An anti-beta2gpi antibody or antigen-binding fragment thereof, and a preparation method and application thereof

Abstract

This disclosure belongs to the field of biotechnology, specifically relating to an anti-β2GPI antibody or its antigen-binding fragment, its preparation method and application. The anti-β2GPI antibody or its antigen-binding fragment binds to the β2GPI-D1 epitope and can effectively activate β2GPI-CAAR-T cells and enhance their effector molecule secretion and killing function.

Description

Technical Field

[0001] This disclosure relates to the field of biomedical technology, specifically to an anti-β2GPI antibody or its antigen-binding fragment, its preparation method, and its application. Background Technology

[0002] Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterized by arterial and venous thrombosis and / or adverse pregnancy outcomes. Its core immunological feature is the presence of antiphospholipid antibodies (aPL), which can induce vascular endothelial damage, abnormal activation of the coagulation cascade, and amplified inflammatory responses. Numerous studies have shown that β2-glycoprotein I (β2GPI) is one of the most important autoantigens in APS, and anti-β2GPI antibodies, especially those recognizing β2GPI domain 1 (D1), are closely related to the thrombotic risk and severity of APS.

[0003] Currently, clinical treatment for APS (autoimmune diseases) still primarily relies on anticoagulation (such as warfarin and heparin). However, anticoagulation therapy usually requires long-term maintenance, which carries risks such as bleeding, significant individual variability, and a high risk of relapse in some patients. Simultaneously, traditional immunosuppressive therapy struggles to precisely eliminate pathogenic autoreactive B cell clones, failing to fundamentally block the continuous production of pathogenic antibodies. In recent years, immunotherapy based on chimeric antigen receptor (CAR) T cells has shown promise in the field of autoimmune diseases. Among these, chimeric autoantibody receptor T cells (CAAR-T) selectively recognize and eliminate autoreactive B cells expressing specific B cell receptors (BCRs) by displaying autoantigen fragments on their surface, achieving the therapeutic goal of "precise elimination by clone." However, CAAR-T therapy targeting β2GPI still faces key technological requirements. For example, there is a lack of stable and reproducible functional anti-β2GPI antibodies as tool molecules to verify the effectiveness of CAAR-T recognition, activation, and killing. At the same time, full-length IgG antibodies may pose potential risks through Fc-mediated effects or coagulation interference, affecting their safe application in scenarios of functional enhancement or bridging activation.

[0004] Therefore, there is an urgent need to provide an anti-β2GPI antibody that binds to key β2GPI epitopes and has clear pathogenic functional characteristics, thereby providing a reliable standardized tool for the construction and in vitro / in vivo evaluation of β2GPI-CAAR-T cells, and providing new candidate molecules and application pathways for the precision immunotherapy of APS. Summary of the Invention

[0005] To address one of the aforementioned technical problems in the prior art, this disclosure provides an anti-β2GPI antibody or its antigen-binding fragment, its preparation method, and its application.

[0006] A first aspect of this disclosure provides an anti-β2 glycoprotein I (β2GPI) antibody or an antigen-binding fragment thereof, said anti-β2GPI antibody or antigen-binding fragment comprising: a1) having HCDR1, HCDR2, and HCDR3 included in the heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 1; and / or having LCDR1, LCDR2, and LCDR3 included in the light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 2; or a2) Having one or more amino acid substitutions, deletions or additions compared to HCDR1, HCDR2 and HCDR3 as shown in a1); and / or having one or more amino acid substitutions, deletions or additions compared to LCDR1, LCDR2 and LCDR3 as shown in a1). The CDR is defined according to the Kabat, Chothia, IMGT, Contact, or AbM numbering system.

[0007] In some embodiments, the anti-β2GPI antibody or its antigen-binding fragment comprises: b1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 3, HCDR2 having the amino acid sequence shown in SEQ ID NO: 4, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 5; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 6, LCDR2 having the amino acid sequence shown in SEQ ID NO: 7, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 8; or b2) VH including the following three CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in b1); and / or VL including the following three CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in b1); Wherein, the CDR is defined according to the Kabat numbering system; or The anti-β2GPI antibody or its antigen-binding fragment includes: c1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 9, HCDR2 having the amino acid sequence shown in SEQ ID NO: 10, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 11; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 12, LCDR2 having the amino acid sequence shown in SEQ ID NO: 13, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 14; or c2) VH including the following three CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in c1); and / or VL including the following three CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in c1); Wherein, the CDR is defined according to the Chothia numbering system; or The anti-β2GPI antibody or its antigen-binding fragment includes: d1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 15, HCDR2 having the amino acid sequence shown in SEQ ID NO: 16, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 17; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 18, LCDR2 having the amino acid sequence shown in SEQ ID NO: 19, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 20; or d2) VH including the following 3 CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in d1); and / or VL including the following 3 CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in d1); Wherein, the CDR is defined according to the IMGT numbering system; or The anti-β2GPI antibody or its antigen-binding fragment includes: e1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 21, HCDR2 having the amino acid sequence shown in SEQ ID NO: 22, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 23; and / or a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 24, LCDR2 having the amino acid sequence shown in SEQ ID NO: 25, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 26; or e2) VH including the following three CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in e1); and / or VL including the following three CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in e1); Wherein, the CDR is defined according to the Contact numbering system; or The anti-β2GPI antibody or its antigen-binding fragment includes: f1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 27, HCDR2 having the amino acid sequence shown in SEQ ID NO: 28, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 29; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 30, LCDR2 having the amino acid sequence shown in SEQ ID NO: 31, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 32; or f2) VH including the following three CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in f1); and / or VL including the following three CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in f1); The CDR is defined according to the AbM numbering system.

[0008] Those skilled in the art should understand that the above-mentioned amino acid substitutions are conservative substitutions.

[0009] In some embodiments, the heavy chain variable region of the anti-β2GPI antibody or its antigen-binding fragment further includes a framework region of the heavy chain variable region.

[0010] In some embodiments, the framework region of the heavy chain variable region includes the framework region of the heavy chain variable region of immunoglobulin derived from mouse, primate, cow, horse, pig, sheep, goat, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose or a mutant thereof; more preferably, it includes the framework region of the heavy chain variable region of immunoglobulin derived from mouse or a mutant thereof.

[0011] In some embodiments, the light chain variable region of the anti-β2GPI antibody or its antigen-binding fragment further includes a framework region of the light chain variable region.

[0012] In some embodiments, the framework region of the light chain variable region includes the framework region of the light chain variable region of immunoglobulin derived from mouse, primate, cow, horse, pig, sheep, goat, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose, or a mutant thereof; more preferably, it includes the framework region of the light chain variable region of immunoglobulin derived from mouse, or a mutant thereof.

[0013] In some embodiments, the anti-β2GPI antibody or its antigen-binding fragment comprises: The heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and / or the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0014] In some embodiments, the anti-β2GPI antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region.

[0015] In some embodiments, the heavy chain constant region includes at least a portion of the heavy chain constant region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; more preferably, it includes at least a portion of the heavy chain constant region or a mutant thereof derived from mouse immunoglobulins.

[0016] In some embodiments, according to the EU numbering system, the heavy chain constant region includes mutations at one or more sites of positions 234, 235, 265, 297, and 329, more preferably including one or more mutations selected from L234A, L235A, D265A, N297G / N297A, and P329G.

[0017] In some implementations, the heavy chain constant region does not include the CH2 region.

[0018] In some embodiments, the light chain constant region includes at least a portion of the light chain constant region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; more preferably, it includes the light chain constant region or a mutant thereof derived from mouse immunoglobulins.

[0019] In some embodiments, the heavy chain constant region includes a heavy chain constant region derived from IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 or IgM immunoglobulin; more preferably, it includes a heavy chain constant region derived from IgG1 immunoglobulin.

[0020] In some embodiments, the light chain constant region includes light chain constant regions derived from κ and λ immunoglobulins.

[0021] In some embodiments, the anti-β2GPI antibody or its antigen-binding fragment may be a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody; more specifically, it may be a murine antibody.

[0022] In some embodiments, the anti-β2GPI antibody or its antigen-binding fragment may include, but is not limited to, monoclonal antibodies, bispecific antibodies, multispecific antibodies, nanobodies, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fv fragments, single-chain Fv (scFv), dsFv, or Fd fragments.

[0023] In some embodiments, the anti-β2GPI antibody or its antigen-binding fragment comprises: g1) Heavy chain, comprising the amino acid sequence shown in SEQ ID NO: 33, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and / or, light chain, comprising the amino acid sequence shown in SEQ ID NO: 34, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; or g2) Heavy chain, comprising the amino acid sequence shown in SEQ ID NO: 35, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and / or, light chain, comprising the amino acid sequence shown in SEQ ID NO: 36, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0024] In some embodiments, the anti-β2GPI antibody or its antigen-binding fragment specifically binds to domain 1 of the β2GPI protein.

