Products and methods for treating thrombocytopenia

Abstract

The present invention provides a product and method for treating thrombocytopenia. The technical solution of the present invention can fundamentally solve the problem of thrombocytopenia caused by autoimmune mechanism.

Description

[Technical field]

[0001] The present invention relates to the fields of biology and pharmacology, more specifically, the present invention relates to the application of platelet surface antigens or truncations thereof in the treatment of thrombocytopenia-related diseases. [Background technology]

[0002] Immune thrombocytopenia (or idiopathic thrombocytopenia), also known as immune thrombocytopenic purpura (ITP), is an autoimmune disease that causes bleeding due to a decrease in peripheral platelets, and accounts for approximately one-third of all bleeding disorders. Currently, in clinical practice, approximately one-third of ITP patients respond poorly to drug treatment, and ultimately progress to chronic ITP (cITP), which has a high mortality rate.

[0003] The main pathogenesis of immune thrombocytopenia is the proliferation of autoreactive B lymphocytes in the body and the secretion of autoantibodies (PA Abs) against specific glycoproteins on the platelet membrane surface. The specificity of these PA Abs in ITP patients is as high as 90%, and these antibodies bind to platelets, causing them to be phagocytosed and removed by the reticuloendothelial system in the spleen, impairing normal hemostatic function. At the same time, PA Abs also bind to megakaryocytes, affecting platelet maturation and differentiation, which is the main etiology leading to the development of ITP. ITP patients who are PA Abs positive have a higher bleeding risk and worse efficacy and prognosis than those who are PA Abs negative.

[0004] Current clinical treatments for ITP are mainly aimed at improving the symptoms of patients. Among them, the first-line treatments include glucocorticoids, gamma globulin, infusion of immunoglobulin preparations, platelet transfusion, etc., but the efficacy is relatively limited, the side effects (hormones) are significant, the proportion of relapsed and refractory patients is high, and the supply of blood products is extremely scarce, which poses the risk of infection. The second-line treatments include the use of immunosuppressants, splenectomy, platelet production promotion, and non-specific removal of B lymphocytes (CD20 antibody), etc. The use of immunosuppressants and CD20 can indiscriminately destroy the immune system, which was originally dysregulated, and can cause problems such as secondary infection. On the other hand, the platelets produced by the stimulation of platelet production promotion agents continue to further activate autoimmunity as antigens, which causes the platelets produced to be rapidly removed, resulting in unstable efficacy and a double risk of inducing thrombosis and bleeding. In addition, such drugs may cause serious drug side effects such as myelofibrosis and liver dysfunction. Therefore, it is urgent to find a more effective treatment strategy in clinical practice. Summary of the Invention

[0005] The present invention aims to provide an application of a truncated platelet surface antigen in the preparation of a therapeutic agent for immune thrombocytopenia-related diseases.

[0006] Another object of the present invention is to provide a chimeric autoantibody receptor (CAAR) prepared using the truncated form or a mutant thereof, and applications thereof.

[0007] In a first aspect of the present invention, there is provided the use of the platelet surface antigen GpIbα, its truncations, its variants, or polynucleotides encoding them, in (a) the preparation of a competing antigen that binds to an anti-platelet antibody or a construct or composition comprising said antigen, (b) the preparation of a construct or composition that targets and removes reactive B lymphocytes, (c) the preparation of a construct or composition that elevates platelets, and (d) the preparation of a construct or composition that alleviates or treats immune thrombocytopenic purpura (ITP).

[0008] In one or more embodiments, the GpIbα truncation or mutant thereof has the following properties: it has a binding site for platelet autoimmune antibodies in vivo, and preferably, the GpIbα truncation or mutant thereof is derived from the extracellular domain of the GpIbα protein.

[0009] In one or more embodiments, the truncated form comprises a protein having an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:21; In one or more embodiments, the construct is a Chimeric AutoAntibody Receptor (CAAR).

[0010] In one or more embodiments, the anti-platelet antibodies include B cell surface anti-platelet BCR and / or free anti-platelet antibodies.

[0011] In another aspect of the present invention, there is provided a truncated form or variant of the platelet surface antigen GpIbα, or a polynucleotide encoding the same, wherein the truncated form or variant of GpIbα is derived from the extracellular domain of the GpIbα protein and has a binding site for an in vivo platelet autoimmune antibody; preferably, the truncated form or variant is a variant having a reduced binding ability due to a mutation in the binding domain that binds to von Willebrand factor (vWF); more preferably, the truncated form or variant has a mutation at position 233 of the amino acid sequence corresponding to SEQ ID NO:2 from G to K (G233K); more preferably, the truncated form or variant comprises a protein having an amino acid sequence as set forth in one or more of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10, SEQ ID NO:17, SEQ ID NO:19 or SEQ ID NO:21.

[0012] In one or more embodiments, the truncated form or mutant form of the platelet surface antigen GpIbα binds to anti-platelet antibodies expressed by reactive B lymphocytes or free anti-platelet antibodies secreted by plasma cells via platelet autoantigens, thereby reducing platelet killing by the antibodies and increasing platelet count.

[0013] In one or more embodiments, the binding is competitive binding.

[0014] In one or more embodiments, the truncated form or mutant form of the platelet surface antigen GpIbα does not act as an antigen to reactivate B lymphocytes to produce more autoimmune antibodies.

[0015] In one or more embodiments, the truncated form or mutant form of the platelet surface antigen GpIbα specifically kills plasma cells that secrete free autoantibodies against platelet antigens as competing antigens or B lymphocytes that express anti-platelet BCRs, while leaving B lymphocytes that express normal BCRs unaffected.

[0016] In one or more embodiments, the removal includes, but is not limited to, reduction, inhibition, reduction, lysis, or blocking; preferably, a statistically significant reduction, inhibition, reduction, lysis, or blocking, such as reducing reactive B lymphocytes and / or free anti-platelet antibodies to less than 90%, 80%, 60%, 50%, 30%, 20%, 10%, 5%, or 2% of the original content.

[0017] In one or more embodiments, the truncated form is encoded by a polynucleotide having the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20, or a degenerate nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:16, SEQ ID NO:18, or SEQ ID NO:20.

[0018] In one or more embodiments, the mutant consists of: (1) a protein formed by substituting, deleting or adding one or more (e.g., 1 to 20; preferably 1 to 15; more preferably 1 to 10, e.g., 5, 3, 1) amino acid residues in the amino acid sequence of the truncated product, and having the function of the truncated product; (2) a protein having 80% or more (preferably 85% or more; more preferably 90% or more; more preferably 95% or more, e.g., 98%, 99%) homology with the amino acid sequence of the truncated product, and having the function of the truncated product; and (3) a fusion protein containing the truncated product.

[0019] In one or more embodiments, the amino acid sequence of the variant is as set forth in SEQ ID NO:17, SEQ ID NO:19 or SEQ ID NO:21.

[0020] In one or more embodiments, the decrease in binding ability is a statistically significant decrease, e.g., a 5%, 10%, 15%, 20%, 30%, 50%, 70%, 80%, 90%, 95% or greater decrease.

[0021] In another aspect of the present invention, a chimeric autoantibody receptor is provided comprising a truncation or mutant thereof of the platelet surface antigen GpIbα as an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain; preferably, the intracellular signaling domain comprises a costimulatory molecule and / or a signal transduction domain.

[0022] In one or more embodiments, the transmembrane domain comprises the hinge region and transmembrane region of CD8 or CD28; preferably, the transmembrane domain comprises the hinge region and transmembrane region of CD8; more preferably, the hinge region has the amino acid sequence shown in positions 291 (T) to 335 (D) of SEQ ID NO: 11, and the transmembrane domain has the amino acid sequence shown in positions 336 (I) to 359 (C) of SEQ ID NO: 11.

[0023] In one or more embodiments, the intracellular signal domain comprises an intracellular signal region selected from 4-1BB, CD3ζ, FcεRIγ, CD27, CD28, CD134, ICOS, or GITR; preferably, the intracellular signal region comprises 4-1BB and CD3ζ; more preferably, the 4-1BB has the amino acid sequence shown in positions 360 (K) to 401 (L) of SEQ ID NO: 11, and the CD3ζ has the amino acid sequence shown in positions 402 (R) to 513 (R) of SEQ ID NO: 11.

