UA91004C2 - Method of preventing or reducing a t cell-mediated immune response - Google Patents

Abstract

The invention relates to a method of preventing or reducing a T cell-mediated immune response in an individual by administering to the individual a multimeric compound that binds to at least two P-Selectin Glycoprotein Ligand 1 (PSGL-1) proteins on the surface of a T cell or NK cell, wherein the multimeric compound comprises a P-Selectin or E-selectin binding domain that binds to PSGL-1 and is fused with a heterologous amino acid sequence corresponding to human Ig1 Fc region. The invention further relates to a kit comprising a compound that binds to PSGL-1 on the surface of a T cell, wherein the binding of the compound to PSGL-1 on the surface of the T cell induces a signal transduction pathway that results in the death of the T cell; and instructions for use of the compound to treat autoimmunity, transplant rejection, an allergic condition, or a T cell cancer.

Description

modulators of the function of RUSSIA-1 to prevent or reduce T-cell-mediated immune response, as well as methods of selection of modulators of the function of PO-1.

In one aspect, the invention relates to a method of preventing or reducing a T cell-mediated immune response in a subject. The method includes the following steps: selection of a subject diagnosed with or at risk of developing a condition characterized by an excessive or unwanted T-cell-mediated immune response; and administering to the subject a compound that binds to RUSSIA-1 on the surface of the T cell, wherein the binding of the compound to RBSI-1 on the surface of the T cell triggers a signal transduction pathway that ultimately results in the death of the T cell, thereby preventing or reducing a T-cell mediated immune response in the subject's body.

The compound used according to this method may include an antibody or a fragment of its binding to an antigen that specifically binds to ROI-1. In one example, the compound is a monoclonal antibody that specifically binds to ROSY-1. In one embodiment, the method includes an additional step of introducing an agent that binds to the monoclonal antibody and causes the crosslinking of a certain number of ROS-1 antigens on the surface of the T-cell.

In some embodiments, the method includes causing cross-linking of a certain number of ROS-1 antigens on the surface of a T-cell, and the cross-linking triggers a signal transduction pathway that ultimately leads to the death of the T-cell.

In some embodiments, the method includes the following steps: (ii) selecting a subject diagnosed with or at risk for a condition characterized by an excessive or unwanted T-cell-mediated immune response; and (her) administering to the subject a multimeric compound that binds to at least two RB(Y-1) proteins on the surface of a T cell, wherein the multimeric compound comprises two polypeptide chains, each polypeptide chain comprising (a) a binding domain , which binds to RBOI-1, and (B) a heterologous amino acid sequence in which the polypeptide chains are linked through the heterologous amino acid sequence to form a multimeric compound, and binding of the multimeric compound to at least two RBOI-1 proteins on the surface The T-cell induces a signal transduction pathway that eventually leads to the death of the T-cell, thus preventing or reducing the T-cell-mediated immune response in the subject's body.

A multimeric compound can be a homomultimeric compound or a heteromultimeric compound. The binding domain may optionally contain the extracellular domain of P-Selectin or its RBOI-1-binding fragment, the extracellular domain of E-Selectin or its RBOI-1-binding fragment, the extracellular domain

Y-Selectin or its RUSSIA-1-binding fragment, an antibody against RBAI-1 or its RUSSIA-1-binding fragment, RBAI-1-binding polypeptide selected from phage libraries, or a combination of any of them

In some embodiments, the multimeric compound does not include an antibody against RUSSIA-1 or an antibody fragment that binds to ROOI-1.

A heterologous amino acid sequence may optionally contain a site for binding to a cell surface receptor, for example, a constant site of an immunoglobulin heavy chain. In some embodiments, the polypeptide chains are covalently linked, for example disulfide linked, through a heterologous amino acid sequence to form a multimeric compound.

In some embodiments, the method may include an additional step of administering to the subject an agent that binds to the multimeric compound through a heterologous amino acid sequence and causes crosslinking of a certain number of ROS-1 antigens on the surface of the T-cell.

In some embodiments, the method described by the authors includes the stage of selecting a subject diagnosed with an autoimmune disease. In another example, the method includes the step of selecting a subject diagnosed with an inflammatory disease. In another example, the method includes the step of selecting a subject who has received or is to receive a xenogeneic transplant. In another example, the method includes the step of selecting a subject diagnosed with an allergic disease. In another example, the method includes the step of selecting a subject who is diagnosed with T-cell cancer.

In some embodiments, the T cell is an activated T cell. In one example, the T cell is a CO4- T cell. In another example, the T cell is a COv'- T cell.

In some embodiments, the method includes the step of detecting the number of T cells in a first biological sample taken from the subject prior to administration of the compound (eg, a multimeric compound) and comparing the results to the number of T cells in a second biological sample taken from the subject after the introduction of a compound (eg, a multimeric compound).

In some embodiments, the method includes the step of detecting the biological activity of T cells in a first biological sample taken from the subject before administration of the compound (eg, a multimeric compound) and comparing the results with the biological activity of T cells in a second biological sample taken in the sub object after the introduction of a compound (for example, a multimeric compound).

In some embodiments, the administration results in the depletion of at least 1095 activated T cells in the subject In some embodiments, the administration results in the depletion of at least 1095, 20905, 3090, 4095, 50905 or more activated T cells in the subject's body object

In some embodiments, the antibody or antigen-binding fragment thereof or the multimeric compound causes the death of at least 1095 activated T cells in the subject's body after exposure to the antibody or antigen-binding fragment thereof or the multimeric compound. In some embodiments, the administration causes the death of at least 1095, 2095, 3095, 4095, 5095 or more activated T cells in the subject's body. Cell death can be measured at any time, for example, one, two, three, four, five, six, seven or more days after exposure to the antibody or antigen-binding fragment or multimeric compound.

In another aspect, the invention relates to a method of inducing the death of a T-cell or natural killer (NK) cell. The method includes steps: providing a T-cell or MK-cell that expresses RBOII-1 on the cell surface; and contacting the T-cell or MK-cell with a compound that binds to ROS-1 on the surface of the T-cell or

MK-cells, and the binding of the compound to ROSY-1 on the surface of the T-cell or MK-cell causes a signal transduction pathway, which eventually leads to the death of the T-cell or MK-cell.

The compound used according to this method may include an antibody or a fragment of it binding to an antigen that specifically binds to ROSI-1. In one example, the compound is a monoclonal antibody that specifically binds to RBOI-1. In one embodiment, the method includes the step of contacting the monoclonal antibody with an agent that binds to the monoclonal antibody and causes crosslinking of a certain number of ROS-1 antigens on the surface of the T-cell or MK-cell.

In one embodiment, the method includes the following steps: (i) providing a T-cell or MK-cell that expresses RBOII-1 on the cell surface; and (iii) contacting the T-cell or MK-cell with a multimeric compound that binds to at least two RBOII-1 proteins on the surface of the T-cell or MK-cell, wherein the multimeric compound comprises two polypeptide chains, each of the polypeptide chains comprising (a) a binding domain that binds to ROBI-1, and (B) a heterologous amino acid sequence in which the polypeptide chains are linked through a heterologous amino acid sequence to form a multimeric compound, wherein binding of the multimeric compound to at least by two RBOI-1 proteins on the surface of a T-cell or MK-cell causes a signal transduction pathway, which ultimately leads to the death of the T-cell or MK-cell.

A multimeric compound can be a homomultimeric compound or a heteromultimeric compound. The binding domain may optionally contain the extracellular domain of P-Selectin or its RBOI-1-binding fragment, the extracellular domain of E-Selectin or its RBOI-1-binding fragment, the extracellular domain

Y-Selectin or its RUSSIA-1-binding fragment, an antibody against RUSSIA-1 or its RUSSIA-1-binding fragment, a peptide selected from a phage library, or a combination of any of them.

A heterologous amino acid sequence may optionally contain a site for binding to a cell surface receptor, for example, a constant site of an immunoglobulin heavy chain. In some embodiments, the polypeptide chains are covalently linked, for example disulfide linked, through a heterologous amino acid sequence to form a multimeric compound.

In some embodiments, the method includes an additional step of contacting the multimeric compound with an agent that binds to the multimeric compound through a heterologous amino acid sequence and causes crosslinking of a certain number of ROI-1 antigens on the surface of the T-cell.

In some embodiments, the method includes the step of causing crosslinking of a certain number of PI-1 antigens on the surface of a T-cell or MK-cell, and the crosslinking triggers a signal transduction pathway that ultimately leads to the death of the T-cell or MK-cell.

In some embodiments of the methods described by the authors, the T cell is an activated T cell. In one example, the T cell is a CO4- T cell. In another example, the T cell is a COv'- T cell.

In some variants of implementation of the methods described by the authors, the method includes the step of determining viability

T cells or MK cells after contact with a compound (eg, a multimeric compound).

