EA005236B1 - Composition for treating and/or preventing disorders promoting hemostasis and method for treating such disorders - Google Patents

Abstract

1. A composition for treating and/or preventing disorders which promotes hemostasis comprising at least one procoagulation agent and at least one inhibiting agent which inhibits a costimulatory molecule on the surface of antigen-presenting cells and a corresponding receptor in a T cell such that the immune response is downmodulated to thereby treat a hemostatic disorder. 2. The composition of claim 1, further comprising a pharmaceutically acceptable carrier. 3. The composition of claim 1, wherein the procoagulation agent is factor VIII. 4. The composition of claim 1, wherein the procoagulation agent is a B-domain deleted variant of factor VIII. 5. The composition of claim 1, wherein the procoagulation agent is factor IX. 6. The composition of claim 1, wherein the procoagulation agent is Von Willebrand factor. 7. The composition of claim 1, wherein the inhibiting agent is a soluble form of a costimulatory molecule. 8. The composition of claim 7, wherein the inhibiting agent is a soluble form of CTLA4. 9. The composition of claim 7, wherein the inhibiting agent is a soluble form of B7-1, a soluble form of B7-2, or a combination thereof. 10. The composition of claim 8, wherein the inhibiting agent is CTLA4Ig. 11. The composition of claim 9, wherein the inhibiting agent is B7-lIg or B72Ig. 12. The composition of claim 1, wherein the inhibiting agent is an antibody which binds to a costimulatory molecule. 13. The composition of claim 12, wherein said antibody is selected from the group consisting of an anti-B7-1 antibody, anti-B7-2 antibody, and a combination thereof. 14. The composition of claim 12, wherein the antibody does not transduce a costimulatory signal in a T cell. 15. A method of treating a hemostatic disorder in a subject comprising administering to the subject a therapeutically effective composition of any one of claims 1-14. 16. The method of claim 15, wherein the subject has a preexisting immune response to the procoagulation agent. 17. The method of claim 15, wherein the subject does not have a preexisting immune response to the procoagulation agent. 18. The method of claim 15, further comprising administering the subject a composition comprising an additional immunosuppressive agent. 19. The composition of claim 15, wherein the subject has a hemostatic disorder of hemophilia A, hemophilia B, and von Willebrand's disease. 20. A method for treating disorders promoting hemostasis comprising administering a therepeutically effective composition to the subject at least one procoagulation agent and at least inhibiting agent which inhibits a costimulatory molecule on the surface of antigen-presenting cells and a corresponding receptor in a T cell such that the immune response is downmodulated to thereby treat a hemostatic disorder. 21. The method of claim 20, wherein the procoagulation agent is factor VIII. 22. The method of claim 20, wherein the procoagulation agent is a B-domain deleted variant of factor VIII. 23. The method of claim 20, wherein the procoagulation agent is factor IX. 24. The method of claim 20, wherein the procoagulation agent is Von Willebrand factor. 25. The method of claim 20, wherein the inhibiting agent is a soluble form of a costimulatory molecule. 26. The method of claim 25, wherein the inhibiting agent is a soluble form of CTLA4. 27. The method of claim 26, wherein the inhibiting agent is a soluble form of CTLA4Ig. 28. The method of claim 25, wherein the inhibiting agent is a soluble form of B7-1, a soluble form of B7-2, or a combination thereof. 29. The method of claim 28, wherein the inhibiting agent is B7-1Ig, B7-2Ig or a combination thereof. 30. The method of claim 20, wherein the inhibiting agent is an antibody which binds to a costimulatory molecule. 31. The method of claim 30, wherein said antibody is selected from the group consisting of an anti-B7-1 antibody, an anti-B7-2 antibody and a combination thereof. 32. The method of claim 30, wherein the antibody does not transduce a costimulatory signal in a T cell. 33. The method of claim 20, wherein the subject has a hemostatic disorder of hemophilia A, hemophilia B, and von Willebrand's disease. 34. The method of claim 20, wherein the subject has a significant titer of antibodies which bind to the procoagulation agent.

Description

FIELD OF THE INVENTION

The invention relates to the field of immunology and medicine and relates to the treatment of hemostasis disorders by introducing foreign therapeutic proteins to the subject with negative modulation of the immune responses to them in the subject.

State of the art

One of the primary limitations of therapeutic treatment using biological proteins is the immune response that the body produces in response to the presence of foreign substances in it. This immune response is a particular problem when foreign substances must be re-introduced in order to obtain their optimal effectiveness.

One example of this situation is the re-administration of agents for the treatment of hemostasis disorders, such as diseases associated with factor VIII deficiency (for example, classical hemophilia A and von Willebrand disease) or factor IX deficiency, also known as hemophilia B. Classical hemophilia, hemophilia A is an X-linked disorder that affects one in 10,000 men. Von Willebrand disease is the most common hereditary bleeding disease that occurs in one of 800-1000 subjects. Hemophilia B, also known as Christmas disease, occurs in approximately 1 out of 100,000 men (see Monograph “The Principles of Harrison's Therapy” (Nutcop'k ΡΓίηοίρΙοδ о £ Ш1етпа1 Мсbyшс) under the editorship of Nksyasist et al., 13th ed., 1994. .MsOga ^ -NS Ν.Υ., Ν.Υ.

Factor VIII is a single chain protein of molecular weight 265 kD, which circulates in the blood in complex with von Willebrand factor (ν ^ Ρ). Factor VIII is an important regulatory protein in the blood coagulation cascade. After thrombin activation, it increases the rate of factor X activation by activated factor IX (factor Ka), ultimately leading to the formation of a fibrin clot. The ν \ νΡ molecule is an adhesion glycoprotein that plays a major role in platelet agglutination. It serves as a carrier for factor VIII in plasma and facilitates platelet interactions with the vessel wall, VVΡ consists of many, possibly identical subunits, each of which has a molecular weight of approximately 230 kD. VVΡ is synthesized in endothelial cells and megakaryocytes. Factor IX is a 55 kD single-chain proenzyme of molecular weight that is converted to an active protease (Ka) by factor XP1 or a tissue factor-SC complex. Activated factor IX and activated factor VIII then activate factor X.

Repeated administration of foreign proteins can lead to an immune response to these proteins in the recipient's body. In the T cell response to foreign proteins, antigen-presenting cells (APC) should deliver two signals to resting T-cells (see articles 1spksh8 M. and 8c11 \ vag1x I., I. Εχρ. Msb., 165, 302-319, (1987) ; Mis11sg, I.L. et al., I. Ittiηo1., 144, 3701-3709, (1990)). The first signal, which determines the specificity of the immune response, is transduced through the T-cell receptor (TSJ) after recognition of a foreign antigenic peptide presented in the context of the main histocompatibility complex (MHC). The second signal, called co-stimulation, induces the proliferation and emergence of T-cell functionality (see article Lsp5c1u \\ · et al., Appi. Yasu. Itipo1., 14: 233, (1996)). Costimulation is neither antigen-specific nor MHC restricted and is believed to be provided by one or more different cell surface molecules expressed by APC (see articles 1spksh8 MK and so on., I. Ittipo.., 140, 3324-3330 , (1988); Lysmu, S. 8. et al., I. Εχρ. Msb., 173, 721-730, (1991); 01tt1 S.I. et al., Proc. No. 11. Asab. 8c1. I8A , 88, 6575-6579, (1991); ^ο ^ IV. Et al., I. S. Shush. 90, 229-237, (1992); Koioua L. et al., I. Εχρ. Msb., 173 , 759-762, (1991); Yassb, N. et al., Proc. Νη11. Asab. 8sk I8A, 89, 271-275, (1992); UAP-8Susp1st O.A. et al., I. !, 144, 4579-4586, (1990); La8a11c 1M. Et al., I. It type 1., 147, 774-80, (1991); Oikip M.1 et al., I. Εχρ. Msb .. 169, 503, (1989); AttIads Ya et al., No. 1its, 357, 80 -82, (1992); Yi, и. Et al., I. Εχρ. Msb., 175, 437-445, (1992)).

Proteins SI80 (B7-1) and CO86 (B7-2) expressed on APC are critical costimulatory molecules (see the articles of Rgestap et al., I. Εχρ. Msb., 174: 625, (1991); Rgsstap and et al., I. Ittipo., 143: 2714, (1989); Ahita et al., No. C.1gs. 366: 76, (1993); Rgstap et al., 8c1spss, 262: 909, (1993)).

B7-2 is thought to be more important in primary immune responses, while B7-1, which is upregulated in the later stages of the immune response, may be important for prolonging primary T-cell responses or co-stimulating secondary T-cell responses ( see Vyuyops article. Ittishu, 2: 555, (1995)).

B7-1 and B7-2 are contraceptors for two ligands expressed on T-lymphocytes. One ligand, which binds B7-1 and B7-2, SI28, is constitutively expressed on resting T cells, and its expression increases after activation. After a signal through a T-cell receptor, ligation of SI28 and transduction of a costimulatory signal induce proliferation of T-cells and their secretion of IL-2 (interleukin-2) (see articles L881eu P.8. Et al., 1. Exp. Meb., 173 , 721730, (1991); С1sht1 S.I. et al., Proc. No. 111. Asab. 8s1. I8A, 88, 6575-6579, (1991); 1ipe S.N. et al., 1ttypo1. Tobau, 11, 211-6, (1990); Nagbtd, E.A. et al., Iaige, 356, 607-609, (1992)). The second ligand, called STBA4 (SI152), is homologous to SI28, but is not expressed on resting T cells and appears after activation of T cells (see article Vgipe! EE. Et al., Iaige, 328, 267-270, (1987 )). STBA 4. Apparently, it is critical in terms of the negative regulation of Tcellular responses (see the article ^ a1egoyo8e et al., 8c1epse, 270: 985, (1995)). STBA4 blockade was found to eliminate inhibitory signals, while STBA4 aggregation was found to provide inhibitory signals that negatively regulate (suppress) T-cell responses (AShkop and Kgitte1, 8s1epse, 270: 932, (1995)). B7 molecules have a higher affinity for STBA4 than for SI28 (see the article YnDeu R. 8 et al., 1. Exp. Meb No. 174, 561-569, (1991)), and B7-1 and B7 -2, as it was shown, bind individual sections of the STBA4 molecule and have different binding kinetics with STBA4 (see the article IpNeu et al., 1ttyyu, 1: 793, (1994)).

10–25% of hemophilia patients develop an immune response to factor VIII. In these patients, inhibitors are formed, usually 1dC antibodies, which neutralize the activity of factor VIII, which thus interferes with effective therapy. Two types of inhibitors have been identified. High-response patients with type I inhibitors exhibit an anamnestic response to factor VIII, resulting in an increased antibody titer against factor VIII. Patients with a low level of response, having a type II inhibitor, show a low antibody titer, which does not increase with the introduction of factor VIII. Modern strategies aimed at smoothing the response of antibodies in these patients are only very successful. Moreover, the improvement of antibodies against substitute proteins is an urgent problem that must be solved if gene therapy is nevertheless effective in the treatment of hemophilia and other diseases associated with defects (see Soppe11u 8. et al., B1ob, 88: 3846 , (1996); Kipa 8.N. et al., B1ob, 91: 784, (1998)).

SUMMARY OF THE INVENTION

The present invention, inter alia, provides compositions and methods that allow the administration of a therapeutic protein to treat a disorder while reducing the production and / or development of an immune response to a therapeutic protein.

In one aspect, the invention relates to compositions for treating and / or preventing disorders leading to abnormal bleeding, comprising at least one coagulation factor and at least one agent that inhibits the interaction of a co-stimulating molecule on the surface of an antigen-presenting cell and its corresponding receptor on a T cell, leading to the generation of an immune response to the specified factor.

In one embodiment of the invention, these compositions further comprise a pharmaceutically acceptable carrier.

In one preferred embodiment, the procoagulation factor is factor VIII. In another embodiment, it is a variant of factor VIII with a deletion of the B domain. In a further embodiment, the procoagulation factor is factor IX. In another embodiment, it is von Willebrand factor.

In a preferred embodiment, the inhibitory agent is a soluble form of a costimulatory molecule. In a preferred embodiment, the inhibitory agent is a soluble form of STBA4. In another preferred embodiment, the inhibitory agent is a soluble form B7-1, or a soluble form B7-2, or a combination of a soluble form B7-1 and a soluble form B7-2. In a more preferred embodiment, the inhibitory agent is (or contains) STEA4- [d. In another more preferred embodiment, it is (or contains) B7-11d or B7-21d.

In a further embodiment, the inhibitory agent is an antibody that binds a costimulatory molecule. In a preferred embodiment, the inhibitory agent is selected from the group consisting of an anti-B7-1 antibody, an anti-B7-2 antibody, and a combination of anti-B7-1 and B7-2 antibodies. In one embodiment, the antibody does not transduce a costimulatory signal into the T cell.

In addition, the invention relates to methods for treating disorders leading to abnormal bleeding in a subject, comprising administering to the subject these compositions so that a therapeutic effect arises with respect to said disorders leading to abnormal bleeding.

In one embodiment, the subject has a high titer of antibodies that bind a procoagulation factor. In another embodiment, the subject lacks a high titer of antibodies that bind procoagulation factor.

In one embodiment, the hemostasis disorder present in a subject is hemophilia A or hemophilia B, or von Willebrand disease.

In another aspect, the invention relates to methods for treating hemostasis disorders in a subject, comprising administering to the subject at least one procoagulation factor and at least one agent that inhibits the interaction of a molecule on the surface of an antigen-presenting cell and its corresponding receptor on a T cell, leading to the generation of an immune response to the specified factor.

In one embodiment, the procoagulation factor is represented by factor VIII. In another embodiment, the procoagulation factor is a variant of factor VIII with a deletion of the B domain. In another embodiment, the procoagulation factor is factor IX. In another embodiment, it is von Willebrand factor.

In a further embodiment, the inhibitory agent is a soluble form of a costimulatory molecule. In a preferred embodiment, it is a soluble form of CTLA4. In a more preferred embodiment, the agent is CTBA4-1d. In another preferred embodiment, it is a soluble form of B7-1, a soluble form of B7-2, or a combination of both B7-1 and B7-2. In another more preferred embodiment, the inhibitory agent is B7-11d, B7-21d, or a combination of both B7-11d and B7-21d.

In yet another embodiment, the inhibitory agent is an antibody that binds a costimulatory molecule. In another embodiment, the agent is selected from the group consisting of an anti-B7-1 antibody, an anti-B7-2 antibody, and a combination of anti-B7-1 and B7-2 antibodies. In yet another embodiment, the antibody does not transduce a costimulatory signal into the T cell.

In one embodiment, the hemostasis disorder present in a subject is hemophilia A, or hemophilia B, or von Willebrand disease.

