JP5808879B2 - Modifier of P-selectin glycoprotein ligand 1 - Google Patents

Description

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

BACKGROUND OF THE INVENTION Control of undesirable immune responses is an important issue in the treatment of diseases such as inflammatory diseases, autoimmune diseases, graft rejection, allergic diseases, and T cell-derived cancer. It is. Overly aggressive T cell activity can be controlled by immunosuppression or by induction of immune tolerance. Tolerance is defined as a condition in which the immune system becomes unresponsive to an antigen, but immunosuppression that suppresses the immune response to the antigen generally requires continued use of the drug. In organ transplantation, T cells play an essential role in the immune response to alloantigens. Current immunosuppressive regimes generally corticosteroids, cyclosporine or rapamycin that block transcription of IL-2, which is an important growth factor for T cells, or inhibit IL-2-dependent proliferation Includes the use of However, as T cell depleting agents (eg, CD3, CD4, CD8) or inhibitors of cytokine signaling or T cell co-stimulatory pathways (eg, CD25, B7-1, B7-2, CD152) Many monoclonal antibodies acting either as CTLA4) have been shown to be effective in suppressing the occurrence of rejection while limiting side effects or toxicity. Some of these drugs have shown some success in treating autoimmune diseases and extending graft survival.

  Apoptosis is widely thought to be crucial to maintaining the proper functioning of the immune system and the primary mechanism of removing unwanted cells (Kabelitz et al. Immunol. Today 14: 338-340 (1993 Raff, Nature: 356: 397-399 (1992)). Various signals derived from the inside or outside of a cell affect cell survival and death. Antibodies against T cell surface molecules such as Fas (or CD95, MW = 43 kD), TNFR2 (MW = 75 kD), CD2 (MW = 45 kD) and CTLA-4 (MW = 33 kD) induce T cell 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 were hampered by the fact that these two molecules are expressed not only in immune cells but also in some other important organ systems such as the liver. . This expression pattern potentially 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)).

  Selectins, integrins and immunoglobulin (Ig) superfamily members are the three major anchoring molecules important in the interaction of leukocytes and platelets with themselves or with the extracellular matrix and vascular endothelium (Springer, Nature 346: 425 (1990); Osborn, Cell 62: 3 (1990); Hynes, Cell 69:11 (1992)). Anchor molecules on one cell type often bind to other anchor molecules expressed on different cell types to form a ligand-receptor pair.

  The selectin family includes P-selectin (also known as CD62, CD62P, GMP140, and PADGEM), E-selectin (also known as ELAM-1 and CD62E), and L-selectin (LECAM-1 , Mel-14, LAM-1, and CD62L). The selectins are highly similar, with quite a number of similarities found in the 120 amino acid N-terminal domain, EGF-like domain, complement regulatory proteins. It is composed of multiple short consensus repeat (SCR) domains followed by a transmembrane domain and a short cytoplasmic tail (Siegelman et al., Science 243: 1165-1172 (1989); Laskey 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); Bevilancqua 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 (1983), Camerini et al., Nature 280: 496-498 (1989)). The selectins have overlapping portions but different 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); Breg 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 initial leukocyte-endothelial cell and platelet-leukocyte adhesive interactions during inflammation (Bevilacqua et al., 1993, supra). All three selectins have been shown to be involved in the initial “rolling” interaction between activated endothelium and leukocytes (von Andrian et al., Proc. Natl. Acad. Sci. USA 88: 7538- 7542 (1992); 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 on activated platelets and endothelial cells binds to cell surface proteins on most leukocytes (McEver et al., J. Biol. Chem. 250: 9799-9804 (1984)). E-selectin expressed on cytokine-activated endothelial cells (eg, after 6-8 hours of TNF-α or IL-1 stimulation) binds to cell surface proteins on 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 on most leukocytes binds to cell surface proteins on some endothelial cells and other leukocytes (Gallatin et al., Nature 304: 30-34 (1983); Breg et al. , Immunol. Rev. 108: 5-18 (1989); Breg 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 have been shown to bind to PSGL-1, a cell surface protein whose expression is largely restricted to leukocytes, particularly T cells and NK cells. Post-translational modification of PSGL-1 is required for binding to P-selectin, E-selectin, and L-selectin (McEver et al., J. Clin. Invest., 1997, supra).

SUMMARY OF THE INVENTION The present invention is based on the finding that T cells can be depleted by the involvement of the T cell surface antigen P-selectin glycoprotein ligand-1 (PSGL-1) and / or can be induced to undergo apoptosis. Is. T cell depletion may be particularly effective in the treatment of conditions associated with excessive or undesirable T cell mediated immune responses or excessive or undesirable T cell proliferation. For example, T cell depletion is an undesirable T cell activity associated with inflammatory disease, autoimmune disease, transplant rejection, allergic disease, and / or T cell-derived cancer, or May cause inhibition or elimination of proliferation. The present invention encompasses methods for using PSGL-1 function modifiers to prevent or suppress T cell-mediated immune responses as well as screening methods for PSGL-1 function modifiers.

  In one aspect, the invention features a method for preventing or suppressing an immune response mediated by a T cell in an individual. The method includes the following steps: selecting an individual having a condition characterized by or at risk of developing an immune response mediated by excess or undesirable T cells And binding of the compound to PSGL-1 on the surface of the T cell induces a signaling pathway that results in T cell death, thereby preventing or suppressing a T cell mediated immune response in the individual, Administering to the individual a compound that binds to PSGL-1 on the surface of T cells.

  The compound used in such a method includes an antibody or antigen-binding fragment thereof that specifically binds to PSGL-1. In one example, the compound is a monoclonal antibody that specifically binds to PSGL-1. In one embodiment, the method includes the further step of administering an agent that binds to the monoclonal antibody and induces cross-linking of multiple PSGL-1 antigens on the surface of the T cells.

  In some embodiments, the method includes inducing cross-linking of multiple PSGL-1 antigens on the surface of the T cell that induces a signaling pathway that results in T cell death.

In some embodiments, the method includes the following steps: (i) diagnosing having or at risk of developing a condition characterized by an immune response mediated by excess or undesirable T cells And (ii) two polypeptide chains, each of which comprises (a) a binding domain that binds to PSGL-1, and (b) the polypeptide chain is a multimeric compound administering to the solid the multimeric compound that binds to at least two PSGL-1 proteins on the surface of the T cell having a heterologous amino acid sequence linked by a heterologous amino acid sequence to form a multimeric compound In this case, the multimeric compound binds to at least two PSGL-1 proteins on the surface of the T cell, thereby causing the death of the T cell. Induced signal transduction pathway dripping, whereby the T cells in the individual to prevent or suppress the immune response mediated.

  The multimeric compound may be a homomultimeric compound or a heteromultimeric compound. The binding domain may be a P-selectin extracellular domain or a PSGL-1 binding fragment thereof, an E-selectin extracellular domain or a PSGL-1 binding fragment thereof, an L-selectin extracellular domain or a PSGL-1 binding thereof, as necessary. A fragment, an anti-PSGL-1 antibody or a PSGL-1-binding fragment thereof, a PSGL-1-binding polypeptide selected from a phage display library, or any combination of the above may be included.

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

  The heterologous amino acid sequence may optionally include a cell surface receptor binding domain such as an immunoglobulin heavy chain constant domain. In some embodiments, the polypeptide chain is covalently linked by a heterologous amino acid sequence, eg, a disulfide bond, to form the multimeric compound.

  In some embodiments, the method comprises the additional step of administering to the individual an agent that binds to the multimeric compound by a heterologous amino acid sequence and induces cross-linking of multiple PSGL-1 antigens on the T cell surface. May be included.

  In some embodiments, the methods described herein include selecting an individual diagnosed with an autoimmune disease. In other examples, the method includes selecting an individual diagnosed with an inflammatory disease. In other examples, the method includes selecting an individual who has received or will receive an allogeneic or xenograft. In another example, the method includes selecting an individual diagnosed with an allergic disease. In other examples, the method includes selecting an individual diagnosed with a T cell cancer.

  In some embodiments, the T cell is an activated T cell. In one example, the T cell is a CD4 + T cell. In other examples, the T cell is a CD8 + T cell.

  In some embodiments, the method detects the number of T cells in a first biological sample collected from an individual prior to administration of the compound (eg, multimeric compound) and after administration of the compound (eg, multimeric compound). Comparing the results with the number of T cells in a second biological sample collected from the individual.

  In some embodiments, the method detects T cell biological activity in a first biological sample collected from an individual prior to administration of the compound (eg, multimeric compound) and administers the compound (eg, multimeric compound). Comparing results with T cell biological activity in a second biological sample taken from a later individual.

  In some embodiments, the administration results in a depletion of at least 10% of the individual's activated T cells. In some embodiments, the administration results in at least 10%, 20%, 30%, 40%, 50%, or more depletion of the activated T cells of the individual.

  In some embodiments, the antibody or antigen-binding fragment or multimeric compound induces death of at least 10% of the individual's activated T cells after exposure to the antibody or antigen-binding fragment or multimeric compound. In some embodiments, such administration induces death of at least 10%, 20%, 30%, 40%, 50%, or more of the activated T cells of the individual. Cell death is measured at any time after exposure to the antibody or antigen-binding fragment or multimeric compound, eg, 1, 2, 3, 4, 5, 6, 7 or more days. May be.

