RU2407539C2 - P-selectin glycoprotein ligand 1 modulators - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to immunology, and concerns P-selectin glycoprotein ligand 1 modulators. Substance of the invention involves a multimeric compound which is bound with at least two (PSGL-1) proteins on a T-cell surface; said multimeric compound contains two polypeptide chains each of which contains a binding domain which is bound with PSGL-1 and which contains a P-selectin extracellular domain or its binding fragment, or E-selectin extracellular domain or its binding fragment, and also a heterologous amino acid sequence; two polypeptide chains are combined by the heterologous amino acid sequence to produce the multimeric compound.

EFFECT: advantage of the invention consists in the development of the agent to be used to induce T-cell depletion and/or apoptosis.

30 cl, 14 ex, 12 dwg

Description

Technical field

The invention relates to compositions and methods for controlling immune responses.

State of the art

Control of unwanted immune responses is important in the treatment of diseases, such as inflammatory diseases, autoimmune diseases, organ transplant rejection, allergic diseases and tumors, the origin of which is associated with T cells. The activity of overly aggressive T cells can be regulated by immunosuppression or by inducing immunological tolerance. Tolerance is defined as a condition where the immune system is made insensitive to an antigen, while immunosuppression, which reduces the immune response to antigens, usually requires prolonged use of therapeutic agents. In organ transplantation, T cells play an exceptional role in the formation of an immune response to alloantigens. Current immunosuppressive techniques typically include the use of corticosteroids, cyclosporine or rapamycin, which block the transcription of IL-2, a key growth factor for T cells, or inhibit IL-2 dependent proliferation. However, some monoclonal antibodies that act either as T-cell depleting agents (e.g., CD3, CD4, CD8), or as inhibitors of cytokine signaling or co-stimulating T-cell metabolism (e.g., CD25, B7-1, B7-2, CD 152, CTLA4) have been shown to be effective in reducing rejection with limited side effects or toxicity. It has been shown that some of these drugs are successfully used in the treatment of autoimmune diseases or in prolonging survival in transplantology.

It is widely believed that apoptosis is vital for maintaining the proper function of the immune system and is the primary mechanism for removing unwanted cells (Kabelitz et al. Immunol. Today 14: 338-340. (1993); Raff, Nature: 356: 397-399 (1992 )). Various signals emanating from the outside or from the inside of the cell affect the life and death (death) of the cell. Antibodies specific to the substances of the cell surface of T cells, such as Fas (or CD95, M.M. = 43 kDa), TNFR2 (M.M. = 75 kDa), CD2 (M.M. = 45 kDa) and CTLA -4 (M.M. = 33 kDa) induce T-cell apoptosis (Osbome, Curr. Opin. Immunol. 8: 245-248 (1996); Lin et al. J. Immunol. 158: 598-603 (1997) ; Zhang et al. Nature: 377: 348-350 (1995); Lai et al. Eur. J. Immunol. 25: 3243-3248 (1995); Mollereau et al. J. Immunol. 156: 3184-3190 (1996) ); Gribben et al. Proc. Natl. Acad. Sci. USA 92: 811-815 (1995)).

Attempts to use Fas and TNFR2 to control unwanted T cells have been hampered by the fact that these two substances are expressed not only by immune cells, but also by other important organ systems, such as the liver. Such an expression example substantially limits the therapeutic use of these two antibodies (Ogasawara et al. Nature 364: 806-809 (1993); Pfeffer et al. Cell: 73: 457-467 (1993); Engelmann et al. J. Biological Chemistry 265: 14497-14504 (1990)).

The selectins, integrins and members of the immunoglobulin superfamily (Ig) family are the three main classes of adhesive substances that are important for the interaction of leukocytes and platelets, either with each other or with the extracellular matrix and vascular endothelium (Springer, Nature 346: 425 (1990); Osborn, Cell 62: 3 (1990); Hynes, Cell 69:11 (1992)).

An adhesive substance of one type of cell often binds to another adhesive substance expressed by another type of cell to form a ligand-receptor pair.

The selectin family consists of P-selectin (also known as CD62, CD62P, GMP140 and PADGEM), E-selectin (also known as ELAM-1 and CD62E) and L-selectin (also known as LEGAM-1, Mel-14, LAM -1 and CD62L). These selectins are highly homologous, they include an N-terminal lectin domain, consisting of 120 amino acid residues, an EGF-like domain, an unstable (changing) number of domains, consisting of numerous short consensus sequences (SCR) and homologous to those found in complement regulatory proteins, followed by a transmembrane domain and a short cytoplasmic tail (Siegelman et al., Science 243: 1165-1172 (1989); Lasky et al., Cell 56: 1045-1055 (1989); Tedder et al., J. Exp .Med. 170: 123-133 (1989); Johnson et al., Cell 56: 1033-1044 (1989); Bevilacqua et al., Proc. Natl. Acad. Sci. USA 84: 9238-9242 (1987) , Bevilacqua et al., Science 243: 1160-1165 (1989); Bevilacqua et al., J. Clin. Invest. 91: 379-387 (1993), Camerini et al. Nature 280: 496-498 (1989)) .

The selectin sequences partially overlap but differ in specificity for cell surface receptors (Bevilacqua et al., J. Clin. Invest. 91: 379-387 (1993); Feize, Current Opinion in Struct Biol. 3: 701-710 (1993 ); Berg et al., Biochem Biophys. Res. Comm. 184: 1048-1055 (1992); Foxall et al., J. Cell Biol. 117: 895-902 (1992); Larsen et al., J. Biol Chem. 267: 11104-11110 (1992); Polley et al., Proc. Natl. Acad. Sci. USA 88: 6224-6228 (1991)).

P-selectin, E-selectin, and L-selectin promote adhesive cell interactions: the first leukocyte-endothelial cell and platelet-leukocyte during inflammation (Bevilacqua et al., 1993, ibid.). It has been shown that all three selectins are involved in the unfolding initial interaction of leukocytes with activated endothelium (von Andrian et al., Proc. Natl. Acad. Sci. USA 88: 7538-7542 (1991); Ley et al., Blood 77: 2553 -2555 (1991); Abassi et al., J. Cin. Invest. 92: 2719-2730 (1993); Dore et al., Blood 82: 1308-1316 (1993); Jones et al., Biophys. J. 65: 1560-1569 (1993); Mayadas et al., Cell 74: 541-554 (1993)).

P-selectin expressed by activated platelets and endothelial cells binds to the cell surface proteins of most leukocytes (McEver et al., J. Biol. Chem. 250: 9799-9804 (1984); Hsu-Lin et al., J. Biol Chem. 264: 8121-9126 (1984)).

E-selectin expressed by cytoxin-activated endothelial cells (e.g., after 6-8 hours of TNF-alpha or IL-1 stimulation) binds to cell surface proteins of most leukocytes (McEver et al, J. Clin. Invest. 100: 485-492 (1997); Bevilacqua et al., 1987, supra; Bevilacqua et al., 1989, ibid.).

L-selectin, expressed by most leukocytes, binds to the cell surface proteins of part of the endothelial cells and other leukocytes (Gallatin et al., Nature 304: 30-34 (1983); Berg et al., Immunol. Rev. 108: 5-18 (1989); Berg et al., J. Cell. Biol. 114: 343-349 (1991), Hallman et al., Biochem. Biophys. Res. Comm. 174: 236-243 (1991); Smith et al. , J. Clin. Invest. 87: 609-618 (1991); Spetrini et al., J. Immunol. 147: 2565-2573 (1991)).

All three selectins have been shown to bind to cell surface protein, PSGL-1, whose expression is limited mainly to leukocytes, especially T cells and NK cells. In order to bind to P-selectin, E-selectin and L-selectin, post-translational modifications of PSGL-1 are necessary (McEver et al., J. Clin. Invest, 1997, ibid.).

SUMMARY OF THE INVENTION

The invention is based on the discovery that T cells can be depleted and / or may be forced to undergo apoptosis if T-cell surface antigen-glycoprotein ligand-1 P-selectin (PSGL-1) is involved. T cell depletion can be, in particular, used in the treatment of conditions associated with an increased or undesired T cell mediated immune response or increased or undesired T cell proliferation. For example, T-cell depletion can cause a decrease or elimination of undesired T-cell activity or proliferation associated with inflammatory diseases, autoimmune diseases, rejection of transplanted organs and / or tumors, the origin of which is associated with T-cells. The invention also includes methods of using PSGL-1 function modulators to prevent or reduce the level of T-cell mediated immune response, as well as methods of screening PSGL-1 modulator functions.

In one aspect, the invention provides a method for preventing or decreasing a T cell mediated immune response in a subject. This method includes the following steps: selecting an individual in respect of whom the presence or risk of achieving a condition characterized by an excessive or undesirable T-cell mediated immune response is established; administering to the individual a compound that binds to PSGL-1 on the surface of the T cell, the binding of this compound to PSGL-1 on the surface of the T cell induces a special signal transduction pathway that leads to the death of the T cell, thereby preventing T cell mediated immune response or reducing its level in an individual.

The compound used in this method may include an antibody or antigen binding fragment thereof that specifically binds PSGL-1. In one example, this compound is a monoclonal antibody that specifically binds to PSGL-1. In one embodiment, the method includes an additional step of administering an agent that binds to the monoclonal antibody and promotes cross-linking of a number of PSGL-1 antigens on the surface of the T cell.

In some embodiments, the method includes inducing cross-linking of a number of PSGL-1 antigens on the surface of the T cell, and this cross-linking induces a particular signal transduction pathway that leads to the death of the T cell.

In some embodiments, the method comprises the following steps: (i) selecting an individual in respect of whom there is a risk or presence of a condition characterized by an excessive or undesired T-cell mediated immune response; and (ii) administering to the individual a multimeric compound that binds to at least two PSGL-1 proteins on the surface of the T cell, the multimeric compound containing two polypeptide chains, each polypeptide chain comprising (a) a binding domain that binds to PSGL -1, and (b) a heterologous amino acid sequence, wherein the polypeptide chains are connected by a heterologous amino acid sequence to form a multimeric compound, and the binding of the multimeric compound to at least two PSGL-1 protein E on the surface of T-cells induces a particular signal transduction pathway that results in the death of T-cells, thereby preventing T-cell mediated immune response or reducing its level in the individual.

