KR101159140B1 - Modulators of p-selectin glycoprotein ligand 1 - Google Patents

Abstract

Multiple compounds that bind to P-selectin glycoprotein 1 (PSGL-1) on the surface of T cells or natural killer (NK) cells of the present invention can be used to deplete T cells or NK cells and / or induce apoptosis thereof. have. Multiple compounds and methods of the invention can be used to modulate unwanted mediated immune responses such as inflammatory diseases, autoimmune diseases, transplant rejection and allergic diseases.

Multiple Compound, T Cell, NK Cell, Apoptosis, Depletion

Description

Modulator of P-selectin glycoprotein ligand 1 {MODULATORS OF P-SELECTIN GLYCOPROTEIN LIGAND 1}

The present invention relates to compositions and methods for modulating immune responses.

In treating diseases such as inflammatory diseases, autoimmune diseases, graft rejection, allergic diseases and T cell cancer, the key is to control unwanted immune responses. Excessively aggressive T cell activity can be regulated through induction of immunosuppression or immunological tolerance. Tolerance is defined as a condition in which the immune system is unresponsive to antigens, and immunosuppression means a decrease in the immune response to antigens and generally requires continued use of the drug. In organ transplantation, T cells play an important role in the immune response to allogenegens. Currently, immunosuppressive therapies typically use corticosteroids, cyclosporin or rapamycin, which block the transcription of IL-2, the main growth factor for T cells, or IL-2. Inhibit dependent proliferation. However, as T cell-depleting substances (eg, CD3, CD4, CD8), cytokine signaling inhibitors or co-stimulatory pathway inhibitors of T cells (CD25, B7-1, B7-2, CD152, CTLA4) Many monoclonal antibodies that act have been proven effective in reducing the incidence of rejection with limited side effects or toxicity. Some of these substances have been found to be somewhat successful in the treatment of autoimmune diseases and in prolonging graft survival.

Apoptosis is considered to be critical for the proper functioning of the immune system and the maintenance of a major mechanism for eliminating unnecessary cells (Kabelitz et al. Immunol. Today 14: 338-340 (1993); Raff, Nature: 356). 397-399 (1992). Various signals derived from inside or outside the cell affect the survival and death of the cell. Antibodies to surface molecules of T cells, such as Fas (or CD95, MW = 43 kD), TNFR2 (MW = 75 kD), CD2 (MW = 45 kD), and CTLA-4 (MW = 33 kD), Induces apoptosis (Osborne, 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 molecules to control unwanted T cells have been limited by the fact that the two molecules are expressed not only in immune cells but also in some other major organs, such as the liver. This expression pattern potentially limits the therapeutic application 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)).

Superfamily members of Selectin, Integrin and Immunoglobulin (Ig) are the three main factors that are important for the interaction between leukocytes and platelets or the extracellular matrix of leukocytes and platelets and the vascular endothelium. (Springer, Nature 346: 425 (1990); Osborn, Cell 62: 3 (1990); Hynes, Cell 69: 11 (1992)). Adhesion molecules for one cell type often bind to other adhesion molecules expressed in various cell types, forming ligand-receptor pairs.

The selectin family includes P-selectin (also commonly referred to as CD62, CD62P, GMP140 and PADGEM), E-selectin (also commonly referred to as ELAM-1 and CD62E), and L-selectin (also, LECAM-1, Mel -14, commonly known as LAM-1 and CD62L). These selectins are highly homologous and contain multiple numbers of multiple short consensus repeat (SCR) domains that are complementary to those found in the 120-amino acid N-terminal lectin domain, EGF-based domain, and complement regulatory protein. , Transmembrane domain and 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) ). Selectins overlap and have distinct specificities 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 mediate the adherent interaction of leukocyte-endothelial and platelet-leukocytes first during the inflammatory process (Bevilacqua et al., 1993, supra). These three selectins have been shown to participate in the initial "rolling" interaction of leukocytes with activated endothelial cells (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. Clin. 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 in activated platelets and endothelial cells binds to 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 in endothelial cells activated by cytokines (eg, 6-8 hours after 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, supra. L-selectin expressed in most leukocytes binds to cell surface proteins of some endothelial cells and other leukocytes (Gallatin et al., Nature 304: 30-34 (1983); Berg et al., Immunol. Rev. 108 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); Spertini et al., J. Immunol. 147: 2565-2573 (1991)). All three selectins are known to bind PSGL-1, a surface protein of cells whose expression is largely confined to leukocytes, particularly T cells and NK cells. Post-translational modification of PSGL-1 is essential for binding with P-selectin, E-selectin and L-selectin (McEver et al., J. Clin. Invest., 1997, supra).

DISCLOSURE OF INVENTION

The present invention is based on the discovery that the use of P-selectin glycoprotein ligand-1 (PSGL-1), the surface antigen of T cells, can deplete T cells and / or lead to apoptosis. In particular, depletion of T cells may be useful for the treatment of symptoms associated with excessive or unnecessary T cell mediated immune responses, or for the treatment of symptoms associated with excessive or unnecessary T cell proliferation. For example, depletion of T cells can reduce or eliminate inappropriate T cell activity or proliferation associated with inflammatory diseases, autoimmune diseases, transplant rejection, allergic diseases and / or T cell cancers. The present invention includes a method of using a modulator of PSGL-1 function and a screening method of a modulator of PSGL-1 function to prevent or reduce a T cell mediated immune response.

One aspect of the invention relates to a method for preventing or reducing a T cell mediated immune response in an individual. The method comprises the following steps: selecting a subject having, or diagnosed with, a risk characterized by an excessive or unnecessary T cell mediated immune response; And administering to said individual a multimeric compound that binds to PSGL-1 on the surface of T cells, wherein said multi-compound at the surface of T cells binds to PSGL-1 protein resulting in T cell death. The pathway is induced, thereby preventing or reducing the individual's T cell mediated immune response.

Compounds used in such methods include antibodies. Or antigen-binding fragments thereof that specifically bind to PSGL-1. In one embodiment, the compound is a monoclonal antibody that specifically binds to PSGL-1. In one embodiment, the method includes the additional step of binding a monoclonal antibody and administering a substance that induces crosslinking with a plurality of PSGL-1 antigens on the surface of the T cells.

In some embodiments, the method comprises inducing crosslinking with a plurality of PSGL-1 antigens at the surface of the T cells, wherein the crosslinking induces a signal transduction pathway leading to T cell death.

In some embodiments, the method comprises the steps of: (i) selecting an individual having, or diagnosed with, a symptom characterized by an excessive or unnecessary T cell mediated immune response; step; And (ii) administering to said individual multiple compounds that bind to at least two PSGL-1 on a T cell surface, wherein said multiple compounds each comprise (a) a binding domain that binds PSGL-1 and (b) a heterologous Comprising two polypeptide chains comprising an amino acid sequence of wherein the polypeptide chains are linked through heterologous amino acid sequences to form multiple compounds, wherein the multiple compounds bind to at least two PSGL-1 proteins at the surface of T cells A signal transduction pathway is induced that results in T cell death, thereby preventing or reducing the individual's T cell mediated immune response.

The multiple compound may be a homo-multimeric compound or a hetero-multimeric compound. The binding domain is optionally an extracellular domain of P-selectin or a PSGL-1-binding fragment thereof, an extracellular domain of E-selectin or a PSGL-1-binding fragment thereof, a cell of L-selectin PSGL-1 binding polypeptides selected from foreign domains or PSGL-1-binding fragments thereof, anti-PSGL-1 antibodies or PSGL-1-binding fragments thereof, phage display libraries, or their May include any combination.

In certain instances, the multiple compound does not contain an anti-PSGL-1 antibody or antibody fragment that binds to PSGL-1.

The heterologous amino acid sequence may optionally include a binding site for any cell surface receptor, such as an immunoglobulin heavy chain constant region of an immunoglobulin. In some embodiments, the polypeptide chains bind covalently, such as disulfide bonds, through the heterologous amino acid sequence to form multiple compounds.

In certain instances, the method may comprise the additional step of administering to the subject a substance that binds to multiple compounds via heterologous amino acid sequences and induces crosslinking of a plurality of PSGL-1 antigens at the surface of the T cells.

In some instances, the methods described herein include selecting a subject diagnosed as having an autoimmune disease. In another embodiment, the method includes the step of screening the individual diagnosed as having an inflammatory disease. In another embodiment, the method comprises the step of selecting individuals that have received or will receive allografts or xenografts. In another embodiment, the method includes the step of screening the individual diagnosed as having an allergic disease. In another embodiment, the method comprises selecting for a subject diagnosed as having T cell cancer.

In some embodiments, the T cells are activated T cells. In one embodiment, the T cells are CD4 + T cells. In another embodiment, the T cells are CD8 + T cells.

In some embodiments, the method comprises detecting the number of T cells in a first biological sample taken from the subject prior to administering the compound (eg, multiple compound) and after administering the compound (eg, multiple compound) And comparing the number of T cells of the second biological sample taken from.

In some embodiments, the method comprises detecting a biological activity of T cells in a first biological sample taken from an individual prior to administering the compound (eg, multiple compound) and after administering the compound (eg, multiple compound) Comparing the biological activity of the T cells of the second biological sample taken from the subject.

In some embodiments, the administration depletes at least 10% of the activated T cells of the individual. In certain embodiments, activated T cells in an individual are depleted at least 10%, 20%, 30%, 40%, 50% or more upon administration.

In some embodiments, after exposure to an antibody, antigen-binding fragment thereof, or multiple compounds, the antibody, antigen-binding fragment thereof, or multiple compounds cause at least 10% of activated T cells in the individual to die. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, or more of the activated T cells in the subject upon administration are induced to die. Apoptosis is measured at any time, such as at least one day, two days, three days, four days, five days, six days, seven days, or seven days after exposure to an antibody, antigen binding fragment thereof or multiple compounds. can do.