  In another aspect, the invention features a method of inducing death of T cells or natural killer (NK) cells. The method comprises the following steps: providing TGL or NK cells expressing PSGL-1 on the cell surface; and binding to PSGL-1 on the surface of the T cell or NK cell A signal that causes T cell or NK cell death by binding the compound to PSGL-1 on the surface of the T cell or NK cell. Inducing a transmission path.

  Compounds used in such methods can include antibodies or antigen-binding fragments thereof that specifically bind to PSGL-1. In one example, the compound is a monoclonal antibody that specifically binds to PSGL-1. In some embodiments, the method includes contacting the monoclonal antibody with an agent that binds to the monoclonal antibody and induces cross-linking of multiple 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 its cell surface; and (ii) two polypeptide chains. Each of the polypeptide chains has (a) a binding domain that binds to PSGL-1, and (b) a heterologous amino acid sequence that is linked by a heterologous amino acid sequence so that the polypeptide chain forms a multimeric compound. Contacting the solid with the multimeric compound that binds to at least two PSGL-1 proteins on the surface of the T cell or NK cell, wherein at least on the surface of the T cell or NK cell Binding of the multimeric compound to two PSGL-1 proteins induces a signaling pathway that leads to death of the T cell or NK cell. .

  The multimeric compound may be a homomultimeric compound or a heteromultimeric compound. The binding domain may be a P-selectin extracellular domain or a PSGL-1 binding fragment thereof, an E-selectin extracellular domain or a PSGL-1 binding fragment thereof, an L-selectin extracellular domain or a PSGL-1 binding thereof, as necessary. A fragment, an anti-PSGL-1 antibody or a PSGL-1-binding fragment thereof, a PSGL-1-binding polypeptide selected from a phage display library, or any combination of the above may be included.

  The heterologous amino acid sequence may optionally include a cell surface receptor binding domain such as an immunoglobulin heavy chain constant domain. In some embodiments, the polypeptide chain is covalently linked by a heterologous amino acid sequence that forms the multimeric compound, such as a disulfide bond.

  In some embodiments, the method includes the additional step of administering to the individual an agent that binds to the multimeric compound by a heterologous amino acid sequence and induces cross-linking of multiple PSGL-1 antigens on the T cell surface. To do.

  In some embodiments, the method induces cross-linking of multiple PSGL-1 antigens on the surface of the T cell or NK cell, and the cross-linking induces a signaling pathway that results in death of the T cell or NK cell. Includes stages.

  In some embodiments of the methods described herein, the T cell is an activated T cell. In one example, the T cell is a CD4 + T cell. In other examples, the T cell is a CD8 + T cell.

  In some embodiments of the methods described herein, the method includes assessing the viability of the T cells or NK cells after contact with the compound (eg, a multimeric compound).

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

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

  In another aspect, the invention features a method for screening for a modulator of PSGL-1 function. The method includes the following steps: providing a cell expressing PSGL-1 on the cell surface; contacting the cell with a test substance; and after contacting the cell with a test substance Measuring the viability of the cells and determining whether the test substance is a modulator of PSGL-1 function.

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

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

  In one embodiment, the method includes inducing cross-linking of a plurality of PSGL-1 antigens on the surface of the cell that induces a signaling pathway that results in cell death.

  In one embodiment, the T cell is an activated T cell. In one example, the T cell is a CD4 + T cell. In other examples, the T cell is a CD8 + T cell.

  In one embodiment, the method includes the step of producing a quantity of the test substance and formulating the test substance in a pharmaceutically acceptable carrier.

  In another embodiment, the present invention relates to PSGL-1 on the surface of the T cell that induces a signaling pathway that results in T cell death by binding of the compound to PSGL-1 on the surface of the T cell. Compounds that bind; and symptoms associated with excessive or undesirable T cell-mediated immune responses or excessive or undesirable T cell proliferation, such as inflammation, autoimmunity, graft rejection, allergic symptoms, or T cell cancer Features a kit that includes instructions for using the compound to treat.

  In one embodiment, the kit comprises (i) two polypeptide chains, each of which is (a) a binding domain that binds to PSGL-1, and (b) the polypeptide chain is a multimeric compound. Induces a signal transduction pathway that results in T cell death by binding to at least two PSGL-1 proteins on the surface of the T cell. The multimeric compound that binds to at least two PSGL-1 proteins on the surface of the T cells; and (ii) excess or such as inflammation, autoimmunity, graft rejection, allergic symptoms, or T cell cancer Compounds for treating conditions associated with unwanted T cell mediated immune responses or excessive or unwanted T cell proliferation Including the instructions for use.

  An advantage of the present invention is that it can deplete T cells and / or induce T cell apoptosis without causing an associated undesirable or deleterious immune response. For example, in some embodiments, administration of an anti-PSGL-1 antibody or multimeric compound to an individual described herein does not cause an undesirable increase in the level of inflammatory cytokines such as IL-2 or TNF-α. .

  Another advantage of the present invention is that it results in T cell depletion through the use of agonist compositions that induce T cell apoptosis. Thus, the present invention relates to antagonist compositions that act by binding immune receptors and preventing immune activation mediated by the receptors (eg, antagonistic anti-PSGL-1 antibodies or antagonistic soluble selectin fragments) ) Provides an active immunosuppressive method rather than passive immunosuppression.

Another advantage of the present invention is that it is possible to target the cell surface protein, PSGL-1, whose expression is fairly limited to leukocytes, and in particular to T cells and NK cells. Thus, the compounds described herein do not induce significant levels of apoptosis in other cell types such as liver cells. T cells and NK cells aimed at selective depletion (significant CD3 ⁻ for graft rejection) without significantly inducing a systemic cytokine response that threatens survival or damaging other organ systems Targeting of cell types is a desirable property of immunosuppressive agents.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and 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 described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of a jargon mismatch, the present specification will control. In addition, the materials and methods described are exemplary only and do not limit the invention in any way.

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

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows the results of an experiment over time that examined when activated T cells gained sensitivity to apoptotic signals mediated by TAB4 (anti-PSGL-1 monoclonal antibody).

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

  FIG. 3 shows PSGL-1 antigen expression on splenic CD4 + T cells, CD8 + T cells, CD19 + B cells, and NK cells.

^4, CD4 ^+, CD8 ^+, and ^CD4 + ^{8 +,} and CD4 ^- 8 ^- shows the expression of PSGL-1 antigens on thymocytes.

  FIG. 5 shows mixed lymphocytes using spleen cells isolated from Balb / c mice treated with TAB4 (or hamster Ig) as a responder and H2 mismatched C3H spleen cells as a stimulator. It shows IL-2 levels produced by sphere incubation.

  FIG. 6 shows that (A) the protein immunoprecipitated with the TAB4 antibody can be recognized by a commercially available anti-PSGL-1 antibody, and (B) the T cell soluble product is precleared with the anti-PSGL-1 antibody. This shows Western blot analysis showing that the protein recognized by TAB4 can be depleted.

  FIG. 7 shows the percentage of surviving grafts in C57BL / 6 mice that received skin grafts from Balb / c mice and were treated with anti-PSGL-1 antibody (filled diamonds) or control antibody (open squares). ).

  FIG. 8 shows the time course of the percentage of apoptotic T cells after treatment of activated human peripheral blood mononuclear cells with anti-human PSGL-1 antibody.

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

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

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

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

DETAILED DESCRIPTION The present invention relates to a method for modulating T cell activity by modulating the function of PSGL-1 molecules on the surface of T cells. Binding of the agonist compositions described herein to PSGL-1 can cause T cell depletion and / or induce T cells to undergo apoptosis. Accordingly, these agonist compositions are useful as therapeutic agents to control immunity related conditions such as inflammatory diseases, autoimmune diseases, transplant rejection, allergic symptoms, and / or T cell induced cancers. is there. The agonist compositions are also useful for depleting T cells from biological samples where the presence or activity of T cells is not desired.

PSGL-1 Protein PSGL-1 is a cell surface anchoring molecule expressed on neutrophils, T and B lymphocytes, NK cells, monocytes, dendritic cells, and primitive human CD34 hematopoietic progenitor cells. Being able to interact with secretin, PSGL-1 mediates leukocyte rolling at the endothelium and leukocyte extravasation to inflamed tissues. PSGL-1 mediated binding or migration of T cells to E- and P-secretin is specifically regulated. For example, the appearance of CLA (cutaneous lymphocyte antigen) epitopes is thought to be induced on T cells naive to memory transition. Not only activated helper 1 but also helper 2T cells express functional PSGL-1 and allow migration to the inflammatory domain of the skin.

  PSGL-1 is a sialomucin that must be specifically sialylated, fucosylated, and sulfated to bind to P-selectin. The PSGL-1 molecule exists in isoforms characterized by different degrees of glycosylation and sulfation sites at its N-terminus. Resting peripheral blood T and B cells, lymphoid cell lines and in vitro activated peripheral blood T cells express the same level of PSGL-1. However, only activated T cells show a functional form of PSGL-1 and greedyly bind to P-selectin. Such activation-dependent binding activity is thought to be the result of different post-translational modifications, as suggested by increased levels of alpha (1,3) fucosyltransferase activity in activated T cells. The PSGL-1 isoform also exhibits different affinities for L-secretin and E-secretin. For example, human T cells exhibiting CLA positive isoforms can tether and rotate to both E- and P-secretin, but T cells expressing PSGL-1 without the CLA epitope only bind to P-selectin. To do. Furthermore, the binding of P-selectin and PSGL-1 is conditioned on the presence of a terminal decapeptide that includes three tyrosine residues for sulfation and one threonine residue for glycosylation.