The multimeric compound may be a homomultimeric compound or a heteromultimeric compound. The binding domain may optionally contain an extracellular domain of P-selectin or its PSGL-1 binding fragment, an extracellular domain of E-selectin or its PSGL-1 binding fragment, an extracellular domain of L-selectin or its PSGL-1 binding fragment specific for PSGL -1 antibody or its PSGL-1 binding fragment, PSGL-1 binding polypeptide selected from a phage display library, or a combination thereof.

In some embodiments, the multimeric compound does not include an antibody specific for PSGL-1, or a fragment of an antibody that binds to PSGL-1.

The heterologous amino acid sequence may optionally comprise a region that binds a cell surface receptor, for example, an immunoglobulin heavy chain constant region. In some embodiments, the polypeptide chains are covalently linked, for example, by disulfide bonds 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 individual an agent that binds to the multimeric compound using a heterologous amino acid sequence and promotes the crosslinking of a number of PSGL-1 antigens on the surface of the T cell.

In some embodiments, the method described above includes the step of selecting (selecting) an individual for whom the presence of an autoimmune disease has been established. In another exemplary embodiment, the method includes the step of selecting (selecting) an individual to whom an allogeneic or xenogenic transplant is transplanted or will be transplanted. In another case, the method includes the step of selecting an individual who is diagnosed with an allergic disease. In another case, the method includes the step of selecting a patient who is diagnosed with T-cell cancer.

In some embodiments, the T cell is an activated T cell. In one case, the T cell is a CB4 + T cell. In another case, the T cell is a CD8 + T cell.

In some embodiments, the method includes the step of determining the number of T cells in the first biological sample taken from the individual before administration of the compound (e.g., multimeric compound), and comparing the results with the number of T cells in the second biological sample taken from the individual after administration of the compound (e.g. multimeric connection).

In some embodiments, the method includes the step of determining the biological activity of T cells in a first biological sample taken from an individual before administration of a compound (e.g., a multimeric compound), and comparing the results with the biological activity of T cells in a second biological sample taken from an individual after administration of compound (e.g. multimeric connection).

In some embodiments, such administration results in depletion of at least 10% of the activated T cells in the individual. In some embodiments, this administration results in depletion of at least 10%, 20%, 30%, 40%, 50%, or more activated T cells in an individual.

In some embodiments, the antibody or antigen binding fragment or multimeric compound causes the death of at least 10% of activated T cells in an individual after exposure to said antibody or antigen binding fragment or multimeric compound. In some embodiments, the administration of said agents causes the death of at least 10%, 20%, 30%, 40%, 50% or more activated T cells in an individual. The number of dead cells can be measured at any time, for example, one, two, three, four, five, six, seven or more days after exposure to an antibody or antigen-binding fragment or multimeric compound.

In another aspect, the invention includes a method of inducing the death of T cells or a natural killer cell (NK cell). The method includes the steps of: providing a T cell or NK cell expressing PSGL-1 on its surface; contacting the T cell or NK cell with a compound that binds to PSGL-1 on the surface of the T cell or NK cell, wherein binding of the compound to PSGL-1 on the surface of the T cell or NK cell induces a particular signal transduction pathway that kills T cells or NK cells.

The compound used in such a method may include an antibody or an antigen binding fragment thereof that specifically binds to PSGL-1. In one case, this compound may be a monoclonal antibody that specifically binds to PSGL-1. In one embodiment, the method includes the step of contacting the monoclonal antibody with an agent that binds to the monoclonal antibody and promotes the crosslinking of a number of PSGL-1 antigens on the surface of a T cell or NK cell.

In one embodiment, the method comprises the following steps: (i) providing a T cell or NK cell expressing PSGL-1 on its surface; and (ii) contacting the T cell or NK cell with a multimeric compound that binds to PSGL-1 proteins on the surface of the T cell or NK cell, wherein the multimeric compound contains two polypeptide chains, each polypeptide chain comprising (a) a binding domain which binds to PSGL-1, and (b) a heterologous amino acid sequence, wherein the polypeptide chains are connected by a heterologous amino acid sequence to form a multimeric compound, and the binding of the multimeric compound to at least two PS GL-1 proteins on the surface of a T cell or NK cell induce a specific signal transduction pathway that leads to the death of a T cell or NK cell.

The multimeric compound may be a homomultimeric compound or a heteromultimeric compound. The binding domain may optionally contain an extracellular domain of P-selectin or its PSGL-1 binding fragment, an extracellular domain of E-selectin or its PSGL-1 binding fragment, an extracellular domain of L-selectin or its PSGL-1 binding fragment specific for PSGL -1 antibody or its PSGL-1 binding fragment, a peptide selected from a phage display library, or a combination thereof.

The heterologous amino acid sequence may optionally comprise a region that binds a cell surface receptor, for example, an immunoglobulin heavy chain constant region. In some embodiments, the polypeptide chains are covalently linked, for example, by disulfide bonds 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 using a heterologous amino acid sequence and promotes the crosslinking of a number of PSGL-1 antigens on the surface of the T cell.

In some embodiments, the method includes the step of inducing cross-linking of a number of PSGL-1 antigens on the surface of a T cell or NK cell, which cross-linking induces a particular signal transduction pathway that leads to the death of the T cell or NK cell.

In some embodiments, the T cell is an activated T cell. In one case, the T cell is a CD4 + T cell. In another case, the T cell is a CD8 + T cell.

In some embodiments of the methods described herein, the method comprises evaluating the viability of a T cell or NK cell after contacting with a compound (eg, a multimeric compound).

In some embodiments of the methods described herein, the method comprises evaluating the biological activity of a T cell or NK cell after contacting a compound (eg, a multimeric compound).

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

In another aspect, the invention includes a method for screening a PSGL-1 functional modulator. The method includes the steps of: providing for the expression of PSGL-1 by cells on the cell surface; contacting the cell with the test substance and measuring the viability (survival) of the cell after contacting with the test substance, according to which it is determined whether the test substance is a modulator of PSGL-1.

In one embodiment, the method includes the step of detecting cell death caused by the test substance, in accordance with which it is established that the test substance is a modulator of the action of PSGL-1.

In one embodiment, the test substance is an antibody, an antigen binding fragment thereof that specifically binds to PSGL-1. In one case, the test substance is a monoclonal antibody that specifically binds to PSGL-1. In one embodiment, the method includes the step of contacting the monoclonal antibody with an agent that binds to the monoclonal antibody and promotes the crosslinking of a number of PSGL-1 antigens on the cell surface.

In some embodiments, the method includes the step of inducing cross-linking of a number of PSGL-1 antigens on the cell surface, and this cross-linking induces a particular signal transduction pathway that leads to cell death.

In some embodiments, the T cell is an activated T cell. In one case, the T cell is a CD4 + T cell. In another case, the T cell is a CD8 + T cell.

In one embodiment, the method includes the step of producing large quantities of the test substance and combining the test substance with a pharmaceutically acceptable carrier.

In another aspect, the invention relates to a kit containing a compound that binds to PSGL-1 on the surface of a T cell, the binding of the compound to PSGL-1 on the surface of a T cell induces a particular signal transduction pathway that will lead to T cell death, and instructions for the use of a compound for the treatment of a condition associated with an excessive or undesirable T-cell mediated immune response or excessive or undesired proliferation of T cells, for example, inflammation, autoimmunity, transplant rejection, allergic state or T-cell cancer.

In one embodiment, the kit comprises: (i) a multimeric compound that binds to at least two PSGL-1 proteins on the surface of a T cell, the multimeric compound containing two polypeptide chains, each polypeptide chain comprising: a) a binding domain that binds to PSGL-1, and b) a heterologous amino acid sequence, wherein the polypeptide chains are connected by a heterologous amino acid sequence to form a multimeric compound, and binding of the multimeric compound to at least two PSG L-1 proteins on the surface of a T-cell induces a special signal transduction pathway, which leads to the death of T-cells; and (ii) instructions for use of the compound for treating a condition associated with an excessive or undesired T-cell mediated immune response or excessive or undesired proliferation of T cells, for example, inflammation, autoimmunity, transplant rejection, an allergic condition, or T-cell cancer.

An advantage of the invention is that it induces T-cell depletion and / or triggering of apoptosis in T-cells without causing an undesirable or detrimental immune response associated with it. For example, in some embodiments, administering to the individual a PSGL-1 specific antibody or multimeric compound described herein does not undesirably increase levels of inflammatory cytokines, such as IL-2 or TNF-α.

Another advantage of this invention is that it causes the depletion of T cells through the use of agonistic compositions that cause apoptosis of T cells. Accordingly, the invention provides active immunosuppressive methods, rather than passive immunosuppression, which is caused by the use of antagonistic compositions (e.g., antagonistic anti-PSGL-1 antibodies or antagonistic soluble selectin fragments) that act by binding to immune receptors and preventing the activation of the immune system in which these receptors are involved.

Another advantage of this invention is the ability to selectively select a cell surface protein, PSGL-1, the expression of which is limited mainly by leukocytes and, in particular, T cells and NK cells. Therefore, the compounds described herein generally do not cause significant levels of apoptosis of cells of other systems, such as liver cells. Selection of T-cells and NK-cells (important type of CD3 ^- cells involved in the processes or transplantation rejection) for selective depletion, without significantly increasing the life-threatening level of cytokine response and the danger to other systems is an attractive property of an immunosuppressive agent.

Unless otherwise specified, all technical and scientific terms used here have the same specific meaning, which is generally understood by a person skilled in the art to which the invention relates. Although methods and materials similar or equivalent to those described herein may be used in the practice and testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents and other references to information sources are indicated here and are given as references in their entirety. In the event of a conflict over terminology, this description will be subject to verification. In addition, the described materials and methods are not limiting, but rather illustrative.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

Brief Description of the Drawings

Figure 1 shows the results of a lengthy experiment in which it was investigated when activated cells become sensitive to TAV4 (PSGL-1 specific monoclonal antibody) mediated apoptosis signals.

Figure 2 shows the results of cell surface biotinylation and immunoprecipitation of an antigen recognized by TAB4 antibody.

Figure 3 shows the expression of PSGL-1 antigen by CD4 + T cells, CD8 + spleen T cells, and NK cells.

Figure 4 shows the expression of PSGL-1 antigen CD4 +, CD8 +, CD4 ⁺ 8 ⁺ and CD4 ^- 8 ^- thymocytes.

Figure 5 shows the levels of IL-2 produced in a mixed lymphocyte culture using spleen cells obtained from Balb / c mice receiving TAB4 (or hamster Ig) as responders and H2-mismatched C3H spleen cells as a stimulant.