In some variants of implementing the methods described by the authors, the method includes the step of determining the biological activity of a T-cell or MK-cell after contact with a compound (for example, a multimeric compound).

In some embodiments, the method includes inducing the death of an activated T cell.

In another aspect, the invention relates to a method of selecting a ROS-1 function modulator. The method includes steps: providing a cell that expresses RBai-1 on the cell surface; cell contact with the tested substance; and measuring cell viability after contacting the cell with the test substance to determine whether the test substance is a modulator of SHOI-1 function.

In one embodiment, the method includes the step of detecting cell death caused by the test substance to determine that the test substance is a modulator of the function of RBI-1.

In one embodiment, the tested substance is an antibody or a fragment of its binding to an antigen that specifically binds to ROS-1. In one example, the test substance is a monoclonal antibody that specifically binds to ROZI-1. In one embodiment, the method includes the step of contacting the monoclonal antibody with an agent that binds to the monoclonal antibody and causes the crosslinking of a certain number of ROS-1 antigens on the cell surface.

In one embodiment, the method includes the step of causing cross-linking of a certain number of ROS-1 antigens on the cell surface, and the cross-linking causes a signal transduction pathway that eventually leads to cell death.

In one embodiment, the T cell is an activated T cell. In one example, the T cell is a CO4- T cell. In another example, the T cell is a COv'- T cell.

In one embodiment, the method includes the step of obtaining a mass amount of the test substance and prescribing the test substance in a pharmaceutically acceptable carrier.

In another aspect, the invention relates to a kit that includes: a compound that binds to RB(ZI-1) on the surface of a T-cell, and the binding of the compound to ROS-1 on the surface of a T-cell triggers a signal transduction pathway that, as a result results in the death of a T-cell; and instructions for using the compound to treat a condition associated with an excessive or unwanted T-cell-mediated immune response or excessive or unwanted T-cell proliferation, such as inflammation, autoimmunity, transplant rejection, an allergic condition, or T-cell cancer.

In one embodiment, the kit includes: (i) a multimeric compound that binds to at least two RBOI-1 proteins on the surface of a T cell, wherein the multimeric compound comprises two polypeptide chains, each polypeptide chain comprising (a) a binding domain , which binds to RUSI-1, and (p) a heterologous amino acid sequence in which the polypeptide chains are linked through a heterologous amino acid sequence to form a multimeric compound, and binding of the multimeric compound to at least two RBII-1 proteins on the surface T cells trigger a signal transduction pathway that eventually leads to T cell death; and (ii) the use of the compound for the treatment of a condition associated with an excessive or unwanted T-cell mediated immune response or excessive or unwanted T-cell proliferation, such as inflammation, autoimmunity, transplant rejection, an allergic condition, or T-cell cancer .

The advantage of the invention is that it can cause exhaustion of T cells and/or induction of apoptosis in T cells without causing an associated unwanted or harmful immune response. For example, in some embodiments, the implementation of the subject's introduction of antibodies against RBOJ-1 or the multimeric compound described by the authors does not lead to an undesirable increase in the level of inflammatory cytokines, such as II -2 or TME-alpha.

Another advantage of the invention is that it causes depletion of T cells through the use of agonist compositions that cause apoptosis of T cells. Accordingly, the invention provides active immunosuppressive methods, rather than passive immunosuppression, which results from the use of an antagonistic composition (eg, antagonistic antibodies against RBOI-1 or antagonistic soluble fragments of selectin) that act by binding immune receptors and preventing immune activation mediated by such receptors.

Another advantage of the invention is that it allows the targeting of the cell surface protein, ROS-1, the expression of which is largely limited to leukocytes, in particular, T-cells and MK-cells. Thus, the compounds described by the authors generally do not cause a significant level of apoptosis in other cell types, such as liver cells. targeting T cells and MECs (an important POP3 cell type involved in transplant rejection) for selective depletion without significantly inducing life-threatening systemic cytokine responses and damage to other organ systems is a useful feature of an immunosuppressive agent.

Unless otherwise defined, all technical and scientific terms used by the authors have the meanings generally accepted among those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described by the authors are used to practice and test the invention, acceptable methods and materials are described below. All publications, patent applications, patents and other sources cited by the authors are incorporated by reference in their entirety. In case of inconsistency in terminology, this description shall prevail. Furthermore, the described materials and methods are presented for illustrative purposes only and are not intended to be limiting.

Other features and advantages of the invention will become clear from the following detailed description and claims.

A brief description of the figures

Fig. 1 shows the results of the experiment with changes in time, in which it was studied when activated T-cells acquire sensitivity to mediated by TAVA (monoclonal antibody against ROCI -1) apoptotic signals.

Fig. 2 shows the results of biotinylation of the cell surface and immunoprecipitation of the antigen recognized by the TAVA antibody.

Fig. 3 shows the expression of the ROI-1 antigen on CO4 T cells, COv T cells, CO 19 B cells and MK cells of the spleen. Fig. 4 shows the expression of antigen RBOI -1 on thymocytes СО4", СО8" and СО4-8: and SAT.

Fig. 5 shows the level of I/-2 obtained in a mixed culture of lymphocytes with the use of spleen cells taken from the body of Vair/c mice treated with TAVA (or hamster Id) as responders and H2-discordant spleen cells of SZN as a stimulator .

Figure 5 shows Western blot analyzes demonstrating that (A) proteins immunoprecipitated with the TAB4 antibody can be recognized by a serially produced anti-RBCI-1 antibody, and (B) preclearing the T-cell lysate with an anti-RBI-1 antibody can to deplete proteins recognized by TAVA.

Figure 7 shows the percentage of preserved grafts in C57VI/6 mice that received skin grafts from Vair/c mice and received an antibody against RBOIII-1 (colored diamonds) or a control antibody (uncolored squares).

Fig8 shows the change over time in the percentage of apoptotic T-cells after treatment of activated mononuclear cells of human peripheral blood with anti-human ROS-1 antibody.

Fig.9 shows the incidence of diabetes in autoimmune male mice with non-obese diabetes (MY) treated with an antibody against RBOY-1 (colored squares) or a control antibody (uncolored squares).

Fig.10 shows the binding of mouse P-selectin, E-selectin and I-selectin to activated mouse T-cells.

Figures 11A-11C show induction of apoptosis of activated mouse T cells by multimeric forms of E-selectin (Figure 11A), P-selectin (Figure 118B) and I-selectin (Figure 11C).

Fig. 12 shows the induction of apoptosis of activated T cells of the mouse ip me by crosslinking the soluble fusion protein P-selectin-Es.

The invention relates to a method of modulating the activity of T cells by modulating the function of molecules

RBII -1, which are on the surface of the T-cell. The contact of ROI-1 with agonist compositions described by the authors can cause exhaustion of T cells and/or cause apoptosis of T cells. These agonist compositions are thus useful as therapeutic agents for the control of immune-related diseases such as inflammatory diseases, autoimmune diseases, transplant rejection, allergic diseases and/or T-cell cancers.

Agonist compositions are also used to purify T cells from any biological sample in which the presence or activity of T cells is undesirable.

Protein RBOI -1

RBOY-1 is a cell surface adhesion molecule that is expressed on neutrophils, T- and B-lymphocytes,

MK cells, monocytes, dendritic cells and primitive human hematopoietic progenitor cells CO34. Due to its ability to interact with selectins, ROSSI -1 mediates the rolling of leukocytes along the endothelium and the extravasation of leukocytes into inflamed tissues. Differentially regulate RBOI-1-mediated binding of T cells to E- and P-selectin or migration. For example, the appearance of the STA (cutaneous lymphocyte antigen) epitope is thought to be caused by T-cells undergoing the transition from naïve cells to memory cells. Only activated helper 1 but not helper 2 T cells express functional

RBI -1 and are capable of migrating to the inflamed area of the skin.

RBII-1 is a sialomucin that must be specifically sialylated, fucosylated, and sulfated to bind to P-selectin. The RBOI-1 molecule exists in isoforms. which are characterized by sites with varying degrees of glycosylation and sulfation at their M-termini. Resting peripheral blood T- and B-cells, lymphoid cell lines, and ip myo-activated peripheral blood T-cells express a similar level of ROBI-1. However, only activated T-cells have a functional form of RBSI-1 and actively bind to P-selectin . Apparently, such activation-dependent binding activity is the result of differential post-translational modification, as evidenced by the increased level of activity of alpha (1,3) fucosyltransferases in activated T-cells. RBYI-1 isoforms also show differential affinity to I-selectin and E-selectin. For example, human T cells that have the C A-positive isoform can bind to and roll along E- and P-selectin, whereas T cells that express RBSI-1 without the SIA epitope only bind to P -selectin. In addition, the binding of ROI-1 to P-selectin depends on the presence of the final decapeptide, which contains three tyrosine residues for sulfation and one threonine residue for glycosylation.

