Anti-LAIR1 antibodies and uses thereof

Abstract

The present invention discloses anti-LAIR1 antibodies and their uses. This invention belongs to the field of biotechnology and aims to provide an isolated antibody or antigen-binding fragment thereof that binds to human leukocyte-associated immunoglobulin-like receptor 1 (LAIR1). The antibodies or antigen-binding fragments provided herein bind to human LAIR1 and exhibit a number of superior properties.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to an anti-LAIR1 antibody and a use thereof. Background Art

[0002] Immunotherapy has brought great hope to all mankind in overcoming cancer, but the current population of beneficiaries of immunotherapy based on PD-1 / PD-L1 is relatively small and still does not meet clinical needs. Therefore, it is extremely important to find the next immunotherapy target at present in order to usher in a new era of immunotherapy.

[0003] Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), also known as cluster of differentiation 305 (CD305), belongs to the immunoglobulin superfamily and the leukocyte-associated inhibitory receptor family. LAIR1 expresses two proteins, LAIR1 and LAIR2. LAIR1 is a type I transmembrane glycoprotein containing an extracellular C2-type Ig-like domain and two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in its cytoplasmic tail. LAIR1 ligands include collagen, complement component 1q (C1q), and surfactant protein D (SP-D). LAIR2 can inhibit the interaction between LAIR1 and its ligands, including collagen, C1q, and SP-D. Its binding affinity to its ligands is higher than that of LAIR1, and it does not exert a functional immune effect. The affinity of collagen to LAIR2 is approximately twice that to LAIR1, and the affinity of C1q to LAIR2 is approximately 16 times that to LAIR1.

[0004] LAIR1 exerts a series of immunosuppressive functions in the body, thereby regulating the function of immune cells. For example, LAIR1 can inhibit the killing effect of NK cells on target cells, and can still exert a significant inhibitory effect even under some positive activation conditions such as strong activation signals such as CD2, CD16, CD69, CD226, etc. At the same time, LAIR1 is also an inhibitory receptor for T cells, which can inhibit the cytotoxic activity of effector T cells and the proliferation of CD4+T cells when CD3 cross-linked or antigen stimulated, and downregulate the production of IL-2 and IFN-γ, while inducing the secretion of TGF-β. LAIR1 can also inhibit CD28 +LAIR1 crosslinking contributes to the cytotoxic function of T cells, leading to reduced B cell antigen receptor (BCR)-induced calcium mobilization and downregulation of Ig and cytokine production. Furthermore, LAIR1 can inhibit cytokine-mediated signaling. This LAIR1-mediated inhibition occurs through protein tyrosine phosphatases 1 / 2 (SHP-1 / 2) and through recruitment of Src carboxyl-terminal kinase (Csk), which inactivates Src family kinases.

[0005] In normal human tissues, LAIR1 is highly expressed in the appendix, bone marrow, lymph nodes, and placenta, moderately expressed in the lung, spleen, and tonsils, and virtually absent in other tissues. LAIR1 is expressed in nearly all immune system cells, including NK cells, T cells, B cells, monocytes, dendritic cells, and macrophages. Compared with normal tissues, LAIR1 expression is increased in various tumors, including breast cancer, renal cell carcinoma, hepatocellular carcinoma, pancreatic cancer, melanoma, gastric cancer, thymoma, esophageal cancer, and acute myeloid leukemia. High LAIR1 expression in tumor tissue is associated with poor prognosis in various tumors, including renal cancer, breast cancer, hepatocellular carcinoma, pediatric acute lymphoblastic leukemia (ALL), low-grade brain gliomas, colon adenocarcinoma, and ovarian serous cystadenocarcinoma. Patients with high LAIR1 expression have significantly decreased survival compared with those with low LAIR1 expression. Literature suggests that LAIR1 may be a potential marker for resistance to anti-PD-1 / PD-L1 therapy in melanoma and lung cancer. Studies of targets and immune-related mechanisms have shown that within the tumor microenvironment, LAIR1 binds to its ligands, collagen and C1q (collagen and C1q are highly expressed in tumor tissues, with collagen serving as a diagnostic and prognostic marker for various tumors), to exert immune cell inhibitory functions. This includes inhibiting NK cell- and T cell-mediated cytotoxic activity and inducing the differentiation of M2 anti-inflammatory macrophages, thereby promoting tumor growth. Currently, all investigational drugs are being developed based on this mechanism. Both preclinical descriptive and in vivo validation evidence exists for LAIR1 in tumors such as breast cancer, colorectal cancer, and pancreatic cancer. While data on LAIR1 monotherapy are limited, it exhibits synergistic effects when used in combination with PD-1 monoclonal antibodies or TGF-β / PD-L1 dual antibodies. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an isolated antibody or an antigen-binding fragment thereof that binds to human leukocyte-associated immunoglobulin-like receptor 1 (LAIR1).

