separated from the complex by a standard method.

C. Monoclonal antibodies against the co-stimulatory molecule

The term "monoclonal antibodies" or "monoclonal antibody compositions" encompasses a population of antibody molecules that contain only one kind of antigen-binding site capable of immunoreacting with a particular epitope of the co-stimulatory molecule. Thus, the monoclonal antibody composition most often exhibits a single binding affinity for a particular costimulatory molecule protein with which it immunoreacts. Preferably, the monoclonal antibody used in said method is further characterized as immunoreactive with a co-stimulatory molecule obtained from a human.

The monoclonal antibodies useful in the compositions and methods of the invention are directed to the epitope of the antigen by a costimulatory antibody by interacting with the molecule and the antigen by the co-stimulatory ligand (s) of stimulating T ligand. molecules production in culture.

hybridomas,

Milstein that forming a complex between a co-stimulatory molecule inhibits molecules with its natural surface on the immune cell, inhibiting the cells by the co-stimulatory molecule interaction, and monoclonal antibodies to the co-stimulatory epitope can be prepared using a method comprising antibody molecules by a continuous cell line. methods include, but are originally described in Nature 256: 495-497)

These which

1975, are not limited to Kohler and, a more recent hybridoma technique

4: 72), human B cells (Kozbor et al., 1983 Immunol. Today method of EBV-hybridoma (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., Pp. 77- 96 and the trioma method Other methods that can effectively lead to the generation of monoclonal antibodies useful in accordance with the invention include phage display methods (Marks et al., 1992 J. Biol. Chem. 16007-16010).

In one embodiment of the invention, the antibody preparation administered according to the invention is a monoclonal antibody produced by a cell line of hybridomas. Methods for hybridoma fusion are first described in Kohler and Milstein et al., Nature (1975) 256: 495-97, Brown et al., (1981) J. Immunol. 127: 53946; Brown et al. (1980) J. Biol. Chem 255: 4980-83, Yeh et al., (1976) PNAS 76: 2927-31 and Yeh et al., 1982 Int. J. Cancer 29: 269-75). Accordingly, the monoclonal antibody compositions of the invention may be produced as follows:

a) immunizing the animal with a co-stimulatory molecule. Said immunization is typically accomplished by administering an immunogen of the co-stimulatory molecule to an immunologically competent mammal in an immunologically effective amount, i.e., an amount sufficient to elicit an immune response. Preferably, the mammal is a rodent such as a rabbit, rat or mouse. The mammal is then maintained for a time sufficient to produce cells secreting antibody molecules that immunoreact with the immunogen of the co-stimulatory molecule. Such immunoreactivity is detected by testing the resulting antibody molecules for immunoreactivity with an immunogenic protein preparation. Alternatively, it may be desirable to test antibody molecules with a protein preparation in the form in which it is to be detected by the antibody molecules in the assay, for example in the form of a membrane-stimulating molecule. These test methods are well known in the art.

b) A suspension of antibody-producing cells taken from each immunized mammal that secretes the desired antibody is then prepared. After sufficient time, the mouse is sacrificed and somatic antibody-producing lymphocytes are obtained. Antibody producing cells can be obtained from lymph nodes, spleen and peripheral blood of animals. Preferred are spleen cells which can be mechanically divided into individual cells in a physically tolerable medium using methods well known in the art. Mouse lymphocytes show higher with mouse myelomas, as described below, using rat somatic cells, rabbit spleen cells encoding immobilization of myeloma cells, usually in the presence of a fusion polyethylene glycol (PEG). Any line such as stable fusion may be used as the fusion partner of the methods

They can also

Chromosomes of immunoglobulins x63-Ag8.653 or in the culture collection

Rockville, Md.

percentage in text.

and frogs, desired spleen reagents, for example Sp2 / O-Ag14.

“American line

These

Cell type such as by standard number of myeloma cell myeloma P3-NS1 / 1-Ag4-1, P3 myeloma lines are available from Culture Collection (ATCC),

The resulting cells, which include in the selection medium, then allow the growth of which unfused eventually die, are cultured in the parent cell. Only hybridoma limited dilutions, desired hybridomas, such as HAT medium, into isolated clones. In antibodies using clones, dilutions are made from myeloma or lymphocytes, and the desired specificities can be tested to obtain hybridoma supernatants, for example, by the antigen method used in immunization, they can then be subcloned under conditions to produce monoclonal different conventional isolation methods which it deprives them of other monoclonal presence of immunoassays

Positive

There are also monoclonal antibodies, contaminants. Commonly used methods of antibody purification are ammonium sulfate precipitation, ion exchange, and affinity chromatography (see et al., In Monoclonar Hybridoma Antibodies: Applications, Hurell (ed.) Pp. 51-52 (CRC Hybridomas produced according to said restrictive antibody methods). , and protein purification and chromatography, for example, Zola

Techniques and

Press 1982)).

can multiply • 9 9 9 9 9 9 9

9

9 9 9 9 9

9 9 9 9 9

999 999 99 999 99 999 in vitro or in vivo (in ascites fluid) using methods well known in the art.

Generally, individual cell lines can be propagated in vitro, for example in a laboratory culture apparatus, and the culture medium, which then contains a high concentration of a single specific monoclonal antibody, can be isolated by alloying, filtration or centrifugation. Alternatively, the yield of monoclonal antibodies may be increased by introducing a hybridoma sample by injection into a histocompatible animal used to provide somatic or myeloma cells for the original fusion. Immunized animals develop tumors producing a specific monoclonal antibody produced by a fusion cell hybrid. Animal body fluids, such as ascites or serum, provide monoclonal antibodies at high concentrations. When human or EBV hybridomas are used, it is necessary to avoid rejection of the xenograft by injection introduced into an animal such as a mouse. Immunodeficiency or nude mice may be used or the hybridoma may be passaged first to irradiated nude mice to form a solid subcutaneous tumor, cultured in vitro, and then injected intraperitoneally into intact irradiated nude mice to develop ascites tumors that secrete large a number of specific human monoclonal antibodies.

Culture media and animals that can be used in the preparation of these compositions are well known in the art and are readily available and include synthetic culture media, inbred mice and the like. An example of a synthetic culture medium is Dulbecco's Minimum Essential Medium (DMEM, Dulbecco et al. (1995) Virol. 8: 396) supplemented with 4.5 g / L glucose, 20 mM glutamine and 20% fetal serum. An example of an inbred mouse strain is Balb / c.

D. Humanized antibodies against the co-stimulatory molecule

r.

In the case of non-human, a recognized approach in the patient can be that antibodies produced in subjects are used as a medicine in humans, are of varying degrees as foreign substances and can generate an immune response. One eliminates or minimizes said problem, for the general reduction of immunity, is the production of chimeric antibody derivatives, i.e., antibody molecules that combine a non-human animal variable region and a human constant region. Such antibodies are equivalents of the monoclonal and polyclonal antibodies described above, but may be less immunogenic when administered, therefore, they are more likely to be tolerated by humans and patients.

Can stimulatory antibodies can produce chimeric mouse-human monoclonal (i.e., chimeric antibodies) reactive with the molecule, for example, by a method recently developed for chimeric antibodies. Humanized antibody production can be produced, for example, by replacing the immunogenic portion of the antibodies with a corresponding, coding constant region but not an immunogenic portion. Genes (or other mouse species) of antibodies encoding an international anti-costimulatory human constant

Akira et al.,

M., European region molecules are replaced (Robinson et genes et al., PCT / US86 / 02269,

European Patent

Application WO

European Patent Application 184, 187, Taniguchi,

Morrison et al., Patent Application 171,496,

Application 174, 94, Neuberger et al., PCT 86/01533, Cabilly et al., European Patent

Application 125,

023, Better et al. (1988) Science 240: 10411043; Liu et al. (1987) PNAS 84: 3439-3443; Liu et al. (1987) J. Immunol. 139: 3521-3526, Sun et al., (1987) PNAS 84: 214-218, Nishimura et al., (1987) Canc. Res. 47: 999-1005

Wood et al., (1985) Nature 314: 446-449 and Shaw et al., (1988) J. Natl. Cancer Inst. 80: 1553-1559). A general overview of "humanized antibodies can be found in the publication."