The present invention also relates to a method for inducing death of T cells or natural killer (NK) cells. The method includes providing a T cell or NK cell expressing PSGL-1 at the cell surface, and contacting the compound that binds PSGL-1 at the surface of the T cell or NK cell to the T cell or NK cell. In addition, by binding the compound to PSGL-1 on the surface of the T cells or NK cells, a signal transduction pathway that induces death of T cells or NK cells is induced.

The compound used in the method may include an antibody or antigen-binding fragment thereof that specifically binds PSGL-1. In one embodiment, the compound is a monoclonal antibody that specifically binds to PSGL-1. In one embodiment, the method comprises contacting the monoclonal antibody with a substance that binds to the monoclonal antibody and induces cross-linking with a plurality of PSGL-1 antigens on the surface of T cells or NK cells.

In one embodiment, the method comprises the following steps: (i) providing a T cell or NK cell expressing PSGL-1 on the cell surface; And (ii) binds at least two PSGL-1 proteins on the surface of T cells or NK cells, each polypeptide chain containing (a) a binding domain that binds PSGL-1 and (b) a heterologous amino acid sequence. Comprising two polypeptide chains, wherein the polypeptide chains are linked through the heterologous amino acid sequence to form multiple compounds, wherein the multiple compounds bind at least two PSGL-1 proteins at the surface of a T cell or NK cell Contacting multiple compounds with T cells or NK cells, inducing signal transduction pathways that cause death of T cells or NK cells.

Multiple compounds may be homo-multiplexed or hetero-multiplexed. The binding domain may optionally be an extracellular domain of P-selectin or a PSGL-1-binding fragment thereof, an extracellular domain of E-selectin or a PSGL-1-binding fragment thereof, an extracellular domain of L-selectin or PSGL-1-binding fragments thereof, anti-PSGL-1 antibodies or PSGL-1-binding fragments thereof, peptides selected from phage display libraries, or any combination thereof.

The heterologous amino acid sequence may optionally comprise a binding site for the cell surface receptor, such as the heavy chain constant region of an immunoglobulin. In some embodiments, the polypeptide chains are joined covalently, such as disulfide bonds, through the heterologous amino acid sequence to form multiple compounds.

In some instances, the method may comprise the additional step of contacting the multiple compound with a substance that binds to the multiple compound via a heterologous amino acid sequence and causes crosslinking with a plurality of PSGL-1 antigens on the surface of the T cell. Can be.

In some embodiments, the method comprises inducing crosslinking with a plurality of PSGL-1 antigens on the surface of a T cell or NK cell, wherein the crosslinking comprises a signal transduction pathway that results in the death of the T cell or NK cell. cause.

In some embodiments of the invention, the T cells are activated T cells. In one embodiment, the T cells are CD4 + T cells. In another embodiment, the T cells are CD8 + T cells.

In some embodiments of the present invention, the method comprises assessing the viability of T cells or NK cells after contact with the compound (multiple compounds).

In some embodiments of the invention, the method comprises assessing the biological activity of T cells or NK cells after contact with the compound (multiple compounds).

In some embodiments, the method comprises inducing killing of activated T cells.

The present invention also relates to a method for screening a modulator of PSGL-1 functionality. The method comprises providing a cell expressing PSGL-1 on the surface of the cell; Contacting the cell with a test substance; And measuring the viability of the cells after contacting the cells with a test substance to determine whether the test substance is a modulator of PSGL-1 function.

In one example, the method includes detecting the death of cells induced by the test substance and determining whether the test substance is a modulator of PSGL-1 function.

In one embodiment, the test agent is an antibody or antigen-binding fragment thereof that specifically binds PSGL-1. In one embodiment, the test agent is a monoclonal antibody that specifically binds to PSGL-1. In one embodiment, the method comprises contacting the monoclonal antibody with a substance that binds to the monoclonal antibody and induces crosslinking with a plurality of PSGL-1 antigens on the cell surface.

In one embodiment, the method induces crosslinking with a plurality of PSGL-1 antigens at the cell surface, the crosslinking comprising inducing a signal transduction pathway leading to cell death.

In one embodiment, the T cells are activated T cells. In one embodiment, the T cells are CD4 + T cells. In another embodiment, the T cells are CD8 + T cells.

In one embodiment, the method includes preparing a bulk amount of the test substance and formulating the test substance in a pharmaceutically acceptable carrier.

In addition, the present invention provides a compound that binds to PSGL-1 on the surface of T cells and induces signal transduction pathways that cause death of T cells by binding to PSGL-1; And instructions for the use of such compounds to treat excessive or unnecessary T cell mediated immune responses or symptoms associated with excessive or unnecessary T cell proliferation, such as inflammation, autoimmune, transplant rejection, allergic symptoms or T cell cancer. It relates to a kit comprising a.

In one example, the kit comprises (i) at least two PSGL-1 proteins on the surface of the T cells, each polypeptide comprising (a) a binding domain to which PSGL-1 is bound, and (b) a heterologous amino acid sequence. Containing two polypeptide chains, wherein the polypeptide chains are linked through heterologous amino acid sequences to form multiple compounds, wherein the multiple compounds bind to at least two PSGL-1 proteins at the surface of the T cells, thereby killing T cells. Multiple compounds to which signal transduction pathways are induced, and (ii) symptoms associated with excessive or unwanted T cell mediated immune responses or proliferation of excessive or unnecessary T cells, such as inflammation, autoimmunity, transplant rejection. Kits containing instructions for use of said compounds for treating reactions, allergic symptoms or T cell cancers.

An advantage of the present invention is that it can induce depletion and / or apoptosis of T cells without causing unnecessary or harmful related immune responses. For example, administration of an anti-PSGL-1 antibody or multiple compounds herein does not result in unnecessarily elevated levels of inflammatory cytokines such as IL-2 or TNF-alpha.

Another advantage of the present invention is that depletion of T cells is caused by using functional compositions that induce apoptosis of T cells. Accordingly, the present invention arises by using antagonistic compositions (eg, antagonistic anti-PSGL-1 antibodies or soluble antagonistic selectin fragments) that bind to an immune receptor and serve to prevent immune activation mediated by said receptor. It provides a method of active immunosuppression rather than passive immunosuppression.

Another advantage of the present invention is that expression allows the targeting of PSGL-1, a cell surface protein that is usually limited to leukocytes, in particular T cells and NK cells. Thus, compounds herein typically do not induce significant levels of apoptosis of other cell species, such as liver cells. Targeting T cells and NK cells (an important CD3 ^- cell species involved in transplant rejection) for selective depletion that do not induce significant life-threatening systemic cytokine responses and do not cause loss of other organ systems Is a suitable feature as an immunosuppressant.

Unless defined otherwise, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods or materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are disclosed below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference. In case of conflicting terms, the present specification will control. In addition, the described materials and methods are illustrative only and not intended to be limiting.

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

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of modulating the activity of T cells by modulating the function of PSGL-1 molecules present on the surface of T cells. Employment of PSGL-1 with the agent compositions described herein can result in depletion of T cells and / or induce apoptosis of T cells. Such agent compositions are useful as therapeutic agents for controlling immune related diseases such as inflammatory diseases, autoimmune diseases, transplant rejection, allergic diseases and / or T cell derived cancer. Agent compositions are also useful for causing depletion of T cells in any biological sample in which the activity or presence of T cells is undesirable.

PSGL-1 Protein

PSGL-1 is a cell surface adhesion molecule, expressed in neutrophils, T-lymphocytes and B-lymphocytes, NK cells, monocytes, dendritic cells, and primitive human CD34 hematopoietic stem cells. PSGL-1, through its ability to interact with selectin, mediates the rolling of white blood cells in the endothelium and leukocyte outflow into inflammatory tissues. PSGL-1-mediated binding or migration of T cells to E-selectin and P-selectin is differentially regulated. For example, the appearance of a CLA (skin lymphocyte antigen) epitope is thought to induce the propensity of T cells to be memory transformed. Only activated helper 1, not helper 2T cells, expresses functional PSGL-1 and can migrate to the inflammatory zone of the skin.

PSGL-1 is sialomucin that must be specifically sialylated, fucosylated, and sulfated to bind P-selectin. PSGL-1 molecules exist as isoforms, characterized by differences in their degree of glycosylation and sulphation at their N-terminus. Resting peripheral blood T and B cells, lymphoid cell lines, and activated peripheral blood T cells in vitro express similar levels of PGSL-1. However, only activated T cells exhibit the functional form of PSGL-1 and actively bind to P-selectin. This activation-dependent binding activity is believed to be the result of different post-translational modifications, as judged from the increase in alpha (1,3) fucosyltransferase activity level in activated T cells. In addition, PSGL-1 isoforms show different affinity for L-selectin and E-selectin. For example, human T cells showing CLA-positive isoforms can bind to and roll both E-selectin and P-selectin, whereas T cells expressing PSGL-1 without CLA epitopes are only P-selectin To combine. Furthermore, binding of PSGL-1 to P-selectin is conditioned by the presence of terminal decapeptides containing three tyrosine residues for sulfateation and one threonine residue for glycosylation.

PSGL-1 protein can be prepared through recombinant methods and / or isolation of native PSGL-1 protein from biological materials. Recombinant PSGL-1 protein may be produced in vitro or in vivo in prokaryotic or eukaryotic cells. Nucleic acids encoding PSGL-1 can be used for recombinant production of the protein (eg GenBank Accession No. NM_003006 for nucleic acids encoding PSGL-1 polypeptide). Antibodies against PSGL-1 are also known and may be used for purification of antigens (eg, Herron et al. (2000) Science Jun 2; 288 (5471): 1653-56; WO 00/25808) and the methods presented herein. Can be. PSGL-1 also includes, but is not limited to, Sako et al. (1993) Cell 75: 1179; Vachino et al. (1995) J. Biol. Chem. 270: 21966; And Veldman et al. (1995) J. Biol. Chem. 270: 16470, and in the literature.

