TWI359024B - Modulators of p-selectin glycoprotein ligand 1 - Google Patents

Description

1359024 IX. Description of the invention: [Technical field to which the invention pertains] The present invention relates to a pharmaceutical combination for unregulating an immune response. [First prior art] In the treatment of such as inflammation, autoimmune diseases, transplant rejection, allergic diseases In the process of degenerative cancer with tau cells, etc., the control of the immune response that needs to occur is the main key. Excessive aggressive tau cell activity can be controlled by immunosuppressive effects or by induction of immunological tolerance. Tolerance (t0丨e r a n c e) refers to a state in which the immune system does not react to the antigen. The drug can be used to reduce the immune system's response to antigens to achieve immunosuppressive effects. When organ transplantation, T cells play a necessary role in the immune response of allogeneic antibodies (aii〇antigens). Most current immunosuppressive methods use corticosteroids, cyclosp〇rin or rapamycin to inhibit the major T cell growth factor interleukin-2. IL-2)" transcriptional machinery (transcripti〇n), or inhibition of interleukin-dependent cell proliferation (IL-2 dependent proliferation;). Apoptosis is thought to have the physiological role of maintaining proper immune system function' and is the primary mechanism for removing unwanted or defective cells (Kabelitz et al. Immunol. Today 1 4:3 3 8-340 (1 993) ; Raff, Nature: 356: 3 97-3 99 (1 992)). Various signals from inside or outside the cell can affect cell survival or death. The antibody binds to a molecule on the surface of a T cell such as Fas (or 5 1359024 CD95, molecular weight 43kD), TNFR2 (molecular weight 75kD), CD2 (molecular weight 45kD), and CTLA-4 (molecular weight 33kD) - 谤-使-Τ- - cell-fat-sheng-fine. Cell-apoptosis I should - (-〇Vb-

Immunol. 8:245-248 (1996); Lin et al. J. Immunol. 1 58:598-603 (1 997); Zhang et al. Nature: 377:348-350 (1 995); Lai et al. Eur. J. Immunol. 25:3243-3248 (1 995); Mollereau et al. J. Immunol. 1 5 6:3 1 84-3 190 (1996); Gribben et al. Proc. Natl. Acad. Sci. USA 92:811-815 (1995))»But since Fas and TNFR2 are not only expressed on immune cells, they are also expressed in several other important organs (such as the liver), so it is impossible to achieve with Fas and TNFR2 molecules. To control the purpose of excess T cells. This type of expression limits the therapeutic range of the two antibodies that bind Fas and TNPR2 molecules. Selectins, integrin and immunoglobbbulin (Ig) superfamily members are three types of adhesion molecules between white blood cells and platelets, or white jk balls and blood _ small The interaction between plates and the extracellular matrix and vascular end oth el ium is quite important (Springer, Nature 346: 425 (1990); Osborn, Cell 62: 3 (1990); Hynes , Cell 69:11 (1992)). Adhesion molecules expressed on one cell bind to other adhesion molecules on another cell to form a ligand-receptor pair. The selectin family consists of P-selectin (or CD62, CD62P, GMP140 and PADGEM), E-selectin (or ELAM-1 and CD62E) 6 1359024 and L-selectin (or LECAM-l, Mel- 14, LAM-1 and CD62L). The above selectins are highly similar and both contain an N-terminal lectin domain of about 12-theta amino acid, and an endothelial growth factor-like structural region (EGF-like). Domain), and several structural regions similar to the multiple short consensus repeat (SCR) of the complement regulatory protein. And after the above-mentioned structural region is followed by a transmenbrane domain and a short cytoplasmic tail (Siegelman et al., Science 243: 1 1 65-1 172 (1 989); Lasky et al., Cell 56:1 045-1055 (1 989); Tedder et al., J. Exp. Med. 1 70:123-133 (1 989); Johnson et al., Cell 56:1 033- 1 044 (1989); Bevilacqua et al., Proc. Natl. Acad. Sci. USA 84:9238-9242 (1987), Bevilacqua et al., Science 243:1160-1165 (1 989),

Bevilacqua et al., J. Clin. Invest. 9 1:379-387 (1 993), Camerini et al., Nature 280:496-498 (1989)). Regarding the binding properties to cellular receptors, these selectins have some overlap but not the same specificity (Bevilacqua et al., J. Clin. Invest. 91:379-387 (1993); Feize, Current Opinion, In Struct. Biol. 3: 701-710 (1 993); Berg et al., Biochem. Biophys. Res. Comm. 184: 1048-1 055 (1 992); Foxall et al., J. Cell Biol. : 895-902 (1992); Larsen et al., J. Biol. Chem. 267:11104-1 1110 (1992); Polley et al., Proc. Natl. Acad. Sci. USA 88:6224-6228 (1991 ))) 7 1359024 Inflammation, P-selectin, E-selectin and L-selectin regulate the adhesion between the original white blood cells and endothelial cells, and between small plates and white ash balls (Be-vilac-qu-a - et-ah, 1 993V supra) ° έ MW: it ^ ^ iS # are involved in the initial "rolling" interaction between white blood cells and activated endothelial cells (initial "rolling" interaction) (von Andrian et al., Proc. Natl. Acad. Sci. USA 88:7538-7542 (1 991); Ley et al., Blood 77:2553-2555 (1 991 ); Abassi et al.s J. Clin. Invest. 92:2719- 2730 (1993); Dore et al., Blood 82:1 308-1 3 16 ( 1 993); Jones et al., Biophys. J. 65:1560-1 569 (1993); Mayadas et al., Cell 74: 54 1 -5 54 (1 993)). P-selectin, which is expressed on activated platelets and endothelial cells, binds to cell surface proteins on most white blood cells (River 0 ugly 6161 Wang 1.,: [.81〇1. (:116111· 250:9799-9804 (1 984); Hsu-Lin et al., J. Biol. Chem. 264:8 121-9 126 (1984)). E-selectin is expressed on cytokine-activated endothelial cells, such as tumor necrosis factor After 6-8 hours of cytokine stimulation with -a (TNF-a) or interleukin-2, E-selectin on endothelial cells binds to most white blood cells (McEver et al., J. Clin. Invest 100:485-492 (1997); Bevilacqua et al., 1 987, supra;

Bevilacqua et al., 1989, supra). L-selectin, which is expressed on most white blood cells, binds to cell surface proteins on some endothelial cells or other white blood cells (Gallatin et al., Nature 304: 30-34 (1 983); Berg et al., Immunol Rev. 108:5-1 8 (1989); Berg et al., J. Cell. Biol. 1 14:343-349 (1 99 1 ), Hallman et al., Biochem. Biophys. Res. Comm. 174 :236-243 (1991); Smith et al., J. 8 1359024

Clin. Invest. 87: 609-61 8 (1 991 ); Spertini et al., J.

Immunol. 147: 2565-2573 (199 1)). It has been confirmed that all three of these factors can bind to the cell surface protein "P-selectin glycoprotein ligand 1", and the P-selectin glycoprotein ligand 1 is almost exclusively expressed in white blood cells and some special T cells. Posttranslational modification of βρ_selectin listener protein ligand j on natural killer cells for the binding of selectin glycoprotein ligand 1 to P-selectin, E_selectin and L_selectin Is a necessary condition (McEver et al., J. Clin. Invest., 1 997, supra) ° L content of the invention. The present invention relates to the use of a combination of the T cell surface antigen "Bu-selector brewing white ligand 1" Act to deplete tau cells, and / or induce τ fine to go to apoptosis. The method of depleting sputum cells is particularly effective for the treatment of those immune immune responses that have an excessive or non-occurring sputum cell, or that have an excessive and undesired phenomenon of tau cell proliferation. For example, depletion of τ cells can reduce or eliminate cellular activity and proliferation associated with inflammation, autoimmune disease, transplant rejection, per-sex disease, and/or squatting cancer. The present invention encompasses a method for preventing or reducing a T cell-regulated immune response by using a g-regulated g-function of the Ρ-selectin glycoprotein ligand i, and screening for a function of P-selectin-coordination H i Method of adjusting factors. One aspect of the present invention is to provide a method of preventing or reducing a T-regulated immune response in a patient. The method comprises the steps of: selecting a patient who is guilty of or may be suffering from excessive or unwanted spleen cell regulatory immunity and 9 1359024; such that the patient takes a T, 4 X cell surface P-selectin glycoprotein ligand 1 bound compound. Gan A, ^ early. Among them, the composition and the P-selection-Qin-sugar-egg-white-match-position-body...the eleven knots are combined with the sound-sounding-Ψ~. 々 praise a signal transmission path and cause T cells Death, which in turn can prevent or reduce ~ T% of patients with T cell regulatory immune response. Compounds useful in such methods of treatment comprise an antibody or an antigen binding fragment having specific binding to p-selectin protein ligand 1. In some preferred embodiments, the compound may be a monoclonal antibody that specifically binds to the P-selectin listener ligand. In a preferred embodiment, the method of the invention comprises the use of an agent that binds to a single antibody and renders the surface of the T cell. Cross-linking occurs between the selectin glycoprotein ligands 1 (cross-linking > » In some preferred embodiments, the method of the invention comprises causing P-selectin glycoprotein ligand 1 on the surface of T cells to occur a cross-linking reaction in which the cross-linking reaction induces an information transfer path leading to T cell death. In some preferred embodiments, the method of the present invention comprises the following steps: (i) selecting one diagnosed as having or possibly a patient who develops a T cell regulatory immune response that is excessive or undesired; (ii) applies to the patient a multimeric complex compound that binds to at least two P-selectin glycoprotein ligands 1 on T cells, Wherein, the multimeric complex compound contains two polypeptide chains, and each of the multi-peptide chains contains (a) a binding region that can bind to the P-selectin gamma protein ligand 1 (binding domain) And (b) a heterologous amino acid sequence, wherein the two multi-peptide chains are joined to form a poly 10 1359024 complex compound by attachment of a heterologous amino acid sequence, and Poly The complex compound binds to at least two Ρ-selectin-loaded protein ligands 1 on the surface of the tau cells, which induces a ----------------------------------------------- The τ I® cell-regulated immune response. The poly-complex compound can be a homo-multimeric compound or a hetero-multimeric compound. Optionally, the p-selectin extracellular domain or one of the P-selectin glycoprotein ligand 1 binding fragment, the E-selectin extracellular domain or one of the p_selectin-flavored protein ligands 1 binding fragment, L-selectin extracellular domain or one of p-selectin glycoprotein ligand 1 binding fragment, anti-P-selectin glycoprotein ligand 1 antibody or P-selectin glycoprotein thereof The ligand 1 binding fragment, a P-selectin protein ligand 1 selected from the phage display library, binds to the multipeptide or any combination of the above. In certain preferred embodiments, Polycomplex compounds do not contain anti-P-selectin sugar a white ligand 1 antibody or an antibody fragment that binds to P-selectin glycoprotein ligand 1. The heterologous amino acid sequence may optionally comprise a cell surface receptor binding region ), for example, the immunoglobulin heavy chain constant region. In certain preferred embodiments, the multi-peptide chain is permeable to a covalent linkage of a heterologous amino acid sequence, such as a disulfide linked, to form a multimeric complex compound. In certain preferred embodiments, the method comprises the additional step of administering to the patient an agent that utilizes a heterologous amino acid sequence to bind to the multimeric complex compound and embroider on the surface of the T cell. Several P_selectin glycoprotein ligands 1 undergo cross-linking reactions. ...

In certain preferred embodiments, the method of the invention comprises the step of selecting a patient who may or may have an autoimmune disease. In another embodiment, the method comprises the step of selecting a patient suffering from an inflammatory disease. In yet another preferred embodiment, the method comprises the step of selecting a patient who is receiving or anticipating an allogeneic or xenogeneic transplant. In another preferred embodiment, the method comprises the step of selecting a patient who may have an allergic disease. In another preferred embodiment, the method comprises the step of selecting a patient diagnosed with T cell-derived cancer. In some preferred embodiments, the T cells are activated T cells. In a preferred embodiment, the T cell is a CD4+ T cell. In another preferred embodiment, the T cell is a CD8+ T cell.