PSGL-1 protein can be prepared by recombinant methods and / or by isolating native PSGL-1 protein from biomaterials. Recombinant PSGL-1 protein can be produced in prokaryotic or eukaryotic cells, either in vitro or in vivo. A nucleic acid encoding PSGL-1 can be used for recombinant production of proteins (see, eg, GenBank ^™ accession number NM_003006 for an example of a nucleic acid encoding a PSGL-1 polypeptide). Antibodies against PSGL-1 are also known and can be used for antigen purification (see, eg, Herron et al. (2000) Science Jun 2; 288 (5471): 1653-56; WO 00/25808) and And / or can be used in the methods described herein. PSGL-1 is further, but 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.

  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. In addition or alternatively, recombinant production of PSGL-1 may be achieved by co-transfection with a nucleic acid encoding PACE and / or a nucleic acid encoding tyrosine sulfotransferase to remove the propeptide. .

  Anti-PSGL-1 antibodies can be used for the isolation and purification of PSGL-1 antigen from biomaterials. For example, cell types that express PSGL-1 protein, such as T cells or T cell lines from an individual, can be used as the protein source. Once purified, the protein can be used in various methods described herein. For example, purified PSGL-1 protein can be used for in vitro screening of modulators of PSGL-1 function on T cells or as an immunogen for preparing antibodies against the protein.

Anti-PSGL-1 Antibodies PSGL-1 polypeptides (or immunogenic fragments or analogs thereof) can be used to produce antibodies useful in the methods of the invention. As described above, PSGL-1 polypeptide or peptide fragments thereof can be produced by recombinant techniques and can be synthesized using solid phase synthesis methods. Recombinant PSGL-1 polypeptide or peptide fragments thereof can be used as an immunogen to produce anti-PSGL-1 antibodies. In addition, an anti-PSGL-1 antibody, such as a TAB4 monoclonal antibody, can then be used as an immunogen to produce an anti-PSGL-1 antibody, eg, a PSGL-1 poly, such as a native structure PSGL-1 polypeptide. It can be used to purify peptides.

  An antibody of the invention may be an antibody engineered to specifically bind to a monoclonal, polyclonal, or PSGL-1 polypeptide. For example, an antibody that “specifically binds” to a particular antigen, such as a PSGL-1 polypeptide, will not substantially recognize or bind other molecules in the sample. Thus, the present invention also provides for contacting a polypeptide with a test compound and determining whether the polypeptide binds to the test compound (eg, direct detection of the binding, the polypeptide and the test compound). A method of identifying a test compound (eg, an antibody) that binds to a polypeptide of the invention, by detecting a competitor molecule that inhibits binding of the protein and / or detecting binding using an assay for apoptosis-inducing activity) .

  Usually, the PSGL-1 polypeptide can be coupled with a carrier protein, such as KLH, mixed with an adjuvant, and injected into the host mammal. The antibodies produced in the animal can then be purified by peptide antigen affinity chromatography.

  In particular, various host animals can be immunized by injection of PSGL-1 polypeptide or antigenic fragment thereof. Commonly used host animals include rabbits, mice, guinea pigs, and rats. Depending on the host species, various adjuvants that can be used to increase the immunological response include Freund's adjuvant (complete and incomplete), inorganic gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols , Polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Potentially useful human adjuvants include BCG (Bacille Calmette-Guerin) and Corynebacterium parvum. Polyclonal antibodies are a heterogeneous population of antibody molecules contained in the sera of immunized animals.

  Thus, antibodies encompassed by the present invention are produced using polyclonal antibodies, as well as monoclonal antibodies, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F (ab ′) 2 fragments, and Fab expression libraries. Including molecules.

  Monoclonal antibodies, which are homogenous populations of antibodies to specific antigens, are produced by the PSGL-1 polypeptide described above 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]; see Hammerling et al., Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY [1981]) can do.

  In particular, monoclonal antibodies enable the production of antibody molecules by continuous cell lines in incubations as described in Kohler et al., Nature 256: 495 (1975), 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 have any immunoglobulin class such as IgG, IgM, IgE, IgA, IgD, and subclasses thereof. The hybridoma producing the mAb of this invention may be incubated in vitro or in vivo. The ability to produce high titer mAbs in vivo is a particularly useful production method.

  Once manufactured, polyclonal or monoclonal antibodies are tested for specific PSGL-1 recognition by Western blot or immunoprecipitation analysis by standard methods such as those described in Ausubel et al., Supra. Antibodies that specifically recognize and bind to PSGL-1 are useful in the present invention. For example, anti-PSGL-1 antibodies that bind to PSGL-1 antigen on the surface of T cells such as CD3 + cells and induce T cell depletion and / or apoptosis in an individual are particularly useful.

  The antibodies are desirable, such as, for example, immune responses mediated by T cells, such as associated with conditions such as inflammatory diseases, autoimmune diseases, transplant rejection, allergic diseases, and cancers induced by T cells. Can be used as part of a therapeutic regimen (to suppress or eliminate immune responses). The antibodies can also be used in screening assays to measure the ability of candidate compounds to bind to PSGL-1.

  In addition, a technology developed for the production of `` chimeric antibodies '' by splicing genes from mouse antibody molecules with suitable antigen specificity together with genes from human antibody molecules with suitable biological activity ( 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]). A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable domain derived from a murine monoclonal antibody and a human immunoglobulin constant domain.

  Alternatively, the techniques described for the production of single chain antibodies (US Pat. Nos. 4,946,778, 4,946,778, and 4,704,692) are available as single chain antibodies against PSGL-1 polypeptides. Or the production of fragments thereof. Single chain antibodies are formed by cross-linking the heavy and light chains of the Fv domain via amino acid bridges so that a single chain polypeptide is obtained.

  Antibody fragments that recognize and bind to specific epitopes can be produced by known techniques. For example, such fragments include, but are not limited to, F (ab ′) 2 fragments that can be produced by pepsin digestion of antibody molecules, and disulfide bridges of F (ab ′) 2 fragments. Fab fragments obtained by this are mentioned. Alternatively, Fab expression libraries may be constructed so that monoclonal Fab fragments with the desired specificity can be quickly and easily identified (Huse et al., Science 246: 1275 [1989]).

  The antibody may be humanized by methods known in the art. For example, monoclonal antibodies with the desired binding specificity can be commercially humanized (Scotgene, Scotland; Oxford Molecular, Palo Alto, Calif.). Sufficient human antibodies, such as those expressed in transformed animals, are also embodiments of the invention (Green et al., Nature Genetics 7:13 [1994]; and US Pat. Nos. 5,545,806 and 5). , 569,825).

Multimeric compounds Multimeric compounds that bind to multiple PSGL-1 proteins on the surface of T cells or NK cells can be used to induce apoptosis in the cells. The multimeric compound comprises at least two polypeptide chains. Each of the polypeptide chains has (i) a binding domain that binds to PSGL-1, and (ii) a heterologous amino acid sequence.

  In general, multimeric compounds bind to at least two different PSGL-1 proteins on a given cell surface. However, multimeric compounds can be formulated to have 3, 4, 5, 6, 7, 8, 9, 10, 12, or more different PSGL-1 binding domains, whereby the multimeric compound is , Induce binding to 3, 4, 5, 6, 7, 8, 9, 10, 12, or more different PSGL-1 proteins on a given cell surface.

  The binding domain may have any amino acid sequence that binds to PSGL-1 (or any amino acid sequence that has modifications such as, for example, glycosylation and / or sulfation). The binding domain can correspond to either a natural or non-natural amino acid sequence. For example, the binding domain can comprise the PSGL-1 binding domain of a selectin (eg, P-selectin, E-selectin, or L-selectin). A polypeptide comprising a PSGL-1 binding domain of selectin may optionally comprise (i) an extracellular domain of a selectin (eg, P-selectin, E-selectin, or L-selectin); (ii) a selectin (eg, A calcium-dependent lectin domain of P-selectin, E-selectin, or L-selectin; or (iii) a selectin that mediates binding to PSGL-1 (eg, P-selectin, E-selectin, or L-selectin) It may contain a fragment of the extracellular domain. In addition to the natural amino acid sequence, one or more amino acid changes may be introduced into the natural PSGL-1 binding domain to obtain a non-natural sequence that retains the PSGL-1 binding function. For example, the extracellular domain of any (i) selectin (eg, P-selectin, E-selectin, or L-selectin) that is bound to the polypeptide PSGL-1; (ii) the selectin (eg, P-selectin) , E-selectin, or L-selectin); or (iii) extracellular of a selectin (eg, P-selectin, E-selectin, or L-selectin) that mediates binding to PSGL-1. It may include an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to a fragment of the domain. Standard molecular biological mutagenesis techniques can be used to induce changes in the nucleic acid sequence encoding the PSGL-1 binding domain. The modified binding domain can then be assessed for its ability to bind to PSGL-1, such as, for example, immobilized PSGL-1 or PSGL-1 on the cell surface. Further, the binding domain is bound to the PSGL-1 binding domain of a polypeptide selected from an anti-PSGL-1 antibody or a phage display library, or PSGL-1, and from the anti-PSGL-1 antibody or the phage display library The selected polypeptide can include an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 98% identical to the PSGL-1 binding domain.