In another embodiment, the subject has a high titer of antibodies that bind procoagulation factors.

List of drawings and other materials

In FIG. 1 shows the experimental design used in Example 1 to test the inhibition of the primary antibody responses to factor VIII.

In FIG. 2 shows that although mice that did not receive STEA4- [d. had high titers of antibodies, starting from such an early date as day 20 (C-1), in mice that received

ST ^ A4- [d. antibodies were not produced until day 82 (C-2 and C-3).

In FIG. 3 shows the experimental design used in Example 2 to test the inhibition of secondary antibody responses to factor VIII.

In FIG. 4 shows that animals that did not receive CT ^ A4- [d. had high titers of antibodies against factor VIII (C-1), while mice that received CT ^ A4-Id (C-2) (except 1 mouse) did not develop a secondary immune response to factor VIII.

In FIG. Figure 5 shows the effect of tST ^ A4-Id on the formation of antibodies against factor VIII.

In FIG. Figure 6 shows the effect of repeated administration of tST ^ A4-Id on the formation of antibodies against factor VIII.

In FIG. 7 shows the effect of the simultaneous administration of tST ^ A4-Id and factor VIII.

In FIG. Figure 8 shows the effect of tST ^ A4-Id on the secondary immune response against factor VIII.

In FIG. Figure 9 shows the role of B7-1 and B7-2 in the response of antibodies against factor VIII.

In FIG. Figure 10 shows the response of T cells to factor VIII for mice with hemophilia A / B7-1 ^{- / -} and hemophilia A / B2 ^{- / -} .

Information confirming the possibility of carrying out the invention

This invention represents a significant advance in the treatment of hemostasis disorders, by providing compositions and methods that allow administration of a therapeutic protein for treating a disease while reducing the production and / or development of an immune response to a therapeutic protein.

Before further describing the invention, a number of terms used in the description, examples and appended claims are compiled in this section for convenience.

I. Definitions.

As used in this context, the expression violation of hemostasis includes disorders that lead to abnormal bleeding and / or thrombosis. Normal hemostasis limits blood loss through a series of interactions between components of the walls of blood vessels, platelets, and plasma proteins. Hemostasis disorders occur, for example, due to the inability of platelets to aggregate and / or form a fibrin clot, which can lead to inappropriate reactions to a disease or injury, for example, uncontrolled bleeding. These disorders can be established, for example, by determining bleeding time, partial thromboplastin time (PTT), prothrombin time (PT), thrombin time (TT), or by quantifying fibrinogen using methods well known in the art. Examples of hemostasis disorders include hemophilia A, hemophilia B, and von Willebrand disease.

As used in this context, the expression agent that stimulates hemostasis includes a protein or polypeptide that is insufficient or absent in the subject and which, when administered to the subject, alleviates or cures hemostasis disorders. Preferred agents that promote hemostasis include coagulation factors such as factor VIII, factor IX, Y \ UR and their analogs.

The term B7 family or B7 molecules, as used in this context, includes costimulatory molecules that have an amino acid sequence identical to B7 polypeptides, for example, B7-1, B7-2 or B7-3 (recognized by BB-1 antibody). In addition, the B7 family of molecules is characterized by a common function, for example, the ability to bind a ligand of the B7 family (for example, one or more of CE28. STA4 or Κ.Ό8) and the ability to stimulate T cell activation.

B7 polypeptides have the ability to co-stimulate activated T cells, thereby inducing T cell proliferation and / or cytokine secretion, or inhibit T cell co-stimulation, for example, when they are in soluble form. Representatives of the B7 family include B7-1, B7-2 and their soluble fragments or derivatives. In one embodiment, members of the B7 family bind CTLA4, CO28, JuO8 and / or other ligands on immune cells and have the ability to inhibit or induce co-stimulation of immune cells.

Used in the context of this application, the expression an agent that inhibits a co-stimulating signal in a T-cell includes agents that inhibit the signal produced by the interaction of a co-stimulating molecule on an antigen-presenting cell (APC), for example, a molecule of the B7 family, and its counterceptor on the T cell . Co-stimulating molecules on APC (for example, molecules of the B7 family) and related ligands on T cells (for example, CTLA4, CE28, and OO8) are collectively referred to as costimulatory molecules in this context. An agent that inhibits a co-stimulating signal can act either extracellularly to inhibit the interaction between co-stimulating molecules, thereby blocking the formation of intracellular signals, or it can act intracellularly to inhibit co-stimulating signals in the signal transduction pathway. Examples of agents are described in more detail in this context and include, for example, soluble forms of costimulatory molecules and antibodies that bind costimulatory molecules.

As used in this context, the expression negative modulation of the immune response includes a decrease in the immune response (e.g., suppression, attenuation or inhibition) in a patient who currently does not have an immune response, or a decrease in the duration and quantitative value of an existing immune response. The term immune response includes any type of immune response that is triggered or dependent on costimulatory signals, for example, a cellular or humoral response that may appear in a subject in response to a foreign antigen. In one embodiment, the immune response is an antibody response to an agent that stimulates hemostasis (e.g., factor VII, ν \ νΡ or factor IX). The term immunotolerization includes the induction of antigen-specific tolerance, which can be measured using techniques known in the art, for example, by measuring secondary immune responses (e.g., cellular or humoral responses) to an antigen.

II. Agents that stimulate hemostasis.

In one embodiment, an agent that stimulates hemostasis is represented by factor VIII. The term factor VIII, as used in this context, includes proteins exhibiting a procoagulant activity characteristic of factor VIII. In one embodiment of the invention, the proteins of factor VIII are proteins of natural factor VIII. These proteins may be purified from the blood or may be administered as a blood product or an enriched blood product. In one embodiment, highly purified factor VIII can be obtained by adsorption and elution of the factor from a blood product on a monoclonal antibody column. Alternatively, these natural proteins can be produced recombinantly using nucleic acid molecules, preferably naturally occurring nucleic acid molecules. For example, in one embodiment, factor VIII proteins are obtained by expressing a nucleic acid molecule encoding factor VIII in a cell using methods known in the art to form a factor VIII protein. The nucleotide sequence (and the corresponding amino acid sequence) of human factor VIII is known in the art (see, for example, Too1e et al., La1ige, 312: 5992 (1984) or SepVapk registration No. X01179; K01740).

In another embodiment, the hemostasis stimulating agent is a non-naturally occurring factor VIII, e.g., a mutant form of factor VIII that retains a therapeutic function, e.g., factor VIII hemostasis stimulation activity. For example, DNA sequences capable of hybridization with DNA encoding human factor VIII under conditions that avoid hybridization with non-factor VIII genes (for example, under conditions equivalent to 65 ° C in 5X 88C (1 X 88C = 150 mM Ν; · ιΟ1 / (). 15 M citrate no. which are included in the scope of the claims of the invention. As examples, the contents of US patents Nos. 5744446, 5663060, 5583209, 5661008, 5422260 and 5707832 are specifically incorporated into this context by reference.

In another embodiment, the agent that stimulates hemostasis is a factor VIII protein in which there is a deletion of at least one domain (eg, a non-primary domain) of the protein. For example, in one embodiment, the factor VIII protein is a modified factor VIII protein in which there is a deletion or replacement of one or more amino acids between the cleavage sites of 90 and 69 kDa relative to native factor VIII, as described in more detail in US patent No. 4868112, the content which is entered into this context by reference.

In another embodiment, the agent that stimulates hemostasis is an analogue of factor VIII containing a deletion (s) between the 50/40 cleavage site and the 73 kD cleavage site, which can be obtained by methods similar to those described in US patent No. 4868112, the content of which entered into this context by reference. In a preferred embodiment, the factor VIII analogue partially or fully retains a portion of acidic amino acids between cleavage sites of 80 and 73 kD. In another embodiment, the site is partially or fully replaced by the corresponding acidic site directly adjacent to the 50/40 cleavage site. In a number of other embodiments, the factor VIII proteins are analogues (with or without deletions), such as those described in PCT / ϋδ 87/01299 (the contents of which are incorporated herein by reference), for example, in which one or more cleavage sites located between the arginine residues at positions 226, 336, 562, 740, 776, 1313, 1648 or 1721 were made resistant to proteolytic cleavage, for example, by replacing one or more amino acids with other amino acids through mutagenesis cDNA using technologies known in the art, for example, using standard site-directed mutagenesis.

Hemostasis promoting agents also include fusion factor VIII proteins that contain a human factor VIII protein fragment and a factor protein fragment

VIII non-human isolated from an animal of another species (for example, swine factor VIII). These fusion proteins can be obtained using technologies that are known in the art, for example, those presented in US Pat. Nos. 5,744,446, 5,663,060 and 5,583,209.

The contents of US patents No. 5693499, 5681746, 5663060, 5583209, 5563045, 5460951 and 5455031 are also specifically incorporated into this context by reference.

In another embodiment, an agent that stimulates hemostasis is represented by factor IX. As used in this context, the term factor IX includes, but is not limited to, factor IX isolated from plasma, transformed cell lines and recombinantly derived factor IX isolated from the host cell culture medium. Factor IX may be purified from the blood or may be administered as a blood product or an enriched blood product. In one embodiment, highly purified factor IX can be obtained by adsorption and elution of the factor from a blood product on a monoclonal antibody column. Examples of purification methods are also described in US Pat. Nos. 5,639,857, 5,457,181 and 5,286,849. Alternatively, these natural proteins can be produced recombinantly using nucleic acid molecules, preferably naturally occurring nucleic acid molecules. For example, in a number of embodiments, factor IX proteins are obtained by expressing a nucleic acid molecule encoding factor IX in a cell using methods known in the art that produce the factor IX protein. Examples of genetic constructs for the expression of factor IX can be found in US patent No. 5650503 and 4994371.

The nucleotide sequence and the amino acid sequence of factor IX is known in the art (see, for example, Wozyjake et al., Wusyeszkyu, 24: 3726, (1985) or SeiVaik registration No. K02402, A07407, A01819 or X54500).

In other embodiments, the implementation of the factor

IX includes, for example, the proteins described in US patent No. 4994371, 5171569, 5679639, 5621039 and 5714583, the full descriptions of which are incorporated into this context by reference.

In addition to the natural forms of factor IX, the term factor IX also includes non-natural forms, for example, mutant forms of factor IX, which retain therapeutic properties, for example, hemostatic stimulation properties, factor IX. For example, it includes DNA sequences capable of hybridization with DNA encoding human factor IX under conditions that avoid hybridization with non-factor IX genes (for example, under conditions equivalent to 65 ° C in 5 X 88C (1 X 88C = 150 mm No. 1C1 / (). 15 M citrate Να) .In addition, DNA sequences that maintain sequence identity with respect to portions of a nucleic acid molecule encoding protein domains that are important for the function of factor IX can be used to produce factor IX proteins that are iat in the scope of claims of the invention.

Proteins of factor VIII or factor IX can also be purchased commercially. For example, there are concentrated forms of factor VIII, for example, Ittiηaΐe®, Dttipo), Vepa! E® (Weippd); there are forms of factor VIII purified using a monoclonal antibody, for example, Oslapayu-M® (Ryagtaspa), NetoP1 M® (Bax! eg) and Mopoe1a! e-P® (Rgtoyg), and there are also recombinant forms of factor VIII, for example, K ееотЫотЫпа11е® В В В В В В В В В! В В (Wah! и)) and Codedepa! е®® (Wаегегег). There is also a form of factor VIII with a deletion of the B-domain - r-VIII 80® (Pryagtepa and Irsobn, 81osk1y1t). Factor IX can be purchased as Naηoί ^ ν® (KaY Ryagtaspa) or Ittishpe® Dttipo); Factor IX purified by monoclonal antibody is also commercially available under the name Mopospe® (Ltogig). There is also a recombinant factor IX, for example, under the name VepeRK® (Oepeisk Sch81y1e).

Y ^ P is a large multimeric plasma protein, consisting only of glycoprotein subunits. The subunits V \ VΕ are linked by disulfide bonds. In plasma, A ^ P circulates in the form of multimers, among which there are multimers from dimers to multimers of more than 50 subunits. Dimers consist of two subunits, possibly connected at the C-termini with flexible, rod-shaped domains, and are believed to be protomers during multimerization. Protomers are linked via large, possibly Ν-terminal globular domains to form multimers. 'ΫΑΡ, apparently, is formed in the form of a glycosylated molecular weight precursor of 269 kDa, which is sequentially processed and sulfated. After dimerization and multimerization and proteolytic cleavage, the mature protein has a molecular weight of approximately 225 kD. 'ΫΑΡ obtained recombinantly. The nucleotide and amino acid sequences ΫΑΡ are known in the art (see, for example, Ref. 8, et al., Co, 8, ppb, Nag 8, Morning 51, 515, (1986) or Registration No. registration No. 15333 or K03028). The contents of patent EP 0197592 B1 are incorporated herein by reference.

In addition to the natural forms of factor V \ VΕ, the term factor ΫΑΡ also includes non-natural forms, for example, mutant forms of factor ΫΑΡ, which retain therapeutic properties, for example, hemostatic stimulation properties of factor V \ VΕ. For example, it includes DNA sequences capable of hybridization with DNA encoding the human factor 'ΫΑΡ under conditions that avoid hybridization with the genes of the non-factor V \ VΕ (for example, under conditions equivalent to 65 ° C in 5 X 88C (1 X 88C = 150 mM No. C1 / 0.15 M citrate Νι.) In addition, DNA sequences that maintain sequence identity with respect to portions of a nucleic acid molecule encoding protein domains that are important for the function of the ΫΑΡ factor can be used to produce factor ’proteins ΫΑ Which are within the scope of the invention.

In one embodiment, the agents that stimulate hemostasis are, by origin, agents isolated from mammals. In a preferred embodiment, the agents that stimulate hemostasis are porcine in origin. In yet another embodiment, the agents that stimulate hemostasis are human in origin. In another embodiment, the agents that stimulate hemostasis are hybrid molecules.

III. Immunomodulating agents.

In one embodiment, an agent that inhibits a costimulatory signal in a T cell is a natural form of a costimulatory molecule. Natural forms of costimulatory molecules can be purified from cells or obtained recombinantly using techniques known in the art. For example, co-stimulating proteins can be obtained by expressing a nucleic acid molecule encoding a co-stimulating molecule in a cell to form a co-stimulating molecule. The nucleotide sequences of costimulatory molecules are known in the art and can be found in the literature or in a database such as OepWapc. See, for example, regarding B7-2, an article by Rgeetap et al., 8spepse, 262: 909, (1993) or OepVapk registration No. P42081 or A48754; regarding B7-1 article Rgeetap et al., I. Exp. Meb., 174: 625, (1991) or OepVapk registration No. P33681 or A45803; for STBA4, see, for example, Oshkett et al., 8c1eise, 228: 1401, (1985) or SeVaik No. P16410 or 291929 and for SE28, articles Ltiyo and 8eb, Prgos. Ν; ι11. Asab. 8sk, 84: 8573 or SeiVaik registration number 180091, regarding 1CO8 article Ni11o £$ et al., Wing. 397: 263, (1999); AO 98/38216 and related sequences.