In certain preferred embodiments, the method comprises detecting the number of T cells from a first biological sample taken from a patient who has not taken a compound (e.g., a multimeric complex compound). After the same patient has used a compound (such as a poly-compound compound), a second biological sample taken from the patient who has taken the compound, and the number of T cells in the second biological sample is detected. Comparing the detection results of the first biological sample and the second biological sample. In certain preferred embodiments f' the method comprises the step of detecting the biological activity of the D-cell of the first biological sample. The first biological sample is taken from a patient who has not yet taken a compound (such as a multimeric complex compound). The measured result is compared with the T cell activity in the second biological sample. The second biological sample is taken from the patient who has taken a compound (such as a multimeric complex compound) 12 '1359024. In certain preferred embodiments, the patient's activated T cells can be at least - reduced by -1-0% after administration. - At least some 10%, 20%, 30%, 40%, 50% or more of activated T cells are reduced in certain preferred embodiment-Chinese medicine-冢. In certain preferred embodiments, the antibody, the antigen-binding fragment or the multimeric complex compound on the antibody can cause the patient's body when the activated T cell is contacted with the antibody, the antigen-binding fragment or the multimeric complex compound on the antibody. At least 1.0% of activated T cells die. In certain preferred embodiments, the administration induces at least 1 〇〇 / 0, 20%, 30%, 40%, 50% or more of activated T cells to die in the body of the patient. The phenomenon of tau cell death can be detected at any time, for example, one, two, three, four, five, six, seven or more days after the use of the antibody, the antigen-binding fragment on the antibody or the multimeric complex compound. Detection of T cell death. On the other hand, the present invention is characterized by a method of causing T cell or natural killer cell death. The method comprises the steps of: providing a T cell or a natural killer cell that expresses P-selectin glycoprotein ligand 1 on the cell surface, such that T cells and natural killer cells with a P-selection on their cell surface a glycoprotein ligand-binding compound is contacted; wherein the binding reaction of the compound to the p-selectin glycoprotein ligand 1 on the surface of T cells or natural killer cells induces a tau cell or natural killer cell The path of death message. The compound used in the method may comprise an antibody or an antigen-binding fragment of the antibody which binds to ρ-selectin 餹 protein ligand 1. In a preferred embodiment 10, the compound is a 13 1359024 monoclonal antibody that specifically binds to the Ρ-selectin glycoprotein ligand i. In a preferred embodiment, the method comprises the step of contacting a monoclonal antibody with an agent that binds to the monoclonal antibody and causes the tau cells or -------- - Self-random - killer fine-cell-table...face-on-number-one-P-selectin-sugar check- 3 plant occurrence _ cross-linking reaction. In a preferred embodiment, the invention comprises the steps of: (丨) providing a T cell or natural killer cell that expresses P-selectin glycoprotein ligand 1 on the cell surface; (ii) making the T cell Or the natural killer cell is contacted with a multimeric complex compound capable of binding to at least two P-selectin glycoprotein ligand 1 molecules on the surface of the cell; wherein the multimeric complex compound contains two multi-peptide chains Each multi-peptide chain comprises (a) a binding region which binds to P-selectin glycoprotein ligand 1, and (b) a heterologous amino acid sequence, wherein the two multi-peptide chains are permeated Joining of a heterologous amino acid sequence to form the multimeric complex compound, wherein when the multimeric complex compound is combined with two P-selectin glycoprotein ligands 1 on the surface of T cells or natural killer cells, It will induce a message transduction pathway that can kill T cells or natural killer cells. The multi-complex compound may be an isomeric polycomplex compound or a heteropolymeric complex compound. The binding region may optionally comprise a P-selectin extracellular domain or one of the P-selectin glycoprotein ligand 1 binding fragment, the E-selectin extracellular domain or one of the P-selectin glycoproteins Ligand 1 binding fragment, L-selectin extracellular domain or one of P-selectin glycoprotein ligand 1 binding fragment, anti-P-selectin glycoprotein ligand 1 antibody or P-selectin thereof The glycoprotein ligand 1 binding fragment, P-selectin glycoprotein ligand 1 selected from the phage display library, binds to the multi-peptide or any combination of the above. 14 1359024 The heterologous amino acid sequence may optionally contain a cell surface receptor binding region, such as an immunoglobulin long chain constant region β. In certain preferred embodiments, the one-to-polypeptide is passed through the iso-amine-amine. The formation of a multimeric complex compound β by a base-acid 'sequence bond, such as a thiophene cleavage'. In some preferred embodiments, the method comprises the additional step of contacting the multimeric complex compound with an agent. The agent is linked to the multimeric complex compound through the heterologous amino acid sequence, and a plurality of ρ-selectin glycoprotein ligands 1 on the surface of the tau cells are cross-linked. In some preferred embodiments, the method comprises the step of causing a cross-linking reaction of a plurality of ρ-selectin glycoprotein ligand 1 antigens on the surface of a tau cell or a natural killer cell. Among them, this cross-linking reaction triggers a message conduction pathway leading to the death of sputum cells or natural killer cells. In some preferred embodiments of the methods of the invention, the sputum cells are activated tau cells. In the embodiment, the sputum cells are CD4 + T cells. In another embodiment the 'T cell is a cd8 + t cell. In some preferred embodiments of the method, the method comprises the step of assessing the cell viability of T cells or natural killer cells after contact with a compound (e.g., a 'polymeric complex compound). In some preferred embodiments of the method, the method comprises the step of assessing the biological activity of T cells or natural killer cells after contact with a compound (e.g., a multimeric complex compound). In some preferred embodiments, the method comprises causing death of activated T cells. Another aspect of the invention features a method of screening for a regulatory factor for Ρ-selectin sugar egg 15 1359024 white ligand 1. The method comprises the steps of: providing a cell which expresses P-selectin glycoprotein ligand 1 on the cell surface; making the ------- fine-cell and two-species-test-test-quality 1 Γ; The cell survival rate after 1 曰Ιέ and test-physical-mass contact is measured, thereby determining whether the test substance is a regulator of ρ-selectin glycoprotein ligand 1. In one embodiment, the method comprises a step of detecting a cell death caused by the test substance, thereby determining whether the test substance is a regulator of ρ-selectin glycoprotein ligand 1. In one embodiment, the test substance can be an antibody or an antigen-binding fragment of the Ρ-selectin glycoprotein ligand 1 of the antibody. In a preferred embodiment, the test substance is a monoclonal antibody that specifically binds to ρ-selectin glycoprotein ligand 1. In a preferred embodiment, the method comprises contacting a monoclonal antibody with an agent that binds to the monoclonal antibody and causing cross-linking of a plurality of ρ-selectin glycoprotein ligands 1 on the cell surface. In one embodiment, the method comprises the step of cross-linking a plurality of Ρ-selectin glycoprotein ligand 1 antigens on the surface of the cell. Among them, the cross-linking reaction triggers a message conduction pathway leading to cell death. In a preferred embodiment, the sputum cells are activated sputum cells. In one embodiment, the sputum cell is a CD4 + sputum cell. In another embodiment, the sputum cell is a CD8+ T cell. In a preferred embodiment, the method comprises the steps of making a plurality of test substances and formulating the test substance with a pharmaceutically acceptable carrier. Another aspect of the present invention is characterized in that a pharmaceutical kit comprising 16 1359024 has a compound which can bind to P-selectin glycoprotein ligand 1 on the surface of a T cell and instructions for use; wherein the compound Binding to the tau cell surface selectin glycoprotein ligand i triggers a message-transferring pathway that causes tau cell death; the instructions for use indicate the method of use of the compound' to explain that this compound can be used to treat excessive or unwanted A tau cell-regulated immune response' or a treatment of excessive or unwanted tau cell proliferation, such as inflammation, autoimmune disease, transplant rejection, allergic condition, or T cell cancer. In one embodiment, the kit comprises (1) a multimeric complex compound that binds to at least two p-selectin glycoprotein ligands i on the surface of the T cell, wherein the multimeric complex compound has two a multi-peptide chain, each of which contains (a) a binding region capable of binding to P-selectin glycoprotein ligand 1 and (b) a heterologous amino acid sequence. Wherein the multi-peptide chain is formed by the linkage of a heterologous amino acid sequence to form a multimeric complex compound, and the multimeric complex compound binds to at least two P-selectin glycoprotein ligands 1 on the surface of the T cell. The reaction causes a message conduction pathway that causes T cells to die. In addition, the kit also contains (ii) a instructions for use to explain the method of use of the compound, and that the kit can be used to treat excessive or unwanted T cell regulatory immune responses, or to treat excessive treatment. Or unwanted T cell proliferation, such as inflammation, autoimmune disease, transplant rejection, allergic conditions, or tau cell cancer. The present invention has the advantage of causing a phenomenon of depletion of tau cells or tau cells, but does not produce an unwanted or noxious immune response. For example, in certain preferred embodiments, a patient using an anti-P-selectin protein ligand 1 antibody or a multimeric complex compound of the invention does not cause, for example, 17 1359024-2 or 戚The amount of inflammatocyte 〇 cytokine such as tumor necrosis factor-α was abnormally elevated. ------------ The other two I® in the fine-negative-agonistic composition to deplete tau cells. Accordingly, the present invention provides an active immunosuppressive method different from passive immunosuppression to induce active immunosuppressive action; the passive immunosuppressive system is due to the use of an antagonistic composition (antagonistic C〇mpositi〇n) ( For example, 'antagonistic anti-p-receptor glycoprotein ligand 1 antibody or antagonist soluble selectin fragment) utilizes binding to an immunoreceptor to prevent an immune activation response regulated by such receptors. Another advantage of the present invention is that it only acts on the p-selectin listener protein ligand 4 which is almost exclusively expressed on the surface of white gray spheres (especially T cells or natural killer cells). Thus, the compounds of the present invention do not cause significant cytometry of other cells (e.g., hepatocytes). Selective depletion of tau cells or natural killer cells (especially CD3 cells involved in transplant rejection, ', M # ^ a Λ ) without causing fatal fineness: hormonal response and injury to other organs The properties desired by immunosuppressive agents. Unless otherwise defined, the meaning of the phrase in the present invention is well known to those skilled in the art and skilled in the art. At the same time, there are other things that can be used as the present invention, or similar or equal, and the implementation or test demonstration of the material, and the following is mentioned in the text. References are fully incorporated into monthly cases, patents, and other specifications. In the course of the discussion, the present invention is intended to be illustrative, and the methods and materials described herein are not intended to limit the scope of the invention. 18 1359024 Other heat of the invention The following detailed description and application features and advantages will be described by the scope of the patent. [Embodiment] The present invention relates to a method for regulating the diameter of the sputum and the activity of the shoulder cells by using the function of the ρ-selectin glycoprotein ligand 1 on the surface of the τ cell. Content of the present invention

The combination of the described P-selectin glycoprotein coordination hetero 3 A 臼 ligand 1 with a synergistic composition can result in depletion of tau cells and/or progression of tau cells to cells> Therefore, the synergistic composition can be used as a therapeutic agent for controlling immune-related symptoms such as inflammation, autoimmune diseases, transplant rejection, allergic diseases, and/or tau cell-derived cancer. Synergistic compositions can also be used to induce tau cell depletion of any biological sample having an excess of tau cell number or activity. Ρ-selectin glycoprotein ligand 1 (PSGL-1) P-selectin listener protein ligand 1 is a cell surface adhesion molecule' expressed in neutrophils, T lymphocytes and B lymphocytes (T and B-lymphocyte), natural killer cells, monocyte, dendritic cell and primitive human CD 34 hematopoietic progenitor cell. Through the binding of P-selectin glycoprotein ligand 1 to selectin, P-selectin glycoprotein ligand 1 can regulate the rolling movement of white blood cells in endothelium and regulate the penetration of white blood cells into blood vessels. The behavior of an inflamed tissue. The binding of tau cells mediated by P-selectin glycoprotein ligand 1 to sputum-selectin and sputum-selectin or the regulation of sputum cell migration 13 1359024 to differential regulation. For example, when tau cells are transformed from a primitive state to a memory sputum cell, cutaneous lympr〇cyfe~ abtigen (CLX) (epitope) is expressed on the surface of sputum cells. And 'only activated type 1 helper tau cells can exhibit a functional Ρ-selectin glycoprotein ligand 1 and can move to the inflamed area of the skin. The second type of helper sputum cells do not have this function. Ρ-selectin glycoprotein ligand 1 is a sialomucin that undergoes special sialylation, fucosylated and suifated to bind to P-selectin. . P-selectin glycoprotein ligand 1 produces several isoforms due to differences in the degree of saccharification at the N-terminus of the protein chain and the position of the vulcanization. The protein expression of P-selectin glycoprotein ligand 1 in peripheral blood T cells and B cells, lymphocyte strains and in vitro activated peripheral blood T cells was substantially equal. And only activated T cells will exhibit a functional P-selectin glycoprotein ligand 1 and have a high affinity for binding to P-selectin. Activation-dependent binding activity is the result of varying degrees of post-translational modification, for example by increasing alpha (1,3)-trehalose in activated T cells. Transferase (alpha (1,3) fucosyltransferases) activity to alter the extent of saccharification. The isomer of P-selectin glycoprotein ligand 1 also exhibits different affinity for L-selectin and E-selectin. For example, human T cells expressing skin lymphocyte antigen (CLA) positive may adhere to E. - Selectin and P-selectin and roll. T cells expressing P-selectin glycoprotein ligand 1, but not having a skin lymphocyte epitope, will only bind to P-selectin. 20 1359024 Furthermore, the binding of P-selectin glycoprotein ligand 1 to P-selectin is determined at the terminal ten amino acid decarboxypeptides, and t contains three sulfur----- ----- --------- --------------- The road to a - amino acid - (iyr〇-sTney. -3⁄4-友-一^ Thionine 〇 can utilize the recombinant method and/or isolate the native P-selectin glycoprotein ligand 1 from biological tissues to obtain P-selectin glycoprotein ligand 1. The recombinant protein of ρ-selectin glycoprotein ligand 1 can be produced in prokaryotic or eukaryotic cells, either in vivo or in vitro. Recombinant protein production is carried out using a nucleic acid sequence having a Ρ-selectin glycoprotein ligand 1 gene sequence. For the nucleic acid sequence of Ρ-selectin glycoprotein ligand 1 please refer to GenBank Accession NM_003006. At the same time, antibodies for identifying P-selectin glycoprotein ligand 1 are well known to those skilled in the art and can be used to purify antigens and/or as described herein (Herron et al. (2000) Science Jun 2; 288 (5471): 1653-56; WO 0 0/25808). A further detailed description of P-selectin glycoprotein ligand 1 can also be found in Sako et al. or other literature (1993, Cell 75: 1179; Vachino et al. (1995) J. Biol. Chem. 270 : 21966; and Veldman et al. (1 995) J. Biol. Chem. 270: 1 6470). Expression of P-selectin glycoprotein ligand 1 and β(1,3)-trehalose transferase (Fuc-T VII) during the production of recombinant protein of P_selectin glycoprotein ligand 1 The modification of the Ρ-selectin glycoprotein ligand 1 needs to be carried out simultaneously in order to exhibit a functional P-selectin glycoprotein ligand 1 . And in the manufacturing process of P-selectin glycoprotein ligand 1 recombinant protein 21 Ϊ 359024, 'cotransfaction' can be selected to remove the propeptide, and/or Transfection of a portion of uryltransferase