99 9 9 9 9 999

9 9 9 9 9 9 999

9 9 9 9 9 9 99

9 · 9 9 9

999 999 99 999 99999

Morrison, S.L. (1985) Science 229: 1202-1207 and Oi et al., (1986) BioTechniques 4: 214. Such methods include isolating, manipulating, and expressing nucleic acid sequences that encode all or part of an immunoglobulin variable region from at least one of the heavy and light chains. Sources of such nucleic acid are well known in the art and can be obtained, for example

from a hybridoma produce antibodies against co-stimulatory molecule. Chimeric cDNA can then cloning to appropriate expressive vector. Appropriate “Humanized the antibodies are can v j otherém case produce by substitution of CDR or CEA (Winter U.S. Pat. Patent

No. 5,225,539 to Jones et al. (1986) Nature 321: 552-525, Verhoeyan et al., (1988) Science 239: 1534, and Beidler et al., (1988) J. Immunol. 141: 4053-4060.

E. Combination Antibodies against the Co-Stimulation Molecule

Both the monoclonal and polyclonal antibody compositions of the invention can also be produced by other methods well known in the art of DNA recombination. In order to identify and isolate antibody fragments that exhibit a certain antigen specificity, an alternative method of "imaging of combination antibodies" has been developed. This method can be used to produce monoclonal antibodies against a costimulatory molecule, as well as to prepare a population of polyclonal antibodies against a co-stimulatory molecule (Sastry et al., (1989) PNAS 86: 5728, Huse et al., (1989) Science 246: 1275 and Orlandi et al., (1989) PNAS 86: 38833). After immunizing the animal with an immunogen of the co-stimulatory molecule as described above, the antibody repertoire of the resulting B cells is cloned. Methods for directly obtaining the variable regions of the DNA sequence of different populations of immunoglobulin molecules are generally known. These are PCR and the use of oligomeric primers. For example, mixed oligonucleotide primers corresponding to the 5 'leader sequences (signal peptide) and / or core structural sequences 1 (FR1) as well as a primer corresponding to the primer

The 3 ' constant regions can be used to amplify the heavy and light chain variable region PCRs derived from a variety of murine antibodies (Larrick et al., (1991) Biotechniques)

11: 152-156. A similar strategy can be used to amplify the heavy and light chain variable regions derived from human antibodies (Larrick et al., (1991)

Methods:

Companion to Methods

Enzymology 2:

106-110).

Cloning of human immunoglobulin V genes in the formation of the repertoire

The ability of particular human importance in light of the advancement of antibodies in transgenic animals (see, for example, Bruggmen et al. (1993) Year Immunol.

7: 33-40; Tuaillon et al. (1993) PNAS

90: 3720-3724; Bruggeman et al., (1991) Eur. J.

Immunol. 21:

1323-1326 and Wood et al.,

PCT WO 91/00906).

of the invention of bone cells of one embodiment

B, such as marrow or protocol formulations (e.g., U.S. Pat.

3833-3837, Sastry et al., RNA cells are isolated from peripheral spleen, standard in Orlando.

PNAS

1275-1281).

activated blood cells using

683,202 et al., PNAS (1989) 86:

86: 5728-5732 and Huse et

First strand cDNA with primers specific for constant al., (1989) Science 246:

synthesizes using the heavy chain region and each of the κ and λ light chains, as well as using primers

Using PCR primers variable for the signal sequence.

regions are amplified by heavy and light chain variable regions, either individually or in combination, and ligated into suitable vectors for further manipulation in forming imaging packages. Oligonucleotide primers useful in amplification may be unique or degenerate, or inosine may be incorporated at degenerative positions. Restriction enzyme sequences may also be included in the primers, allowing cloning of the amplified fragment into a vector in a predetermined reading frame for expression.

The V-gene library cloned from antibodies, by immunization, can be expressed by a population that has formed imaging packages, preferably obtained from filamentous phage to form a library that contains a number of different ideal screening for large screening rapid isolation antibodies. In a system that allows antibodies that produce libraries, after each cycle of affinity separation and easy antibody gene from purified imaging packages. In addition to commercially available kits for creating libraries of phage display (e.g., Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01 and Surf Stratagene ZAP ^(TM), catalog and reagents particularly suitable for the library displaying antibodies No. 240612), examples of methods used in the preparation various (Ladner et al., U.S. Pat.

No. 5,223,409 to Kang et al., International Application No. WO

91/17271, against a co-stimulatory molecule

92/18619,

Winter et

Dower et al., International Application No. WO

al., International Application WO 92/20791, Markland et al., international application No. WO 92/15679, Breitling et al., international application WO 93/01288, McCafferty et al., international application C. WO 92/01047, Garrard et al., international application C. WO 92/09690, Ladner et al., international application No. WO 90/02809, Fuchs et al. (1991) Bio / Technology 9: 1370- -1372, Hay et al. , (1992) Hum. Antibod. Hybridomas 3 Huse et al., (1989) Science 246: 1275- 1281, Griffths et al., (1993: ) EMBO J. 12: 725-734. Hawkins et et al., (1992) J. Mol. Biol. 226: 889-896 , Clackson et al.,

(1991)

Nature 352: 624-628, Gram et al., (1992) PNAS 89: 35763580,

Garrad et al. (1991) Bio / Technology 9: 1373-1377.

Hoogenboom et al., (1991) Nuc. Acid Res. 19. 4133-4137 a

Barbas et al. (1991) PNAS 88: 7978-7982.

In certain embodiments of the invention, the heavy or light chain V region domains can be expressed on the same polypeptides linked by a flexible linker behind a single-chain Fv fragment, and the scFV gene is subsequently cloned into the desired expression vector or cue genome. As generally described in McCafferty et al., Nature (1990) 348: 552-554, the entire V _H and V _L regions of the antibody linked by a flexible linker (Gly ₄ -Ser) ₃ can be used to produce a single chain antibody that can The protein may be separable depending on the antigenic affinity. Isolated scFV antibodies that are an immunoreactive co-stimulatory molecule can then be incorporated into a pharmaceutical composition for use in the invention.

F. Hybridomas and Methods of Preparation

The fibridomas useful according to the invention are those characterized by monoclonal immunoreacting with the antibody, the co-stimulators exhibiting the ability to produce which will be immunospecifically further on the molecule. As described herein, hybridoma cells producing antibodies against a costimulatory molecule can be directly implanted into a recipient animal to obtain a constant source of antibody.

The use of an immuno-isolating device for hybridoma culture may allow cells to encapsulate a response against implanted uncontrolled proliferation of hybridoma cells by the host. A preferred hybridoma to prevent immunogenic as well as prevent immunocompromised according to the invention is characterized by the production of antibody molecules that specifically immunoreact with a co-stimulatory molecule expressed on the surface of activated human B cells.

Methods generating hybridomas that produce, for example, secrete, antibody molecules having the desired immunological specificity, i. have the ability to bind to a particular co-stimulatory molecule and / or an identifiable epitope of the co-stimulatory molecule are known. Particularly useful is the hybridoma technology described in Niman et al. (1983) PNAS 80: 4949-4953 and Galfre et al. (1981) Meth. Enzymol. 73:

3-46.

In another exemplary method, transgenic mice bearing human antibodies can be immunized with a human co-stimulatory molecule. Splenocytes from these immunized transgenic mice can then be used to generate hybridomas that secrete human monoclonal antibodies that specifically react with the human costimulatory molecule (see, for example, Wood et al. PCT Application WO 91/00906, Kucherlapati et al., PCT WO 91 / 10741, Lonberg et al., PCT WO 92/03918, Kay et al., PCT 92/03917, Lonberg, N. et al., (1994) Nature 368: 856-859, Green, LL et al. , (1994) Nature Genet 7: 13-21, Morrison et al., SL et al., (1994) Proc Natl Acad Sci USA 81: 6851-6855, Bruggeman et al., (1993) Year Immunol., 7: 33-40, Tuaillon et al., (1993) PNAS 90: 3720-3724 and Bruggeman et al., (1991) Eur. J. Immunol. 21: 1323-1326).

The term "antibodies" includes fragments thereof that are also specifically reactive with the co-stimulatory molecule of the invention. Antibodies can be divided into fragments using conventional methods, and the fragments tested for use for the same purposes as whole antibodies. For example, F (ab ') ₂ fragments can be generated by treating the antibody with pepsin. The resulting F (ab ^z ) ₂ fragment can be treated to reduce disulfide bridges to produce Fab 'fragments.

Antibodies obtained by toutor or other methods can be tested for inhibiting the co-stimulatory signal in a T cell using the methods described below.