[0025] A second aspect of this disclosure provides biological materials related to the antibody or antigen-binding fragment thereof of the first aspect of this disclosure, said biological material comprising any one of n1)-n9): n1) A nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof of the first aspect of this disclosure; n2) contains an expression cassette containing the nucleic acid molecule described in n1); n3) A carrier containing the nucleic acid molecule described in n1); n4) A carrier containing the expression box described in n2); n5) A cell containing the nucleic acid molecules described in n1); n6) Cells containing the expression cassette described in n2); n7) Cells containing the carrier described in n3); n8) contains cells containing the carrier described in n4); n9) Cells containing an antibody or an antigen-binding fragment thereof of the first aspect of this disclosure; None of the cells described in n5)-n9) contain reproductive material.

[0026] Those skilled in the art will understand that nucleotides in nucleic acid molecules can be substituted based on codon degeneracy. In some embodiments, the nucleotide sequence of the nucleic acid molecule is codon-optimized.

[0027] In some embodiments, the nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the first aspect of the present disclosure comprises a nucleic acid molecule encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof of the first aspect of the present disclosure and a nucleic acid molecule encoding the light chain variable region of the antibody or antigen-binding fragment thereof of the first aspect of the present disclosure.

[0028] In some embodiments, the nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the first aspect of the present disclosure comprises a nucleic acid molecule of a heavy chain encoding the antibody or antigen-binding fragment thereof of the first aspect of the present disclosure and a nucleic acid molecule of a light chain encoding the antibody or antigen-binding fragment thereof of the first aspect of the present disclosure.

[0029] In some embodiments, any of the vectors n3)-n4) can be expression vectors. In some embodiments, the expression vector may include eukaryotic expression vectors and / or prokaryotic expression vectors. In some embodiments, the eukaryotic expression vector includes, for example, but not limited to, yeast expression vectors, mammalian expression vectors, and insect expression vectors. For example, the expression vector may include, but is not limited to, plasmids, retroviral vectors, lentiviral vectors, bacteriophage vectors, adenovirus vectors, adeno-associated vectors, or herpes simplex vectors.

[0030] In some embodiments, the carrier may be selected from nanoparticles, liposomes, exogenous bodies, microbubbles, or gene guns.

[0031] In some embodiments, any of the cells (n5)-n9) can be host cells conventionally used in the art, provided that the expression vector stably expresses the carried nucleic acid molecule as the antibody or antigen-binding fragment of the present disclosure. In some embodiments, the host cell can be a prokaryotic cell and / or a eukaryotic cell. The prokaryotic cell may include, for example, *Escherichia coli*, and the eukaryotic cell may include, for example, CHO cells, HEK293 cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, HeLa cells, Vero cells, Expi293 cells, hybridoma cells, yeast cells, and insect cells.

[0032] A third aspect of this disclosure provides a method for preparing an antibody or antigen-binding fragment thereof according to the first aspect of this disclosure, the method comprising: culturing the cells of the third aspect of this disclosure, and collecting the antibody or antigen-binding fragment thereof from the cultured cell culture.

[0033] A fourth aspect of this disclosure provides a conjugate comprising an antibody or an antigen-binding fragment thereof of the first aspect of this disclosure; and a conjugation portion.

[0034] In some implementations, the coupling portion may include, but is not limited to, a detectable marker or a therapeutic agent.

[0035] In some embodiments, the detectable marker can be any substance detectable by means of fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, chemical, etc. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridine esters, magnetic beads, calorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) microbeads, and biotin for binding avidin (e.g., streptavidin) modified with the above markers. In some embodiments, such markers are suitable for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable marker is selected from radioactive isotopes, fluorescent substances, luminescent substances, colored substances, or enzymes. In some embodiments, the detectable markers described above can be linked to the antibodies or antigen-binding fragments of this disclosure using linkers of different lengths to reduce potential steric hindrance.

[0036] In some embodiments, the detectable marker may include, but is not limited to, enzymes (e.g., horseradish peroxidase), radionuclides, fluorescent dyes, luminescent substances (e.g., chemiluminescent substances), colored substances, biotin, etc.

[0037] In some embodiments, the therapeutic agent may include, for example, but not limited to, drugs for the prevention and / or treatment of β2GPI-related diseases or symptoms.

[0038] In some embodiments, the coupling portion is selected from substances that can improve the biological properties of the antibody (e.g., increase serum half-life), such as chemical groups, such as polyethylene glycol (PEG), methyl, ethyl, or glycosyl groups.

[0039] A fifth aspect of this disclosure provides a pharmaceutical composition comprising: an antibody or antigen-binding fragment thereof of the first aspect of this disclosure, a biological material of the second aspect or a conjugate of the fourth aspect; and a pharmaceutically acceptable carrier.

[0040] In some embodiments, the pharmaceutical composition may also include additional pharmaceutically active agents.

[0041] In some embodiments, the additional pharmaceutically active agent may be a biologically active drug, such as a drug capable of preventing and / or treating β2GPI-related diseases or symptoms.

[0042] In some embodiments, the antibody or its antigen-binding fragment is provided as a separate component or as a mixed component with the additional pharmaceutically active agent.

[0043] In some embodiments, the pharmaceutical composition further includes any one of A1)-A6): A1) Chimeric autoantibody receptor (CAAR), the chimeric autoantibody receptor comprising an extracellular domain, and optionally a hinge region, a transmembrane domain, a co-stimulatory intracellular domain and / or a signaling domain, wherein the extracellular domain is a β2GPI domain. A2) encodes the polynucleotide of the chimeric autoantibody receptor described in A1); A3) A carrier containing the polynucleotides described in A2); A4) Cells containing the carrier described in A3); A5) Genetically modified cells, said genetically modified cells comprising the chimeric autoantibody receptor described in A1); A6) Genetically modified T cells, wherein the variable region of the TCR of the genetically modified T cells contains a β2GPI domain, and the β2GPI domain comprises the amino acid sequence shown in SEQ ID NO.40 or SEQ ID NO.41, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0044] In some embodiments, the β2GPI domain described in A1) includes any of the amino acid sequences shown in SEQ ID NO.37 to SEQ ID NO.41, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0045] In some embodiments, the hinge region in A1) is selected from any one of the CD8α hinge region, the CD28 extracellular hinge region, and the (Gly-Ser) repeat linker peptide; the transmembrane domain is selected from any one of the CD8α transmembrane domain and the CD28 transmembrane domain; the co-stimulatory intracellular domain is a domain of a co-stimulatory molecule; the co-stimulatory molecule is selected from any one of MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137); and / or, the signaling domain is a CD3ζ signaling domain.

[0046] In some embodiments, the hinge region described in A1) is the extracellular hinge region of human CD28, comprising the amino acid sequence shown in SEQ ID No. 42, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; the transmembrane domain is the human CD28 transmembrane domain, comprising the amino acid sequence shown in SEQ ID No. 43, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; the co-stimulatory intracellular domain is the human 4-1BB intracellular co-stimulatory domain, comprising the amino acid sequence shown in SEQ ID No. 43. The amino acid sequence shown in SEQ ID No. 44, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and / or, the signal domain is a CD3ζ signal domain comprising the amino acid sequence shown in SEQ ID No. 45, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0047] In some embodiments, the chimeric autoantibody receptor described in A1) comprises a β2GPI domain, a hinge region, a transmembrane domain, a co-stimulatory intracellular domain, and a signaling domain sequentially linked together.

[0048] In some embodiments, the polynucleotide described in A2) may be DNA or RNA (e.g., mRNA).

[0049] In some embodiments, the vector described in A3) is an expression vector containing a polynucleotide encoding the aforementioned CAAR. The vector can be a lentiviral vector carrying a constitutive promoter such as EF-1α to drive CAAR expression for long-term, stable T-cell expression; it can also be an RNA vector or use electroporation of mRNA for short-term CAAR expression, suitable for applications with higher safety requirements or requiring short-term intervention; it can also be a transposon system (such as Sleeping Beauty, PiggyBac) for stable integration; or it can be other vectors such as retroviruses, adenoviruses, or AAVs. According to this disclosure, in addition to the CAAR coding sequence, the vector may also contain: a nucleotide sequence encoding a selection marker gene (such as a drug resistance gene or a fluorescent protein gene) for screening transduced positive cells; a nucleotide sequence encoding a reporter gene (such as GFP or luciferase) for monitoring expression and signaling pathway activation; an inducible promoter or a synthetic Notch (synNotch) system to control CAAR expression with exogenous small molecules or specific ligands, thereby achieving "on / off" safety control; and / or a nucleotide sequence encoding a signal peptide to guide newly synthesized CAAR protein into the secretory pathway, enabling it to reach the cell membrane surface. Signal peptide sequences known in the art can be used, such as human IgG signal peptide, CD8α signal peptide, CD116 (also known as GM-CSFRα) leader sequence, etc. One embodiment of this disclosure is a CD116 (GM-CSFRα) leading sequence, which comprises an amino acid sequence as shown in SEQ ID No. 46, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0050] In some embodiments, the cells in A5) are selected from: helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, γδ T cells, natural killer cells, cytokine-induced killer cells, T memory stem cells, T cells differentiated from pluripotent stem cells, and T cell lines used for screening and in vitro validation. According to this disclosure, the cells can be derived from the patient themselves, from other donors of the same species, or from cells from other species. Cells derived from the patient themselves, after genetic modification, will not cause rejection when reinfused into the patient, exhibiting high safety. If cells derived from other donors are used and infused into the patient after genetic modification, graft-versus-host disease (GVHD) may occur. Therefore, if cells derived from other donors are used, to avoid rejection of the host cells, it is preferable to interfere with the function of the TCR complex of the cells. This can be done using various methods known in the art, including but not limited to knocking out the T cell receptor (TCR) complex in the cells, knocking out the T cell receptor α constant region protein in the cells, and / or knocking out the T cell receptor β constant region protein in the cells, etc.