Figure 6 shows the results of a Western blot analysis, demonstrating that (A) proteins immunoprecipitated with a TAB4 antibody can be recognized by a commercially available PSGL-1 specific antibody, and (B) preliminary purification (clearance) of the T cell lysate with using anti-PSGL-1 antibodies can deplete proteins recognized by TAB4.

Fig. 7 shows the percentage of grafts that have taken root in C57BL / c mice that received skin grafts from BALB / c mice and who were injected with antibodies specific for PSGL-1 (indicated as a line with shaded diamonds), or control antibodies (line with unshaded squares).

On Fig shows the dependence of the percentage of apoptotic T cells on time obtained by processing the activated peripheral mononuclear cells of a human blood with a human antibody specific for PSGL-1.

Figure 9 shows the spread of diabetes in male (NOD) mice with autoimmune non-obese diabetes that received an anti-PSGL-1-1 antibody (a line with hatched squares) or a control antibody (a line with hatched squares).

Figure 10 shows the binding of murine P-selectin, E-selectin and L-selectin to activated mouse T cells.

11A-11C show the induction of apoptosis of activated mouse T cells with multimeric forms of E-selectin (FIG. 11A), P-selectin (FIG. 11B) and L-selectin (FIG. 11C).

12 shows in vitro induction of apoptosis of activated mouse T cells by crosslinking of soluble P-selectin-Fc-fusion protein.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to methods for modulating the activity of T cells by affecting the function of PSGL-1 molecules located on the surface of T cells. Compound PSGL-1 with the agonist compositions described herein can cause T cell depletion and / or induce T cell apoptosis. Therefore, these agonistic compositions can be used as therapeutic agents for the regulation of immune-dependent conditions, such as inflammatory diseases, autoimmune diseases, transplant rejection, allergic diseases and / or T-cell cancer. Agonist-based compositions can also be used to cause T cell depletion in any biological sample where the presence or activity of T cells is undesirable.

PSGL-1 protein

PSGL-1 is a cell surface adhesive that is expressed by neutrophils, T, B lymphocytes, NK cells, monocytes, dendritic cells and primitive human CD34 hematopoietic progenitor cells. Due to its ability to interact with selectins, PSGL-1 is involved in the passage of leukocytes in the endothelium and extravasation of leukocytes in inflamed tissues. PSGL-1-mediated binding of T cells to E- and P-selectin, or migration, is regulated differently.

For example, it is believed that the appearance of CLA (skin lymphogen antigen) of the epitope is induced by T cells that undergo a transition from the primary state to memory (to the antigen). Only Helper 1-, but not Helper 2- T cells express functional PSGL-1 that can migrate to the area of skin inflammation.

PSGL-1 is sialomucin, which needs specific sialylation, fucosylation and sulfation in order to possess the ability to bind P-selectin. PSGL-1 exists in the form of isoforms characterized by different positions of glycosylation and sulfation sites at their N-ends. Dormant peripheral blood T and B cells, lymphoid cell lines, and in vitro activated peripheral blood T cells express approximately the same level of PSGL-1. However, only activated T cells give the functional form of PSGL-1 and rapidly bind to P-selectin. Apparently, this activation-dependent ability to bind is the result of post-translational modification, which can be assumed based on the increased activity of alpha (1, 3) -fucosyltransferases in activated T cells.

Isoforms of PSGL-1 also show different affinities for L-selectin and E-selectin. For example, human T cells in which a CLA-positive isoform is detected can bind and interact with both E- and P-selectin, while T cells expressing PSGL-1 without a CLA epitope bind only P-selectin. Moreover, the binding of PSGL-1 to P-selectin is possible in the presence of a terminal decapeptide that contains three tyrosine residues for sulfation and one threonine residue for glycosylation.

PSGL-1 protein can be obtained using recombinant technology and / or isolation of native PSGL-1 protein from biological material. Recombinant PSGL-1 protein can be obtained in prokaryotic and eukaryotic cells, either in vitro or in vivo. Nucleic acids encoding PSGL-1 can be used to recombinantly produce a protein (see, for example, GenBank ™ Accession NM 003006 for an example of a nucleic acid encoding a PSGL-1 polypeptide). Antibodies to PSGL-1 are also well known and can be used to purify antigen (see, for example, Herron et al. (2000) Science Jun 2; 288 (5471): 1653-56; WO 00/25808) and / or used in the methods described herein. In addition, PSGL-1 is described, for example, in sources including Sako et al. (1993) Cell 75: 1179; Vachino et al. (1995) J. Biol. Chem. 270: 21966; Veldman et al. (1995) J. Biol. Chem. 270: 16470.

When recombinantly producing PSGL-1, functional expression of PSGL-1 may require simultaneous expression of both PSGL-1 and its alpha (1, 3) -fucosyltransferase, Fuc-TVII. In addition to or instead, recombinant production of PSGL-1 may be accompanied by cotransfection of a nucleic acid encoding PACE to remove a propeptide and / or nucleic acid encoding tyrosine sulfotransferase.

The anti-PSGL-1 antibody can be used to isolate and purify the PSGL-1 antigen from biological material. Any type of cell expressing a PSGL-1 protein, for example T cells obtained from any individual, or a T cell line, can be used as a source of this protein. When purified, this protein can be used in the various methods described herein. For example, purified PSGL-1 protein can be used in the in vitro screening of PSGL-1 modulators of T-cell function or as an immunogen in the production of antibodies specific for this protein.

PSGL-1 specific antibodies

PSGL-1 polypeptides (or immunogenic fragments or analogs thereof) can be used to produce antibodies suitable for use in the methods of the present invention. As described above, PSGL-1 polypeptides or their peptide fragments can be obtained by recombinant techniques or synthesized by solid phase synthesis methods. PSGL-1 polypeptides or a peptide fragment thereof can be used as an immunogen to produce antibodies specific for PSGL-1. In addition, antibodies to PSGL-1, for example TAB4 monoclonal antibody, can be used to purify a PSGL-1 polypeptide, for example a PSGL-1 polypeptide in its natural conformation, which can then be used as an immunogen to produce subsequent anti-PSGL-1 antibodies.

According to the invention, the antibody may be a monoclonal, polyclonal or engineered antibody, all of which specifically bind to the PSGL-1 polypeptide. An antibody that “specifically binds” to a specific antigen, such as a PSGL-1 polypeptide, should not substantially recognize or bind other substances in the sample. So the invention also includes methods for identifying a test substance (e.g., an antibody) that binds a polypeptide of the invention by contacting a polypeptide with a test substance and determining if the polypeptide binds to a test substance (e.g., by direct binding determination, by determining a competitor, which disrupts the binding of the test substance to the polypeptide, and / or the determination of binding by the study of apoptosis-inducing activity).

In general, PSGL-1 polypeptides can be coupled to a carrier protein, such as KLH, mixed with an adjuvant, and administered to a recipient mammal. Antibodies derived from this animal can then be isolated by peptide antigen affinity chromatography.

In particular, a wide variety of animal recipients can be immunized with the introduction of the PSGL-1 polypeptide or its antigenic fragment. Typically, rabbits, mice, guinea pigs, and rats are used as such. Various adjuvants that can be used to enhance the immune response depend on the type of host animal and include Freund's adjuvant (complete and incomplete), mineral gels, such as aluminum hydroxide, surfactants, such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, hemocyanin isolated from reynell limpet, and dinitrophenol. Adjuvants including BCG (Calmette-Guerin bacilli) and Corynebacterium parvum may be potentially useful for humans. Polyclonal antibodies are heterogeneous populations of antibody molecules that are found in the serum of immunized animals.

Thus, in the scope of the invention, antibodies include polyclonal antibodies and, in addition to them, monoclonal antibodies, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F (ab ') 2 fragments and substances obtained from the Fab expression library.

Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, can be obtained using the PSGL-1 polypeptides described herein and standard hybridoma technology (see, for example, Kohler et al., Nature 256: 495 [1975]; Kohler et al., Eur J Immunol 6: 511 [1976]; Kohler et al., Eur J Immunol 6.292 [1976]; Hammerling et al., Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY [1981]).

In particular, monoclonal antibodies can be obtained by any method that enables the production of antibodies using cell lines in a continuous culture, such as those described by Kohler et al., Nature 256: 495 (1975) and US Pat. No. 4,376,110; hybridoma technology using human B cells (Kosbor et al., Immunology Today 4:72 [1983]; Cole et al., Proc Natl Acad Sci USA 80: 2026 [1983]) and EBV hybridoma technology (Cole et al. , Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 [1983]). Such antibodies can belong to any class of immunoglobulins, including IgG, IgM, IgE, IgA, IgD, and any subclass thereof. A hybridoma producing the mAb of the invention may be cultured in vitro or in vivo. The ability to produce high titers of mAb in vivo makes this method a particularly acceptable method of preparation.

The resulting polyclonal or monoclonal antibodies are tested for the specificity of recognition of PSGL-1 by Western blot analysis or immunoprecipitation analysis using known methods, such as those described by Ausubel et al. above. Antibodies that recognize and bind to PSGL-1 are used in the present invention. Anti-PSGL-1 antibodies that bind to the PSGL-1 antigen on the surface of T cells, for example CD3 + cells, and induce depletion and / or apoptosis of T cells in an individual are particularly useful.

These antibodies can be used, for example, as part of a course of therapy (for example, to reduce or eliminate an unwanted immune response, such as a T-cell mediated immune response associated with conditions such as inflammatory diseases, autoimmune diseases, graft rejection, allergic diseases and T cell cancer). Antibodies can also be used in screening to determine the ability of a candidate substance to bind PSGL-1.

In addition, methods developed to produce “chimeric antibodies” (Morrison et al., Proc Natl Acad Sci USA 81: 6851 [1984]; Neuberger et al., Nature 312: 604 [1984]; Takeda et al., Nature 314 : 452 [1984]) by splicing the genes of a mouse antibody with the corresponding antigenic specificity together with the genes of a human antibody with the corresponding biological activity, can be used. A chimeric antibody is a molecule (substance) in which different parts are derived from different species of animals, for example, one that has a variable region from a murine monoclonal antibody and a constant region of a human immunoglobulin.

Alternatively, the methods described for the production of single chain antibodies (US Patent Nos. 4,946,778, 4,946,778 and 4,704,669) can be adapted to produce single chain antibodies to the PSGL-1 polypeptide or fragment thereof. Single chain antibodies are prepared by linking fragments of the heavy and light chains of the Fu region using an amino acid bridge, resulting in a single chain polypeptide.