The RBOI-1 protein is obtained by recombinant methods and/or by separating the natural protein

RBOY-1 from biological material. Recombinant protein RBSI-1 can be produced in prokaryotic or eukaryotic cells, IP myo or IP mimo. Nucleic acids that encode PO(I-1) are used for recombinant protein production (see, for example, SepVapk"M Assezvsiop MM 003006 for an example of a nucleic acid that encodes a RBII-1 polypeptide). Antibodies directed to ROB(I-1 , are also well known and can be used for antigen purification (see, for example, Ne!top ei ai. (2000)

Zsieepse dip 2:;288(5471): 1653-56; MO 00/25808) and/or applied according to the methods described by the authors.

RBOY-1 is also described in sources, which, among others, include Zako ei ai. (1993) SeiI 75:1179; Masnipo her a. (1995) 9. Axis. Spent 270:21966; and MeIdtap her a. (1995) 9. Axis. Snet 270:16470.

Recombinant production of RBSI-1 may require simultaneous expression of RB(I-1) and its modifying alpha (1,3) fucosyltransferase, Eis-TMII, for functional expression of ROSI-1. Additionally or alternatively, recombinant production of ROSI-1 may be accompanied cotransfection with nucleic acid encoding RACE to remove the propeptide and/or nucleic acid encoding tyrosine sulfotransferase.

The antibody against RUSSIA-1 is used to isolate and purify the antigen RB(Y-1) from biological material. Any type of cell that expresses the protein RBIA-1, for example, T-cells taken from an organism or a line

T cells can be used as a source of protein. Immediately after purification, the protein is used in various methods described by the authors. For example, the purified protein of RUSSIA-1 can be used in IP miigo selection of modulators of the function of RUSSIA-1 on T-cells or as an immunogen for obtaining antibodies directed against the protein.

Antibodies against RBOI -1

RBIII-1 polypeptides (or their immunogenic fragments or analogs) can be used to produce antibodies that are used in methods according to the invention. As described above, RUSSIA-1 polypeptides or their peptide fragments are obtained by recombinant methods or synthesized using solid phase synthesis methods. Recombinant ROY-1 polypeptides or their peptide fragments are used as an immunogen for the production of antibodies against ROY-1. In addition, an antibody against RBOY-1, such as the monoclonal antibody TAVA, is used to purify the RB(I-1 polypeptide, for example, the ROSI-1 polypeptide in its natural conformation, which can then be used as an immunogen for the production of additional antibodies against Ra - 1.

The antibody according to the invention can be a monoclonal, polyclonal or engineered antibody that specifically binds to the RBSII-1 polypeptide. An antibody that "specifically binds" to a specific antigen, for example, the RUSSIA-1 polypeptide, largely does not recognize or bind to other molecules in the sample. Thus, the invention also relates to methods of determining a test compound (e.g., an antibody) that binds to a polypeptide of the invention by contacting the polypeptide with the test compound and determining whether the polypeptide binds to the test compound (e.g., by direct detection binding, detection of a competitor molecule that disrupts the binding of the test compound to the polypeptide, and/or detection of binding using an apoptosis-inducing activity assay.

In general, ROSII-1 polypeptides can be bound to a carrier protein, such as KIN, mixed with an adjuvant, and injected into a mammalian host. Antibodies produced in the body of this animal are then purified by affinity chromatography with a peptide antigen.

In particular, various host animals are immunized by injection of the ROSI-1 polypeptide or its antigenic fragment. Commonly used host animals are rabbits, mice, guinea pigs, and rats. Various adjuvants used to enhance the immunological response depend on the host species and include Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols , polyanions, peptides, oil emulsions, snail lymph hemocyanin and dinitrophenol. Potentially useful human adjuvants include BSO (bacillus CaIteicke-

Siepp) and Sogupebasieput rag/it. Polyclonal antibodies are heterogeneous populations of antibody molecules contained in the serum of immunized animals.

Thus, antibodies within the scope of the invention include polyclonal antibodies, as well as monoclonal antibodies, "humanized" or chimeric antibodies, single-chain antibodies, Rab fragments, fragments

K(iabr)2 and molecules obtained using the Bar expression library.

Monoclonal antibodies, which are homogenous populations of antibodies to a particular antigen, are produced using the above-described ROI-1 polypeptides and standard hybridoma technology (see, e.g., Kopieg et al., Mashte 256:495 (1975); Kopieg et al., Yeig U Ittipoi! 6:511 (1976); Kopieg ei ai., Yeig U Ittipoi! 6:292 119761;

Nattetgivo ei a!., Moposiopa! Apiirodiez apa T Sei! Nurgidotav, Eizemieg, M.U. (19811).

In particular, monoclonal antibodies are prepared by a method that provides for the production of antibody molecules by continuous cell lines in culture, as described in Kopieg et al., Mayte 256:495 (1975), and

US Patent Me4,376,110; using human B-cell hybridoma (Kozbog ei ai., IttypoYodu Todau 4:72 (1983); Soye ei a!i., Rhos Mai! Asai 5si OBA 80:2026 (1983) and EVM-hybridomas (Soye ei ai! ., Moposiopa! Apiirodiyev apa Sapseg Tpegaru, Aap V. Iiv, Ips., pp. 77-96 (19831). Such antibodies can belong to any class of immunoglobulins, including Ids, DM, CHE, IdA, dO and any -what is its subclass The hybridoma producing tAr according to the present invention is cultured ip migo or ip mimo The ability to generate high titers of tAbrz ip mimo makes this method of production particularly useful.

Immediately after production, polyclonal or monoclonal antibodies are tested for specific recognition of RBSI-1 by Western blotting or immunoprecipitation, which are carried out by standard methods, for example, as described above in the publication of Alizybeya ei a). According to the invention, antibodies are used that specifically recognize and bind to RBOY-1. Particularly useful are antibodies against RBSI-1 that bind to the RBSI-1 antigen on the surface of a T-cell, such as a POP cell, and cause exhaustion and/or apoptosis of T-cells in the subject's body.

Antibodies are used, for example, within a therapeutic regimen (eg, to reduce or avoid an unwanted immune response, such as a T-cell-mediated immune response, associated with conditions such as inflammatory diseases, autoimmune diseases, transplant rejection, allergic diseases, and T - cellular cancer). Antibodies can also be used in a screening assay to measure the ability of a test compound to bind to ROY-1.

In addition, technologies developed for the production of "chimeric antibodies" are used (Moitizop ei ai., Rgos

Maya! Asai bsi OBA 81:6851 (1984); Meibregdeg eyi aII, Mashge 312:604 (1984); Takeada et al., Mashge 314:452 (19841) by splicing genes from a mouse antibody molecule with appropriate antigen specificity together with genes from a human antibody molecule with appropriate biological activity. A chimeric antibody is a molecule in which different parts are taken from different animal species, such as those that have a variable region from a rodent monoclonal antibody and a constant region from human immunoglobulin.

Alternatively, the technologies described for the production of a single-chain antibody (US Pat

MoMo4,946,778, 4,946,778 and 4,704,692), can be adapted to produce a single-chain antibody against the RUSSIA-1 polypeptide or its fragment. Single-chain antibodies are formed by linking heavy- and light-chain fragments of the Em site through an amino acid bridge, resulting in a single-chain polypeptide.

Antibody fragments that recognize and bind to specific epitopes can be produced by known methods. For example, such fragments include, among others, E(ab)2 fragments, which can be obtained by pepsin cleavage of an antibody molecule, and Rab fragments, which can be formed by reducing the disulfide bridges of E(ar)2 fragments. Alternatively, Rab expression libraries can be constructed (Nize ei ai.. bsiepse 246:1275 (19891), which allow quick and easy identification of monoclonal Rab fragments with the desired specificity.

Antibodies are humanized by methods known to those skilled in the art. For example, monoclonal antibodies with the desired binding specificity can be humanized on an industrial scale (Soidepe, 5soyMapa; Ohio MoiiIesshiag,

Raio Ayu, Saiyyi). Fully human antibodies, such as those expressed in transgenic animals, are also features of the invention.

Multimeric compounds

Multimeric compounds that bind to many of the RUSSIA-1 proteins on the surface of a T-cell or MK-cell are used to induce apoptosis in the cell. A multimeric compound contains at least two polypeptide chains. Each of the polypeptide chains contains (i) a binding domain that binds to ROS-1, and (ii) a heterologous amino acid sequence.

In general, a multimeric compound binds to at least two different RBOII-1 proteins on the surface of a given cell. However, a multimeric compound can be created that has 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more individual binding domains of ROBY-1, causing the multimeric compound to bind to 3, 4 , 5,6, 7, 8, 9, 10, 11, 12 or more different RIS-1 proteins on the surface of this cell.