  The PSGL-1 binding domain may comprise an amino acid sequence corresponding to a PSGL-1 binding fragment of P-selectin. Examples of polypeptide chains (of the multimeric compounds described herein) comprising such amino acid sequences include the following components: (i) CD33 signal peptide (Met1-Ala16); (ii) mouse P-selectin Recombinant mouse P-selectin / Fc chimera (R & D SystemMinneapolis) comprising (Trp42-Ala709 of extracellular domain); (iii) IEGRMD (SEQ ID NO: 1); and (iv) human IgG1 (Pro100-Lys330). Available from MN). Other examples of polypeptide chains encompassing such amino acid sequences include the following components: (i) human P-selectin (Met1-Ala771, extracellular domain); (ii) IEGRMD (SEQ ID NO: 1) And (iii) recombinant human P-selectin / Fc chimera (available from R & D Systems Mineneapolis, MN), including human IgG1 (Pro100-Lys330).

  The PSGL-1 binding domain may comprise an amino acid sequence corresponding to a PSGL-1 binding fragment of E-selectin. Examples of polypeptide chains (of the multimeric compounds described herein) comprising such amino acid sequences include the following components: (i) mouse E-selectin (Met1-Pro557, extracellular domain); Recombinant mouse E-selectin / Fc chimera (R & D Systemmminneapolis) comprising: (1) IEGRMD (SEQ ID NO: 1); (iii) human IgG1 (Pro100-Lys330); Available from MN). Other examples of polypeptide chains comprising such amino acid sequences include the following components: (i) human E-selectin (Met1-Pro556, extracellular domain); (ii) IEGRMD (SEQ ID NO: 2); A recombinant human E-selectin / Fc chimera (available from R & D SystemMinneapolis, MN), including (iii) human IgG1 (Pro100-Lys330); and (iv) HHHHHH (SEQ ID NO: 2).

  The PSGL-1 binding domain may comprise an amino acid sequence corresponding to a PSGL-1 binding fragment of L-selectin. An example of a polypeptide chain (of a multimeric compound described herein) comprising such an amino acid sequence includes the following components: (i) mouse L-selectin (Met1-Asn332, extracellular domain); Recombinant mouse L-selectin / Fc chimera (R & D SystemMinneapolis) comprising: IEGRMD (SEQ ID NO: 1); (iii) human IgG1 (Pro100-Lys330); Available from MN). Other examples of polypeptide chains comprising such amino acid sequences include the following components: (i) human L-selectin (Met1-Asn332, extracellular domain); (ii) IEGRMD (SEQ ID NO: 2); A recombinant human L-selectin / Fc chimera (available from R & D SystemMinneapolis, MN) comprising (iii) human IgG1 (Pro100-Lys330); and (iv) HHHHHH (SEQ ID NO: 2).

  Multimeric compounds can be formulated as homomultimeric compounds or heteromultimeric compounds. Homomultimeric compounds contain only polypeptide chains having the same PSGL-1 binding domain. For example, a homomultimeric compound can comprise a polypeptide chain comprising a PSGL-1 binding domain fragment of P-selectin. Heteromultimeric compounds include polypeptide chains having different PSGL-1 binding domains. For example, the heteromultimeric compound can comprise a first polypeptide chain comprising a PSGL-1 binding domain fragment of P-selectin and a second polypeptide chain comprising a PSGL-1 binding domain fragment of E-selectin.

  The heterologous amino acid sequence may be any amino acid sequence. However, the amino acid sequence of the polypeptide chain described herein does not match the sequence of the native protein. A heterologous amino acid sequence includes one or more amino acids that allow the polypeptide chains to bind. For example, the one or more amino acids can be covalently bound to the polypeptide chain, such as by a disulfide bond. An example of a heterologous sequence is an immunoglobulin heavy chain constant domain. Disulfide bonds between the Fc domains of two polypeptide chains can result in the formation of dimeric compounds.

  In addition to contributing to the binding of the polypeptide chain, the heterologous amino acid sequence may also include a cross-linking domain such as, for example, a cell surface receptor binding domain. In the binding of drugs to the binding domain, cross-linking of the polypeptide chains and the cell surface PSGL-1 proteins to which they bind can result. The immunoglobulin heavy chain constant domain comprises an Fc receptor binding domain. For example, a cross-linker can be an antibody that specifically binds to a cross-linking domain of a heterologous amino acid sequence (eg, an anti-Fc antibody).

Screening Assay for Compounds that Modulate PSGL-1 Function The present invention relates to compounds that induce T cell depletion and / or T cell apoptosis upon binding to PSGL-1 (but are not limited to such compounds) Also included are methods of identifying compounds that interact with PSGL-1 (or a PSGL-1 domain) comprising Also included are compounds that modulate PSGL-1 interaction with transmembrane, extracellular, or intracellular proteins that modulate PSGL-1 activity and compounds that modulate PSGL-1 activity.

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

  Such compounds include, but are not limited to, for example, but not limited to, those from random peptide libraries; (Lam et al., Nature 354: 82 [1991]; Houghten et al., Nature 354: 84 [1991]) such as soluble peptides and combinatorial chemistry-derived molecular libraries made from D- and / or L-amino acids, phosphopeptides (restricted below) Including, but not limited to, those of random or partially denatured directed phosphopeptide libraries; Songyang et al., Cell 72: 767 [1993]), antibodies (not limited to Are polyclonal, monoclonal, humanized, anti-idiotype, chimeric or single chain antibodies, and FAb, F (ab ′) 2 and FAb expression library fragments, and epitoids thereof It encompasses flop binding fragment), as well as organic or inorganic small molecule.

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

  Computer modeling and exploration techniques can identify compounds or modify already identified compounds that can modulate PSGL-1 expression or activity. If such a compound or composition has been identified, the active site or domain is identified. In general, such active sites may be binding sites for active natural modifiers. The active site may be identified using methods known in the art such as, for example, studying the amino acid sequence of a peptide, the nucleotide sequence of a nucleic acid, or a complex of a related compound or composition with its natural ligand. In the latter case, chemical or X-ray crystallisation methods can be used to find the active site by knowing where the modifier (or ligand) is found on that factor.

  Natural products or synthetic chemicals relating to compounds that bind to PSGL-1 protein and induce T cell depletion and / or apoptosis of T cells, as described above with reference to the design and production of compounds capable of altering binding A library of biologically active substances including known compounds and proteins may be screened.

  In vitro systems may be designed to identify compounds that can interact with PSGL-1 (or a domain of PSGL-1). The identified compounds are useful, for example, for modulating T cell activity, as described herein, and thus, such as inflammation, autoimmunity, graft rejection, allergic disease, or T cell cancer Useful for the treatment of immune responses mediated by excessive or undesirable T cells or conditions associated with excessive or undesirable T cell proliferation.

  The assay principle used to identify compounds that bind to PSGL-1 is sufficient for the two components to interact and bind, and thus sufficient to form a complex that can be removed and / or detected in the reaction mixture. Preparing a reaction mixture of PSGL-1 (or its domain) and test compound under various conditions and time. The PSGL-1 species used can vary based on the goal of the screening assay. In some cases, it is preferred to use a peptide corresponding to a domain of PSGL-1 fused to a suitable heterologous protein or polypeptide in an assay system (eg, labeling, isolation, etc. of the resulting complex).

  The screening assay can be performed in a variety of ways. For example, one method for performing such an assay is to attach PSGL-1 protein, polypeptide, peptide or fusion protein or test compound on the solid phase and attach PSGL-1 / A method for detecting a test compound complex is mentioned. In one embodiment of such a method, the PSGL-1 reactant may be anchored to the solid surface and the test compound that is not anchored may be labeled directly or indirectly.

  In practice, a microtiter plate may simply be used as a solid phase. The anchored component may be immobilized by non-covalent or covalent bonds. Non-covalent binding may be achieved by simply coating the solid surface with a protein solution and drying. Alternatively, an immobilized antibody, preferably a monoclonal antibody, specific for the protein to be immobilized may be used for protein anchoring to the solid surface. The surface may be pre-prepared and stored.

  To perform the assay, the non-immobilized component is added to the coated surface containing the anchoring component. After the reaction is complete, unreacted components are removed (eg, by washing) under conditions such that the formed complex continues to be immobilized on the solid surface. Detection of complexes anchored on a solid surface can be accomplished in a number of ways. If a component that has not been previously immobilized is pre-labeled, detection of the label immobilized on the surface indicates that a complex has formed. If the pre-immobilized component is not pre-labeled, use indirect labeling; for example, using a labeled antibody specific for the pre-immobilized component (and this antibody Can be directly labeled or indirectly labeled with a labeled anti-Ig antibody) and can detect complexes anchored on the surface.

  Alternatively, the reaction may be carried out in the liquid phase, separating the reaction product from unreacted components, for example, PSGL-1 protein, polypeptide, peptide or fusion to anchor the complex formed in solution. The complex may be detected using an immobilized antibody specific for the protein or test compound and a labeled antibody specific for other components of the complex possible to detect the anchored complex.

  Alternatively, cellular assays may be used to identify compounds that interact with PSGL-1. In this case, a cell line expressing PSGL-1 or a cell line genetically engineered to express PSGL-1 can be used. Cell based assays are particularly useful for assessing the functional effects of compounds identified by the screening described herein. For example, once a compound has been identified based on its ability to bind to the PSGL-1 protein, the compound is then tested for its ability to induce apoptosis of, for example, in vitro or in vivo T cells or to deplete T cells in vitro or in vivo. May be.