In one embodiment, the agent that inhibits the costimulatory signal in a T cell is an antibody that binds to the B7-1 and B7-2 receptors. For example, portions of the extracellular region that are conserved between two co-stimulatory molecules can be used as an immunogen to prepare such antibodies (see, for example, Metzler et al., 1997 Nat. Struct. Biol. 4: 527).

In one embodiment of the invention, the agent that inhibits the co-stimulatory signal in a T cell is an antibody that binds to the B7-1 receptor. Such antibodies are known in the art or can be prepared as described above using a B7-1 molecule or a portion thereof as an immunogen and tested using the methods described above or other standard methods. Examples of B7-1 antibodies include those disclosed in U.S. Patent No. 5,747,034 and McHugh et al., 1998, Clin. Immunol. Immunopathol. 87: 50 or Rugtveit et al., 1997. Clin. Exp. Immunol. 110: 104.

In another embodiment of the invention, the agent that inhibits the co-stimulatory signal in a T cell is an antibody that binds to the B7-2 receptor. Such antibodies are known in the art or can be prepared as described above using the B7-2 molecule or a portion thereof as an immunogen and assayed using the methods set forth above or other standard methods. Examples of B7-2 antibodies include those described in Rugtveit et al., 1997. Clin. Exp. Immunol. 110: 104.

In one embodiment of the invention, the agent that blocks the costimulatory signal in a T cell is a combination of an antibody that binds to the B7-1 receptor and an antibody that binds to the B7-2 receptor.

π In another embodiment of the invention, the agent that inhibits the co-stimulatory signal in a T cell is an antibody that is »

binds to CD28 but does not transmit a co-stimulatory signal to a T cell (e.g., a Fab28 fragment of CD28 antibody. Such antibodies are known in the art or can be prepared as described above using a CD28 molecule or portion thereof as an immunogen and assayed using the methods described above or other standard methods Examples of known antibodies against CCD28 include those described in Darling et al., 1997. Gene ther. 4: 1350.

In preferred embodiments of the invention, Fab fragments of an antibody that bind to CD28 can be used. It was found that

99 ···· · ··· · 9 * 9

• ·

4 that such antibody fragments that are unable to bind to CD28 on the surface of a T cell block T-cell co-stimulation (Walunas et al., 1994. Immunity

In other embodiments, according to a stimulation signal in a T cell,

CTLA4 and blocks the negative signal

CTLA4). Turns out

1: 405).

is an antibody agent that inhibits those stimulating into a T cell that the cell is bound to by introducing a surface agonist T signal in T (i.e., crosslinking CTLA4 cells inhibits the proliferation and production of IL-2 Allison. 1996. J. Exp. Med. 183: 2533). Such may prepare CTLA4 molecules, or portions thereof, using the methods set forth above standard methods. Examples (Krummel and antibodies are as described above using as immunogen and tested in Vandenborre)

In another embodiment, it inhibits co-stimulatory et al., According to the text or antibodies

1998. Am.

invention

J.

using others, Pathol. 152: 963.

is an antibody-binding agent that blocks the co-stimulatory signal. Such antibodies can be prepared as described above using the ICOS molecule or tested using the methods set forth above by other standard methods.

the signal in the T cell to be

T cell.

describes parts as an immunogen and in the text or

IV. Reagents,

In another stimulatory expression expression interaction that regulates the expression of co-stimulatory molecules, an embodiment of the invention is a signal in T co-stimulatory between CD40 on the cell, molecule.

cells, a CD40 ligand (CD40L) for enhancing or extending the antigen-containing agent,

She found out which one

T cells that inhibit which interfere, for example, with antigen, and important in promoting, or B7-2 on B7-1 expression cells, lead to enhanced co-stimulation (Van

Gool, et al., 1996. Immunol. Roar. 153: 47, Klaus et al., 1994.

J. Immunol. 152: 5643).

• ·· · « • • • · • · • • · ·· * • • • • · • • · • • • • • • · ·· ♦ ·· ·· ··

In one embodiment of the invention, the agent that blocks expression of the co-stimulatory molecule and thus blocks the co-stimulatory signal in the T cell is a soluble form of CD40 or CD40L. DNA sequences encoding CD40 and CD40L are known in the art (see, e.g., GenBank Accession No. Y10507 or Stamenlovic et al., 1988. EMBO J. 7: 1053-1059 for CD40 or Gauchat et al. 1993. FEBS 315 (3): 259266, Graf et al., 1992. Eur J Immunol 22: 3191-3194, Seyama 1996. Hum Genet 97: 180-185 or GenBank accession numbers L07414, X67878, X96710 for CD40L .

In one embodiment of the invention, the agent that blocks expression of the co-stimulatory molecule and thus blocks the costimulatory signal in the T cell is a soluble form of CD40 or CD40L. In one embodiment, the soluble CD40 or CD40L protein comprises the entire protein. In preferred embodiments, the soluble CD40 or CD40L protein comprises an extracellular region of the protein. For example, a soluble recombinant form of the extracellular region of the CD40 or CD40L protein, or a portion thereof, can be prepared as a fusion protein comprising at least a portion of CD40 or CD40L such that the interaction between CD40 on APC and CD40L on T cells is interrupted and inhibits the introduction . Such a soluble recombinant form of the CD40 or CD40L protein comprises at least a portion of a molecule essential for binding to its receptor and at least a portion of a second protein that is not CD40 or CD40L.

In a preferred embodiment of the invention, the CD40 or CD40L fusion protein comprises an extracellular region of CD40 or CD40 that is fused to the amino terminus with a signal peptide, for example from oncostatin M (see, for example, WO 93/00431).

In a particularly preferred embodiment of the invention, the soluble form of CD40 or CD40L is a fusion protein comprising an extracellular region of CD40 or CD40L fused to a portion of an immunoglobulin molecule (e.g. Chen et al., 1995. J. Immunol. 155: 2833).

Such a CD40Ig or CD40LIg fusion protein can be prepared using methods well known in the art (see, for example, Linsley 1994. Perspectives in Drug Discovery and Design 2: 221, Linsley WO 93/00431, US Patent 5,770,197 and US Patent 5 580 756).

Furthermore, anti-CD40 ligand antibodies have been found to be synergistic with agents that inhibit the co-stimulatory signal in a T cell to promote graft tolerance (Kirk et al., 1997. Proc. Natl. Acad. Sci. USA 94: 8789, Larsen et al., 1996. Nature 381: 434).

Thus, in one embodiment of the invention, anti-CD40 or CD40L antibodies that bind to these molecules but which do not induce the expression of co-stimulatory molecules can be used as an agent that blocks the co-stimulatory signal in a T cell.

V. Reagents that act intracellularly to inhibit the costimulatory signal

In one embodiment of the invention, an agent that inhibits a co-stimulatory signal in a T cell is an agent that acts intracellularly to inhibit such a signal. T cell stimulation via CD28 surface receptors (i.e., a co-stimulatory signal) results in the production of phosphoinositides D-3 in the T cell. Thus, in one embodiment of the invention, the production of phosphoinositides D-3 can be inhibited in a T cell, inhibiting the co-stimulatory signal, thereby inhibiting the T cell response, as measured, for example, by T cell proliferation and / or cytokine production. The term "D-3 phosphoinositides includes phosphatidylinositol derivatives that are phosphorylated at the D 3 position of the inositol ring and contain phosphatidylinositol (3) -monophosphate (Ptdlns (3) P), phosphatidylinositol (3,4) -biphosphate (Ptdlns (3,4)" compounds P ₂₎ and phosphatidylinositol tol (3, 4, 5) triphosphate (PtdIns (3, 4, 5) P3).

Phosphoinositides D-3 are formed intracellularly by the activity of phosphatidylinositol-3-kinase (PI3K). PI3K is a heterodimer containing a 85,000 subunit that binds to tyrosylphosphorylated proteins via SH2 regions and a catalytic subunit of 113,000. PI3K was first identified as a lipid kinase that phosphorylates at the D-3 position of the phosphatidylinositol inositol ring (PtIns (4)) P and Ptdns (4,5) P2 Two recent studies have demonstrated that PI3K is in fact a dual specificity kinase that exhibits both lipid and serine kinase activities (Dhand R., et al., (1994) EMBO J. 13: 522 and Carpenter, CL et al (1993) Mol Cell Biol 13: 1657).