The binding domain can contain any amino acid sequence (or any amino acid sequence with modification, for example, glycosylation and/or sulfation) that binds to RBI - 1. The binding domain can correspond to a natural or non-natural amino acid sequence . For example, the binding domain may contain a ROS-1 selectin binding domain (for example, P-selectin, E-selectin or I-selectin). A polypeptide that contains the binding domain of Re(I-1 selectin) may optionally include: (i) an extracellular domain of a selectin (eg, P-selectin, E-selectin, or I-selectin); (ii) a calcium-dependent lectin- the binding domain of a selectin (eg, P-selectin, E-selectin, or I1-selectin); or (ii) a fragment of the extracellular domain of a selectin (eg, P-selectin, E-selectin, or I1-selectin) that mediates binding with RBAI-1. In addition to these natural amino acid sequences, one or more amino acid changes in the natural binding domain of RBAI-1 are possible, resulting in a non-natural sequence that retains the RBAI-1 binding function. For example, a polypeptide may contain an amino acid sequence that binds to PO-1 and is at least 8095, 85905, 9095, 9595, or 9895 identical to any of the following domains: (i) the extracellular domain of a selectin (eg, P-selectin, E-selectin or I-selectin); (ii) calcium-dependent lectin-binding domain en selectin (for example, P-selectin, E-selectin or I-selectin); or (ii) a fragment of the extracellular domain of a selectin (for example, P-selectin, E-selectin or I-selectin), which mediates binding to ROI-1.

Standard methods of mutagenesis in molecular biology are used to make changes in the nucleic acid sequence that encodes the binding domain of RBOY-1. Modified binding domains in this case are tested for the ability to bind to ROI-1, for example, immobilized ROI-1 or RBOI-1 on the cell surface. The binding domain may also comprise the binding domain of RBOY-1 antibody against RBOY-1 or a polypeptide selected from a phage library or an amino acid sequence that binds to POOSI-1 and is at least at 8095, 8595, 9095, 9595 or 9895 identical to the binding domain of RBai-1 antibody against POOI-1 or a polypeptide selected from the phage library.

The RBAI-1 binding domain may contain an amino acid sequence that corresponds to the RBAI-1 binding fragment of P-selectin. An example of a polypeptide chain (a multimeric compound described by the authors), which contains such an amino acid sequence, is a recombinant mouse P-selectin/Pc chimera (from NGO Buvieev, Mippearoykh, MM), which contains the following components: () SOZ33 signal peptide (Mei-AIa16) ; (ii) mouse P-selectin (Tgr42-AIa709 extracellular domain); (ii) IEEASAMO (5EO IO MO 1); and (him) human yes (Rgo100-I uh330). The second example of a polypeptide chain that contains such an amino acid sequence is a recombinant chimera human P-selectin/Bs (from N.A. Zuviet, Mippearoykh, MM), which contains the following components: (i) human P-selectin (Mei-AlIa771, extracellular domain) ; (her) IESAMO (5EO IYU

MO-1); and (iii) the human Idai (Rgo100-I uz330).

The RBAI-1 binding domain may contain an amino acid sequence that corresponds to the RBAI-1 binding fragment of E-selectin. An example of a polypeptide chain (a multimeric compound described by the authors) containing such an amino acid sequence is the recombinant chimera mouse E-selectin/Es (from NAO Zuvieetv, Mippearoiis, MM), which contains the following components: (i) mouse E-selectin (Mei-

Pgo557, extracellular domain); (i) IESAMO (5EO IO MO 1); (iii) Nytap Idai (Rgo100-I uz330); and (m) NNNNNN (5EO

And MO:2). The second example of a polypeptide chain that contains such an amino acid sequence is a recombinant chimera human E-selectin/Bs (from N.A. Busietv, Mippearoi5, MM), which contains the following components: (i) human E-selectin (Main-Rho556, extracellular domain) ; (her) IESAMO (5EO IO MO:2); (her) human Idai (Rgo100-I uz330); and (m) NNNNNN (5EO IO MO:2).

The RBAI-1 binding domain may contain an amino acid sequence that corresponds to the RBAI-1 binding fragment of I-selectin. An example of a polypeptide chain (a multimeric compound described by the authors) containing such an amino acid sequence is the recombinant I-selectin/Pc chimera (from AAO Zuvietv5, Mippearoykh, MM), which contains the following components: () mouse I-selectin (Meiy-Avp332, extracellular domain); (iii) IESAMO (5EO IO MO:1); (ii) Nytap da (Rgo100-I uz330); and (m) NNNNNN (5EBO IU

MO:2). The second example of a polypeptide chain that contains such an amino acid sequence is a recombinant chimera I-selectin/Bs (from NGO Buziev5, Mippearoykh, MM), which contains the following components: (Her) human I-selectin (Mei-A5zp332, extracellular domain); (ii) I(EZAMO (5EO IYU MO:1); (ii) human Id (Rgo100-I uz330); and (m) NNNNNN (5EO I MO:g).

A multimeric compound can be formed as a homomultimeric compound or a heteromultimeric compound.

A homomultimeric compound contains only polypeptide chains that have identical РБай-1 binding domains. For example, a homomultimeric compound can contain polypeptide chains that contain identical РОС-1-binding fragments of P-selectin. The heteromultimeric compound contains polypeptide chains that have different RBOY-1 binding domains. For example, a heteromultimeric compound may contain a first polypeptide chain that contains a РОС-1-binding fragment of P-selectin and a second polypeptide chain that contains a Ра-1-binding fragment of E-selectin.

The heterologous amino acid sequence can be any amino acid sequence. However, the amino acid sequence of the polypeptide chains described by the authors does not correspond to the sequence of the natural protein. A heterologous amino acid sequence contains one or more amino acids that allow binding of polypeptide chains. For example, one or more amino acids can covalently bind, for example, through a disulfide bond, polypeptide chains. One example of a heterologous sequence is the constant region of an immunoglobulin heavy chain. As a result, disulfide bonding between Es sections of two polypeptide chains can ensure the formation of a dimeric compound.

In addition to participating in the binding of polypeptide chains, a heterologous amino acid sequence may also contain a cross-linking site, for example, a site for binding to a cell surface receptor. As a result of the binding of the agent to such a binding site, cross-linking of polypeptide chains and proteins may occur

RBOY-1 surfaces of cells with which they bind. The constant section of the immunoglobulin heavy chain contains the binding site of the Es receptor. The cross-linker can be, for example, an antibody (for example, an antibody against

Es), which specifically binds to the crosslinking site of the heterologous amino acid sequence.

Assays with the selection of compounds that modulate the function of RBOI -1

The invention also encompasses methods of recognizing compounds that interact with RUSSIA-1 (or the RBAI-1 domain), including, but not limited to, compounds that cause T-cell exhaustion and/or T-cell apoptosis upon binding to RBAI-1. It also encompasses compounds that modulate the interaction of RBai-1 with transmembrane, extracellular, or intracellular proteins that regulate ROS-1 activity, and compounds that modulate the activity

FIGHTS--1.

Compounds that can be selected according to the invention include, but are not limited to, peptide, antibodies and fragments thereof, and other organic compounds that bind to RB(CI-1) and modulate the biological function mediated by RBII-1, as described by the authors .

Such compounds may include, but are not limited to, peptides such as, for example, soluble peptides, including, but not limited to, those belonging to random peptide libraries; (at ei ai!., Matthew 354:82 (1991); Noshoniep ei ai,

Maishge 354:84 (19911), and a combinatorial chemically derived molecular library consisting of O- and/or I-configuration amino acids, phosphopeptides (including, but not limited to, those belonging to libraries of randomly or partially degenerate, directed phosphopeptides; bZopduapd ei ai., Se! 72:767 (19931), antibodies (including but not limited to polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single-chain antibodies and an expression library of Rar-, E(ab)2- and Bar-fragments and their fragments that bind to epitopes) and small organic or inorganic molecules.

Other compounds that can be selected according to the invention include, among others, small organic molecules that affect the activity of the ROOI-1 protein, as described by the authors.

Computer modeling and search technologies allow to identify compounds or to improve already identified compounds that can modulate the expression or activity of ROBA-1. Once such a compound or composition is identified, active sites or sites are identified. Such active sites are usually binding sites for a natural activity modulator. The active site is identified using methods known to specialists, including, for example, determination from among the amino acid sequences of peptides, nucleotide sequences of nucleic acids, or based on the study of complexes of the corresponding compound or composition with its natural ligand. In the latter case, chemical or X-ray crystallographic methods are used to find the active site by determining the location on the modulator factor (or ligand).

Although the above has been directed to the design and production of compounds that can alter binding, it is also possible to screen libraries of known compounds, including natural products or synthetic chemicals and biologically active materials, including proteins, for compounds that bind to the protein

RBII-1 and induce T-cell exhaustion and/or induce T-cell apoptosis.