In addition to the natural forms of costimulatory molecules, the term costimulatory molecule also includes non-natural forms, for example, mutant forms of costimulatory molecules that retain the function of a costimulatory molecule, for example, the ability to bind a related contraceptor. For example, DNA sequences capable of hybridization with DNA encoding a B7 molecule, an STA4 molecule, CE28 molecule or a 1CO8 molecule under conditions that avoid hybridization with genes of non-stimulating molecules (for example, under conditions equivalent to 65 ° C in 5 X 88C (1 X 88C = 150 mM No. C1 / 0.15 M citrate (α) are costimulatory molecules that are within the scope of the invention Alternatively, DNA sequences that maintain sequence identity with respect to portions of a nucleic acid molecule encoding protein domains that are which are important for the function of the costimulatory molecule, for example, the functions of binding to other costimulatory molecules, can be used to obtain costimulatory proteins that can be used as agents that inhibit the costimulatory signal in the T cell. Preferably, when the non-natural costimulatory molecules possess significant (for example, exceeding 70%, preferably exceeding 80% and more preferably exceeding 90-95%) amino acid identity with the natural amino acid sequence e stratsellyularnogo domain of a costimulatory molecule.

To determine the amino acid residues of the costimulatory molecule, which are probably important for the binding of the costimulatory molecule to its counterceptor, we can compare the amino acid sequences containing extracellular domains of costimulatory molecules of various animal species, e.g., mouse and human, and note conservative (e.g., identical) residues . This can be done, for example, using any standard comparison program, such as MedAIdp (ΌΝΑ 8TAB). These conservative or identical residues are apparently necessary for the proper binding of costimulatory molecules to their receptors, and thus are probably not susceptible to change.

Specific residues of costimulatory molecules that are important for binding have also been identified. For example, the CE28 fragment, which is critical for interaction with B7-1 and B7-2, was determined using site-directed mutagenesis, epitope mapping using monoclonal anti-SE28 antibody, receptor-based adhesion assays, and direct binding of 1d fusion proteins to cell surface receptors. The portion of the proline-rich sequence in CE28 ΜΥΡΡΡΥ, as was shown, is necessary for the function of this protein (see the article by Tttsiy et al., Mo1. 1ttio1., 33: 321, (1996)). Similarly, portions of the B7-1 molecule, which are important for mediating the functional interaction with CE28 and CTLA4, were identified by mutation. It was found that two hydrophobic residues in the V-like domain of B7-1, including residue Υ87, which is conserved in all B7-1 and B7-2 molecules cloned from organisms of various species, are necessary (see the article by Ratdeak et al., 1 Exp.Meb., 182: 667, (1995)). Using data or similar methods, it is possible to determine the amino acid residues of the extracellular domains of costimulatory molecules, which are necessary and therefore should not be subjected to changes.

Costimulatory molecules can be expressed in soluble form or used as immunogens to produce antibodies. These soluble costimulatory molecules or antibodies are used as agents that inhibit the costimulatory signal in the T cell, as described in more detail in this context.

A. Agents with extracellular action, inhibiting co-stimulating signal in the T-cell.

1. Soluble forms of costimulatory molecules.

In one embodiment, an agent that blocks a co-stimulating signal in a T cell is a soluble form of a T-cell co-stimulating molecule (e.g., STBA4, CE28 and / or 1CO8) that is capable of blocking transduction of a co-stimulating signal in a T cell.

In one embodiment, the T-cell blocking co-stimulating signal is a soluble form of STBA4. DNA sequences encoding human or murine STBA4 protein are known in the art, see, for example, Papuyuy et al., Eig. 1. 1ttypo1., 18 (12), pp. 1901-1905, (1988); High 1 ΙΕ. et al., Kirta, (1987); Wow! !R. et al. 1ttio1. Neu. 103: 21-36, (1988) and Rgeetai OE. et al., 1. 1ttio1., 149: 3795-3801, (1992). In a number of embodiments, the CTLA4 soluble protein contains the entire CTL4 protein. In a preferred embodiment, the STL4 soluble protein contains the extracellular domain of the STL4 protein. For example, the soluble recombinant form of the extracellular domain of CTL4 was expressed in yeast (see Setyshauet et al., REB8 Le11., 407: 63, (1997)). In other embodiments, the CTL4 soluble proteins comprise at least a fragment of the extracellular domain of the CTL4 protein that retains the ability to bind B7-1 and / or B7-2.

In one embodiment, the CTL4 soluble protein or fragment thereof is a fusion protein containing at least a portion of CTL4 that binds B7-1 and / or B7-2, and at least a portion of a second protein other than CTL4. In preferred embodiments, the CTL4 fusion protein contains an extracellular domain of CTL4, which is fused at the amino terminus with a signal peptide, for example, from oncostatin M (see, for example, application ΧνΟ 93/00431).

In a particularly preferred embodiment, the soluble form of CTL4 is a fusion protein containing the extracellular domain of CTL4 fused to a fragment of an immunoglobulin molecule. This CTBA4-1d fusion protein can be obtained using methods known in the art (see, for example, article 1shk1еу Prospects for the discovery and creation of drugs (Retkresyuek ίη Aegid Oschsuotu apb Oeydp). 2: 221, (1994); bshk1еу Application XVO 93/00431 and U.S. Patent 5770197).

In one embodiment, an agent that blocks a co-stimulating signal in a T cell is a soluble form of a co-stimulating molecule of an antigen-presenting cell (for example, a molecule of the B7 family, such as B7-1, B7-2 and / or ligand 1CO8). For example, in one embodiment, the soluble form of the costimulatory molecule comprises a soluble form B7-1 or a soluble form B7-2 or a combination of a soluble form B7-1 and a soluble form B7-2. DNA sequences encoding B7 proteins are known in the art, see, for example, Rteeshap et al., 8c1epse, 262: 909, (1993) or SepVapk registration No. P42081 or A48754, relative to B7-1 article, regarding B7-2 Rteshap et al., 1. Exp. Meb., 174: 625, (1991) or SepVapk registration No. P33681 or A45803. In a number of embodiments, the soluble B7 protein contains the complete B7 protein. In preferred embodiments, the soluble B7 protein comprises the extracellular domain of the B7 protein. For example, a soluble recombinant form of the extracellular domain of CTLA4 was expressed in yeast (see article

Setyshauet et al., REV8 LeI., 407: 63, (1997)). In other embodiments, the soluble B7 proteins comprise at least a fragment of the extracellular domain of the B7 protein that retains the ability to bind CTLA4 and / or CE28.

In one embodiment, the soluble B7 protein or fragment thereof is a fusion protein containing at least a portion of B7 that binds CO28 and / or CTBA4 and at least a portion of a second protein other than B7. In preferred embodiments, the B7 fusion protein contains an B7 extracellular domain that is fused at the amino terminus with a signal peptide, for example, from oncostatin M (see, for example, νθ 93/00431).

In a most preferred embodiment, the soluble form of B7 is a fusion protein containing an extracellular domain of B7 fused to a fragment of an immunoglobulin molecule. This B71d fusion protein can be obtained using methods known in the art (see, for example, the article “Prospects for the Detection and Creation of Drugs” (Retcresyueck Otid P1xowu apb Oeydp). 2: 221, (1994); 93/00431, US Patent 5770197 and US Patent 5580756).

2. Antibodies that bind costimulatory molecules.

In a number of embodiments, the co-stimulating signal blocking agent in the T cell is represented by an antibody that binds a co-stimulating molecule. When producing antibodies that bind co-stimulating molecules, it is possible to use a co-stimulating protein, a fragment of a co-stimulating protein (for example, a peptide isolated from a co-stimulating protein) or a fusion protein that includes the whole or part amino acid sequence of the co-stimulating molecule to create a polyclonal antiserum or monoclonal anti-protein antibodies and / or against the peptide using standard methods. The term antibody, as used in this context, is intended to include whole antibodies, as well as fragments thereof. Antibody fragments (e.g., fragments of PaB ', fragments of P (aB') ₂ or single chain antibodies) can be obtained using methods well known in the art. The term antibody also includes chimeric and humanized antibodies.

For example, mammals (in particular, a mouse, hamster, or rabbit) can be immunized with the immunogenic form of a costimulatory protein or peptide that elicit an antibody response in the mammal. An immunogen can be represented, for example, by a recombinant protein of a co-stimulating molecule or fragment thereof, a synthetic peptide fragment, or a cell that expresses a co-stimulating molecule on its surface. The cell may be, for example, an antigen-presenting cell or a Cell or a cell transfected with a nucleic acid encoding a co-stimulating molecule, wherein the co-stimulating molecule is expressed on the cell surface. Host cells transfected in such a way as to obtain expression of the peptides can be represented by any prokaryotic or eukaryotic cell. For example, a peptide having co-stimulating molecule activity can be expressed in bacterial cells such as E. soy, insect cells (baculovirus), in yeast or mammalian cells such as Chinese hamster ovary (CHO) cells and N80 cells. Other suitable cell hosts and expression vectors can be found in Soebbe1 (1990) Kirg, or they are known to those skilled in the art. Examples of vectors for expression in yeast 8. espubas include pUep8ec1 (see Ba1bap et al., Etio 1., 6: 229-234, (1987)), pMEA (see Kig | ap and Neg5ko \\ 'x. Се11, 30: 933-943, (1982)), РЖУ88 (see article 8сйи11х et al., Sepe, 54: 113-123, (1987)) and рУЕ82 Дпуйгодеп Согога! пο п, 8ап И1едо, СА). Available baculovirus vectors for expression of proteins in cultured insect cells (8E 9 cells) include the pAC series (see article 8th et al., Mo1. Ce11Bu1., 3: 21562165, (1983)) and the series pUB (see article Liskote V .A. And 8ittegk M.I., U1go1odu, 170: 31-39, (1989)). Basically, for the time amplification / expression in mammalian cells, C08 cells are used (see article C1khtap Υ., Ce11, 23: 175-182, (1981)) in combination with vectors such as pCIM8 (see article 8eb B., No. 1! Ighe, 329: 840, (1987)), while CHO cells (Chinese hamster ovum bGG cells) are used with vectors such as pMT2RS (see KaiGgpap et al., EMB0 1., 6: 187-195, (1987)), for stable amplification / expression in mammalian cells. The preferred cell line for the production of the recombinant protein is the N80 myeloma cell line available at ECACC (catalog No. 85110503) and described in the article by b. C. and M11k! Es S. ((1981) Meobob w Ephutododu, 73 (13): 3 -46 and monographs. Strategy and methods for producing monoclonal antibodies (Rheraga! Ju rG Mopos1op1 ApyoFek: 8! Ha! Ed1e8 apb Rgosebigek), Asabetyu Rgekk, Ν.Υ., Ν.Υ). The DNA vector can be introduced into mammalian cells using accepted technologies, such as coprecipitation with calcium phosphate or calcium chloride, IEEA-dextran-mediated transfection, lipofection or electroporation. Suitable methods for the transformation of host cells can be found in the monograph of 8goig et al. Laboratory Manual for Molecular Cloning (Mo1ci1ag C1ogspd: Aiog! Ogu Mapia1), 2nd ed., Co1b 8rgd Nagyog ogiogu ogge (1989)) and other laboratory manuals. When used in mammalian cells, control functions of expression vectors are often provided by viral material. For example, commonly used promoters are isolated from polyoma, adenovirus 2, cytomegalovirus, and, most often, 40 monkey viruses.

Peptides with costimulatory molecule activity were expressed in mammalian cells, or else they could be purified according to standard procedures in the art, including ammonium sulfate precipitation, fractionation on chromatographic columns (e.g. ion exchange chromatography, gel filtration, electrophoresis , affinity chromatography, etc.) with the final purification by crystallization (see, in general, the work Enzyme purification and similar technologies (Ephute RschGyu! ju apb Ke1a! eb Tesishd Iek), Me!

Specialists in this field will understand that the scope of their competence includes the production of antibodies against human costimulatory molecules using standard technologies. Antibodies can be represented by either polyclonal or monoclonal antibodies, or antigen-binding fragments of these antibodies. Of particular importance in terms of therapeutic use are antibodies that inhibit the binding of a co-stimulating molecule to its natural ligand (s) on the surface of immune cells, thus inhibiting the co-stimulation of the immune cell. Preferred antibodies against costimulatory molecules are antibodies that are capable of inhibiting or downregulating T cell mediated immune responses by binding to B7-1 or B7-2 on the surface of the lymphocytes and preventing interaction with CTLA4 and / or C.P28. Other preferred antibodies against costimulatory molecules are antibodies which, in combination with a second antibody that binds another costimulatory molecule, increase the level of inhibition of T-cell co-stimulation compared to the action of only the first antibody, for example, a combination of antibodies against B7-1 and against B7 -2.

A. Immunogen.

The term immunogen is used in this context to describe a composition containing a peptide having the activity of a costimulatory molecule as an active ingredient which is used to produce antibodies against a costimulatory molecule. When a peptide having a costimulatory molecule activity is used to induce antibodies, it should be understood that a single peptide or peptide can be used that is coupled to the carrier as a conjugate or as a peptide polymer.

To obtain suitable antibodies against a costimulatory molecule, the immunogen must contain an effective immunogenic amount of a peptide having the activity of a costimulatory molecule, usually in the form of a conjugate bound to a carrier. The effective amount of peptide / unit dose depends, inter alia, on the type of inoculated animal, the body weight of the animal, and the chosen immunization schedule, as is well known in the art. An immunogenic preparation will typically contain a peptide at a concentration of from about 10 μg to about 500 mg / immunization dose, preferably from about 50 μg to about 50 mg / dose. The immunization preparation may also include an adjuvant as part of a diluent. Adjuvants, such as Freund's complete adjuvant (CPA), Freund's incomplete adjuvant (1PA) and alum, are materials well known in the art and commercially available from various sources.

Competent specialists in this field will appreciate that instead of using natural forms of a costimulatory molecule for immunization, synthetic peptides can alternatively be used against which antibodies can be formed for the use of this invention. Both soluble and membrane-bound costimulatory molecules or peptide fragments are suitable for use as an immunogen and can also be isolated by immunoaffinity purification. Thus, a purified form of a protein of a costimulatory molecule, such as can be isolated as described above or as is known in the art, can be used as an immunogen, or, alternatively, be coupled to a suitable carrier protein by conventional methods, including methods chemical binding, as well as genetic engineering methods using the cloned gene of the costimulatory molecule.