Pharmaceutical Compositions Where the purpose of the invention is to alter the immune response in an individual, for example, antibodies, multimeric compounds, small molecules, or other compounds that specifically bind to a PSGL-1 polypeptide. The pharmaceutical composition containing it is also 1 aspect of this invention. In a preferred example, the compound functions as an agonist of PSGL-1.

  The pharmaceutical composition used in accordance with the present invention may be formulated in a known 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 by various routes of administration.

  The compounds may be formulated for parenteral administration by injection, for example, bolus injection or continuous infusion. Formulations for injection may be provided in the form of unit dosage forms, such as ampoules or multidose containers, with the addition of preservatives. The composition may take the form of a suspension, solution or emulsion in an oily or aqueous medium, and may contain formulations such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be in powder form before use, for example with a suitable medium such as sterile and pyrogen-free water.

Methods for Controlling T Cell-Mediated Immune Responses and Methods for Depleting T Cell Populations Similar compounds as described in the screening assays described herein include, for example, biology mediated by PSGL-1 polypeptides. May be useful in the regulation of functional function and / or in the treatment of diseases associated with excessive or undesirable immune responses, such as T cell mediated immune responses. These compounds include, but are not limited to, peptides, antibodies and fragments thereof that bind to PSGL-1 on the surface of T cells and induce signaling pathways that lead to T cell death, and Other organic compounds are mentioned. The method of the present invention may include adding a cross-linking agent that induces cross-linking of PSGL-1 on the cell surface, if necessary. The compounds described herein can be used where depletion or elimination of T cell activity is desired. Particularly useful conditions that can be treated with the compounds of the present invention include inflammatory diseases, autoimmune diseases, transplant rejection, allergic diseases, and cancers induced by T cells.

  Symptoms that can be treated with the anti-PSGL-1 compounds described herein include, but are not limited to, diabetes mellitus, arthritis (rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, and psoriatic arthritis) ), Multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosus, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczema dermatitis), psoriasis, Sjogren's syndrome, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, type I diabetes, inflammatory bowel disease, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis , Proctitis, drug eruption, leprosy reversal reactions, erythema nodosum erythema, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensory nerve Sexual hearing loss , Aplastic anemia, erythrocytic anemia, idiopathic thrombocytopenia, polychondritis, Wegner granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves' disease, sarcoidosis , Primary biliary cirrhosis, posterior uveitis, interstitial pulmonary fibrosis, graft-versus-host disease, bone marrow transplantation, liver transplantation or transplantation of any organ or tissue such as allogeneic or xenogeneic tissue Including allografts used), allergies such as atopic allergy, AIDS, and T cell neoplasms such as leukemia and / or lymphoma.

  The methods of the invention can be used to deplete T cells from a cell population, either in vitro or in vivo. For example, depleting T cells in vitro from a biological sample from an individual by contacting the sample with an anti-PSGL-1 compound described herein, and optionally with a cross-linking agent. it can. The method can be useful, for example, in enhancing non-T cells in a cell population, and in reducing or eliminating T cell activity from the cell population.

  The following are examples of the invention. The following examples are not to be construed as limiting the concept of the invention.

Example 1: Preparation of Anti-T Cell Apoptosis-Inducing Protein ("TAIP") Monoclonal Antibody A monoclonal antibody specific for TAIP can be used to produce hybridomas that secrete the desired antibody by Kohler and Milstein ((1976) European Journal of Obtained by applying the known cell fusion method of Immunology 6: 511-519). Antibody producing cells from hamsters injected with splenic T cells of Concanavalin A (ConA) activated Bab / c were fused with the myeloma cell line to form hybridomas that secrete antibodies. Two populations of these cells were fused with polyethylene glycol, and the resulting antibody-producing cells were cloned and expanded by standard tissue incubation methods. One hybridoma obtained according to these methods secreted a monoclonal antibody, termed TAB4, capable of inducing T cell apoptosis in vitro and depleting T cells in vivo. The protein recognized by TAB4 was called T cell apoptosis-inducing protein (TAIP).

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

  The supernatant of the concentrated incubation solution of TAB4 hybridoma was centrifuged at 20,000 × g for 10 minutes, and the supernatant was diluted with a binding buffer (0.1 M sodium acetate, pH 5.0) at a ratio of 1: 1. The protein G column (about 1 ml bed volume) was washed 3 times with 3-5 ml binding buffer. The clear incubation supernatant was placed on a protein G column, and the fluid was collected and placed on the column again. The column was washed with 6-10 ml binding buffer and the bound antibody was eluted from the column with 5 ml elution buffer (0.1 M glycine-HCl, pH 2.8). Each fraction contained 1 ml of eluted antibody and the elution fractions were adjusted to neutral pH by mixing each 1 ml fraction with 50 μl of 1M Tris-HCl, pH 7.5. Fractions containing the antibody were pooled and dialyzed 3 times for 2 hours with 2 liters of PBS, pH 7.4 for each dialysis. Protein concentration in antibody samples was measured using the Bio-Rad Protein Assay (BIO-RAD, Hercules, Calif.) As described by Bradford.

Example 2 Preparation of Mouse Spleen Cell Suspension and Activation and Enhancement of T Cells Mouse spleen is immersed in 8 ml Hanks' solution (HBSS), gently chopped with a sterilized cover glass, and a 15 ml centrifuge tube. (Costar), and further 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 wall. Contaminated red blood cells (RBC) were lysed by adding 1 ml of RBC lysis buffer (0.6 M NH ₄ Cl, 0.17 M Tris-base, pH 7.65) and then incubated for 2 minutes at room temperature, Quenched quickly with 9 ml HBSS. Cells were pelleted at 200 xg for 5 minutes, washed twice and resuspended in RPMI incubation. The concentration and viability of cells in the mixture were measured with a hemocytometer (Cambridge Scientific Inc.) and Trypan blue exclusion.

Spleen cells were adjusted to a final concentration of 3 × 10 ⁶ / ml in RPMI medium and concanavalin A was added to a final concentration of 2 μg / ml to activate T cells. Cell suspensions were transferred to 6-well incubation plates (5 ml / well) or 10 cm incubation dishes (10 ml / dish) and incubated for 48 hours at 37 ° C., 5% CO ₂ before collection. Activated spleen cells, including activated T cells, were resuspended in 5 ml HBSS and carefully placed on top of a 5 ml 55% cushion of percoll solution in a centrifuge tube. Care was taken not to disturb the separated layers. The cells were centrifuged at 1,900 × g for 13 minutes at 25 ° C. without stopping. Enriched T cells were collected from the bilayer interface, washed twice with HBSS, and prepared for experiments.

Example 3: Apoptosis of activated T cells Activated T cells (see Example 2) were re-applied in RPMI medium containing 5 ng / ml IL-2 to a final concentration of 5 x 10 ⁵ cells / ml. Suspended and treated with control Ig, TAB4, or anti-CD3 according to the conditions shown in Table 1.

After 18-24 hours of incubation, the extent of apoptosis in each incubation was measured using a 7-AAD apoptosis assay. Treated cells were transferred to a FACS tube (Falcon), washed twice with ice-cold FACS solution (1% fetal bovine serum in PBS, 0.05% sodium azide) and pelleted at 200 × g at 4 ° C. did. The 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 at a final concentration of 2 μg / ml and then incubated at 4 ° C. for 20 minutes in the dark. Finally, the stained cells were washed twice with ice-cold FACS solution, resuspended in 0.5 ml FACS solution and analyzed with a BD LSR flow cytometer (Beckton Dickison).

  FIG. 1 shows the results of a representative time course experiment examining when activated T cells gain sensitivity to TAB4 (anti-TAIP) mediated apoptotic signals. Mouse splenocytes were activated with Con-A and maintained in IL-2 containing medium. Activated T cells were collected, resuspended and challenged with TAB4 monoclonal antibody or control hamster IgG in the presence of anti-hamster IgG antibody as a cross-linker. It was clear on day 1 that TAIP crosslinking could induce low levels (6.5%) of apoptotic cell death. However, the degree of apoptosis induced by TAB4 increased from 17% on day 2, peaked at 52% on day 4, and decreased to 44% on day 6. Control hamster IgG did not induce specific apoptotic T cell death compared to incubations administered IL-2 alone. Anti-CD3 (as a positive control) induced 38% T cell apoptosis 48 hours after activation (data not shown).

Example 4: Expression of TAIP antigen in different tissues Cells were washed twice with ice-cold FACS solution (1% fetal bovine serum in PBS, 0.05% sodium azide) and at 4 ° C in a FACS tube (Falcon). Centrifuge at 200 xg. Cells are resuspended in ice-cold FACS solution to a final concentration of 1 × 10 ⁷ cells / ml and 0.1 ml aliquots of resuspended cells in FACS tubes (Falcon) are used for each assay. did. To stain the surface, TAB monoclonal antibody or 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 minutes in the dark. After the cells were washed once with ice-cold FACS solution, (1) for splenocytes, cychrome-conjugated anti-CD3 antibody (2 μg / ml), FITC-conjugated anti-hamster Ig in 100 μl ice-cold FACS solution, and PE-conjugated anti-CD8 / CD4 / CD19 / CD11b / pan-NK / IA / IE / Mac-3 antibody (2 μg / ml); and (2) for thymocytes, FITC in 100 μl ice-cold FACS solution Stained with conjugated anti-hamster Ig, PE-conjugated anti-CD8 antibody, and cychrome-conjugated anti-CD4 antibody (2 μg / ml). The reaction was carried out in the dark at 4 ° C. for 30 minutes. Finally, the stained cells were washed twice with ice-cold FACS solution, resuspended in 1 ml FACS solution and analyzed with a BD LSR flow cytometer (Beckton Dickison).