In one embodiment of the invention, the agent that inhibits the co-stimulatory signal in a T cell is an agent that inhibits PI3K activity. A preferred agent that inhibits PI3K activity in a T cell is the fungal metabolite wortmanin or derivatives or analogs thereof. Wortmanin is a potent PI3K inhibitor obtained from T. wortmannii (Kyowa Hakko Kohyo Co. Ltd.) or P. fumiculosum (Sigma). Wortmanin derivatives or analogs include compounds that are structurally related to wortmanin and that retain the ability to inhibit PI3K and T cell response. Examples of wortmanin derivatives and analogs are described in Wiesinger, D. et al., (1974) Experientia 30: 135-136, Closse, A., et al., (1981) J. Med. Chem. 24: 1465-1471 and Baggiolini, M. et al., (1987) Exp. Cell Res. 169: 408-418. Another PI3K activity inhibitor that can be used is the bioflavenoid quercetin or derivatives or analogs thereof. Quercetin derivatives or analogs include compounds that are structurally related to quercetin and retain the ability to inhibit T cell response. Examples of quercetin derivatives and analogs are described in Vlahos et al., C. J. et al., (1994) J. Biol. Chem. 269: 5241-5284. A preferred quercetin derivative that inhibits PI3K activity is LY294002 (2- (4-morpholinyl) -8-phenyl-4H-1-benzopyran-4-one, Lilly Indianapolis).

IN (Vlahos et al., C.J. et al., (1994) J. Biol. Chem. 269: 5241-5284).

Stimulation of CD28 has also been shown to result in phosphorylation of a tyrosine protein in a T cell (see, for example, VAndenberghe, P. et al. (1992), J. Exp. Med. 175: 951-960; Lu, Y. et al. (1992) J. Immunol. 149: 24-29).

In one embodiment of the invention, an agent that inhibits I co-stimulatory signal in a T cell inhibits tyrosine phosphorylation in a T cell. A preferred protein tyrosine kinase inhibitor is one that inhibits src protein tyrosine kinase.

In one embodiment of the invention, the protein tyrosine kinase inhibitor is src herbimycin A or an analogue or derivative thereof. The derivatives and analogs of herbimycin A include compounds that are structurally related to herbimycin A and retain the ability to inhibit protein tyrosine kinase activity.

In one embodiment of the invention, the agent that inhibits protein tyrosine phosphorylation is protein tyrosine phosphatase or a protein tyrosine phosphatase activator. By increasing the tyrosine phosphatase activity in the T cell, the crude amount of protein tyrosine phosphorylation is reduced. The protein tyrosine phosphatase may be a cellular protein tyrosine phosphatase in a T cell, such as CD45 or Hcph. Cell surface tyrosine phosphatase activity on a T cell can be activated when the T cell comes into contact with a phosphatase-binding molecule and stimulates its activity. For example, an anti-CD45 directed antibody can be used to stimulate tyrosine phosphatase activity in a T cell that expresses CD45 on its surface. Thus, in one embodiment of the invention, an agent that inhibits protein tyrosine phosphorylation in a T cell is an anti-CD45 antibody or fragment thereof that retains the ability to stimulate CD45 activity. Examples of antibody fragments include Fab and F (ab ') 2 fragments.

In addition, ligation of CD28 has been associated with increased phospholipase C activity (see, e.g., Nunes, J. et al., (1993) Biochem. J. 293: 835-842) and increased levels of intracellular calcium (see, for example, Ledbetter, JA et al., (1990) Blood 75: 1531-1539 and in the examples). Thus, an agent that acts intracellularly to inhibit the co-stimulatory signal in a cell may act by inhibiting phospholipase C activity and / or by inhibiting an increased amount of intracellular calcium. For example, the herbrosine A tyrosine kinase inhibitor also inhibits CD28-induced calcium flow in T cells.

Protein serine kinase and serine-

threonine kinase se share i on the runway signaling transduction connected with CD28 (Siegel, J. N. et al. (1993) J. Immunol. 151: 4116-4127, Pai, ST. et al. (1994) J. Immunol. 24: 2364, Parry et al. , 1997, Eur J. Immunol. 27: 2495) . IN j otherém

thus, an embodiment of the invention, an agent that acts intracellularly and inhibits a co-stimulatory signal in a T cell, inhibits serine or serine-threonine kinase activity.

VI. Other agents that block co-stimulation of T cells

Other agents that block the co-stimulatory signal in the T cell can be identified using standard methods. For example, these agents can be identified based on their ability to inhibit T cell proliferation and / or cytokine production. For example, a co-stimulating assay system may be used. In such a system, human CD28 T cells were isolated, for example, by depleting immunomagnetic particles using monoclonal antibodies directed against B cells, natural K cells, and macrophages as described by Gimmi, C. D. et al. (1993) Proc. Nati. Acad. Sci USA 90, 6586-6590). Cells containing antigen, such as whole spleen cells, or purified B cells or B7-1 transfected COS cells, or

B7-2 may be irradiated or treated with mitomycin C (e.g. at a concentration of 25 µg / ml) for 1 hour and then washed extensively to inhibit propagation. CD28 ⁺ T cells at 10 ⁵ for example can be incubated with 10 ⁵ to 10 ⁴ APCs (e.g., COS cells transfected with a B7 molecule). In this assay, one population of T cells received the primary activation signal alone (e.g., the T cell receptor signal), another population of T cells received the co-stimulation signal alone, another population of T cells received the primary activation signal and the co-stimulation signal, and another population. The primary activation signal and the co-stimulation signal were applied to the T cells in the presence of an agent being tested for the ability to block the co-stimulation signal in the T cell. The primary activation signal can be introduced, for example, by a presentogenic dose of PMA (eg, 1 ng / ml), a presentogenic dose of mitogen, a suboptimal dose of antigen, or a presentogenic dose of an antibody against T cell receptors. Signal 2 is introduced by antigen-containing cells that carry the B7 molecule. Potential blocking agents may be tested over a range of concentrations. For example, potential blocking antibodies can be used as hybridoma supernatants or as purified antibodies (e.g., at an approximate concentration of 10 µg / ml). T cell proliferation can then be measured by incorporating ³ H thymidine (1 pCi) for the last 12 to 18 hours of a 72 hour incubation. The introduction of the primary activation signal should result in proliferation, but T cells that have received the primary activation and co-stimulation signal 2 should be maximally proliferated.

Blocking agents have been identified based on the ability to reduce the maximum proliferation induced by the co-stimulatory signal.

As an alternative to or in addition to measuring T cell proliferation, cytokine production by a T cell can be measured using methods well known in the art. For example, IL-2 and IL-4 produced in a T cell culture can be assayed in culture supernatants collected 24-72 hours after initiation of the cultures using routinely using culture supernatants. An available ELISA (R&D Systems, Minneapolis, MN and BioSource, Camarillo, CA). As mentioned above, blocking agents can be identified by their ability to reduce the maximum cytokine production induced by the co-stimulatory signal.

In the case of antibodies, any of the "blocking antibodies identified using said or other assay can be further tested to determine the co-stimulatory molecule to which it binds, using methods known in the art. For example, the ability of a blocking antibody to reduce binding of a labeled antibody to a known ligand can be measured.

VII. Other agents that suppress immune responses

In certain embodiments of the invention, the compositions and methods of the invention may include additional agents or other agents may be used to enhance immunotolerance to an agent that promotes hemostasis. In one embodiment of the invention, an agent that promotes immunotolerance but does not inhibit the co-stimulatory signal in a T cell may be added to the compositions or may be used in said method. For example, an anti-CD40 ligand (e.g., a monoclonal antibody against human CD40 ligand, 5C8 (Kirk et al.,

1997. Proc. Nati. Acad. Sci. USA 94: 8789)). CD40 and its T cell-based ligand, CD40L (CD154), play an important role in the positive regulation of B7 and in establishing B cell activity (U.S. Patent No. 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, Lederman et al., 1992. J.

Immunol. 149: 3817). Antibodies against the ligand were found

CD40 are synergistic with agents that inhibit the co-stimulatory signal in a T cell while promoting graft tolerance (Kirk et al., 1997. Proc. Natl. Acad. Sci. USA 94: 8789, Larsen et al., 1996. Nature 381: 434). In another embodiment, an agent may be included in the composition of the present invention which acts to effect 999999 9999999 9999 99999 9999999

999 999 ·· 999 9999 9 intracellular to promote immunotolerance but which does not inhibit the co-stimulatory signal in the T cell. For example, in one embodiment, cyclosporin A (CSA), FK506, rapamycin, or another agent that inhibits immune responses may be included or administered as part of a method of the invention (Sigal et al., 1992. Annv. Rev. Immunol. 10 : 519, Ruhlmann et al., 1997 Immunology. 198: 192, Shaw et al., 1996. Clin. Chem. 42: 1316).