The peptide or protein selected for immunization can be modified to increase its immunogenicity. For example, technologies for imparting immunogenicity to a peptide include conjugation with carriers or other methods well known in the art. Any peptide selected for immunization can also be synthesized. In a number of embodiments, these peptides can be synthesized in the form of branched peptides to enhance immune responses, as is known in the art (see, for example, the monograph Peptides (Reryek) under the editorship of Vegie Siye, Asayetk Rgeck, pp. 456-493, ( 1995)).

The purified protein of the costimulatory molecule may also be covalently or non-covalently modified with non-proteinaceous materials such as lipids or carbohydrates in order to increase immunogenicity or solubility. Alternatively, the purified protein of the costimulatory molecule can be coupled to or incorporated into a viral particle, a replicating virus, or other microorganism to enhance immunogenicity. A protein of a costimulatory molecule can, for example, be chemically attached to a viral particle or microorganism, or an immunogenic part thereof.

In an illustrative embodiment, the purified protein of a costimulatory molecule or a peptide fragment having activity of a costimulatory molecule (for example, obtained by limited proteolysis or recombinant DNA technologies) is conjugated to a carrier that is immunogenic for animals. Preferred carriers include 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, hemocyanin saucer, egg ovalbumin and cow gamma globulins. Synthetic polyamino acids, such as polylysine or polyarginine, are also effective carriers. Regarding the covalent attachment of a protein of a costimulatory molecule or peptide fragment to a suitable immunogenic carrier, there are a number of chemical cross-linking agents that are known to those skilled in the art. Preferred crosslinking agents are heterofunctional crosslinking linkers that can be used for stepwise protein binding. A wide range of heterobifunctional cross-linking linkers is known in the art, including succinimidyl 4- (Y-maleimidomethyl) cyclohexane-1-carboxylate (ZMSS), t-maleimidobenzoyl-Y-hydroxysuccinimide ester (MVZ); Ν-succinimidyl (4-iodoacetyl) aminobenzoate (З1АВ), succinimidyl 4- (p-maleimidophenyl) butyrate (ЗМРВ), 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride (BIS); 4-succinimidyloxyacarbonyl-a-methyl-a- (2pyridyldithio) toluene (ЗМРТ), Ν-succinimidyl-3- (2-pyridyldithio) propionate (8РЭР). succinimidyl-6- [3- (2-pyridyldithio) propionate] hexanoate (BS-ZRIR).

It may also be desirable to simply immunize with whole cells that express a costimulatory molecule protein on their surface. Various cell lines can be used as immunogens to generate monoclonal antibodies against the antigen of a costimulatory molecule, including, but not limited to, activated B cells. For example, pancreatic B cells can be obtained from a subject and activated with anti-immunoglobulin. Alternatively, a B cell line can be used provided that the costimulatory molecule is expressed on the cell surface, such as a Var cell line (VigxI B cell lymphoma. See, for example, Prgetap

6.1. et al., 8c1epse, 262: 909-911, (1993)) or the line of lymphoblastoid cells ΡΥ B (see, for example, article A by M. M. et al., Mishe, 366: 76-79, (1993)). Whole cells that can be used as immunogens to produce antibodies with specificity for a costimulatory molecule also include recombinant transfectants. For example, CO8 and CHO cells were reconstructed by transfection of cDNA of a costimulatory molecule as described in Kpibzop et al. (ΡΝΑ8, 90: 4003-4007, (1993)); Tauetpot et al. Cttipodes, 37: 474-477, (1993));

Ooidyyu et al. (1. Exp. Meb., 174: 1-5, (1991)) and Atiyo et al. (Ce11, 61: 1303-1313, (1990)), in order to obtain an intact costimulatory molecule on the cell surface. These transfectant cells can then be used as an immunogen to produce antibodies against a costimulatory molecule with a given specificity. Other examples of transfectant cells are known, in particular eukaryotic cells capable of glycosylating a protein of a costimulatory molecule, but any method suitable for expressing transfected genes of a costimulatory molecule on a cell surface could be used to produce an immunogen in the form of a whole cell.

B. Polyclonal antibodies against costimulatory molecules.

Polyclonal antibodies against a purified protein of a costimulatory molecule or peptide having activity of a costimulatory molecule can mainly be obtained in animals using a variety of subcutaneous (ss) or intraperitoneal (1p) injections of the immunogen in the form of a costimulatory molecule, such as the extracellular domain of the protein of the costimulatory molecule, and adjuvant. For example, as described above, conjugation of a costimulatory molecule (including fragments containing a particular epitope (s) of interest) to a protein that is immunogenic to the immunized species, such as a saucer hemocyanin, serum albumin, can be effective.

In a method and a scheme for immunizing an animal host or cultured antibody producing cells isolated from it, developed and adopted technologies for stimulation and production of antibodies can generally be used. In an exemplary embodiment, animals are typically immunized against immunogenic conjugates of a costimulatory molecule or derivatives by mixing 1 μg to 1 mg of the conjugate with Freund's complete adjuvant and intradermal injection of the solution into various parts of the body. After one month, the animals are re-immunized with 1 / 5-1 / 10 of the initial amount of conjugate in Freund's complete adjuvant (or other suitable adjuvant) by subcutaneous injection into many parts of the body. After 7-14 days, the animals were bled and the serum was evaluated for a titer of molecules against a costimulatory molecule. Animals are re-immunized until the titer reaches a plateau. Preferably, the animal is re-immunized with a conjugate of the same protein of a costimulatory molecule, but conjugated to another protein and / or using another cross-linking agent. Conjugates can also be obtained in recombinant cell culture as fusion proteins. In addition, aggregating agents such as alum can be used to enhance the immune response.

The data generated by mammals of a population of antibody molecules is called polyclonal because the population contains antibodies with different immunospecificity and affinity for the costimulatory molecule. Then the antibody molecules are collected from the mammalian organism and isolated by well-known methods, such as, for example, using ΌΕΑΕ Berjabeh to obtain the 1d6 fraction. To increase the specificity of antibodies, these antibodies can be purified by immunoaffinity chromatography using an immunogen bound to a solid phase. The antibody contacts the immunogen bound to the solid phase for a time that is sufficient to immunoreact the immunogen with the antibody molecules to form a solid phase immunocomplex. Bound antibodies are separated from the complex by standard methods.

C. Monoclonal antibodies against costimulatory molecules.

The term monoclonal antibodies or a composition of monoclonal antibodies, as used in this context, refers to a population of antibody molecules that contains only one type of antigen binding center that is capable of immunoreaction with a particular epitope of a costimulatory molecule. Thus, the composition of monoclonal antibodies usually exhibits one binding affinity for a particular protein of the costimulatory molecule with which it enters into an immunoreaction. Preferably, when the monoclonal antibody used in this method is further characterized as entering into an immunoreaction with a co-stimulating molecule isolated in humans.

The monoclonal antibodies used in the compositions and methods of the invention are directed to an antigen epitope of a costimulatory molecule, so that the formation of a complex between an antibody and a costimulatory molecule inhibits the interaction of the costimulatory molecule with its natural ligand (s) on the surface of immune cells, thus inhibiting co-stimulation of the T cells by interactions of a costimulatory molecule and a ligand. A monoclonal antibody against an epitope of a costimulatory molecule can be obtained using technology that provides the formation of antibody molecules in a culture of stable cell lines. These technologies include, but are not limited to, hybrid technologies, originally described in the article by Koiég and MIjesh (No. 11ige. 256: 495-497, (1975)), an earlier technology using human B-cell hybridoma (see the article by Kohlog et al., 1tipo 1. Tobau, 4:72, (1983)), technology using an EBU hybridoma (see Co1e et al., Monoclonal Antibodies and Cancer Therapy (Mopos1op1 Lpbob1e8 apb Sapseg Tyegaru), A1ap B. kx 1ps., P. 77-96, (1985)), as well as technology using triomes. Other methods that can ensure the efficient production of monoclonal antibodies used in this invention include phage presentation technologies (see Matk et al., 1. Vu1. Sit., Pp. 16007-16010, (1992)).

In one embodiment, the antibody preparation used in this method is a monoclonal antibody formed by a hybridoma cell line. The methods of fusion with the formation of a hybridoma were first introduced by Koieg and M1181esh (see Koieg et al., No. 11ige. 256: 495-97 (1975); Vgotep et al., 1. 1tipo1, 127: 539-46, (1981); Vgotep et al., 1. Vyu 1. Szet., 255: 4980-83, (1980); Way and so on., ΡΝΑ8, 76: 2927-31, (1976) and Waye et al., 1p1. 1. Sapseg, 29: 269-75, (1982)). Thus, the monoclonal antibody compositions of this invention can be prepared by the following method, which comprises the steps of (a) immunizing an animal with a costimulatory molecule. Immunization is usually carried out by administering an immunogen to a costimulatory molecule to an immunocompetent mammal in an immunologically effective amount, i.e. in an amount sufficient to obtain an immune response. Preferably, the mammal is a rodent, such as a rabbit, rat or mouse. The mammal is then maintained for a time that is sufficient for the formation in the mammal of cells secreting antibody molecules immunoreactive with the immunogen of a costimulatory molecule. This immunoreaction is determined by screening the antibody molecules thus obtained for immunoreactivity using an immunogen protein preparation. Optionally, but it may be desirable to screen the antibody molecules using a protein preparation in the form in which it should be determined using antibody molecules during analysis, for example, in a membrane-bound form of a costimulatory molecule. These screening methods are well known to those skilled in the art;

(B) a suspension of antibody producing cells taken from each immunized mammal is then prepared. After a sufficient period of time, the mouse is sacrificed and somatic lymphocytes producing the antibody are obtained. Antibody-producing cells can be isolated from the lymph nodes, spleens, and peripheral blood of loaded animals. Spleen cells are preferred, and they can be mechanically separated into individual cells in a physiologically tolerated medium using methods known in the art. Mouse lymphocytes give a higher percentage of stable fusions with murine myeloma cells, described below. It is also possible to use somatic cells of a rabbit, rat and frog. Chromosomes of spleen cells encoding the desired immunoglobulins are immortalized by fusion of spleen cells with myeloma cells, mainly in the presence of a fusion promoting agent such as polyethylene glycol (PEC). As a partner for fusion, any of the number of myeloma cell lines can be used in accordance with standard methods, for example, Ρ3-Νδ1 / 1-Α§4-1 myeloma lines, P3-x63-Ad8.653 or 8p2 / 0-Ad14. These myeloma lines are available in the American Type Culture Collection (Atepsap Tour SyJige SoNesyop) (ATSS), Voskushe, Mb.

The resulting cells, which include the desired hybridomas, are then grown in a selective medium, such as NAT medium, in which the unfused parental myeloma or lymphocyte cells eventually die. Only hybridoma cells survive and can be grown under restrictive dilution in order to obtain isolated clones. Hybridoma supernatants are screened for the presence of an antibody of the desired specificity, for example, by immunoassay methods using the antigen that was used for immunization. Positive clones can then be subcloned under restrictive dilution conditions and the generated monoclonal antibodies can be isolated. There are various traditional methods for the isolation and purification of monoclonal antibodies, aimed at releasing them from other proteins and other impurities. Commonly used methods for purifying monoclonal antibodies include ammonium sulfate precipitation, ion-exchange chromatography, and affinity chromatography (see, for example, the section written by Sokol et al. In the monograph, Methods for the Preparation and Use of Monoclonal Hybridoma Antibodies Ed. Nige11, pp. 51-52 (SIS Rgskk (1982)). Hybridomas purified according to these methods can be propagated by w νίΙΐΌ or w y1uo (in ascites fluid) using methods known in the art.

In general, a single cell line can be propagated by w νίΙΐΌ, for example, in laboratory culture vessels, and culture media containing high concentrations of monospecific monoclonal antibodies can be obtained by decantation, filtration or centrifugation. Alternatively, the monoclonal antibody yield can be increased by injecting a hybridoma sample of a histocompatible animal of the type that was used to produce somatic and myeloma cells for the initial fusion. Tumors secreting a specific monoclonal antibody were obtained by developing a hybrid fusion cell in the body of an injected animal. Animal body fluids, such as ascites fluid or serum, provide monoclonal antibodies in high concentrations. When using human hybridomas or EBU hybridomas, it is necessary to avoid rejection of the xenograft injected into animals such as mice. It is possible to use immunodeficient or nude mice, or you can pass the hybridoma first on irradiated nude mice as a solid subcutaneous tumor, cultivate w ν ш, and then intraperitoneally inject irradiated nude mice with ascendant tumors that secrete large amounts of specific human monoclonal antibodies.

The media and animals used to prepare these compositions are well known in the art, are manufactured on an industrial scale, and include synthetic media for cultivation, inbred mice, etc. An example of a synthetic medium is Dulbecco's Minimum Basic Medium (EIBssso) (see IMEM; article Ebssso et al., ¥ 1go1., 8: 396, (1959)) with the addition of 4.5 g / l glucose, 20 mM glutamine and 20% fetal feline serum. An example of an inbred line of mice is Ba1b / s.

Ό. Humanized antibodies against a costimulatory molecule.

When antibodies produced by non-human subjects are used for human therapy, they are recognized to varying degrees as foreign, and an immune response may be generated in the patient. One approach to minimize or eliminate this problem, which is preferred for general immunosuppression, is to produce chimeric derivatives of antibodies, i.e. antibody molecules that combine the variable region of an animal and a constant region of a person. These antibodies are equivalent to the above-described monoclonal and polyclonal antibodies, but may be less immunogenic when administered to humans and, therefore, it is more likely that they will be transferred by the patient.

Mouse-human chimeric monoclonal antibodies (i.e., chimeric antibodies) that are reactive with a costimulatory molecule can be prepared, for example, by newly developed methods for producing chimeric antibodies. Humanized antibodies can be obtained, for example, by replacing the immunogenic portion of an antibody with an appropriate but non-immunogenic portion. Accordingly, genes encoding the constant regions of a mouse (or other animal species) antibody against a costimulatory molecule are replaced with genes encoding the human constant regions. (See Kotkop et al., International patent publication PCT / I8 86/02269; Ak1ga et al. European patent application 184187; M. Tashdisy European patent application 171496; Motkop et al. European patent application 173494; Hsibsgdsg et al., PCT AO 86/01533; SaBu et al. European Patent Application 125023; Articles Vsbsg et al., 8c1spss, 240: 1041-1043, (1988); bsh et al. RIA8, 84: 3439-3443, (1987); bsh et al. 1. Ittipo1., 139: 3521-3526, (1987); 8ip et al. RIA8, 84: 214-218, (1987); McKitiga et al., Saps. Isk., 47: 999-1005, (1987); Aob et al., JaShgs, 314: 446-449 and 81ι; · ι \ ν et al., 1. No. 111. Sappsg ΡικΙ., 80: 1553-1559, (1988)). General reviews of humanized chimeric antibodies are presented by Motcop 8.b, 8c1spss, 229: 12021207, (1985), and O1 et al., WuTssystdisk, 4: 214, (1986). These methods include the isolation, manipulation and expression of nucleic acid sequences that encode a fully or partially variable region of an immunoglobulin from at least one heavy or light chain. The sources of this nucleic acid are well known to those skilled in the art and, for example, can be obtained from a hybridoma producing an antibody against a costimulatory molecule. The chimeric cDNA can then be cloned into a suitable expression vector.