FIGS. 3 and 4 show the distribution of TAIP antigen in various subpopulations of splenocytes and thymocytes by FACS analysis. As shown in FIG. 3, CD19 ⁺ B cells expressed a low but detectable amount of TAIP protein on the surface. Significantly higher amounts of TAIP protein were detected in the CD3 ⁺ T cell and NK cell fractions. Most CD4 ⁺ , CD8 ⁺ , and CD4 ⁺ 8 ⁺ thymic T cells expressed significant amounts of TAIP protein. In contrast, TAIP proteins, CD4 ^- 8 ^- was not expressed only in a small population of thymocytes T cells (Fig. 4).

  Tissues from B6 and Balb / c mice, such as brain, thymus, heart, lung, liver, stomach, kidney, spleen, and skin, are collected and fixed in 10% formaldehyde overnight at room temperature and wrapped in paraffin block Buried. Tissue sections 4 μm thick were prepared from paraffin blocks with a Leica RM2135 microtome, spread in water at 45 ° C. and mounted on coated slides. Slides were dried at 37 ° C. and prepared for the next experiment.

  Slides containing tissue paraffin sections were dewaxed and dried with xylene-100% ethanol series according to standard protocols and finally kept in 100% ethanol. Sections were rehydrated from continuous incubation of 100% ethanol-90% ethanol-85% ethanol-70% ethanol-PBS to a final PBS solution according to standard protocols. All the following reactions were carried out in a humidification box. Nonspecific binding was blocked by incubating tissue sections in blocking buffer (1% normal goat serum) for 1 hour at room temperature (or overnight at 4 ° C.). Blocking buffer was removed, TAB4 or normal hamster Ig (1: 200 dilution) was added to the sections and incubation continued for an additional hour at room temperature (or overnight at 4 ° C.). Sections were washed twice with PBS for 5 minutes each to remove primary antibody, reacted with 1: 250 diluted alkaline phosphatase conjugated goat anti-hamster Ig and incubated for 1 hour at room temperature. The sections were again washed twice with PBS for 5 minutes each to remove the antibody-enzyme conjugate and the color reaction was developed with BCIP / NBT substrate solution in the dark for 30 minutes at room temperature. The sections were washed again with PBS to remove excess enzyme substrate, dehydrated with PBS-ethanol-xylene series and subjected to microscopy.

  The results showed that TAIP protein expression was detected and tested only in bone marrow derived tissues and not in the remaining tissues.

Example 5: Cell surface biotinylation and immunoprecipitation of TAIP antigen 5 × 10 ⁷ RL male 1 or NIH-3T3 cells were treated with 0.5 mg / ml sulfo-NHS-biotin (30 μm on ice for 30 minutes). Surface biotinylation in 1 ml PBS containing Pierce). The reaction was terminated by incubating the cells on ice for 10 minutes with 0.5 ml Dulbecco's Modified Eagle Medium (Life Technologies, Inc.). The cells were washed once with 1 ml Dulbecco's modified Eagle medium and twice with 1 ml phosphate buffer.

Labeled cells can be lysed with cold lysis buffer (1% Triton X-100, 20 mM Tris-HCl, pH 8.0, 160 mM NaCl, 1 mM CaCl, including complete protease inhibitor cocktail (Roche). ₂ ) Lysed at a density of 5.0 × 10 ⁷ cells / ml in 15) for 15 minutes and pelleted insoluble material at 10,000 × g for 10 minutes; these and all subsequent steps were performed at 4 ° C. It was. For immunoprecipitation, the lysate was preincubated with 50 μl of packed protein G-Sepharose (Amersham Pharmacia Biotech) for 30 minutes to remove non-specifically bound proteins. The beads are pelleted and an aliquot of the supernatant (constantly corresponding to 5.0 × 10 ⁷ cells) is incubated with 20 μl protein G-Sepharose preloaded with 10 μg mAb TAB4 or IgG from normal hamster serum. did. After 4 hours of incubation at 4 ° C., 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). Washed twice with the same wash buffer, including 250 mM instead of 400 mM NaCl. Proteins that specifically bind to TAB4 were eluted with 50 μl of 1 × SDS sample buffer. The eluted protein was separated by 8% SDS-PAGE and transferred to a nitrocellulose membrane (Millipore). Filters were analyzed for biotinylated proteins with peroxidase-conjugated avidin (PharMingen) and developed with chemiluminescent reagents (NEN ^™ Life Science Products).

As shown in FIG. 2, a biotinylated surface protein having a molecular weight of about 120 kD was identified by TAB4 in RL1 cells (TAIP ⁺ T cells) but not in 3T3 cells (TAIP ⁻ T cells). There wasn't. In contrast, protein G sepharose coated with normal hamster serum could not extract this 120 kD protein. These results suggest that this 120 kD protein is an antigen recognized by the monoclonal antibody TAB4 on the cell surface of T cells.

Example 6: In vivo T cell depletion To test the effects of TAB4 on T cells and other cell populations in vivo, mice were injected intraperitoneally with 300 μg TAB4 or control hamster Ig. On day 4, splenocytes, thymocytes, and peripheral blood mononuclear cells were collected for whole cell counting and for analysis of cell surface markers by FACS.

In the FACS assay, cells were fixed with 2% paraformaldehyde for 20 minutes at 4 ° C., washed twice and resuspended in ice-cold FACS solution to a final concentration of 1 × 10 ⁷ cells / ml. . A 100 μl aliquot of resuspended cells in a FACS tube (Falcon) was used for each assay. A final concentration of 2 μg / ml TAB4 or control hamster Ig was added to the cells and the mixture was incubated in the dark at 4 ° C. for 30 minutes. Cells were washed once with ice-cold FACS; (1) For splenocytes, cychrome-conjugated anti-CD3 antibody (2 μg / ml), FITC-conjugated anti-hamster Ig and PE-conjugated anti-antigen in 100 μl ice-cold FACS solution CD8 / CD4 / CD19 / CD11b / pan-NK / IA / IE / Mac-3 antibody (2 μg / ml); and (2) for thymocytes, FITC-conjugated anti-hamster in 100 μl ice-cold FACS solution Reacted with Ig, PE-conjugated anti-CD8 antibody, and cychrome-conjugated anti-CD4 antibody (2 μg / ml). The reaction was carried out in the dark at 4 ° C. for 30 minutes. Finally, the stained cells were washed twice with ice-cold FACS solution, resuspended in 1,000 μl FACS solution and analyzed with a BD LSR flow cytometer (Beckton Dickison).

Four days after 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). . TAB4 treatment slightly reduced the total number of splenocytes. However, in TAB4-treated mice, the number of CD3 ⁺ T cells was reduced by 62%, the number of NK cells was reduced by 50%, and the total number of CD19 ⁺ B cells was slightly increased. The total number of thymocytes recovered from TAB4-treated mice was only 48% of the level seen in controls (52% reduction). Moreover, other than CD4 ⁺ T cells, all other CD8 ⁺ , CD4 ⁺ CD8 ⁺ , and CD4 ⁻ CD8 ⁻ T cells were reduced, with the CD4 ⁺ CD8 ⁺ subpopulation being most significantly affected ( 64.7% decrease).

Example 7: Anti-TAIP antibody does not induce IL-2 or TNF-α secretion Balb / c mice (H-2d) were injected intraperitoneally with 300 μg TAB4 or control hamster Ig. Splenocytes were isolated 7 days after injection and used as responders in incubations with mitomycin C-treated C3H (H-2k) splenocytes (as stimulators). After 3 days, the incubate supernatant was collected and IL-2 content was measured by ELISA set (PharMingen). As shown in FIG. 5, IL-2 production was suppressed in responder cells from TAB4-treated mice compared to that of control mice. When plasma levels of IL-2 and TNF-α were also analyzed, there was no significant difference in IL-2 (or TNF-α) levels in sera of control and TAB4-treated mice. Since IL-2 production is important for T cell activity, these results indicate that antibodies specific for TAIP, such as TAB4, manipulate T cells and are associated with autoimmune diseases and graft rejection. It is shown that it can be used in vivo to control immune responses mediated by undesirable T cells such as those.

Example 8: Use of anti-TAIP antibody to prevent graft rejection 8-12 week old mice (obtained from Jackson Laboratory) were treated with Acepromazin maleate (Fermenta Animal Health Co., Kansas). City, MO) was anesthetized. Prior to skin transplantation, recipient thymectomized recipient C57BL / 6 mice (H-2 ^b ) were injected intraperitoneally with 500 μg TAB4 or isotype control antibody 7 days prior to skin transplant surgery. Seven days later, the flank of the skin of a fully allogeneic mismatched Balb / cj mouse (H-2 ^d ) was transplanted into the flank of a C57BL / 6 mouse previously treated with the antibody. Seven days after transplantation, the mice were again injected with 500 μg of TAB4 or isotype control antibody. Mice were monitored daily after transplantation. If 50% of the donor's skin was necrotic, the graft was considered rejected. The survival rate (%) of the graft is shown in FIG. 7 (n = 8). The data show that treatment with TAB4 antibody prolongs the survival of allogeneic skin grafts.