The anti-P-selectin glycoprotein ligand 1 antibody can be used for isolating and purifying the P-selectin glycoprotein ligand in the biological neoplasm, and any cell line expressing the p-selectin glycoprotein ligand 1 is T cells obtained from patients or T cell strains are all sources of P-selectin glycoprotein ligand 1. The purified P-selectin glycoprotein ligand 1 can be used to carry out the various experiments described in the present invention. For example, purified Ρ-selected de-glycoprotein ligand 1 can be used to perform in vitro screening of τ cell-selectin glycoprotein ligand 1 functional regulators or to identify Ρ-selectin glycoproteins Immunogen of the antibody of ligand i (immunogen) anti-P-selectin glycoprotein ligand 1 antibody

P-selectin glycoprotein ligand 1 (or immunogenic fragment or analog thereof) can be used to produce antibodies in the methods of the invention. As described above, recombinant recombination technology or solid phase synthesis method can be used to produce recombinant P-selectin glycoprotein ligand 1 or its peptide fragment e p_selectin glycoprotein ligand 1 The multi-peptide or its peptide fragment can be used as an immunogen to produce an anti-P-selectin glycoprotein ligand 1 antibody. In addition, an anti-P-selectin glycoprotein ligand 1 antibody, such as a TAB4 monoclonal antibody, can be used to purify p_selectin-flavored protein ligand 1. For example, an anti-P-selectin glycoprotein ligand 1 antibody can be used to purify a P-selectin glycoprotein ligand of a natural structure, and a purified P-selectin glycoprotein ligand i can be used as a production resistance.卜22 1559024 The immunogen of the glycoprotein ligand 1 antibody. The antibody of the present invention may be a single antibody, a multi-drug antibody (polyclonal) - a poorly-selected saccharide egg self-contained cylinder factory has a singularity of 1 liters of 3 human body (engineered antibody). An antibody having a specific binding activity to a specific antigen (e.g., ρ•selectin 2 protein 1) does not recognize or bind to other molecules in the test sample. It is therefore a feature of the present invention to provide several methods of identification for finding test compounds (e.g., antibodies) that bind to the multi-peptide of the present invention. The method of the present invention is to contact a test compound with the multi-peptide of the present invention, and to directly block the binding force, or to add a competitor molecule to hinder the binding of the test compound to the multi-peptide, or to detect the molecule by using an apoptosis activity test. A method such as binding force is used to judge whether the test compound can bind to the multi-peptide. The P-selectin glycoprotein ligand i can be mixed with a carrier protein such as cardinal limpet hemocyanin (KLH) and mixed with an adjuvant to be injected into a mammalian host. Subsequently, the antibodies produced in this mammal can be purified using peptide antigen affinity chromatography. If necessary, P-selectin vinegar protein ligand 1 or an antigenic fragment thereof can be injected into various animals to cause an immune response. Commonly used animals include rabbits, mice, guinea pigs and rats. The adjuvant used to increase the immune response needs to be selected in accordance with the animal germline used, including Freund's adjuvant (complete adjuvant and incomplete adjuvant), mineral gel such as aluminum hydroxide. , surface active substances such as lysolecithin (or lysolecithin), pluronic polyols, polyanions, 23 1359024 peptide oil emulsions, Jk cyanide protein and dinitrophenol. Human adjuvants that may be useful include bacille Cklmette-GiiVrin (BCG) 3 Short rods. Corynebacterium parvum ° Multi-strain system refers to heterogeneous populations present in the serum of immunized animals. antibody. The antibody of the present invention comprises a plurality of antibodies, monoclonal antibodies, humanized or chimeric antibodies, single chain antiodyses, Fab fragments, F(ab')2 fragments and expression using Fab The product produced by the library. A monoclonal antibody system refers to a homologous polulation that recognizes a specific antigen, which can be prepared using the above-described P-selectin glycoprotein ligand 1 multi-peptide and standard hybridoma technology (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, N.Y. [1981]). In some cases, antibody-producing methods for culturing a permanent cell line can be used to obtain monoclonal antibodies, for example, in the case of Kohler et al., in Journal of Nature 256:495 (1975) and U.S. Patent No. 4,376,110. Human B cell fusion tumor technology described in (Kosbor et al, Immunology Today 4:72 [1 983]; Cole et al., Proc Natl Acad Sci USA 80:2026 [1983]), and EBV fusion tumor technology ( Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, 24 1359024

Inc., ρρ· 7 7-9 6 [1983]). Such antibodies may be any of the immunoglobulin classes 'including IgG, IgE, IgM, IgA, IgD and subclasses of any immunoglobulin. The fusion tumor of the present invention for producing a single corpus callosum can be cultured in vivo or in vitro. The in vivo method is a more effective method for the production of high-concentration individual strains. The obtained monoclonal antibody or multiple antibodies need to be determined by Western blot or immunosuppression; immunoprecipitation analysis to determine the specific recognition of p-selectin glycoprotein ligand i, The detection method can be referred to a paper such as Ausubel et al. An antibody having specific discriminating power and binding ability to P-selectin glycoprotein ligand 1, in particular, capable of binding to P-selectin glycoprotein ligand 1 antigen on the surface of T cells (such as CD3 + cells), The anti-P-selectin glycoprotein ligand 1 antibody which can cause T cell depletion or dying in a patient can be used for the present invention. The above antibodies have a variety of uses, for example, can be used to formulate medical compositions to reduce or eliminate undesired immune responses, such as concomitant inflammation, autoimmune diseases, transplant rejection, allergic diseases, and T cell-derived cancer T cells. The T cell regulatory immune response. The above antibodies can also be used in the screening assay to test the binding ability of the test compound to P-selectin glycoprotein ligand 1. In addition, chimeric antibodies can be produced by cleavage of a single antibody gene having appropriate antigen specificity and a human antibody gene having appropriate biological activity, and combining the two to produce a chimeric antibody (Morrison et al_, Proc Natl Acad Sci USA 81 :685 1 [1 984]; Neuberger et al., Nature 312:604 25 1359024 [1984]; Takeda et al., Nature 3 14·452 [1984]). A chimeric anti-system refers to a molecule from which different parts of the molecule are derived from different animal species. For example, a molecule with both mouse monoclonal antibodies and a constant region of human immunoglobulin®

The single-chain antibody production technique (U_S. Pat. Nos. 4,946,778, 4,946'778, and 4,704,692) can also be used to prepare a single-chain antibody against p-selectin glycoprotein ligand 1, or a single-chain antibody fragment thereof. The single-stranded anti-system utilizes an amino acid to bridge the heavy chain and light chain fragments in the anti-Zhao Fv region to form a single-chain polypeptide. Techniques for the preparation of antibody fragments that recognize a particular epitope are well known to those skilled in the art. For example, pepsin can be used to decompose an antibody to obtain an F(ab')2 fragment. The disulfide bond on the F(ab,)2 fragment can also be reduced to obtain a Fab fragment. Or use the Fab expression library to achieve the purpose of easily and quickly identifying a single Fab fragment with a specific affinity.

Conventional techniques can be utilized to anthropomorphize antibodies. For example, a single-body antibody with specific __ binding can be used for commercial-scale anthropomorphic modification (Sc〇tgene, Scotland; Oxford Molecular, Palo Alto, Calif.) 0 fully anthropomorphic antibody (eg in a gene-transforming animal body) Internally expressed antibodies) are also one of the features of the present invention (Green et al., Nature Genetics 7:13 [1994]; and US Pat. Nos. 5,545,806 and 5,569,825) ° Polyplex compounds can be associated with T cells or naturally The poly-complex compound of p-selectin glycoprotein ligand 1 protein binding on the surface of killer cells can be used to induce cells > week 26 1359024. The polyplex compound contains at least two multi-peptide bonds. 1 ^3 The peptide chain contains (i) a region that can bind to the P-selectin glycoprotein ligand 1 box, and (π)-heterologous amino acid. Preface > ^ ^*· In summary, a multimeric complex compound binds to two different P-selectin glycoprotein ligand white 1 proteins on a specific cell surface. Non-4,5,6' polymeric complex compounds can also be designed to have 3, 7, 8, 9, 10, 11, 12 or more different P-selectin listeners a region such that the multimeric complex compound is capable of interacting with 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more P-selectin glycoprotein ligands on a particular cell surface Combine. The binding region comprises a 15'.33⁄4 amino acid sequence which is capable of binding to the P-selectin glycoprotein ligand 1 or any modified amino acid sequence' such as a sulfonation or sulfidation reaction. The binding region can be identical to a naturally occurring amino acid sequence or a naturally occurring amino acid sequence. For example, the binding region may comprise a Ρ-selectin glycoprotein ligand 1 binding region of a selectin such as Ρ-selectin, Ε-selectin or L-selectin. The multi-peptide chain comprising the selectin-selectin glycoprotein ligand 1 binding region may optionally comprise: (i) a selectin membrane outer region (eg, Ρ_selectin, Ε-selectin or L- Selective elemental membrane); (Π) a selectin (such as ρ_selectin, Ε-selectin or .....,, a % dependent lectin domain; or (ίϋ An extra-membranous region fragment of a selectin (P-selectin, E-selectin or L-selectin) that is used to regulate the binding ability of a selectin to a glycosidin ligand 1. In addition, In the case of an amino acid sequence, one or more amino acids in the binding region of the natural p-selective k-sugar egg ligand 1 can be altered to distinguish the sequence from the 27 1359024 sequence, and the unnatural amino acid sequence is retained. Its function to bind to p-selectin glycoprotein ligand 1. For example, a multi-peptide chain contains a segment that can bind to p.-selection-option-trick egg-white-match-position-one-1 The amine tomb column and the amino acid sequence has at least 80%, 85%, 90%, 95% or

98% similarity. These arbitrary fragments include: (0-selectin (such as P-selectin, E-selectin or L-selectin) extramembranous region; (ii) a selectin (such as P-selectin, E-selectin or a calcium-dependent lectin structural region of L-selectin; or (iii) a selectin (such as P-selectin, E-selectin or L-selectin) for regulating selectin versus P-selectin The extra-membranous region fragment of the binding ability of glycoprotein ligand 1. The general molecular biology mutagenesis technique can be used to change the nucleic acid sequence of the binding region of the P-selectin glycoprotein ligand 1. The modified binding region Subsequent use

The P-selectin glycoprotein ligand 1 or the P-selectin glycoprotein ligand 1 on the cell surface was tested for its binding ability to P-selectin glycoprotein ligand 1. The binding region may also comprise an anti-P-selectin glycoprotein ligand 1 antibody, or a p-selectin glycoprotein ligand 1 binding region selected from a peptide of a phage display library, or a segment capable of - Selecting the p-selectin glycoprotein ligand 1 binding region of the amino acid sequence to which the protein ligand 1 binds. Therefore, the amino acid sequence needs to have at least 80 binding to the P-selectin glycoprotein ligand 1 antibody of the anti-P-selectin glycoprotein ligand 1 antibody or a P-selectin glycoprotein ligand 1 selected from the phage display library. %, 85%, 90%, 95% or 98% similarity" P_selectin glycoprotein ligand 1 binding region may comprise an amino-based fluorene sequence, the amino acid sequence and P on the p•selectin - The select glycoprotein ligand i binding fragment is identical. For example, the poly-compound 28 1359024 % chain of the poly-complex compound described herein contains a mouse P-selectin/Fc recombinant conjugate (R&D Systems, Minneapolis, MN). This segment of mouse P-selectin/Fc ----------- want-complex-package-containing-column structure-mKi fights alanine 16, MeU-Alal6); (Π) small Murine-selectin (fragment of tryptophan 42 to alanine 709 in the extracellular domain, Trp42-Ala709 of extracellular domain); (iii) IEGRMD (SEQ ID NO: 1); and (iv) human immunoglobulin Gl (proline 100-isamino acid 330, Prol00-Lys330). Another preferred example of a multi-peptide chain can contain human P-selectin/Fc recombinant enantiomers.