For recombinant production of PSGL-1, co-expression of PSGL-1 and its modified alpha- (1,3) fucosyltransferase Fuc-TVII may be required for functional expression of PSGL-1. Additionally or alternatively, recombinant production of PSGL-1 can be achieved by co-transforming a nucleic acid encoding PACE to remove a nucleic acid encoding a tyrosine sulfotransferase and / or a propeptide.

Anti-PSGL-1 antibodies can be used for the isolation and purification of PSGL-1 antigens from biological materials. Any cell that expresses a PSGL-1 protein, eg, a T cell derived from an individual or a T cell line, can be used as a source of the protein. After purification, the protein can be used in the various methods presented herein. For example, the purified PSGL-1 protein may be screened in vitro for modulators of PSGL-1 function on T cells or used as an immunogen to prepare antibodies to the protein.

Anti-PSGL-1 antibody

PSGL-1 polypeptides (or immunogenic fragments or analogs thereof) can be used in the production of antibodies useful in the methods of the invention. As described above, the PSGL-1 polypeptide or peptide fragment thereof may be produced by recombinant technology or synthesized by solid phase synthesis. The recombinant PSGL-1 polypeptide or peptide fragment thereof can be used as an immunogen to produce anti-PSGL-1 antibodies. In addition, anti-PSGL-1 antibodies, such as TAB4 monoclonal antibodies, can be used to purify PSGL-1 polypeptides, eg, PSGL-1 polypeptides in natural configuration, followed by additional anti-PSGL-1 It can be used as an immunogen for producing an antibody.

Antibodies of the invention can be monoclonal, polyclonal, or engineered antibodies that specifically bind to PSGL-1 polypeptide. An antibody that "specifically binds" to a particular antigen, eg, a PSGL-1 polypeptide, does not substantially recognize or bind other molecules in one sample. Accordingly, the present invention provides a method for preparing a test compound comprising contacting a polypeptide with a test compound; Determining whether the polypeptide binds to the test compound (eg, direct detection of binding, detection of a competition molecule that breaks the binding of the test compound to the polypeptide, and / or binding using an apoptotic induction activity assay) Sex detection), and methods of identifying test compounds (eg, antibodies) that bind to the polypeptide.

In general, the PSGL-1 polypeptide may be mixed with an adjuvant such as KLH, coupled with a carrier protein, and injected into a mammalian host. Antibodies produced in such animals can then be purified by peptide antigen affinity chromatography.

In particular, various host animals can be immunized by infusion of PSGL-1 polypeptide or antigenic fragments thereof. Commonly used host animals include rabbits, mice, guinea pigs, and rats. The various adjuvant that can be used to increase the immune response depends on the type of host, the adjuvant of Freund (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolexitin, flu Ronin polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenols. Potentially useful human adjuvant include bacille Calmette-Guerin (BCG) and Corynebacterium parvum . Polyclonal antibodies are a collection of heterologous antibody molecules contained in the serum of an immunized animal.

Thus, antibodies belonging to the present invention include molecules produced using polyclonal and monoclonal antibodies, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F (ab ') 2 fragments, and Fab expression libraries.

Monoclonal antibodies that are homologous to a particular antigen cluster include the PSGL-1 polypeptide and standard hybridoma technology (eg, 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 include the production techniques of antibody molecules by continuous cultured cell lines, such as those described in Nature 256: 495 (1975) to Kohler et al. And US Pat. No. 4,376,110; Human B-cell hybridoma technology (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 may be antibodies of any immunoglobulin class and subclass thereof, including IgG, IgM, IgE, IgA, IgD. Hybridomas producing the mAb of the present invention can be cultured in vitro or in vivo. The ability to produce high titers of mAbs in vivo is a particularly useful method of production.

After preparation, the polyclonal or monoclonal antibodies are tested for specific PSGL-1 cognition by Western blot or immunoprecipitation assays, for example, by standard methods as set forth in the aforementioned literature by Ausubel et al. Antibodies that specifically recognize and bind to PSGL-1 are useful in the present invention. Particularly useful are anti-PSGL-1 antibodies that bind to PSGL-1 antigen on the surface of T cells, eg, CD3 + cells, and induce depletion and / or apoptosis of T cells in the individual.

Antibodies can be used, for example, to reduce or eliminate inappropriate immune responses, such as treatment regimens (eg, T cell mediated immune responses associated with symptoms such as inflammatory diseases, autoimmune diseases, transplant rejection, allergic diseases, and T cell derived cancers). Can be used as part of The antibody can also be used in screening assays to determine the binding capacity of the candidate compound with PSGL-1.

In addition, techniques developed to produce "chimeric antibodies" by splicing genes of human antibody molecules with appropriate biological activity with genes of mouse antibody molecules with appropriate antigen specificity (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)) can also be used. Chimeric antibodies are one molecule in which each portion is derived from a different animal species, such as having a variable region derived from a murine monoclonal antibody and a constant region of human immunoglobulin.

Alternatively, techniques for producing single chain antibodies (US Pat. Nos. 4,946,778, 4,946,778 and 4,704,692) can be modified to produce single chain antibodies or fragments thereof against the PSGL-1 polypeptide. Single-chain antibodies are formed by linking the heavy and light chain fragments of the Fv region through amino acid bonds to produce one single-chain polypeptide.

The antibody fragment which recognizes and binds a specific epitope can be manufactured by a well-known technique. For example, such fragments include, but are not limited to, F (ab ') 2 fragments that can be produced by pepsin cleavage of antibody molecules and Fab fragments that can be prepared by reducing disulfide bonds of F (ab') 2 fragments. . Alternatively, Fab expression libraries (Huse et al., Science 246: 1275 (1989)) can be constructed 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 the desired binding specificities can be commercially humanized (Scotgene, Scotland; Oxford Molecular, Palo Alto, Calif.). In addition, fully human antibodies, such as those expressed in transgenic animals, are also a feature of the invention (Green et al., Nature Genetics 7: 13 (1994); US Pat. Nos. 5,545,806 and 5,569,825).

Multiple compound

Multiple compounds that bind to a plurality of PSGL-1 proteins on the surface of T cells or NK cells can be used to induce apoptosis of these cells. Multiple compounds contain at least two polypeptide chains. Each polypeptide chain comprises (i) a binding domain that binds PSGL-1 and (ii) a heterologous amino acid sequence.

In general, multiple compounds bind to at least two different PSGL-1 proteins at specific cell surfaces. However, multiple compounds have 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more distinct PSGL-1 binding domains, resulting in 3, 4, 5, 6 at the surface of specific cells. Can be designed to allow multiple compounds to be combined with, 7, 8, 9, 10, 11, 12 or more other PSGL-1 proteins.

The binding domain may comprise any amino acid sequence that binds PSGL-1 (any amino acid sequence modified such as glycosylation and / or sulfate). The binding domain may correspond to either naturally formed or unnaturally formed amino acid sequence. For example, the binding domain may comprise a PASL-1 binding domain of a selectin (eg, P-selectin, E-selectin or L-selectin). Polypeptides containing the PSGL-1 binding domain of the selectin may optionally include (i) the extracellular domain of the selectin (eg, P-selectin, E-selectin or L-selectin); (ii) a calcium dependent selectin domain of a selectin (eg, P-selectin, E-selectin or L-selectin); Or (iii) a fragment of the extracellular domain of a selectin (eg, P-selectin, E-selectin or L-selectin) that mediates binding to PSGL-1. In addition to these naturally occurring amino acid sequences, one or more amino acid changes can be introduced into a naturally formed PSGL-1 binding domain to produce an unnaturally formed sequence that maintains PSGL-1 binding function. For example, a polypeptide may comprise an extracellular domain of any (i) selectin (eg, P-selectin, E-selectin or L-selectin); (ii) a calcium dependent selectin domain of a selectin (eg, P-selectin, E-selectin or L-selectin); Or (iii) at least 80%, 85%, 90%, 95% or 98% of a fragment of an extracellular domain of a selectin (eg, P-selectin, E-selectin or L-selectin) that mediates binding to PSGL-1 Homologous and may contain an amino acid sequence that binds to PSGL-1. Standard molecular biology mutagenesis techniques can be used to introduce modifications into nucleic acid sequences encoding the PSGL-1 binding domain. The modified binding domain can then be tested for its binding ability to PSGL-1, such as immobilized PSGL-1 or PSGL-1 on the cell surface. In addition, the binding domain may be a polypeptide selected from the PSGL-1 binding domain or phage display library of an anti-PSGL-1 antibody, or a PSGL-1 binding domain or phage of an anti-PSGL-1 antibody that binds to PSGL-1 and binds to PSGL-1. And at least 80%, 85%, 90%, 95%, or 98% homologous amino acid sequences with polypeptides selected from the display library.

The PSGL-1 binding domain may comprise an amino acid sequence corresponding to the PSGL-1 binding fragment of P-selectin. An example of a polypeptide chain (of multiple compounds herein) containing this amino acid sequence is a recombinant mouse P-selectin / Fc chimera (R & D Systems, Minneapolis, MN) containing the following components: (i) CD33 signal Peptide (Metl-Alal 6); (ii) mouse P-selectin (Trp42-Ala709 in extracellular domain); (iii) IEGRMD (SEQ ID NO: 1); And (iv) human IgGl (ProlO0-Lys330). A second example of a polypeptide chain containing this amino acid sequence is a recombinant human P-selectin / Fc chimera (R & D Systems, Minneapolis, MN) containing the following components: (i) Human P-selectin (Metl-) Ala771, extracellular domain); (ii) IEGRMD (SEQ ID NO: 1); And (iii) human IgG1 (ProlOO-Lys330).