Example 9: Identification of TAIP as PSGL-1 P-selectin glycoprotein ligand-1 (PSGL-1), also referred to as CD162, is the major P-selectin ligand expressed in leukocytes, such as 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, such as its molecular weight and dimer susceptibility, suggested that TAB4 may be similar to PSGL-1. In order to investigate the relationship between these two antigens, the following tests were conducted: 1) whether the antigen precipitated by TAB4 can be recognized by a commercially available anti-PSGL-1 antibody; and 2) the anti-PSGL-1 antibody is expressed in cells. Whether TAB4 can be depleted from the lysate.

RL male 1 T cells in lysis buffer (1% Triton X-100, 20 mM Tris-HCl, pH 8.0, 160 mM NaCl, 1 mM CaCl ₂ ) containing a complete protease inhibitor cocktail. Was lysed at a density of 1.0 × 10 ⁸ cells / ml for 1 hour and insoluble material was pelleted at 10,000 × g for 10 minutes. These and all subsequent steps were performed at 4 ° C. Lysate corresponding to 5.0 × 10 ⁷ cells was preloaded with 10 μg of anti-PSGL-1 mAb (clone 2PH1, PharMingen, San Diego, Calif.), Anti-TAIP mAb, TAB4, or IgG from normal hamster serum. And incubated with 20 μl of protein G-Sepharose. After 4 hours incubation at 4 ° C., the beads were 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). , And twice with the same wash buffer containing 250 mM instead of 400 mM NaCl. The bound protein was eluted with 40 μl of 1 × SDS sample buffer. The eluted protein was separated by 6% SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were immunoblotted with anti-PSGL-1 mAb and developed by peroxidase-conjugated goat anti-rat IgG (H + L) and further by chemiluminescence (Renaissance, NEN).

Surface biotinylated RL male 1 T cells were lysed at a density of 1.0 × 10 ⁸ cells / ml in lysis buffer. Cell extracts were incubated with 20 μg of antibody conjugated to 40 μl protein G-Sepharose overnight at 4 ° C. Depletion was performed with anti-PSGL-1 mAb (2PH1) or control rat IgG using TAB4 or control normal hamster serum. The depleted lysate was further immunoprecipitated with TAB4 or anti-PSGL-1 mAb, respectively. Immunoprecipitates were separated on 6% SDS-polyacrylamide gel and detected by fluorescent screen indirect imaging. As shown in FIG. 6, anti-PSGL-1 antibody can deplete TAIP protein from T cell lysates. In addition, proteins immunoprecipitated with anti-TAIP antibody (TAB4) can be recognized by anti-PSGL-1 antibody by Western analysis.

Example 10: Induction of apoptosis in human T cells by anti-PSGL-1 antibody To determine the role played by PSGL-1 in the apoptosis of human T cells, a time course experiment was conducted to determine when activated human T cells It was investigated whether the sensitivity to the apoptosis signal mediated by PSGL-1 was obtained. Human T cells were stimulated with phytohemaglutinin (PHA) mitogen and further expanded in a medium containing IL-2. Activated T cells were collected and then challenged with anti-PSGL-1 in the presence of IL-2 and cross-linked antibody.

  Human peripheral blood was collected from healthy adults, heparinized, and peripheral blood mononuclear cells (PBMC) based on different densities were enhanced using Ficoll-Paque Plus (Pharmacia Biotech). PBMC were activated with 1% PHA (Life Technologies, GibcoBRL) for 48 hours and then maintained in recombinant human IL-2 (5 ng / ml) for the duration of the assay. In order to evaluate the apoptosis-inducing ability of anti-human PSGL-1 antibody, activated cells were: (1) 1 μg / ml anti-PSGL-1 antibody clone KPL-1 (BD PharMingen) cross-linker rabbit Anti-mouse Ig (0.5 μg / ml) (Jackson ImmunoResearch Laboratories) added; (2) Cross-linked rabbit anti-mouse Ig added to isotype control purified mouse Ig; or (3) Cross-linked rabbit anti-antibody Mouse Ig alone was treated. After 6 hours of treatment, the percent of early apoptotic cells was measured by FACS and stained with anti-Annexin V (BD PharMingen) and PI (Sigma).

  As shown in FIG. 8, signaling caused by PSGL-1 using anti-PSGL-1 antibody plus a cross-linked product is significant in PHA-activated human PBMC (mainly T cells). Caused a certain level of apoptosis. The percentage of apoptotic cells increased from 8.5% on day 3 to 24% on day 8 in anti-PSGL-1 treated incubations. Neither isotopic-matched control or cross-linked antibody alone had any effect on these cells.

Example 11 Use of Anti-PSGL-1 Agonist Antibody to Treat Autoimmune Diseases A well-recognized autoimmune diabetic animal, non-obese diabetic (NOD) mice, is Reared under conditions. Spontaneous diabete developed in NOD mice about 20 weeks old. In the experimental group, mice were intraperitoneally administered 300 μg anti-PSGL-1 antibody (TAB4) three times per 14, 15 and 17 week old mice. Two more injections of the same dose were given at 24 and 26 weeks of age. The control group received the same dose of hamster Ig. Mice were monitored for urinary glucose by Medi-Test Glucose strip (Macherey-Nagel, Germany) twice weekly after 15 weeks of age. Non-fasting urinary glucose levels above 300 mg / dl in two consecutive measurements were considered diabetic.

  As shown in FIG. 9, TAB4 (anti-PSGL-1) antibody treatment provided significant protection compared to control antibody treatment. Therefore, treatment with anti-PSGL-1 antibody can suppress the activity of 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 Binding activity of selectins (P-selectin, E-selectin, and L-selectin) to activated T cells To measure, splenocytes freshly prepared from C57BL / 6 were activated and collected on days 2, 4, and 6. Non-activated T cells (ie freshly prepared splenocytes on day 0) were also analyzed. Day 2 samples contained 3 × 10 ⁶ cells / ml splenocytes activated with 2 μg / ml Concanavalin A (Con A) in DMEM + 10% FBS for 2 days. Viable cells were isolated by ficoll gradient separation. Samples on day 4 were obtained from cells that were activated with Con A for 3 days and maintained in an incubation medium containing 5 ng / ml IL-2 for 1 day. Samples from day 6 were obtained from cells that were activated with Con A for 3 days and maintained in an incubation solution containing 5 ng / ml IL-2 for an additional 3 days.

To assay the sample by FACS analysis, 2 × 10 ⁵ cells per well from day 0, 2, 4 and 6 were obtained in a concentration range of 20 μg / ml to 0.156 μg / ml by a 2-fold serial dilution method. Then, 40 μl / well of mouse P-selectin, E-selectin, or L-selectin fused to the Fc domain of human IgG1 (R & D System, Minneapolis, Minn.) Was used and incubated at 4 ° C. for 30 minutes. After the incubation, the cells were washed with 1 × FACScan buffer (Biochrom AG, Berlin, 1 × PBS and 2% FBS without calcium and magnesium ions). Samples were used with 95 μl / well anti-Thy1.2 and a second reagent (FITC anti-human IgG specific for the Fc fragment obtained from Jackson Immuno Research Laboratories, Inc., West Grove, PA). Further incubation at 25 μg / ml for 30 minutes at 4 ° C. followed by washing with 1 × FACScan buffer.

  The results of FACSCalibur analysis are shown in FIG. At 20 μl / ml, the binding of P-selectin to mouse activated T cells increased gradually, maximizing from day 2 to day 4 and slightly decreasing on day 6. The binding of E-selectin increased significantly from day 2 to day 4 and maintained its peak on day 6. The binding of L-selectin to mouse activated T cells was not clear and did not change throughout the activation period, ie from day 0 to day 6. The observations with L-selectin are due to the apparent low binding ability of L-selectin to its ligand. Similar results were observed in the above experiment when the three selectins were at low concentrations.

Example 13: Multimeric forms of E-selectin and P-selectin induce apoptosis of activated T-cells 96-well plates (NUNC) with 50 μl of 20 μl / ml anti-human FcIg in 1 × PBS at 4 ° C. Overnight coated, blocked with 1% BSA for 2 hours at 37 ° C. and incubated with 50 μl selectin-human Fc fusion (0.063-5 μg / ml) for 2 hours at room temperature. In all experimental steps, each well was washed thoroughly 5 times with 1 × PBS. Then 2 × 10 ⁵ T cells pre-activated with Con A for 4 days were added to each well and before centrifuging the plate at 200 × g for 5 min at 4 ° C. Incubated at 37 ° C for 5 hours. The resulting pellet containing activated T cells was incubated with annexin V-biotin conjugate for 15 minutes at room temperature and then 37 with avidin conjugate (SA-β-gal at a dilution of 1: 5000). Incubated for an additional 30 minutes at 0 ° C. In any binding reaction, each well was washed three times with annexin V binding buffer. Incubation of 110 μl Z-buffer mixture (54 μl 2-mercaptoethanol in 20 ml Z-buffer) and 30 μl ONPG (0.04 g / 10 ml) overnight at 4 ° C. produced color development. The optical density reading at 420 nm was recorded.