[0024] In one or more embodiments, the chimeric autoantibody receptor comprises a structure in which a truncated form or mutant form of the platelet surface antigen GpIbα, the hinge region and transmembrane region of CD8 or CD28, 4-1BB, and CD3ζ are linked in this order; preferably, the chimeric autoantibody receptor has an amino acid sequence as set forth in any of SEQ ID NOs: 11 to 15.

[0025] In one or more embodiments, in the chimeric autoantibody receptor, the truncated form of the platelet surface antigen GpIbα preferably has the amino acid sequence shown in SEQ ID NO:11.

[0026] In one or more embodiments, in the chimeric autoantibody receptor, the truncated form of the platelet surface antigen GpIbα is preferably a mutant having a reduced binding ability due to a mutation in the binding region that binds to von Willebrand factor (vWF); more preferably, in the mutant, position 233 of the amino acid sequence corresponding to SEQ ID NO: 2 is mutated from G to K (G233K).

[0027] In one or more embodiments, the chimeric autoantibody receptor does not act as an antigen to reactivate B lymphocytes to produce more autoimmune antibodies.

[0028] In one or more embodiments, the chimeric autoantibody receptor specifically targets and kills plasma cells secreting free autoantibodies against platelet antigens or B lymphocytes expressing anti-platelet BCRs, but does not affect B lymphocytes expressing normal BCRs, thereby reducing platelet killing by autoimmune antibodies and increasing platelet counts.

[0029] In one or more embodiments, the chimeric autoantibody receptor has limited or no toxicity to healthy cells.

[0030] In another aspect of the invention, there is provided an expression construct comprising (i) a polynucleotide encoding the chimeric autoantibody receptor, or (ii) a polynucleotide encoding a truncated form or mutant form of the platelet surface antigen GpIbα.

[0031] In one or more embodiments, the expression construct comprises an expression vector.

[0032] In one or more embodiments, the vector is an RNA vector.

[0033] In one or more embodiments, the expression vector comprises a viral vector or a non-viral vector.

[0034] In one or more embodiments, the expression vector is a viral vector.

[0035] In one or more embodiments, the viral vector includes, but is not limited to, a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.

[0036] In one or more embodiments, the virus includes, but is not limited to, a lentivirus, an adenovirus, or an adeno-associated virus.

[0037] In one or more embodiments, the expression construct comprises a promoter operably linked to the chimeric autoantibody receptor polynucleotide.

[0038] In one or more embodiments, the expression construct comprises a promoter operably linked to the polynucleotide of the truncated form of the platelet surface antigen GpIbα or a mutant thereof.

[0039] In one or more embodiments, the promoter is a constitutive expression promoter.

[0040] In one or more embodiments, the promoter is an inducible promoter, including a spatiotemporally inducible promoter, a tissue-inducible promoter, and the like.

[0041] In another aspect of the present invention, there is provided a virus (including but not limited to a lentivirus) comprising the expression vector.

[0042] In another aspect of the invention, there is provided an immune effector cell into which the chimeric autoantibody receptor polynucleotide or the expression construct has been introduced or which expresses the chimeric autoantibody receptor.

[0043] In one or more embodiments, the immune effector cells comprise cells selected from the group consisting of T cells, natural killer cells (NK), NKT cells, or cytokine-induced killer cells (CIK), or a combination thereof; more preferably, the immune effector cells are T cells; more preferably, the T cells comprise helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, γδ T cells, T memory stem cells, or T cells derived from pluripotent stem cells.

[0044] In one or more embodiments, the T cells are cytotoxic immune effector T cells.

[0045] In one or more embodiments, the chimeric autoantibody receptor expressed by the immune effector cells has high affinity for the autoantibody-based B lymphocyte receptor (BCR) on B lymphocytes.

[0046] In one or more embodiments, the immune effector cells also express cytokines that have immune modulating activity or can enhance immune effector cell function; preferably, the cytokines include, but are not limited to, IL-12 (stimulates immune cell proliferation), IL-15 (activates immune cells), IL-21 (increases immune cell activity), IL-2 (stimulates and maintains T cell differentiation and proliferation), IL-4 (modulates immune cell activity), IL-7 (modulates immune cell maturation, activation, proliferation, immunomodulation), IL-9 (modulates immune cell growth or activity), IL-17 (promotes immune cell activation), IL-18 (promotes immune cell proliferation), IL-23 (promotes immune cell proliferation).

[0047] In another aspect of the invention, there is provided the use of said immune effector cells in (a) the preparation of a composition for neutralizing anti-platelet antibodies or free antibodies, (b) the preparation of a composition for targeting and depleting reactive B lymphocytes, (c) the preparation of a composition for elevating platelets, and (d) the preparation of a composition for alleviating or treating immune thrombocytopenic purpura.

[0048] In another aspect of the present invention, there is provided a pharmaceutical composition or drug box comprising said immune effector cells for (a) neutralizing anti-platelet antibodies or free antibodies, (b) targeting and eliminating reactive B lymphocytes, (c) elevating platelets, and (d) alleviating or treating immune thrombocytopenic purpura.

[0049] In one or more embodiments, the pharmaceutical composition also includes a pharma- ceutically acceptable pharmaceutical carrier or excipient.

[0050] In one or more embodiments, the medication box comprises a container / package and the immune effector cells or a pharmaceutical composition comprising the immune effector cells contained in the container / package.

[0051] In another aspect of the invention, there is provided a method for (a) neutralizing anti-platelet antibodies or free antibodies, (b) targeting and depleting reactive B lymphocytes, (c) elevating platelets, or (d) alleviating or treating immune thrombocytopenic purpura, said method comprising administering to a subject an effective amount of said immune effector cells or said pharmaceutical composition.

[0052] In one or more embodiments, the immune thrombocytopenic purpura (ITP) is a disease caused by a variety of primary or secondary causes.

[0053] In one or more embodiments, a thrombocytopenic subject is one whose peripheral platelet count is statistically lower than the normal peripheral platelet count, e.g., 1%, 2%, 3%, 5%, 8%, 10%, 12%, 15%, 20%, 25% or more.

[0054] In one or more embodiments, the thrombocytopenic (thrombocytopenic) subject is a relapsed / refractory subject.

[0055] In one or more embodiments, the relapsed / refractory subject has undergone at least one treatment for ITP.

[0056] Other aspects of the invention will be apparent to those of ordinary skill in the art as a result of the disclosure herein. [Brief description of the drawings]