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

In one embodiment of the invention, the compositions and / or agents are administered to a subject suffering from a hemostatic disorder and has previously been administered an agent that promotes hemostasis. In another embodiment of the invention, the disclosed compositions and / or agents are administered to subjects who have not previously administered an agent that promotes hemostasis.

In another embodiment of the invention, the compositions and / or agents described herein are administered to a subject that has not yet developed an immune response to an agent that promotes hemostasis. In other embodiments of the invention, the compositions and / or agents are administered to subjects that already exhibit an immune response to an agent that promotes hemostasis. Whether a subject exhibits an already existing immune response can be determined by measuring the titer of antibodies in the subject that react with a hemostasis promoting agent using methods well known in the art. If the subject exhibits a measurable titer of such antibodies (e.g., a statistically significant titer) as compared to titers from control individuals, the subject may be said to exhibit a pre-existing immune response to an agent that promotes hemostasis. Alternatively, a cellular immune response to an agent that promotes hemostasis can be measured to determine whether the subject

shows an immune response to an agent that promotes hemostasis. Such methods are well known in the art.

In one embodiment of the invention, a first agent that promotes hemostasis and a second agent that inhibits or blocks a co-stimulatory signal in a T cell is used in the treatment of haemotasis disorders. In another embodiment, compositions comprising a combination of a first agent that promotes hemostasis and a second agent that inhibits a co-stimulatory signal in a T cell may be used to treat hemostasis disorders.

Administration of the compositions and / or agents described herein can be carried out in any pharmaceutical form which comprises a therapeutically active amount of the agent and a pharmaceutically acceptable carrier. Administration of a therapeutically active amount of agents and / or compositions is defined as an amount effective at dosages and for a period of time necessary to achieve treatment of a hemostatic disorder for a hemostasis promoting agent and to achieve immunotolerance to a hemostasis promoting agent or a co-inhibitory agent. stimulating signal in T cell. The therapeutically active amount of the agent or composition may vary based on factors such as the stage of the disease, the age, sex and body weight of the individual and whether the individual already has an immune response to the agent that promotes hemostasis. Such amount is readily determined by one skilled in the art.

The optimum course of administration of the agent and / or composition may also vary depending on the subject to be treated. In certain embodiments, the subject will require treatment with both agents at a time. In this case, it will be desirable to administer an agent that promotes hemostasis and concurrently an agent that inhibits the co-stimulatory signal, for example in the form of a composition comprising both agents.

In other embodiments of the invention, it may be desirable to administer the agents separately, for example, to promote the stability of the agents or to facilitate sequential administration of the agents. In one embodiment, sequential administration may be desirable to achieve the optimal therapeutic effect of a hemostasis promoting agent while optimally inhibiting the immune response to the agent, with an antibody response being preferred. For example, the agent that inhibits the co-stimulatory signal may be administered alone or may be administered alone for several days after administration of the hemostasis promoting agent.

In one embodiment of the invention, the agent that blocks the costimulatory signal in the cell can be administered sustainably, for example, each time a hemostasis promoting agent is administered. In another embodiment of the invention, the agent that blocks the co-stimulation signal in the T cell is administered sporadically. For example, a subject may require regular treatment with an agent that promotes hemostasis, or may require treatment with an agent that inhibits a co-stimulatory signal in a T cell only periodically. For example, treatment with an agent that promotes hemostasis can be performed, while only one or two times a subject is treated with an agent that blocks a co-stimulatory signal in a T cell; additional application of an agent that blocks the co-stimulation signal may not be necessary. In a preferred embodiment of the invention, the agent that blocks the co-stimulation signal in the cell is applied at appropriate intervals for at least 6 months.

In one embodiment of the invention, the compositions or agents are administered to the patient if the patient has been found to be predisposing to antibodies. In another embodiment of the invention, the compositions or agents are administered to the patient, wherein the patient has not been pre-treated, i. It does not show pre-existing antibodies. In another embodiment of the invention, the agents and compositions can be administered to patients who have been previously treated but do not exhibit antibody titers against an agent that promotes hemostasis.

It is possible to determine a dosage regimen to achieve an optimal therapeutic response in each subject without a 444 · 444 · 444 44 Φ Φ φ φ φ · · Φφφφ • ΦΦ φφφ φφ φφφ 44 ΦΦΦ excessive experimentation. For example, the antibody titer to an agent that promotes hemostasis can be measured to determine whether a subject develops an immune response to the agent, and a dosage regimen can be determined accordingly. For example, if antibody titers against an haemostasis promoting agent increase, multiple doses of an agent that blocks the co-stimulatory signal in the T cell may be administered.

If agents or compositions are administered by other than parenteral routes, it may be necessary to coat or apply them with a material that prevents their inactivation. The agent or composition of the invention may be administered to an individual with a suitable carrier or diluent or enzyme inhibitors or with a suitable carrier, such as liposomes. Pharmaceutically acceptable diluents include saline and aqueous buffers. Enzyme inhibitors include a pancreatic trypsin inhibitor, diisopropyl fluorophosphate (DEP), and trasylol. Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes (Strejan et al., (1984) J. Neuroimmunol. 7: 27).

The active agent or composition may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, in liquid polyethylene glycol in mixtures thereof and in oils. Under normal conditions of storage and use, these preparations may contain preservatives which prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (when soluble in water) or dispersions and sterile powders when the injectable solutions or dispersions are prepared just prior to administration. In all cases, the agent or composition must be sterile and must be fluid to the extent that easy syringability exists. The composition must also be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Appropriate 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 surfactants. Prevention of the action of microorganisms can be achieved by various bactericidal or fungicidal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it is preferred that the compositions include isotonic agents such as sugars, polyalcohols such as mannitol, sorbitol, sodium chloride. Delayed adsorption of injectables can be achieved by including agents that delay absorption such as aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active agent or agent in the required amount in a suitable solvent with a single component or, if desired, combining the listed active ingredients followed by filter sterilization. Generally, dispersions are prepared by incorporating the active ingredient into a sterile vehicle that contains a basic dispersion medium and adding the other agent included in the above group. In the case of sterile powders suitable for the preparation of a sterile injectable solution, the preferred method of preparation is vacuum drying and lyophilization resulting in a powder of the active ingredient (e.g., agent or composition) plus any additional desired ingredient from a previously sterile filtered solution thereof.

When the active agent or composition is suitably protected as described above, the protein may be administered orally, for example, as an inert diluent or an assimilable edible carrier. The term "pharmaceutically acceptable carrier includes any solvent; or"

9 9 9 999 • 999999

9

9 · 99

All solvents, dispersion media, coatings, bactericidal or fungicidal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. The use of any conventional medium or reagent is contemplated if it is not incompatible with the active compound. Other active agents may also be included in the compositions.

It is particularly advantageous to provide parenteral compositions in unit dosage form which provide for easy dosing, ease of administration and uniformity of dosage. Dosage unit form herein refers to physically discrete units suitable as unitary dosages for the mammalian subject to be treated, each unit containing a predetermined amount of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification of the unit dosage forms of the invention is directly dependent on (a) the unique characteristics of the active agent and the particular therapeutic effect to be achieved, and (b) the concomitant limitations known in the art to produce such active agents or compositions useful in the treatment of individuals.

The term "pharmaceutically acceptable carrier" includes all solvents, dispersion media, coatings, bactericidal or fungicidal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art.

The practice of the invention will employ conventional methods of cell biology, cell culture, molecular biology, microbiology, DNA recombination, and immunology, which are well known in the art. Such methods are described, for example, in Genetics, Molecular Cloning A Laboratory Manual, 2 ^nd Ed., Ed. Sambrook, J. et al., (Cold Spring Harbor Laboratory Press (1989)), Short Protocols in Molecular Biology, 3 ^rd Edi. , ed. Ausubel, F. et al. (Wiley, NY (1995)), DNA Cloning, Volumes I ♦ φ φ φ φ φ • ♦ φ φ φ φ · · φ φ φ φ φ φ φ φ and (D. G Glover ed., 1985), Oligonucelotide Synthesis (J. J. Gait ed. (1984)), Mullis et al., US Patent No. 4,683,195, Nucleic Acid Hybridization (BD Hames and SJ Higgins eds). (1984)), Methods In Enzymology (Academic Press, Inc., NY), Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds. Academic Press, London (1987)), Handbook of Experimental Immunology, volumes I- IV (DM Weir and CC Blackwell, eds. (1986)), and Miller, J. Experiments in Molecular Genetics (Cold Spring Harbor Press, Cold Spring Harbor, NY (1972)).