(R&D Systems, Minneapolis, MN). This human p_selectin/FC recombinant chimera contains the following structures: (i) human P-selectin (a fragment of methionine 1 to alanine 771 in the extracellular domain, Metl-Ala771); (ii) IEGRMD (SEQ ID NO: 1); and (iii) human immunoglobulin gi (proline 1 〇〇 lysine 330). The P-selectin glycoprotein ligand 1 binding region may comprise an amino acid sequence. The amino acid sequence is identical to the P-selectin glycoprotein ligand 1 binding fragment on E-selectin. For example, the multi-peptide chain of the poly-complex compound described herein comprises a small fluoro E-selectin/Fc recombinant chimera (R&D Systems, Minneapolis, MN). This segment of mouse E-selectin/Fe-adherent contains the following structures: (1) mouse E_selectin (fragment of methionine 1 to amino acid 557 in the extracellular domain 'Metl_Pr〇557); oligopeptide IEGRMD ( SEQ ID NO: 1); (Ui) human immunoglobulin g1 (proline lysine 330, Pr 〇 100_Lys33 ;); and (iv) oligopeptide HHHHHH (SEQ ID NO: 2). Alternatively, the preferred multi-stranded chain may comprise a human E-selectin/Fc recombinant chimera (R&D Systems, Minneap(R) H, 29 1359024 MN). This human E-selectin/Fc recombinant chimera contains the following structure: (i) human E-selectin (purine thiol in the extracellular domain! to fragment of proline 556, Metl-pr〇556); (Π) oligopeptide IEGRMD (SEQ ID ΝΟ: Γ); (iii) human immunoglobulin G1 (proline 100-isamino acid 33 0); and (iv) oligopeptide HHHHHH (SEQ ID NO: 2). The P-selectin glycoprotein ligand 1 binding region may comprise an amino acid sequence which is identical to the P-selectin glycoprotein ligand 1 binding fragment on L-selectin. For example, the multi-peptide chain of the poly-complex compound as set forth herein contains a mouse L-selectin/Fc recombinant chimera (R&D Systems, Minneapolis, MN). This mouse L-selectin/Fc chimera comprises the following structures: (i) mouse L-selectin (fragment of methionine 1 to aspartic acid 332 in the extracellular region, Metl-Asn332); (ii) oligopeptide IEGRMD (SEQ ID NO: 1); (iii) human immunoglobulin G1 (proline 100-isamino acid 3 30 , Prol〇〇-Lys330); and (iv) oligopeptide HHHHHH (SEQ ID NO : 2). Another preferred embodiment of the multi-peptide chain may comprise a human L-selectin/Fc recombinant chimera (r&d Systems, Minneapolis, MN). This human L-selectin/Fc recombinant chimera contains the following structure: (i) human L-selectin (a fragment of thiol acid 1 to aspartic acid 3 32 in the extracellular region, Metl-Asn332); (ii) oligopeptide IEGRMD (SEQ ID NO: 1); (out) human immunoglobulin G1 (proline 100- lysine 330); and (iv) oligopeptide HHHHHH (SEQ ID NO: 2). The multimeric complex compound can be prepared as a homopoly complex compound or a heteropoly complex compound. The multi-peptide chain of the same polymeric complex compound has only the same P-selectin gamma protein ligand 1 binding region. For example, the 30 1359024 homopoly complex compound may contain a multi-peptide chain identical to the sequence of the P-selectin P-selectin glycoprotein ligand 1 binding region. One of the heteropolymeric complex compounds has a different -p_-selecto-glycoprotein ligand 1 binding region on the poly-peptide-chain. For example, the first multi-peptide chain of the heteropolymeric compound has the P-selectin glycoprotein ligand i binding region of p-selectin, while the second multi-peptide chain has E-selectin P-selectin glycoprotein ligand! Consolidation zone. The heterogeneous amino acid sequence may be any of the linked acid sequences. However, the amino acid sequence of this segment of the polypeptide chain described herein may not be similar to the sequence of any naturally occurring protein. The heterologous amino acid sequence contains one or more amino acids for use as a link between the multi-peptide chains. For example, one or more amino acids can be covalently bonded to a multi-peptide bond by, for example, a disulfide bond. For example, an immunoglobulin long bond can be used as the heterologous amino acid sequence. The disulfide bond in the constant region (Fc) can link two multi-peptide chains to form a dimeric compound. In addition to the function of linking the multi-peptide chain, the heterologous amino acid sequence may further comprise a cross-linker bind region, such as a cell surface receptor binding region, when the cross-linking molecule is selected. After binding to a single agent, the combination of the multi-peptide chain and the ρ·selectin glycoprotein ligand 1 protein on the cell surface can be caused by a cross-linking reaction. The immunoglobulin long chain region contains an Fc receptor binding region. The cross-linking molecule may be an antibody, such as an anti-Fc antibody, which has a specific binding ability to the cross-linking molecule binding region on the heterologous amino acid sequence. 31 1359024 Compound screening assay for modulating the function of P-selectin glycoprotein ligand 1 The present invention also encompasses a ligand having a P-selectin glycoprotein 1 (or p-selection of an egg-white coordination-Zhao —1 I: The identification method of the combination of the ability of the zone and the stone. The above compounds include at least the ability to induce T cell depletion and T cell apoptosis by binding to the Ρ-selectin glycoprotein ligand i. a compound, a compound that modulates the binding reaction of P-selectin glycoprotein ligand 1 to a transmembrane protein, an intercellular protein or an intracellular protein that controls the activity of P-selectin glycoprotein ligand 1, and a regulatory P-selection Compounds that are liganded for activity. The compounds screened according to the methods of the invention may include peptides, antibodies or partial fragments of antibodies and other organic compounds, and as described herein, 'such organic compounds It can bind to Ρ-selectin glycoprotein ligand 1 and can regulate the biological functions regulated by Ρ-selectin glycoprotein ligand 1. Compounds having the above functions may include a triumphant, such as a soluble peptide, Including Various peptide molecules in the peptide library (Lam et al., Nature 354: 82 [1991]; Houghten et al., Nature 354: 84 [1991]). And the compound also includes D- and/or L- a combination of a chemically derivatized molecular library, a phosphorylated peptide, an antibody and an organic or inorganic mol'ecules, wherein the acidified peptide is included in the phosphorylated peptide library. Random or partially degenerate molecules (Songyang et al., Cell 72:767 [1993])» and antibodies include multiple antibodies, monoclonal antibodies, anthropomorphic antibodies, anti-idiotypic antibodies, chimeric antibodies or single-chain antibodies, FAb, F(ab')2, FAb expression library fragments, and antigenic epitope binding fragments of the above various types of antibodies. 32 1359024, other compounds selected according to the method of the present invention further comprise organic small knives, such small organic molecules Will affect the ρ·selectin glycoprotein ligands. Egg computer simulation and data search techniques can be used to identify compounds or compounds that have been identified as modulating the amount or activity of P-selectin glycoprotein ligands. Make improvements. When & The active site or region can be identified by identification of the compound or composition. The active site is generally a regulatory factor and a binding region. The method can be utilized, such as the amino acid sequence from the peptide: The nucleic acid sequence on the nucleic acid, or the complex formed by the ligand of the compound and a similar compound or composition of the compound to identify the active site. In the following, 'using chemical methods or x-ray crystal diffraction method

CryStall0graphic method), and find the active site of the compound by looking for the regulatory factor or the binding position of the ligand to the molecule. Although it is a method to design and manufacture a compound having binding ability with reference to the above. Another method is to select from known compounds, including natural products, synthetic chemicals and biologically active substances including proteins, to bind to the P-selectin glycoprotein ligand and to produce a decrease in T cells. , or trigger the apoptosis of T cells. An in vitro assay can be designed to identify whether a compound binds to p-selectin glycoprotein ligand 1 (or a structural region on the P-selectin glycoprotein ligand 1 protein). The identified compounds may have regulatory T cells. The function of activity may therefore be suitable for the treatment of symptoms associated with excessive or unwanted T cell regulatory immune responses or T cell hyperplasia, such as inflammation, autoimmune disease, transplant rejection, allergic disease or T cell carcinoma. . 33 1359024 The test method of the compound of the present invention which can be combined with the compound of the P-selectin glycoprotein ligand 1 includes: preparation for the preparation of a glycoprotein ligand containing P-selectin 1 (or its structural region fragment) and hui-bubble # _ ) to obtain a mixture of compounds, and the mixture is placed under appropriate reaction conditions for a sufficient time to make the two components in the mixture Interact and combine. With you.. - Then the mixture will produce a complex that can be removed and/or detected. The type of the quinone-selectin glycoprotein ligand 1 used in the tube _ by μ # ^ Λ ^ Du Guan can be changed according to the purpose of the screening test. In some cases, a peptide having the same sequence as the structural region of the Ρ-selectin. glycoprotein ligand i can be used, and the peptide can be fused with another protein or multiple peptides for convenient experimentation. The purpose, for example, as a calibration mark (labeHng), or to separate the resulting complex from the mixture. In addition, different methods can be used for the screening test, for example, the p_selectin glycoprotein ligand 1 protein, the multi-peptide, the short peptide, the fusion protein or the test compound can be immobilized on a solid phase. And after the end of the reaction, 'detect the amount of the P-selectin glycoprotein ligand 丨/test compound complex remaining on the solid surface. In a preferred embodiment of the method, the p-selectin protein ligand 1 is immobilized on a solid surface, and the test compound is not immobilized on a solid surface. And the test compound can be labeled by direct or indirect methods. In the preferred embodiment, microtiter plates can be used as solid surfaces. Further, a non-covalent linkage or a covalent linkage can be used to immobilize the component to be immobilized on a solid surface. A non-covalent linkage can be achieved by coating a solid surface with a protein solution containing the protein to be fixed. Alternatively, the protein can be immobilized on a solid surface using a solid 34 1359024 identifiable antibody that recognizes the protein to be immobilized. The antibody to be purified is preferably a monoclonal antibody. The solid surface can be stored as one-to-one after the above--storage 1---------------------------------- The non-fixed ingredients are then added to have a fixed build. After the reaction is completed, under the condition of the surface of the composite body produced after the reaction, by means of rinsing, the method of detecting the remaining on the solid surface can be detected by various methods. Mark, after the reaction, if the mark signal is detected, it means that the complex is generated. If the labeling process is not fixed, an indirect labeling method can be used to invert the complex, for example, using a labeled antibody, the immobilized component has a specific binding force, and the labeled antibody is recorded again, or the labeled antibody is utilized. Anti-immunoglobulin antibodies are ligated to the label. The above reaction can also be carried out in the liquid phase, and the unreacted components are separated and the complexes are determined to be 4, and a pair of Ρ-selectin glycoprotein ligands 1 are integrated, multi-peptide, short A peptide fragment, a fusion protein, or a complex produced in a liquid to be tested. And using a labeled antibody that has a specific binding ability to detect complex sputum or a cell activity screening method (cen-based binding to P-selectin glycoprotein ligand 1) - selecting a cell line of the glycoprotein ligand 1, or modifying the P-selectin glycoprotein ligand 1 to be used after the immobilization method, the solid surface of the first treatment can still be immobilized on the solid reaction component I. The combination of the components on the non-solid surface is not pre-measured in the solid-labeled antibody to the non-available direct label of the product after the antibody is applied. For example, the immobilized immobilized compound To capture the other quantities in the anti-body, the assay is used to screen the material. The use of the gene cell line will be used 35

I screening test. The cell viability screening method is obtained by evaluating the use of the sieves described in the text. _ No JB » For example, ~ roots ~ according to a P _ 萚 1 鲧 ~ 2 functional effects are particularly effective . For example, after a compound, the binding ability of the ligand 1 can be extracted and the sulfonate can be combined with the effect of the sulfonate, for example, whether the number of T cells can be exhausted in a body cell screening test. Induction of T cell apoptosis or pharmaceutical composition in @ or in vitro assays Another preparation of the present invention + ▲ is to provide a pharmaceutical composition for the purpose of altering a patient's immune response. This ^ composition contains such as antibodies, poly-complex

Body compound, small fraction + &#4_ U bis-selectin glycoprotein ligand 1 has a specific,,, 〇 力 之 他 他 他 在一 在一 在一 在一 在一 在一 在一 在一 在一 在一The compound has the efficacy of a ruthenium-selectin glycoprotein ligand. The pharmaceutical composition of the present invention can be formulated by a conventional method using - or more than a physiologically acceptable carrier or adjuvant. Because of &, the compound and its physiologically acceptable salts and solvates can be prepared according to the needs of the different modes of administration. The compounds of the present invention can be prepared in a dosage form for parenteral administration, e.g., by rapid injection or continuous instillation. Injectable Formulations Formulations can be prepared in a single dosage form such as a small glass vial or in a multi-dose package with a preservative. The compositions of the present invention can be formulated as emulsions in suspensions, oily or aqueous solutions. And the formulation may contain a formulation such as a suspending agent, a stabilizer or a dispersing agent. Further, the active ingredient can be prepared into a powder form and used in a suitable solvent (e.g., sterile pyrogen-free water) 36 1359024 before use. Method for controlling T cell regulatory immune response and depleting the number of tau cells

The compounds in the above screening assays can be used to modulate biological functions regulated by p-selectin glycoprotein ligand 1, or to treat diseases that are over or not required to have an immune response, such as a tau cell-regulated immune response. These compounds contain peptides, antibodies, or partial fragments of antibodies that bind to other cells that bind to the Ρ-selector protein ligand 1 on the cell surface and induce a message-transferring pathway that allows sputum cells to die. The method of the present invention selectively adds a crosslinking agent which crosslinks the Ρ-selectin glycoprotein ligand 1 on the cell surface. The compounds described herein are useful in a variety of conditions where it is desirable to reduce or eliminate the activity of sputum cells. The compounds of the present invention are excellent in the treatment of diseases such as inflammation, autoimmune diseases, transplant rejection, allergic diseases, and sputum-derived cancers.