Suitable humanized antibodies can alternatively be obtained by substituting CEE or CEA (see US Pat. No. 5,225,539; Articles 1pek et al., 32G: 321: 552525, (1986); Uetoeuoyai et al., 8c1epse,

239: 1534, (1988) and BeI1et et al., 1. 1tipo1., 141: 4053-4060, (1988)).

E. Combinatorial antibodies against a costimulatory molecule.

Compositions of both monoclonal and polyclonal antibodies of the invention can also be prepared by other methods well known to those skilled in the art of recombinant DNA technology. An alternative method, called the presentation of combinatorial antibodies, was developed to identify and isolate antibody fragments with a specific specificity for antigens, and can be used to obtain monoclonal antibodies against a costimulatory molecule, as well as a polyclonal population against a costimulatory molecule (see Articles 8act. ., ΡΝΑ8, 86: 5728, (1989); Nike et al., 8c1epse, 246: 1275, (1989) and Ot1apb1 et al., ΡΝΑ8, 86: 3833, (1989)). After immunization of the animal with an immunogen in the form of a co-stimulating molecule, as described above, the spectrum of antibodies from the obtained set of B cells is cloned. Basically, methods for directly obtaining the DNA sequence of variable regions of a heterogeneous population of immunoglobulin molecules using a mixture of oligomeric primers and PCR (polymerase chain reaction) are known. For example, mixed oligonucleotide primers corresponding to 5'-leader sequences (signal peptide sequences) and / or skeletal sequences 1 (EC1), as well as a primer of a conserved 3'-constant region, can be used for PCR amplification of variable regions of the heavy and light chains of a number of murine antibodies (see article Laglsk et al., Vyujesishdiek, 11: 152-156, (1991)). A similar strategy can also be used to amplify the variable regions of the heavy and light chains of human antibodies (see the work of Bagps et al. Methods: addition to the methods of enzymology (Me11yubk: Sotrashop 1o MeByubk sh En7utoodu), 2: 106-110, (1991)) . The ability to clone human immunoglobulin V genes is of particular importance in light of the success in creating sets of human antibodies in transgenic animals (see, for example, the articles by Wygdetap et al., Wheat 1tipo1., 7: 33-40, (1993);

TiaShop et al., ΡΝΑ8, 90: 3720-3724, (1993);

Vgiddetap et al., Eig. 1.

In an illustrative embodiment, RNA is isolated from activated Cells, for example, from peripheral blood, bone marrow, or spleen preparations using standard protocols (for example, US Pat. No. 4,683202; articles Og1apb1 et al., No. 8, 86: 3833-3837 (1989); Saktu et al., ΡΝΑ8, 86: 5728-5732 (1989) and Nike et al. 8άepse, 246: 1275-1281 (1989)). The cDNA of the first strand is synthesized using primers specific for the constant region of the heavy chain (s) and each of the k- and λ-light chains, as well as primers for the signal sequence. Using PCR primers for the variable region, the variable regions of both the heavy and light chains are amplified (each of them individually or in combination) and ligated into the corresponding vectors for further manipulations to create packaging for presentation. Oligonucleotide primers used in amplification protocols may be unique or degenerated, or include inosine at degenerated positions. Restriction enzyme recognition sequences can also be included in the primers to allow the amplified fragment to be cloned into a vector into a predetermined reading frame for expression.

A ν gene library cloned from the spectrum of antibodies isolated by immunization can be expressed by a population of representative phages, preferably isolated from a filamentous phage, to form a library representing antibodies. Ideally, the presentation package contains a system that allows one to obtain samples of the presentation libraries of very different antibodies, carry out quick sorting after each affinity separation step and quickly isolate the antibody gene from the purified presentation packages. In addition to the kits for creating phage presentation libraries produced in industry (for example, ^ а ^ ^ ас К К ес ес ес!!!!!! 8 8,,,,,,,,,,,,,,,,,,, catalog number 27-9400-01 and kit for the presentation of phages 81ta1adepe δυτΕΖΑΡ ™ catalog number 240612), examples of methods and reagents , especially suitable for use in creating a library, presenting a wide range of antibodies against a costimulatory molecule can be found, for example, in US patent No. 5223409, issued by Lubpet et al .; international publication Kapd et al. νθ 92/18619; international publication Iotete et al. νθ 91/17271; International publication \ Ush1eg et al. νθ 92/20791; international publication Magkapap et al. \ νθ 92/15679; international publication VGSYShd et al. XV O 93/01288; international publication MsSayeyu et al. νθ 92/01047; international publication Saggagb et al. νθ 92/09690; International Publication, Abpeg et al. νθ 90/02809; Risijk et al., B1o / Tesipoduod, 9: 1370-1372, (1991); Nau et al., Neath. Ljyob. Nuypbotak, 3: 81-85, (1992); Nike et al., 8c1epse, 246: 1275-1281, (1989); SpGPyk et al., EMBO 1., 12: 725-734, (1993); To \\ 'kshk et al., 1. Mo1. Vu1., 226: 889-896, (1992); C1askcop et al., No. 11 352: 624-628, (1991); Sgat et al., ΡΝΑ8, 89: 3576-3580, (1992); Saggab et al., Vyu / Teispoodu, 9: 13731377, (1991); Noodepyoot et al., Ms. Lab Century., 19: 4133-4137, (1991) and Vagyak et al., ΡΝΑ8, 88: 7978-7982, (1991).

In a number of embodiments, the domains of the Y region of the heavy and light chains can be expressed on the same polypeptide linked by a flexible linker to form a single-stranded fragment of фрагν, and the csRU gene is then cloned into the desired expression vector or phage genome. As basically described in the article, MsSayeyu et al. (Ba1ige, 348: 552554, (1990)), the complete domains of the _{H H} and Y _b antibodies bound by a flexible liquor (C1u ₄ -8g) ₃ can be used to produce a single chain antibody that can provide separation of the present package by affinity for antigen. From the isolated xcRU antibodies immunoreactive with a costimulatory molecule, a pharmaceutical preparation can be prepared for use in this method.

R. Hybridomas and methods of preparation.

The hybridomas used in this invention are hybridomas characterized by the ability to form a monoclonal antibody that is specifically immunoreactive with a costimulatory molecule. As described below, a hybridoma cell producing an antibody against a costimulatory molecule can be directly implanted into an animal recipient to obtain a constant source of antibody. The use of immuno-isolating devices for encapsulating a hybridoma culture can prevent an immunogenic response to implanted cells, as well as prevent uncontrolled proliferation of hybridoma cells in the body of an immunocompromising host. A preferred hybridoma of the invention is characterized by the production of antibody molecules that are specifically immunoreactive with a costimulatory molecule expressed on the cell surfaces of activated human B cells.

Methods for creating hybridomas that produce, for example, secrete, antibody molecules having the desired immunospecificity, i.e. having the ability to bind a particular costimulatory molecule and / or an identifiable epitope of a costimulatory molecule are well known in the art. Especially suitable for use is the hybridoma technology described in the works of No. Tap et al., ΡΝΑ8, 80: 49494953, (1983) and Site et al., Me1y. En / uto 73: 3-46, (1981).

In another example of the method, transgenic mice carrying spectra of human antibodies can be immunized with a human costimulatory molecule. The splenocytes of these immunized transgenic mice can be used to create hybridomas that secrete human monoclonal antibodies that are specifically reactive with the human costimulatory molecule (see, for example, PCT application publication Vooав et al. Νθ 91/00906, PCT application publication Kisieg1aray et al. Νθ 91/10741; publication of the PCT application by Lopeygd et al. Νθ 92/03918; publication of the application by PCT Kau et al. 92/03917; articles by Lopjjegd et al., No. Iige, 368: 856-859, (1994); Sgiep b .B et al., No. Shgs Sepe!., 7: 1321, (1994); Motkop 8.b et al., Proc. No. I. Lsab. 8s1. δΆ, 81: 6851-6855, (1994); Biddetap et al., Weag 1tipo1., 7: 33-40, (1993); TiaShop et al., ΡΝΆ8, 90: 3720-3724, (1993) and Biddetap and et al., Eig 1. 1ttypo1., 21: 1323-1326, (1991)).

The term antibody, as used in this context, includes the inclusion of fragments of antibodies that are also specifically reactive with a costimulatory molecule, as described in this context. Antibody fragments can be obtained using accepted methods, and fragment screening for use is carried out in the same manner as described above for whole antibodies. For example, fragments P (ai ') ₂ can be obtained by treating the antibody with pepsin. The resulting fragment P (ai) ₂ can be processed to restore disulfide bridges to obtain fragments of Paradise ¹ .

Antibodies obtained using certain methods can be tested to determine if they inhibit a costimulatory signal in a T cell using the methods described below.

In one embodiment, an agent that inhibits a costimulatory signal in a T cell is an antibody that binds both B7-1 and B7-2. When creating such an antibody, for example, fragments of the extracellular domain, which are conserved in two costimulatory molecules, can be used as an immunogen. See, for example, the article Me1x1eg et al., Ba1. 81gis1. ΒίοΙ., 4: 527, (1997)).

In one embodiment, an agent that inhibits a costimulatory signal in a T cell is an antibody that binds B7-1. These antibodies are known in the art or can be prepared as described above using a B7-1 molecule or fragment thereof as an immunogen by screening using the above methods or other standard methods. Examples of antibodies against B7-1 include antibodies presented in US patent No. 5747034 and articles MSCD, et al., Syp. Ittiηο1. [References 87:50, (1998), or RiSgey et al., Cl. Exp Ittipo., 110: 104, (1997).

In another embodiment, an agent that inhibits a costimulatory signal in a T cell is an antibody that binds B7-2. These antibodies are known in the art or can be prepared as described above using a B7-2 molecule or fragment thereof as an immunogen by screening using the above methods or other standard methods. Examples of antibodies against B7-2 include antibodies presented in the article RiSgey et al., Syp. Exp Ittipo., 110: 104, (1997).

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

In a number of other embodiments, an agent that inhibits a co-stimulating signal in a T cell is an antibody that binds CE28 but does not transduce a co-stimulating signal into a T cell (eg, an Eb fragment or anti-CE28 antibody). These antibodies are known in the art or can be prepared as described above using an SE28 molecule or fragment thereof as an immunogen by screening using the above methods or other standard methods. Examples of known antibodies against SE28 include antibodies presented in the article Oag1shd et al., Sepe Tieg., 4: 1350, (1997).

In preferred embodiments, fragments of an Eb antibody that binds CE28 can be used. It was found that these antibody fragments that are unable to cross-link SE28 on the surface of a T cell block the co-stimulation of the T cell (see article Va1ia-5 et al., Iptipyu, 1: 405, (1994)).

In a number of other embodiments, an agent that inhibits a costimulatory signal in a T cell is an antibody that binds STBA4 blocking a cos stimulating signal in a T cell by delivering a negative signal to a T cell (i.e., that which is an agonist STBA4). For example, cross-linking of STBA4 on the surface of a T cell has been shown to inhibit the proliferation and production of IL-2 (interleukin-2) (see Kgitte1 and AShkoy,

1. Exp. Meb., 183: 2533, (1996)). These antibodies can be obtained, as described above, using the STBA4 molecule or its fragment as an immunogen by screening using the above methods or other standard methods. Examples of antibodies are also described in the article Vaenbogogge et al., At. 1. Ra11u1., 152: 963, (1998).

In yet another embodiment, an agent that inhibits a co-stimulatory signal in a T cell is an antibody that binds JuO8 blocking a co-stimulatory signal in a T cell. These antibodies can be obtained, as described above, using a molecule of Κ.Ό8 or its fragment as an immunogen by screening using the above methods or other standard methods.

IV. Agents that regulate the expression of costimulatory molecules.

In another embodiment, an agent that inhibits a costimulatory signal in a T cell is an agent that disrupts the expression of a costimulatory molecule. For example, it was found that interactions between CE40 on antigen-presenting cells and CE40 ligand (CO40b) on T cells are important for maintaining, enhancing or prolonging the expression of B7-1 or B7-2 on antigen-presenting cells and lead to increased costimulation (see the articles by Vai Soo1, et al., Ittiyo1. Veg., 153: 47, (1996); K1ai5 and soavt., 1. Ittiyo1., 152: 5643, (1994)).

In one embodiment, an agent that blocks the expression of a costimulatory molecule, thereby blocking the costimulatory signal in the T cell, is a soluble form of ί.Ό40 or CO40B. DNA sequences encoding SE40 and CO40b data are known in the art, see, for example, SepBapk registration No. 10507 or Articles 81atep1og1c et al., EMBO 1., 7: 1053-1059, (1988) regarding SE40 or Saisya! et al., EEB8, 315 (3): 259266, (1993); 6 ha! et al., Eig. 1. ^ types, 22: 3191-3194, (1992); 8th, Neath. bepek, 97: 180-185, (1996) or SepVapk registration No. B07414, X67878, X96710 regarding СО40Ь.

In one embodiment, an agent that blocks expression of a costimulatory molecule, thereby blocking a costimulatory signal in a T cell, is a soluble form of CE40 or

СО40Б. In one embodiment, the soluble ΟΌ40 protein or SE40B protein contains a complete protein. In preferred embodiments, the soluble ΟΌ40 or CO40B protein contains the extracellular domain of the protein. For example, a soluble recombinant form of the extracellular domain of the CE40 protein. or ΟΌ40Η or a fragment thereof can be obtained as a fusion protein containing at least a portion of ΟΌ40 or SE40B. in this case, the interaction between ΟΌ40 on APC and SE40B on the T cell is disrupted and the delivery of the costimulatory signal to the T cell is inhibited. This soluble recombinant form of the ΟΌ40 or CE40b protein contains at least a fragment of the molecule sufficient to bind to its contraceptor, and at least a fragment of a second protein other than CE40 or CE40b. In preferred embodiments, the CE40 or CE40b fusion protein contains an extracellular domain of CE40 or CO40E which is fused at the amino terminus to a signal peptide, for example, from oncostatin M (see, for example, publication XV O 93/00431).