Overview of the drawings

Figure 1 shows the design of the experiment used in Example 1 to test inhibition of the primary antibody response to factor VIII.

Figure 2 shows that while CTLA4Ig-free mice showed high antibody titers as early as day 20 (GI), CTLA4Ig-treated mice showed no antibodies until day 82 (G-2 and G-3). ).

Figure 3 shows the design of the experiment used in Example 2 to test the inhibition of the secondary antibody response to Factor VIII.

Figure 4 shows that while animals that did not receive CTLA4Ig showed a high titer of anti-Factor VIII (G1) antibodies, mice given CTLA4Ig (G-2) except one mouse did not show a secondary immune response to Factor VIII .

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

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

Figure 7 shows the effect of co-administration of mCTLA4-Ig on the production of anti-Factor VIII antibodies.

• · · · · · · · · · · 9 9 999 994 · · 4 · · · 4444

999 999 99 999 99999

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

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

Figure 10 shows T cell response to factor VIII in haemophilia A (B7-1 ⁾ and haemophilia A (B7-2 ⁾ in mice.

DETAILED DESCRIPTION OF THE INVENTION

In the examples, a mouse haemophilia A model was used to evaluate novel methods suitable for preventing and treating inhibitor formation. Mice with haemophilia A generated by target exon 16 disruption of the Factor VIII gene do not show detectable factor VIII activity in their plasma (Bi L, Nature Genetics 10: 119, 1995) and are similar in this way to patients with severe haemophilia A. As expected, mice with haemophilia And show in vivo signs of a coagulation pathway with fatal bleeding if they cut off their tail without using hemostatic measures and developed subcutaneous and intramuscular bleeding after handling or temporary immobilization (Qian J, Borokov M, Bi L, Kazazian HH, Hoyer L. Thromb Haemost 81: 940 (1999), Evans GL et al., Proc. Natl. Acad. Sci USA 95: 5734, 1998).

Intravenous infusion of 0.2 μg of human factor VIII, the dose equivalent given to patients with haemophilia A, resulted in minimal or no antibody response in these haemophilia A mice after a single injection, but repeated infusions resulted in a high titer of inhibitory anti- factor VIII (Qian J et al., Thromb Haemost 81: 940, 1999). In addition, a factor VIII specific T cell proliferative response was detected three days after the first exposure to human factor VIII, before antibodies were detected.

Example 1: Inhibition of primary immune response to factor

VIII

The experimental design for this example is shown in Figure 1. Three groups of mice were injected intravenously with Factor VIII on day 0. One group of mice was also injected with CTLA4-Ig the day before and the day after the injection of factor VIII. The second group of mice received the same CTLA4-Ig treatment (intraperitoneally), followed by daily administration of Factor VIII injection on days 2 to 12. The third group received no CTLA4-Ig. All mice then received two additional injections of factor VIII on days 23 and 44. Animals were sampled on days 20, 37, 58, and 82. While mice not receiving CTLA4Ig showed high antibody titers, which started to form on day 20, CTLA4Ig-treated mice do not develop antibodies until day 82 (Figure 2).

Example 2: Inhibition of secondary response to factor VIII

The experiment proposal for this example is shown in the figure

In this example, mice received multiple intravenous Factor VIII injections at two-week intervals and then divided into two groups. Mice were re-injected with Factor VIII on days 1, 20, and 37. One group of mice was injected with CTLA4-Ig on day -1 and day +1 relative to day 1, 20, and 37. Animals not treated with CTLA4Ig, showed high titers of anti-Factor VIII antibodies, whereas CTLA4Ig-treated mice (except one mouse) did not show a secondary immune response to Factor VIII (Figure 4).

The following methods and materials were used in Examples 3 to 6.

Animals

The characteristics of the exon-16 (E-16) strain of hemophilic mice are described in Bi L., et al., Nature Genetics 10: 119,

1995, Bi L, et al., Blood 88: 3446, 1996. Adult male and homozygous female E-16 mice of age were used in this study.

up to 20 weeks. Blood samples were obtained by bleeding the orbital venous girdle and the serum was separated by centrifugation at 600 g for 3 minutes. Serum samples were stored at -20 ° C and then tested. To avoid severe bleeding and death of animals, ear tags were not used to identify mice in some experiments. Therefore, the sequence data of individual mice is not shown in Figure 5.

The generation of E-16 mice that do not transcribe B7-1 and B72 genes was performed by crossing E-16 mice with mice that did not transcribe B7-1 genes and mice that did not transcribe B7-2 genes (Borriello F., et al. (1997) Immunity 6: 303).

Homozygous mice with knockout E-16 / B7-1 and E-16 / B7-2 genes were identified by genotype determination (Bi L. et al., Nature Genetics 10: 119, 1995, Borrielo F, et al., Immunity 6: 303,

Limited factor VIII activity was verified using the Coatest chromogenic bioassay (Chromogenix, Molndal, Sweden (Bi L., et al., Blood 88: 3446, 1996). Factor VIII activity is in E-16 / B7-1 and E16 / B7-2 deficient mice less than 1%.

Antigens

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

mCTLA-4Ig

An expression plasmid containing the murine CTLA4-Ig cDNA was prepared by ligating the leader and extracellular regions of CTLA4 to join, mutating the CH2 and CH3 regions of IgHg2a to remove effector functions as described by Streurer et al., J. Immunol. 155: 1165, 1995. The insert was cloned into the pED expression vector and stably transfected into CHO cells as described by Lollar P., et al., J. Clin. Invest 93: 2497, 1994. The concentrated conditioned medium was loaded onto a fast flow chromatography column with Sepharose and Protein A (Amersham Pharmacia Biotech, Piscataway, NJ). The column was washed with PBS pH 7.1 and mCTLA4-Ig was eluted with 20 mM citrate at pH 3.0. The peak was neutralized with 1M Tris pH 8.0 to a final pH of 7.5 and transferred to PBS at pH 7.1 using a stirred Amicon cell with a YM30 membrane. mCTLA4-Ig was depyrogenated using a Poros PI chromatography column (Perceptive Biosystems) and the product eluted from the column with a linear NaCl gradient from 0 to 1 M NaCl in 25 mM Tris pH 7.5. mCTLA-4-Ig was then transferred to PBS pH 7.1 using a stirred Amicon cell with a YM30 membrane.

Measurement of antibodies

The anti-factor VIII titer was determined by ELISA (Qian J., et al., Inhibitor Antibody Development and Cell Response to Human Factor VIII in Murine Hemophilia A. Thromb Haemost 81: 940, 1999). The ELISA was performed using recombinant human Factor VIII coated microtiter wells at a concentration of 0.8 µg / ml in 0.05 M carbonate-bicarbonate at pH 9. Then, the mouse plasma samples were incubated overnight in the wells at 4 ° C and then washed, alkaline phosphatase-conjugated goat anti-mouse IgG (Southern Biotechnology Associates Inc, Birmingham, AL) was added and the reaction was incubated at room temperature for 2 hours. After washing, P-nitrophenyl phosphate (Sigma, St. Louis, MO), 2 mg / ml in 100 mmol / l glycine, 1 mmol / l Mg ₂ Cl 2, 2 mmol / l ZnCl ₂ pH 10.4 was then added and then measured absorbance at 410 nm using an automatic microplate reader for ELISA. The concentration of anti-Factor VIII antibodies was estimated from a standard curve obtained using monoclonal mouse anti-human Factor VIII IgGs that bind to the A2 region (Mab413) (Lollar P et al., J. Clin. Invest 93: 9497, 1994). The titer was calculated from points falling within the linear area of the test standard curve.