[0057] [Figure 1]The effect of the G233K point mutation on the aggregation ability of CAAR-T cells and vWF was detected by agglutination reaction. The aggregation ability of Jurkat cells stably expressing CAAR1, CAAR4, and CAAR5 mutants and human vWF was detected by agglutination reaction under the action of ristocetin. As a result, the aggregation ability of Jurkat cells expressing CAAR1, CAAR4, and CAAR5 and vWF was significantly reduced (light transmittance was reduced) compared with that of non-mutated Jurkat cells, indicating that the G233K point mutation obviously reduced the binding ability of Jurkat-CAAR-T cells and human vWF. [Diagram 2] The binding ability of CAAR-T cells to anti-GpIbα positive hybridoma cells was screened by flow cytometry. The detection results showed that different CAAR-T cells selectively recognized and bound to anti-GpIbα positive hybridoma cells (Gvb1-Gvb4), and compared with CAAR2-T cells and CAAR3-T cells, CAAR4-T cells and CAAR5-T cells had stronger binding ability to the four positive hybridoma cells of Gvb1-Gvb4, and the G233K point mutation did not affect the specific binding between CAAR-T cells and anti-GpIbα positive hybridoma cells. [Diagram 3] The competitive killing effect of CAAR-T cells against anti-GpIbα positive hybridoma cells was detected by flow cytometry. The results showed that GpIbα-CAAR-T cells could selectively recognize and competitively kill positive hybridoma cells with different antigen epitopes, and that CAAR1-T cells had a stronger competitive killing effect against all four positive hybridoma cells, Gvb1 to Gvb4, compared to CAAR2-T cells to CAAR5-T cells. [Figure 4] The killing and lysis effects of CAAR-T cells against anti-GpIbα positive hybridoma cells were detected by LDH release experiments. The results showed that, at the same ET ratio, CAAR1-T cells had a killing effect against all four positive hybridoma cells, Gvb1 to Gvb4, and had the strongest ability to lyse the cells, suggesting that CAAR1-T cells have stronger cytotoxicity against target cells than other CAAR-T cells. [Diagram 5] The release of cytokines (IL-2 and IFN-γ) from CAAR-T cells against anti-GpIbα positive hybridoma cells was detected by ELISA. The results showed that CAAR1-T cells had a killing effect against all four positive hybridoma cells, Gvb1 to Gvb4, and had the strongest ability to release the cytokines IL-2 and IFN-γ, suggesting that CAAR1-T cells have stronger toxicity to target cells than other CAAR-T cells. [Figure 6] The killing effect of CAAR-T on target cell Gvb1 in vivo is visually observed with a Luciferase fluorescent expression system. To detect the proliferation and metastasis of Gvb1 cells in real time, the Gvb1 cell line virus is packaged and stably transfected with GFP fluorescent reporter gene. Gvb1-GFP cells / mouse, CAAR-T cells and control T cells are injected into the tail vein of NCG mice, respectively. The survival status of the mice is observed daily, and the Gvb1-GFP fluorescent reporter in vivo of the mice is photographed on the 7th, 14th and 21st days. According to the photographing results and statistical data, on the 14th day, the NTD mice in the control group already showed significant Gvb1-GFP amplification and metastasis, while the mice in the experimental group injected with CAAR-T cells had significantly lower Gvb1-GFP cell amplification and metastasis than the control group. On day 21, the control NTD mice already showed significant Gvb1-GFP amplification and metastasis, while the experimental mice injected with CAAR-T cells showed significantly lower amplification and metastasis of Gvb1-GFP cells than the control group, indicating a clear killing effect of CAAR-T cells on Gvb1 cells. [Figure 7]Construction of GpIbα-CAAR-T cells with different truncations. A certain number of T cells were prepared and isolated from the peripheral blood of volunteers, washed and activated in an ultraclean environment, cultured in a medium containing cytokines necessary for survival and proliferation, and activated in vitro using CD3 and CD28 antibodies. GpIbα-CAAR gene fragments containing different truncations were infected into activated T cells using a lentiviral vector, and after culturing for a certain period of time, the results of flow cytometry detection showed that all of the GpIbα-CAAR lentiviruses with different truncations could infect human T cells at a certain rate, and GpIbα-CAAR-T cells containing different truncations could be constructed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] As a result of extensive and detailed research, the present inventors unexpectedly discovered for the first time that extracellular protein fragments derived from the platelet surface antigen GpIbα, multiple truncations and modified antigen fragments thereof can competitively bind to autoimmune antibodies (antiplatelet antibodies) expressed by reactive B lymphocytes and free antibodies secreted by plasma cells in vivo in patients with immune thrombocytopenia (immune thrombocytopenic purpura), and do not bind to natural ligands in vivo and do not affect blood coagulation; using the competing antigens, the inventors unexpectedly discovered that extracellular protein fragments derived from the platelet surface antigen GpIbα, multiple truncations and modified antigen fragments thereof can competitively bind to platelet autoantigens in vivo in patients with immune thrombocytopenia (immune thrombocytopenic purpura), and do not bind to natural ligands in vivo and do not affect blood coagulation; The Fc fusion protein can be further prepared to specifically kill plasma cells secreting free autoantibodies against platelet antigens or B lymphocytes expressing anti-platelet BCR, while not affecting B lymphocytes expressing normal BCR, thus reducing the killing of platelets by autoimmune antibodies and increasing platelet count, fundamentally solving the problem of thrombocytopenia caused by autoimmune mechanism, and the competing antigen and its modified chimeric CAAR-T cells or Fc fusion protein do not act as antigens to reactivate B lymphocytes to produce more autoimmune antibodies. Based on this, the present invention has been completed.

[0059] term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0060] In the present invention, when referring to measurable values ​​such as amounts, durations, and the like, "about" as used herein is intended to encompass ±20%, ±10%, ±5%, ±2%, or ±1% of the stated value, and also means an error of ±0.5% or 0.1% in some cases.

[0061] As used herein, "limited toxicity" means that the truncates (peptides), polynucleotides, chimeric autoantibody receptors, or cells of the invention exhibit essentially no negative biological effects or physiological symptoms on healthy, non-diseased, non-target cells, or cell populations thereof, in vitro or in vivo.

[0062] As used herein, "autoantibody" means an antibody produced by specific B lymphocytes against a self-antigen.

[0063] As used herein, an "autoimmune disease" is defined as a disorder or condition resulting from an autoimmune response mediated by antibodies against self-antigens. Autoimmune diseases cause the inappropriate and / or excessive production of self-antigens or auto-antibodies against self-antigens.

[0064] As used herein, "chimeric autoantibody receptor" or "CAAR" refers to a modified receptor expressed on T cells or other effector cell types that have cell-mediated cytotoxicity. The CAAR contains an antigen or fragment thereof that is specific for a pathogenic autoantibody. The CAAR also contains a transmembrane domain, an intracellular domain, and a signaling domain.

[0065] As used herein, "effective amount" or "therapeutically effective amount" are used interchangeably and refer to an amount of a compound, formulation, material or composition effective to achieve a particular biological result as described herein.

[0066] As used herein, "operably linked" or "operably linked" refers to a functional link between a regulatory sequence and a heterologous nucleic acid sequence, resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first and second nucleic acid sequences are in a functional relationship. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Usually, operably linked DNA sequences are contiguous and, where necessary, link two protein coding regions in the same reading frame.

[0067] As used herein, a "subject" includes organisms in which an immune response can be elicited, such as mammals, including humans.

[0068] As used herein, "transmembrane domain" means the portion or region of a molecule that spans the lipid bilayer membrane.

[0069] As used herein, the term "test sample" or "test sample" refers to a sample to be detected. In the present invention, the sample to be detected may be plasma (e.g., from peripheral blood), a separated sample containing platelets, or a processed sample, etc.

[0070] As used herein, the term "diagnosis" refers to a method used by those skilled in the art to assess and / or determine the likelihood that an individual will suffer from a particular disease, condition, or syndrome. The term "diagnosis" also encompasses detecting a propensity to suffer from a disease, condition, or syndrome, determining the therapeutic efficacy of drug treatment, or predicting a response to drug treatment. The diagnostic methods of the present disclosure may be performed alone or in combination with other diagnostic and / or staging methods for analyzing a disease, condition, or syndrome.

[0071] Truncated forms of GpIbα In most patients with immune thrombocytopenia (immune thrombocytopenic purpura), one or more autoantibodies against platelet membrane antigen epitopes can be detected, among which the membrane protein GPIbα is one of the major antigen epitopes for autoantibody production. The present invention provides a series of truncations of the extracellular domain of GpIbα that competitively bind to platelet autoantigens in vivo with autoimmune antibodies (antiplatelet antibodies) expressed by reactive B lymphocytes and free antibodies secreted by plasma cells, and do not bind to natural ligands in vivo and do not affect blood coagulation. The truncations of GpIbα are derived from the extracellular domain of GpIbα protein and have binding sites for autoimmune antibodies in vivo; preferably, the truncations are characterized in that they include proteins with the amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:17, SEQ ID NO:19, or SEQ ID NO:21.

[0072] The present invention also includes active fragments, derivatives and analogs of the truncations. As used herein, the terms "active fragment," "derivative" and "analog" refer to a polypeptide that essentially retains a biological function or activity homologous to the truncation.

[0073] The protein fragment, derivative or analog of the present invention may be (i) a protein in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and the substituted amino acid residues may or may not be encoded by the genetic code, (ii) a protein having a substitution at one or more amino acid residues, or (iii) a protein formed by fusing an additional amino acid sequence to the protein sequence (e.g., a leader sequence, a secretory sequence, a sequence for purifying the protein or a protein constituent sequence, or a fusion protein). According to the definitions in this specification, these fragments, derivatives and analogs are within the scope well known to those skilled in the art.

[0074] In the present invention, the "GpIbα truncation product" also includes (but is not limited to) those obtained by deleting, inserting and / or substituting a small number of amino acids (usually 1-20, preferably 1-10, more preferably 1-8, 1-5, 1-3 or 1-2) and adding or deleting one or more amino acids (usually 20 or less, preferably 10 or less, more preferably 5 or less) at the C-terminus and / or N-terminus. For example, in the present field, substitution with amino acids having close or similar properties usually does not change the function of the protein. Also, for example, addition of one or more amino acids to the C-terminus and / or N-terminus usually does not change the function of the protein.

[0075] In a preferred embodiment, the amino acid sequence of the GpIbα truncates or their active fragments, derivatives and analogues described in the present invention is mutated, preferably at discrete sites, thereby resulting in a mutation in the binding region for binding to vWF and reducing the binding ability; more preferably, said mutants have a G233K mutation.