In a particularly preferred embodiment, the fused form of CO40 or CO40B is a fusion protein containing the extracellular domain of CE40 or CO40C which is fused to a fragment of an immunoglobulin molecule (see, for example, Sbey et al., 1. 1ttypo1., 155: 2833, (1995) ) Such a fusion protein SE40-1d or СО40B- [d can be obtained by methods known in the art (see, for example, Ipk1eu Prospects for the discovery and development of drugs (Retkresbuyek ίη Aegid Oxotegu apb Peydp), 2: 221, (1994); Ipk1eu , international application νθ 93/00431, US patent No. 5770197 and US patent No. 5580756).

In addition, it was shown that antibodies against the CO40 ligand have a synergistic effect with agents that inhibit the costimulatory signal in the T cell, contributing to the development of transplant tolerance (see Kik et al., Prg. Yab. Asab. 8ά. 8A. 94: 8789, (1997); Lathkep et al., LaShge, 381: 434, (1996)).

Therefore, in one embodiment, antibodies against CE40 or CE40b that bind these molecules but do not induce expression of costimulatory molecules can be used as an agent that blocks a costimulatory signal in a T cell.

V. Agents that act intracellularly to inhibit a costimulatory signal.

In one embodiment, an agent that inhibits a costimulatory signal in a T cell is an agent that acts intracellularly to inhibit a given signal. Stimulation of the T cell via the CO28 surface receptor (i.e., a costimulatory signal) leads to the formation of Ό-3 phosphoinositides in the T cell. Consequently, in one embodiment, the formation of β-3 phosphoinositides can be inhibited in a T cell in order to inhibit a costimulatory signal so as to inhibit the T cell response, which is measured, for example, by proliferation and / or cytokine production in a T cell. The term Ό-3 phosphoinositides includes the inclusion of phosphatidylinositol derivatives phosphorylated at the position of the Ό-3 cycle of inositol, and encompasses the compounds phosphatidylinositol (3) monophosphate (P1b1pk (3) P), phosphatidylinositol (3.4) bisphosphate (P1b1pk (3.4 ) P ₂ ) and phosphatidylinositol (3,4,5) -triphosphate (P1b1pk (3,4,5) P ₃ ).

Ό-3 phosphoinositides are formed intracellularly under the action of phosphatidylinositol 3 kinase (P13K). P13K is a heterodimer formed by a 85 kD subunit of molecular weight that binds tyrosylphosphorylated proteins through its 8H2 domains and a 110 kD catalytic subunit of molecular weight. P13K was first identified as lipid kinase, which phosphorylates the Ό-3 position of the inositol phosphatidylinositol, P! B1pc (4) P and P1b1pk (4,5) P ₂ . Two recent studies have shown that P [3K is actually a double specificity kinase that has both lipid and serine kinase activities (see ENapb V. et al., EMBO 1., 13: 522, (1994 ) and Satreshet S.L. et al. Mo1. Ce11 Vu1., 13: 1657, (1993)).

Accordingly, in one embodiment, an agent that inhibits a costimulatory signal in a T cell is an agent that inhibits P [3K. A preferred agent that inhibits the activity of P [3K in the T cell is represented by the fungal metabolite wortmannin or its derivatives or analogues. Wortmannin is a potent inhibitor of P [3K, isolated from T. poitappb (Ku opa Nakko Ko bu o So. Yb.) Or from R. Gipbsyokit (81dta). Derivatives or analogues of vortamannin include compounds similar in structure to wortmannin, which retain the ability to inhibit P [3K and T-cell responses. Examples of derivatives and analogues of wortmannin are described in articles XV ^ Excl. Ό. et al., Exrepepba, 30: 135-136, (1974); S1okke A. et al., 1. Meb. Sc., 24: 1465-1471, (1981) and Waddubsch, M. et al., Exp. Ce11 Century., 169: 408-418, (1987). Another inhibitor of P [3K activity that can be used is quercetin bioflavonoid or its derivatives or analogues. Derivatives and analogues of quercetin include compounds that are similar in structure to quercetin, which retain the ability to inhibit P [3K and inhibit T-cell responses. Examples of derivatives and analogues of quercetin are described in article U1a1yu5 C.1. et al., 1. Vu1. Siet., 269: 5241-5284, (1994). A preferred quercetin derivative that inhibits the activity of RKK is LY294002 (2- (4morpholinyl) -8-phenyl-4H-1-benzopyran-4-one, LIl IηύίαηαροΙίδ, ΓΝ) (described in Y1a1105 et al., Kirg et al.).

It was also shown that stimulation of SB28 leads to protein tyrosine phosphorylation in the T-cell (see, for example, articles WaibeybegdBe R. et al., 1. Exp. Meb., 175: 951-960, (1992); et al., 1. Ittii 1., 149: 24-29, (1992)). Accordingly, in one embodiment, an agent that inhibits a costimulatory signal in a T cell inhibits tyrosine phosphorylation in a T cell. A preferred protein tyrosine kinase inhibitor is an inhibitor that inhibits 5gs protein tyrosine kinase. In one embodiment, the 5gs protein tyrosine kinase inhibitor is herbimycin A or a derivative or analog thereof. Derivatives and analogues of herbimycin A include compounds that are similar in structure to herbimycin A and retain the ability to inhibit the activity of protein tyrosine kinases. In another embodiment, an agent that inhibits protein tyrosine phosphorylation is a protein tyrosine phosphatase or a protein tyrosine phosphatase activator. With an increase in tyrosine phosphatase activity in the T cell, the absolute amount of protein tyrosine phosphorylation decreases. Protein tyrosine phosphatase can be a T protein cell protein tyrosine phosphatase, such as SB45 or Hcp1. The action of cell surface tyrosine phosphatase on a T cell can be activated by contacting the T cell with a molecule that binds phosphatase and stimulates its activity. For example, an antibody directed against SB45 can be used to stimulate tyrosine phosphatase activity in a T cell expressing SB45 on its surface. Accordingly, in one embodiment, an agent that inhibits protein tyrosine phosphorylation in a T cell is represented by an anti-SB45 antibody or fragment thereof that retains the ability to stimulate SB45 activity. Examples of antibody fragments include fragments EaB and E (ab ') ₂ . Antibodies or fragments thereof can be obtained in a stimulating form, for example, multimerized or immobilized, etc.

In addition, ligation of SB28 was associated with increased activity of phospholipase C (see, for example, article Speck 1. et al., Wusbet. 1., 293: 835-842, (1993)) and increased intracellular calcium levels (see. for example, the article by Lebeig EL. et al., Blob, 75: 1531-1539, (1990), as well as examples).

Accordingly, an agent that acts intracellularly to inhibit a costimulatory signal in a T cell can act by inhibiting phospholipase C activity and / or inhibiting an increase in intracellular calcium levels. For example, the tyrosine kinase inhibitor herbimycin A also inhibits SB28-induced calcium flux into T cells.

It has also been shown that protein-serine and serine-threonine kinases are involved in signal transduction pathways associated with SB28 (see Articles 81ede1, et al., 1. ^ Tiok, 151: 41164127, (1993); Ra1 8.Υ. et al., 1. Ittiyo1., 24: 2364, (1994); Raggu et al., Eig. 1. Ittiyo1., 27: 2495, (1997)). Thus, in another embodiment, an agent that acts intracellularly to inhibit a costimulatory signal in a T cell inhibits the activity of serine or serine threonine kinase.

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

Other agents that block the costimulatory signal in the T cell can be identified using standard methods. For example, these agents can be identified by their ability to inhibit proliferation and / or cytokine production in T cells. In particular, a costimulation analysis system can be used. In this system, human SB28 ⁺ T cells are isolated, for example, by depletion using immunomagnetic particles using monoclonal antibodies directed against B cells, natural killer cells and macrophages, as described previously (see article C1tt1

S.B. et al., Proc. No. I1. Asab. 8cg I8A, 90, 6586-6590, (1993)). Antigen-presenting cells, for example, whole spleen cells or purified B cells or CO8 cells transfected with B7-1 or B7-2, can be irradiated or treated with mitomycin C (for example, at a concentration of 25 μg / ml) for 1 h, and rinse thoroughly to inhibit proliferation. 10 ⁵ SB28 ⁺ T cells can be incubated, for example, with 10 ⁵ -10 ⁴ APC (for example, CO8 cells transfected with the B7 molecule). In the example of this analysis, one T-cell population receives only a primary activation signal (for example, a T-cell receptor signal); another T-cell population receives only a co-stimulating signal, another T-cell population receives both a primary activation signal and a co-stimulating signal in the presence of an agent that is tested for its ability to block a co-stimulating signal in a T-cell. The primary activation signal can be delivered, for example, with a submitogenic dose of PMA (e.g. 1 ng / ml), a submitogenic dose of mitogen, a suboptimal dose of antigen, or a submitogenic dose of anti-T cell receptor antibody. Signal 2 is delivered using antigen-presenting cells carrying the B7 molecule. Potential blocking agents can be tested in a concentration range. For example, potential blocking antibodies can be used as supernatants of a hybridoma or as a purified antibody (for example, at a concentration of about 10 μg / ml). T-cell proliferation can be measured by incorporation of ³ H-thymidine (1 μCi) during the last 12-18 hours of the 72-hour incubation period. Delivery of the primary activation signal should lead to some proliferation, but in T cells that received both the primary activation signal and costimulatory signal 2, proliferation should take place at the maximum level. Blocking agents are identified by their ability to reduce maximal costimulatory signal-induced proliferation.

In addition to or as an alternative to measuring T cell proliferation, cytokine production by T cells can be measured using methods well known in the art. For example, IL-2 and IL-4 produced by T-cell cultures can be analyzed in supernatants of cultures collected 24-72 hours after the start of cultivation using the industrial EY8A (E & 0 8y51et5. M1paro118, ΜΝ and Vyuoigse, Satagh, CA). As indicated above, blocking agents can be identified by their ability to reduce the maximum costimulatory signal-induced cytokine production.

In the case of antibodies, any blocking antibodies identified by this or another analysis method can be tested further to determine the costimulatory molecule that they bind using methods known in the art. For example, the ability of blocking antibodies to reduce the level of binding of a labeled antibody to a known ligand can be measured.

VII. Additional agents for negative modulation of immune responses.

In some embodiments, the compositions and methods of the invention may include additional agents or the use of additional agents to enhance immunotolerance to an agent that stimulates hemostasis. In one embodiment, an agent that promotes immunotolerance but does not act by inhibiting a costimulatory signal in a T cell can be added to or included in these compositions. For example, an anti-SE40 ligand may be included in the composition (for example, a monoclonal antibody against the human SE40, 5C8 ligand (see Kik et al., Propos. No. 11. Asab. 8cE I8A, 94: 8789, (1997)). CO40 and its T-cell ligand CO40 (CE154) play an important role in upregulating (stimulating) B7 and stabilizing the activity of Cells (see U.S. Patent 5683693; Articles Waid et al., 8c1epse, 273: 1862, (1996); Cge \ \ 'a1 et al., 8c1epse, 273: 1864, (1996); Beiugtap et al.,

I. Exp. Meb., 175: 1091, (1992); Bebegshap et al., I. Ittipo1., 149: 3817, (1992)). It was found that antibodies against the SE40 ligand have a synergistic effect with agents that inhibit the costimulatory signal in the T cell, promoting the development of transplant tolerance (see Kik et al., Proc. No. 11. Asab. 8sk I8A, 94: 8789, (1997); Bagzep et al., ΝιιΙιιιό, 381: 434, (1996)). In another embodiment, an agent can be used in these compositions that acts intracellularly, stimulating immunotolerance, but does not inhibit the costimulatory signal in the T cell. For example, in one embodiment, cyclosporin A (C8A), EC506, rapamycin, or another agent that inhibits immune responses can be incorporated into or included as part of these methods. (See, for example, 81da1 et al., Appu. Keu. Ittiηo1., 10: 519, (1992); EiTapp et al., Itipo ^ o1odu, 198: 192, (1997) or 81ι; · ι \ ν and et al., S1sh. Sbet., 42: 1316, (1996)).

Viii. Methods of using compositions that contain a therapeutic protein and immunotolerizing agent.

In one embodiment, the compositions and / or agents described herein are administered to subjects who have hemostasis disorders and who have previously been treated with a hemostasis stimulating agent. In another embodiment, these compositions and / or agents described herein are administered to subjects who have not yet been treated with an agent that stimulates hemostasis.

In yet another embodiment, these compositions and / or agents described herein are administered to subjects who have not yet developed an immune response to an agent that stimulates hemostasis. Whether a subject has a pre-existing immune response can be ascertained by measuring the titer of the subject's antibody that reacts with an agent that stimulates hemostasis using methods well known in the art. If a given subject has a measurable titer of such antibodies (for example, a statistically significant titer) compared to the titers of control subjects, then it can be said that the subject has a pre-existing immune response to an agent that stimulates hemostasis. Alternatively, the cellular immune response to an agent that stimulates hemostasis can be measured to determine whether a subject has a developing immune response with respect to an agent that stimulates hemostasis. These methods are well known in the art.

In one embodiment, the first agent that stimulates hemostasis and the second agent that inhibits or blocks the costimulatory signal in the T cell are used to treat hemostasis disorders. In another embodiment, compositions containing a combination of a first agent that stimulates hemostasis and a second agent that inhibits a costimulatory signal in a T cell can be used to treat hemostasis disorders.

The administration of the compositions and / or agents described herein can be carried out in any pharmacological form that includes a therapeutically active amount of an agent and a pharmaceutically acceptable carrier. The introduction of a therapeutically active amount of these agents and / or compositions is defined as the amount effective in dosages and periods of time that are necessary to treat hemostasis disorders when using an agent that stimulates hemostasis, and to achieve immunotolerance in relation to an agent that stimulates hemostasis, in when using an agent that inhibits a costimulatory signal in a T cell. The therapeutically active amount of the agent or composition may vary in accordance with factors such as the condition of the disease, age, gender and body weight of the subject, as well as whether the subject has already developed an immune response with respect to the agent that stimulates hemostasis. This amount can be easily ascertained by one of ordinary skill in the art.

The optimal schedule for the administration of agents and / or compositions may also vary depending on the subject being treated. In some embodiments, a subject may require treatment with both agents at the same time. In this case, simultaneous administration of an agent that stimulates hemostasis and an agent that inhibits the costimulatory signal of T cells, for example, in the form of a composition containing both agents, will be desirable.

In other embodiments, separate administration of the agents will be desirable, for example, in order to ensure the stability of the agents or to facilitate the phased administration of the agents. In one embodiment, phased administration may be desirable to achieve the optimal therapeutic effect of an agent that stimulates hemostasis, while optimally inhibiting the immune response, preferably the immune response, to the agent. For example, an agent that inhibits a costimulatory signal may be administered as monotherapy prior to administration of an agent that stimulates hemostasis, or may be administered as monotherapy several days after administration of an agent that stimulates hemostasis.