Antibody fragments that recognize and bind to specific epitopes can be created using known methods. For example, such fragments include (but are not limited to) F (ab ') 2 fragments that can be obtained by pepsin digestion of an antibody molecule, and Fab fragments can be obtained by reducing the disulfide bridges of F (ab') 2 fragments. Alternatively, Fab expression libraries can be constructed (Huse et al., Science 246.1275 [1989]) to quickly and easily identify monoclonal Fab fragments with the desired specificity.

Antibodies can be humanized by methods known in the art. For example, monoclonal antibodies with desired binding specificity can be humanized and commercially available (Scotgene, Scotland; Oxford Molecular, Palo Alto, Calif). Complete human antibodies, such as those expressed by transgenic animals, are also used in this invention (Green et al., Nature Genetics 7:13 [1994]; US patents No. 5545806, 5569825).

Multimeric compounds

Multimeric compounds that bind to a number of PSGL-1 proteins on the surface of a T cell or NK cell can be 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 PSGL-1, and (ii) a heterologous amino acid sequence.

In general, the multimeric compound binds to at least two different PSGL-1 proteins on the surface of the cell. However, the multimeric compound may include 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more different PSGL-1 binding domains, which causes the binding of the multimeric compound 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more different PSGL-1 proteins on the surface of a given cell.

The binding domain may contain any amino acid sequence (or any amino acid sequence with a modification, such as, for example, glycosylation and / or sulfation) that binds to PSGL-1. The binding domain may correspond to the amino acid sequence of both natural origin and non-natural origin. For example, the binding domain may comprise a PSGL-1 binding domain of selectin (e.g., P-selectin, E-selectin or L-selectin). A polypeptide containing a PSGL-1 selectin binding domain may optionally include: (i) an extracellular selectin domain (eg, P-selectin, E-selectin or L-selectin); (ii) a calcium-dependent lectin domain of selectin (e.g., P-selectin, E-selectin or L-selectin); or (iii) a fragment of the extracellular domain of selectin (e.g., P-selectin, E-selectin or L-selectin) that is involved in the binding of PSGL-1. In addition to these naturally occurring amino acid sequences, one or more amino acid substitutions can be included in the PSGL-1 binding domain of natural origin, resulting in a sequence of a non-natural nature, but maintaining the PSGL-1 binding function. For example, a polypeptide may contain an amino acid sequence that binds to PSGL-1 and is at least 80%, 85%, 90%, 95%, or 98% identical to any of the following sequences: (i) an extracellular domain of selectin (e.g. P-selectin, E-selectin or L-selectin); (ii) a calcium-dependent lectin domain of selectin (e.g., P-selectin, E-selectin or L-selectin); or (iii) a fragment of the extracellular domain of selectin (e.g., P-selectin, E-selectin or L-selectin) that is involved in the binding of PSGL-1. Standard molecular biological mutagenesis methods can be used to make changes to the nucleic acid sequence encoding the PSGL-1 binding domain. Modified binding domains can then be tested for the ability to bind PSGL-1, for example, immobilized PSGL-1 or PSGL-1 on the cell surface. The binding domain may also contain a PSGL-1 binding domain of an antibody specific for PSGL-1, or a polypeptide selected from a phage display library, or an amino acid sequence that binds to PSGL-1 and at least 80%, 85%, 90%, 95% or 98% is identical to the PSGL-1 binding domain of an anti-PSGL-1 antibody or polypeptide selected from a phage display library.

The PSGL-1 binding domain may contain an amino acid sequence that corresponds to the PSGL-1 binding fragment of P-selectin. An example of a polypeptide chain (multimeric compound described herein) containing such an amino acid sequence is a recombinant murine P-selectin and Fc chimeric compound (marketed by R&D Systems, Minneapolis, MN) containing the following components: (i) CD33 signal peptide ( Met 1-Ala 16); (ii) murine P-selectin (Trp 42-Ala 709 extracellular domain); (ii) IEGRMD (SEQ ID NO: l); and (iv) human IgG1 (Pro 100-Lys 330). A second example of a polypeptide chain containing such an amino acid sequence is a recombinant chimeric compound of human P-selectin and Fc (supplied by R&D Systems, Minneapolis, NM) containing the following components: (i) human P-selectin (Met 1 -Ala 771, extracellular domain); (ii) IEGRMD (SEQ ID NO: 1) and (iii) human IgG1 (Pro 100-Lys 330).

The PSGL-1 binding domain may contain an amino acid sequence that corresponds to the PSGL-1 binding fragment of E-selectin. An example of a polypeptide chain (multimeric compound described herein) containing such an amino acid sequence is a recombinant chimeric compound of mouse E-selectin and Fc (supplied by R&D Systems, Minneapolis, MN) containing the following components: (i) mouse E-selectin (Met 1 -Pro 557, extracellular domain); (ii) IEGRMD (SEQ ID NO: 1); (iii) human IgG1 (Pro 100 - Lys 330); and (iv) HHHHHH (SEQ ID NO: 2). A second example of a polypeptide chain containing such an amino acid sequence is a recombinant chimeric compound of human E-selectin and Fc (supplied by R&D Systems, Minneapolis, MN) containing the following components: (i) human E-selectin (Met 1 - Pro 556, extracellular domain); (ii) IEGRMD (SEQ ID NO: 2); Human IgG1 (Pro 100 - Lys 330) and (iv) HHHHHH (SEQ ID NO: 2).

The PSGL-1 binding domain may contain an amino acid sequence that corresponds to the PSGL-1 binding fragment of L-selectin. An example of a polypeptide chain (multimeric compound described herein) containing such an amino acid sequence is a recombinant chimeric compound of murine L-selectin and Fc (supplied by R&D Systems, Minneapolis, MN) containing the following components: (i) mouse L-selectin (Met 1 - Pro 557, extracellular domain); (ii) IEGRMD (SEQ ID NO: 1); (iii) human IgG1 (Pro 100 - Lys 330); and (iv) HHHHHH (SEQ ID NO: 2). A second example of a polypeptide chain containing such an amino acid sequence is a recombinant chimeric compound of human L-selectin and Fc (supplied by R&D Systems, Minneapolis, MN) containing the following components: (i) human L-selectin (Met 1 - Pro 556, extracellular domain); (ii) IEGRMD (SEQ ID NO: 2); Human IgG1 (Pro 100 - Lys 330) and (iv) HHHHHH (SEQ ID NO: 2).

The multimeric compound may be a homomultimeric compound or a heteromultimeric compound. A homomultimeric compound contains only polypeptide chains that have identical PSGL-1 binding domains. For example, a homomultimeric compound may contain polypeptide chains containing identical PSGL-1 binding fragments of P-selectin. The heteromultimeric compound contains polypeptide chains that have different PSGL-1 binding domains. For example, a heteromultimeric compound may comprise a first polypeptide chain that contains a PSGL-1 binding fragment of P-selectin and a second polypeptide chain that contains a PSGL-1 binding fragment of E-selectin.

A heterologous amino acid sequence may be an amino acid sequence. However, the amino acid sequence of the polypeptide chains described herein does not match the sequence of the natural protein. A heterologous amino acid sequence contains one or more amino acids that enable polypeptide chains to bind. For example, one or more amino acids can covalently bind, for example, polypeptide chains using disulfide bonds. One example of a heterologous sequence is an immunoglobulin heavy chain constant region. Disulfide binding between the Fc regions of two polypeptide chains can lead to the formation of a dimeric compound.

In addition to participating in the bonding of the polypeptide chains, the heterologous amino acid sequence may also contain a region that binds to a “cross-linker”, for example, a region that binds to a cell surface receptor. When the agent binds to such a binding region, cross-linking (crosslinking) of the polypeptide chains and PSGL-1 of the cell surface proteins with which they interact can result. The immunoglobulin heavy chain constant region contains an Fc receptor binding region. The crosslinker may, for example, be an antibody that specifically binds to a region of a heterologous amino acid sequence that binds to the crosslinker.

Tests for screening compounds that are able to modulate the action of PSGL-1

The invention also includes methods for detecting compounds that interact with PSGL-1 (or with the PSGL-1 domain), including but not limited to compounds that contribute to T cell depletion and / or T cell apoptosis upon binding to PSGL- one. This also includes substances that modulate the interaction of PSGL-1 with transmembrane, extracellular or intracellular proteins that affect PSGL-1 activity, or substances that modulate activity.

Substances that can be screened in accordance with the invention include, but are not limited to: peptides, antibodies and fragments thereof, and other organic compounds that bind to PSGL-1 and modulate the biological effect produced by PSGL-1, as described above.

Such compounds may include (but not limited to): peptides, for example, soluble peptides, including (but not limited to) representatives from arbitrary peptide libraries (Lam et al., Nature 354: 82 [1991]; Houghten et al., Nature 354: 84 [1991]) combinatorial chemically obtained molecular libraries consisting of amino acids of the D and / or L configuration, phosphopeptides (including, but not limited to, libraries of arbitrary or partially degenerate complete phosphopeptides; Songyang et al., Cell 72: 767 [1993]), antibodies (including, but not limited to), polyclonal, monoclonal specific, humanized, anti-idiotypic, chimeric or single-chain antibodies, and Fab, F (ab ') 2 and FAb fragments from libraries and their epitope-binding fragments), as well as small organic and inorganic substances.

Other substances that can be screened in accordance with the invention include (but not limited to) small organic substances that affect the activity of the PSGL-1 protein as described above.

Computer modeling and search technologies allow you to identify substances or improve already identified substances that can simulate PSGL-1 expression or activity. With such a substance or composition identified, it is possible to identify its active centers or regions. Such active centers, as a rule, are a binding site of a natural modulator of activity. Such an active center can be detected using methods known in the art based on the amino acid sequences of peptides, nucleotide sequences of nucleic acids, or by studying the complexes of the corresponding substance or composition with its natural ligand. In the latter case, chemical or X-ray crystallographic methods can be used to find the active center by establishing under what condition a modulator (ligand) is detected.

Despite what has been described above with regard to the development and preparation of substances that can alter binding, a library of already known substances, including natural and synthetic chemicals and materials, including proteins, can be checked to find compounds that bind to the PSGL-1 protein and cause depletion T cells and / or induce apoptosis of T cells.

In vitro systems can be developed to search for substances capable of interacting with PSGL-1 (or the PSGL-1 domain). The detected substances can be used, for example, to modulate the activity of T cells, as described above, and as a result can be used to treat conditions associated with an excessive or undesirable T-cell mediated immune response or excessive or undesired proliferation of T cells, for example, inflammation, autoimmunity, transplant rejection, allergies, or T-cell cancer.