The PSGL-1-binding domain may comprise an amino acid sequence corresponding to the PSGL-1 binding domain of E-selectin. An example of a polypeptide chain (of multiple compounds herein) containing this amino acid sequence is a recombinant mouse E-selectin / Fc chimera (R & D Systems, Minneapolis, MN) containing the following components: (i) mouse E-selectin (Metl-Pro557, extracellular domain); (ii) IEGRMD (SEQ ID NO: 1); (iii) human IgGl (ProlO0-Lys330); And (iv) HHHHHH (SEQ ID NO: 2). A second example of a polypeptide chain containing this amino acid sequence is a recombinant human E-selectin / Fc chimera (R & D Systems, Minneapolis, MN) containing the following components: (i) Human E-selectin (Metl-) Pro556, extracellular domain); (ii) IEGRMD (SEQ ID NO: 2); (iii) human IgGl (ProlO0-Lys330); And (iv) HHHHHH (SEQ ID NO: 2).

The PSGL-1-binding domain may comprise an amino acid sequence corresponding to the PSGL-1 binding domain of L-selectin. An example of a polypeptide chain (of multiple compounds herein) containing this amino acid sequence is a recombinant mouse L-selectin / Fc chimera (R & D Systems, Minneapolis, MN) containing the following components: (i) Mouse L Selectin (Metl-Asn332, extracellular domain); (ii) IEGRMD (SEQ ID NO: 1); (iii) human IgG1 (ProlO0-Lys330); And (iv) HHHHHH (SEQ ID NO: 2). A second example of a polypeptide chain containing this amino acid sequence is a recombinant human L-selectin / Fc chimera (R & D Systems, Minneapolis, MN) containing the following components: (i) Human L-selectin (Metl-) Asn332, extracellular domain); (ii) IEGRMD (SEQ ID NO: 1); (iii) human IgGl (ProlO0-Lys330); And (iv) HHHHHH (SEQ ID NO: 2).

Multiple compounds can be envisioned as homo-multiplexed or hetero-multiplexed. Homo-multiple compounds are just polypeptide chains having identical PSGL-1 binding domains. For example, the homo-multiple compound may comprise a polypeptide chain containing the same PSGL-1 binding fragment of P-selectin. Hetero-multiple compounds include polypeptide chains having various PSGL-1 binding domains. For example, the hetero-multiple compound may comprise a first polypeptide chain containing the PSGL-1 binding domain of P-selectin and a second polypeptide chain containing the PSGL-1 binding domain of E-selectin.

The heterologous amino acid sequence may be any amino acid sequence. However, the amino acid sequences of the polypeptide chains described herein are not consistent with the sequences of naturally formed proteins. Heterologous amino acid sequences include one or more amino acids that tolerate linkage of a polypeptide chain. For example, one or more amino acids can link a polypeptide chain covalently, eg, via disulfide bonds. An example of a heterologous sequence is the heavy chain constant region of an immunoglobulin. Disulfide bonds between the Fc region of two polypeptide chains can form a dimeric compound.

In addition, the heterologous amino acid sequence may not only contribute to the linkage of the polypeptide chain, but may also include cross-linker binding sites, such as binding sites to receptors on the cell surface. The binding of a substance to this binding site results in the formation of a crosslinked link between the polypeptide chain and the PSGL-1 protein on the cell surface that binds to it. The heavy chain chain constant region of an immunoglobulin comprises an Fc receptor binding site. Cross-linkers can be, for example, antibodies (eg, anti-Fc antibodies) that specifically bind to cross-linker binding sites of heterologous amino acid sequences.

Screening Analysis of Compounds That Modulate PSGL-1 Function

In addition, the invention interacts with PSGL-1 (or PSGL-1 domain), which binds to PSGL-1 and includes, but is not limited to, compounds that induce depletion of T cells and / or apoptosis of T cells. Includes identification methods. The invention also encompasses transmembrane, extracellular or intracellular proteins that modulate PSGL-1 activity, and compounds that modulate PSGL-1 activity with compounds that modulate PSGL-1 activity.

Compounds that can be screened according to the present invention include, but are not limited to, peptides, antibodies and fragments thereof, and other organic compounds that bind to PSGL-1 and modulate biological activity mediated by PSGL-1 as described herein. It is not limited.

Such compounds include, but are not limited to, peptides such as, for example, soluble peptides, and members of random peptide libraries (Lam et al., Nature 354: 82 (1991); Houghten et al., Nature 354: 84 (1991)). And D-coordinating and / or L-coordinating amino acids, phosphopeptides (including but not limited to random or some variants, members of the direct phosphopeptide library; Songyang et al., Cell 72: 767 (1993)), Antibodies (including but not limited to polyclonal antibodies, monoclonal antibodies, humanized antibodies, anti-idiotype antibodies, chimeric antibodies or single chain antibodies, and FAb, F (ab ') 2 and FAb expression library fragments, and epitope-binding thereof Fragments), and molecular libraries obtained through chemical combinations including small organic or inorganic molecules.

Other compounds that can be screened in accordance with the present invention include, but are not limited to, small organic molecules that affect the activity of the PSGL-1 protein as described above.

Computer modeling and retrieval techniques enable the identification of compounds capable of modulating PSGL-1 expression or activity, or the improvement of already identified compounds. By identifying such compounds or compositions, active sites or regions are identified. Such active sites will typically be binding sites to the active natural modulator. Active sites can be identified using methods known in the art, for example, from studies of amino acid sequences of peptides, nucleotide sequences of nucleic acids, or complexes of related compounds or compositions and natural ligands. In the latter case, chemical or X-ray crystallography can be used to find the active site by revealing the site where the modulator (or ligand) is found on the factor.

While the design and fabrication of compounds capable of modifying binding have been described above, those skilled in the art have described natural products or synthetic chemistry that bind to PSGL-1 protein to induce T cell depletion and / or induce apoptosis of T cells. Known compounds, including substances, and libraries of biologically active substances, including proteins, may be screened.

In vitro systems can be designed to identify compounds that can interact with PSGL-1 (or domain of PSGL-1). Identified compounds may be useful in the regulation of T cell activity, for example, as described herein, and therefore, excessive or unnecessary T cell mediated immune responses such as inflammation, autoimmunity, transplant rejection, allergic symptoms, and T cell cancer. It may be useful for treating symptoms associated with excessive or unnecessary T cell proliferation.

The principle of analysis used in the identification of compounds that bind to PSGL-1 is based on conditions sufficient to allow formation of complexes that can be removed and / or detected in the reaction mixture by allowing the two components to bind and interact with each other, and Under time, preparing a reaction mixture of PSGL-1 (or a domain thereof) and a test compound. The PSGL-1 species used may vary depending on the purpose of the screening assay. In some cases, it is desirable to use a peptide corresponding to the domain of PSGL-1 fused with a heterologous protein or polypeptide that provides an advantage to the assay system (eg, labeling, isolation of the resulting complex, etc.).

Screening analysis can be performed in a variety of ways. For example, a method of analysis comprising immobilizing a PSGL-1 protein, polypeptide, peptide or fusion protein or test substance on a solid phase, and identifying the PSGL-1 / test compound complex immobilized on the solid phase at the end of the reaction. Can be done. As one example of such a method, the PSGL-1 reactant may be immobilized on a solid surface, and the unimmobilized test compound may be directly or indirectly labeled.

In fact, the microtiter plate can be conveniently used as a solid phase. The immobilized component can be immobilized by non-covalent or covalent attachment. Non-covalent attachment can be achieved by simply coating the solid surface with a protein solution and drying. Alternatively, immobilized antibodies, preferably monoclonal antibodies, specific for the protein to be immobilized can be used to immobilize the protein on a solid surface. Surfaces can be manufactured and stored in advance.

To conduct the assay, an unimmobilized component is added to the coated surface containing the immobilized component. After the end of the reaction, the unreacted components are removed (eg by washing) under conditions in which any complexes formed remain fixed to the solid surface. Detection of complexes immobilized on a solid surface can be accomplished in a variety of ways. If the previously unimmobilized component has been previously labeled, detection of the immobilized label on the surface suggests that a complex has been formed. If a component that has not been previously immobilized is not pre-labeled, an indirect label can be used, for example, a label antibody specific for the pre-immobilized component (an antibody that can be directly or indirectly labeled with a labeled anti-Ig antibody). Can be used to detect complexes immobilized on the surface.

Alternatively, the reaction may be directed to a test compound that immobilizes the reaction product and the detected complex isolated from the unreacted components, eg, PSGL-1 protein, polypeptide, peptide or fusion protein or any complex formed in solution. Immobilized antibodies specific for, and labeled antibodies specific for other components of the complexes that allow detection of immobilized complexes, can be used in the liquid phase.

Alternatively, cell assays may be used to identify compounds that interact with PSGL-1. To this end, cell lines expressing PSGL-1, or cell lines genetically engineered to express PSGL-1, can be used. Cellular assays are particularly useful for evaluating the functional effects of compounds identified by the screenings presented herein. For example, the ability to identify compounds based on their ability to bind to PSGL-1 protein and then induce apoptosis of T cells, or deplete T cells in vitro or in vivo, for example. The compound can be tested for.

Pharmaceutical composition

It is an object of the present invention to vary the immune response of an individual, for example a pharmaceutical composition containing an antibody, multiple compounds, small molecules, or other compounds that specifically bind to a PSGL-1 polypeptide is also a feature of the present invention. . In a preferred embodiment, the compound acts as an agonist of PSGL-1.