IP myto systems can be designed to identify compounds capable of interacting with РОС-1 (or the РБи-1 domain). The identified compounds may be useful, for example, in modulating T-cell activity as described by the authors, and thus may be useful in the treatment of conditions associated with an excessive or undesirable T cell-mediated immune response or excessive or undesirable T cell proliferation -cells, such as inflammation, autoimmunity, transplant rejection, an allergic condition or

T-cell cancer.

The principle of the assays used to identify compounds that bind to ROZI-1 involves the preparation of a reaction mixture of ROZI-1 (or its domain) and the test compound under conditions and for a time that are sufficient for the interaction and binding of the two components. thus forming a complex that can be removed and/or detected in the reaction mixture. Types of RBII -1 can be different, depending on the purpose of the analysis with selection. In some situations, preference is given to the use of a peptide that corresponds to the PO-1 domain, fused to a heterologous protein or polypeptide, which allows you to take advantage of the analytical system (for example, labeling, isolation of the resulting complex, etc.).

Analyzes with selection are carried out in different ways. For example, one method of performing such an analysis includes anchoring the RBOY-1 protein, polypeptide, peptide or fusion protein or test substance on a solid phase and detecting complexes of ROZI - / test compound anchored on the solid phase at the end of the reaction. In one embodiment of this method, the ROY-1 reagent can be anchored on a solid surface, and the tested compound, which is not anchored, is directly or indirectly labeled.

In practice, it is convenient to use microtiter tablets as a solid phase. The anchored component is immobilized by non-covalent or covalent binding. Non-covalent binding is carried out by simply covering a solid surface with a protein solution and drying. Alternatively, an immobilized antibody, preferably a monoclonal antibody specific to the protein to be immobilized, is used to anchor the protein to a solid surface. Surfaces are prepared in advance and stored.

To carry out the analysis, the non-immobilized component is added to the covered surface containing the anchored component. After completion of the reaction, unreacted components are removed (for example, by washing) under conditions in which any complexes formed remain immobilized on the solid surface. Detection of complexes anchored on a solid surface is carried out in various ways. If the previously non-immobilized component is pre-labeled, detection of the label immobilized on the surface means that complexes have formed. If the non-immobilized component is not pre-labeled, an indirect label is used to detect surface-anchored complexes; for example, using a labeled antibody specific to a previously non-immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with a labeled anti-Id antibody).

In an alternative version, the reaction is carried out in the liquid phase, the reaction products are separated from the unreacted components and complexes are detected; for example, using an immobilized antibody specific for the RBSII-1 protein, polypeptide, peptide or fusion protein or test compound to anchor any complexes formed in solution and a labeled antibody specific for another component of the potential complex to detect the anchored complexes.

Alternatively, cell-based assays are used to identify compounds that interact with ROOI-1. For this, cell lines that express Ра-1 or cell lines subjected to genetic engineering to express RBOI-1 are used. Cell-based assays are particularly useful for assessing the functional impact of a compound identified by the screening described by the authors. For example, immediately after identifying a compound based on its ability to bind to ROSY-1 white matter, the compound is tested for its ability, for example, to induce apoptosis of ip myo or ip mimo T cells or depletion of ip miigo or ip mimo T cells.

Pharmaceutical compositions

If the purpose of this invention is to change the immune response in the subject's body, a pharmaceutical composition containing, for example, antibodies, multimeric compounds, small molecules or other compounds that specifically bind to RUSSIA-1 polypeptides, is also a feature of the invention. In an optimal example, the compound functions as an agonist of RESZI -1.

Pharmaceutical compositions for use according to the present invention are formulated in a traditional way, using one or more physiologically acceptable carriers or fillers. Thus, the compounds and their physiologically acceptable salts and solvates are formulated for administration by various routes.

The compounds are formulated for parenteral administration by injection, for example by bolus injection or continuous infusion. Compositions for injections can exist in the form of dosage units, for example, in ampoules or multi-dose containers, with the addition of a preservative. The compositions may be in the form of suspensions, solutions or emulsions in oily or aqueous carriers and may contain formulation agents such as suspending agents, stabilizers and/or dispersants. Alternatively, the active ingredient may be in the form of a powder for mixing prior to administration. application with a suitable carrier, for example, sterile pyrogen-free water.

Ways to control the T-cell-mediated immune response and depletion of T-cell populations

Compounds such as those detailed in connection with the selection may be useful, for example, in modulating a biological function mediated by the RBai-1 polypeptide and/or in the treatment of disorders associated with an excessive or unwanted immune response, for example, a T-cell-mediated immune response. These compounds include, among others, peptides, antibodies and their fragments, as well as other organic compounds that bind to PO(Y-1 on the surface of T cells and trigger a signal transduction pathway that ultimately leads to the death of T- cells. Methods according to the invention do not necessarily include the addition of a crosslinking agent that causes crosslinking of RUSSIA-1 on the cell surface. The compounds described by the authors can be used in any case where depletion or elimination of T cell activity is required. For conditions, for treatment for which compounds according to the invention are particularly suitable include inflammatory diseases, autoimmune diseases, transplant rejection, allergic diseases and T-cell cancers.

Examples of conditions amenable to treatment with the anti-RBAI-1 compounds described by the authors include, but are not limited to, diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, and psoriatic arthritis; multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosus lupus, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjögren's syndrome, Crohn's disease, aphthous ulcer, inflammation of the iris, conjunctivitis, keratoconjunctivitis, type I diabetes, inflammatory bowel disease, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy adverse reactions, egulteta podozitis, ergositis, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, true red blood cell anemia , idiopathic thrombocyto penia, polychondritis, granulomatosis

Wegener's, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic trophic aphthae, lichen planus, Based's disease, sarcoidosis, primary biliary cirrhosis, posterior uveitis, interstitial fibrosis of the lungs, graft-versus-host reaction, transplantation cases (including transplantation with allogeneic or xenogeneic tissues) such as bone marrow transplantation, liver transplantation or transplantation of any organ or tissue, various types of allergies such as atopic allergy, AIDS and

T-cell neoplasms such as leukemias and/or lymphomas.

Methods according to the invention are used to remove T cells from a population of cells, ip migo or ip mimo.

For example, a biological sample taken from the subject's body can be freed from T cells immediately by contacting the sample with the anti-ROS-1 compound described by the authors, optionally together with a crosslinking agent. This method may be useful by considering, for example, an increase in non-T cells in a cell population, as well as by reducing or eliminating T cell activity in a cell population.

Examples of practical implementation of the invention are presented below. They should not be construed as limiting the scope of the invention in any way.

Examples

Example 1: Preparation of a monoclonal antibody against a protein that causes apoptosis of T cells ("TA!IR")

The TAIR-specific monoclonal antibody was obtained by using the well-known methods of cell fusion according to Kohler and Milstein (Kopieg apa Miivivip (1976) Eigoreap doshgpai! oii Ittypoiodu 6:511-519)) to obtain a hybridoma that secretes the desired antibodies. Antibody-producing cells taken from a hamster injected with concanavalin A (Sop A) activated splenic Wair/s T-cells were fused with a myeloma cell line to produce an antibody-secreting hybridoma. The two cell populations were fused with polyethylene glycol, and the resulting antibody-producing cells were cloned and propagated by standard tissue culture methods. One hybridoma produced according to these methods secreted a monoclonal antibody called TAVA that was capable of inducing apoptosis of ip myo T cells and depleting ip mimo T cells. The protein that was recognized by the TABA4 antibody was defined as the protein that induces apoptosis of T cells (TAI!P).

Mice S5781/62U (86) and VA! The air force was purchased from Iab (Vagh Nabog, ME) from Daskbvop. Syrian hamsters were purchased from Apit! Soge Rasiyu, National Taiwan University College of Medicine (Maiyopai! Taimap

Opimegzyu Meais! CoPede).

The concentrated supernatant of the TAVA hybridoma culture was centrifuged at 20,000 x g for 10 minutes and the supernatant was diluted 1:1 with binding buffer (0.1 M sodium acetate, pH 5.0).

The column with C-protein (layer volume approximately 1 ml) was washed three times with 3-5 ml of binding buffer. The purified culture supernatant was applied to a Cs-protein column and the flow-through liquid was collected and re-applied to the column.

The column was washed with 6-10 ml of binding buffer and the bound antibody was eluted from the column with 5 ml of elution buffer (0.1 M glycine-HCl, rng,8). Each fraction contained iml of eluted antibody, and the eluted fraction was brought to neutral pH by mixing each iml fraction with 50 microliters of 1M

Tgiv-NSY, pH 7.5. The fractions containing the antibody were combined and dialyzed three times with 2 liters of РВ5, рН7 4 for three hours for each dialysis. The concentration of protein in the antibody samples was determined using the procedure described

Bradford (Vgadyuga) using the Vio-Vaa protein assay (VIO-NAB, Negsshev, CA).