The anti-purine-selectin glycoprotein ligand 1 compound described in the present invention can also be used for the treatment of diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis). Juvenile rheumatoid arthritis, osteoarthritis and psoriatic arthritis, multiple sclerosis, encephalomyelitis, myasthenia gravis, Systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and wet sputum 37 1359024 eczematous dermatitis) , psoriasis, Sjogren's Syndrome 'Crohn's disease, aphthous-sexual ulcer--(#hthoiisulcery :-;& 辕itis (iritis), #膜烫( Conjunctivitis), keratoconjunctivitis, type I diabetes, inflammatory bowel Inflammatory bowel diseases, ulcerative colitis, asthma (asth'ma), allergic asthma, cutaneous lupus erythematosus, scleroderma , vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis ( Autoimmune uveitis), allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, regeneration Aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic activity Chronic active hepatitis Stevenson-Johnson syndrome, idiopathic sprue, lichen planus, Graves' di sease, sarcoidosis, original Primary biliary

3S 1359024 cirrhosis), uveitis posterior, interstitial lung fibrosis 'graft versus host disease' (gr a; fr- vefs \IsviiM plant dis e is e plant - official transplant (ITU7 exhibits a ϋϋ-ϋϋ or allogeneic tissue, such as bone marrow transplant liver transplantation or other woven or organ transplants), allergies (such as atopic allergies), AIDS and T-cell neoplasms (T-cell neoplasms) Such as leukemias and / or lymphomas ° Whether in vivo or in vitro, the method of the invention can deplete the number of T cells in a cell population. For example, in an in vitro test, the organism will be taken from the patient. The sample is contacted with the anti-P-selectin glycoprotein ligand 1 compound of the present invention and selectively used in combination with a crosslinking agent to reduce the number of T cells in the sample. The method of the present invention can also effectively increase the cell population. The effect of reducing the number of non-T cells to reduce or eliminate the T cell activity of the cell population. The following is a preferred embodiment of the invention. It is for illustrative purposes only and is not sufficient to limit the scope of the invention. BEST MODE FOR CARRYING OUT THE INVENTION The first preferred embodiment: preparation of a monoclonal antibody against T cell apoptosis-inducing protein (TAIP) "T is melted by K〇hler and Milstein cells familiar to those skilled in the art. Method (1976) , European Journal of Immunology 6:5 1 1 - 5 1 9) to produce ia to secrete the fusion antibody cells of the specified antibody, in order to produce a T cell-induced protein-specific monoclonal antibody. Antibody-producing cells can be obtained by injecting Balb/c spleen tau cells (Con A-activated Balb/c spleen 39 1359024 T cells) into hamsters. The obtained antibody-producing cells and myeloma cell lines (myeI〇ma) Ce〗 1 line) Cell-fusion------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ After the cell strain is fused, the antibody-producing cells are obtained and propagated by a common tissue culture method. The fusion cell produced by the above method can secrete a monoclonal antibody "TAB4". The TAB4 monoclonal antibody can be induced in vitro. The T cell is apoptotic and can deplete the number of T cells in the body. The protein that can be recognized by the TAB4 antibody is the T cell apoptosis-inducing protein (ΤΑΪΡ). C57BL/6J (B6) and BALB/ used in the experiment. c mice were purchased from Jackson lab (B to r Harbor, ME). Syrian hamsters are available from the National Taiwan University Medical College (Animal Core Facility, National Taiwan University Medical College). The concentrated culture supernatant of TAB4 fusion tumor cells was centrifuged at 20, OOOxg for 1 minute, and then the supernatant was adjusted to 1 by binding buffer (0.1 Μ sodium acetate, pH 5.0). Dilute the ratio of 1:1. Prepare a G-protein column with a bed volume of about 1 ml, and wash the G-protein column three times with 3-5 ml of binding buffer, and then inject the clarified culture supernatant into the G-protein column. The supernatant was allowed to flow out through the column. The supernatant from the outflow column was collected and the effluent was refilled into the column. After the supernatant was passed through the G-protein column, the column was washed with 6_1 ml of binding buffer, and then 5 ml of elution buffer (eiuti〇n buffer, 〇·1 μglycine-hydrochloric acid ( ")^1^-11(:1), 1)112.8) The antibody bound to the column was washed away and the eluate containing the antibody was collected in a manner of 1 ml per tube. 40 1359024 Add 50 μl of Tris-HCl (pH 7.5) solution to 1 ml of antibody eluate per tube to bring the pH of the antibody to neutral. After the neutralized antibody solution was concentrated, the antibody solution was dialyzed three times for 3 hours each with 2 liters of phosphate-buffered saline (PBS). After completion of the dialysis step, the protein concentration of the obtained antibody sample was measured by Bio-Rad Protein Assay (BIO-RAD, Hercules, CA). Second preferred embodiment: Preparation of mouse spleen cell suspension and T cell activation and proliferation The mouse spleen is immersed in 8 ml of Hank's balanced salt solution (HBSS) and sterilized The tablets gently shred the mouse spleen. The mouse spleen fragments were then placed in a 15 ml centrifuge tube (Costar) and centrifuged at 20 Torr for 5 minutes. After centrifugation, the supernatant was discarded and the pelleted cells were resuspended in a residual buffer by tapping the tube wall. Add to the cell suspension! Mp of erythrocyte lysis buffer (RSC lysis buffer, 0.6 M gasification record (NEUC1), 0.17 M Tris-base, pH 7.65) to dissolve red blood cells doped in the cells. The cell suspension was then allowed to stand at room temperature for 2 minutes and then rapidly cooled by the addition of 9 ml of Hank's balanced salt solution. After centrifugation at 200 xg for 5 minutes to precipitate the cell sedimentation, the pellet was washed twice with RPMI medium, and the cells were resuspended in RPMI medium. Finally, the spleen cells obtained by measuring the cell concentration in the suspension and the cell 41 1359024 survival rate using a hemocytometer (hemocytometer, Cambridge Scientific Inc.) and Trypan blue exclusion were adjusted to 3 cell/ by RPMI culture. Mr's fine running concentration' and in the cell suspension disambiguation - add Concanavalin (white Concanavalin A in the cell suspension concentration of 2 μβ / 丨 A ' l〇' T cells. Suspend the cells The solution was injected into a 6-well culture plate at a dose of 5 ml/well, or the culture was poured into a β-cm culture dish at a dose of 10 ml/plate, and placed at 37. : 5%C02 of 2 loops were cultured for 48 hours and cells were collected. The lived leaf cells containing T cells were resuspended in 5 ml of Hank's balanced salt solution and placed in a centrifuge tube.

Carefully float 5 ml of 55% cell separation solution (ρ6κ〇ιι solution) on the surface of the suspension » Keep the conditions of the two separation layers undisturbed, and at 25 ° > c, the cell suspension at 1,900 x g The speed was continuously centrifuged for 1 3 minutes. After centrifugation, T cells can be collected between the interfaces of the two stratified liquids. The collected tau cells are washed twice with Hank's balanced salt solution and used in subsequent experiments. Third preferred embodiment: cell death reaction of activated tau cells Resuspended activated sputum cells (see the second preferred embodiment) in RPMI medium containing 5 ng/ml of interleukin-2 And adjust the cell concentration to 5 X 1 05 cell/ml. Subsequently, a control immunoglobulin (c〇ntr〇1 Ig), a TAB4 antibody or an anti-CD3 antibody was separately added to the activated tau cell suspension under the conditions shown in Table 1.

Table 1 Experimental group treatment method * ~ Negative control group 3 ug/ml hamster immunization 琢 5 ng/ml interleukin-2 42 3 ug/ml cross-linking antibody (anti-hamster immunoglobulin) ΤΑΒ4 3 ug/ml TAB4 hamster Antibody 5 _.ng/ml Interleukin-2 3 ug/ml Cross-linking antibody (anti-hamster immunoglobulin) Positive control group 1 ug/m丨anti-CD3 monoclonal antibody 5 ng/ml interleukin-2 1 Ug/ml cross-linking antibody (anti-mouse immunoglobulin) The final concentration of the specified drug in the culture medium is 1359024. The cell mixture in each group in Table 1 is reacted for 8-24 hours, using 7-AAD cell apoptosis assay. To determine the degree of apoptosis in each cell mixture. The 7-ADD apoptosis assay system moves the treated cells into the FACS camp (Falcon). Subsequently, a solution of 1 〇 / 0 fetal bovine serum and 0.05% sodium azide was added to the phosphate buffer solution, and the cells were washed twice with ice-cold FACS solution at 200 x g. At a rotational speed, the cells were pelleted at 4 ° C. The obtained cells were resuspended in ice-cold FACS solution and the cell concentration was adjusted to 1 to 2 cells/ml. Mix 1 ml of the cell suspension with the 7-ADD reagent so that the concentration of the 7-ADD reagent in the cell suspension becomes 2 ug/ml. Subsequently, the mixture of the cells and the 7-ADD reagent was allowed to stand in the dark at 4 ° C for 20 minutes to carry out a cell staining reaction. Finally, the stained cells were washed twice with ice-cold FACS solution. The stained cells were then resuspended in 0.5 ml FACS solution' and analyzed by BD LSR flow cytometer (Beckton Dickison). Figure 1 is a time-course experiment showing the sensitivity of activated T cells to TAB4 (anti-T cell apoptosis-inducing protein) regulation 43 1359024 apoptotic signal. In this experiment, mouse spleen cells were activated with concanavain abundance (Con-A) and maintained in a viable state by containing interleukin-2 and 0 nutrient solution. After the activated T cells were resuspended and the T cells were resuspended, 'TAB4 monoclonal antibody or control hamster immunoglobulin G was added to the T cell suspension, and anti-hamster immunoglobulin antibody was added for the treatment. The molecule is reacted. T cell apoptosis was measured on the first day of the experiment. The white cross-linking reaction caused a low degree (6.5%) of cell death. However, the degree of apoptosis induced by ΤΑΒ4 will rise to .I? 0/ on the next day. , 〇 ' rose to 52% on the fourth day and fell to 44% on the sixth day. The control hamster immunoglobulin g was unable to induce τ cell apoptosis compared to cells treated with interleukin-2 alone. The addition of anti-C D 3 antibody (positive control group) caused apoptosis of 38% of activated sputum cells after 48 hours (data not shown). The fourth preferred embodiment: the expression of apoptosis-inducing proteins in various tissues is washed with ice-cold FACS solution (containing 1% calf serum and 〇·〇5 % sodium azide filling buffer) The cells were obtained by centrifugation at 2 0 0 X g at 4 ° C in a FACS tube (Falcon). The obtained cells were resuspended in an ice-cold FACS solution to a cell concentration of 1 x i 〇7 cells/ml. Dispense 1 1 ml of 'cell suspension in a FACS tube and set aside. Cell surface staining was performed by adding TAB4 monoclonal antibody or control hamster immunoglobulin adjusted to 2 pg/ml to the above-packed cell suspension, and the mixture was allowed to react at 4 ° C in the dark. 30 minutes. After the reacted cells were washed once with ice-cold FACS solution, the cells were subjected to the following treatments: (1) cychrome 44 1359024-labeled anti-CD3 antibody (2 gg/ml) was added to 100 μl of ice-cold FACS solution, FITC-labeled Anti-hamster immunoglobulin and PE-labeled anti-CD8/CD4/CD19/CD1 lb/ yaH: NK7l-A/i-E7MaC: 3... Tick TT~Dong spleen-贜 fine staining; (2) at 1〇〇μΙ FITC-labeled anti-hamster immunoglobulin, PE-labeled anti-CD8 antibody and cychrome-labeled anti-CD4 antibody (2 ug/ml) were added to the ice-cold FACS solution for thymus cell staining. The reaction was placed at 4 ° C and allowed to react in the dark for 30 minutes. Finally, the stained cells were washed twice with ice-cold FACS solution, and the stained cells were resuspended in 1 ml of FACS solution and analyzed by BD LSR flow cytometry (Beckton Dickison). Figures 3 and 4 show the analysis of T cell apoptosis-inducing protein antigens on different subpopulations of spleen cells and pleural cells by FACS. In Figure 3, the apoptosis of tau cells on the surface of CD 19 + B cells induced a very low protein content, but was still detectable. On the cell surface of natural killer cells and cd3 + t cells, a higher level of T cell apoptosis-inducing protein was detected. And most CD4, CD8 + and CD4 + 8 + pleural T cells can express a large number of T cell apoptosis-inducing proteins. Compared to the results in Figure 3, Panel 4 shows that T cell apoptosis-inducing proteins are only expressed on a small fraction of cd4_8-thorax T cells. Collect B6 and BALB/c small brain, thymus, heart, lung, liver, stomach, kidney, spleen and skin. The tissue sample was 10 〇/ at room temperature. Formaldehyde treatment - fixed tissue at night. After embedding the tissue with paraffin, it was placed under 45^, and the tissue was cut into thin slices of thickness 4 by a slicer (Leica RM2135 microtome, spread). 45 1359024 Place the tissue slide on the treated slide, then dry the slide at 37 °C and set aside. The paraffin wax of the tissue sarcophagus on the slide was removed by using xylenes in the same order, and the tissue section was dried with 1 〇〇〇/〇 ethanol, and the slide containing the tissue was stored. 1 ()% in ethanol. Referring to the standard procedure, tissue sections were sequentially immersed in 1 〇〇〇 / 0 ethanol and phosphate buffer containing 90% ethanol · 85% ethanol _7 〇〇 / 〇 ethanol, and finally using phosphate buffer to make tissue sections The aqueous phase is rehydrated. Subsequent experiments were performed in wet containers. The tissue sections were immersed in blocking buffer (containing 1% normal goat serum) for one hour at room temperature (or soaked at 4 °C for one night) to avoid non-specific binding. After removing the blocking buffer, 'Dilute the TAB4 antibody or normal hamster immunoglobulin at a ratio of ι:2〇〇, add to the tissue section and continue to react at room temperature for one hour (or one night at 4 °C) . Tissue sections were washed twice with phosphate buffer for 5 minutes each time to remove primary antibodies. Goat anti-hamster immunoglobulin antibody with alkaline phosphatase labeling (alkaline)