In one embodiment, an agent that blocks a costimulatory signal in a T cell can be administered continuously, for example, each time an agent that stimulates hemostasis is administered. In another embodiment, an agent that blocks a costimulatory signal in a T cell is administered irregularly. For example, a subject may require regular treatment with an agent that stimulates hemostasis, but may only require periodic treatment with an agent that inhibits a costimulatory signal in the T cell. For example, treatment with an agent that stimulates hemostasis may be permanent, while just one or two administrations of an agent that blocks a costimulatory signal in a T cell may be sufficient for the subject; further administration of an agent that blocks the costimulatory signal in the T cell may not be required. In a preferred embodiment, an agent that blocks a costimulatory signal in a T cell is administered at appropriate intervals for at least about 6 months.

In one embodiment, the implementation of these agents or compositions is administered to patients, if it is found that the patients have pre-existing antibodies. In another embodiment, these agents or compositions are administered to patients who have not previously been treated, i.e. not having pre-existing antibodies. In yet another embodiment, these agents or compositions are administered to patients who have previously been treated but who lack antibody titers against an agent that stimulates hemostasis.

The dosage regimen can be selected for each patient to obtain the optimal therapeutic response without undue experimentation. For example, antibody titers against an agent that stimulates hemostasis can be measured in order to determine whether an immune response to an agent develops or does not develop in a subject, and accordingly choose a dosage schedule. For example, if antibody titers for an agent that stimulates hemostasis increase, higher doses of an agent that blocks the costimulatory signal in the T cell can be administered.

For the introduction of these agents or compositions other than the parenteral method, it may be necessary to cover them with a material that prevents inactivation, or co-administration with this material. An agent or composition of the invention may be administered to a subject in a suitable carrier or diluent, administered together with enzyme inhibitors, or in a suitable carrier such as liposomes. Pharmaceutically acceptable diluents include saline and aqueous buffers. Enzyme inhibitors include a pancreatic trypsin inhibitor, diisopropyl fluorophosphate (EER) and trasilol. Liposomes include emulsions of the type water-oil-water, as well as traditional liposomes (81g) ap et al., I. No. igottypo1. 7:27, (1984)).

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

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if soluble in water) or dispersions, and sterile powders for preparation immediately before use of sterile injectable solutions or dispersions. In all cases, the agent or composition must be sterile and must be fluid to the extent that it is administered by syringe. They must be stable under the conditions of preparation and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, a polyhydric alcohol (e.g. glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Proper fluidity can be achieved, for example, by using a coating such as lecithin, maintaining the required particle size in the case of dispersion and the use of surfactants. Prevention of the activity of microorganisms can be achieved using various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyhydric alcohols such as mannitol and sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be obtained by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by introducing the active composition or agent in the required amount into a suitable solution together with one of the above ingredients or a combination thereof, followed by sterilization by filtration. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and other necessary ingredients from the above. In the case of sterile powders, sterile injectable solutions are prepared by vacuum drying and lyophilization, whereby the active ingredient powder is obtained from a pre-sterilized solution solution of the ingredient.

When the active agent or composition is properly protected, as described above, the protein can be administered orally, for example, with an inert diluent or an assimilable edible carrier. As used in this context, the expression pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. The use of these media and agents for pharmaceutically active substances is well known in the art . Except in cases of incompatibility of any known medium or agent with the active compound, the use of any of them in therapeutic compositions is contemplated. Additional active compounds may also be incorporated into the compositions.

Particularly promising is the preparation of parenteral compositions in the form of a unit dosage form for ease of administration and dosage standardization. The term unit dosage form, as used in this context, refers to physically separate units serving as single doses for mammalian subjects that are being treated; wherein each unit contains a pre-determined amount of the active compound, calculated so as to obtain the desired therapeutic effect, in combination with the necessary pharmaceutical carrier. The description of the unit dosage forms corresponding to the invention is dictated and directly depends on (a) the unique characteristics of the active compound and the specific therapeutic effect that is expected to be achieved, and (b) the limitations inherent in the field of preparation of this active agent and composition for treating subjects.

As used in this context, the term pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of these media and agents for pharmaceutically active substances is well known in the art. Additional active compounds may also be incorporated into the compositions.

In the practical implementation of this invention will be used, unless otherwise indicated, traditional technologies of cell biology, cell cultivation, molecular biology, microbiology, recombinant DNA and immunology, which are within the purview of this technical field. These technologies are fully explained in the literature. See, for example, the monographs Laboratory Manual on Genetics and Molecular Cloning (Seiysk; Mo1eci1ag C1osid A baoga! Ogu Maiaia1), ed. 8th Century 1. et al., 2nd ed. (Co1B 8Ryid NagGoLaGaoga! Og Rgekk (1989)); A Brief Description of Molecular Biology Techniques (8th Prgo! Oso1k w Mo1esiag Wu1odu), ed. AikiLe1 R. et al., 3rd ed. (Ayeu, ΝΥ (1995)); DNA Cloning (TOA C1shid), Volumes I and II, ed. Ό.Ν. C1oueg (1985); Synthesis of Oligonucleotides (Oydois1eoybe 8ui! Eek1k), ed. M.1. Sack (1984)); U.S. Patent No. 4,683,195, issued to Mi. et al .; monograph Hybridization of Nucleic Acids (Hlc1e1cAc1b Hbg1b | / a11oi), ed. V.E. Natek and 8.1. Shddtk (1984)); treatise Methods of Enzymology (Ме11юбк 1и Еихуто1оду) (Asabetk Rgekk, 1is., Ν.Υ.); Monographs Immunochemical methods in cell and molecular biology (1 type 11 net 1 Me! 1 ob 1 and Ce 11 Aib Mo1ec1ag Vu1od), ed. Maueg and Aa1keg, Asabetk Rgeck, Lauiboy (1987)), Manual of Experimental Immunology (Naibooc O £ Extretei! A1 1thio1od), vol. Ι-ΐν, ed. EM. Aey and S.S. B1ask \\ 'e11, (1986) and Marsh I. Experiments in molecular genetics (Extretei! T t M1ci1ag Seieisk), (Co1b 8rgd NagGr Rgeck, Co1b 8rgd NagGr, Ν.Υ. (1972)).

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

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

Examples

In the examples, a mouse hemophilia A model is used to evaluate new methods for the prevention and treatment of inhibitor formation. In mice with hemophilia A obtained by targeted destruction of exon 16 of the factor VIII gene, there is no detectable plasma factor VIII activity (see article B1, No. 11 and Seijesk, 10: 119 (1995)), and in this regard they are close to patients with severe form of hemophilia A. In accordance with expectations, mice with hemophilia A exhibit signs of a defect in the coagulation pathway, manifested in lethal bleeding when the tail is cut without the use of hemostatic agents and in the development of subcutaneous and intramuscular bleeding after they are kept in RU kah or temporarily immobilized (see articles O | ai I., Voguok M., B1 b, Ka7a71ai NN, Noueg b.Tygotnayetok!., 81: 940 (1999); Euaik S. et al., Prgos No. I. Asab. 8sk I8A, 95: 5734 (1998)).

Intravenous infusions of 0.2 μg of human factor VIII, a dose equivalent to body weight to the dose administered to patients with hemophilia A, lead to a minimal antibody response in these mice with hemophilia A after a single injection or there is no response, but repeated injections result in a high titer inhibitory molecule against factor VIII (see article O | ai I. et al., Tygot. Nayatok!., 81: 940 (1999)). In addition, a factor VIII specific proliferative T cell response is detected three days after the first administration of human factor VIII before antibody detection.

Example 1. Inhibition of the primary immune response against factor VIII.

The experimental design for this example is shown in FIG. 1. Three groups of mice are intravenously injected with factor VIII on day 0. One group of mice is also given CTEA4- [d one day before and one day after the administration of factor VIII. The second group of mice is administered with the same CT ^ Α4-Id (intraperitoneal) followed by daily injections of factor VIII from day 2 to day 12. The third group is not administered CT ^ Α4-Id in any form. All mice received two additional injections of factor VIII on the 23rd and 24th day. Animals take blood on days 20, 37, 58, and 82. While mice that do not receive CT ^ Α4-Id have high titers of antibodies, starting from an early date such as day 20, in mice, which receive CT ^ Α4-Id, antibodies do not appear until the 82nd day (see Fig. 2).

Example 2. Inhibition of the secondary response to factor VIII.

The experimental design for this example is shown in FIG. 3. In this example, mice are given many intravenous injections of factor VIII at two-week intervals, and then divided into two groups. Mice are given a repeated injection of factor VIII on day 1, 20 and 37. CT ^ Α4-Id on day -1 and day +1 relative to factor VIII injections on day 1, 20 and 37 are administered to one group of mice. In animals that do not receive CT ^ Α4- [d. there is a high titer of antibodies against factor VIII, while mice that receive CT ^ Α4-Id (with the exception of 1 mouse) do not develop a secondary immune response to factor VIII (see Fig. 4).

In examples 3-6, the following methods and materials are used.

Animals. The characteristics of exon 16 (E16) line of hemophilia mice are presented in articles B1 b. et al., No. 11ige Seiyesk, 10: 119 (1995); B1 b. et al., B1ob, 88: 3446 (1996). In these studies, adult males and E-16 homozygous female mice aged 10-20 weeks are used. Blood samples are obtained by bleeding from the orbital venous plexus and serum is separated by centrifugation at 600 d for 3 minutes. Serum samples are stored at -20 ° C until analysis. To avoid severe bleeding and death of animals, ear tags are not used to identify mice in a number of experiments. For this reason, in FIG. 5 do not indicate sequential data for individual mice.

The generation of doubly knocked out (having two knocked out (inactivated) genes) mice E-16 / B7-1 and B7-2 is carried out by cross-crossing the line E-16 with mice knocked out according to B7-1 and B7-2 (see article Vote11 R. et al. 1shtipyu, 6: 303 (1997)). Homozygous mice with double knockout E-16 / B7-1 and E-16 / B7-2 are identified by the definition of genotype (see articles B1, et al., No. 11ig Sepeisk, 10: 119 (1995); R. Vote11o and et al., 1shtyu, 6: 303 (1997)). The reduced activity of factor VIII is checked using the chromogenic bioassay Coa1ec1 (Sygotodesch, Mo1ba1, 8 ^ ebep) (see article B1 et al., B1oob, 88: 3446 (1996)). The activity of factor VIII is less than 1% in mice with a deficiency of both E-16 / B7-1 and E-16 / B7-2.

Antigens. Recombinant human factor VIII is obtained from the company Nu1apb Όίνίkyup oG Vakh! Ne Neisyag Sogr. (C1epba1e, CA).

tSTAShd. A plasmid for the expression of mouse CTE-A4- [d] cDNA is prepared by ligation of the leader and extracellular domains of mouse CTBA4 with hinge, CH2 and CH3 domains of Nd2a, into which a mutation is introduced to remove effector functions, as described by 81geigeg et al. (see article 81geigeg, I. Ittiηo1, 155: 1165 (1995)). The insertion is cloned into the PEE expression vector and stably transfected with CHO cells, as previously described (see article L. P. 11 et al., I. Syp. I1νec !. 93: 2497 (1994)). Concentrated conditioned medium is introduced into the chromatographic column of the HrGo! E1o \ y (Ategkyat Ryagtaaa Vu! Es, P1ksa! A ^ ay, N1). The column is washed with PB8 (phosphate buffered saline) with a pH of 7.1 and elute with hBST ^ Id 20 mM citrate with a pH of 3.0. The combined peaks are neutralized with a 1M Tris solution at pH 8.0 to a final pH of 7.5 and the preparation is obtained in PB8 pH 7.1 using an Atuop mixing chamber with a ΥΜ30 membrane. tSTB ^ Id is made pyrogen-free using a Rogok PI chromatographic column (Regser! Ae Vyukuk! etk) and the product is eluted from the column with a linear gradient No. 1C1 from 0 to 1M No. 1C1 in 25 mM Tris with pH 7.5. Then, tSTEA4Chd was prepared in PB8 pH 7.1 using an Atuop mixing chamber with a ΥΜ30 membrane.

Antibody Measurements Antibody titer against factor VIII is determined using EB18A (enzyme-linked immunosorbent assay) (see article Οίηΐ'ΐ I. et al. The appearance of inhibitory antibodies and the cellular response to human factor VIII in hemophilia A in mice (п й ог ап η η!!! about 1 staggas! VIII w tippe Yetor A), Tygot. Nayatok!., 81: 940 (1999)). EB18A methods are carried out using microtiter wells coated with recombinant human factor VIII at a concentration of 0.8 μg / ml in 0.05 mol / ml carbonate-bicarbonate, pH 9. After incubating plasma samples in wells at 4 ° C. overnight, followed by washing add alkaline phosphatase-conjugated goat ^ C against the mouse (8o! yeppe Vu! espoiodo Akkoaa! ek Sch., Wyddnat. AB) for 2 hours at room temperature. Then, after washing, p-nitrophenyl phosphate (81 dtA, 8 !. bosh, MO) is added at a concentration of 2 mg / ml in 100 mmol / L glycine, 1 mmol / L MDCl ₂ , 2 mmol / L ₂ pC1 ₂ , pH 10.4 and measured absorbance at 410 nm using an automated reader for EB18A on microtiter boards. The concentration of antibodies against factor VIII is determined by the standard curve obtained using a monoclonal mouse antibody C against human factor VIII, which binds the domain A2 (Mab 413) (see article Lb11ag R. et al., 1. Syp. yghekR, 93: 9497 (1994)). The titer is calculated from the points that are on the linear portion of the standard curve for analysis.

The titers of the antibody inhibitor against factor VIII in units of Be! 1ekba (VI) are measured using the analysis of Be! 1ekba (see the article Kakreg S.K., Tygot. E! 1 + 1111. Nahat., 30: 263 (1973)).

T cell proliferation assays. A spleen is used as a source of T cells for proliferation assays. Then, spleen cells were cultured (5 x 10 ⁵ / well) in 96 well flat bottom plates. Recombinant factor VIII is added in varying amounts to the culture medium, which contains the complete NR-I-1640 medium containing 0.5% serum from hemophilia mice. After 72 hours of cultivation at 37 ° C, 37 kBp of ³ H-thymidine / well (6.7 Ci / mmol, Κ, Ν й аг та се се се се,,, CA) was added. Cultures were harvested after 16 hours using a Mayah 9600 (Raskagb, Maybep, CT). The results are expressed as the average of three replicates of the well the number of pulses per minute (CPT), incorporated into insoluble DNA.

Example 3. tSTEA4-k | blocks the induction of response against factor VIII.