  The level of selectin-induced apoptosis of Con-A activated T cells was increased with increasing concentrations of P-selectin (FIG. 11A) or E-selectin (FIG. 11B) fused with human IgG1 Fc (from 0.063 μl / ml to 5 μl / ml). ml). The hamster antibody TAB4 induced apoptosis of activated T cells (see Example 1) and was used as a positive control in the experiment. As negative controls, anti-human Fc, human Ig (HIg), and BSA did not induce apoptosis. Consistent with the fact that L-selectin did not bind well to activated T cells (Example 12), no significant apoptosis was observed in the presence of L-selectin human Fc fusion protein ( FIG. 11C). In summary, plate-bound fusion proteins, including PSGL-1 binding fragments of P-selectin or E-selectin and human Fc fragments, induce apoptosis of activated T cells.

Example 14: Crosslinking of soluble P-selectin-Fc fusion induces apoptosis of activated T cells Mouse selectins (P-selectin, E-selectin, and L-selectin) are as detailed above. It was fused to the Fc domain of human IgG1 to form a soluble dimeric fusion protein. In order to evaluate whether or not the soluble selectin can induce apoptosis of activated T cells, Example 13 shows that plate-bound anti-human Fc Ig is omitted except that plate-bound anti-human Fc Ig is omitted. Detailed experiments were conducted. In the presence of the soluble form of P-selectin fusion protein (dimer) alone, negligible or low levels of activated T cell apoptosis occurred (FIG. 12). However, with the addition of the cross-linked product (anti-human Fc), the apoptotic activity was greatly increased and increased to almost the same level as that induced in the presence of the plate-bound antibody. None of the anti-human Fc, human Ig (HIg), or BSA induced apoptosis.

  In the case of the E-selectin-Fc fusion protein, the same results as in the case of the P-selectin-Fc fusion protein were obtained. Furthermore, in agreement with the results obtained with plate-bound (multimeric forms) of L-selectin, the soluble form of L-selectin fusion protein is responsible for the apoptosis of activated T cells. Not induced.

Other Embodiments Although the present invention has been described in conjunction with the detailed description, it is understood that the description is intended to explain in detail and not to limit the concept of the invention. Other aspects, advantages, and modifications of the invention are within the scope of the claims.

2 shows representative results of an experiment over time that examined when activated T cells gained sensitivity to apoptotic signals mediated by TAB4 (anti-PSGL-1 monoclonal antibody). The results of biotinylation and immunoprecipitation on the cell surface of the antigen recognized by the TAB4 antibody are shown. FIG. 5 shows PSGL-1 antigen expression on splenic CD4 + T cells, CD8 + T cells, CD19 + B cells, and NK cells. CD4 ^+, CD8 ^+, and ^CD4 + ^{8 +,} and CD4 ^- 8 ^- shows the expression of PSGL-1 antigens on thymocytes. Produced by mixed lymphocyte incubation with spleen cells isolated from Balb / c mice treated with TAB4 (or hamster Ig) as responder and H2 mismatched C3H spleen cells as stimulator The IL-2 level is shown. (A) The protein immunoprecipitated with the TAB4 antibody can be recognized by a commercially available anti-PSGL-1 antibody, and (B) the T cell lysate is precleared with the anti-PSGL-1 antibody. FIG. 6 shows a Western blot analysis showing that the protein recognized by can be depleted. Shows the percentage of surviving grafts in C57BL / 6 mice that received skin grafts from Balb / c mice and were treated with anti-PSGL-1 antibody (filled diamonds) or control antibody (open squares) It is. FIG. 5 shows the time course of the percentage (%) of apoptotic T cells after treating activated human peripheral blood mononuclear cells with anti-human PSGL-1 antibody. FIG. 5 shows the incidence of diabetes in autoimmune non-obese diabetic (NOD) male mice treated with anti-PSGL-1 antibody (black squares) or control antibody (open squares). FIG. 3 shows the binding of mouse P-selectin, E-selectin, and L-selectin to mouse activated T cells. 11A-11C show the induction of apoptosis of mouse activated T cells by multimeric forms of E-selectin (FIG. 11A), P-selectin (FIG. 11B), and L-selectin (FIG. 11C). ing. FIG. 6 shows in vitro induction of mouse activated T cell apoptosis by cross-linking of soluble P-selectin-Fc fusion protein.

Claims (14)

To prevent or suppress a T cell mediated immune response in an individual having a condition characterized by or at risk of developing an immune response mediated by excess or undesirable T cells Use of a multimeric compound that binds to at least two P-selectin glycoprotein ligand 1 (PSGL-1) proteins on the surface of T cells in the manufacture of Two polypeptide chains, each of which has (i) a binding domain that binds to PSGL-1, and (ii) a heterologous amino acid sequence;
In this regard, binding of the multimeric compound to at least two PSGL-1 proteins on the surface of the T cell induces a signaling pathway that results in T cell death, thereby causing an immune response mediated by the T cell in the individual. Prevent or suppress,
The binding domain is a PSGL-1 binding domain of P-selectin or a PSGL-1 binding fragment thereof, a P-selectin extracellular domain or a PSGL-1 binding fragment thereof, a PSGL-1 binding domain of E-selectin or a PSGL-1 thereof. A binding fragment, or an E-selectin extracellular domain or a PSGL-1 binding fragment thereof,
The heterologous amino acid sequence comprises an immunoglobulin heavy chain constant region;
Use of a multimeric compound wherein the polypeptide chain is bound by the heterologous amino acid sequence to form a multimeric compound, and the compound is not an anti-PSGL-1 antibody or an antibody fragment that binds to PSGL-1.   The use according to claim 1, wherein the multimeric compound is a homomultimeric compound or a heteromultimeric compound.   3. Use according to claim 1 or 2, wherein the polypeptide chain is covalently linked to form the multimeric compound with a heterologous amino acid sequence.   4. Use according to claim 3, wherein the covalent bond is a disulfide bond.   The drug according to any one of claims 1 to 4, further comprising an agent that binds to the multimeric compound by a heterologous amino acid sequence and induces cross-linking of a plurality of PSGL-1 antigens on the surface of the T cell. Use as described in.   The individual is diagnosed as having an inflammatory disease, diagnosed as having an autoimmune disease, undergoing allogeneic or xenotransplantation or scheduled to undergo such transplantation, diagnosed as having an allergic disease, or T cell cancer 6. Use according to any one of claims 1 to 5, which has been diagnosed as having.   The use according to any one of claims 1 to 6, wherein the T cell is an activated T cell. Use of a multimeric compound that binds to at least two PSGL-1 proteins on the surface of T cells or NK cells in the manufacture of a medicament for inducing death of T cells or natural killer (NK) cells, comprising: The multimeric compound comprises two polypeptide chains, each of which has (i) a binding domain that binds PSGL-1, and (ii) a heterologous amino acid sequence;
In this process, the multimeric compound binds to at least two PSGL-1 proteins on the surface of the T cell or NK cell to induce a signal transduction pathway that leads to death of the T cell or NK cell,
The binding domain is a PSGL-1 binding domain of P-selectin or a PSGL-1 binding fragment thereof, a P-selectin extracellular domain or a PSGL-1 binding fragment thereof, a PSGL-1 binding domain of E-selectin or a PSGL-1 thereof. A binding fragment, or an E-selectin extracellular domain or a PSGL-1 binding fragment thereof,
The heterologous amino acid sequence comprises an immunoglobulin heavy chain constant region;
Use of the polypeptide chains are coupled by said heterologous amino acid sequence to form a multimeric compound, and the compound is not an antibody fragment that binds to the anti-PSGL-1 antibody or PSGL-1, multimeric compound. Use according to claim 8, wherein the multimeric compound is a homomultimeric compound or a heteromultimeric compound. 10. Use according to claim 8 or 9, wherein the polypeptide chain is covalently linked to form the multimeric compound with a heterologous amino acid sequence. Use according to claim 10, wherein the covalent bond is a disulfide bond. 9. The multimeric compound is prepared for administration of an agent that binds to the multimeric compound by a heterologous amino acid sequence and induces cross-linking of a plurality of PSGL-1 antigens on the T cell surface. use according to any one of 11. 13. Use according to any one of claims 8 to 12, comprising inducing death of activated T cells. At least two PSGL-1 proteins on the surface of a T cell, comprising two polypeptide chains, each of which has (i) a binding domain that binds to PSGL-1, and (ii) a heterologous amino acid sequence induce a signal transduction pathway that results in the death of T cells by binding to, it binds to at least two PSGL-1 proteins on the surface of the T cell multi monomer compound; and inflammation, autoimmunity, graft rejection Instructions for using the multimeric compound to treat allergic symptoms, or T-cell cancer,
In this case, the binding domain is a PSGL-1 binding domain of P-selectin or a PSGL-1 binding fragment thereof, a P-selectin extracellular domain or a PSGL-1 binding fragment thereof, a PSGL-1 binding domain of E-selectin or a thereof A PSGL-1 binding fragment, or an E-selectin extracellular domain or a PSGL-1 binding fragment thereof,
The heterologous amino acid sequence comprises an immunoglobulin heavy chain constant region;
A kit wherein the polypeptide chain is bound by the heterologous amino acid sequence to form a multimeric compound, and the compound is not an anti-PSGL-1 antibody or an antibody fragment that binds to PSGL-1.

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