[0076] The polypeptide sequences described herein are preferably polypeptides comprising the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10. More preferably, the G233K mutation is also present. Said polypeptides comprising the G233K mutation further comprise the sequence shown in SEQ ID NO:17, SEQ ID NO:19 or SEQ ID NO:21.

[0077] The amino or carboxyl terminus of the GpIbα truncations of the present invention may also comprise one or more polypeptide fragments as a protein tag. Any suitable tag may be used in the present invention. For example, the tag may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE, and Ty1. These tags may be used for protein purification. To secrete or express (e.g., secrete outside the cell) the translated protein, a signal peptide sequence, such as the pelB signal peptide, may be added to the amino terminus of the amino acid of the GpIbα truncation. The signal peptide may be cleaved during the secretion of the polypeptide from the cell.

[0078] The present invention also provides a polynucleotide sequence encoding the GpIbα truncation or its conservative variant protein of the present invention. The polynucleotide may be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The polynucleotide encoding the mature protein of the truncation includes a coding sequence encoding only the mature protein; a coding sequence of the mature protein and various additional coding sequences; a coding sequence of the mature protein (and any additional coding sequences) and non-coding sequences. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence of the polynucleotide provided in the examples or may be a degenerate variant. As used herein, a "degenerate variant" refers to a nucleic acid sequence encoding the truncation of the present invention in the present invention but different from the coding region sequence of the polynucleotide provided in the examples.

[0079] The present invention also relates to variants of the above polynucleotides that code for proteins or protein fragments, analogs and derivatives having the same amino acid sequence as the present invention. The variants of the polynucleotides may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, allelic variants are interchangeable forms of a polynucleotide, which may include substitutions, deletions or insertions of one or more nucleotides, but do not substantially alter the function of the protein encoded thereby.

[0080] The full length nucleotide sequence of the GpIbα truncated fragment of the present invention or a fragment thereof can usually be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers are designed according to the relevant nucleotide sequence disclosed in the present invention and amplified to obtain the relevant sequence. Alternatively, the relevant sequence may be synthesized by artificial synthesis. The DNA sequence is then introduced into various existing DNA molecules (e.g., vectors) and cells known in the art. Alternatively, mutations may be introduced into the protein sequence of the present invention by chemical synthesis.

[0081] The present invention also relates to vectors which include polynucleotides of the invention, host cells genetically engineered with the vectors or GpIbα truncation coding sequences of the invention and methods for producing the proteins of the invention by recombinant techniques.

[0082] The polynucleotide sequences of the invention can be used to express or produce recombinant GpIbα truncations by conventional recombinant DNA techniques, generally comprising the steps of (1) transforming or transducing a suitable host cell with a polynucleotide (or variant) of the invention encoding a GpIbα truncation, or a recombinant expression vector containing the polynucleotide; (2) culturing the host cell in a suitable medium; and (3) isolating and purifying the protein from the medium or cells.

[0083] The present invention also relates to nucleic acid constructs comprising the polynucleotides described herein and one or more regulatory sequences or sequences required for homologous recombination of a genome operatively linked to these sequences. The polynucleotides of the present invention may be manipulated in various ways to ensure expression of said polypeptides or proteins. The nucleic acid construct may be manipulated according to the differences or requirements of the expression vector before it is inserted into a vector. Techniques for modifying polynucleotide sequences by recombinant DNA methods are known in the art.

[0084] In some embodiments, the nucleic acid construct is a vector. The polynucleotide sequence of the GpIbα truncation can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors well known in the art. In short, any plasmid or vector can be used as long as it can replicate and is stable in the host. Important features of an expression vector are that it usually contains an origin of replication, a promoter, a marker gene, and a translation control element.

[0085] Methods well known to those skilled in the art can be used to construct expression vectors containing the coding DNA sequence of the GpIbα truncation and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, etc. The DNA sequence may be operatively linked to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector further comprises a ribosome binding site utilized for translation initiation and a transcription terminator. In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for the selection of transformed host cells.

[0086] A vector containing an appropriate DNA sequence and an appropriate promoter or control sequence as described above can be used to transform an appropriate host cell to express the protein. The host cell can be a prokaryotic cell such as a bacterial cell, a lower eukaryotic cell such as a yeast cell, or a higher eukaryotic cell such as a plant cell, a fungal cell, an insect cell, or a mammalian cell. Representative examples include prokaryotic cells such as Escherichia coli, Bacillus subtilis, Streptomyces, Agrobacterium, and fungal cells such as yeast cells (e.g., Pichia pastoris, Saccharomyces cerevisiae). Transformation of a host cell with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. The truncated form of the present invention can be expressed by culturing the obtained transformant.

[0087] Chimeric Autoantibody Receptor (CAAR) The truncated form of GPIbα described herein can be used to prepare a chimera with killing effect by targeting cell-dependent platelet antibodies, which can be applied to, but is not limited to, the construction of CAAR-T cell immunotherapeutic drugs, etc., and plays a role in targeting and killing reactive B lymphocytes and reducing the titer of autoantibodies, thereby meeting the clinical need to target and eliminate reactive B lymphocytes that produce PA Abs.

[0088] Thus, the present invention provides a CAAR expressed on the surface of immune effector cells, which consists of an extracellular binding domain, a transmembrane domain, and an intracellular signal domain, which are linked in sequence, and by combining these domains and expressing the CAAR in immune effector cells, the immune effector cells have a highly specific cytotoxic effect against reactive B lymphocytes (B lymphocytes that cause autoimmune diseases). The extracellular binding domain includes the truncated form of the present invention or a mutant thereof.

[0089] A linker may or may not be included between the extracellular binding domain, the transmembrane domain, and the intracellular signal domain.

[0090] Examples of hinge and / or transmembrane domains include the hinge and / or transmembrane domains of the α, β or ε chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIR, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS(CD278), 4-1BB(CD137), GITR, CD40, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80(KLRF1), CD1 60, CD19, IL2Rβ, L2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD 11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA -1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96, CEACAM1, CRTAM, Ly9 (CD229), CD1 60 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0091] In one preferred embodiment, the transmembrane region of CAAR can be selected from the transmembrane region of proteins such as CD8 or CD28. Human CD8 protein is a heterodimer consisting of two chains of αβ or γδ. In a preferred embodiment of the present invention, the transmembrane region is selected from the transmembrane region of CD8 (CD8a) or CD28. Since the CD8 hinge region is a flexible region, the CD8 or CD28 transmembrane region and hinge region can be used to link the target recognition domain scFv of CAAR with the intracellular signal region.

[0092] In one preferred embodiment, the nucleic acid sequence of the transmembrane domain encodes a CD8 hinge and / or transmembrane domain.

[0093] The cytoplasmic domain or other intracellular signal region (intracellular signal domain) of the CAAR of the present invention is responsible for the activation of at least one normal effector function of immune cells having CAAR. By "effector function" is meant the function of cell specification. For example, the effector function of T cells can be cytolytic activity or auxiliary activity, including the secretion of cytokines. Thus, the term "intracellular signal region" means a part of a protein that transmits a signal of effector function and instructs the cell to perform a function of specification. The entire intracellular signal region can be used, but in many cases it is not necessary to use the entire domain. For truncated portions of the intracellular signal region, such truncated portions can be used instead of the complete domain, so long as they transmit a signal of effector function. Thus, "intracellular signal region" is meant to include any truncated portion of the intracellular domain sufficient to transmit a signal of effector function.

[0094] Examples of intracellular signaling regions for use in the CAAR of the present invention include, but are not limited to, the cytoplasmic portion of the T cell receptor (TCR) and co-receptors, as well as any derivatives or variants of these elements, and any synthetic sequences having the same functional capabilities. Examples of intracellular signaling regions include fragments or domains from one or more molecules or receptors, such as CD3zeta, CD3gamma, CD3delta, CD3epsilon, CD86, common FcRgamma, FcRbeta (FcεR1b), CD79a, CD79b, FcγRIIa, DAP10, DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligand specific for CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TN Costimulatory molecules include, but are not limited to, FR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96, CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, as well as derivatives, variants or fragments thereof, any synthetic sequence of a costimulatory molecule having the same functional capability, and any combination thereof.Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are necessary for lymphocytes to respond effectively to antigens.