Phosphatase-conjugated goat anti-hamster Ig) was diluted 1:250 and reacted with tissue sections for 1 hour at room temperature. Wash the tissue with linoleic acid buffer twice twice for 5 minutes to remove the enzyme. Labeled antibody. Finally, the reaction of the BCIP/NBT reagent solution with the tissue section in a dark environment at room temperature for 30 minutes is completed. The color reaction of the tissue section is washed with a buffer solution to wash away excess enzyme reagent on the cut surface. Phosphate buffer, ethanol, and xylene were used to remove the water of the tissue sections and then placed on a microscope to observe β 46 1359024. The results showed that they could only be detected in b〇ne marr〇w derived-tissues. To T cell death induced protein performance. In other groups, it was impossible to detect the apoptosis of sputum cells. A fifth preferred embodiment: immunohistochemical analysis of cell surface biotin labeling and tau cell apoptosis-inducing protein on ice, so that 5 x 107 RL (?1 cells or ΝΙΗ-3Τ3 cells with 1 ml of 0.5 mg/ml sulfide) - Amber sulphide biotin (Sulfo-NHS-biotin, Pierce) phosphate buffer was reacted for 3 , to biotinylated the cell surface. The cells were incubated with 0.5 ml of DMEM on ice ( Dulbecco's modified Eagle's medium, Life Technologies, Inc. reacted for 1 minute to stop the biotin modification reaction. Wash the cells once with 1 ml of DMEM medium and wash the cells twice with 1 ml of phosphate buffer. Use 1% Triton Cell-adjusted X-100, 20 mM Tris-HCl (pH 8.0), 160 mM sodium chloride (160 mM NaCl) and 1 mM chlorinated mother (1 mM. CaCl2) in ice-cold lysis buffer The cell concentration of 5.〇xl〇7 cell/ml was added and the protease mixed inhibitor was added and allowed to stand for 15 minutes to dissolve the cells. The lysate was centrifuged at 10 000 x g for 10 minutes to precipitate. Insoluble matter in the lysate. The above steps The subsequent steps were performed at 4 ° C. For immunoprecipitation analysis, the lysate was first reacted with 50 μM of G protein Sepharose (Amersham Pharmacia Biotech) for 30 minutes to remove the non- Specificity binding protein. 47 1359024 Subsequently, the G protein agarose gel particles were removed by centrifugation, and the supernatant was divided into several portions, and the protein content of each portion was equivalent to the protein amount of 5·〇χ107 cells. i into a 10 pg of 单4 monoclonal antibody or normal hamster serum felidin globulin 20 μΐ G-protein agarose gel particles and the supernatant was reacted for 4 hours under the armpit. 4. After the reaction, use contains 0.05〇/〇Triton Χ-100, 50 mM Tris-HCl (pH 8.5), 400 mM sodium sulphate, 1 mM calcium chloride and 1 mg/mi

After washing the gelatin particles four times with albumin (ovalbumin) washing buffer, the gel particles were washed twice with the above-mentioned washing buffer with a sodium carbonate concentration of 250 mM. The protein bound to the TAB4 antibody on the colloid was eluted with 50 μM of 1XSDS sample buffer. The removed protein was analyzed by 8% S D S electrophoresis gel, and the protein on the electrophoresis gel was transferred to a nitrocellulose membrane (nitr〇cellul〇se membrane, Millipore). The biotinylated protein on the nitrocellulose membrane was subsequently analyzed by peroxidase-conjugated Avidin (PharMingen) and chemiluminescence reagent (NENTM Life Science Products).

Fig. 2 shows that TAB4 recognizes a biotinylated surface protein having a molecular weight of about 12 〇 kD in RL ancient j cells (tau cell apoptosis-inducing protein phenotype T cells). 3T3 cells (T-cell apoptosis-inducing protein non-phenotype cells) were absent. The G protein agarose gel granules treated with normal hamster serum could not capture this protein with a molecular weight of 120 kD. The results obtained suggest that the 120 kD size protein is an antigen on the surface of T cells that can be recognized by tab4 monoclonal antibodies. 48 1359024 The sixth preferred embodiment: depletion of T cells in vivo by intraperitoneal injection to provide 300 ng of ΤΑΒ4 antibody or control mmi·white 四 出 出 出 、 、 、 Cells and peripheral blood mononuclear cells were counted for total cell number, and markers on the cell surface were analyzed by FACS analysis to test the role of TAB4 antibodies in T cells or other cell populations.

The FACS assay was performed at 4 ° C. The cells were fixed with 2% paraformaldehyde for 20 minutes to fix the cells. The cells were then washed twice with ice-cold FACS solution and the cells were resuspended and adjusted to a cell concentration of 1 x ;! 07 cells/ml. The suspended cells are packed in a volume of 1 〇〇 μ! per tube.

After 'backup. ΤΑΒ4 antibody at a concentration of 2 pg/ml or control hamster immunoglobulin was added to 100 μm of each tube, and the antibody was mixed with the cells, and then allowed to react in the dark at 4 ° C for 30 minutes. After the reaction, the cells were washed once with ice-cold FACS solution, and the cells were subjected to the following treatments: (1) cychrome-labeled anti-CD3 antibody (2 pg/ml), FITC-labeled antibody was added to 丨〇〇μ1 ice-cold FACS solution. Resistance of hamster immunoglobulin to pE marker

CD8/CD4/CD19/CDllb/pan-NK/IA/IE/Mac-3 antibody (2 pg/mi) to stain spleen cells; (2) Add FITC label to 1 〇〇μΐ ice-cold FACS solution Chest cells were stained with anti-hamster immunoglobulin antibody, PE-labeled anti-CD8 antibody and cychrome-labeled anti-CD4 antibody (2 ug/ml). The above reaction was placed at 4t and reacted in the dark for 3 minutes. Finally, the stained cells were washed twice with ice-cold FACS solution, and the stained cells were resuspended in 1 ml of FACS solution and analyzed by BD LSR 49 1359024 flow cytometer (Beckton Dickison). Four days after antibody injection, the percentage of CD3 + T cells in peripheral blood leukocytes (PBL) of mice treated with TAB4 antibody decreased from 36.7% of normal mice to 4.1% (see Table 2). Treatment of the TAB4 antibody only resulted in a slight decrease in the total number of spleen cells. However, in TAB4 antibody-treated mice, the number of CD3+ T cells was reduced by 62%, the number of natural killer cells was reduced by 50%, and a slight increase in the total number of CD 19 + B cells was caused. The total number of pleural cells in TAB 4 antibody-treated mice was only 48% of the control mice (ie, a 52% reduction). In addition, in addition to CD4 + T cells, the number of other CD8+, CD4 + CD8 + and CD4 - CD8_T cells decreased. Among them, the population of CD8 + CD4 + had the most obvious effect (down 64.7%). Table 2 Spleen X 10b No treatment Normal hamster Immunoglobulin TA-B4-Depletion after treatment (%) Total number of 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 2.4 1.80 50 Peripheral blood leukocytes untreated normal hamster immunoglobulin TA - B 4 - post-depletion amount (%) CD3+ T cells 3 6.7 % 3 6 % 4.1% 8 8.8% 50 chest X 10° No treatment normal Hamster immunoglobulin TA-B4-^ After treatment ~ Depletion amount (%) - -- - - Total number of chest line cells 94 229 45 52.1 9.3 28.4 ~Γ〇Τ9~~~~ -16.6 CD8 + 5.2 7.7 3.6 30.3 CD4 + CD8 + 73.8 182 26~^~ 64.7 ~CDV~~ CD8' 5.6 10.5 4.5 19.3 (The above data are representative data obtained from three experiments) 1359024 The seventh preferred embodiment: single anti-τ cell apoptosis-inducing protein Strains of antibodies were unable to induce secretion of interleukin-2 or tumor necrosis factor-α. 300 mg of the ΤΑΒ4 antibody or control hamster immunoglobulin was injected into the peritoneal cavity of Balb/c mice (H-2d). Seven days after the injection, the spleen cells of the mouse were isolated as a reaction cell, and cultured with C3H (H-2k) spleen cells (mitomycin C-treated) treated with mitomycin which is used as a stimulating cell. After three days of culture, the culture supernatant was collected, and the ELISA reagent group (pharMingen) was used to measure the content of interleukin-2 in the culture supernatant. Fig. 5 is a comparison with the control mice. The amount of interleukin-2 in the reaction cells obtained from TAB4-treated mice was inhibited. Analysis of plasma-mediated interleukin-2 and tumor necrosis factor levels in bloody sputum of control mice and ΤΑΒ4 treated mice There was no significant difference in the content of interleukin-2 (or tumor necrosis factor-CX). 51 1359024 Since the production of interleukin-2 is an indicator of the activity of T cells, the results show that the apoptosis-induced protein-specific antibodies (such as ΤΑΒ4) can be used to control the cells in the body, and can control the immune response of the τ-cell regulation-free immune response that does not occur, for example, accompanied by autoimmune diseases and transplant rejection. : Using anti-tuberculosis apoptosis Inducing protein antibodies to prevent transplant rejection Acepromazin maleate K Fermenta Animal Health Co. (Kansas City, MO) was used to anesthetize mice aged 8 to 12 weeks (available from Jackson Laboratory). Seven days before surgery, C5 7BL/6 mice (H-2b), which had not been resected from the thymus, were given 500 pg of TAB4 antibody or isotype of control antibody by intraperitoneal injection. After 7 days of antibody injection, The flank skin of Balb/cj mice (H-2d), which was completely heterologously paired with non-matching, was removed and transplanted into the flanks of C57BL/6 mice injected with antibodies. After 7 days of skin transplantation, the c57BL/ 6 Mice were reinjected with 500 pg of TAB4 antibody or a control antibody of the same type. The mice that received skin transplantation were observed daily. If 5% of the skin of the implanted skin was necrotic, then the transplanted skin was rejected. Figure 7 shows the percentage of graft skin survival (n = 8). The data in the figure show that the treatment of tab4 antibody can prolong the survival time of xenograft tissue. The 9th preferred implementation is identified as ρ_selection. Glycoprotein ligand 1 The Ρ-selectin glycoprotein ligand called CIM62 is a P-selectin ligand on the cell surface of 52 1359024 white blood cells (including T cells) (Sak〇 et al. (1 993) Cell 75: 1 1 79; Vachino et al. (1995) J Biol