The main idea of studies conducted to identify substances that bind to PSGL-1 involves the preparation of a reaction mixture of PSGL-1 (or domain PSGL-1) and the test substance under conditions and for a time sufficient for the interaction and binding of these two components to form a complex that can be separated and / or determined in the reaction mixture. The types of PSGL-1 used may vary depending on the purpose of the screening. In some cases, it is preferable to use a peptide corresponding to the PSGL-1 domain, fused to a heterologous protein or polypeptide, which gives advantages in the study (for example, by introducing a label, isolating the resulting complex, etc.).

Such identification studies can be carried out in different ways. For example, one way of carrying out such a study involves fixing the PSGL-1 protein, polypeptide, peptide or fusion protein or test substance to a solid phase and determining complexes based on PSGL-1 / test substance to this solid phase after completion of the reaction. In one embodiment of this method, the PSGL-1 reagent can be fixed on a solid surface, and the test substance, which is not fixed, can be labeled, indirectly or directly.

In practice, microtiter plates are used as the solid phase. The fixed component can be immobilized by covalent or non-covalent binding. Non-covalent binding can be achieved by applying a protein solution to a solid surface and drying. Alternatively, an immobilized antibody, preferably a monoclonal antibody specific for the protein to be immobilized, can be used to fix the protein to a solid surface. Such solid phase surfaces can be prepared in advance and stored.

For this, in order to conduct this study, an immobilized component is added to the coated surface containing the fixed component. After the reaction is completed, unreacted components are removed (for example, by washing) under conditions when the complexes formed remain on the surface of the solid phase in an immobilized state. The determination of complexes fixed on a solid surface can be carried out in different ways. If the non-immobilized pre-component is pre-labeled, the determination of a label immobilized on the surface indicates that the complexes were obtained. If the non-immobilized pre-component is not pre-labeled, then direct labeling can be used to determine the complexes attached to the surface, for example, using a labeled antibody specific for the pre-non-immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with labeled anti-IgG antibodies).

Alternatively, the reaction can be carried out in the liquid phase, reaction products are separated from unreacted components and complexes are determined; for example, using an immobilized antibody specific for a PSGL-1 protein, polypeptide, peptide or fusion protein or test substance to fix (on the surface) any complexes formed in solution, and a labeled antibody specific for another component of a possible complex to determine the attached complexes.

Alternative cell-based studies can be used to identify compounds that interact with PSGL-1. For this purpose, cell lines that express PSGL-1, or cell lines that are created by genetic methods for the expression of PSGL-1, can be used. Studies based on the use of cells are used, in particular, in assessing the functional action of a substance identified in the search described above. For example, a substance is tested for its ability to bind to PSGL-1 protein, then the ability of this substance is tested, for example, to induce T-cell apoptosis in vitro or in vivo or to deplete T cells in vitro or in vivo.

Pharmaceutical Compositions

Provided that the object of the invention is to alter the immune response of an individual, a pharmaceutical composition comprising, for example, antibodies, multimeric compounds, small molecules or other substances that specifically bind to PSGL-1 polypeptides also characterizes the invention. In a preferred example, such a substance manifests itself as a PSGL-1 agonist.

Pharmaceutical compositions for use in the present invention can be formulated in the usual way using one or more physiologically acceptable carriers or excipients. Thus, based on the compounds and their physiologically acceptable salts and solvates, compositions for administration by various methods can be created.

The compositions may be formulated for parenteral administration by injection, for example, bolus injection or continuous infusion. Compositions for injection can be in the form of single doses, for example in ampoules or in multi-dose containers, with the addition of preservatives. The compositions may be in the form of suspensions, solutions or emulsions in oil or aqueous media and may contain formulating agents, such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, for example sterile pyrogen-free water, before use.

Methods for regulating T cell mediated immune response and depletion of T cell populations

Substances, for example, disclosed in the screening experiments described herein, can be used, for example, to modulate the biological function mediated by the PSGL-1 polypeptide, and / or to treat diseases associated with an excessive or undesirable immune response, for example, mediated by T cells immune response. These substances include (but are not limited to) peptides, antibodies, and fragments thereof, as well as other organic compounds that bind to PSGL-1 on the surface of T cells and induce a specific transduction pathway, which leads to the death of T cells. The methods of the invention optionally include the addition of a cross-linking agent that induces cross-linking of PSGL-1 on the cell surface. The substances described herein can be used in any case where depletion or elimination of T-cell activity is desired. Specific conditions that can be treated with the compounds of this invention include inflammatory diseases, autoimmune diseases, transplant rejection, allergic diseases, and T-cell cancer.

Examples of conditions that can be treated with the PSGL-1 specific substances described herein include, but are not limited to: diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriasis arthritis), multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosus, autoimmune thyroiditis, dermatitis (including atonic dermatitis, eczematous dermatitis), psoriasis, Sjogren's syndrome, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, type I diabetes, inflammatory intestinal obstruction levania, ulcerative colitis, asthma, allergic asthma, lupus erythematosus, scleroderma, vaginitis, proctitis, drug dermatitis, leprosy reverse reactions, leprosy erythema nodosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic leukemia, and biluberulopathic encephalopathy anemia, RBC anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Jones syndrome on, idiopathic sprue, lichen planus, Graves disease, sarcoidosis, primary biliary cirrhosis, uveitis of the posterior eyeball choroid, interstitial pulmonary fibrosis, graft-versus-host condition, cases of transplantation (including tissue transplantation of allogeneic or xenogenic type ), including bone marrow transplantation, liver transplantation or transplantation of any organ or tissue, allergies such as atonic allergies, AIDS and T-cell tumors such as leukemia and / or imfomy.

The methods of this invention can be used to deplete T cells in cell populations either in vitro or in vivo. For example, in a biological sample taken from an individual, T cells can be depleted in vitro by contacting the sample with the anti-PSGL-1 substance described herein, optionally together with a cross-linking agent. This method is suitable, for example, if a decrease in the number of T cells in the cell population is allowed, as well as a decrease or elimination of the activity of T cells in the cell population.

The following are examples of the invention. They are not intended to limit the scope of the invention in one way or another.

Examples

Example 1: Preparation of Protein Specific Monoclonal Antibody (“TAIP”) Inducing T Cell Apoptosis

A TAIP-specific monoclonal antibody was prepared using known cell fusion methods developed by Kohler and Mistein ((1976) European Journal of Immunology 6: 511-519) to produce the desired antibodies secreted by hybridomas.

Antibody-producing hamster cells that have previously been injected with Balb / c spleen T cells activated by concanavalin A (Con A) are fused to the myeloma cell line to obtain an antibody-secreting hybridoma. Two cell populations were fused with polyethylene glycol, and the resulting antibody producing cells were cloned and propagated by conventional culture methods. One hybridoma obtained in this way secreted a monoclonal antibody designated TAB4, which was able to induce apoptosis of T cells in vitro and deplete T cells in vivo. The protein recognized by TAB4 has been designated as a protein (protein) inducing T cell apoptosis (TAIP).

C57 BL / 6J (B6) and BALB / c mice were purchased from Jackson Laboratory (Bar Harbor, ME). Syrian hamsters were acquired at Animal Core Facilitu, National Taiwan University Medical College.

The concentrated culture supernatant from TAB4 hybridoma was centrifuged at 20,000 xg for 10 minutes, then the supernatant was diluted 1: 1 with binding buffer (0.1 M sodium acetate, pH 5.0). The G-protein column (approximately 1 ml by volume) was washed three times with 3-5 ml of binding buffer. The purified culture supernatant was applied to a G-protein column, flow fractions were collected and reloaded into 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, pH 2.8). Each fraction contained 1 ml of eluted antibody and the pH was adjusted to a neutral value in the eluted fraction by mixing each 1 ml of the fraction with 50 microliters of 1M Tris-HCl, pH 7.5. Antibody-containing fractions were collected, pooled and dialyzed against 2 L PBS, pH 7.4, three times for 3 hours for each dialysis operation. Protein concentration in antibody samples was determined according to the method described by Bradford using the Bio-Rad Protein Assay (BIO-RAD, Hercules, CA).

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

The mouse spleen was immersed in 8 ml of Hanks balanced salt solution (HBSS), carefully milled with a sterile coverslip and transferred to a 15 ml centrifuge tube (Costar), centrifuged at 200 xg for 5 minutes. The supernatant was separated and the cell pellet was resuspended in the remaining buffer, gently shaking. Contaminating red blood cells (RBS) were lysed by adding 1 ml of RBC lysis buffer (0.6 M NH ₄ Cl, 0.17 M Tris base, pH 7.65), followed by 2 min incubation at room temperature and rapid addition of 9 mm HBSS. Cells were besieged at 200 xg for 5 minutes, washed twice and resuspended in RPMI medium. The concentration and viability of the cells in the mixture was determined using a hemocytometer (Cambridge Scientific Inc) with the exception of Trypan blue.

The spleen cell content was adjusted to a final concentration of 3 × 10 ⁶ / ml by adding RPMI medium, Concanavalin A was added to a final concentration of 2 microorganisms to activate T cells. The cell suspension was transferred to a 6-well culture plate (5 ml / well) or to a 10-cm culture dish (10 ml / cup) and incubated at 37 ° C. in an atmosphere of 5% CO ₂ for 48 hours before harvesting. Activated spleen cells, including activated T cells, were resuspended in 5 ml of HBSS and carefully layered on the surface of 5 ml of the top layer of a 55% Percall solution in a centrifuge tube. Care must be taken not to move the layers. The cells are centrifuged at 1900 xg for 13 minutes at 25 ° C without stirring (layers). Concentrated T cells were collected from the surface of two layers; after three washes with HBSS, they were ready for use in experiments.

Example 3: Apoptosis of Activated T Cells

Activated T cells (see Example 2) were resuspended to a final concentration of 5 × 105 cells / ml in RPMI medium containing 5 ng / ml IL-2 and treated with control Ig, TAB4 or anti-CD3 in accordance with the conditions indicated in table 1.