[0007] In a first aspect, the present invention provides an antibody or antigen-binding fragment thereof having binding specificity to human leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, and wherein:

[0008] The amino acid sequence of the HCDR1 is shown in SEQ ID NO: 2, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 3, the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 4, the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 6, the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 7, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO: 8.

[0009] In some embodiments, in the above-mentioned antibodies or antigen-binding fragments thereof, the heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 1, 13, 14, 15, and 16, or an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 1, 13, 14, 15, and 16, or a sequence that has one or more amino acid substitutions (e.g., conservative substitutions), deletions, or insertions, or any combination thereof, compared to the amino acid sequence shown in any one of SEQ ID NOs: 1, 13, 14, 15, and 16; and / or

[0010] The light chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 5, 17, 18, and 19, or an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 5, 17, 18, and 19, or a sequence having one or more amino acid substitutions (e.g., conservative substitutions), deletions, or insertions, or any combination thereof, compared to the amino acid sequence shown in any one of SEQ ID NOs: 5, 17, 18, and 19.

[0011] In some embodiments, in any of the above antibodies or antigen-binding fragments thereof:

[0012] 1) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 1; the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 5; or

[0013] 2) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 13; the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19; or

[0014] 3) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 14; the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19; or

[0015] 4) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 15; the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19; or

[0016] 5) The heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 16; the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19.

[0017] In some embodiments, in any of the above antibodies or antigen-binding fragments thereof, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.

[0018] In some embodiments, in any of the above antibodies or antigen-binding fragments thereof, the light chain constant region is a kappa chain or a lambda chain constant region.

[0019] In some embodiments, in any of the above antibodies or antigen-binding fragments thereof, the heavy chain constant region is selected from the IgG, IgM, IgA, IgE or IgD classes.

[0020] In some embodiments, in any of the above antibodies or antigen-binding fragments thereof, the heavy chain constant region is a heavy chain constant region selected from the IgG1, IgG2, IgG3 or IgG4 subclass.

[0021] In some embodiments, in any of the above antibodies or antigen-binding fragments thereof, the antibody or antigen-binding fragment thereof further comprises a human IgG1 heavy chain constant region or a variant thereof, and / or a human κ light chain constant region or a variant thereof.

[0022] In some embodiments, any of the above antibodies or antigen-binding fragments thereof is a chimeric antibody or a humanized antibody.

[0023] In some embodiments, in any of the above antibodies or antigen-binding fragments thereof, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 9; and / or

[0024] The amino acid sequence of the light chain constant region is shown in SEQ ID NO: 10.

[0025] In some embodiments, in any of the above antibodies or antigen-binding fragments thereof, the antigen-binding fragment is a Fab, Fv or scFv fragment.

[0026] In some embodiments, any of the above antibodies or antigen-binding fragments thereof is a monoclonal antibody (including a full-length monoclonal antibody), a polyclonal antibody, or a multispecific antibody (such as a bispecific antibody).

[0027] In a second aspect, the present invention provides a nucleic acid molecule encoding any of the above-described antibodies or antigen-binding fragments thereof;

[0028] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0029] In a third aspect, the present invention provides a recombinant vector comprising the above nucleic acid molecule.

[0030] In a fourth aspect, the present invention provides a recombinant cell comprising the aforementioned nucleic acid molecule and / or the aforementioned recombinant vector, or expressing any of the aforementioned antibodies or antigen-binding fragments thereof.

[0031] In a fifth aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof of the present invention, which comprises culturing a recombinant cell containing a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the present invention under conditions suitable for expressing the antibody.

[0032] In some embodiments, the method further comprises recovering the antibody or antigen-binding fragment thereof from the recombinant cell or culture medium.

[0033] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to the present invention, and a pharmaceutically acceptable carrier.

[0034] In some embodiments, the above-mentioned pharmaceutical composition further comprises a second therapeutic agent; in some embodiments, the second therapeutic agent is a checkpoint inhibitor; in some embodiments, the checkpoint inhibitor is selected from one or more of the following: T cell checkpoint inhibitors, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, anti-LAG-3 antibodies, and anti-TGF-β antibodies.