* · * 9 · • · * • • • · · • • • • • · a ♦ · • • • · • • • · 9 • • a « • • 3 « 9 • • ··· • · ·· ·

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

T cell proliferation assays

Spleen was used as a source of T cells for proliferation assays. The spleen cells were then cultured (5 x 10 ⁵ cells per well) in 96-well flat-bottom plates. Different amounts of recombinant factor VIII were added to the culture medium containing complete RPMI-1640 and 0.5% hemophilic mouse serum. After 72 hours of culture at 37 ° C, 37 kBp ³ Hthymidine / well (6.7 Ci / mmol), ICN Pharmaceuticals Irvine, CA) was added. The culture was harvested after 16 hours using a Matrix 9600 (Packard, Meriden, CT). Data were expressed as the mean values of three wells in cpm units incorporated into insoluble DNA.

Example 3: mCTLA4-Ig blocks the induction of a factor VIII response

Control mice developed factor VIII inhibitory antibodies by repeated intravenous injections of 1 µg of recombinant human factor VIII at three week intervals. In this example, four groups of mice with haemophilia A were injected with recombinant human factor VIII on days 0, 23, 44, and 66 (1 microgram intravenously administered on the first injection and 0.2 microgram administered on the second, third and fourth injections) . Mice in the G-3 and G-4 groups were also injected with 0.2 µg of Factor VIII on Days 2-12 by intraperitoneal injection. Blood samples tested for anti-factor VIII were obtained on days 20, 37, 58, and 82. Control groups G1 and G-3 (indicated by open circles) were injected with only factor VIII. Groups G-2 and G-4 (solid circles) were also injected with mCTLA4-Ig (250 µg, i.p.) on the day before or the day after

Λ • 4 ···· 44 44 • • • 4 · 44 • • • 44 »4 • · • 4 4 · · • • 4 • 4 · • · 444 44 ··· ·· ··

the first injection with factor VIII. The concentration of anti-factor VIII antibodies was determined by ELISA. The anti-Factor VIII antibody concentration was shown to be less than 0.16 μρ / πιΐ, a value similar to plasma samples obtained from non-immunized mice suffering from haemophilia A. The results of this experiment are shown in Figure 5 (note that that some of the data shown in Figure 2 is repeated in Figure 5, but there are extra data).

Anti-Factor VIII antibodies were detected in four of the five mice on the twentieth day after the first injection, and all control mice show a high titer of anti-Factor VIII antibodies after receiving two to four injections. The average amount of inhibitor after four injections is 1,860 Bethesda Units (BU). Factor VIII antibody production decreases markedly in mice treated intraperitoneally with 250 μς of mouse CTLA4-Ig on the day before and the day after the first injection of factor VIII (group G-2, shown in Figure 5), although three subsequent Factor VIII injections on days 23, 44 and 66 did not administer mCTLA4-Ig. Anti-Factor VIII antibodies were not detected in any of the G-2 mice after the first or second Factor VIII injection. Three weeks after the third injection of factor VIII, a weak immune response was detected in two of the six mice in the G-2 group.

To investigate whether the limited duration of no response is the result of the short half-life of human factor VIII in these mice (4-5 hours in hemophilia A mice) (Evans GL., Et al., Proc. Natl. Acad. Sci USA 95: 5734, 1998), control mice and mCTLA4-Ig-treated mice (groups G-3 and G-4, shown in Figure 5) received intravenous injections with 1 μρ of factor VIII on day 0 followed by daily dosing intraperitoneal injections with 1 μρ of factor VIII on days 2 to 12. A high titer of anti-factor VIII antibodies occurred in control mice on day 20 (group G-3), with ELISA values greater than 350 μρ / ml and mean titer the inhibitor is 649 BU. In contrast, a group of G-4 mice injected with mCTL4-Ig on the day before and the day after the first factor VIII exposure did not show detectable anti-factor VIII antibodies on day 20. The delayed anti-factor VIII antibody response after three additional factor VIII injections was in these mice the same as the response in animals in the G-2 group. Thus, the limited persistence of factor VIII in plasma following CTLA4-Ig injections is not a reason for the limited duration of non-response time in CTLA4Ig treated mice.

Example 4: Effects of repeated administration of CTLA4-Ig

Since delayed response to Factor VIII was detected after repeated infusions of Factor VIII when mCTLA4-Ig was administered only at the time of the first exposure to Factor VIII, it was investigated whether mCTLA4-Ig could hinder the development of anti-Factor VIII antibodies when administered with each factor VIII infusion. In this experiment (shown in Figure 6), haemophilia A mice were co-infused six times at three week intervals with factor VIII and mCTLA4-Ig. Mice with haemophilia A were injected intravenously with 1 gg of Factor VIII and 250 μς mCTLA4Ig at three-week intervals (filled circles) or with Factor VIII alone after the first injection containing Factor VIII and mCTLA4-Ig (labeled with open circles). Serum samples for the anti-factor VIII assay were obtained 4 weeks after the sixth injection of factor VIII. No anti-Factor VIII antibodies were detected in any of the ten mice treated as described above, tested four weeks after the sixth Factor VIII injection. In contrast, in sera from mice receiving only one injection with mCTL4-Ig (at the time of the first exposure to invoice VIII), followed by five injections with factor VIII alone, a high anti-factor VIII titer was found.

These mCTLA4-Ig-treated mice were then tested to determine if they would show an immune response afterwards.

9 9 9

999 9 factor VIII injections without mCTLA4-Ig. After two intravenous injections at three-week intervals, none of the five mice developed anti-Factor VIII antibodies, whereas two of the four control mice that had not previously been exposed to either Factor VIII or mCTLA4-Ig (shown in Figure 7) were low. the value of anti-factor VIII antibodies. In this experiment, mice with haemophilia A treated as described in Figure 6, received six injections of Factor VIII and mCTLA4-Ig subsequently received six intravenous injections of 0.2 μς of Factor VIII at three-week intervals without the administration of additional mCTLA4- Ig (solid circles). In parallel, control mice not previously exposed to Factor VIII (labeled with open circles) were immunized. Serum samples for anti-Factor VIII antibody testing were obtained 3 weeks after the second and sixth injections. After six injections of Factor VIII alone, the average Factor VIII titer was 93 μς / ιη myší in mice previously treated with both Factor VIII and mCTLA4-Ig, whereas the mean titer in the control mouse is 155 μς / πιΐ. These data document the limitation of antigen-specific immune suppression following repeated administration of mCTLA4-Ig with factor VIII.

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

To determine whether mCTLA4-Ig modifies the secondary immune response to factor VIII, mCTLA4-Ig was injected at the same time as factor VIII was administered to hemophilia A mice that already show anti-factor antibody

VIII. Initially, all mice with haemophilia A were injected three times with 0.2 µg of factor VIII and the amount of anti-factor VIII antibodies was determined by ELISA. Control mice received three additional injections of the remaining mice at the same time, the first of the three additional injections of factor VIII, while, as when given them with factor VIII,

mCTLA4-Ig. While many mice died as a result of large blood loss during these experiments because repeated injections and blood samples were taken during the experiment, the results were clearly different in both groups.

In this example, all mice were initially given 3 intravenous injections with 0.2 µg FVIII at two-week intervals. Control mice (open circles) were then treated three more times with Factor VIII to obtain blood samples for the assay (upper part of the figure). Other mice (filled circles) received mCTLA4-Ig (250 µg, intraperitoneally) the day before and the day after the fourth injection of factor VIII (indicated by the arrow); followed by two additional injections of factor VIII alone at three week intervals. The number of Factor VIII injections prior to sample testing to determine the presence of anti-Factor VIII antibodies is shown on the X-axis.

An increase in anti-Factor VIII antibody titer was recorded after the fourth injection of Factor VIII in control mice, with an average titer ranging from 16 to 230 pg / ml (shown in Figure 8A). After the fifth injection with factor VIII, the anti-factor VIII antibody titer was greater than 350 pg / ml in the four remaining control mice. In contrast, mice treated with mCTL4-Ig at the fourth injection of factor VIII show little or no increase in anti-factor VIII antibodies (shown in Figure 8B and C). Administration of mCTLA4-Ig inhibits said secondary immune response to Factor VIII in mice already showing relatively high levels of anti-Factor antibodies

VIII, corresponding to 5 to 90 BU inhibitor titers (Qian J. et al., Thromb Haemost 81: 940, 1999) (shown in Figure 8C) as well as mice with minimal anti-Factor VIII antibodies after three initial injections where the inhibitor titer is less than 5 BU (shown in FIG.

8B).