Pharmaceutical compositions for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. Thus, the compounds and their physiologically acceptable salts and solvates may be formulated for administration via various routes of administration.

The compounds may be formulated for parenteral administration by injection, eg, pill injection or continuous infusion. Injectable formulations may be present in a single dosage form such as, for example, ampoules, with a preservative added, or stored in a multi-dose container. The composition may take the form of a suspension, solution, or emulsion in an oil-based or aqueous vehicle, and may contain formulating agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the active ingredient may be in powder form consisting of a suitable vehicle, eg, sterile pyrogen-free water, before use.

How to Regulate T Cell-Mediated Immune Response and Depletion of T Cell Community

The screening assays presented herein include, for example, for the regulation of biological functions mediated by PSGL-1 polypeptides and / or for the treatment of diseases associated with excessive or unnecessary immune responses, eg, T cell-mediated immune responses. As such, the compounds specifically presented may be useful. These compounds include, but are not limited to, peptides, antibodies and fragments thereof, and other organic compounds that bind to PSGL-1 on the surface of T cells and induce signal transduction pathways leading to T cell death. The method of the invention optionally comprises the step of adding a crosslinker to induce crosslinking of PSGL-1 on the cell surface. The compounds presented herein may be useful where activity depletion or removal of T cells is desired. Particularly useful symptoms that can be treated with the compounds of the present invention include inflammatory diseases, autoimmune diseases, transplant rejection, allergic diseases, and cancers derived from T cells.

Examples of symptoms that can be treated with the anti-PSGL-1 compounds presented herein include, but are not limited to, diabetes, arthritis (including rheumatoid arthritis, child rheumatoid arthritis, osteoarthritis and psoriatic arthritis), multiple sclerosis, encephalomyelitis, severe Myasthenia Gravis, Systemic Lupus Erythema, Autoimmune Thyroiditis, Dermatitis (Atopic Dermatitis and Eczema Dermatitis), Psoriasis, Sjogren's Syndrome, Crohn's Disease, Afta Ulcers, Iris, Conjunctivitis, Corneal Conjunctivitis, I Type diabetes mellitus, inflammatory bowel disease, ulcerative colitis, asthma, allergic asthma, skin lupus erythema, scleroderma, vaginitis, proctitis, drug rash, leprosy conduction reaction, leprosy nodule erythema, autoimmune uveitis, allergic encephalomyelitis, acute moving hemorrhagic Myelitis, idiopathic progressive bilateral sensorineural hearing loss, aplastic anemia, pure erythrocyte cellular anemia, idiopathic hypoplateletosis, Fusitis, Wegener's granulomaltosis, Chronic active hepatitis, Stevens-Johnson syndrome, Idiopathic sprue, Squamous lichen, Graves' disease, Sarcoidosis, Early biliary hardening, Beds of grafts (including grafts with isomorphic or heterotypic tissue), such as posterior uveitis, interstitial pulmonary fibrosis, graft-versus-host disease, bone marrow transplantation, liver transplantation, or transplantation of any organ or tissue, such as atopic allergy Allergy, AIDS, and T-cell tumors such as leukemia and / or lymphoma.

The methods of the present invention can be used to deplete T cells from cell populations in vitro or in vivo. For example, a biological sample derived from an individual can optionally be depleted of T cells in vitro by contacting the sample with an anti-PSGL-1 compound provided herein along with a crosslinker. Such methods may be useful, for example, for enrichment of non-T cells in a cell population and for reducing or eliminating T cell activity from the cell population.

1 shows the results of experiments over time when activated T cells responded to TAB4 (anti-PSGL-1 monoclonal antibody) -mediated apoptosis signals.

2 shows the results of biotinylation and immunoprecipitation of the cell surface of antigens recognized by TAB4 antibodies.

Figure 3 shows the expression of PSGL-1 antigen in spleen CD4 ⁺ T cells, CD8 ⁺ T cells, CD19 ⁺ B cells, and NK cells.

4 is a CD4 ^+, CD8 ^+, and CD4 ⁺ 8 ^+, and ^CD4-a diagram of a PSGL-1 antigen expression in thymus cells ^- 8.

Figure 5 shows the splenocytes isolated from Balb / c mice treated with TAB4 (or hamster Ig) as a responder and H2-mismatched C3H spleen cells as promoters of IL-2 produced in mixed leukocyte medium. The figure which shows the level.

6 shows that (A) proteins that are immunoprecipitated with TAB4 antibodies can be recognized by commercially available anti-PSGL-1 antibodies, and (B) pre-cleaning of T cell lysates with anti-PSGL-1 antibodies. Western blot analysis demonstrating that it can deplete the protein recognized by TAB4.

FIG. 7 shows the survival rate (%) of grafts in C57BL / 6 mice transplanted into skin from Balb / c mice and treated with anti-PSGL-1 antibody (black diamond) or control antibody (white diamond). .

Figure 8 shows the apoptosis rate of T cells after treatment of peripheral blood monoclonal cells activated with anti-human PSGL-1 antibody.

FIG. 9 shows the incidence of diabetes in diabetic (NOD) male mice in autoimmune non-obese state treated with anti-PSGL-1 antibody (black square) or control antibody (white square).

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

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

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

Hereinafter, embodiments for carrying out the present invention. These examples do not limit the scope of the invention in any way.

Example 1: Preparation of Apoptosis Inducing Protein ("TAIP") Monoclonal Antibodies of Anti-T Cells

To produce hybridomas that secrete the desired antibody, TAIP-specific monoclonal antibodies were prepared using well-known cell fusion methods of Kohler and Milstein (1976) European Journal of Immunology 6: 511-519). Antibody-secreting hybridomas were prepared by fusion of hamster-derived antibody-producing cells infused with concanovalin A (ConA) -activated Balb / c splenic T cells with a myeloma cell line. Cells from both populations were fused using polyethylene glycol, and the resulting antibody producing cells were cloned and expanded by standard tissue culture methods. One hybridoma prepared according to this method secretes a monoclonal antibody designated TAB4, which can induce apoptosis of T cells in vitro and can deplete T cells in vivo. The protein recognized by TAB4 was termed apoptosis inducible protein (TAIP) of T cells.

C57BL / 6J (B6) and BALB / c mice were purchased from Jackson Laboratories (Bar Harbor, ME). Syrian hamster was purchased from the Animal Core Facility (National Taiwan University Medical College).

The concentrated culture supernatant of TAB4 hybridomas was spun at 20,000 × g for 10 min, and then the supernatant was diluted 1: 1 in binding buffer (0.1 M sodium acetate, pH5.0). The Protein G column (approximately 1 ml fill volume) was washed three times with 3-5 ml of the binding buffer. The washed culture supernatant was injected into a Protein G column, and column flow-through was collected and injected again into the column. The column was washed with 6-10 mL of binding buffer and the bound antibody was eluted with 5 mL of elution buffer (0.1 M glycine-HCl, pH 2.8). Each fraction contained 1 ml of eluted antibody and 1 ml of each fraction was mixed with 50 μl of 1M Tris-HCl (pH 7.5) to adjust the pH of the eluted fractions to neutral. Fractions containing the antibody were mixed and dialyzed three times with 3 hours of dialysis each time against 2 L of PBS, pH 7.4. The concentration of protein in the antibody samples was measured using a Bio-Rad protein assay (BIO-RAD, Hercules, Calif.) Following the procedure proposed by Bradford.

Example 2: Preparation of Spleen Cell Suspensions in Mice and Activation and Proliferation of T Cells

Mouse spleens were immersed in 8 ml of Hanks' balanced salt solution (HBSS), sliced with sterile cover slips and transferred to 15 ml centrifuge tubes (Costar) and centrifuged at 200 × g for 5 minutes. The supernatant was discarded and the cell pellet was resuspended in the remaining buffer by gently tapping the centrifuge tube wall. 1 ml of erythrocyte lysis buffer (0.6 M NH ₄ Cl, 0.17 M Tris-base, pH 7.65) was added to contaminated erythrocytes (RBC), incubated for 2 minutes at room temperature, and then 9 ml of HBSS Quickly quenched with. These cells were pelleted at 200 × g for 5 minutes, washed twice and resuspended in RPMI medium. Cell concentration and viability in the mixture were assessed by hemocytometer (Cambridge Scientific Inc.) and Trypan blue exclusion.

T cells were activated by adjusting the final concentration of the spleen cells in RPMI medium to 3 × 10 ⁶ / ml and adding concanoovalin A to a final concentration of 2 μg / ml. The cell suspensions were transferred to 6-well culture plates (5 mL / well) or 10 cm culture dishes (10 mL / dish) and harvested by incubating for 48 hours at 37 ° C., 5% CO ₂ . Activated spleen cells, including activated T cells, were resuspended in 5 ml of HBSS and carefully stacked on top of 5 ml of 55% Percoll solution in a centrifuge tube. At this time, care was taken not to collapse the separation layer. The cells were centrifuged for 13 min at 1,900xg at 25 ° C. Proliferated T cells were collected at the interface of the two layers and washed twice with HBSS to prepare for the experiment.

Example 3: Apoptosis of Activated T Cells

Activated T cells (Example 2) were resuspended in RPMI medium containing 5 ng / ml IL-2 to a final concentration of 5 × 10 ⁵ cells / ml, and control Ig, according to the conditions shown in Table 1, Treatment with TAB4 or anti-CD3.