[0035] In a seventh aspect, the present invention provides use of any of the above-described antibodies or antigen-binding fragments thereof, any of the above-described nucleic acid molecules, any of the above-described recombinant vectors, any of the above-described recombinant cells, and / or any of the above-described pharmaceutical compositions in the preparation of any of the following products:

[0036] (1) Testing of LAIR1 products;

[0037] (2) Products that stimulate or enhance immune responses;

[0038] (3) Products for the prevention and / or treatment of diseases;

[0039] Preferably, the disease is selected from one or more of the following: breast cancer, renal cell carcinoma, hepatocellular carcinoma, colorectal cancer, pancreatic cancer, melanoma, gastric cancer, thymoma, esophageal cancer, acute myeloid leukemia, and lung cancer.

[0040] The antibodies or antigen-binding fragments thereof provided by the present invention bind to human LAIR1 and exhibit many excellent properties, including the following:

[0041] 1. The antibodies or antigen-binding fragments of the present invention bind to human LAIR1, cynomolgus monkey LAIR1, primary human CD3 + High-affinity binding of T cells can modulate immune responses and suggests that cynomolgus monkeys may be a relevant species for preclinical studies;

[0042] 2. The antibodies of the present invention do not bind to LAIR2 of the same family. LAIR2 is a soluble protein. Not binding to LAIR2 can accurately target LAIR1, avoiding the reduction of drug efficacy caused by off-target effects;

[0043] 3. The antibodies of the present invention can block the binding to collagen. Collagen transmits inhibitory signals through LAIR1 (such as in the tumor microenvironment). Antibody blocking can relieve immunosuppression and enhance anti-tumor immunity.

[0044] 4. Can activate primary human CD3 + T cell proliferation and IL-2 and IFN-γ secretion, thereby stimulating T cell activation to trigger tumor immune response;

[0045] 5. Activate collagen-polarized monocyte-derived dendritic cells to secrete CCL4. CCL4, as an inflammatory cytokine, can recruit more lymphocytes to the tumor inflammation site to exert an immune response. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1A It represents the binding activity of mouse monoclonal antibody against human LAIR1 to human LAIR1 protein.

[0047] Figure 1B The binding activity of the mouse monoclonal antibody against human LAIR1 to the cynomolgus monkey LAIR1 protein.

[0048] Figure 1C The binding activity of mouse monoclonal antibody against human LAIR1 to human LAIR2 protein.

[0049] FIG2 shows the binding activity of anti-human LAIR1 mouse monoclonal antibody to human LAIR1-expressing cell lines.

[0050] Figure 3 The binding activity of the mouse monoclonal antibody against human LAIR1 to the cell line expressing cynomolgus monkey LAIR1 is shown.

[0051] Figure 4 This is a mouse monoclonal antibody against human LAIR1 that blocks the interaction between human LAIR1 protein and collagen type I.

[0052] Figure 5 The binding activity of anti-human LAIR1 mouse monoclonal antibodies mAb003 and mAb013 to the CHOK1 cell line expressing human LAIR1 is shown.

[0053] Figure 6 The mouse monoclonal antibodies mAb003 and mAb013 against human LAIR1 were combined with primary human CD3 + T cell binding activity.

[0054] Figure 7A mAb003 and mAb013, mouse monoclonal antibodies against human LAIR1, block the interaction between LAIR1 and collagen type I.

[0055] Figure 7B mAb003 and mAb013, mouse monoclonal antibodies against human LAIR1, block the interaction between LAIR1 and collagen type III.

[0056] Figure 7CmAb003 and mAb013, mouse monoclonal antibodies against human LAIR1, block the interaction between LAIR1 and collagen type IV.

[0057] Figure 8 The anti-human LAIR1 positive control antibody NGM438 and the anti-human LAIR1 mouse monoclonal antibodies mAb003 and mAb013 inhibited the IL-2 secretion activity of T cells activated by CD3 (OKT3) and anti-CD28.

[0058] FIG9A shows the binding activity of anti-human LAIR1 chimeric antibody ch75A81E9 to cell lines expressing human LAIR1.

[0059] Figure 9B The binding activity of the anti-human LAIR1 chimeric antibody ch75A81E9 to the cell line expressing cynomolgus monkey LAIR1 is shown.