[0051] The genetically modified cells in A5) can bind to the antibody or its antigen-binding fragment from the first aspect. Upon binding to the antibody or its antigen-binding fragment from the first aspect, the genetically modified cells are activated, exhibiting CD25 activation in the cells. + CD69 + The proportion of cells with this gene is increased. Upon binding to the antibody or its antigen-binding fragment from the first aspect, the genetically modified cells are activated and produce effector functions, manifested by increased secretion levels of interferon-γ (IFNγ), tumor necrosis factor-α (TNFα), and granzyme B (GrB). The targeted killing activity of the genetically modified cells against B cells expressing β2GPI autoantibodies is further enhanced in the presence of the antibody or its antigen-binding fragment from the first aspect.

[0052] In some embodiments, the β2GPI domain described in A6) is inserted into the variable region of the T cell's TCR, or the β2GPI domain replaces the variable region of the T cell's TCR. The T cells may be derived from the patient themselves, from other donors of the same species, or from cells from other species, etc.

[0053] The genetically modified T cells in A6 can also bind to the antibody or its antigen-binding fragment from the first aspect. Upon binding to the antibody or its antigen-binding fragment from the first aspect, these genetically modified T cells are activated and produce effector functions, manifested by increased secretion levels of interferon-γ (IFNγ), tumor necrosis factor-α (TNFα), and granzyme B (GrB).

[0054] In some embodiments, the pharmaceutical composition can be administered via, for example, parenteral, subcutaneous, sublingual, rectal, nasal, intravenous, intramuscular, oral, ocular, or topical routes.

[0055] In some embodiments, the pharmaceutical composition is in the form of, for example, an aqueous solution, suspension, powder, tablet, capsule, granule, powder, pill, disintegrant, syrup, spray, gel, emulsion, injection, elixir, lozenge, suppository, etc.

[0056] In some implementations, β2GPI-related diseases or symptoms include antiphospholipid syndrome.

[0057] A sixth aspect of this disclosure provides a diagnostic or therapeutic kit comprising: an antibody or antigen-binding fragment thereof of the first aspect of this disclosure, a biological material of the second aspect, a conjugate of the fourth aspect, or a pharmaceutical composition of the fifth aspect.

[0058] In some embodiments, the kit may also include instructions and / or a drug delivery device.

[0059] In some embodiments, the kit can be used to diagnose β2GPI-related diseases or symptoms, detect the presence or level of β2GPI, its domain 1 protein, or fusion proteins containing its domain 1 protein in a sample, and / or develop or screen drugs for the prevention and / or treatment of β2GPI-related diseases or symptoms.

[0060] In some embodiments, the kit can be used to prevent and / or treat β2GPI-related diseases or symptoms.

[0061] In some implementations, β2GPI-related diseases or symptoms include antiphospholipid syndrome.

[0062] The seventh aspect of this disclosure provides the use of the antibody or antigen-binding fragment thereof of the first aspect, the biological material of the second aspect, the conjugate of the fourth aspect, or the pharmaceutical composition of the fifth aspect in any one of m1)-m8): m1) Prepare products for diagnosing β2GPI-related diseases or symptoms; m2) Prepare products for the prevention and / or treatment of β2GPI-related diseases or symptoms; m3) Prepare products for detecting the presence or level of β2GPI or proteins containing the D1 domain of β2GPI in samples; m4) detects the presence or level of β2GPI or proteins containing the D1 domain of β2GPI; m5) to prepare products for drug development or screening, said drug for the prevention and / or treatment of β2GPI-related diseases or symptoms; m6) Drug development or screening, wherein the drug is intended to prevent and / or treat β2GPI-related diseases or symptoms; m7) Prepare products for research on the pathogenesis mechanisms of β2GPI-related diseases or symptoms; (m8) Construct a disease or symptom model related to β2GPI.

[0063] In some implementations, the applications described in m4) and m6) do not involve the diagnosis or treatment of diseases.

[0064] In some embodiments, the protein containing the D1 domain of β2GPI may be a fusion protein or may consist of the D1 domain of β2GPI.

[0065] In some embodiments, the sample is selected from at least one of the body fluids, tissues, cells, and excretions of the subject.

[0066] In some embodiments, the body fluid includes at least one of blood and lymph.

[0067] In some embodiments, the blood includes at least one of serum, plasma, dried blood spots, and whole blood.

[0068] In some embodiments, the excrement includes at least one of urine, feces, and tears.

[0069] In some implementations, the test subject includes mammals such as humans, non-human primates (e.g., orangutans, apes), rodents (e.g., rats, mice, guinea pigs), pets (e.g., cats, dogs), and livestock (e.g., horses, cattle, sheep, pigs, rabbits).

[0070] In some implementations, the subject of the test includes humans.

[0071] In some embodiments, the product is a pharmaceutical product, a reagent, or a reagent kit.

[0072] In some implementations, β2GPI-related diseases or symptoms include antiphospholipid syndrome.

[0073] The eighth aspect of this disclosure provides a method for preventing and / or treating β2GPI-related diseases or symptoms, the method comprising administering to a subject in need an effective amount of an antibody or antigen-binding fragment thereof from the first aspect of this disclosure, a biological material from the second aspect, a conjugate from the fourth aspect, or a pharmaceutical composition from the fifth aspect.

[0074] In some implementations, the subjects include mammals such as humans, non-human primates (e.g., orangutans, apes), rodents (e.g., rats, mice, guinea pigs), pets (e.g., cats, dogs), and livestock (e.g., horses, cattle, sheep, pigs, rabbits).

[0075] In some implementations, the subjects include humans.

[0076] A ninth aspect of this disclosure provides a method comprising contacting a sample with an antibody or antigen-binding fragment of the first aspect of this disclosure, wherein the formation of the complex is detected, provided that the antibody or antigen-binding fragment thereof of the first aspect of this disclosure is allowed to form a complex with β2GPI or a protein containing a D1 domain of β2GPI. The method is used for any one of f1)-f3): f1) Diagnose β2GPI-related diseases or symptoms; f2) Detect the presence or level of β2GPI or proteins containing the D1 domain of β2GPI in the sample; f3) Develop or screen drugs for the prevention and / or treatment of β2GPI-related diseases or symptoms.

[0077] In some embodiments, the sample is selected from at least one of the body fluids, tissues, cells, and excretions of the subject.

[0078] In some embodiments, the body fluid includes at least one of blood and lymph.

[0079] In some embodiments, the blood includes at least one of serum, plasma, dried blood spots, and whole blood.

[0080] In some embodiments, the excrement includes at least one of urine, feces, and tears.

[0081] In some implementations, the β2GPI-related diseases or symptoms include antiphospholipid syndrome.

[0082] In some embodiments, the protein containing the D1 domain of β2GPI may be a fusion protein or may consist of the D1 domain of β2GPI.

[0083] The beneficial effects of this disclosure are: This disclosure provides an anti-β2GPI antibody that binds to the β2GPI-D1 epitope, which can be used for APS pathogenesis research, in vitro functional evaluation, and model construction. By removing the CH2 domain to obtain a modified antibody, it can effectively activate β2GPI-CAAR-T cells and enhance their effector molecule secretion and killing function while avoiding CH2-mediated complement activation. This provides a safer activation and / or effector enhancement molecule for β2GPI-targeted CAAR-T therapy. Furthermore, the anti-β2GPI antibody provided in this disclosure can serve as a tool antibody for β2GPI-related autoantibody detection and β2GPI epitope research. Attached Figure Description

[0084] Figure 1 The results of SDS-PAGE assays for P3-7 antibody and P3-7ΔCH2 antibody are shown.

[0085] Figure 2 The BLI binding curves and fitting results of P3-7 antibody and P3-7△CH2 antibody for different β2GPI truncated fragments are shown.

[0086] Figure 3 The study demonstrated that P3-7 antibody and P3-7ΔCH2 antibody activated β2GPI-CAAR-T cells and enhanced their cytotoxic efficacy. Detailed Implementation

[0087] To make the objectives, technical solutions, and advantages of this disclosure clearer, the following detailed description is provided in conjunction with embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of this disclosure in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in numerous publications.

[0088] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure pertains. For purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0089] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0090] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0091] As used in this article, "antibody" refers to a globulin produced by plasma cells, which are formed from the proliferation and differentiation of B lymphocytes in response to antigen stimulation. Antibodies specifically bind to the corresponding antigens and mediate immune effects. They are mainly found in serum and body fluids and are important immune molecules mediating humoral immunity. Antibodies can encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific, trispecific, or tetraspecific antibodies), single-chain molecules, and antigen-binding fragments. The chemical basis of antibodies is immunoglobulin (Ig).