[0095] In one alternative embodiment, the intracellular signal region can be selected from the intracellular signal regions of CD3ζ, FcεRIγ, CD27, CD28, 4-1BB, CD134, ICOS, and GITR proteins, and combinations thereof. The CD3 molecule is composed of five subunits, among which the CD3ζ subunit (also called CD3 zeta, abbreviated as Z) contains three ITAM motifs, which are important signal transduction regions of the TCR-CD3 complex. FcεRIγ is mainly distributed on the surface of mast cells and basophils, contains one ITAM motif, and is similar to CD3ζ in structure, distribution, and function. In addition, CD28, 4-1BB, and CD134 are costimulatory signal molecules, and the costimulatory effect generated by their intracellular signal segments after binding to their respective ligands can cause the continuous proliferation of immune effector cells (mainly T lymphocytes), increase the secretion levels of cytokines such as IL-2 and IFN-γ from immune effector cells, and at the same time improve the survival cycle of CAAR immune effector cells in vivo. In a preferred embodiment, the intracellular signaling region of CAAR comprises a CD3ζ signaling domain and a 4-1BB costimulatory domain.

[0096] In the CAAR according to the present invention, the truncated form can be operatively linked to a hinge region sequence and a transmembrane region sequence, and can further be operatively linked to an intracellular signal region.

[0097] In the embodiment of the present invention, the present inventor provides the preferred CAAR. It should be understood that the present invention also includes CAARs that are modified or altered based on the optimized CAAR of the present invention. For example, the CAAR of the present invention can be obtained from the specific sequential linkage of truncated or mutant-CD8-CD3, truncated or mutant-CD8-CD137-CD3, truncated or mutant-CD28a-CD28b-CD3, or truncated or mutant-CD28a-CD28b-4-1BB-CD3, but is not limited thereto.

[0098] In an alternative embodiment of the present invention, the CAAR also comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, which is optionally cleaved from the antigen recognition domain (e.g., scFv) during cellular processing and localization of the CAAR to the cell membrane.

[0099] The present invention also includes nucleic acids encoding said CAAR. The nucleic acid sequences of the present invention may be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The nucleic acid codons encoding the amino acid sequence of the CAAR protein of the present invention may be degenerate, i.e., multiple degenerate nucleic acid sequences encoding the same amino acid sequence are all included within the scope of the present invention. Degenerate nucleic acid codons encoding the corresponding amino acids are well known in the art.

[0100] The present invention also includes variants of the above polynucleotides that code for polypeptides having the same amino acid sequence as the present invention, or fragments, analogs and derivatives of the polypeptides. The variants of the polynucleotides may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, allelic variants are interchangeable forms of a polynucleotide, and may include substitutions, deletions or insertions of one or more nucleotides, but do not substantially alter the function of the polypeptide encoded thereby.

[0101] The present invention also provides a genetically modified immune effector cell transduced with a nucleic acid of the invention or a recombinant plasmid comprising said nucleic acid of the invention or a virus comprising said plasmid, said cell being a cell or a cell population comprising said cell, preferably a T cell or a cell population comprising T cells.

[0102] Any nucleic acid transduction method conventional in the art can be used in the present invention, including non-viral and viral transduction methods. Non-viral based transduction methods include electroporation and transposon methods.

[0103] The present invention also provides an expression construct (vector) comprising the above-described nucleic acid encoding a CAAR protein to be expressed on the surface of immune effector cells, including a viral vector or a non-viral vector.

[0104] The non-viral vector system, such as the Sleeping Beauty system or the PiggyBac transposon system, has a higher transduction efficiency than conventional electroporation, and the combined use of the Sleeping Beauty transposon system with the nucleofector transfector has already been reported [Davies JK., et al. Cancer Res, 2010, 70(10): OF1-10.], which not only has a high transduction efficiency but also realizes site-specific integration of the target gene. In addition, mRNA transfection technology can also be applied.

[0105] For example, various vectors for virus packaging can be used in the present invention, including lentivirus vectors, adenovirus vectors, adeno-associated virus vectors, etc., as well as virus vectors formed by modifying these virus vectors. In one specific embodiment of the present invention, the backbone vector used in the present invention is a lentivirus plasmid vector. In this vector, the expression of CAAR molecule is transcriptionally driven by EF1 promoter, and the CAAR lentivirus vector plasmid can be packaged as a lentivirus with CAAR molecule by co-transfecting virus-producing cells (e.g., 293T cells) in the presence of helper packaging plasmid. In addition, although a specific lentivirus vector is preferred in the present invention, it should be understood that other types of vectors can also be used as long as active CAAR and immune effector cells modified thereby can be obtained in the end.

[0106] The present invention further includes viruses packaged in a viral vector. The viruses may be lentiviruses, adenoviruses, adeno-associated viruses, etc., and also include viruses formed from these viruses through further modification. The viruses of the present invention include infectious packaged viruses, but also packaged viruses that contain the components necessary for packaging as infectious viruses. Although lentiviruses are preferred for use in the present invention, it is understood that other viruses and their corresponding plasmid vectors known in the art that can be used to transduce foreign genes into immune effector cells can also be used in the present invention.

[0107] In one embodiment of the present invention, the transduction method for achieving CAAR gene-modified immune effector cells is a virus-based transduction method such as lentivirus. This method has the advantages of high transduction efficiency, stable expression of foreign genes, and shortening the time it takes for in vitro cultured immune effector cells to reach clinical levels. On the surface of the transgenic immune effector cells, the transduced nucleic acid is expressed on the surface through transcription and translation.

[0108] The immune cells according to the present invention can also carry coding sequences for exogenous cytokines, including but not limited to IL-12, IL-15, or IL-21. These cytokines have immunomodulatory activity and can enhance the function of effector T cells and activated NK cells. Therefore, those skilled in the art can understand that the use of these cytokines can contribute to better function of the immune cells.

[0109] The immune cells according to the present invention can also express a safety switch, preferably comprising iCaspase-9, Truncated EGFR, or RQR8. As mentioned above, current CA therapy is still challenged due to its complex production and adverse events related to cell activity, such as cytokine storm (CRS). Therefore, drugs or therapies that effectively regulate CAAR-T, such as setting a safety switch, are more preferable.

[0110] Pharmaceutical Compositions The genetically modified immune effector cells of the present invention can be applied to the preparation of compositions, particularly pharmaceutical compositions. In addition to an effective amount of said immune effector cells, said compositions can contain pharma- ceutically acceptable vectors. The term "pharma-ceutically acceptable" means that the molecular entities and compositions do not produce undesirable, allergic, or other adverse reactions when appropriately administered to animals or humans.

[0111] The truncated product of the present invention or a fusion protein containing the same can be used as an antigen and can also be applied to the preparation of a composition, such as a pharmaceutical composition. In addition to an effective amount of the truncated product, the composition can contain a pharma- ceutically acceptable vector.

[0112] Specific examples of some substances that can be used as pharma- ceutically acceptable vectors or components thereof include polyols such as propylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween®; wetting agents such as sodium lauryl sulfate; colorants; flavorings; tableting agents, stabilizers; antioxidants; preservatives; unheated raw water; isotonic salt solutions; sugars such as lactose, glucose, sucrose, and the like; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and methylcellulose; tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa oil; and phosphate buffers.

[0113] The composition of the present invention can be made into various dosage forms as needed, and the doctor can determine and administer the beneficial dose to the patient based on factors such as the type, age, weight, disease state, and administration method of the patient, which can be, for example, injection or other treatment methods.

[0114] When considering the "immunologically effective amount", "anti-autoantibody effective amount", "autoimmune disease suppression effective amount" or "therapeutic amount", the exact amount of the composition of the present invention to be administered can be determined by a physician taking into consideration individual differences in age, weight, and condition of the patient (subject). Usually, the pharmaceutical composition containing the immune effector cells is administered in an amount of 10 3 -10 10 (For example, 10 4 , 10 5 , 10 6 , 10 7 or 10 8 ) cells / kg, and in some embodiments, for example, 10 5 -10 8The compositions may be administered in doses of 1000 to 2000 cells / kg. The compositions may also be administered multiple times at these doses. The cells may be administered using injection techniques commonly known in immunotherapy. The optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical arts by monitoring the patient for disease symptoms and adjusting treatment accordingly.

[0115] The compositions of the invention can also be packaged in a suitable medication box for use by a clinician. Preferably, the medication box also includes instructions explaining how to use the compositions of the invention.