Table 1 Groups for the experiment Treatment* Negative control 3 mcg / ml hamster Ig 5 ng / ml IL-2 3 μg / ml antibodies "cross-linker" - (specific for hamster Ig) TAB4 3 μg / ml TAB4 mAb hamster 5 ng / ml IL-2 3 μg / ml "cross-linker" - antibodies (specific for Ig hamster) Positive control 1 μg / ml anti-CDS mAb 5ng / ml IL-2 1 μg / ml cross-linker antibody (mouse Ig specific) *: Final concentration of the indicated reagents in the medium

After an incubation of 18-24 hours, excess apoptosis was determined in each culture using a 7-AAD apoptosis study. The treated cells were transferred to FACS tubes (Falcon), washed twice with ice-cold FACS solution (1% calf serum, 0.05% sodium azide in PBS), centrifuged at 200 xg at 4 ° C. Cells were resuspended in ice-cold FACS solution to a final concentration of 1-2 × 10 ⁷ cells / ml. For staining, 0.1 ml of resuspended cells were mixed with 7-AAD to a final concentration of 2 μg / ml and then incubated at 4 ° C in the dark for 20 minutes. Finally, stained cells were washed twice with ice-cold FACS solution, resuspended in 0.5 ml FACS solution, and analyzed using a BD LSR flow cytometer (Beckton Dickinson).

Figure 1 shows the results of a representative experiment lasting for a certain time, in which it was revealed when activated T cells acquire sensitivity to TAB4 (anti-TAIP) mediated apoptosis signals. Mouse splenocytes were activated by Con-A and maintained in IL-2-containing medium. Activated T cells were harvested, resuspended, and exposed to a TAB4 monoclonal antibody or a control hamster IgG in the presence of a “cross-linker”, a hamster IgG specific antibody. The possibility of cross-linking using TAIP influenced the induction of a rather low level (6.5%) of apoptotic cell death, which was evident on the first day. However, the degree of TAB4-induced apoptosis increased to 17% on day 2, peaked at 52% on day 4, and decreased to 44% on day 6. The control hamster IgG did not cause specific T cell death as a result of apoptosis compared with cultures that received only IL-2. Anti-CD3 (as a positive control) caused apopotosis in 38% of T cells 48 hours after activation (data not shown).

Example 4: Expression of TAIP Antigen in Various Tissues

Cells were washed twice with ice-cold FACS solution (1% fetal calf serum, 0.05% sodium azide in PBS) and centrifuged at 200xg at 4 ° C in a FACS tube (Falcon). Cells were resuspended in ice-cold FACS solution to a final concentration of 1 × 10 ⁷ cells / ml and an aliquot of 0.1 ml of all cells resuspended in the FACS tube (Falcon) was used for each assay. For surface staining of TAB4, monoclonal antibodies or a control hamster IgG at a final concentration of 2 μg / ml was added to the cells and the mixture was incubated at 4 ° C for 30 minutes in the dark. The cells were washed with ice-cold FACS solution and then stained: (1) for spleen cells, a zichrom-conjugated anti-CD3 antibody (2 μg / ml), FITC-conjugated anti-hamster hamster and PE-conjugated anti-CD8 / CD4 / CD19 / CD11 b / pan-NK / IA / I-E / Mac-3 antibody (2 μg / ml) in 100 μl of ice-cold FACS solution; and (2) for thymocytes, a FITC-conjugated anti-Ig hamster, a PE-conjugated anti-CD 8 and a chichrom-conjugated anti-CD4 antibody (2 μg / ml) in 100 μl of ice-cold FACS solution. The reaction was carried out at 4 ° C for 30 minutes in the dark. Finally, stained cells were washed twice with FACS solution, resuspended in 1 ml of FACS solution and analyzed on a BD LSR flow cytometer (Beckton Dickison).

Figures 3 and 4 show the results of FACS analysis of the distribution of TAIP antigen in various subpopulations of splenocytes and thymocytes. As shown in FIG. 3, CD19 ⁺ B cells expressed low but detectable amounts of TAIP proteins on their surface. Significantly higher amounts of TAIP proteins were detected for CD3 ⁺ T cells and NK cell fractions. Most CD4 ⁺ , CD8 ^+, and CD4 ⁺ 8 ⁺ thymic T cells expressed significant amounts of TAIP proteins. Thus TAIP proteins were expressed only a small population of CD4 ^- 8 ^- thymus T cells (Figure 4).

Tissues from B6 and BALB / c mice, including the brain, thymus, heart, lung, liver, stomach, kidney, spleen and skin, were collected, fixed with 10% formaldehyde overnight at room temperature and immersed in paraffin blocks. Tissue sections 4 μm thick were prepared from paraffin blocks using a Leica RM2135 microtome, straightened at 45 ° C in water and placed on a coverslip (slide). These slides were dried at 37 ° C and stored ready for subsequent experiments.

Slides containing tissue sections in paraffin were cleaned of paraffin and dried by passing through xylenes and 100% ethanol according to the standard procedure and finally kept in 100% ethanol. Sections were dehydrated by incubating them repeatedly in 100% ethanol - 90% ethanol - 85% ethanol - 70% ethanol - PBS according to the standard procedure until the final step in PBS. The following reactions were performed in a humidification box. Nonspecific binding was blocked by incubation of tissue sections in blocking buffer (1% standard goat serum) for 1 hour at room temperature (or 4 ° C. overnight). The blocking buffer was removed, TAB4 or standard hamster Ig was added to the sections (1: 200 dilution) and incubated for another hour at room temperature (or at 4 ° C overnight). Sections were washed twice with PBS, each time for 5 minutes to remove the primary antibody, reacted with 250 times diluted conjugate of alkaline phosphatase and goat anti-hamster immunoglobulin antibodies, then incubated at room temperature for 1 hour. The sections were then again washed twice with PBS, 5 minutes each time, to remove the conjugate of the antibody and enzyme, after which the reaction mixture was stained using a BCIP / NBT substrate solution at room temperature and in the dark for 30 minutes. Sections were washed again with PBS to remove excess enzyme substrate, dehydrated using PBS ethanol xylene, and prepared for microscopy.

The results showed that the expression of TAIP proteins was detected only in bone marrow tissues, and was not detected in the rest.

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

5 × 10 ⁷ RL ♂ 1 or NIH-3T3 cells were surface biotinylated in 1 ml PBS containing 0.5 mg / ml Sulfo-NHS-biotin (Pierce) for 30 minutes on ice. The reaction was stopped by incubating the cells with 0.5 ml of Dulbecca's medium modified with Eagle (Life Technologies, Inc.) for 10 minutes on ice. Cells were washed once with 1 ml of Dulbecco's medium, modified with a Needle, and 2 times with 1 ml of phosphate-buffered saline.

Labeled cells were lysed at a density of 5.0 × 10 ⁷ cells / ml in cold lysis buffer (1% Triton X-100, 20 mM Tris-HCl, pH 8.0, 160 mM NaCl, 1 mM CaCl ₂ ) containing a complete mixture of protease inhibitors (cocktail) (Roche), for 15 minutes, insoluble material precipitated at 10,000 xg for 10 minutes; these and subsequent steps were carried out at 4 ° C. For immunoprecipitation, the lysate was incubated for 30 min with 50 µl of G-protein coupled Sepharose (Amersham Pharmacia Biotech) to remove non-specific binding proteins. Grains (sepharoses) were separated, aliquots of the supernatant (usually corresponding to 5.0 × 10 ⁷ cells) were incubated with 20 μl protein G-Sepharose, pre-loaded with 10 μg TAB4 mAb or standard hamster serum IgG. After incubation at 4 ° C for 4 hours, the resin was washed 4 times with wash buffer (0.05% Triton X-100, 50 mm Tris-HCl, pH 8.5, 400 mm NaCl, 1 mm CaCl ₂ , 1 mg / ml ovalbumin), twice - with a similar composition wash buffer containing 250 mM instead of 400 mM NaCl. Proteins specifically bound to TAB4 eluted with 50 μl of 1 × SDS sample buffer. Eluted proteins were isolated with 8% SDS-PAGE and transferred onto a nitrocellulose membrane (Millipore). The filters were then analyzed for biotinylated proteins using a peroxidase-avidin conjugate (PharMingen) and treated with Chemilumenescense reagent (NEN ^™ Life Science Products).

As shown in Figure 2, a biotinylated surface protein with a molecular weight of approximately 120-kD was identified by TAB4 in RL.1 cells (TAIP ⁺ T cells), but not in 3T3 cells (TAIP ^- cells). In contrast, this 120-kDa protein did not elute with FG Sepharose protein coated with standard hamster serum. These results suggest that this 120-kDa protein is an antigen recognized by the TAB4 monoclonal antibody on the surface of T cells.

Example 6: T cell depletion in vivo

In order to reveal the effect of TAB4 on T-cell populations and other cells in vivo, mice were injected with 300 μg of TAB4 or a hamster Ig Ig by intraperitoneal injection and on the 4th day splenocytes, thymocytes, mononuclear peripheral blood cells were taken for total cell counting and analysis using FACS cell surface markers.

For FACS testing, cells were fixed with 2% paraformaldehyde at 4 ° C for 20 minutes, washed twice and resuspended in ice-cold FACS solution to a final concentration of 1 × 10 ⁷ cells / ml. A 100 μg aliquot of the resuspended cells in the FACS tube (Falcon) was used for each assay. TAB4 or a control hamster Ig at a final concentration of 2 μg / ml was added to the cells and the mixture was incubated at 4 ° C for 30 min in the dark. The cells were washed once with ice-cold FACS solution and reacted with: (1) for spleen cells: cichrome conjugated antibody specific for CD3 (2 μg / ml) conjugated to FITC specific for hamster Ig and PE-specific for CD8 / CD4 / CD 19 / CD 11b / pan - NK / IA / I-E / Mac-3 antibody (2 μg / ml) in 100 μg of ice-cold FACS solution; and (2) for thymic cells: FITC conjugated hamster Ig specific antibody, PE conjugated CD8 specific antibody and cichrome conjugated CD4 specific antibody (2 μg / ml) in 100 μg ice-cold FACS solution. The reaction was carried out at 4 ° C for 30 minutes in the dark. Finally, stained cells were washed twice with ice-cold FACS solution, resuspended in 1000 μl of FACS solution and analyzed using a BD LSR cytometer (Beckton Dickison).