[0092] As used herein, the term "monoclonal antibody" refers to antibodies derived from a substantially homogeneous group of antibodies, meaning that, apart from possible trace amounts of variant antibodies (e.g., containing naturally occurring mutations or generated during the production of the monoclonal antibody formulation, typically present in small quantities), the individual antibodies within the group are identical and / or bind to the same epitopes. Unlike polyclonal antibody formulations, which typically comprise different antibodies targeting different antigenic determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen.

[0093] As used herein, the term "multispecific antibody" is used in its broadest sense to encompass antibodies exhibiting multi-epitope specificity. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit exhibits multi-epitope specificity; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable regions, each single variable region binding to a different target or a different epitope of the same target; full-length antibodies, antibody fragments, bispecific antibodies, and trispecific antibodies, antibody fragments covalently or non-covalently linked, etc.

[0094] The terms “full-length antibody” and “intact antibody” as used herein are used interchangeably to refer to antibodies that are structurally similar to natural antibodies. “Natural antibody” refers to a naturally occurring immunoglobulin molecule. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH) (also called a variable heavy chain domain or heavy chain variable domain) and three constant domains (CH1, CH2, and CH3) (also called heavy chain constant regions, CH). From the N-terminus to the C-terminus, each light chain has a variable region (VL) (also called a variable light chain domain or light chain variable domain) and a light chain constant domain (CL) (also called light chain constant region). The heavy chain of an antibody can be one of five types: α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), and can be further subdivided into subtypes such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chain of an antibody, based on the amino acid sequence of its constant domain, can be one of two types: the κ (kappa) light chain and the λ (lambda) light chain.

[0095] Within the light and heavy chains, variable and constant regions are linked by a "J" region containing approximately 12 or more amino acid residues, and the heavy chain also contains a "D" region containing approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0096] The term "Fd fragment" as used herein refers to an antibody fragment consisting of VH and CH1 domains. The term "dAb fragment" as used herein refers to an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544 546 (1989)). The term "Fab fragment" as used herein refers to an antibody fragment consisting of VL, VH, CL, and CH1 domains. The term "F(ab')2 fragment" as used herein refers to an antibody fragment containing two Fab fragments linked by disulfide bridges on the hinge region. The term "Fab' fragment" as used herein refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light and heavy chain Fd fragment (composed of VH and CH1 domains). The term "Fab'-SH" as used herein refers to a Fab fragment containing free thiol groups.

[0097] As used in this article, the term "Fv fragment" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. The Fv fragment is generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as the Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0098] As used herein, the term "scFv" refers to a single polypeptide chain containing VL and VH domains linked by a linker. In some cases, a disulfide bond may also exist between the VH and VL domains of the scFv.

[0099] As used herein, the term "dsFv" refers to a disulfide-stabilized Fv fragment. In a dsFv, VH and VL are linked by interdomain disulfide bonds. To generate such molecules, one amino acid in each of the framework regions of VH and VL is mutated to a cysteine ​​residue, which in turn forms a stable interchain disulfide bond. Typically, position 44 in VH and position 100 in VL are mutated to cysteine ​​residues. The term dsFv can encompass so-called dsFv (molecules in which VH and VL are linked by interchain disulfide bonds rather than linker peptides) or scdsFv (molecules in which VH and VL are linked by both linker and interchain disulfide bonds).

[0100] As used herein, the term "variable region" or "variable domain" refers to the domain of the antibody heavy or light chain involved in the binding of the antigen-binding molecule to the antigen. The variable regions (VH and VL) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FR1-4) and three hypervariable regions (HVR1-3), arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL domain can confer antigen-binding specificity. The three HVRs within the VH and VL together constitute the antigen-binding site of Ig, which can bind complementary to the corresponding antigenic epitope; therefore, HVRs are also called complementarity-determining regions (CDRs), denoted as CDR1, CDR2, and CDR3, respectively. The VH or VL chain of an antibody may further contain all or part of the constant regions of the heavy or light chain.

[0101] The CDR of the antibody or antigen-binding fragment thereof disclosed herein can be determined according to various numbering systems known in the art. In some embodiments, the CDR contained in the antibody or antigen-binding fragment thereof disclosed herein is preferably determined by the IMGT, Kabat, Contact, Chothia, or AbM numbering system.

[0102] As used in this article, the term "variable" refers to the fact that certain segments of the variable region are generally different in sequence between antibodies. Variable domains mediate antigen binding and define the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed throughout the variable region, but is concentrated in three segments called hypervariable regions (HVRs) within the variable regions of the light and heavy chains. The relatively highly conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, mostly in a β-sheet configuration, linked by three HVRs that form loops and, in some cases, form part of a β-sheet structure. The HVRs in each chain are tightly held together by the FRs and, together with the HVRs of other chains, contribute to the formation of the antibody's antigen-binding site.

[0103] Constant regions do not directly participate in antibody-antigen binding but have other effector functions, such as participating in antibody-dependent cytotoxicity (ADCC). The numbering of constant regions is determined according to the EU numbering system.

[0104] Antibody "classes" refer to the types of constant structural domains or constant regions possessed by the antibody's heavy chain. Based on differences in heavy chain structure and antigenicity, they can be classified into five classes: μ chain, γ chain, α chain, δ chain, and ε chain. Immunoglobulins composed of different heavy and light chains are respectively called IgA, IgD, IgE, IgG, and IgM. Even within the same class of Ig, the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain differ, thus allowing for further subclassing of the same class of Ig. For example, human IgG can be divided into IgG1–IgG4; IgA can be divided into IgA1 and IgA2. Based on differences in light chain structure and antigenicity, immunoglobulin (Ig) light chains are divided into κ (kappa) chains and λ (lambda) chains, thus classifying Ig into two types: κ and λ.

[0105] Chimeric antibodies are a novel type of antibody created by combining antibody fragments from different sources using genetic engineering techniques. Typically, the variable region of a chimeric antibody is derived from a murine monoclonal antibody, responsible for recognizing and binding to specific antigens; while its constant region is derived from a human antibody, used to reduce immunogenicity in the human body. Theoretically, the variable region of a chimeric antibody can originate from various species such as humans, mice, rats, dogs, rhesus monkeys, chickens, rabbits, and camels, while the constant region can also originate from these species. The two can be combined arbitrarily according to requirements to meet different research and application needs. For example, chimeric antibodies are formed by concatenating the variable region of a mouse or rat-derived antibody with a human-derived constant region (see Proc. Natl. Acad. Sci. U.S.A., 81, 6851-6855, (1984)).

[0106] "Humanized antibodies" comprise amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, humanized antibodies comprise at least one, typically two, variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to the HVRs of the non-human antibody, and all or substantially all FRs correspond to the FRs of the human antibody. Humanized antibodies may optionally comprise at least a portion of the antibody constant region derived from a human antibody. Antibodies in a "humanized form," such as non-human antibodies, refer to antibodies that have undergone humanization.

[0107] As used herein, the terms “polynucleotide,” “nucleic acid,” or “nucleotide sequence” refer to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA), virus-derived RNA, or plasmid DNA (pDNA). Polynucleotides may contain conventional phosphodiester bonds or unconventional bonds (such as amide bonds, as found in peptide nucleic acids (PNAs)). The term “nucleic acid molecule” refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide.

[0108] An "antibody fragment" or "antigen-binding fragment" contains a portion of a complete antibody that retains the antibody's antigen-binding activity. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv; bisomatic antibodies, trisomatic antibodies, tetrasomatic antibodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments and single-domain antibodies (single-domain antibodies).

[0109] As used herein, the terms "antigen-binding domain" or "antigen-binding site" refer to the portion of an antigen-binding molecule that specifically binds to an antigenic determinant. More specifically, the term "antigen-binding domain" refers to a portion of an antibody containing a region that specifically binds to and is complementary to a portion or all of the antigen. In cases where the antigen molecule is large, the antigen-binding molecule may bind only a specific portion of the antigen, called an epitope. The antigen-binding domain may be provided by, for example, one or more variable domains (also called variable regions). Preferably, the antigen-binding domain comprises a variable region (VL) of the antibody light chain and a variable region (VH) of the antibody heavy chain. In one aspect, the antigen-binding domain is capable of binding its antigen and blocking or partially blocking the function of said antigen.

[0110] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site on a polypeptide macromolecule (e.g., a continuous amino acid sequence or a conformation composed of different regions of non-continuous amino acids) to which an antigen-binding moiety binds, thereby forming an antigen-binding moiety-antigen complex. Antigenic determinants can be present, for example, on the surface of tumor cells, on the surface of microbially infected cells, on the surface of other diseased cells, on the surface of immune cells, in serum, and / or in the extracellular matrix (ECM). Unless otherwise stated, proteins used as antigens in this disclosure can be any naturally occurring form of protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). Antigens can also be human proteins, or antigens can be "full-length," unprocessed proteins, and any form of protein produced by intracellular processing, or naturally occurring protein variants, such as splice variants or allelic variants.