(Table 1)

 Experimental group Treatment ^*  Negative control group 3 μg / ml hamster Ig
5 ng / ml IL-2
3 μg / ml crosslinked antibody (anti-hamster Ig)  TAB4 3 μg / ml TAB4 hamster mAb
5 ng / ml IL-2
3 μg / ml crosslinked antibody (anti-hamster Ig)  Positive control 1 μg / ml of anti-CD3 mAb
5 ng / ml IL-2
1 μg / ml crosslinked antibody (anti-mouse Ig) ^* : Final concentration of the specified reagent in the medium

After culturing for 18 to 24 hours, the degree of apoptosis of each culture was evaluated using 7-AAD apoptosis assay. Treated cells were transferred to FACS tubes (Falcon), washed twice with cold FACS solution (1% fetal bovine serum, 0.05% sodium azide in PBS) and pelleted at 200 × g at 4 ° C. Resuspend in cold FACS solution so that the final concentration of the cells is 1-2 × 10 ⁷ cells / ml. To stain this, 0.1 ml of resuspended cells were mixed with 7-AAD to a final concentration of 2 μg / ml, followed by incubation for 20 minutes in the dark at 4 ° C. Finally, the stained cells were washed twice with cold FACS solution, resuspended in 0.5 ml FACS solution and analyzed by BD LSR flow cytometer (manufactured by Beckton Dickison).

1 shows experimental results over time when activated T cells responded to TAB4 (anti-TAIP) -mediated apoptosis signals. Splenocytes of mice were activated with Con-A and maintained in IL-2 containing medium. Activated T cells were drained and resuspended and contacted with control hamster IgG in the presence of anti-hamster IgG antibody as TAB4 monoclonal antibody or crosslinker. On the first day, the ability of TAIP crosslinking to induce low levels (6.5%) of apoptotic cell death was demonstrated. However, TAB4-induced apoptosis increased to 17% at day 2, peaked at 52% at day 4, and decreased to 44% at day 6. Compared with cultures treated only with IL-2, the control hamster IgG did not specifically induce apoptotic death of T cells. Anti-CD3 (positive control) induced apoptosis of T cells at 38% 48 hours after activation (data not shown).

Example 4: Expression of TAIP Antigens in Different Tissues

Cells were washed twice with cold FACS solution (1% bovine fetal serum in PBS, 0.05% sodium azide) and pelleted at 200 ° C. at 4 ° C. in a FACS tube (Falcon). The cells were resuspended in cold FACS solution at a final concentration of 1 × 10 ⁷ cells / ml, and 0.1 ml of the resuspended cell aliquots in FACS tubes (Falcon) were used for each assay. By surface staining, TAB4 monoclonal antibody or control hamster Ig was added to the cells at a final concentration of 2 μg / ml and the mixture was incubated for 30 min in 4 ° C. dark. The cells were washed once with cold FACS, then (1) spleen cells were chromium-conjugated anti-CD3 antibody (2 μg / ml), FITC-conjugated anti-hamster Ig in cold 100 μl FACS solution, and Stained with PE-conjugated anti-CD8 / CD4 / CD19 / CD11b / pan-NK / IA / IE / Mac-3 antibody (2 μg / ml), and (2) thymic cells in cold 100 μl FACS solution Staining was performed using FITC-conjugated anti-hamster Ig, PE-conjugated anti-CD8, and cytochrome-conjugated anti-CD4 antibodies (2 μg / ml). The reaction was carried out for 30 minutes in the dark at 4 ℃. Finally, the stained cells were washed twice with cold FACS solution, resuspended in 1 ml FACS solution and analyzed using a BD LSR flow cytometer (manufactured by Beckton Dickison).

3 and 4 show the distribution of TAIP antigen in splenocytes and thymic cells of various subpopulations by FACS analysis. As shown in FIG. 3, CD19 ⁺ B cells expressed a small amount of TAIP protein on the surface at detectable levels. Large amounts of TAIP protein were detected in the CD3 ⁺ T cell and NK cell fractions. A large amount of TAIP protein was expressed in most of CD4 ⁺ , CD8 ⁺ and CD4 ⁺ 8 ⁺ thymic T cells. On the other hand, TAIP proteins CD4 ^- was expressed only small clusters of thymus T cells (Fig. 4) ^- 8.

Tissues from B6 and BALB / c mice, including brain, thymus, heart, lung, liver, stomach, kidney, spleen and skin, were collected and fixed overnight at room temperature with 10% formaldehyde and embedded in paraffin blocks. . 4 μm thick tissue sections were prepared from the paraffin block using a Leica RM 2135 microtome, dispersed in 45 ° C. water, and placed on coated slides. This slide was dried at 37 ° C. and prepared for the next experiment.

The wax of the slide containing paraffin sections of the tissue was removed, dried by successive passage of xylene-100% ethanol according to standard protocols and finally stored in 100% ethanol. The sections were sequentially passed through 100% ethanol-90% ethanol-85% ethanol-70% ethanol-PBS according to standard protocols and rehydrated in the final PBS solution. The following reactions were all carried out in a humid box. The crude sections were incubated in blocking buffer (1% normal goat serum) for 1 hour at room temperature (or overnight at 4 ° C.) to block nonspecific binding. The blocking buffer was removed and TAB4 or normal hamster Ig (diluted at 1: 200) was added to the tissues and continued incubation at room temperature for 1 hour (or overnight at 4 ° C.). The sections were washed twice with PBS for 5 minutes each time to remove the primary antibody, reacted with an anti-hamster Ig of alkaline phosphatase-conjugated goat diluted at a ratio of 1: 250, and incubated for 1 hour at room temperature. . Again, the sections were washed twice with PBS twice for 5 minutes each time to remove antibody-enzyme conjugates and developed for 30 minutes in the dark at room temperature using BCIP / NBT substrate solution. The sections were washed again with PBS to remove excess enzyme substrate, dehydrated by passing through PBS-ethanol-xylene in turn and mounted on the microscope.

As a result, it was shown that TAIP protein was expressed only in bone marrow-derived tissue among the tissues used in the experiment.

Example 5: Biotinylation of Cell Surfaces and Immunoprecipitation of TAIP Antibodies

5 × 10 ⁷ RL♂1 or NIH-3T3 cells were placed in 1 ml PBS containing 0.5 mg / ml sulfo-NHS-biotin (Pierce) for 30 minutes on ice to biotinylate the surface. The cells were placed on ice with 0.5 ml of DMEM (Dulbecco's modified Eagle's medium, Life Technologies, Inc.) for 10 minutes to terminate the reaction. Cells were washed once with 1 ml of DMEM and then twice with 1 ml of PBS (phosphate-buffered saline).

Labeled cells were treated with cold lysis buffer (1% Triton X-100, 20 mM Tris-HCl, pH 8.0, 160 mM NaCl, 1 mM CaCl ₂ ) containing the complete protease inhibitor cocktail (Roche). For 15 minutes, the cells were lysed at a density of 5.0 × 10 ⁷ cells / ml, and the insoluble material was pelleted at 10,000 × g for 10 minutes. All of the above and below steps were performed at 4 ° C. Upon immunoprecipitation, the lysate was left in advance for 30 minutes with 50 μl of filled protein G-Sepharose (Amersham Pharmacia Biotech) to remove nonspecific binding proteins. Beads were pelleted and aliquots of supernatant (typically equivalent to 5.0 × 10 ⁷ cells) were placed with 20 μl of protein G-Sepharose pre-injected with 10 μg of mAb TAB4 or IgG obtained from normal serum of hamsters. . It was left at 4 ° C. for 4 hours and then the resin was washed 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) Bumin) and washed twice with a similar wash buffer containing 250 mM NaCl instead of 400 mM NaCl. Proteins that specifically bound TAB4 were eluted with 50 μl of 1 × SDS sample buffer. Eluted protein was separated by 8% SDS-PAGE and transferred to nitrocellulose membrane (Millipore). From the membrane, biotinylated proteins were analyzed using peroxidase-conjugated Avidin (PharMingen) and developed with a chemiluminescence reagent (NEN ^™ Life Science Products).

As shown in FIG. 2, biotinylated surface protein of about 120 kD molecular weight was identified in RL.1 cells (TAIP ⁺ T cells) by TAB4, but not in 3T3 cells (TAIP ⁻ cells). In contrast, the protein of 120 kDa was not detected in protein G Sepharose coated with normal serum of hamsters. These results suggest that 120 kDa protein is recognized as antigen by the monoclonal antibody TAB4 at the surface of T cells.

Example 6: Depletion of T Cells In Vivo

To investigate the in vivo effects of TAB4 on T cells and other cell populations, 300 μg of TAB4 or control hamster Ig was injected intraperitoneally of mice and on day 4 the total cell count and cell surface markers by FACS were analyzed. To do this, splenocytes, thymic cells and peripheral blood mononuclear cells were harvested.

For FACS analysis, the cells were fixed with 2% paraformaldehyde for 20 minutes at 4 ° C., washed twice and resuspended in cold FACS solution at a final concentration of 1 × 10 ⁷ cells / ml. For each assay 100 μl of resuspended cell aliquots in FACS tubes (Falcon) were used. TAB4 or control hamster Ig was added to the cells at a final concentration of 2 μl / mg and the mixture was left in the dark at 4 ° C. for 30 minutes. The cells were washed once with cold FACS and (1) spleen cells were cytochrome-conjugated anti-CD3 antibody (2 μg / ml), FITC-conjugated anti-hamster Ig, and PE in 100 μl of cold FACS solution. Reacted with conjugated anti-CD8 / CD4 / CD19 / CD11b / pan-NK / IA / IE / Mac-3 antibody (2 μg / ml), and (2) thymic cells were FITC-conjugated in 100 μl of cold FACS solution Were reacted with anti-hamster Ig, PE-conjugated anti-CD8, and cytochrome-conjugated anti-CD4 (2 μg / ml). The reaction was carried out for 30 minutes in the dark at 4 ℃. Finally, the stained cells were washed twice with cold FACS solution, resuspended in 1,000 μl FACS solution and analyzed using a BD LSR flow cytometer (manufactured by Beckton Dickison).