[0060] FIG10A shows the binding activity of anti-human LAIR1 humanized antibodies to human LAIR1 protein.

[0061] FIG10B shows the binding activity of anti-human LAIR1 humanized antibodies to cynomolgus monkey LAIR1 protein.

[0062] Figure 10C shows the interaction between humanized antibodies against human LAIR1 and primary human CD3 + T cell binding activity.

[0063] FIG11A shows the binding activity of anti-human LAIR1 humanized antibodies to cell lines expressing human LAIR1.

[0064] Figure 11B The binding activity of the humanized antibody against human LAIR1 to the cell line expressing cynomolgus monkey LAIR1 is shown.

[0065] Figure 12A Humanized antibodies against human LAIR1 activate primary human CD3 + T cell (donor LP230111009) proliferation activity.

[0066] Figure 12B Humanized antibodies against human LAIR1 activate primary human CD3 + T cell (donor LP230112007) proliferation activity.

[0067] Figure 12C Humanized antibodies against human LAIR1 activate primary human CD3 + IL-2 secretion activity of T cells (donor LP230111009).

[0068] Figure 12D Humanized antibodies against human LAIR1 activate primary human CD3 + IL-2 secretion activity of T cells (donor LP230112007).

[0069] Figure 12E Humanized antibodies against human LAIR1 activate primary human CD3 + IFN-γ secretion activity of T cells (donor LP230111009).

[0070] Figure 12F Humanized antibodies against human LAIR1 activate primary human CD3 + IFN-γ secretion activity of T cells (donor LP230112007).

[0071] Figure 13 The activity of a humanized antibody against human LAIR1 on CCL4 secretion by collagen-polarized monocyte-derived dendritic cells. DETAILED DESCRIPTION

[0072] The present invention will be further described below with reference to specific examples. It should be understood that the following examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Unless otherwise specified, the techniques used in the examples are conventional in the art, or in accordance with the experimental methods recommended by the kit and instrument manufacturers. The reagents and biological materials used in the examples are all commercially available unless otherwise specified.

[0073] Abbreviations and definitions

[0074] Unless otherwise specified, the following terms shall have the meanings set forth below. Other terms or abbreviations have meanings commonly known in the art.

[0075] As used herein, the term "antibody" is generally an immunoglobulin molecule consisting of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as kappa (κ) and lambda (λ) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 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 domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating 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. The VH and VL regions can also be subdivided into regions with high variability (called complementarity determining regions (CDRs)), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen binding site. The allocation of amino acids to various regions or domains can follow the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883. An "antibody" refers to any form of an antibody that exhibits a desired biological activity (e.g., inhibition of ligand binding to its receptor or by inhibiting ligand-induced receptor signaling). Therefore, "antibody" is used in its broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies), fully human, humanized, primatized, chimeric antibodies, single-chain antibodies, and the like.

[0076] "Hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region comprises the following amino acid residues: amino acid residues from the "complementarity determining regions" or "CDRs" as defined by sequence alignments. "Framework" residues or "FR" residues are the variable domain residues other than the hypervariable region residues as defined herein.

[0077] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003).

[0078] "Antigen-binding fragment" refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of their structure, antibody fragments bind to the same antigen recognized by the intact antibody. The term "antigen-binding fragment" includes aptamers, Spiegelmers, and diabodies. The term "antigen-binding fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by binding to a specific antigen to form a complex.

[0079] A "Fab fragment" consists of a light chain and the CH1 and variable region of a heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0080] The "Fc region" contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domain.

[0081] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0082] A "single-chain Fv antibody" (or "scFv antibody") refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see U.S. Patent No. 6,423,538.

[0083] Those skilled in the art will appreciate that antibody heavy chains are classified into gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with several subclasses (e.g., γ1-γ4). The properties of these chains determine the "class" of the antibody, IgG, IgM, IgA, IgD, or IgE, respectively. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, and IgG4, are well characterized, and the functional specificities they confer are also known. All immunoglobulin classes are within the scope of the present disclosure. In some embodiments, the immunoglobulin molecule is of the IgG class. IgG typically comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of approximately 53,000-70,000 daltons. These four chains are connected by disulfide bonds in a "Y" configuration, with the light chains originating at the mouth of the "Y" and continuing through the variable region to surround the heavy chains. Antibodies in the IgG1 form are a subclass of IgG, and their heavy chains are of the γ1 subtype. In some embodiments, the antibodies disclosed herein are IgG1.