[0111] The specific “binding strength” or “affinity” of an antibody or its antigen-binding fragment to an antigen refers to the strength of the non-covalent interaction between a single binding site and its binding ligand (e.g., antigen), and can be distinguished from unwanted or non-specific binding. The ability of an antigen-binding molecule to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) and conventional binding assays. In one embodiment, for example, as measured by SPR, the degree of binding of the antigen-binding molecule to an unrelated protein is less than about 10% of the degree of binding of the antigen-binding molecule to the antigen. Binding affinity is typically expressed as an equilibrium dissociation constant (KD or ka), which is the ratio of the dissociation rate constant to the binding rate constant (kd and ka, respectively); the KD value is inversely proportional to the intermolecular affinity, i.e., the smaller the KD value, the higher the intermolecular affinity. In some embodiments, the equilibrium dissociation constant (KD) of the molecule that binds to the antigen is 10⁻⁶ M or less, 10⁻⁷ M or less, preferably 10⁻⁸ M or less, and even more preferably 10⁻⁹ M or less.

[0112] As used herein, the terms "vector" or "expression vector" and "expression construct" are used interchangeably to describe a DNA molecule to which a specific gene, operatively linked, is introduced into a target cell and directed for expression. The vector includes a vector as a self-replicating nucleic acid structure and a vector incorporated into the genome of the host cell into which it has been introduced. The expression vectors of this disclosure comprise expression cassettes. Expression vectors can be transcribed into large amounts of stable mRNA. Once the expression vector is within the target cell, cellular transcription and / or translation mechanisms generate a ribonucleic acid molecule or protein encoded by the gene. In one embodiment, the expression vector of this disclosure comprises an expression cassette containing a polynucleotide sequence encoding an antibody of this disclosure or an antigen-binding fragment thereof. The term "expression cassette" of this disclosure refers to a recombinant or synthetically generated polynucleotide having a series of nucleic acid elements that allow a specific nucleic acid to be transcribed in a target cell. Recombinant expression cassettes can be introduced into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, in addition to other sequences, the recombinant expression cassette portion of the expression vector includes the nucleic acid sequence to be transcribed and a promoter. In some embodiments, the expression cassette of this disclosure contains a polynucleotide sequence encoding an antibody of this disclosure or an antigen-binding fragment thereof.

[0113] As used herein, the terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” including primary transformed cells and their derived progeny. The nucleic acids of the progeny may not be completely identical to those of the parent cells and may contain mutations. Host cells are any type of cell that can be used to generate the disclosed antibody or its antigen-binding fragment, such as prokaryotic and / or eukaryotic cells. Host cells include cultured cells, such as cultured prokaryotic cells, such as Escherichia coli cells, cultured mammalian cells such as CHO cells, HEK293 cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, HeLa cells (human cervical cancer cells), Vero cells (African green monkey kidney cells), Expi293 cells (human embryonic kidney cells), or hybridoma cells, yeast cells, insect cells, and plant cells, and also include cells contained within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0114] As used herein, the term "conjugate" includes "antibody conjugated to a drug," also known as "antibody-drug conjugate" or "ADC," referring to a binding protein (such as an antibody or its antigen-binding fragment) chemically linked to one or more chemical drugs. In a preferred embodiment, an ADC includes a binding protein, a drug / therapeutic agent, and a connector linking the binding protein to the drug / therapeutic agent.

[0115] As used herein, the term "linker" or "connector" refers to a (peptide) linker of natural and / or synthetic origin, composed of linear amino acids. Domains in the antibodies or antigen-binding fragments of this disclosure may be linked by linkers, wherein each linker is fused to and / or otherwise linked (e.g., via peptide bonds) with at least two polypeptides or domains. In some embodiments, all linkers present in the antibodies or antigen-binding fragments of this disclosure have the same amino acid sequence. In other embodiments, at least two linkers present in the antibodies or antigen-binding fragments of this disclosure have different amino acid sequences. Linkers should have a length suitable for linking two or more monomeric domains in this manner, ensuring that the different domains they are linked to fold correctly and are properly presented to perform their biological activities. In various embodiments, linkers have a flexible conformation. Suitable flexible linkers include, for example, those having glycine, glutamine, and / or serine residues. In some embodiments, the linker is selected from one or more of the following: the amino acid sequence shown in (GnS)m, Gn1(Gn2S)m, (Gn1S)m1(Gn2S)m2, (EAAAK)n, wherein m, m1, m2, n, n1 and n2 are each independently selected from integers from 1 to 5.

[0116] As used herein, the term "chimeric autoantibody receptor" or "CAAR" refers to an engineered receptor expressed on cells (e.g., T cells) or any other effector cell type (e.g., effector cell types capable of cell-mediated cytotoxicity) containing an antigen or fragment thereof against BCR and / or autoantibodies.

[0117] An "effective amount" of a drug is the amount necessary to produce physiological changes in the cells or tissues to which it is administered. An "effective amount" includes the amount sufficient to improve or prevent the symptoms or signs of a medically diagnosed disease. An effective amount also means the amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of any side effects. An effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0118] The "therapeutic effective amount" of a drug (such as a pharmaceutical composition) refers to the amount necessary to effectively achieve the desired therapeutic or preventive effect in terms of dosage, dosing intervals, and time. For example, a therapeutically effective amount of a drug can eliminate, mitigate / reduce, delay, minimize, or prevent the adverse effects of a disease.

[0119] As used herein, the term “individual” or “subject” refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). Specifically, an individual or subject is a human being.

[0120] As used herein, the term "pharmaceutical composition" refers to a mixture containing one or more antibodies or antigen-binding fragments of the present disclosure, along with other chemical components, such as physiologically / pharmaceutically acceptable carriers or excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity.

[0121] As used herein, the term "pharmaceuticalally acceptable excipient" refers to a component in a pharmaceutical composition that, apart from the active ingredient, is non-toxic to the subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers, and / or preservatives.

[0122] As used herein, the term "treatment" refers to the administration of an oral or topical therapeutic agent, such as a composition comprising any antibody of this disclosure or an antigen-binding fragment thereof, or a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof, to a patient having one or more diseases or symptoms associated with β2GPI, and the therapeutic agent having a therapeutic effect on these diseases or symptoms. Typically, the therapeutic agent is administered in a treated patient or population in an amount that effectively relieves one or more diseases or symptoms, to induce regression of such symptoms, or to inhibit the development of such symptoms to any clinically measurable extent.

[0123] As used herein, the term "prevention" refers to delaying, suppressing, or preventing the onset of β2GPI-related diseases in mammals where the initiation of cancer or tumorigenesis has not been confirmed, but a susceptibility to cancer has been identified, for example, through genetic screening or other methods. The term also includes treating mammals with precancerous lesions to halt the progression of the precancerous lesions to malignancy or to induce their regression.

[0124] As used herein, the term "detectable marker" encompasses a marker that can be detected directly or indirectly, either attached to the antibody or the bispecific binding protein, or present independently in the kit. Suitable markers include, but are not limited to, any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, chemical, or other methods. Suitable markers include, but are not limited to, fluorescent dyes (e.g., GFP and its variants, FITC, TRITC, fluorescein, phycoerythrin (PE), Texas red, quantum dots, cyanine dye derivatives (e.g., Cy7, Alexa 750), and rhodamine), electron-dense reagents (e.g., gold), enzymes (e.g., horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, β-galactosidase, urease, catalase, glucosylase, etc.), molecules containing radionuclides (i.e., radioisotopes, e.g., 3H, 125I, 35S, 14C, 32P, etc.), chemiluminescent molecules (e.g., acridine esters, luminol or its variants, acridine sulfonamides, etc.), electrochemiluminescent molecules (e.g., terpyridine ruthenium complexes, terpyridine osmium complexes, iridium complexes, metal nanoclusters, quantum dots, etc.), etc. Antibodies or their antigen-binding fragments or bispecific binding proteins in this disclosure are linked to a "detectable marker," thus being detectably labeled. In some embodiments, such markers are suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable marker is selected from radioactive isotopes, fluorescent substances, luminescent substances, colored substances, or enzymes.

[0125] The "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies are not nucleotides or amino acids and are not counted in the target sequence. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are counted, but those from the reference sequence are not.

[0126] The percentage of sequence identity can be calculated as follows: determine the number of positions in both sequences where the same amino acid residue or nucleic acid base appears (the number of matching positions), divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for protein and nucleotide sequence alignment. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparing two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0127] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997)), which are incorporated herein by reference.

[0128] The following embodiments and accompanying drawings are provided to aid in understanding this disclosure. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of this disclosure is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of this disclosure. The reagents and / or kits used in the following examples are commercially available or can be synthesized by known methods.

[0129] It should be noted that, unless specific conditions are specified in the examples, experimental conditions should be performed according to standard conditions, manufacturer recommendations, or publicly reported experimental conditions. Reagents or instruments whose manufacturers are not specified are all commercially available, standard products. For reagents whose manufacturers are specified, similar products from other manufacturers are substitutes.