After 4 days of injection, the percentage of CD3 ⁺ T cells in peripheral blood leukocytes (PBL) decreased from 36.7% in control mice to 4.1% in mice treated with TAB4 (Table 2). Upon treatment with TAB4, the total number of splenocytes decreased slightly. However, the number of CD3 ⁺ T cells in mice treated with TAB4 decreased by 62%, NK cells by 50%, and the total number of CD19 + B cells slightly increased. The total number of thymic cells recovered from TAB4-treated mice was only 48% (52% reduction) of the levels seen in the control group. In addition, all CD8 +, CD4 + CD8 + and CD4-CD8-T cells, except for CD4 + T cells, were reduced and the CD4 + CD8 + subpopulations were significantly affected (64.7% reduction).

(Table 2)

spleen × 10 ⁶ Untreated Normal Hamster Ig TA-B4 treatment Depletion Rate (%) Whole spleen cells 123 93.3 105 14.6 CD3 ⁺ T Cells 32.8 28.4 12.4 62.2 CD3 ^- CD19 ⁺ 72.2 53.4 72.9 -0.8 CD3 ^- NK ⁺ 3.6 5.4 1.80 50

Peripheral Blood White Blood Cells Untreated Normal Hamster Ig TA-B4 treatment Depletion Rate (%) CD3 ⁺ T Cells 36.7% 36% 4.1% 88.8%

Thymus × 10 ⁶ Untreated Normal Hamster Ig TA-B4 treatment Depletion Rate (%) Whole thymocyte 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

(Data from three experiments)

Example 7: Anti-TAIP Antibodies Do Not Induce Secretion of IL-2 or TNF-α

Balb / c mice (H-2d) were injected intraperitoneally with 300 μg of TAB4 or control hamster Ig. Seven days after injection, splenocytes were isolated and used as responders in culture with C3H (H-2K) splenocytes (as stimulant) treated with mitomycin C. After 3 days, the culture supernatants were harvested and the IL-2 content was measured by ELISA set (PharMingen). As shown in FIG. 5, production of IL-2 was further inhibited in responder cells derived from mice treated with TAB4 compared to control mice. In addition, as a result of analyzing the plasma levels of IL-2 and TNF-a, no significant difference in the level of IL-2 (or TNF-a) was observed in the serum of control and TAB4 treated mice. Since the production of IL-2 is pivotal to T cell activity, these results suggest that TAIP-specific antibodies, such as TAB4, may mediate unnecessary T-cell mediation of T cells and controls associated with autoimmune disease and transplant rejection in vivo. It can be seen that it can be used to modulate the immune response.

Example 8 Use of Anti-TAIP Antibody to Prevent Transplant Rejection

Mice from 8 to 12 weeks old (obtained from the Jackson Laboratory) were anesthetized with aceprozin maleate (Acepromazin maleate, Fermenta Animal Health Co., Kansas City, Mo.). Seven days prior to skin transplantation into the mice, 500 μg of TAB4 or isotype control antibody was intraperitoneally administered to recipient C57BL / 6 mice (H-2 ^b ) that did not excise the thymus. Seven days after dosing, the lateral flanks of the antibody-pretreated C57BL / 6 mice were implanted with lateral flank skins of the fully heterogeneous mismatched Balb / cj mice (H-2 ^d ). Seven days after transplantation, the mice were again injected with 500 μg of TAB4 or isotype control antibody. After transplantation, the mice were observed daily. If 50% of the donor skin was necrotic, the transplant was considered rejected. 7 shows graft survival (n = 8). The results show that TAB4 antibody treatment prolongs the survival of allogeneic skin grafts.

Example 9: TAIP Identification as PSGL-1

P-selectin glycoprotein ligand-1 (PSGL-1), also referred to as CD162, is the major P-selectin ligand expressed on 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). Biochemical properties such as the molecular weight and dimerization trend of TAIP suggest that TAB4 may be similar to PSGL-1. To investigate the relationship between these two antigens, 1) whether the antigen precipitated by TAB4 can be recognized by commercially available anti-PSGL1 antibodies, and 2) the anti-PSGL-1 antibody is directed to TAB4 from cell lysate. It was tested whether it could be exhausted.

RL♂1 T cells were lysed in 1% Triton X-100, 20 mM Tris-HCl, pH 8.0, 160 mM NaCl, containing a complete protease inhibitor cocktail at a density of 1.0 × 10 ⁸ cells / ml. , 1 mM CaCl ₂ ) was dissolved for 1 hour, and the insoluble material was pelleted at 10,000 × g for 10 minutes. Both the above and subsequent steps were performed at 4 ° C. Melt equivalents of 5.0 × 10 ⁷ cells were previously obtained from 10 μg of anti-PSGL-1 mAb (clone 2PH1, PharMingen, San Diego, CA), anti-TAIP mAb, TAB4, or IgG from normal hamster serum. Place with 20 μl of injected protein G-Sepharose. It was placed at 4 ° C. for 4 hours and then washed 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). The beads were washed 5 times and again washed twice with the same wash buffer containing 250 mM NaCl instead of 400 mM NaCl. Bound proteins were eluted with 40 μl of 1 × SDS sample buffer. Eluted protein was separated by 6% SDS-PAGE and transferred to nitrocellulose membrane. The membrane was immunoblotted with anti-PSGL-1 mAb and visualized with peroxidase-conjugated goat anti-rat IgG (H + L) followed by chemiluminescent agent (Renaissance, NEN).

Surface-biotinylated RL♂1 T cells were lysed in lysis buffer at a density of 1.0 × 10 ⁸ cells / ml. The cell extracts were incubated overnight at 4 ° C. with 20 μg of antibody bound to 40 μl of protein G-Sepharose. Depletion was carried out using anti-PSGL-1 mAb (2PH1) or IgG from control rats and normal serum from TAB4 or control hamsters. Depleted lysates were further immunoprecipitated using either TAB4 or anti-PSGL-1-mAb, respectively. Immunoprecipitates were separated on 6% SDS-polyacrylamide gels and detected by fluorography. As shown in FIG. 6, anti-PSGL-1 antibodies may deplete TAIP protein from T cell lysate. In addition, proteins that have been immunoprecipitated with anti-TAIP antibodies (TAB4) can be recognized by anti-PSGL-1 antibodies in Western assays.

Example 10 Induction of Apoptosis of Human T Cells by Anti-PSGL-1 Antibody

In order to confirm the role of PSGL-1 in the apoptosis of human T cells, experiments were performed over time when activated human T cells responded to PSGL-1-mediated apoptosis signals. Human T cells were stimulated with phytohemagglutinin (PHA) mitogen and proliferated in medium containing IL-2. Activated T cells were collected and then administered anti-PSGL-1 in the presence of IL-2 and crosslinked antibodies.

Peripheral blood was collected from healthy adults, heparin was added, and the mononuclear cells (PBMC) of peripheral blood were enriched on the basis of differential density using Ficoll-Paque Plus (Pharmacia Biotech). The PBMCs were activated for 48 hours with 1% PHA (Life Technologies, GibcoBRL) and then maintained in human recombinant IL-2 (5 ng / ml) for the duration of the assay. To assess the apoptosis induction ability of the anti-human PSGL-1 antibody, the activated cells were (1) 1 μg of anti-PSGL-1 antibody clone KPL-1 (BD PharMingen) and anti-mouse in rabbits crosslinked. Ig (0.5 μg / ml) (Jackson ImmunoResearch Laboratories); (2) purified mouse Ig, a control group of the isotype, and rabbit anti-mouse Ig, a crosslinker; Or (3) anti-mouse Ig alone of the rabbit as a crosslinker. After 6 hours of treatment, the percentage of early apoptotic cells was measured according to FACS staining with anti- Annexin V (BD PharMingen) and PI (Sigma).

As shown in FIG. 8, signaling induced by PSGL-1 using anti-PSGL-1 antibodies and crosslinkers resulted in significant levels of apoptosis in PHA-activated human PBMCs (usually T cells). . The percentage of apoptotic cells in anti-PSGL-1 treated medium increased from 8.5% on day 3 to 24% on day 8. Both matched control and isotype crosslinked antibodies of the isotype had no effect on these cells.

Example 11 Use of Anti-PSGL-1 Agonist Antibodies to Treat Autoimmune Diseases

NOD-obesed mice, which are animals susceptible to autoimmune diabetes, were raised under standard conditions. Spontaneous diabetes developed in about 20 weeks old NOD mice. As an experimental group, anti-PSGL-1 antibody (TAB4) was administered intraperitoneally of mice three times at 300 μg per 14, 15 and 17 week old mice. Mice at 24 and 26 weeks of age were injected two additional doses. The same amount of hamster Ig was also administered to the control group. After these mice were 15 weeks old, the glucose uria was monitored twice weekly by a Medi-test glucose strip (Macherey-Nagel, Germany). If fasting urine glucose concentration was 300 mg / l or more in two consecutive measurements, diabetes was considered.

As shown in Figure 9, TAB4 (anti-PSGL-1) antibody treatment group showed a significant protective effect, compared to the control antibody treatment group. Therefore, anti-PSGL-1 antibody treatment can suppress autoimmune T cells and delay the onset of type I diabetes.