Chem. 2 70:21966; Veldman et al. (1 995) J. Bi〇l " Chem 270:16470). T cell apoptosis-inducing proteins, due to their molecular weight and tendency to form biochemical properties such as dimers, suggest that TAB4 may be an analog of P-selectin 靡 protein ligand 1. To investigate the relationship between the two antigens, the assay was performed as follows: 1) Testing whether the antigen precipitated by the TAB4 antibody was recognized by the commercially available anti-P-selectin glycoprotein ligand 1 antibody; Test whether the anti-P-selectin glycoprotein ligand 1 antibody can deplete the TAB4 antibody in the cell lysate. The following steps were performed at 4 ° C with lysate solution (1% Triton X-100, 20 mM Tris-HCl, pH 8.0, 160 mM sodium chloride, 1 mM calcium chloride) added with protease mixing inhibitor. After the RLd11 T cells were adjusted to a cell density of 1 · 0 χ 1 08 ce11 s/m 1 , they were allowed to stand for 1 hour to dissolve the cells. The lysate was then centrifuged at 10 000 X g for 10 minutes to remove insoluble material from the lysate. 10 μg of anti-Ρ-selectin glycoprotein ligand 1 monoclonal antibody (cl〇ne 2PH1, PharMingen, San Diego, CA) and anti-T cell apoptosis were added to each 20 μM G protein agarose gel pellet. After inducing the antibody of the monoclonal antibody, the TAB4 antibody or the immunoglobulin in the serum of the normal hamster, the lysate corresponding to the protein amount of 5·〇χ107 cells is added to the G protein agarose gel particle containing the antibody. . The lysate was reacted with the G protein agarose gel particles containing the antibody at 4 ° C for 4 hours, followed by washing buffer

Washing G protein ring grease (0.05% Triton X-100, 50 mM Tris-HCl, pH 8.5, 400 mM sodium chloride, 1 mM calcium chloride, 1 mg/ml ovalbumin) 53 1359024

Sugar gel particles five times. After adjusting the sodium sulphate in the washing buffer to a concentration of 250 mM, the G protein cyclolipose granules were washed twice, and then eluted on the protein agarose gel granules with 4 〇 μΐ of lxSDS sample buffer. The H-mass was followed by separation of the washed protein with 6% SDS electrophoresis gel and the protein on the electrophoresis gel was broken onto the nitrocellulose membrane. Anti-p_selectin 黯 protein ligand 1 monoclonal antibody was used to immunoblotted the protein on the membrane and labeled with peroxidase-conjugated goat anti-mouse immunoglobulin _ G(H + L) The chemiluminescent reagent (Renaissance, NEN) was used to develop the protein on the film.

The cell surface biotinylated RLc? 1 T cells were adjusted to a density of 1 · 〇χ 108 cell / ml with lysate and the cells were lysed. The obtained cell extract was mixed with 20 pg of antibody bound to 40 plG protein agarose gel particles and reacted at 4 ° C for one night. The antibody used in the above reaction may be an anti-P-selectin glycoprotein ligand 1 monoclonal antibody (2PH1) or a control mouse immunoglobulin G, or a TAB4 antibody or a control normal hamster serum. The cell extract obtained by the above treatment was subjected to an immunoprecipitation reaction using a TAB4 antibody or an anti-P-selectin glycoprotein ligand 1 monoclonal antibody, respectively. The obtained immunoprecipitates were separated by 6% SDS-polyacrylamide gel, and the results were examined by fluo.rography. Figure 6 shows that the anti-p-selectin listener protein ligand 1 monoclonal antibody can remove T cell apoptosis-inducing proteins in T cell extracts. In addition, Western blotting methods can be used to detect that proteins that are immunoprecipitated with anti-T cell apoptosis-inducing protein monoclonal antibody (TAB4) can be resistant. _ Selectin glycoprotein ligand 1 was identified by monoclonal antibodies. 54 1359024 第ίο preferred embodiment: using anti-P_selectin glycoprotein ligand j antibody to induce # ΪΙ 乂 ΐ I cell of the cell ''two' seven-anti-watt-------- --------------------—To understand the role of P-selectin glycoprotein ligand 1 in the apoptotic transduction of human T cells, A one-time experiment was performed to investigate the sensitivity of activated human T cells to the cell apoptosis signal regulated by p-selectin glycoprotein ligand i. A plant phytohemagglutinin mitogen (PHA) was added to the culture medium containing interleukin-2 to stimulate human T cells. Activated tau cells are harvested and the activated human sputum cells are treated with an anti-Ρ-selectin glycoprotein ligand 1 antibody containing interleukin and a cross-linking antibody. Peripherai blood mononuclear cells (PBMC) were collected from healthy adults by treating peripheral blood with human peripheral blood and treating them with hepatic cadherin. Ficoll-Paque reagent (Pharmacia Biotech) was used to collect peripheral blood mononuclear cells (PBMC). ). After 1 hour of phytohemagglutinin (Life Technologies, GibcoBRL) was used to treat peripheral blood mononuclear cells for 48 hours' and the activated peripheral blood mononuclear spheres were preserved in the presence of recombinant human interleukins throughout the experiment. In the condition of 1-2 (5 ng/ml). In order to evaluate the apoptosis-inducing ability of the anti-human P-selectin glycoprotein ligand 1 antibody, the activated peripheral blood mononuclear spheres were treated with various substances described below. The materials used were: (1) lpg/ml anti-purine-selectin glycoprotein ligand 1 plant KPL_ 1" (bd PharMingen) plus cross-linking rabbit anti-mouse immunoglobulin (0.5 pg/ml) ( Jackson ImmunoResearch Laboratories); (2) Purified isotype control with mouse immunoglobulin plus cross-linking rabbit anti-mouse immunization 55 1359024 globulin, or (3) paternal rabbit anti-mouse immunoglobulin. When the activated peripheral blood mononuclear spheres are treated for 6 hours by the above three reagents, the FACS cup system, the decidual egg quail egg: V-splitting system (5ntT_Xnn-exln flying Bf)-PharMingen) and propidium iodide can be used. PI, Sigma) staining to detect the rate of early apoptotic cells. Figure 8 shows human peripheral blood mononuclear cells that do not utilize anti-P-selectin glycoprotein ligand 丨 antibody plus cross-linking molecules to cause P-selectin glycoprotein ligand 1 to signal and induce PHA activation. Significant apoptosis occurred in the predominantly tau cells. In cells treated with anti-P-selectin glycoprotein ligand 丨 antibody, the ratio of apoptosis cells was 8.5 from the third day. / On the 8th day, it will rise to 24〇/〇. Neither the isotype control antibody nor the cross-linking antibody has any effect on the cells. Eleventh Preferred Embodiment·Using anti-P-selectin glycoprotein ligand 1 synergistic antibody to treat autoimmune diseases, feeding non-obese diabetic (N〇D) mice in a general manner (ie, autoimmune) Diabetic animals). At about 20 weeks of age, non-obese urinary mice spontaneously develop diabetes. In the experimental group, Xiaoqi was given an anti-ρ of 300 μμ per mouse by intraperitoneal injection at a dose of 3 〇〇μ§ for a total of three times at 14 weeks, 15 weeks, and 17 weeks of age. Select a glycoprotein ligand 1 antibody (ΤΑΒ4 antibody). Mice were then given the same dose of ΤΑΒ4 antibody at 24 and 26 weeks of age, respectively. Mice were injected with hamster immunoglobulin in the same dose and in the same manner as a control group. The glucose content of T in the urine of the mice was followed by Medi-Test grape 56 1359024 sugar test paper (Macherey-Nagel, Germany) twice a week after the age of the rats. After two consecutive measurements of non-fasted urinary glucose levels above ____ ____ ' 3 〇 (mg/dl, the demon mouse was judged to have a sugar exhibition, and the ninth image is strict, compared to the control With the antibody, the TAB4 antibody can produce significant protection, so the use of the anti-P-selectin glycoprotein ligand 1 antibody can finely automate the activity of autoimmune T cells and delay the onset of type 1 diabetes. Twelfth preferred embodiment: binding of P-selectin, E-selectin and L-selectin to activated T cells in order to determine selectins (P-selectin, E-selectin and L_selectin*) *τ Activated sputum cell binding ability, activated from s__ fresh spleen cells in C57BL/6 mice, and collected on the 6th and 4th day of activation in the activation process and not activated yet. The sputum cells (the first day when the spleen cells were prepared) were subjected to an analytical test. Two days after the spleen cells were cultured using DMEM medium containing 2 pg/ml of concanavalin a and sputum/calf serum. The cells collected were the cell samples of the 2nd day and survived by the F ic ο 11 specific gravity gradient separation method. Liver cells. Cells were activated with concanavalin A for three days, and then cultured in a medium containing 5 ng/ml of A-beta-2 for one day to obtain cell samples on day 4. The cell sample was obtained by activating the cells with concanavalin A for three days and then culturing for 3 days in a nutrient solution containing 5 ng/ml of interleukin-2. The cell sample was then detected by FACS analysis. The cell samples of Dijon, 2nd, 4th and 6th day were taken out and 2×10 5 cells were placed in different wells, and then a volume of 40 μΐ was added to each well, and the concentration was from 57 1359024. 20 gg/ml 2-fold serial dilution to 0.156 Hg/ml of mouse P-selectin, E-selectin or L. selectin fusion protein (R&D Systems, Minneapolis, μθ). - Some of these - selection - selection The fusion of the prime-combined protein [-small-n-human-fluorine mm G1 Fc constant region. The mouse selectin fusion protein is mixed with the cells, and then reacted at 4 ° C for 30 minutes. After the reaction, Wash with lxFACScan buffer (1% phosphate buffer (Biochrom AG) with calcium and magnesium ions plus 2% calf serum) The anti-Thy 1.2 and the second reagent (FITC-anti-human immunoglobulin G constant region fragment, Jackson ImmunoResearch Laboratories, Inc.) were then added to each well plate at a concentration of 3.25 pg/m and a volume of 95 μM. , West Grove, PA) was mixed with sample cells and allowed to react at 4 ° C for 30 minutes, then washed in 1 x FACScan buffer. Figure 10 shows the results of the FACSCalibur analysis. When P-selectin at a concentration of 20 pg/ml was used to react with activated T cells, the binding force of P-selectin to activated cells gradually increased with the increase of the number of days, and the binding force of the fourth day was the highest. In six days, the binding force decreased slightly. The binding force of E-selectin remained unchanged until the sixth day after the 2nd to 4th day. The binding capacity of L-selectin to mouse activated T cells was not significant' and there was no change throughout the activation', e.g., no change in binding from day to day 6 to day 6. This result may be due to the fact that L-selectin has a very low affinity for its ligand. When the three selectants were tested using lower concentrations, the experimental results obtained were similar to those obtained using higher concentrations. Thirteenth preferred embodiment·· p-selectin, E-selectin and L-selectin poly 58 5859024 complex Zhao can cause apoptosis of tau cells after activation at a concentration of 20 pg/at 4 ° C A 96-well plate (NUNC) was treated with ml, a volume of 5 μl of anti-human ectopic immunoglobulin antibody for one night. The treated 96-well plate was then treated with 1% calf serum albumin (BSA) at 37 °C.