[0130] The sequence information involved in the embodiments of this disclosure is shown in Table 1: Table 1: Sequence Information

[0131] Clinical data and serum samples in this disclosed embodiment were obtained from subjects who were treated at Peking Union Medical College Hospital and included in the study. All APS patients met the 2023 ACR / EULAR classification criteria. All subjects signed written informed consent forms. All experiments involving human materials followed institutional guidelines and were reviewed and approved by the Ethics Committee of Peking Union Medical College Hospital (HS-3309).

[0132] Example 1: Preparation of full-length and truncated fragments of recombinant human β2GPI with different structural domains The gene encoding amino acids 1-345 of human β2GPI (Uniprot: P02749) was synthesized by a commercial company and inserted between the BamHI and XhoI restriction endonuclease sites in the pcDNA3.1 expression vector. Sequencing confirmed its correctness after construction. Using this full-length β2GPI expression plasmid as a template, five different truncated β2GPI fragments (D1, D12, D123, D1234, and D12345) were further constructed using a homologous recombination strategy. All constructs were verified by sequencing. These five recombinant β2GPI fragments were transiently transfected into HEK293T cells at 37°C and expressed for 6 days. After expression, the culture supernatant was collected and automatically purified using immobilized metal affinity chromatography (IMAC) on an ÄKTA avant 25 system (GE Healthcare). Specifically, the culture supernatant was first dialyzed with PBS to replace the buffer system. Then, the β2GPI fragments D1, D12, D123, D1234, and D12345 with C-terminal His tags were loaded into 5 mL HisTrap Ni columns, and the target proteins were eluted with 300 mM imidazole under PBS conditions. Further purification was then performed by size exclusion chromatography (HiLoad 16 / 60 Superdex 75 column, PBS, pH 7.4). The purity and integrity of the final protein were assessed by SDS-PAGE analysis.

[0133] Example 2: β2GPI-specific memory B cell sorting and single B cell antibody gene acquisition Peripheral blood samples were collected from patients with antiphospholipid syndrome (APS). Peripheral blood mononuclear cells (PBMCs) were separated using density gradient centrifugation. Specifically, whole blood was diluted with an equal volume of PBS (Gibco, 10010023), slowly plated onto a Ficoll-Paque PLUS (Cytiva, 17-1440-03) plate, and centrifuged to separate the mononuclear cell layer. After collection, PBMCs were washed twice with PBS and counted. PBMCs could be used directly for subsequent flow cytometry sorting or cryopreserved using cryopreservation buffer (90% FBS + 10% DMSO): fetal bovine serum was treated with heat-inactivated FBS (Gibco, 10082147) and DMSO was applied using cell culture grade (Sigma-Aldrich, D2650), followed by programmed cooling and liquid nitrogen storage.

[0134] To enrich β2GPI-binding specific B cells, a dual-probe strategy was used to construct β2GPI-PE and β2GPI-APC. The β2GPI antigen was derived from recombinant β2-Glycoprotein I (Sino Biological, 11221-H08H). β2GPI protein was fluorescently conjugated using the Lightning-Link R-PE labeling kit (Bio-Techne / Novus, 703-0004) and the Lightning-Link APC labeling kit (Bio-Techne / Novus, 705-0010), respectively. After conjugation, free dye was removed using a desalting column (Thermo Scientific Zeba Spin Desalting Columns, 89882), and the protein was quantified according to concentration and then aliquoted for storage.

[0135] Frozen PBMCs were rapidly thawed in a 37°C water bath and immediately added to pre-chilled FACS buffer (PBS + 2% FBS). The cells were centrifuged at 300g for 8 min, the supernatant was discarded, and the suspension was resuspended in FACS buffer. The cell suspension was filtered through a 40μm cell sieve to remove aggregated cells, and the cell concentration was adjusted for staining. Negative control and single-stain compensation tubes were prepared for fluorescent staining; approximately 1 × 10^6 cells were collected from each tube. First, the cells were incubated with the live / dead dye LIVE / DEAD™ Fixable Blue Dead Cell Stain (Thermo Fisher Scientific, L34957) at 4°C in the dark for 20 min to remove dead cells. After washing, the human Fc receptor blocking reagent Human TruStain FcX™ (BioLegend, 422302) was added, and the cells were incubated at 4°C for 10 min to reduce non-specific binding. Subsequently, surface antibody staining was performed, and the cells were incubated at 4°C in the dark for 30 min.

[0136] The antibodies used were as follows: anti-human CD3 Pacific Blue (BioLegend, 300419, cloneOKT3), anti-human CD14 Pacific Blue (BioLegend, 325621, clone HCD14), anti-humanCD16 Pacific Blue (BioLegend, 302031, clone 3G8), anti-human CD19 PE-Cy7 (BioLegend, 302215, clone HIB19), anti-human CD27 BV605 (BioLegend, 302835, clone O323), anti-human IgG PerCP / Cy5.5 (BioLegend, 410713, clone M1310G05), anti-human IgA FITC (BioLegend, 411005, clone IS11-8E10). The preparation methods for β2GPI-PE and β2GPI-APC are as described above.

[0137] Cells were mixed with antibodies / probes and incubated at 4°C in the dark for 30 min, followed by washing twice with 500 μL FACS buffer and resuspending for sorting. Sorting was performed using a high-speed flow cytometer with the following gating strategy: first, lymphocyte populations were selected from forward and side-scatter patterns; then, cell aggregates were excluded based on the relationship between forward scatter height and forward scatter area; next, live / dead dye-positive cells were excluded to obtain a viable cell population. Based on this, T cells, monocytes, and NK / myeloid cells were removed using CD3, CD14, and CD16 labeled with the same fluorescent channel. In lineage-negative cells, memory B cells were identified based on CD19 and CD27 expression; further, IgG and IgA staining was used to differentiate B cell subsets of different immunoglobulin isotypes. Finally, β2GPI-specific B cells were strictly defined as double-positive cells that simultaneously bind two β2GPI fluorescently labeled antigen probes.

[0138] The sorted β2GPI-specific memory B cells were single-cell sorted into 96-well PCR plates, each well containing a lysis / reverse transcription reaction system. Single-cell cDNA synthesis was performed using the SuperScript IV First-Strand Synthesis System (Invitrogen, 18091050) with a nuclease-free water-based preparation system. Immunoglobulin variable region sequences were obtained using one of two strategies: multiplex PCR amplification: separately amplifying the IgH, Igκ, and Igλ variable regions; and 5' RACE amplification: using the SMARTer RACE 5' / 3' Kit (Takara / Clontech, 634858) to obtain the complete V(D)J sequence. PCR amplification used Q5 High-Fidelity DNA Polymerase (NEB, M0491) to reduce the amplification error rate. After purification, the amplified products were subjected to Sanger sequencing to screen for candidate β2GPI-binding antibody sequences, ultimately yielding the P3-7 antibody sequence.

[0139] Example 3: Design, vector construction, expression, and purification of full-length P3-7 antibody and P3-7ΔCH2 antibody with CH2 domain removed. Based on the sequenced heavy chain variable region (VH) and light chain variable region (VL) sequences (amino acid sequences are shown in Table 1), expression vectors were constructed by ligating them to the constant regions of human antibodies. Specifically, the P3-7 VH was ligated to the human IgG1 constant region (CH1-CH2-CH3) to obtain the P3-7 heavy chain expression cassette; the P3-7 VL was ligated to the human κ light chain constant region to obtain the P3-7 light chain expression cassette. These expression cassettes were inserted into the mammalian cell pcDNA3.1 expression vector and verified by sequencing. To obtain the P3-7△CH2 antibody with the CH2 domain removed, the coding sequence corresponding to the CH2 domain was deleted using the full-length P3-7 heavy chain expression vector as a template, so that CH1 and the hinge region were directly linked to the CH3 domain, thus forming the P3-7△CH2 heavy chain expression cassette; its light chain expression vector is the same as the P3-7 light chain. The modified heavy chain vector was verified to be correct by sequencing. The light and heavy chain amino acid sequences of P3-7 antibody and P3-7△CH2 antibody are shown in Table 1.