[0116] Clinical Diagnosis / Typing The truncations of GPIbα described in the present invention can be used to prepare diagnostic antigens for defining autoantibody binding epitopes and relative titers in immune thrombocytopenia (ITP) patients, thereby enabling the identification of patients with different clinical subtypes.

[0117] As an alternative embodiment, a reagent that can be used for detecting an autoantibody can be designed based on the principle of indirect enzyme immunoassay. For example, the truncated product described in the present invention is linked to a solid-phase carrier, and the autoantibody in the test sample is bound to it to form a solid-phase antigen-test autoantibody complex, and then an enzyme-labeled secondary antibody is bound to the autoantibody in the solid-phase antigen-test autoantibody complex to form a solid-phase antigen-test autoantibody-enzyme-labeled secondary antibody complex, and the absorbance after adding a substrate is measured to determine the level of the test autoantibody.

[0118] The truncated form is immobilized on a solid support as an antigen protein, for example, at a concentration of 10 nM to 100 uM, and the concentrations used for different antigen proteins may be the same or different. Preferably, the truncated form is immobilized on the surface of a solid support using a covalent immobilization method, a specific concentration of human anti-tag peptide is used as a calibrator for quantitative detection, and the autoantibody is detected by detecting the signal intensity using a colorimetric / fluorescent / chemiluminescent method. The signal detection method used for the detection includes, but is not limited to, visible light colorimetric method, fluorescent method, chemiluminescent method, etc.

[0119] Based on the novel discoveries of the present invention, kits for the diagnosis, prognosis, or typing of ITP are also provided.

[0120] In one embodiment, the kit includes a solid-phase carrier coated with the antigen protein. The detection kit can be designed to be suitable for detection by visible light colorimetry, fluorescence, or chemiluminescence. The solid-phase carrier can be, but is not limited to, magnetic particles, microspheres, plastic beads, liquid-phase chips, microwell plates, affinity membranes, slides, or test strips. For example, the antigen protein is immobilized on the solid-phase carrier using a covalent immobilization method. Preferably, the detection by visible light colorimetry, fluorescence, and / or chemiluminescence is achieved by coating the antigen on a microwell plate, such as a 96-well plate.

[0121] The method for immobilizing the antigen protein includes a direct coating method and an indirect coating method. In a specific embodiment, the antigen is activated by mixing and incubating with an appropriate premix, and then added to a solid support, incubated, and blocked. In the indirect coating method, the antigen protein is indirectly immobilized on a solid support by a specific reaction between biotin and streptavidin, for example.

[0122] According to the present invention, the kit also includes diluents (such as sample and antibody diluents), secondary antibodies, washing solutions, humanized tag peptide antibody calibrators, humanized tag peptide antibody quality control products, substrates, stop solutions, luminescence solutions, etc. Preferably, the secondary antibodies carry a detectable marker, such as horseradish peroxidase.

[0123] The present invention will be further described below with reference to specific examples. Note that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. In the following examples, the experimental methods for which no specific conditions are specified generally follow conventional conditions such as those described in Molecular Cloning: A Laboratory Manual, 3rd Edition, edited by J. Sambrook et al., Scientific Press, or follow the conditions recommended by the manufacturer. EXAMPLES

[0124] Design of gene fragments for different truncations of GpIbα In order to determine the binding site of GpIbα and autoimmune antibodies in vivo, it was considered to clarify the antigen-antibody binding site by investigating the binding site of GpIbα and autoantibodies. Therefore, based on the extracellular terminal base sequence of human GpIbα, several sets of specific primer sequences were designed and PCR technology was used to obtain truncated gene fragments for different antigen epitopes of GpIbα. Five truncated fragments, whose antigen epitopes on GpIbα are 1H-290R, 265P-414P, 265P-509F, 1H-509F, and 1H-414P (1#~5#), respectively, were screened, and the specific nucleotide / amino acid sequences were as follows:

[0125] The nucleotide sequence of truncated form 1#:1H-290R (GPIbα-1#) is shown in SEQ ID NO:1, the amino acid sequence is shown in SEQ ID NO:2, the amino acid sequence containing the G233K mutation is shown in SEQ ID NO:17, and the corresponding coding sequence is shown in SEQ ID NO:16. The nucleotide sequence of truncated form 2#:265P-414P (GPIbα-2#) is shown in SEQ ID NO:3, and the amino acid sequence is shown in SEQ ID NO:4. The nucleotide sequence of truncated 3#:265P-509F (GPIbα-3#) is shown in SEQ ID NO:5, and the amino acid sequence is shown in SEQ ID NO:6. The nucleotide sequence of truncated 4#:1H-509F (GPIbα-4#) is shown in SEQ ID NO:7, the amino acid sequence is shown in SEQ ID NO:8, the amino acid sequence containing the G233K mutation is shown in SEQ ID NO:19, and the corresponding coding sequence is shown in SEQ ID NO:18. The nucleotide sequence of truncated 5#:1H-414P (GPIbα-5#) is shown in SEQ ID NO:9, the amino acid sequence is shown in SEQ ID NO:10, the amino acid sequence containing the G233K mutation is shown in SEQ ID NO:21, and the corresponding coding sequence is shown in SEQ ID NO:20. EXAMPLES

[0126] Construction and screening of GpIbα-CAAR lentiviral vectors with different truncations (1) Synthesis of second-generation CAR structural gene Nucleotide fragments of the CAR structural gene encoding the intracellular segment including the CD8 hinge and transmembrane domain, as well as the 4-1BB costimulatory domain and the CD3ζ signal domain were obtained by PCR. The second-generation CAR structural gene and the previously screened truncated gene fragments against different antigen epitopes of GpIbα were spliced ​​by overlap PCR to obtain the GpIbα-CAAR sequence. At present, five different truncated GpIbα-CAAR fragments, 1# to 5#, have been successfully constructed as follows:

[0127] The amino acid sequence of CAAR corresponding to CAAR1#:1H-290R is shown in sequence number 11, in which positions 1 to 290 are the antibody recognition sequence of GpIbα, a G233K mutation occurs at position 233 of sequence number 11, positions 291 to 335 are the CD8 hinge, positions 336 to 359 are the CD8 transmembrane region, positions 360 to 401 are 4-1BB, and positions 402 to 513 are the CD3ζ signal domain. The amino acid sequence of CAAR corresponding to CAAR2#:265P-414P is shown in sequence number 12, in which positions 1 to 150 are the antibody recognition sequence (265P-414P sequence), and positions 151 to 373 are the CD8 hinge, CD8 transmembrane region, 4-1BB, and CD3ζ signal domain. The amino acid sequence of CAAR corresponding to CAAR3#:265P-509F is shown in sequence number 13, in which positions 1 to 245 are the antibody recognition sequence (265P-509F sequence), and positions 246 to 468 are the CD8 hinge, CD8 transmembrane region, 4-1BB, and CD3ζ signal domain. The amino acid sequence of CAAR corresponding to CAAR4#:1H-509F is shown in sequence number 14, in which positions 1 to 509 are the antibody recognition sequence (1H-509F sequence), a G233K mutation occurs at position 233 of sequence number 14, and positions 510 to 732 are the CD8 hinge, CD8 transmembrane region, 4-1BB, and CD3ζ signal domain. The amino acid sequence of CAAR corresponding to CAAR5#:1H-414P is shown in sequence number 15, in which positions 1 to 414 are the antibody recognition sequence (1H-414P sequence), a G233K mutation occurs at position 233 of sequence number 15, and positions 415 to 637 are the CD8 hinge, CD8 transmembrane region, 4-1BB, and CD3ζ signal domain. (2) Construction of Pre-Lenti-EF1-GpIbα-CAAR lentiviral vector The GpIbα-CAAR gene sequence was analyzed, the enzyme cleavage site was selected and matched with the polyclonal site of the lentiviral vector Pre-Lenti-EF1-MCS. After enzyme cleavage, recombination, transformation, clone selection, bacterial liquid amplification, plasmid extraction and sequencing, the Pre-Lenti-EF1-GpIbα-CAAR lentiviral vector was constructed by homologous recombination.