[0084] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region comprises at least one of the following: a CH1 domain, a hinge (e.g., an upper hinge region, a middle hinge region, and / or a lower hinge region) domain, a CH2 domain, a CH3 domain, or variants or fragments thereof. For example, an antigen-binding polypeptide for use in the present disclosure may comprise: a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain; or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the present disclosure comprises a polypeptide chain comprising a CH3 domain. Furthermore, an antibody for use in the present disclosure may lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). As described above, those of ordinary skill in the art will appreciate that the heavy chain constant region can be modified such that it differs in amino acid sequence from a naturally occurring immunoglobulin molecule.

[0085] An "isolated antibody" is an antibody that has been separated from all or part of the components of its natural environment. Contaminating components of its natural environment are substances that may interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified to the following degree: (1) greater than 95% by weight of the antibody, such as greater than 99% by weight, as determined by the Lowry method; (2) a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by spinning cup sequenator; or (3) homogeneity as determined by SDS-PAGE under reducing or non-reducing conditions with Coomassie blue or silver staining. An isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will be absent. An isolated antibody is typically prepared by at least one purification step. In some embodiments, the isolated antibody is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99% pure, or a range (including the endpoints) between any two of these values, or any value therein.

[0086] "Nucleic acid" or "polynucleotide" refers to a polymeric molecule composed of the individual nucleotides adenine (A), cytosine (C), guanine (G), thymine (T) (or uracil (U) in RNA), such as DNA, RNA, or modifications thereof. A nucleic acid molecule can be a natural nucleic acid molecule or a synthetic nucleic acid molecule, or a combination of one or more natural nucleic acid molecules and one or more synthetic nucleic acid molecules. Examples of nucleic acids include, but are not limited to, genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.

[0087] An "isolated nucleic acid molecule" is a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule. An isolated nucleic acid molecule is different from its naturally occurring form or environment. Thus, an isolated nucleic acid molecule is distinguished from the nucleic acid molecule present in its natural cell. However, an isolated nucleic acid molecule includes nucleic acid molecules contained in cells that normally express an antibody, for example, where the nucleic acid molecule is located in a chromosomal location that is different from the chromosomal location of the natural cell.

[0088] As used herein, the term "identity" can be assessed visually or using computer software (e.g., the software programs described in Ausubel et al., eds. (2007), in Current Protocols in Molecular Biology). When a position in the compared sequences is occupied by the same base or amino acid, the molecules are identical at that position. Identity between two or more sequences can be expressed as a percentage (%), which can be used to assess identity between related sequences. A polynucleotide sequence or amino acid sequence that has a certain percentage (e.g., 90%, 95%, 98%, or 99%) of "sequence identity" with another sequence means that, when the sequences are aligned, that percentage of bases or amino acids are the same in the two sequences being compared.

[0089] "Monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, the individual antibodies comprising the population being identical. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include multiple different antibodies directed against multiple different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0090] The term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is derived from one source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0091] "Immune cells" include cells that have hematopoietic origin and play a role in immune responses. Immune cells include: B lymphocytes, T lymphocytes, natural killer cells, monocytes, macrophages, eosinophils, mast cells, basophils and granulocytes.

[0092] As used herein, a sequence "variant" refers to a sequence that differs from the indicated sequence at one or more amino acid residues but retains the biological activity of the resulting molecule.

[0093] "Amino acids" refer to organic compounds containing both an amino group and a carboxyl group, such as α-amino acids, which can be encoded by nucleic acids directly or in the form of precursors. A single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). The fact that the same amino acid can be encoded by different codons is known as the "degeneracy of the genetic code." Amino acids include both natural and unnatural amino acids. Natural amino acids include alanine (three-letter code: Ala, one-letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0094] "Conservatively substituted variants" or "conservative amino acid substitutions" are amino acid substitutions known to those skilled in the art that are made without generally altering the biological activity of the resulting molecule. In general, it is recognized by those skilled in the art that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter the biological activity. Conservative substitutions can be made with amino acids containing side chains with similar chemical properties, such as: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; and 6) acidic side chains: aspartic acid and glutamic acid.

[0095] As used herein, the term "about" refers to a value that is within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which value depends in part on how the value is measured or determined (i.e., the limitations of the measurement system). Alternatively, "about" can mean a range of up to ±20%, such as ±10%, ±5%, or ±1%. Unless otherwise indicated, when a particular value appears in this application and claims, the meaning of "about" should be assumed to be within an acceptable error range for that particular value.