[0140] This embodiment uses recombinant expression to prepare antibodies. Specifically, the nucleic acid sequences encoding the heavy and light chains of the target antibody were cloned into the eukaryotic expression vector pcDNA3.4 (ampicillin-resistant, universal sequencing primers). The signal peptide MGWSCIILFLVATATGVHS (SEQ ID NO: 47) was linked to the front end of the sequences, and a recombinant plasmid was constructed by using NotI / XbaI restriction endonuclease sites. Chinese hamster ovary cells (CHO cells) were used as the expression host. Electroporation was performed using an electroporator with a 1 mL electroporation cuvette. After the recombinant plasmid was introduced into the cells, they were cultured in suspension at 37 ℃, 8% CO2, and 120 rpm for expression. After transfection, the cells were aliquoted into shake flasks containing 20 mL of DMEM high-glucose medium (10% FBS) and incubated for about 40 min. Then, they were cultured for another 24 h, and sodium butyrate, penicillin, and streptomycin were added. The cells were cultured for about 4 days to allow the antibody protein to be secreted into the culture supernatant. After collecting the culture supernatant, Protein A was used to analyze the antibody protein secreted into the culture supernatant. Antibody purification was performed using a pre-packed affinity chromatography column. Specifically, the column was equilibrated with 1×PBS (flow rate 1 mL / min, volume approximately 20 mL), followed by washing with 1×PBS and elution with sodium citrate buffer at pH 3.4. The eluent was collected in separate tubes, and the absorbance at 280 nm was measured using a NanoDrop instrument. Subsequently, the high-concentration antibody solution was placed in a dialysis bag and dialyzed with 1×PBS. Quality control of the purified antibody was performed, including SDS-PAGE (10 µL loading, 180 V, 45 min) to analyze antibody purity and integrity. Size exclusion chromatography (SEC-HPLC) was also performed using an LC-20AT high-performance liquid chromatography system combined with a gel chromatography column. The detection conditions were: flow rate 1 mL / min, column temperature 35 ℃, injection volume 20 µL, detection wavelengths of 214 nm and 280 nm, and acquisition time 15 minutes. The ratio of antibody monomers and aggregates was assessed by incubation at 37 °C for 60 min. In addition, the endotoxin was detected by the Limulus amebocyte lysate (LAL) method. The test results showed that the test sample was clear and transparent and did not coagulate under the condition of constant temperature incubation at 37 °C for 60 min, thus obtaining an antibody preparation with high purity, stable structure and endotoxin level that meets the requirements.

[0141] The purified antibody was detected by SDS-PAGE, and the results are as follows: Figure 1 As shown, in the reduced state, the light and heavy chains of P3-7 antibody and P3-7△CH2 antibody can be detected, while in the non-reduced state, the full-length antibody containing both light and heavy chains can be detected.

[0142] Example 4: Results of in vitro binding assay (BLI) and epitope localization Biolayer Interference (BLI): The binding affinity of P3-7 antibody to different truncated β2GPI fragments was determined using a FortéBio Octet Red 96 instrument. First, β2GPI fragments with a 6×His tag (D1, D12, D123, D1234, and D12345 prepared in Example 1) were immobilized on Ni-NTA-coated biosensors. Then, the probes were immersed in antibody solutions (P3-7 antibody, P3-7ΔCH2 antibody, or control antibody P1-117 antibody (see DOI: 10.1111 / j.1538-7836.2011.04212.x)) and the binding process was monitored for 1 minute. Subsequently, the dissociation process was monitored for 1 minute in buffer solution. After baseline correction and reference subtraction using a blank buffer control, the obtained sensor images were analyzed using Octet software, based on a 1:1 Langmuir combined model and employing local fitting, to calculate the kinetic parameters kon, koff, and the equilibrium dissociation constant KD. Figure 2 As shown in Figure A, the P3-7 antibody exhibits specific binding to both the β2GPI fragment containing the D1 domain and fragments containing only the D1 domain, with KD values ​​all less than 1 × 10^-12, suggesting that the binding epitope of the P3-7 antibody is located in the D1 domain of the β2GPI. The P3-7ΔCH2 antibody also shows a binding spectrum consistent with P3-7 with the aforementioned β2GPI fragment, with KD values ​​all less than 1 × 10^-12, indicating that the removal of the CH2 domain does not affect its antigen recognition ability. According to literature reports, the control group P1-117 antibody recognizes the D12 region, therefore it does not bind to D1, but binds to D12, D123, D1234, and D12345. Figure 2 As shown in Figure B, the gradient binding curves of P3-7 antibody and P3-7△CH2 antibody to D12345 are shown.

[0143] Example 5: Detection and Results of β2GPI-CAAR-T Cell Activation and Effector Function 1. Construction of β2GPI-CAAR-T cells 1.1 Construction of CAAR Construct The β2GPI-CAAR construct was expressed using the fourth-generation lentiviral vector pCCL (provided by Delivectory Biosciences Inc.). Genes encoding amino acids 20–345 of human β2GPI (Integrated DNA Technologies), flanked by NheI sites, were synthesized and cloned into a second-generation CAR backbone. This backbone contained the GM-CSFRα leader sequence (SEQ ID No. 46), the human CD28 extracellular hinge (SEQ ID No. 42), a transmembrane domain (SEQ ID No. 43), the human 4-1BB intracellular co-stimulatory domain (SEQ ID No. 44), and the CD3ζ signaling domain (SEQ ID No. 45), followed by the linking of a P2A peptide sequence and EGFP, and the entire structure was placed in the pCCL transfer vector. Each β2GPI variant (D1, D12, D123, D1234, and D12345) was inserted between the GM-CSFRα leader sequence and the CD28 hinge. Five β2GPI-CAAR constructs were ultimately obtained: D1-CAAR contains β2GPI domain 1 (SEQ ID No. 37); D12-CAAR contains domains 1-2 (SEQ ID No. 38); D123-CAAR contains domains 1-3 (SEQ ID No. 39); D1234-CAAR contains domains 1-4 (SEQ ID No. 40); and D12345-CAAR contains domains 1-5 (SEQ ID No. 41).

[0144] 1.2. Lentiviral Preparation Lentiviral viruses were prepared in HEK293T cells using a fourth-generation packaging system. When cells reached approximately 90% confluence, they were transfected with the pCCL-EF1α-target gene transfer vector (i.e., a pCCL transfer vector containing any one of the five β2GPI-CAAR constructs), the envelope plasmid pMD2.G (Addgene, 12252), and the packaging plasmids pRSV-Rev (Addgene, 12253) and pMDLg / pRRE (Addgene, 12251) using jetPRIME (Polyplus). The supernatant was collected 24–48 h post-transfection, filtered through a 0.45 μm PES filter, concentrated by centrifugation at 12000 × g for 12 h at 4 °C, aliquoted, and stored at -80 °C.

[0145] 1.3. In vitro transduction and expansion of β2GPI-CAAR-T cells Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors, separated by density gradient centrifugation, and cultured in T cell culture medium (RPMI 1640 + 10% FBS + 2 mM L-glutamine + 200 U / ml human recombinant IL-2 + 100 U / ml Pen / Strep). CD3 / CD28 co-stimulation (1×10^6 PBMCs / 10 μL TransAct) was performed according to the manufacturer's instructions for 48 h using T Cell TransAct (Miltenyi, 130111160). 24 h after activation, lentivirus with an MOI of 5 was added, along with 5 μg / mL protamine sulfate, and the mixture was centrifuged at 32°C and 800×g for 90 min, followed by overnight incubation. The culture medium was refreshed 24 h after transduction, and static amplification continued until day 9-10, with fresh culture medium added approximately every two days. β2GPI-CAAR-T cells were obtained by detecting the expression of β2GPI-CAAR on human T cells using flow cytometry.

[0146] 2. CAAR-T cell activation experiment: CAAR-T cell activation assays were performed using β2GPI-CAAR-T cells containing D12345-CAAR. In 96-well plates, 2.5 × 10^4 β2GPI-CAAR-T cells were co-incubated with either P3-7 antibody or P3-7ΔCH2 antibody at a final concentration of 50 μg / mL for 24 h. A human IgG1κ isotype control (MCE, HY-P99001) was used as a control group. The expression levels of CD25 (Biolegend, 356114) and CD69 (Biolegend, 310910) were then detected by flow cytometry. After 24 hours of co-culture, the supernatant was collected, and the levels of IFN-γ, TNF-α and GrB were measured using the human IFN-γ one-step ELISA kit (S0C3005), the human TNF-α one-step ELISA kit (S0C3024) and the human granzyme B (GrB) one-step ELISA kit (S0C3041) according to the instructions. All samples were tested in triplicate.

[0147] The results showed that both P3-7 antibody and P3-7ΔCH2 antibody could significantly increase CD25 in β2GPI-CAAR-T cells. + CD69 + Proportion( Figure 3 (A), and promotes the secretion of IFN-γ, TNF-α and GrB ( Figure 3 (B) suggests that both can effectively activate β2GPI-CAAR-T cells and enhance their effector function.

[0148] 3. CAAR-T cell killing assay: CAAR-T cell killing assay: In the NanoLuc luciferase-based cytotoxicity assay, engineered Nalm-6 target cells (COBIOER, CBP30231L) expressing NanoLuc were seeded at 2.5 × 10^4 cells / well in white flat-bottomed 96-well plates, and β2GPI-CAAR-T cells with an effector-to-target ratio of 16:1 were added and co-cultured for 24 h. Subsequently, the NanoLuc substrate fluorofurimazine (IMMOCELL, IMC-910) was added, and the luminescence signal was detected on a plate reader. Specific cytotoxicity was calculated using the following formula: Specific killing rate = 1 - (luminescence signal in T cell treatment group / luminescence signal in untransduced T cell control group).

[0149] like Figure 3 As shown in Figure C, both P3-7 and P3-7△CH2 can enhance the killing efficacy of β2GPI-CAAR-T against target cells.

[0150] The above description is only a preferred embodiment of this disclosure. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principles of this disclosure, and these improvements and modifications should also be considered within the scope of protection of this disclosure.