Example 2: Preparation of mouse spleen cell suspension and activation and enrichment of T cells

The spleen of mice was immersed in 3 ml of Hank's balanced salt solution (HB55), slightly crushed with a sterile cover glass, transferred to a 15 ml centrifuge tube (Soviai) and centrifuged at 200 x g for 5 minutes. The supernatant was drained and the sediment with cells was resuspended in the residual buffer by lightly tapping the walls. Contaminating erythrocytes (ABCs) were lysed by adding 1 ml of lysis buffer for ABCs (0.6 M MNAaCl, 0.17 M Tgiz-base, pH7.65), after which a 2-minute incubation at room temperature and rapid quenching with Uml HB55 was carried out. Cells were precipitated in the amount of 200 x g for 5 minutes, washed twice and resuspended in ARMI medium. The concentration and viability of cells in the mixture was determined using a hemocytometer (Satbgidde Zsiepiyis Ips.) and the exclusion of trypan blue.

Spleen cells were brought to a final concentration of Х X 10b/ml using VRMI medium and concanavalin A was added to a final concentration of 2 micrograms/ml for T-cell activation. The cell suspension was transferred to a 6-well plate (5ml/well) or 10-cm culture dishes (100ml/dish) and incubated at 37°C, 595°C for 48 hours before harvesting. Activated splenic cells, including activated T- cells were resuspended in Zml HB55 and carefully transferred to a 5-ml layer of Percoll 5595 substrate in a centrifuge tube, taking care not to disturb the separated layers.

Cells were continuously centrifuged at 1900 x g for 13 minutes at 25°C. Enriched T-cells were collected from the contact surface between the two layers, washed twice with HB55, and then they were ready for experiments.

Example 3: Apoptosis of activated T cells

Activated T-cells (see Example 2) were resuspended to a final concentration of 5 x 10 cells/ml. in ARMI medium containing 5 ng/ml 1I--2 and treated with control Id, TAVA, or anti-COZ according to the conditions shown in Table 1.

Table 1

Experimental Processing" groups

Negative /|Zmkg/ml Id hamster control Bng/ml 1-2

Zmkg/ml of cross-linker antibody

Id vs Hamster)

TAVAA Zmkg/ml TAVA tAbB hamster

Bng/ml 1-2

Zmkg/ml of cross-linker antibody

Id vs Hamster

Positive | μg/ml anti-SOZ tAb control 5ng/ml I -2 1Tmkg/ml cross-linker antibody (Id against mouse) 7; The final concentration of the indicated reagents in the environment.

After an incubation period of 18-24 hours, the degree of apoptosis in each culture was determined using the 7-AAA apoptosis assay. The treated cells were transferred to RAS5 test tubes (Rysop), washed twice with ice-cold BAS5 solution (195 fetal calf serum, 0.0595 sodium azide in РВ5), precipitated in the amount of 200 x g at 4"C. Cells were resuspended in ice-cold EAS5 solution to the final concentration 1-2x10" cells/ml. For staining, 0.1 ml of resuspended cells were mixed with 7-AAO to a final concentration of 2 μg/ml, and then incubated at 4°C in the dark for 20 minutes. And finally, the stained cells were washed twice with ice-cold ЕАС5 solution, resuspended in 0.5 ml of ГАС5 solution and analyzed by flow cytometry for VO I 5A (Weskup Oiskizop).

Fig. 1 shows the results of a representative experiment with changes in time, in which it was studied when activated T-cells become sensitive to mediated TAVA (anti-TAIR) apoptotic signals.

Splenocytes of mice were activated by Sop-A and kept in a medium containing II-2. Activated T-cells were collected, resuspended, and tested for immunity with a TABA monoclonal antibody or a hamster control in the presence of a hamster anti-Ids antibody as a cross-linker. The ability of TA!IR cross-linking to induce a low level (6,595) of apoptotic cell death was detected on the first day. However, the extent of TAB4A4-induced apoptosis increased from 17905 on day 2, peaked at 5295 on day 4, and decreased to 4495 on day 6. Control

Hamster Id did not induce specific apoptotic death of T cells compared to cultures treated with only

And--2. Api-COZ (as a positive control) induced apoptosis in 3895 T cells after 48 hours of activation (data not shown).

Example 4: TAIR antigen expression in different tissues

Cells were washed twice with ice-cold EAS5 solution (195 fetal calf serum. 0.0595 sodium azide in PB5) and centrifuged at 200 x g at 4"С in an EASZ5 tube (Rysop). Cells were resuspended in ice-cold EASZ5 solution to a final concentration of 1 x 10 "cells/ml and an aliquot of 0.1 ml of resuspended cells in an EAS5 tube (Rysop) was used for each analysis. For surface staining, TAVA monoclonal antibody or control hamster Id was added to cells at a final concentration of 2 μg/ml and the mixtures were incubated at 4°C for 30 minutes in the dark. Cells were washed once with ice-cold EAS5B and then stained: (1) for cells spleens - with cychrome-conjugated antibody against POPs (2μg/ml), EITO-conjugated Id against hamster and RE-conjugated antibody against СО8/С04/С2019/С011р/rap-МК/-А/- E/Mas-3 (2μg/ml) in 100μl of ice-cold BAS solution, and (2) for thymus cells, with RITO-conjugated anti-hamster Id, PE-conjugated anti-CO8, and cychrome-conjugated anti-CO4 antibodies (2 μg/ml) in 100 μl of ice-cold solution

EAS5. The reaction was carried out at 4"C for 30 minutes in the dark. Finally, the stained cells were washed twice with ice-cold EAS5 solution, resuspended in 1 ml of ГАС5 solution and subjected to analysis by flow cytometry on VO I 5A (Veskup Oiskizop). Fig. 3 and 4 are shown through analysis of EAS5 distribution of TAIR antigen on different subpopulations of splenocytes and thymocytes. As shown in Fig. 3, CO19" B cells expressed a low but detectable amount of TAIR proteins on the surface. A significantly higher amount of TAIR proteins was detected on SOYUZ T-cells and MK-cell fractions. Most T-cells of the thymus gland CO", CO8" and CO4787 expressed a significant amount of TAIR proteins.

Instead, TAIR proteins were expressed only on a small population of T cells CO4-8: thymus (Fig. 4).

Tissues from Wb and YAI B/s mice, including brain, thymus, heart, lung, liver, stomach, kidney, spleen, and skin, were collected, fixed in 1095 formaldehyde until the next day at room temperature, and embedded in paraffin blocks. Sections of tissues 4 μm thick were taken from paraffin blocks with the help of a microtome I Yeiss AM2135, spread in 457 water and placed on a glass slide. The slides were dried at 37"C, and after that they were ready for further experiments.

Slides containing paraffin-embedded tissue sections were deparaffinized and dried in xylene-10095 ethanol series according to standard protocol and finally kept in 10095 ethanol. Sections were rehydrated by serial incubations in 10095 ethanol-9095 ethanol-8595 ethanol-7095 ethanol-PB5 according to the standard protocol to the final solution in PB5. All the reactions presented below were carried out in a humidified chamber.

Nonspecific binding was blocked by incubating tissue sections in blocking buffer (195 normal goat serum) for 1 hour at room temperature (or 4°C until the next day). Blocking buffer was removed and TAVA or normal hamster Id (dilution 1:200) and incubation was continued for another hour at room temperature (or 4"C until the next day). Sections were washed twice in RVB5, each for 5 minutes, to remove the primary antibody, reacted with diluted 1:250 alkaline phosphatase-conjugated goat anti-hamster Id and incubated at room temperature for 1 hour. The sections were again washed twice with PB5, for 5 minutes each, to remove the antibody-enzyme conjugate, and the color reaction was shown with the substrate solution VSIR/MVT at room temperature for 30 minutes in the dark.

Sections were washed again with PB5 to remove excess enzyme substrate, dehydrated in series

PB5-ethanol-xylene and fixed on glass for a microscope.

The results showed that the expression of TAIR proteins was detected only in tissues derived from the bone marrow, but not in the rest of the tested tissues.

Example 5: Biotinylation of the cell surface and immunoprecipitation of TAIR antigen

5 x 107 VI 41 or MIN-3T3 cells were subjected to surface biotinylation in 1 ml of PB5 containing 0.5 mg/ml sulfo-MH5-biotin (Riegse) for 30 minutes on ice. The reaction was stopped by incubating the cells with 0.5 ml of modified Dulbecco's Igla medium (Ge Tesppoiodiev, Ips.) for 10 minutes on ice. Cells were washed once with 1 ml of Eagle's modified Dulbecco's medium and twice with 1 ml of phosphate-buffered saline.

Labeled cells were lysed at a density of 5.0 x 10 cells/ml in cold lysis buffer (195 Type X-100, 20 mM Tiiv-HCl, rna,0, 160 mM Mas, 1 mM Sasi) containing a complete protease inhibitor cocktail ( NKospe) for 15 minutes and the insoluble material was precipitated in an amount of 10,000 x g for 10 minutes; these and all subsequent steps were carried out at 4"C. For immunoprecipitation, the lysate was pre-incubated for 30 minutes with 5 μl of C-protein-sepharose (Atehepat RNagtasia Vioiesii) to remove proteins that bind non-specifically.