Four days after injection, the proportion of CD3 ⁺ T cells in peripheral blood leukocytes (PBL) decreased from 36.7% for control mice to 4.1% for TAV4-treated mice (Table 2). TAB4 treatment caused a slight decrease in the total number of splenocytes. However, in TAB4-treated mice, there was a 62% decrease in the number of CD3 ⁺ T cells, a 50% decrease in the number of NK cells and a slight increase in the total number of CD19 + B cells. The total number of thymocytes obtained again from TAV4-treated mice was only 48% of the level obtained in the control (less than 52%). In addition, with the exception of CD4 ⁺ T cells, the numbers of all other CD8 ⁺ , CD4 ⁺ CD8 ⁺ and CD4 ^- CD8 ^- T cells were smaller, and especially in the CD4 ⁺ CD8 ⁺ subpopulations, which is clearly visible (64.7% decrease) .

table 2 Spleen × 10 ⁶ No processing Standard Ig hamster TA-B4-treated Depletion (%) The total number of splenocytes 123 93.3 105 14.6 CB3 ^- CB19 ⁺ 72,2 53,4 72.9 -0.8 CD3 ^- NK ⁺ 3.6 2,4 1.80 fifty Peripheral blood leukocytes No processing Standard Ig hamster TA-B4-treated Depletion (%) CD3 ⁺ T cells 36.7% 36% 4.1% 88.8% Thymus × 10 ⁶ No processing Standard Ig hamster TA-B4-treated Depletion (%) Total thymocyte count 94 229 45 52.1 CD4 ⁺ 9.3 28,4 10.9 -16.6 CD8 ⁺ 5.2 7.7 3.6 30.3 CD4 ⁺ CD8 ⁺ 73.8 182 26 64.7 CD4 ^- CD8 ^- 5,6 10.5 4,5 19.3 (representative data from three experiments)

Example 7: Specific TAIP antibody does not induce the secretion of IL-2 or TNF-alpha

Balb / c mice were injected intraperitoneally with 300 μg TAB4 or a standard hamster Ig. Splenocytes were isolated 7 days after the injection and used as responders in a culture with mitomycin C 3H (H-2k) treated splenocytes (as stimulants). Three days later, culture supernatants were collected and the IL-2 content was determined using an ELISA kit (PharMingen). As shown in FIG. 5, IL-2 production is inhibited in responder cells obtained from TAB4-treated mice compared to control mice. The levels of IL-2 and TNF-alpha were also checked, and there was no significant difference in the content of IL-2 (or TNF-alpha) in the serum of control and TAB4-treated mice. Since the reproduction of IL-2 is fundamental in terms of T-cell activity, the results show that a TAIP-specific antibody, such as TAB4, can be used in vivo to manipulate T-cells and regulate undesired T-cell-mediated immune responses, such as those associated with with autoimmune diseases and transplant rejection.

Example 8: Use of TAIP-Specific Antibodies to Prevent Graft Rejection

Mice (obtained from Jackson Laboratory) aged 8-12 weeks were anesthetized with Acepromazine maleate (Fermenta Animal Health Co., Kansas City, MO).

Prior to skin transplantation, recipient C57BL / 6 mice (H-2 ^b ) (which did not have a thymus removed) received intraperitoneal injections of 500 μg TAB4 or isotypic control antibodies seven days before the skin grafting operation. Seven days later, skin from the lateral region from completely allogeneic mismatched Balb / cj mice (H-2 ^d ) was transplanted into the lateral region of pre-treated C57BL / 6 mice. Seven days after transplantation, mice were again injected with 500 μg of TAB4 or isotypic control antibodies. Mice were examined daily after transplantation. The conclusion about transplant rejection was made if the condition of 50% of the donor skin was necrotic. The proportion of transplanted grafts shown in Fig.7 (n = 8). These data show that treatment with TAB4 antibody prolongs the viability of allogeneic skin grafts.

Example 9: Identification of TAIP as PSGL-1

P-selectin glycoprotein ligand-1 (PSGL-1), also called CD 162, is the main P-selectin ligand expressed by white blood cells, including T cells (Sako et al. (1993) Cell 75: 1179; Vachino et al. ( 1995) J. Biol. Chem. 270: 21966; Veldman et al. (1995) J. Biol. Chem. 270: 16470). The biochemical properties of TAIP, for example its molecular weight and its tendency to dimerize, suggest that TAB4 may be similar to PSGL-1. In order to investigate the relationship between the two antigens, the following was examined: 1) whether the antigen precipitated with TAB4 could be recognized by a suitable commercial antibody specific for PSGL-1; and 2) whether a PSGL-1 specific antibody can deplete TAB4 from a cell lysate.

RL ♂ T cells were lysed at a density of 1.0 × 10 cells / ml in lysis buffer (1% Triton X-100, 20 mM Tris-HCl, pH 8.0, 160 mM NaCl, 1 mM CaCl ₂ ) containing complete "Cocktail" of protease inhibitors, for 1 hour, insoluble material was besieged at 10,000 xg for 10 minutes. This and subsequent steps were performed at 4 ° C. A lysate with 54.0 × 10 ⁷ cells was incubated with 20 μl of G-Sepharose protein, pre-loaded with 10 μg anti-PSGL-1 mAb (clone 2HP1, PharMingen, San Diego, CA), anti-TAIP mAb, TAB4 or standard serum IgG hamster. After 4 hours of incubation at 4 ° C, the grains were washed five times with washing buffer (0.05% Triton X-100, 50 mM Tris-HCl, pH 8.5, 400 mM NaCl, 1 mM CaCl ₂ , 1 mg / ml ovalbumin) and twice with a similar wash buffer containing 200 mM instead of 400 mM NaCl. Bound proteins were eluted with 40 ml of 1 × SDS sample buffer. Eluted proteins were separated with 6% SDS-PAGE and transferred onto a nitrocellulose membrane. These membranes were immunoblotted with a PSGL-1 specific mAb and detected with a goat anti-rat IgG (H + L) peroxidase conjugate followed by chemiluminescence (Renaissance, NEN).

Surface biotinylated RL ♂ T cells were lysed at a density of 1.0 × 10 ⁸ cells / ml in lysis buffer. The cell extract was incubated with 20 μg of antibody bound to 40 μl of protein G-Sepharose at 4 ° C. overnight. Depletion was achieved with anti-PSGL-1 mAb (2HP1) or control rat IgG, with TAB4 or control standard hamster serum. The depleted lysates were further immunoprecipitated with TAB4 or mAbs specific for PSGL-1, respectively. Immunoprecipitates were isolated on a 6% SDS-polyacrylamide gel and detected by fluorography. As shown in FIG. 6, antibodies specific for PSGL-1 can deplete TAIP protein from T-cell lysates. In addition, proteins immunoprecipitated with anti-TAIP antibodies (TAB4) are recognized by antibodies specific for PSGL-1 by Western analysis.

Example 10: Induction of apotosis of human T cells with an antibody specific for PSGL-1

To determine the role that PSGL-1 plays in apoptosis of human T cells, experiments were carried out for certain periods of time to determine when activated human T cells become sensitive to PSGL-1-mediated apoptosis signals. Human T cells were stimulated with the mitogen phytohemagglutinin (PHA) and then kept in an IL-2 containing medium. Activated T cells were harvested and dissolved in anti-PSGL-1 in the presence of IL-2 and cross-linking antibodies.

In healthy adults, peripheral blood was sampled, heparinized, and peripheral blood mononuclear cells (PBMCs) were enriched (concentrated) based on the density difference using Ficoll-Pague Plus (Pharmacia Biotech). PBMCs were activated with 1% PHA (Life Technologies, GibcoBRL) for 48 hours and sequentially cultured in recombinant human IL-2 (5 ng / ml) throughout the study period. In order to evaluate the apoptosis inducing ability of a human PSGL-1 specific antibody, the activated cells were treated with: (1) 1 μg / ml PSGL-1 specific antibody, KPL clone (BD PharMingen) plus rabbit cross-linking anti-mouse Ig (0.5 μg / ml) (Jackson ImmunoResearch Laboratories); (2) an isotypic control purified mouse Ig plus a cross-linking rabbit anti-mouse Ig; or (3) one rabbit cross-linking anti-mouse Ig. After six hours of FACS treatment, the proportion of cells of the initial stage of apoptosis was determined by staining with anti-Annexin V (BD PharMingen) and PI (Sigma).

As shown in FIG. 8, signaling triggered by PSGL-1 using a PSGL-1 specific antibody, plus triggered by a cross-stapler, resulted in significant apoptosis of PHA-activated human PBMCs (mainly T cells). The percentage of apoptotic cells increased from 8.5% on the 3rd day to 24% by the 8th day in cultures treated with a specific PSGL-1 antibody. Neither isotypically similar control nor cross-linking antibodies alone yielded any result on the above cells.

Example 11: Use of an agonistic antibody specific for PSGL-1 for the treatment of autoimmune diseases

Mice with lean diabetes (NOD), animals with well-known autoimmune diabetes, were bred. Spontaneous diabetes developed in NOD mice at the age of about 20 weeks. In the experimental group of mice received three doses of anti-PSGL-1 antibodies (TAB4) intraperitoneally at 300 μg per mouse at the age of 14, 15 and 17 weeks. Two additional injections of the same doses were given at the age of 24 and 26 weeks. The control group received the hamster immunoglobulin in the same doses. Urine glucose was regularly determined in mice using Medi-Test Glucose strips (Macherey-Nagel, Germany) twice a week every 15 weeks. A non-decreasing urinary glucose level in excess of 300 mg / dl during the following two consecutive determinations led to the conclusion about the presence of diabetes.

As shown in FIG. 9, treatment with TAB4 antibody (specific for PSGL-1) resulted in significant protection compared with treatment with antibodies in the control. Thus, treatment with a specific antibody specific for PSGL-1 can inhibit the activity of autoimmune T cells and stop the onset of type I diabetes.

Example 12: Binding of P-selectin, E-selectin and L-selectin to activated T cells

To determine the ability of selectins (P-selectin, E-selectin and L-selectin) to bind to activated T cells, freshly obtained splenocytes from C57BL / 6 mice were activated and collected on days 2, 4 and 6. Inactive T cells (i.e., freshly prepared splenocytes on day 0) were also analyzed. On day 2, a sample containing 3 × 10 ⁶ cells / ml splenocytes was activated with 2 μg / ml Concanavalin A (Con A) in DMEM + 10% FBS for 2 days. Living cells were isolated by separation using a Ficoll gradient. On day 4, a sample was obtained from cells that were activated with Con A for 3 days and grown in medium containing 5 ng / ml IL-2 for another day. On day 6, a sample was obtained from cells activated with Con A for 3 days and cultured at 5 ng / ml IL-2 for 3 days.