[0128] (3) Packaging and titer detection of GpIbα-CAAR lentivirus 293T cells were transfected with GpIbα-CAAR:PsPAX2:PMD2G in a ratio of 3:2:1, and the viral supernatant was collected 48 and 72 hours later. The virus was concentrated with PEG8000, and an appropriate amount of the viral concentrate was taken, the viral titer was measured using a Takara lentivirus kit, and the virus was frozen and stored at -80°C. EXAMPLES

[0129] Preparation of GpIbα-CAAR-T cells Human peripheral blood PBMC cells were obtained using Ficoll density gradient centrifugation. Then, T cells were obtained from PBMCs using immunomagnetic bead selection. The obtained T cells were added to a T75 culture flask coated with 5 μg / mL CD3 and CD28 antibodies, and 1000 U / mL IL2 and 100 U / mL IFN-γ were added to activate the T cells. Two days later, GpIbα-CAAR virus solutions containing different truncations were added to the activated T cells for virus packaging. After 48 h to 72 h of culture, the virus solution was removed, and the virus infection efficiency was detected by flow cytometry to prepare GpIbα-CAAR-T cells. In addition, a portion of the activated T cells was taken and cultured and grown normally without transfection with CAAR to serve as negative control T cells. EXAMPLES

[0130] Construction of GpIbα-CAAR-T cells carrying a mutation in the binding domain of von Willebrand Factor (vWF) (1) Construction of GpIbα-CAAR plasmid carrying a point mutation in the vWF binding domain To reduce possible off-target effects after injecting GpIbα-CAAR-T cells into the body, we performed a point mutation in the vWF-binding region of GpIbα based on the successfully designed GPIbα-CAAR plasmid, and constructed a GpIbα-CAAR-G233K mutant plasmid by mutating glycine (G) at position 233, where the GpIbα epitope binds to the vWF-A1 functional domain, to lysine (K).

[0131] (2) Detection of the binding ability of the GpIbα-CAAR-G233K mutant to WF The constructed GpIbα-CAAR-G233K mutant plasmid was lentivirally infected into Jurkat cells to generate a stable transfected cell line. Jurkat cells stably expressing CAAR1, CAAR4, and CAAR5 mutants were allowed to interact with human vWF under the action of ristocetin, and the cell aggregation ability was detected (the measurement result was reflected according to the light transmittance).

[0132] The results, as shown in Figure 1, showed that in the presence of vWF, the aggregation levels of Jurkat cells expressing CAAR1, CAAR4, and CAAR5 mutants were significantly reduced (light transmittance was reduced) compared to Jurkat cells without point mutations, indicating that the ability of Jurkat cells stably expressing CAAR1, CAAR4, and CAAR5 mutants to bind to human vWF was clearly reduced. Therefore, expression of the GpIbα-G233K mutant can reduce the off-target effects of CAAR-T cells in vivo after infusion, and the present invention successfully prepared a series of CAAR-T cells carrying mutants with mutations in the vWF binding region. EXAMPLES

[0133] Preparation of anti-human GpIbα monoclonal hybridoma cells (1) Immunization of BALB / c mice with human GpIbα protein Human-derived GpIbα protein was injected into BALB / c mice via the footpad to immunize them. The first injection of immunization of the animals was made by emulsifying GpIbα antigen in complete Freund's adjuvant, and the second or third injection was made by using incomplete Freund's adjuvant or no adjuvant, and the antigen emulsion prepared with a syringe was pushed into the mouse's footpad from one side. In general, each mouse was injected with 50-100 μg of protein antigen per injection, with the antigen volume not exceeding 300 μL per injection, and spleen cells were harvested and fused on the third day after the last booster immunization.

[0134] (2) Preparation of hybridoma cells by cell fusion Splenic B cells from mice immunized three times were fused with myeloma cells SP2 / 0 using PEG-1500, and hybridoma cells were screened using HAT medium. Successfully fused cells were able to grow in HAT medium, but unsuccessful B cells died within 5 to 7 days.

[0135] (3) Screening of monoclonal hybridoma cells The surviving hybridoma cells in HAT medium were counted and the concentration was adjusted to about 200 cells / mL. 4 mL of semi-solid medium was plated in a 6-well plate, and about 500 μL of hybridoma cell suspension was added to each well. After 7 days of culture, a single clone was selected and transferred to a new 6-well plate for continued culture, allowing the cells to proliferate gradually.

[0136] (4) Selection of anti-human GpIbα positive monoclonal hybridoma cells Monoclonal hybridomas were isolated, and the binding between the hybridoma culture supernatant and GpIbα protein was detected using the Elisa method, and hybridoma cells with positive Elisa results were selected. The hybridoma cells produced anti-human GpIbα antibody (anti-human platelet antibody).

[0137] (5) Verification of binding of GpIbα-CAAR-T cells to anti-GpIbα-positive hybridoma cells GPIbα-CAAR-T cells carrying different truncations designed as described above were co-incubated with the obtained anti-GPIbα-positive hybridoma cells, and the level of binding with the antibodies was evaluated by flow cytometry to detect the mutual binding ability between GpIbα-CAAR-T cells and the antibodies in the culture supernatant of monoclonal hybridoma cells carrying different antigen epitopes.

[0138] The experimental results are shown in Figure 2. The different CAAR-T cells designed by the present inventors can selectively bind to anti-GpIbα positive hybridoma cells (Gvb1-Gvb4) with different antigen epitopes, and the binding abilities are also different. Among them, CAAR2-T cells and CAAR3-T cells have the ability to bind to Gvb4 hybridoma cells, CAAR4-T cells and CAAR5-T cells can both strongly bind to the four hybridoma cells Gvb1-Gvb4, and the G233K point mutation did not affect the specific binding of CAAR-T cells to anti-GpIbα positive hybridoma cells, so all of the different CAAR-T cells containing the G233K point mutation retained the ability to recognize target B cells, among which CAAR1-T cells had the strongest recognition ability. EXAMPLES

[0139] In vitro killing effect of GpIbα-CAAR-T cells against anti-GpIbα-positive hybridoma cells (1) Competitive killing level experiment CellTracker TM Deep Red Dye and CellTracker TM Red CMTPX Dye was used to label anti-GpIbα positive hybridoma cells and negative control hybridoma cells, respectively. The two were mixed at a certain ratio and GpIbα-CAAR-T cells were added. After co-culture of the three for 16 hours, the proportion of the two hybridoma cells with different fluorescent labels was detected using flow cytometry, which reflected the killing effect of CAAR-T cells on target cells.

[0140] The results are shown in Figure 3. CAAR1-T~CAAR5-T (corresponding to 1#~5#CAAR-T, respectively) cells can selectively kill positive hybridoma cells with different antigen epitopes, and reduce the proportion of positive hybridoma cells to different degrees. Among them, CAAR2-T cells and CAAR3-T cells have killing effect on Gvb4 hybridoma cells, CAAR4-T cells and CAAR5-T cells have strong killing effect on Gvb1 and Gvb2 hybridoma cells, and CAAR1-T cells have strong competitive killing effect on all four positive hybridoma cells of Gvb1~Gvb4, which was statistically significant compared with the blank control group. This indicates that CAAR-T cells with different antigen epitopes can specifically recognize and kill anti-GpIbα positive target cells, and among them, CAAR1-T cells have strong competitive killing effect on all target cells.

[0141] (2) Detection of LDH release levels To evaluate the specific cytotoxicity of GpIbα-CAAR-T cells, we examined the release of LDH after co-culture of CAAR-T cells containing different antigen epitopes with anti-GpIbα hybridoma cells for 16 h.

[0142] As shown in Figure 4, the constructed CAAR-T cells could well recognize positive hybridoma cells producing antibodies against different antigen epitopes of GpIbα and had a certain degree of killing effect. Among them, CAAR1-T cells had killing effect against all four positive hybridoma cells Gvb1 to Gvb4 at different ET ratios, and the cytolytic effect was stronger than that of CAAR4-T cells and CAAR5-T cells.

[0143] (3) Detection of cytokine release levels GpIbα-CAAR-T cells and anti-GpIbα-positive hybridoma cells were mixed at a certain ratio and co-cultured for 24 h, after which the levels of IL12 and IFN-γ released in the supernatant samples were detected using Elisa, which reflected the killing ability of CAAR-T cells against target cells.