The spheres were precipitated and aliquots of the supernatant (which usually correspond to 5.0 x 10 cells) were incubated with 20 μl (- protein-sepharose with the previous introduction of 1 μg of tAr TAV4 or IdS;i from normal hamster serum. After incubation for 4 hours at 4"C the resin was washed four times with a washing buffer (0.0595 Typeop X-100, 50 mM Thiez-HCl, pH 5, 400 mM Masi, imm Sasi", Img/ml egg albumin), twice - with a similar washing buffer, which contained 250 mM instead of 400 mM Masi . Proteins specifically bound to TAVA were eluted from 50 μl of 1x505 sample buffer. Eluted proteins were separated using 895 505-RACE and transferred to a nitrocellulose membrane (MiSroghe). Filters were analyzed for biotinylated proteins by peroxidase-conjugated avidin (RpagMipdep ) and developed with a chemiluminescent reagent (MEM"M |Me Zsiepse

Rhodysiv).

As shown in Fig. 2, a biotinylated surface protein with a molecular weight of approximately 120 kDa was identified by TAVA in VI 21 cells (TAYIR" T- cells), but not in 373 cells (TAYIR" cells).

In contrast, C-protein-Sepharose coated with normal hamster serum cannot detect this 120-kDa protein.

These results indicate that this 120-kDa protein is an antigen recognized by the monoclonal antibody TAVA on the cell surface of T cells.

Example 6: Depletion of T cells by ip mimo

To investigate the effect of TABA4 on the population of T cells and other cells, mice were injected intraperitoneally with 30 μg of TABA4 from a control ID hamster, and on the 4th day, splenocytes, thymocytes, and peripheral blood mononuclear cells were collected for counting the total number of cells and for analysis. cell surface markers using EAS5.

For BAS analyses, cells were fixed with 295 paraformaldehyde at 4°C for 20 minutes, washed twice and resuspended in ice-cold EAS5 solution to a final concentration of 1x10 cells/ml. For each analysis, an aliquot of 100 μl of resuspended cells was used in an EAS5 test tube (RaiYsop). Added to the cells

TAVA or control hamster Id at a final concentration of 2 μg/ml and the mixture was incubated at 4"C for 30 minutes in the dark. Cells were washed once with ice-cold EASA and reacted: (1) for spleen cells - with a cichrome-conjugated antibody against POPs (2μg/ml), EITO-conjugated Id against hamster and RE-conjugated antibody against СО8В/С04/С019/2011Б/rap-МК/-А/-Е/Мас-3 (2μg/ ml) in 100 μl of ice-cold EAS5 solution; and (2) for thymus cells - with EITO-conjugated anti-hamster Id, RE-conjugated anti-CO8 and cychrome-conjugated anti-CO4 antibodies (2 μg/ ml) in 100 μl of ice-cold EAS5 solution. The reaction was carried out at 4-7C for 30 minutes in the dark. | finally, the stained cells were washed twice with ice-cold EAS5 solution, resuspended in 1000 μl of HAS5 solution and subjected to analysis by flow cytometry on VO I 5A (Veskuop Oiskizop).

Four days after the injection, the percentage of SOYUZ T cells in the peripheral blood leukocytes (PBW) decreased from 36.796 in control mice to 4.195 in TABA-treated mice (Table 2). TABA treatment caused a small decrease in the total number of splenocytes. However, in mice treated with TABA4 showed a 6290 decrease in the number of SOYUZ" T cells, a 5095 decrease in the number of MK cells and a slight increase in the total number of CO19- B cells. The total number of thymocytes taken from TABA-treated mice was only 4,895 from the level observed in control animals (a decrease of 5,295). In addition, with the exception of СО4- T-

cells, all other СО8-, СО4-С0О08- and С04-СО08- T-cells decreased, with the СО4-С08- subpopulation being most severely affected (decrease by 64,790).

Table 2 USSR | 72e | 777777117534. |771711177298 11111068 11111111. | Unprocessed | Normal hamster | TA-BA processing Depletion (9) (representative data of three experiments)

Example 7: Antibody against TAI!R does not cause II-2 or TME-alpha secretion

Vair/s (H-2a) mice were injected intraperitoneally with 300 micrograms of TAVA or a control Id hamster. Splenocytes were isolated 7 days after injection and used as responders in culture with mitomycin C SZN (H-2K) splenocytes (as stimulators). After three days, the culture supernatants were collected and the II-2 content was measured using EGISA (RpagmMmipdep). As shown in Fig.5, production

I/-2 was suppressed in responder cells taken from TAB4-treated mice compared to control mice. Plasma levels of II-2 and TME-alpha were also analyzed, and no significant differences were observed in the level of II-2 (or TME-alpha) in the serum of control mice and mice treated with TAB4.

Since the production of 1-2 is a key driver of T-cell activity, the results show that specific to

A TAIIR antibody such as TAB4A can be used ip to manipulate T cells and control unwanted T cell-mediated immune responses, such as those associated with autoimmune diseases and transplant rejection.

Example 8: Use of anti-TAIIR antibody to prevent transplant rejection

8- to 12-week-old mice (purchased from Chaskwop I arogaiyug) were anesthetized with acepromazine maleate (Heptepia Apita! Neaip So., Kapzaz Xu, MO). Before skin graft transplantation, recipient C57VI/6 (H-252) mice, in which the thymus gland was not removed, were injected intraperitoneally with 50 μg of TAVA or isotype control antibodies seven days before skin graft surgery.

After seven days, the skin from the barrel of completely allogeneic non-matching Vair/si (N-27) mice was transplanted onto the barrel of C57VI/6 mice, which had previously been injected with an antibody. Seven days after transplantation, mice were again injected with 500 μg of TAB4 or isotype control antibody. Mice were observed daily after transplantation. Grafts were considered rejected if 5095 donor skins were necrotic.

The percentage of graft survival is shown in Fig. 7 (n-8). The data show that the introduction of the TAVA antibody increased the survival of allogeneic skin grafts.

Example 9: Identification of TA!IR as RBOII -1

P-selectin glycoprotein ligand-1 (RBOBI-1), also known as CO162, is the major P-selectin ligand expressed on leukocytes, including T cells (Zako et al. (1993) Cell 75:1179; Masnipo (1995). Biochemical characteristics

TA!IR, such as its molecular weight and its tendency to dimerize, indicated the possibility that

TAV4 may be similar to ROSYA-1. In order to investigate the connection between these two antigens, it was found out: 1) whether the antigen precipitated with TAVA can be recognized by an antibody against RBOI 1 of serial production; and 2) whether the anti-P5OYI 1 antibody can remove TAVA from the cell lysate.

VI 41 T-cells were subjected to lysis at a density of 1.0x10 cells/ml in lysis buffer (195 Type X-100, 20 mM

Tyv-NSI, rna,0, 160mM Masi, 1mM Sasi"), which contained a complete protease inhibitor cocktail, for 1 hour and the insoluble material was precipitated in an amount of 10,000 x g for 10 minutes. These and all subsequent stages were carried out at 42C. The lysate, which corresponded to 5.x107 cells, was incubated with 20 μl of C-protein-sepharose with the previous introduction of 10 μg of anti-RBaI-I tAbB (clone 2RNI, RpagMipdep, Zap Oiedo, SA), anti-TAIR tAB, TAVA or da from normal hamster serum. After incubation for 4 hours at 4°C, the beads were washed five times with washing buffer (0.0595 Top X-100, 50 mM Ttiz-HCII, rna,5, 400 mM Mass, 1 mM Sasi, 1 mg/ml egg albumin) and twice - a similar washing buffer, which contained 250 mM instead of 400 mM Masi. The bound proteins were eluted from 40 μl of 1x505 sample buffer. The eluted proteins were separated using 695 505-RACE and transferred to a nitrocellulose membrane. The membranes were subjected to immunoblotting using anti-P5III - 1 tAB and detected with the help of peroxidase-conjugated IdsS goat anti-rat (H-Y) followed by chemiluminescence (Nepaiizzapse, MEM).

Surface biotinylated VI 41 T-cells were subjected to lysis at a density of 1.0 x 108 cells/ml in lysis buffer. The cell extract was incubated with 2 µg of antibody bound to 40 µg of C-protein-Sepharose at 4°C until the next day. Depletion was carried out using anti-P5αi-I tAB (2RNI) or control rat Ida, with TAVA or control normal human serum. which. The depleted lysates were further subjected to immunoprecipitation using TAVA or anti-P5C21-1 tAB, respectively. The immunoprecipitates were separated on a 695 505- polyacrylamide gel and detected by fluorography. As shown in Fig.b, the anti-ROSI-1 antibody can remove the TAIIR protein from of T-cell lysates.In addition, proteins subjected to immunoprecipitation using an anti-TAIR antibody (TAVA4) can be recognized by an anti-RUSSIA-1 antibody by Western blot analysis.