[0096] "Specific" binding, when referring to a ligand / receptor, antibody / antigen, or other binding pair, refers to a binding reaction that determines the presence or absence of a protein, such as LAIR1, in a heterogeneous population of proteins and / or other biological agents. Thus, under specified conditions, a particular ligand / antigen binds to a particular receptor / antibody and does not bind in significant amounts to other proteins present in the sample.

[0097] The term "encoding" when applied to a polynucleotide refers to a polynucleotide that, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and / or translated to produce mRNA that produces a polypeptide and / or fragment thereof. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced therefrom.

[0098] When used in reference to an animal, human, subject, cell, tissue, organ, or biological fluid, "administering" and "treating" refer to contacting an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administering" and "treating" can refer to, for example, therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating a cell includes contacting an agent with a cell and contacting an agent with a fluid, wherein the fluid is contacted with the cell. "Administering" and "treating" also mean the in vitro and ex vivo treatment of a cell, for example, by an agent, diagnostic agent, binding composition, or by other cells.

[0099] The term "treating" includes amelioration or cessation of a disorder or its symptoms. Treating includes inhibiting, e.g., reducing the overall frequency of occurrence of a disorder or its symptoms.

[0100] The term "preventing" includes avoiding the initiation of a disorder or its symptoms.

[0101] As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of an anti-LAIR1 antibody or antigen-binding fragment thereof that is effective in preventing or alleviating the disease or condition to be treated when it is administered alone or in combination with another therapeutic agent to a cell, tissue or subject. A therapeutically effective dose further refers to an amount of the antibody or antigen-binding fragment thereof sufficient to cause symptom relief, such as treatment, cure, prevention or alleviation of a related medical condition, or to increase the rate of treatment, cure, prevention or alleviation of the symptoms. The effective amount for a specific subject may vary depending on a variety of factors, such as the disease to be treated, the patient's overall health, the method, route and dose of administration, and the severity of side effects. The effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects. When administered to an individual, the therapeutically effective amount refers to the individual ingredient. When administered in combination, the therapeutically effective amount refers to the combined amount of the active ingredients that produces a therapeutic effect, regardless of whether they are administered in combination, sequentially or simultaneously.

[0102] Pharmaceutical composition

[0103] The present invention also provides a pharmaceutical composition comprising an effective dose of an antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

[0104] In some embodiments, the term "pharmaceutically acceptable carrier" refers to a substance approved by a governmental regulatory agency or listed in other generally recognized pharmacopoeias for use in animals (particularly in humans). In addition, a "pharmaceutically acceptable carrier" will generally be any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary.

[0105] The term "carrier" refers to a diluent, adjuvant, excipient or vehicle for treatment used together with the active ingredient. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including oils of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In some embodiments, when the pharmaceutical composition is administered intravenously, the carrier can be water. Saline solutions and aqueous glucose solutions and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein by reference. Such compositions will contain a clinically effective dose of an antibody or antibody fragment, together with a suitable carrier, to provide a dosage form suitable for the patient. The preparation should be suitable for administration mode. The preparation can be packaged in an ampoule, a disposable syringe or a multidose vial made of glass or plastic.

[0106] In some embodiments, the pharmaceutical compositions of the present invention may be administered by any suitable route known in the art, including but not limited to oral, nasal, intradermal, subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, and / or cerebrospinal.

[0107] In some embodiments, the composition is formulated according to conventional procedures as a pharmaceutical composition suitable for intravenous injection into humans. Compositions for intravenous administration are typically solutions in a sterile isotonic aqueous buffer. Pharmaceutical compositions may also include a solubilizer and a local anesthetic, such as lidocaine, to relieve pain at the injection site. Generally, the active ingredients are supplied individually or mixed together in unit dosage form, such as as a dry lyophilized powder or anhydrous concentrate in a sealed container (such as an ampoule or pouch) indicating the amount of active agent. When the composition is administered by infusion, the composition can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, an ampoule of sterile water or saline for injection can be used so that the active ingredients can be mixed prior to administration.

[0108] The antibodies or antigen-binding fragments thereof of the present invention include salts thereof. Pharmaceutically acceptable salts include salts derived from anions such as hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and salts derived from cations such as sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, and procaine.

[0109] DX26 was purchased from BD Pharmingen™, Cat. No. 550810, and is an anti-LAIR1 antibody.