Example 12 Binding of P-Selectin, E-Selectin and L-Selectin to Activated T Cells

To determine the binding ability of selectin (P-selectin, E-selectin and L-selectin) to activated T cells, freshly prepared splenocytes were activated from C57BL / 6 mice and recovered on days 2, 4 and 6. It was. Inactivated T cells (ie freshly prepared splenocytes on day 0) were also analyzed. Day 2 samples consisted of 3 x 10 ⁶ cells / ml splenocytes activated with 2 ug / ml concanavalin A (ConA) dissolved in DMEM + 10% FBS for 2 days. Living cells were isolated by Ficoll concentration gradient separation. Day 4 samples were obtained from cells that were activated with Con A for 3 days and maintained in medium containing 5 ng / ml of IL-2 the next day. On day 6 samples were obtained from cells activated for 3 days with Con A and then maintained in medium containing IL-2 5 ng / ml for 3 days.

To verify the samples by FACS analysis, 2 x 10 ⁵ 0, 2, 4 and 6 cells per well were collected from Fc of human IgG1 at 2-fold serial dilution concentrations from 20 ug / ml to 0.156 ug / ml. Mouse P-selectin, E-selectin or L-selectin (R & D Systems, Minneapolis, MN) fused with the portion was placed at 40 ul / well for 30 hours at 4 ° C. After incubation, cells were lx FACScan buffer (1 × PBS and 2% FBS free of calcium and magnesium ions, from Biochrom AG of Berlin). Samples were prepared with a second reaction of anti-Thy1.2 95 ul / well and 3.25 ug / ml (FITC-anti human IgG, which is specific to Fc fragment, purchased from Jackson hnmunoResearch Laboratories, Inc., West Grove, PA). An additional 30 minutes at 4 ° C. was followed by washing with 1 × FACS buffer.

FACSCalibur analysis results are shown in FIG. 10. At a concentration of 20 ug / ml, binding of P-selectin to activated mouse T cells gradually increased, peaking at 4 days, slightly decreasing at 6 days. Binding of E-selectin increased significantly between 2 and 4 days, maintaining peaks on day 6. No L-selectin binding to activated mouse T cells was observed, and no change was observed during the activation period, ie between 0 and 6 days. The result of L-selectin may be due to the low binding affinity of L-selectin for its ligand. Similar results were obtained when three kinds of selectins were used in the experiment at low concentration.

Example 13: Multiple Forms of E-Selectin and P-Selectin Induce Apoptosis of Activated T Cells

96-well plates (NUNC) were coated overnight at 4 ° C. with 20 ug / ml human anti-Fc Ig 50 ul dissolved in 1 × PBS, blocked at 37 ° C. for 2 hours with 1% BSA, 50 ul of the selectin-human Fc fusion (0.063-5 ug / ml) was maintained at room temperature for 2 hours. In all experimental steps, each well was thoroughly washed 5 times with 1 × PBS. T cells 2 × 10 ⁵ activated using ConA in advance for 4 days were added to each well and incubated at 37 ° C. for 5 hours, followed by centrifugation at 200 × g at 4 ° C. for 5 minutes. The obtained pellet containing activated T cells was incubated with Annexin V-biotin conjugate for 15 minutes at room temperature, and then incubated with avidin conjugate (SA-beta-gal, 1: 5000 dilution) at 37 ° C for 30 minutes. . For each reaction, each well was washed three times with Annexin V binding buffer. Development was carried out overnight at 4 ° C with 110 ul of Z-buffer mixture (54 ul 2-mercaptoethanol in 20 ml of Z-buffer) and 30 ul of ONPG (0.04 g / ml). Optical density was measured and recorded at 420 nm.

Selectin-induced apoptosis levels of T cells activated with ConA increased the concentration of P-selectin (FIG. 11A) or E-selectin (FIG. 11B) fused with Fc of human IgG1 (0.063 ug / ml-5 ug / ml). Increased by). Hamster antibody TAB4 induced apoptosis of activated T cells (Example 1) and was used as a positive control in this experiment. Negative controls anti-human Fc, human IgG (HIg) and BSA did not cause apoptosis. In the presence of L-selectin human Fc fusion protein (FIG. 11C), L-selectin did not bind well to activated T cells, consistent with failure of binding L-selectin to activated T cells (Example 12).

In sum, the fusion protein bound to the plate containing the 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 Proteins Induces Apoptosis of Activated T Cells

As described above, selectin of mice (P-selectin, E-selectin and L-selectin) was fused to the Fc region of human IgG1 to form an available dimeric fusion protein. To evaluate whether the soluble selectin can induce apoptosis of activated T cells, the same experiment as in Example 13 was performed except that the anti-human Fc Ig bound to the plate was omitted. When the soluble form of the P-selectin fusion protein (dimer) alone was present, apoptosis of almost trace or small amounts of activated T cells was achieved (FIG. 12). However, the addition of cross-linker (anti-human Fc) substantially increased apoptosis activity to the level of apoptosis induced in the presence of antibody bound to the plate. Neither anti-human Fc, human Ig (HIg) nor BSA induced apoptosis.

Similar results as obtained in the P-selectin-Fc fusion protein could be obtained in the E-selectin-Fc fusion protein. In addition, consistent with the results of L-selectin (multiple forms) bound to the plate, the soluble form of the L-selectin fusion protein did not induce apoptosis of activated T cells.

Other embodiments

The present invention is disclosed with the detailed description, but the above description is intended to explain the present invention in detail and does not limit the scope of the present invention. Other aspects, advantages and modifications of the invention are included in the following claims.

Claims (39)

A pharmaceutical composition for the prophylaxis or treatment of a disease induced by a T cell mediated immune response selected from the group consisting of inflammatory diseases, autoimmune diseases, allergic or xenograft rejection, allergic diseases, and T cell cancer, A multimeric compound that binds to at least two P-selectin glycoprotein ligand 1 (PSGL-1) at the T cell surface, The multiple compound comprises two polypeptide chains, each of which comprises (i) a binding domain that binds to PSGL-1, and (ii) a heterologous amino acid sequence. Wherein the two polypeptide chains are linked through the heterologous amino acid sequence to form the multiple compound, By binding the multiple compounds to at least two PSGL-1 proteins on the surface of the T cells, signal transduction pathways are induced that result in the death of T cells, thereby preventing or reducing the T cell mediated immune response in the subject, The multiple compound is not an anti-PSGL-1 antibody or a fragment of an antibody that binds to PSGL-1, The binding domain of the polypeptide comprises a P-selectin extracellular domain or a PSGL-1 binding fragment thereof or a E-selectin extracellular domain or a PSGL-1 binding fragment thereof. Composition. The pharmaceutical composition of claim 1, wherein the multiple compound is a homo-multimeric compound or a hetero-multimeric compound. delete The pharmaceutical composition of claim 1, wherein the heterologous amino acid sequence comprises a binding site for a surface receptor of a cell. delete delete delete delete delete The pharmaceutical composition of claim 1, wherein the polypeptide chains are covalently linked through heterologous amino acid sequences to form multiple compounds. The pharmaceutical composition of claim 10, wherein the covalent bond is a disulfide bond. The pharmaceutical composition of claim 1, wherein the heterologous amino acid sequence comprises an immunoglobulin heavy chain constant region. The pharmaceutical composition of claim 1, further comprising a substance that binds to multiple compounds through heterologous amino acid sequences and induces crosslinking of a plurality of PSGL-1 antigens on the surface of T cells. delete delete delete delete delete The pharmaceutical composition of claim 1, wherein the T cells are activated T cells. delete delete delete As a multiple compound that binds at least two PSGL-1 proteins at the surface of T cells or NK cells, The multiple compound comprises two polypeptide chains, each of which comprises (i) a binding domain that binds to PSGL-1, and (ii) a heterologous amino acid sequence; The polypeptide chains are linked through the heterologous amino acid sequence to form multiple compounds having the purpose of inducing death of T cells or NK cells, By binding the multiple compounds to at least two PSGL-1 proteins on the surface of the T cells or NK cells, signal transduction pathways are induced that result in the death of the T cells or NK cells, The multiple compound is not a fragment of the PSGL-1 antibody or an antibody that binds to PSGL-1, and also The binding domain of the polypeptide comprises a P-selectin extracellular domain or a PSGL-1 binding fragment thereof, or comprises an E-selectin extracellular domain or a PSGL-1 binding fragment thereof. compound. The multiple compound of claim 23, wherein the multiple compound is a homo-multiple compound or a hetero-multiple compound. delete The multiple compound of claim 23, wherein the heterologous amino acid sequence comprises a binding site for a surface receptor of a cell. delete delete delete delete delete The multiple compound of claim 23, wherein the polypeptide chains are covalently linked through a heterologous amino acid sequence to form multiple compounds. 33. The multiple compound of claim 32, wherein said covalent bond is a disulfide bond. The multiple compound of claim 23, wherein the heterologous amino acid sequence comprises an immunoglobulin heavy chain constant region. The multiple compound of claim 23, wherein the multiple compound is administered with a substance that binds to the multiple compound via heterologous amino acid sequences and induces crosslinking of a plurality of PSGL-1 antigens on the surface of T cells. The multiple compound of claim 23, wherein the multiple compound induces death of activated T cells. delete delete A multiple compound that binds to at least two PSGL-1 proteins at the surface of T cells, wherein the multiple compounds comprise two polypeptide chains, each of which comprises: (i) a binding domain that binds to PSGL-1 And (ii) a heterologous amino acid sequence, wherein the polypeptide chains are linked through the heterologous amino acid sequence to form multiple compounds, wherein the multiple compounds form at least two PSGLs on the T cell surface. Binding to -1 proteins leads to signal transduction pathways leading to death of T cells; And A kit comprising instructions for use of said multiple compounds for the treatment of inflammation, autoimmunity, transplant rejection, allergic symptoms, or T cell cancer, The multiple compound is not an anti-PSGL-1 antibody or a fragment of an antibody that binds to PSGL-1, The binding domain of the polypeptide comprises a P-selectin extracellular domain or a PSGL-1 binding fragment thereof or an E-selectin extracellular domain or a PSGL-1 binding fragment thereof. .

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