Example 10: Induction of apoptosis in human T-cells by an antibody against ROS-1

To determine the role that ROSSI-1 plays in human T-cell apoptosis, time-course experiments were performed to investigate when activated human T cells become sensitive to ROSSI-1-mediated apoptotic signals. Human T-cells were stimulated with the mitogen phytohemagglutinin (RNA) and then propagated in a medium containing I/-2. Activated T cells were collected and then tested for immunity using anti-RBI-I in the presence of II-2 and cross-linking antibodies.

Human peripheral blood was taken from healthy adults, heparinized and enriched for peripheral blood mononuclear cells (PBMC) based on differential density using RisoiI-Radne Riv (Rpagtasia Violiesp). PBMCs were activated with 195 RNA (Tespoiodiez, SsirsoVvV|) for 48 hours and then kept in recombinant human 1-2 (Zng/ml) for the duration of the analysis. To evaluate the apoptosis-inducing ability of the anti-human RBAI-1 antibody, activated cells were treated with: (1) 1 μg/ml of the anti-RBAI-1 antibody clone KRI-1 (VO RpatmMmipdep) and cross-linker rabbit anti-mouse Id (0.5 μg/ml) (Chaskwap

IttypoVezeaigsi I arogayugiev); (2) isotype control purified mouse Id and cross-linked rabbit anti-mouse Id; or (3) by cross-linking rabbit vs. mouse Id only. After six hours of treatment, the percentage of early apoptotic cells was determined by EAS5, with anti-Appehip M (VO RnagMipdep) and RI (Zidta) staining.

As shown in Fig.8, the signal caused by RBOI-1 using an antibody against ROS-1 and a cross-linker caused a significant level of apoptosis in RNA-activated human PBMCs (mostly T-cells). The percentage of apoptotic cells increased from 8.595 on the 3rd day to 2495 on the 8th day in the treated anti-P5II-1 cultures.

Neither the isotope-matched control nor the cross-linking antibody alone had any effect on these cells.

Example 11: Use of an antibody against the ROI-1 agonist for the treatment of an autoimmune disease

Non-obese diabetic mice (MBD), carefully selected animals with autoimmune diabetes, were bred under standard conditions. Spontaneous diabetes developed in MY mice at approximately 20 weeks of age. In the experimental group, mice were injected intraperitoneally with three doses of antibodies against RBOII-1 (TAB4) in the amount of ZOOmkg per mouse at the age of 14, 15 and 17 weeks. Two additional injections of the same dose were administered at the age of 24 and 26 weeks. The control group was injected with hamster Id in the same dose. Mice were monitored for glucosuria using Meai-Tevi Scissoze (Maspegeu-Madei, Germany) twice a week after reaching the age of 15 weeks.

A postprandial urine glucose level of more than 300 mg/dL for two consecutive measurements was considered diabetic.

As shown in Fig.9, the introduction of the TAVA antibody (anti-P5II -1) provided significant protection compared to the introduction of the control antibody. Thus, administration of an antibody against ROI-1 can weaken the activity of autoimmune T-cells and delay the onset of type I diabetes.

Example 12: Binding of P-Selectin, E-Selectin and I-Selectin to activated T-cells

To determine the ability of selectins (P-Selectin, E-Selectin and I-Selectin) to bind to activated T-cells, freshly prepared splenocytes of C57VI/6 mice were activated and collected on the 2nd, 4th and 6th days. Non-activated T cells (ie, freshly prepared splenocytes on day 0) were also analyzed.

The sample on the 2nd day included 3x109 cells/ml of splenocytes, which were activated by 2 μg/ml concanavalin A (Sop A) in

OMEM--1095 ЕВ5 within 2 days. Living cells were separated using Risoi! gradient A day 4 sample was obtained from cells that had activated Sop A for 3 days and maintained in medium containing 5 ng/ml

And--2, within one more day. The sample on the 6th day was obtained from cells that activated Sop A for 3 days and kept in 5ng/ml II -2 for 3 days.

To evaluate samples by EAS5 analysis, 2x105 cells per well, taken on days 0, 2, 4, and 6, were incubated at 4"C for 30 minutes with 40 μl/well of mouse P-Selectin, E-Selectin, or I-Selectin, merged with the Es region of human IDS (80 Zuzietv, Mippearoiykh, MM) at a concentration of 20 μg/ml with two-fold serial dilution to 0.156 μg/ml. After incubation, the cells were washed with their EASbsap buffer (1 x PB5 without calcium and magnesium ions from Viospgot As, Vepipe and 295 EB5).The samples were further incubated at 4°C for 30 minutes with 95 µl/well of anti-Tpu1.2 and a secondary reagent (anti-human RITS-Ida, which is specific for the fragment

Es, which was obtained from Chaskhop IttypoVezeagsi and arogayugez, Ips., MUev5i Sgome, RA) in the amount of 3.25μg/ml, and then washed with their EAS5sap buffer.

The results of the analysis of EAS5Sairig are shown in Fig. 10. At 20μg/ml binding of P-selectin with activated

Mouse T cells gradually increased, peaked on the 4th day, and slightly decreased on the 6th day.

E-selectin binding increased significantly from days 2 to 4 and then remained at a peak level on day 6.

Binding of I-selectin to activated T-cells of mice was not detected and did not change during the activation period, that is, from 0 to 6 days. The results observed with I-Selectin could be due to the apparent low binding affinity of I-Selectin to its ligand. Similar results were also obtained when lower concentrations of the three selectins were used in the experiments.

Example 13: Multimeric forms of E-Selectin and P-Selectin cause apoptosis of activated T-cells. A 96-well plate (MOM) was covered with 5 µm of anti-human EsID in the amount of 20 µg/ml in their РВ5 at 4"C until the next day, blocked with 195 B5A at 37"C for 2 hours and incubated with 50 μl of fused selectin-human Es (from 0.063 to 5 μg/ml) at room temperature for 2 hours. At all stages of the experiment, each well was thoroughly washed five times with their РВ5. Then 2x109 pre-activated Sop A for four days of T-cells were added to each well and incubated at 37"C for 5 hours before centrifugation of tablets at 200 x g for 5 minutes at 4"C. The resulting pellet, which contained activated T cells, was incubated with Appehip M-biotin conjugate at room temperature for 15 minutes, and then with avidin conjugate (ZA-reia-da! at a dilution of 1:5000) for another 30 minutes at 37"C.

For each binding reaction, each well was washed three times with binding buffer Appehip M. Color development was achieved by incubating 11 μl of a mixture of 2-buffer (54 μl of 2-mercaptoethanol in 20 ml of 2-buffer) and

ZOmcl OMR (0.04g/1Oml) at 4"C until the next day. The optical density at 420nm was recorded.

The level of selectin-induced apoptosis of activated Sop-A T cells increased with an increase in the concentration (from 0.06 µg/ml to 5 µg/ml) of P-selectin (Fig. 11A) or E-selectin (Fig. 118) fused with ES of human da .

Hamster TAB4A antibody induces apoptosis of activated T cells (see Example 1) and was used as a positive control in these experiments. As a negative control, Es anti-human, human Id (NiId) and B5A did not induce apoptosis. Significant apoptosis was not detected in the presence of a fusion protein of I-selectin and human ES (Fig. 11C), which corresponded to the inability of I|-selectin to properly bind to activated T-cells (Example 12).

Therefore, the fusion protein bound on the plate, which contained the Pα-1-binding fragment of P-selectin or

E-selectin and a fragment of human Es, induced apoptosis of activated T cells

Example 14. Crosslinking of the soluble fusion protein P-Selectin-Es causes apoptosis of activated T cells

Mouse selectins (P-Selectin, E-Selectin, and I-Selectin) were fused to the E site of human IDS as detailed above to form soluble dimeric fusion proteins. To determine whether soluble selectins could induce apoptosis of activated T cells, an experiment was performed as detailed in Example 13, except that plate-bound anti-human EsID was not used Insignificant or low level of apoptosis activated T cells were observed in the presence of only the soluble form of the P-selectin fusion protein (dimer) (Fig. 12). However, after the addition of a cross-linker (human Bs-wires), the apoptotic activity increased significantly to approximately the apoptotic level induced by the presence of the antibody bound on the plate, neither anti-human Es, nor human Id (NId), nor B5A induced apoptosis.

For the E-selectin-E fusion protein, similar results were obtained to those obtained for the fusion protein

P-selectin-Es. In addition, according to the results obtained for the plate-bound (multimeric form) I-selectin, the soluble form of the fusion protein I-selectin did not cause apoptosis of activated T cells.

Other implementation options

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