Line blocking processing (blocking). After completion of the blocking treatment, 5 μl of human Fc-selectin fusion protein (concentration between 0.063 and 5 pg/ml) was added to the well plate, and allowed to react at room temperature for 2 hours. In all of the above experimental steps, each well was washed with 1 time phosphate buffer. Five times. Subsequently, 2 cells were added to each well: < 1 〇 5 cells which had been activated by concanavalin a for four days, and cultured at 37 ° C for 5 hours. This 96-well plate was then centrifuged at 200Xg for 5 minutes at 4 ° C to obtain a cell sedimentation chamber. Adding a biotin-Annexin V-biotin conjugate to a precipitate containing activated cells' After standing at room temperature for 15 minutes, add an avidin conjugate (Beta _ SA beta gal is diluted at 1.000 times and then placed in 37 <>c to continue the reaction for 3 minutes. After each reaction step is completed, 'An Annexin-V binding buffer is used. Clean each well three times. Finally, add 1 z of z-buffer mixture to each well (prepared by adding 54 ul of 2-mercaptoethanol to 20 ml of Z-buffer) and 30 μl of 〇NPG (0_04g). /10ml) After a night at 4C, the color reaction can be completed. A light source with a wavelength of 42 〇 nm is then recorded to read the result. The degree of selectin-regulated apoptosis of activated T cells increases with increasing concentration of P-selectin (Fig. 11A) or E-selectin (Fig. 11B) of the Fc region of human immunoglobulin G1. . The concentration of the selectin 59 1359024 is used in a range from 0. () 63 to (5) to 5 ug / m. ^ ^ A preferred embodiment - a hamster antibody TAB4 can trigger the activation of T cells. , ϋ _ using - nmi ' / in the preferred embodiment - the anti-human antibody, human immunoglobulin and calf serum protein that trigger the cell death of activated sputum cells can be used as other experiments Negative control group. The result of human Fc_L_selectin fusion ", ancient 5丨 significant apoptosis (Fig. 1C)” is in contrast to the result of the combination of L-selectin and activated tau cells in the twelfth preferred embodiment. echo. In the preferred embodiment, it is concluded that the fusion protein of the P_selectin 黯 protein ligand i-binding fragment and the human Fc fragment having P-selectin 4 E-selectin immobilized on the well plate can be induced. Cell apoptosis in activated butyl cells. The 14th preferred embodiment · The cross-linking reaction of the soluble p_selectin_Fc constant region fusion protein causes the cell death reaction of the activated T cells. The mouse selectin is selected according to the above method (ρ·selectin, £_selection) And L-selectin) fused to the Fc region of human immunoglobulin G1 to form a soluble dimeric fusi〇n pr〇tein, and experimentally evaluated whether this soluble selectin can cause activated T cells. The apoptotic reaction, the experimental design details refer to the method described in the preferred embodiment of the present invention, but the step of adding the anti-human Fc immunoglobulin immobilized on the well plate is omitted. As shown in Fig. 12, if only soluble p-selectin fusion protein (dimer) is added, the degree of apoptosis of activated T cells is extremely low, even to a negligible condition. However, if the cross-linking of the anti-human Fc is 60 1359024, the phenomenon of apoptosis of the activated tau cells is greatly enhanced, and the degree of apoptosis-------apoptosis is fixed. The degree of apoptosis induced by the antibody. The anti-human class IV, - human ϋϋ-white one (-^g) or white meal can not cause the cell death reaction of activated tau cells. The same as the Ε-selectin-Fc fusion protein can also give the same results as the Ρ-selectin F fusion protein. Furthermore, the soluble L-selectin fusion protein was also unable to elicit an apoptotic response to activated tau cells, and this result was consistent with the use of L-selectin immobilized on the well (multimer) to activated tau cells. The detailed description of the present invention is intended to be illustrative only and not to limit the scope of the invention. Other objects, advantages and modifications of the present invention are still to be covered by the scope of the following claims. [Simple description of the map]

Figure 1 shows the results of a time-course experiment used to investigate the sensitivity of activated tau cells to a modulating apoptotic signal of the ΤΑΒ4 antibody (anti-Ρ-selectin glycoprotein ligand} monoclonal antibody). Fig. 2 shows the results of biotin labeling of the cell surface and immunoprecipitation of the antigen using the ΤΑΒ4 antibody. Figure 3 shows the expression of Ρ-selectin glycoprotein ligand 1 on spleen CD4 + T cells, CD8+ sputum cells, CD19 + sputum cells and natural killer cells. Figure 4 shows the expression of p-selectin glycoprotein ligand 1 on CD4+, CD8+, CD4 + 8 + and CD4.8-thoracic line 61 1359024 cells. Figure 5 shows the amount of interleukin-2 in the mixed cell culture. The t used to treat the spleen cells of Balb/c mice with TAB4 antibody (3⁄4) Unpaired C3H spleen cells are used as stimulator cells. Figure 6 shows the results of Western blot analysis to illustrate: (A) Immunoassay using the TAB4 antibody to detect the white matter can be recognized by the commercially available anti-P-selectin glycoprotein ligand 1 antibody, and (B) After the T cell lysate is pretreated by the anti-P-selectin glycoprotein ligand 1 antibody, the amount of protein recognized by the TAB4 antibody in the lysate is reduced. Figure 7 shows C57BL/6 mice treated with anti-P-selectin glycoprotein ligand 1 antibody (solid diamond) or control antibody (open square), respectively, after receiving transplanted skin from Balb/c mice. Transplant survival rate. Figure 8 is a time course experiment showing the percentage of T cell apoptosis after treatment of peripheral blood mononuclear cells with activated steroids with anti-human P-selectin glycoprotein ligand 1 antibody. Figure 9 shows the incidence of diabetes in males with autoimmune diabetes (or non-obese diabetic NOD) treated with a TAB4 antibody (closed square) or a control antibody (open square). Figure 10 shows the binding of P-selectin, E-selectin and L-selectin to mouse activated T cells. Figure 11 shows cells of mouse activated T cells using a poly-plex of P-selectin (Fig. 11A), E-selectin (UB map) and L-selectin (Fig. 11C). Apoptosis-induced response. 62 1359024 Figure 12 shows the in vitro apoptosis-inducing response of mouse activated T cells using a soluble P-selectin-Fc constant region fusion protein. [Simplified description of component symbol] None

63

Claims (1)

1359024 X. Patent application scope: 1. An antibody fragment other than the anti-p-selectin glycoprotein ligand 1 anti-Zhao or a determinin glycoprotein ligand 1 can bind to at least two Ρ-selections on the surface of sputum cells. The use of a protein-binding poly-complex compound, wherein the multi-plex compound comprises at least two multi-peptide chains, each of which comprises at least (1) one and p Selecting a binding region of the glycoprotein ligand 1 binding to a (U)-heteroamino acid sequence, wherein the plurality of peptide chains are linked by the heterologous amino acid sequence to form the multimeric complex compound 'and the use is for the manufacture of a drug that is used to prevent or reduce T cell regulatory immune responses in patients diagnosed with or likely to have symptoms of excessive or unwanted sputum regulatory immune responses, where The binding of the poly-compounding compound to at least two P-selectin glycoprotein ligands 1 on the surface of tau cells induces a message-transmission pathway that can cause tau cell death to prevent or reduce tau cell regulation in the patient. Sexual immune response. 2. The use according to claim 1, wherein the multimeric compound is a multimeric complex compound. 3. The use of claim 1, wherein the multimeric compound is a heteropolymeric compound. 4. The use of claim 1, wherein the heterologous 64 1359024 amino acid sequence comprises at least a cell surface receptor binding region. 5. The use according to claim 1, wherein the binding region comprises at least a P-selectin extracellular region or a P-selectin glycoprotein ligand 1 binding fragment of the P-selectin extracellular region. . 6. The use according to claim 1, wherein the binding region comprises at least an E-selectin extracellular region or a P-selectin glycoprotein ligand 1 binding of the E-selectin extracellular region. Fragment. 7. The use of claim 1, wherein the binding region of at least one multi-peptide chain comprises an antigen binding region of an anti-P-selectin glycoprotein ligand 1 antibody or the anti-P-selectin A PSGL-1 binding fragment of the antigen binding region of a glycoprotein ligand 1 antibody. 8. The use of claim 1, wherein the plurality of peptide chains are covalently linked by the heterologous amino acid sequence to form the polymeric composite compound. 9. The use of claim 8 wherein the covalent linkage is a double sulfur linkage. 10. The use of claim 1, wherein the heterologous 65 1359024 amino acid sequence comprises at least one immunoglobulin long chain constant region. 11. The use according to claim 1, wherein the binding of the multimeric complex compound to the at least two P-selectin glycoprotein ligands 1 induces the signal transduction causing T cell death. The pathway further comprises binding to the multimeric complex compound by a drug, the agent is bound to the multimeric complex compound through the heterologous amino acid to cause a plurality of P-selectin glycoproteins on the surface of the tau cell Crosslinking reaction of the ligand 1 antigen. 12. The use of claim 1, wherein the patient is diagnosed with an inflammatory disease. 13. The use of claim 1, wherein the patient is diagnosed as having an autoimmune disease. 14. The use of claim 1, wherein the patient has accepted or is scheduled to undergo an allograft or a xenograft. 15. The use of claim 1 wherein the patient is diagnosed as having an allergic disease. 1 6. The use of claim 1, wherein the patient 66 1359024 is diagnosed as having a tau cell cancer. 17. The use of claim 1, wherein the sputum cell is an activated sputum cell. 18. The use of claim 1, wherein the first biological sample is obtained from a patient who has not used the multimeric complex compound, and Comparing the number of cells in the two biological samples, the second biological sample is taken from a patient who has used the multimeric complex compound. 1 9. The use of claim 1, wherein the biological activity of the first biological sample is measured, the first biological sample being taken from a patient not using the multimeric complex compound, and A comparison of the biological activity of the cells of a second biological sample taken from a patient who has used the multimeric complex compound. 20. The use of claim 1, wherein the result of administering the agent is to reduce the number of activated sputum cells in the patient by at least 10%. 2 1. An anti-Ρ-selectin glycoprotein ligand 1 antibody or an antibody fragment that binds to Ρ-selectin glycoprotein ligand 1 can be combined with sputum cells 67 1359024 Or the use of at least two P-selectin glycoprotein ligand 1 (PSGL-1) proteins on the surface of natural killer (NK) cells to form a poly J complex compound, wherein the multimeric complex compound comprises at least two a multi-peptide chain, each of the plurality of peptide chains comprising at least (i) a binding region that binds to P-selectin glycoprotein ligand 1, and (ii) a heterologous amino acid sequence, wherein The multi-peptide chain is formed by the ligation of the heterologous amino acid sequence to form the multimeric complex compound, and the use is to produce a preparation for inducing T cell or natural killer cell death by providing on the cell surface T cells or natural killer cells expressing P-selectin glycoprotein ligand 1 and contacting the T cells or natural killer cells with the preparation to induce death of T cells or natural killer cells, wherein the multimeric complex Binding of the compound to the T cell or the at least two P-selectin glycoprotein ligands 1 on the surface of the natural killer cell induces a message delivery pathway that can cause death of the T cell or the natural killer cell. 22. The use of claim 21, wherein the polymeric composite compound is a homomeric complex compound. The use according to claim 21, wherein the poly-complex compound is a heteropoly-complex compound. 24. The use of claim 21, wherein the heterologous amino acid sequence comprises at least a cell surface receptor binding region. 68 1359024 y. The use of claim 21, wherein the binding region comprises at least one Ρ-selectin extracellular region or one of the Ρ-selectin extracellular regions Ρ-selectin glycoprotein ligand 1 binding fragment. 26. The use of claim 21, wherein the binding region comprises at least one Ε-selectin extracellular region or one of the Ε-selectin extracellular regions Ρ-selectin glycoprotein ligand 1 binding Fragment. 27. The use of claim 21, wherein the binding region of the at least one poly-peptide chain comprises an antigen-binding region of an anti-plucon-selectin glycoprotein ligand 1 antibody or the anti-sputum- The Ρ-selectin glycoprotein ligand 1 binding fragment of the antigen binding region of the prime glycoprotein ligand 1 antibody was selected. 28. The use of claim 21, wherein the plurality of peptide chains are covalently linked through the heterologous amino acid sequence to form the multimeric compound. 29. The use of claim 28, wherein the covalent linkage is a disulfide linkage. 30. The use of claim 21, wherein the amino acid sequence of at least 13 1359024 comprises at least one immunoglobulin long chain constant region. The use according to claim 21, wherein the binding of the multimeric complex compound to the at least two P-selectin glycoprotein ligands 1 induces the death of T cells or natural killer cells. The message-conducting pathway further comprises contacting the multimeric complex compound with an agent that is conjugated to the multimeric complex compound via the heterologous amino acid sequence to cause a T cell or natural killer cell surface Cross-linking reaction of a plurality of P-selectin glycoprotein ligand 1 antigens. 32. The use of claim 21, wherein the T cell is an activated T cell. 33. The use of claim 21, wherein the survival rate of the T cell or the natural killer cell after contact with the multimeric complex compound is further evaluated. 34. The use of claim 21, wherein the biological activity of the T cell or the natural killer cell after contact with the multimeric complex compound is further evaluated. 3 5. A kit of at least one comprising: an anti-P-selectin glycoprotein ligand 1 antibody or may be associated with P-select 1359024 a multimeric complex compound other than the antibody fragment bound by the glycoprotein ligand 1, which is capable of binding to at least two P-selectin sugar i white ligands on the surface of a tau cell, wherein the multimeric complex compound Comprising at least two multi-peptide chains, each of the plurality of peptide chains comprising at least (i) a binding region, and (ii) a heterologous amino acid sequence, wherein the binding region will coordinate with the P-selectin glycoprotein Binding, the plurality of peptide chains form a multimeric complex compound by linking the heterologous amino acid sequence, wherein the multimeric complex compound and at least two P-selectin glycoproteins on the surface of the T cell The binding of ligand 1 induces a message-conducting pathway that can cause the death of the T cell; and instructions to demonstrate that the multimeric complex compound can be used to treat inflammation, autoimmune disease, transplant rejection, allergic symptoms or T cell cancer. 71 1359024 VII. Designation of representative representatives: (1) The representative representative of this case is the first picture. —(二-) The representative of the genus-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------