Inhibitory antibodies against factor VIII are induced in control mice by repeated intravenous injection of 1 μg of recombinant human factor VIII at intervals of three weeks. In this example, recombinant human factor is injected into four groups of mice with hemophilia A on day 0, 23, 44 and 66 (initially 1 μg intravenously, and then 0.2 μg in the 2nd, 3rd and 4th injections) . Mice in groups C-3 and 6-4 are also injected intraperitoneally with 0.2 μg of factor VIII for 2-12 days. Blood samples for analysis of antibodies against factor VIII receive on 20, 37, 58 and 82 days. Animals of the control groups C-1 and C-3 (white circles) are injected only with factor VIII. Animals of groups C-2 and C-4, black circles, are also injected with tSTEA4- [d (250 μg, intraperitoneally) on the day before and on the day after the first administration of factor VIII. The concentration of antibodies against factor VIII is determined using E8A. The points obtained in the analysis of antibodies against factor VIII, indicated as <0.16 μg / ml, are close to the points obtained for plasma samples isolated from non-immunized mice with hemophilia A. The results of this experiment are presented in FIG. 5 (note that in Fig. 5 some data presented in Fig. 2 is repeated, but additional data is entered).

Antibody against factor VIII is determined in four of five mice 20 days after the first injection, and all control mice have a high titer of antibodies against factor VIII after two to four injections. The average level of inhibitor after four injections is 1860 VeShesb units (VI). The formation of antibodies against factor VIII is significantly suppressed in mice that are injected intraperitoneally with 250 μg of mouse CT ^ Α4- [d per day before and the day after the first injection of factor VIII (group C-2, Fig. 5) even in the absence of further administrations of tSTA4C together with three consecutive injections of factor VIII on days 23, 44 and 66. Antibodies against factor VIII are not detected in any of the C-2 mice after the first or second injection of factor VIII. Three weeks after the third injection of factor VIII, a weak immune response is determined in two of the three mice in the C-2 group.

To investigate whether the limited duration of immunological tolerance is the result of a short half-life of human factor VIII in the organisms of these mice (4-5 hours for hemophilia A in mice (see article P. C. et al., Proc. N11. Asab. Δα. I8A, 95: 5734, (1998)), control and treated with TST ^ 4-Id mice (groups C-3 and C-4, Fig. 5) were injected intravenously 1 μg of factor VIII on day 0 followed by daily intraperitoneal injections of 1 μg factor VIII on days 2-12. A high titer of antibodies against factor VIII was observed in control mice (group C- 3) on day 20, more than 350 μg / ml according to EH8A with an average titer of an inhibitor of 694 VI.In contrast, in group C-4 mice injected with tST ^ Α4-Id on the day before and on the day after the first exposure to factor VIII, detectable activity against factor VIII on day 20. The delayed response of antibodies against factor VIII after three additional injections of factor VIII is the same in these mice as the response in animals of group C-2. Thus, the limited persistence of factor VIII in plasma after CT ^ Α4-Id injection does not cause a limited duration of immunological tolerance in treated CT ^ Α4-Id mice.

Example 4. Effects of repeated administration of CTEA4-C.

Since the delayed response of antibodies against factor VIII is detected after repeated injections of factor VIII, when tSTA4C is administered only during the first exposure of factor VIII, it was found that tTT ^ Α4-Id can inhibit the appearance of antibodies against factor VIII with each infusion of factor VIII. In this experiment (Fig. 6), mice with hemophilia A were simultaneously infused with both factor VIII and tST ^ Α4-Id six times at three-week intervals. Mice with hemophilia A are injected intravenously with either 1 μg of factor VIII or 250 μg of tST ^ Α4-Id at 3-week intervals (black circles) or factor VIII as monotherapy after the first injection, which contains both factor VIII and tST ^ Α4 -Id (white circles). Serum samples for analysis of antibodies against factor VIII receive 4 weeks after the 6th injection of factor VIII. Detected antibodies against factor VIII are not found in any of the ten mice that are treated in this way, when they are tested four weeks after the sixth injection of factor VIII. In contrast, a high titer of antibodies against factor VIII is present in the serum of mice given only one injection of ^ ^ ”CT ^ Α4-Id (during the first exposure of factor VIII) followed by five injections of factor VIII as monotherapy.

The data from treated tST ^ 4-Id mice were then tested to determine if they had an immune response after additional injections of factor VIII in the absence of tSTA4C. After two intravenous injections at intervals of three weeks, none of the five mice developed antibodies against factor VIII, while two of the four control mice that had not previously been exposed to either factor VIII or tST ^ Α4-Id showed a low antibody level against factor VIII (Fig. 7). In this experiment, mice with hemophilia A treated as described for FIG. 6, using six injections of both factor VIII and tCT ^ Α4-Id, then six intravenous injections of 0.2 μg of factor VIII are given at three-week intervals without additional administration of tCT ^ Α4-Id (black circles). Control mice that were not previously exposed to factor VIII are immunized in parallel (white circles). Serum samples for analysis of antibodies against factor VIII receive 3 weeks after the 2nd and 6th injection. After six injections of factor VIII as monotherapy, the average titer of factor VIII is 93 μg / ml for mice that have previously received both factor VIII and tSTGA4- [d. whereas for control mice, the average titer is 155 μg / ml. These results document the limitations of antigen-specific immunosuppression, which is due to the repeated co-administration of tST ^ Α4- [d with factor VIII.

Example 5. tST ^ Α4-Id suppresses the secondary immune response against factor VIII.

To determine whether tCT ^ ^4- [d is a secondary immune response against factor VIII, tCT ^ Α4-Id is injected at the same time that factor VIII is administered to hemophilia A mice that already have anti-factor VIII antibodies. Mice are injected three times with 0.2 μg of factor VIII and the level of antibodies against factor VIII is determined using ETZA. The control mice were then given three additional injections of factor VIII, while the remaining mice were injected with tST ^ Α4- [d at the same time that they were given the first and third additional injections of factor VIII. Although many mice die due to bleeding complications during the experiment due to repeated injections and blood sampling, the results in the two groups are clearly different.

In this example, all mice were initially given 3 intravenous injections of 0.2 μg of factor VIII at 2 week intervals. Then, control mice (white circles) are injected with factor VIII three more times and blood samples are obtained for analysis (upper panel). Other mice (black circles) were injected with tST ^ Α4-Id (250 μg, intraperitoneally) the day before and one day after the 4th injection of factor VIII (as indicated by the arrow) followed by two injections of factor VIII as monotherapy at 3-week intervals. The number of injections of factor VIII before testing blood samples for antibodies against factor VIII is indicated on the horizontal axis.

An increase in the titer of antibodies against factor VIII was observed after the 4th injection of factor VIII for control mice with an average titer of 16 to 230 μg / ml (see Fig. 8A). After the 5th injection of factor VIII, all anti-factor VIII antibody titers were over 350 μg / ml in the four remaining control mice. In contrast, in mice treated with tST ^ ^ 4- [d. at the 4th injection of factor VIII, there is a minimal amount or no antibodies against factor VIII (see Fig. 8B and C). Administration of tSTA4-1d inhibits this secondary immune response against factor VIII in mice that already have relatively high levels of anti-factor VIII antibodies corresponding to inhibitory titers of 5-90 VI (see article I. I. et al., Tyr. Nathoy. 81: 940 (1999)) (see FIG. 8C), as well as in mice with a minimum level of anti-factor VIII antibody, after three initial injections, less than 5 VI (see FIG. 8B).

Example 6. The establishment of the role of B7-1 and B7-2 in the primary immune response against factor VIII.

Next, the roles of costimulatory ligands B7-1 and B7-2 on antigen-presenting cells are determined, since it is their interaction with именно28 that is supposed to be prevented in experiments using tST ^ Α4- [d. To complete the task, mice with hemophilia A are crossed with B7-1 ^{- / -} and B7-2 ^{- / -} mice (see the articles by R. Vote11o et al., Itti ^^, 6.303 (1997); Rgetetp Ο.Ι. et al., 8c1epse, 262: 907 (1993)) and select mice with deficiency both in factor VIII and in either B7-1 or B7-2 based on the analysis of the genotype. Then mice such as hemophilia A / B7-1 ^{- / -} and hemophilia A / B7-2 ^{- / -} are injected intravenously with 0.2 μg of human factor VIII at two-week intervals. Serum samples for analysis of antibodies against factor VIII receive 12 days after the 2nd and 6th injections of factor VIII. After four injections, all nine mice of hemophilia type A / B7-1 ^{- / -} (white circles) develop antibodies against factor VIII with titers of more than 350 μg / ml and an average inhibitor level of 712 VI (see Fig. 9), the values are close results obtained for otherwise normal mice with hemophilia A injected with factor VIII (see article I. I. et al., Tygot Naeta A 81: 940 (1999)). In contrast, none of the eight hemophilia A / B72 ^{- / -} mice (black circles) have detectable antibodies against factor VIII. Similar results are obtained for mice with hemophilia A, which are treated with antibodies against B7-1 and against B7-2.

To assess the T-cell response of these mice with hemophilia A and a deficiency of B7-1 and B7-2 three days after the fifth intravenous injection of factor VIII, spleen cells are obtained. The combined spleen cells of 3 mice are used to obtain proliferation data. White and black squares in FIG. 10 mean cells obtained from untreated mice of hemophilia types A / B7-1 ^{- / -} and hemophilia A / B7-2 ^{- / -,} respectively. White circles indicate the cells of hemophilia type A / B7-1 ^{- / -} mice that are given 5 intravenous injections of factor VIII, and black circles indicate the hemophilia mice of type A / B7-2 ^{- / -} that are given 5 intravenous injections of factor VIII. The concentration of factor VIII in cultures is indicated along the horizontal axis. The proliferative activity of T cells, determined by the incorporation of ³ Nthimidine, shows a dose-dependent factor VIII response in cells obtained from type A / B7-1 ^{- / -} mice (see Fig. 10). In contrast, no T-cell response was determined at any level of factor VIII in spleen cells obtained from A / B7-2 ^{- / -} mice. Thus, B7-2 plays a major role in supporting the immune response against factor VIII, administered intravenously, and in its absence, the formation of a response against factor VIII is prevented.

Equivalent solutions.

Those skilled in the art will determine or are able to establish, with the help of no more than routine experiments, a multitude of equivalent solutions regarding specific compositions and methods described in this context. It is intended that these equivalent solutions fall within the scope of the invention and are encompassed by the following claims.

List of sequences <110> Scan, LaLia <120> Methods of negative modulation of the immune response to therapeutic proteins <130> ΘΙΝ-013 <140> 09 / 158,178 <141> 1998-09-21 <160> 1 <170> Pa1eM 1n 2.0 <210> 1 <211> 6 <212> PAT <213> Noto 5ar1epz <400> 1

Me1 Tug Rgo Rgo Rgo Tug

Claims (34)

CLAIM 1. Composition for the treatment and / or prevention of disorders leading to abnormal bleeding, containing at least one procoagulation factor and at least one agent that inhibits the interaction of a co-stimulating molecule on the surface of an antigen-presenting cell and its corresponding receptor on a T-cell, leading to generate an immune response to the specified factor. 2. The composition according to claim 1, characterized in that it further comprises a pharmaceutically acceptable carrier. 3. The composition according to claim 1, characterized in that the procoagulation factor is a factor VIII. 4. The composition according to claim 1, characterized in that the procoagulation factor is a variant of factor VIII with a deletion of the B domain. 5. The composition according to claim 1, characterized in that the procoagulation factor is a factor IX. 6. The composition according to claim 1, characterized in that the procoagulation factor is a von Willebrand factor. 7. The composition according to claim 1, characterized in that the inhibitory agent is a soluble form of a costimulatory molecule. 8. The composition according to claim 7, characterized in that the inhibitory agent is a soluble form of CTLA4. 9. The composition according to claim 7, characterized in that the inhibitory agent is a soluble form of B7-1, or a soluble form of B7-2, or a combination thereof. 10. The composition of claim 8, characterized in that the inhibitory agent contains C.'TEA4- [d. 11. The composition according to claim 9, characterized in that the inhibitory agent contains B7-Nd or B7-2Td. 12. The composition according to claim 1, characterized in that the inhibitory agent is an antibody that binds a costimulatory molecule. 13. The composition of claim 12, wherein said antibody is selected from the group consisting of an anti-B7-1 antibody, an anti-B7-2 antibody, and a combination thereof. 14. The composition according to p. 12, characterized in that the antibody does not transduce a costimulatory signal into a T cell. 15. A method of treating disorders leading to abnormal bleeding in a subject by administering to the subject a composition according to any one of claims 1-14, with the achievement of a therapeutic effect. 16. The method according to clause 15, wherein the subject is found to have an immune response against procoagulation factors. 17. The method according to clause 15, wherein the subject does not show an immune response in relation to procoagulation factors. 18. The method according to clause 15, wherein the subject is further administered a composition containing an additional immunosuppressive agent. 19. The method according to clause 15, wherein the subject has hemophilia A, or hemophilia B, or von Willebrand disease. 20. A method of treating disorders leading to abnormal bleeding in a subject, characterized in that the subject is administered at least one procoagulation factor and at least one agent that inhibits the interaction of a co-stimulating molecule on the surface of an antigen-presenting cell and its corresponding receptor on a T cell leading to the generation of an immune response to the specified factor, with the achievement of a therapeutic effect. 21. The method according to claim 20, characterized in that the procoagulation factor is a factor VIII. 22. The method according to claim 20, characterized in that the procoagulation factor is a variant of factor VIII with a deletion of the B domain. 23. The method according to claim 20, characterized in that the procoagulation factor is a factor IX. 24. The method according to claim 20, characterized in that the procoagulation factor is a von Willebrand factor. 25. The method according to claim 20, characterized in that the inhibitory agent is a soluble form of a costimulatory molecule. 26. The method according A.25, characterized in that the inhibitory agent is a soluble form of CTLA4. 27. The method according to p, characterized in that the inhibitory agent is a soluble form ΟΤΕ · Λ4- [§. 28. The method according A.25, characterized in that the inhibitory agent is Primary Immune Inhibition of Factor VIII 0.2 mcg / injection, day 44, intravenously Factor VIII 1 mcg / injection daily from day 21, intraperitoneally FIG. 1 soluble form B7-1, or soluble form B7-2, or a combination thereof. 29. The method according to p. 28, wherein the inhibitory agent is a soluble form of B7-PD, or a soluble form of B7Shd, or a combination thereof. 30. The method according to claim 20, characterized in that the inhibitory agent contains an antibody that binds a costimulatory molecule. 31. The method of claim 30, wherein said antibody is selected from the group consisting of an anti-B7-1 antibody, an anti-B7-2 antibody, and a combination thereof. 32. The method of claim 30, wherein the antibody does not transduce a costimulatory signal into a T cell. 33. The method according to claim 20, characterized in that the subject has hemophilia A, or hemophilia B, or von Willebrand disease. 34. The method according to claim 20, characterized in that a significant titer of antibodies binding procoagulation factors is detected in the subject.