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

The roles of the co-stimulatory ligands B7-1 and B7-2 on antigen-containing cells were then evaluated, as these were their interactions with CD28, which were believed to have been avoided during mCTLA4-Ig experiments. In order to do this, mice with hemophilia A were crossed with mouse B7-1. ⁽ B and B7-2 ⁾ (Borriello F, et al., B Immunity 6: 303, 1997,

Freeman GJ et al., Science 262: 907, 1993) and genotypic analysis selected mice that are deficient in both Factor VIII and either B7-1 or B7-2. Mice with haemophilia A B7-1 and B7-2 ^{_ /} 'were then injected intravenously with 0.2 gg of human factor VIII at two week intervals. Serum samples for the Factor VIII antibody assay were obtained 12 days after the second and sixth Factor VIII injections. After four injection, all nine hemophilia A B7-l ^{- / _} (open circles) developed antibodies to factor VIII, the antibody titre is greater than 350 gg / ml and the average quantity of the inhibitor is 712 BU (see FIG. 9), values similar to those normally found in haemophilia A mice injected with factor VIII (Qian J, et al., Thromb Haemost 81: 940, 1999). In contrast, none of the eight mice with haemophilia AlB7-2 ^_ (solid circles) show detection of anti-Factor VIII antibodies. Similar results were obtained in hemophilia A mice treated with antibodies to B71 and B7-2.

- »

To evaluate the T cell response of these B7-1 and B7-2 deficient hemophilia A mice, spleen cells were obtained three days after the fifth intravenous injection of factor VIII. Pooled spleen cells from three mice were used to determine proliferation data. Empty and filled squares in Fig. 10 represents cells from untreated hemophilia A B71 ^{_ /,} respectively. B7-2 ^{- /} . Empty circles indicate cells derived from a hemophilia B7-l ^{'/ _} dosed with 5 * • ···· · · · ·· ··· ···· ♦ • · · · · · · ·· • 9 9 intravenous Factor VIII injections and solid circles apply to hemophilia A / B7-2 ^{_ /} mice that received 5 intravenous injection of factor VIII. The concentration of factor VIII in the cultures is plotted on the X axis T cell proliferative activity determined by 3H-thymidine incorporation showed that the response factor VIII, is dependent on cells derived from a hemophilia A / B7-l ^{_ /,} a dose (Figure No. 10). In contrast, no T cell response was detected in spleen cells from A / B7-2 ^{- /} 'haemophilia mice. It follows that B7-2 plays a major role in promoting the immune response to Factor VIII, which is administered by intravenous injection and, in the absence of anti-Factor VIII antibodies.

Equivalents

One of ordinary skill in the art knows or is able to find, by routine experimentation only, a number of equivalents of the particular compositions and methods described herein. Such equivalents are also within the scope of the invention.

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LIST OF SEQUENCES <110> Qian, Jiahua <120> METHODS OF DAMPING IMMUNE RESPONSE TO THERAPEUTIC PROTEINS <130> GIN-013 <140> 09 / 158,178 <141> 1998-09-21 <160> 1 <170> Patentln Ver. 2.0 <210> 1 <211> 6 <212> PRT <213> Homo sapiens

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ATENTOVE

Claims (1)

Claims 1. 2. 3. 4. 5. 6. 7. 8. The composition, comprising a first agent which The means by The means by The composition by inhibiting the agent according to e further supports haemostasis and a second co-stimulatory signal in the T cell. The claim includes a claim 1, is pharmaceutically 1, comprising an acceptable carrier. The first agent of claim 1 is Factor VIII. with the first region B deleted reagent. The means by The composition of claim 1, v being a variant of the factor The first agent is Factor IX. AND. according to claim 1, characterized in that: A means by stimulating The composition e, the first agent of claim 1, is von The Willebrand factor e is the second agent of the molecule. according to the second being the soluble form of the branch 7, characterized by the agent being the soluble form CTLA4. wherein the second agent is a soluble form B7-1, 9. The composition of claim 7, wherein the soluble form of B7-2 or a combination of the soluble form B7-1 and soluble forms of B7-2. 10. The composition of claim 1, wherein the second agent is a second agent 8, characterized by CTLA4Ig. 11. The composition according to claim 9, wherein the second agent of n is B7-1Ig or B7-2Ig. 12. The composition of claim 1, wherein the second agent is a soluble form of CD40 CD40L. 13. The composition of claim 1, wherein the second agent is an antibody which binds to a co-stimulatory molecule. The composition of claim 13, 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 an anti-B7-1 antibody and an anti-B7-2 antibody. 15. The composition of claim 13, wherein the antibody is a non-activating form of an anti-CD28 antibody. A method of treating a hemostatic disorder in a subject, comprising administering to the subject a composition according to any one of claims 1 to 15, for treating a hemostatic disorder. 17. The method of claim 16, wherein the subject exhibits a pre-existing immune response to the first agent. • φ · • 4 4 4 4 «4 • 18. The method of claim 16 19 Dec 20 May make you immune The method comprises the answer of m including Thus, the composition does not comprise the first agent, comprising the subject. claim 16, further comprising a further pre-administration immunosuppressive agent. The method of claim 16, wherein the haemostatic disorder of haemophilia A, the haemophilia existing agent is selected from the group and von Willebrand's disease. 21. A method for treating a hemostatic disorder in a subject by administering to the subject a first agent that promotes hemostasis and a second agent that inhibits the co-stimulatory signal in T cell by treating a hemostatic disorder. 22. A method of treating a hemostatic disorder in a subject, comprising administering to the subject a first agent that promotes hemostasis and a second agent that inhibits the co-stimulatory signal in a T cell by attenuating the immune response to the first agent thereby treats hemostatic disorder. Way according to claim 21 or 22, p brand u j ici se t i m, f e the first reagent is a factor VIII. Way according to claim 21 or 22, p brand u j ici se t i m, f e the first reagent is a variant of the factor VIII with deleted region B. wherein the first agent is Factor IX. 25. A method according to claim 21 or 22 The method of claim 21 or 22, v y z n and watching with that first the agent is von Willebrand factor. The method of claim 21 or 22, v y z n and watching wherein the second agent is a soluble form of the agent that introduces a co-stimulatory signal into the T cell. 28. The method of claim 27, wherein the agent is a soluble form of CTLA4. 29. The method of claim 28 wherein the agent is CTLA4Ig. 30. The method of claim 27, wherein the agent is a soluble form of B7-1, a soluble form of B7-2, or a combination of a soluble form of B7-1 and a soluble form of B7-2. 31. Method se t B7-Ig according to i m that and B7-2Ig. claim 30, the agent is B7-Ig, mark i c i tec B7-2Ig or kombir 32. Method according to claim 21 or 22, marked u j i c i se t i m that the second agent is antibody, which se binds to . a co-stimulatory molecule. 33. Method according to Claim 32 F marked u j i c i se t i m that second agent Yippee selected from groups comprising an anti-B7-1 antibody, an anti-B7-2 antibody, and a combination of an anti-B7-1 antibody and an anti-B7-2 antibody. wherein the antibody is a non-activating form of an anti-CD28 antibody. 34. The method of claim 32, wherein said method is: 35. The method of claim 21 or 22, wherein the haemostatic disorder is selected from the group consisting of haemophilia A, haemophilia B, and von Willenbrand's disease. 36. The method of claim 21 or 22, wherein the subject exhibits a substantial titer of antibodies that bind to the first agent. > OT £ Inhibition of primary immune response to FVIII Inhibition of immune response to FVIII by mCTLA4 Ig in mouse haemophilia A by 000 o 000 o 00 QO ODO · «oooo OJ> o λ; φ N to bi O ί> to “c to 'o c oj bi ω vrd N bi M O N> Inhibitor treatment using CTLA4 Ig mouse with haemophilia A • ♦♦ · Inhibition of secondary immune response to FVIII by mCTLA4 Ig in mouse haemophilia A> 1 About tsl> • »'’ Τ I oo »·· ♦ · * ♦ • 9 - / (c SLW2Z coo o cro o in co Λ i l · I * l · co CM co CM co CM co O CM L10> 0 (1) N '<0 LI X O CM O O T O V (u u 6 rí ni ni P PPBd-nuy) CT i D ΤΓ <d -j O IF O X. m x <D o (D • i — i C • ··· • · 9 · f · f · 9 9 99 999 9 999 Ή o CD • rn C -H Ή C> CL ω <D -rn 44 • rH H, -1 H λλ H rtS> ti d) ω Υ υ cn 0) H I • | -> · Μ * c <• H hH H what U what> o