For FACS analysis of samples from cultures of 0, 2, 4, and 6 days old, 2 × 10 ⁵ cells were taken per well, incubated at 4 ° C for 30 minutes with 40 μg / ml mouse P-selectin, E-selectin or L -selectin conjugated to the Fc region of human IgG1 (R&D Systems, Minneapolis, MN) at concentrations exceeding 20 μg / ml, with two serial dilutions of up to 0.156 μg / ml. After incubation, the cells were washed with 1 × FACScan buffer (1 × PBS without calcium and magnesium ions from Biochrom AG, Berlin and 2% FBS). Samples were then incubated at 4 ° C for 30 minutes with 95 μl / well of anti-Thy 1.2 and a second reagent (FITC anti-IgG human, which is specific for the Fc fragment, obtained from Jackson Immuno Research Laboratories, Inc., West Grove , PA) at 3.25 μg / ml, and then washed with 1 × FACSsan buffer.

The results of the FACSCalibur analysis are shown in FIG. 10. At 20 μg / ml, the binding of P-selectin to activated mouse T-cells increased sharply, peaked by the 4th day and decreased slightly on the 6th day. E-selectin binding increased significantly from day 2 to day 4, and then remained at a high level on day 6. The binding of L-selectin to activated murine T cells was not explicit and did not change after the activation period, i.e. from day 0 to day 6. The results observed with L-selectin could be such because of the apparent low binding affinity of L-selectin with its ligand. Similar results were also obtained when lower concentrations of all three selectins were used in the experiments.

Example 13: Multimeric forms of E-selectin and P-selectin induce apoptosis of activated T cells.

In a 96-well plate (NUNC), 50 μl of human Fc-specific IgG was applied, 20 μg / ml in 1 × PBS at 4 ° C. overnight, 1% BSA was fixed at 37 ° C. for 2 hours, and incubated with 50 μl of human selectin-Fc fused (0.063 to 5 μg / ml) at room temperature for 2 hours. In all experimental stages, each well was thoroughly washed five times with 1 × PBS. Then 2 × 10 ⁵ T cells, previously activated with Con A for four days, were added to each well and incubated at 37 ° C for 5 hours until the tablet was centrifuged at 200xg for 5 minutes at 4 ° C. The resulting pellet containing activated T cells was incubated with Annexin-V-biotin conjugate at room temperature for 15 minutes and then with an avidin conjugate (SA-beta-gal at a dilution of 1: 5000) for another 30 minutes at 37 ° C. In each binding reaction, each well was washed three times with Annexin V-binding buffer. Color staining occurred during incubation and 110 μl, Z-buffer mixture (54 μl of 2-mercaptoethanol in 20 ml of Z-buffer) and 30 μl of ONPG (0.04 g / 10 ml) at 4 ° C. The values of optical density at 420 nm were recorded.

The level of selectin-induced apoptosis of activated Con-A T cells increased with increasing concentrations (from 0.063 μg / ml to 5 μg / ml) of P-selectin (Fig. 11A) or E-selectin (Fig. 11B) fused to human Fc IgG1. The hamster antibody, TAB4, induces apoptosis of activated T cells (see Example 1) and was used as a positive control in all experiments. As a negative control, anti-human Fc, human Ig (HIg) and BSA did not cause apoptosis. No significant apoptosis level was detected in the presence of the human L-selectin and Fc fusion protein (Fig. 11C), which corresponds to the inability of L-selectin to bind successfully to activated T cells (Example 12).

In conclusion, it should be noted that a stereotype-linked fusion protein containing PSGL-1 binding fragment of P-selectin or E-selectin and human Fc-fragment induced apoptosis of activated T cells.

Example 14: Crosslinking of soluble P-selectin-Fc fusion protein induces apoptosis of activated T cells

Mouse selectins (P-selectin, E-selectin and L-selectin) were fused to the Fc region of human IgG1, which is described in detail above, with the formation of soluble dimeric fusion proteins. In order to evaluate whether soluble selectins can induce apoptosis of activated T cells, an experiment was performed which is described in detail in Example 13, except that stereotypically-bound Ig specific for human Fc was not used. Significantly low or low levels of apoptosis of activated T cells were fixed exclusively in the presence of a soluble form of the fused protein P-selectin (dimer) (Figure 12). However, with the addition of a cross-linking agent (human anti-Fc), apoptosis activity increased substantially, to approximately the levels of apoptosis induced in the presence of a stereotype-linked antibody. Neither human anti-Fc, human Ig (HIg), nor BSA induced apoptosis.

Similar results were obtained for E-selectin-Fc fusion protein, as well as for P-selectin-Fc fusion protein. In addition, according to the results obtained for the bound (multimeric form) of L-selectin, the soluble form of the fused protein of L-selectin does not induce apoptosis of activated T cells.

Claims (30)

1. A method of preventing or reducing T-cell mediated immune response in an individual, comprising;
the choice of an individual in respect of whom the presence or risk of achieving a condition characterized by an excessive or undesirable T-cell mediated immune response has been established; and
administering to the individual a multimeric compound that binds to at least two PSGL-1 proteins on the surface of the T cell, the multimeric compound containing two polypeptide chains, each polypeptide chain comprising (i) a binding domain that binds to PSGL-1, which includes the extracellular domain of P-selectin or its PSGL-1 binding fragment or the extracellular domain of E-selectin or its PSGL-1 binding fragment, and (ii) a heterologous amino acid sequence, the polypeptide chains being connected hetero ogichnoy amino acid sequence to form a multimeric compound, and
binding of the multimeric compound to at least two PSGL-1 proteins on the surface of the T cell induces a special signal transduction pathway that leads to the death of the T cell, thereby preventing the T cell mediated immune response or reducing its level in the individual. 2. The method according to claim 1, characterized in that the multimeric compound is a homomultimeric compound. 3. The method according to claim 1, characterized in that the multimeric compound is a heteromultimeric compound. 4. The method according to claim 1, characterized in that the heterologous amino acid sequence includes a region that binds to a cell surface receptor. 5. The method according to claim 1, characterized in that the binding domain includes the extracellular domain of P-selectin or its PSGL-1 - binding fragment. 6. The method according to claim 1, characterized in that the binding domain includes either the extracellular domain of E-selectin or its PSGL-1 binding fragment. 7. The method according to claim 1, characterized in that the binding domain includes PSGL-1 - a binding polypeptide selected from a phage display library. 8. The method according to claim 1, characterized in that the polypeptide chains are covalently linked by a heterologous amino acid sequence to form a multimeric compound. 9. The method of claim 8, wherein the covalent bond is a disulfide bond. 10. The method according to claim 1, characterized in that the heterologous amino acid sequence includes a constant region of the heavy chain of immunoglobulin. 11. The method according to claim 1, characterized in that it further comprises the step of introducing to the subject an agent that binds a multimeric compound using a heterologous amino acid sequence and promotes the crosslinking of a number of PSGL-1 antigens on the surface of the T cell. 12. The method according to claim 1, characterized in that the T cell is an activated T cell. 13. The method according to claim 1, characterized in that it further includes determining the number of T cells in the first biological sample taken from the individual before the introduction of the multimeric compound, and comparing the results with the number of T cells in the second biological sample taken from the individual after administration of the multimeric compound. 14. The method according to claim 1, characterized in that it further includes determining the biological activity of T cells in the first biological sample taken from the individual before the introduction of the multimeric compound, and comparing the results with the biological activity of T cells in the second biological sample taken in an individual after administration of a multimeric compound. 15. The method according to claim 1, characterized in that the introduction of a multimeric compound leads to the depletion of at least 10% of activated T cells in an individual. 16. A method of inducing the death of a T cell or a natural killer cell (NK), comprising:
the use of a T cell or NK cell expressing PSGL-1 on its surface;
contacting the T cell or NK cell with a multimeric compound that binds to at least two PSGL-1 proteins on the surface of the T cell or NK cell, the multimeric compound containing two polypeptide chains, each polypeptide chain comprising (i) a binding domain that binds to PSGL-1, which includes the extracellular domain of P-selectin or its PSGL-1 - binding fragment or extracellular domain of E-selectin or its PSGL-1 - binding fragment, and (ii) a heterologous amino acid sequence, wherein the polype poultry chains are connected by a heterologous amino acid sequence to form a multimeric compound; and
binding of the multimeric compound to at least two PSGL-1 proteins on the surface of a T cell or NK cell induces a particular signal transduction pathway that leads to the death of the T cell or NK cell. 17. The method according to clause 16, wherein the multimeric compound is a homomultimeric compound. 18. The method according to clause 16, wherein the multimeric compound is a heteromultimeric compound. 19. The method according to clause 16, wherein the heterologous amino acid sequence includes a region that binds to the cell surface receptor. 20. The method according to clause 16, wherein the binding domain includes the extracellular domain of P-selectin or its PSGL-1 - binding fragment. 21. The method according to clause 16, wherein the binding domain includes the extracellular domain of E-selectin or its PSGL-1 - binding fragment. 22. The method according to clause 16, wherein the binding domain includes PSGL-1 - a binding polypeptide selected from the phage display library. 23. The method according to clause 16, wherein the polypeptide chains are covalently linked by a heterologous amino acid sequence to form a multimeric compound. 24. The method according to item 23, wherein the covalent bond is a disulfide bond. 25. The method according to clause 16, wherein the heterologous amino acid sequence comprises an immunoglobulin heavy chain constant region. 26. The method according to clause 16, further comprising contacting the multimeric compound (s) of the agent that binds to the multimeric compound using a heterologous amino acid sequence and promotes the crosslinking of a number of PSGL-1 antigens on the surface of the T cell. 27. The method according to clause 16, wherein the T cell is activated. 28. The method according to clause 16, characterized in that it includes assessing the viability of a T cell or NK cell after contact with a multimeric compound. 29. The method according to clause 16, characterized in that it includes the assessment of the biological activity of a T cell or NK cell after contact with a multimeric compound. 30. A kit for preventing or decreasing a T cell mediated immune response in an individual, comprising:
a multimeric compound that binds to at least two PSGL-1 proteins on the surface of the T cell, the multimeric compound comprising two polypeptide chains, each polypeptide chain comprising (i) a binding domain that binds to PSGL-1, which includes extracellular the P-selectin domain or its PSGL-1 binding fragment or extracellular domain of the E-selectin or its PSGL-1 binding fragment, and ii) a heterologous amino acid sequence, wherein the polypeptide chains are connected by a heterologous amino acid sequence to form a multimeric compound, and the binding of multimeric compound having at least two PSGL-1 proteins on the surface of T-cells induces a particular signal transduction pathway that results in the death of T-cells; and instructions for using the compound.

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