[0144] As shown in Figure 5, the GpIbα-CAAR-T cells were able to recognize anti-GpIbα positive target cells well and had a certain degree of killing effect against them. Among them, CAAR1-T cells had a killing effect against all four positive hybridoma cells, Gvb1 to Gvb4, and the release levels of IL-2 and IFN-γ were significantly higher than those of CAAR4-T cells and CAAR5-T cells. CAAR1-T cells have stronger cytotoxicity against target cells than other CAAR-T cells, suggesting that they can target and eliminate reactive B cells in vivo, thereby reducing the production of antiplatelet antibodies and free antibodies. EXAMPLES

[0145] Construction of a humanized ITP mouse model and in vivo validation We constructed an ITP hybridoma animal model using humanized NSG mice. Specifically, 6-week-old NSG (NOD-Prkdcscid il-2rgtm1 / Bcgen) immunodeficient mice were irradiated to destroy the bone marrow, and then human umbilical cord blood or fetal liver hematopoietic stem cell (HSC) bone marrow was transplanted into the irradiated mice to continuously produce various types of human-derived hematopoietic or immune cells, thereby completing the humanization of the mice. Then, 1 × 10 6 Anti-GpIbα positive hybridoma cells were injected intraperitoneally. The survival status of the mice was observed daily, and changes in the platelet count, PA Abs level in peripheral blood, megakaryocyte count in bone marrow, and coagulation function of the mice were regularly detected, and the changes in these indicators were used to determine whether the mouse ITP animal model was successfully established.

[0146] A certain number of GPIbα-CAAR-T cells were selectively injected into the tail vein of the successfully constructed ITP hybridoma mice, and the killing effect of CAAR-T cells with different cleavage forms on hybridoma cells in vivo was comprehensively determined by detecting the titer of anti-GPIbα autoantibodies, platelet count, coagulation index changes in the mice in vivo, and it was found that CAAR-T cells have an improving effect on the ITP disease model induced in mice. According to the results in Figure 6, on the 14th day, the NTD mice in the control group already showed significant Gvb1-GFP amplification and metastasis, while the mice in the experimental group injected with CAAR-T cells had significantly lower Gvb1-GFP cell amplification and metastasis than the control group. On the 21st day, the NTD mice in the control group already showed significant Gvb1-GFP amplification and metastasis, while the mice in the experimental group injected with CAAR-T cells had significantly lower Gvb1-GFP cell amplification and metastasis than the control group, indicating the obvious killing effect of CAAR-T cells on Gvb1 cells. EXAMPLES

[0147] Selection and application of GpIbα-CAAR-T cells (1) Collection, isolation and activation of T cells A fixed number of T cells were obtained from a volunteer subject using Ficoll separation or cell apheresis, the T cells were washed and activated in an ultraclean environment, and the T cells were cultured in a medium containing the cytokines necessary for survival and proliferation. The most commonly used antibodies to activate T cells in vitro were CD3 and CD28.

[0148] (2) Construction and selection of GpIbα-CAAR-T cells containing different truncations Common methods for transducing GpIbα-CAAR gene fragments containing different truncations into activated T cells include lentiviral vectors, retroviral vectors, transposons, and mRNA electroporation techniques. Based on the types of autoantibodies of ITP patients previously identified, the inventors specifically selected GpIbα-CAAR-T cell therapy containing different truncation fragments.

[0149] (3) In vitro expansion and culture of GpIbα-CAAR-T cells Typically, billions to tens of billions of engineered CAAR-T cells are needed to stimulate and culture the cells in vitro to a certain number, perform cell quality control, and inject them intravenously into the body. Figure 7 shows that GpIbα-CAAR gene fragments containing different truncations were infected into activated T cells using a lentiviral vector and cultured for a certain period of time. The results of flow cytometry detection showed that all of the GpIbα-CAAR lentiviruses containing different truncation fragments could infect human T cells at a certain rate, and that GpIbα-CAAR-T cells containing different truncations could be constructed.

[0150] The above examples only represent some embodiments of the present invention, and although the descriptions thereof are relatively specific and detailed, they should not be construed as limiting the patent scope of the present invention. It should be noted that those skilled in the art may make some modifications and improvements without departing from the idea of ​​the present invention, all of which are within the scope of protection of the present invention. Therefore, the scope of protection of the patent of the present invention should be determined by the appended claims. At the same time, all documents related to the present invention are incorporated by reference in this application as if each document was incorporated by itself.

Claims

1. (a) the preparation of a competing antigen that binds to an anti-platelet antibody or a construct or composition comprising said antigen; (b) the preparation of a construct or composition that targets and eliminates reactive B lymphocytes; (c) the preparation of a construct or composition that elevates platelets; and (d) the use of the platelet surface antigen GpIbα, its truncations, its variants, or the polynucleotides encoding them, in the preparation of a construct or composition that alleviates or treats immune thrombocytopenic purpura.

2. A truncated form or mutant form of the platelet surface antigen GpIbα, or a polynucleotide encoding the same, wherein the truncated form or mutant form of GpIbα is derived from the extracellular domain of the GpIbα protein and has a binding site for an in vivo platelet autoimmune antibody; more preferably, the truncated form or mutant form is characterized in that it comprises a protein having an amino acid sequence shown in one or more of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10, SEQ ID NO:17, SEQ ID NO:19 or SEQ ID NO:

21.

3. A chimeric autoantibody receptor comprising a truncated or mutant form of the platelet surface antigen GpIbα described in claim 2 as an extracellular binding region, a transmembrane domain, and an intracellular signaling domain; preferably, the intracellular signaling domain comprises a costimulatory molecule and / or a signal transduction domain.

4. The transmembrane domain comprises the hinge region and transmembrane region of CD8 or CD28; preferably, the transmembrane domain comprises the hinge region and transmembrane region of CD8; more preferably, the hinge region has the amino acid sequence shown in positions 291 to 335 of SEQ ID NO: 11, and the transmembrane domain has the amino acid sequence shown in positions 336 to 359 of SEQ ID NO: 11; or The chimeric autoantibody receptor according to claim 3, wherein the intracellular signal domain comprises an intracellular signal region selected from 4-1BB, CD3ζ, FcεRIγ, CD27, CD28, CD134, ICOS, or GITR; preferably, the intracellular signal region comprises 4-1BB and CD3ζ; more preferably, the 4-1BB has an amino acid sequence represented by positions 360 to 401 of SEQ ID NO:11, and the CD3ζ has an amino acid sequence represented by positions 402 to 513 of SEQ ID NO:

11.

5. The chimeric autoantibody receptor according to claim 3, characterized in that the chimeric autoantibody receptor comprises a structure in which a truncated form or mutant form of the platelet surface antigen GpIbα described in claim 2 or 3, a hinge region and a transmembrane region of CD8 or CD28, 4-1BB, and CD3ζ are linked in this order; preferably, the chimeric autoantibody receptor has an amino acid sequence as shown in any one of SEQ ID NOs: 11 to 15.

6. (i) a polynucleotide encoding the chimeric autoantibody receptor of claim 3; or (ii) An expression construct comprising a polynucleotide encoding a truncated form or mutant form of the platelet surface antigen GpIbα according to claim 2.

7. An immune effector cell into which the polynucleotide of the chimeric autoantibody receptor of claim 3 or the expression construct (i) of claim 6 has been introduced; or which expresses the chimeric autoantibody receptor of claim 3; Preferably, the immune effector cell comprises a cell selected from the group consisting of a T cell, a natural killer cell, a NKT cell, or a cytokine-induced killer cell, or a combination thereof; more preferably, the immune effector cell is a T cell; more preferably, the T cell is an immune effector cell comprising a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, a γδ T cell, a T memory stem cell, or a T cell derived from a pluripotent stem cell.

8. 10. Use of the immune effector cells of claim 7 in: (a) preparing a composition that neutralizes anti-platelet antibodies or free antibodies; (b) preparing a composition that targets and eliminates reactive B lymphocytes; (c) preparing a composition that elevates platelets; and (d) preparing a composition that alleviates or treats immune thrombocytopenic purpura.

9. A pharmaceutical composition or drug box comprising the immune effector cells of claim 7 for (a) neutralizing anti-platelet antibodies or free antibodies, (b) targeting and eliminating reactive B lymphocytes, (c) elevating platelets, and (d) alleviating or treating immune thrombocytopenic purpura.

10. 11. A method for (a) neutralizing anti-platelet antibodies or free antibodies, (b) targeting and depleting reactive B lymphocytes, (c) elevating platelets, or (d) alleviating or treating immune thrombocytopenic purpura, the method comprising administering to a subject an effective amount of an immune effector cell of claim 7.

11. 10. A method for (a) neutralizing anti-platelet antibodies or free antibodies, (b) targeting and depleting reactive B lymphocytes, (c) elevating platelets, or (d) alleviating or treating immune thrombocytopenic purpura, said method comprising administering to a subject an effective amount of the pharmaceutical composition of claim 9.

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