Claims

1. An anti-β2 glycoprotein I (β2GPI) antibody or its antigen-binding fragment, wherein the anti-β2GPI antibody or its antigen-binding fragment comprises: a1) having HCDR1, HCDR2, and HCDR3 included in the heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 1; and / or having LCDR1, LCDR2, and LCDR3 included in the light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 2; or a2) Having one or more amino acid substitutions, deletions or additions compared to HCDR1, HCDR2 and HCDR3 as shown in a1); and / or having one or more amino acid substitutions, deletions or additions compared to LCDR1, LCDR2 and LCDR3 as shown in a1). The CDR is defined according to the Kabat, Chothia, IMGT, Contact, or AbM numbering system.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The anti-β2GPI antibody or its antigen-binding fragment includes: b1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 3, HCDR2 having the amino acid sequence shown in SEQ ID NO: 4, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 5; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 6, LCDR2 having the amino acid sequence shown in SEQ ID NO: 7, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 8; or b2) VH including the following three CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in b1); and / or VL including the following three CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in b1); Wherein, the CDR is defined according to the Kabat numbering system; or The anti-β2GPI antibody or its antigen-binding fragment includes: c1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 9, HCDR2 having the amino acid sequence shown in SEQ ID NO: 10, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 11; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 12, LCDR2 having the amino acid sequence shown in SEQ ID NO: 13, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 14; or c2) VH including the following three CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in c1); and / or VL including the following three CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in c1); Wherein, the CDR is defined according to the Chothia numbering system; or The anti-β2GPI antibody or its antigen-binding fragment includes: d1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 15, HCDR2 having the amino acid sequence shown in SEQ ID NO: 16, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 17; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 18, LCDR2 having the amino acid sequence shown in SEQ ID NO: 19, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 20; or d2) VH including the following 3 CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in d1); and / or VL including the following 3 CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in d1); Wherein, the CDR is defined according to the IMGT numbering system; or The anti-β2GPI antibody or its antigen-binding fragment includes: e1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 21, HCDR2 having the amino acid sequence shown in SEQ ID NO: 22, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 23; and / or a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 24, LCDR2 having the amino acid sequence shown in SEQ ID NO: 25, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 26; or e2) VH including the following three CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in e1); and / or VL including the following three CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in e1); Wherein, the CDR is defined according to the Contact numbering system; or The anti-β2GPI antibody or its antigen-binding fragment includes: f1) A VH comprising the following three CDRs: HCDR1 having the amino acid sequence shown in SEQ ID NO: 27, HCDR2 having the amino acid sequence shown in SEQ ID NO: 28, and HCDR3 having the amino acid sequence shown in SEQ ID NO: 29; and / or, a VL comprising the following three CDRs: LCDR1 having the amino acid sequence shown in SEQ ID NO: 30, LCDR2 having the amino acid sequence shown in SEQ ID NO: 31, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 32; or f2) VH including the following three CDRs: HCDR1, HCDR2, and HCDR3 having one or more amino acid substitutions, deletions, or additions compared to HCDR1, HCDR2, and HCDR3 as shown in f1); and / or VL including the following three CDRs: LCDR1, LCDR2, and LCDR3 having one or more amino acid substitutions, deletions, or additions compared to LCDR1, LCDR2, and LCDR3 as shown in f1); The CDR is defined according to the AbM numbering system; Preferably, the heavy chain variable region of the anti-β2GPI antibody or its antigen-binding fragment further includes the framework region of the heavy chain variable region; Preferably, the framework region of the heavy chain variable region includes the framework region of the heavy chain variable region of immunoglobulins derived from mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese, or a mutant thereof; more preferably, it includes the framework region of the heavy chain variable region of immunoglobulins derived from mice, or a mutant thereof. Preferably, the light chain variable region of the anti-β2GPI antibody or its antigen-binding fragment further includes the framework region of the light chain variable region; Preferably, the framework region of the light chain variable region includes the framework region of the light chain variable region of immunoglobulin derived from mouse, primate, cow, horse, pig, sheep, goat, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose or a mutant thereof; more preferably, it includes the framework region of the light chain variable region of immunoglobulin derived from mouse or a mutant thereof.

3. The antibody or antigen-binding fragment thereof according to any one of claims 1-2, characterized in that, The anti-β2GPI antibody or its antigen-binding fragment includes: The heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and / or the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; Preferably, the anti-β2GPI antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region; Preferably, the heavy chain constant region includes at least a portion of the heavy chain constant region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; more preferably, it includes at least a portion of the heavy chain constant region or a mutant thereof derived from mouse immunoglobulins. Preferably, according to the EU numbering system, the heavy chain constant region includes mutations at one or more sites selected from positions 234, 235, 265, 297, and 329; more preferably, it includes one or more mutations selected from L234A, L235A, D265A, N297G / N297A, and P329G. Preferably, the heavy chain constant region does not include the CH2 region; Preferably, the light chain constant region includes at least a portion of the light chain constant region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese; more preferably, it includes the light chain constant region or a mutant thereof derived from mouse immunoglobulins. Preferably, the heavy chain constant region includes a heavy chain constant region derived from IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 or IgM immunoglobulin; more preferably, it includes a heavy chain constant region derived from IgG1 immunoglobulin. Preferably, the light chain constant region includes light chain constant regions derived from κ and λ immunoglobulins.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, characterized in that, The anti-β2GPI antibody or its antigen-binding fragment is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody; Preferably, the anti-β2GPI antibody or its antigen-binding fragment includes monoclonal antibodies, bispecific antibodies, multispecific antibodies, nanobodies, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fv fragments, single-chain Fv (scFv), dsFv, or Fd fragments; Preferably, the anti-β2GPI antibody or its antigen-binding fragment comprises: g1) Heavy chain, comprising the amino acid sequence shown in SEQ ID NO: 33, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and / or, light chain, comprising the amino acid sequence shown in SEQ ID NO: 34, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; or g2) Heavy chain, comprising the amino acid sequence shown in SEQ ID NO: 35, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; and / or, light chain, comprising the amino acid sequence shown in SEQ ID NO: 36, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

5. A biological material relating to the antibody or antigen-binding fragment thereof according to any one of claims 1-4, said biological material comprising any one of n1)-n9): n1) A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof as described in any one of claims 1-4; n2) contains an expression cassette containing the nucleic acid molecule described in n1); n3) A carrier containing the nucleic acid molecule described in n1); n4) A carrier containing the expression box described in n2); n5) A cell containing the nucleic acid molecules described in n1); n6) Cells containing the expression cassette described in n2); n7) Cells containing the carrier described in n3); n8) contains cells containing the carrier described in n4); n9) Cells comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-4; None of the cells described in n5)-n9) contain reproductive material.

6. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1-4, the method comprising: The cells of claim 5 are cultured, and the antibody or its antigen-binding fragment is collected from the cultured cell culture.

7. A conjugate comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-4; and a conjugation portion.

8. The coupling according to claim 7, characterized in that, The coupling portion includes a detectable marker or therapeutic agent; Preferably, the detectable markers include enzymes, radionuclides, fluorescent dyes, luminescent substances, colored substances, and / or biotin.

9. A pharmaceutical composition comprising: The antibody or antigen-binding fragment thereof as described in any one of claims 1-4, the biological material as described in claim 5, or the conjugate as described in any one of claims 7-8; and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutical composition further includes any one of A1)-A6): A1) Chimeric autoantibody receptor (CAAR), the chimeric autoantibody receptor comprising an extracellular domain, and optionally a hinge region, a transmembrane domain, a co-stimulatory intracellular domain and / or a signaling domain, wherein the extracellular domain is a β2GPI domain. A2) encodes the polynucleotide of the chimeric autoantibody receptor described in A1); A3) A carrier containing the polynucleotides described in A2); A4) Cells containing the carrier described in A3); A5) Genetically modified cells, said genetically modified cells comprising the chimeric autoantibody receptor described in A1); A6) Genetically modified T cells, wherein the variable region of the TCR of the genetically modified T cells contains a β2GPI domain, and the β2GPI domain comprises the amino acid sequence shown in SEQ ID NO.40 or SEQ ID NO.41, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

11. Diagnostic or therapeutic reagent kits, comprising: The antibody or antigen-binding fragment thereof according to any one of claims 1-4, the biological material according to claim 5, the conjugate according to any one of claims 7-8, or the pharmaceutical composition according to any one of claims 9-10.

12. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-4, the biomaterial according to claim 5, the conjugate according to any one of claims 7-8, or the pharmaceutical composition according to any one of claims 9-10 in any one of m1)-m8): m1) Prepare products for diagnosing β2GPI-related diseases or symptoms; m2) Prepare products for the prevention and / or treatment of β2GPI-related diseases or symptoms; m3) Prepare products for detecting the presence or level of β2GPI or proteins containing the D1 domain of β2GPI in samples; m4) detects the presence or level of β2GPI or proteins containing the D1 domain of β2GPI; m5) to prepare products for drug development or screening, said drug for the prevention and / or treatment of β2GPI-related diseases or symptoms; m6) Drug development or screening, wherein the drug is intended to prevent and / or treat β2GPI-related diseases or symptoms; m7) Prepare products for research on the pathogenesis mechanisms of β2GPI-related diseases or symptoms; (m8) Construct a disease or symptom model related to β2GPI.

Images