[0110] LA235 N297A is LA-235 in US2019 / 0338026A1, which is an anti-LAIR1 antibody.

[0111] Anti-LAIR2 mAb was purchased from Invitrogen, catalog number: MA5-24041.

[0112] NGM438 is Hz47H1.v4 in WO2021 / 262597A2, which is an anti-human LAIR1 antibody.

[0113] Example 1: Generation of mouse monoclonal antibodies against human LAIR1

[0114] 1. Obtaining hybridomas

[0115] Mice were immunized with LAIR1 protein, fused with SP2 / 0 cells, and positive clones were screened by ELISA to obtain hybridomas. The specific method is as follows:

[0116] Animal Immunization: Human LAIR1 Protein, mIgG2a Tag (ACROBiosystems, Catalog No. LA1-H5253) was used as the immunogen. The protein was diluted to 1 mg / mL in saline and mixed with an equal volume of adjuvant (CFA (Sigma, Catalog No. F5881) or IFA (Sigma, Catalog No. F5506)) (CFA was used for the initial immunization, and IFA was used for subsequent immunizations). Balb / c and SJL female mice (6-8 weeks old, purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were immunized by intraperitoneal injection (50 μg / mouse for the first immunization, 25 μg / mouse for each immunization starting from the second immunization). Immunizations were repeated three or more times at 2-week intervals. Fourteen days after the final immunization, 25 μg of the immunogen was injected intraperitoneally for a pulse immunization. Three days later, spleens were harvested for cell fusion.

[0117] Cell fusion: Mouse spleen cells and Sp2 / 0-Ag14 cells (ATCC No. CRL-1581) were electrofused at a cell number ratio of 2:1 (BTX electrofusion instrument: ECM2001 + ), cultured in 96-well culture plates with HAT medium (Sigma, catalog number: H0262), and hybridoma cell supernatant antibody screening was performed after 10 days.

[0118] ELISA screening of positive clones: 100 μL of hybridoma supernatant was added to each well of a 96-well flat-bottom assay plate (Corning, Catalog No. 9018) coated with human LAIR1 protein (Human LAIR1 / CD305 Protein, His Tag (ACROBiosystems, Catalog No. CD5-H52H1)) and incubated at 37°C for 60 minutes. After washing three times with PBST, secondary antibody (Anti-Mouse IgG-HRP (Sigma, Catalog No. A0168)) was added to each well and incubated at 37°C for 45 minutes. After washing three times with PBST and patting dry, 100 μL of TMB (Inc. Biotech, Catalog No. EL0009) was added to each well. After 15 minutes of color development, the reaction was terminated by adding 50 μL of sulfuric acid to each well. Read the OD value on a microplate reader. Mix the cells in the positive clone culture wells and aspirate them into a centrifuge tube. Add an appropriate amount of culture medium. Mix well and aspirate a small amount of cells for counting. Dilute the hybridoma cells to 5 cells per mL based on the count results. Add 0.2 mL of the diluted cell suspension to each well of a 96-well plate. After culturing for one week, select the culture supernatant containing a single cell colony and test again as described above.

[0119] 2. Preparation of mouse monoclonal antibody against human LAIR1

[0120] After culturing hybridoma cells in serum-free medium for 10 days, the supernatant was collected and purified using a Protein A column (Borgron (Shanghai) Biotechnology Co., Ltd., Catalog No. AA0272) to obtain purified mouse monoclonal antibodies. Table 1 shows the purified mouse monoclonal antibodies against human LAIR1 and the corresponding hybridoma clone numbers.

[0121] Table 1 Mouse monoclonal antibodies against human LAIR1

[0122]

[0123] 3. Functional characterization of mouse monoclonal antibodies against human LAIR1

[0124] (1) Binding activity of mouse monoclonal antibodies against human LAIR1 to human LAIR1, cynomolgus monkey LAIR1, and human LAIR2 proteins

[0125] The coating protein was human LAIR1 protein (Human LAIR1 / CD305 Protein, Fc Tag (ACROBiosystems, Catalog No.: LA1-H5252)), cynomolgus monkey LAIR1 protein (Recombinant Cynomolgus LAIR1 Fc Chimera Protein, CF (R&D Systems, Catalog No.: 10226-LR-050)), or human LAIR2 protein (Recombinant Human LAIR2 Fc Chimera Protein, CF (R&D Systems, Catalog No.: 10166-LR-050)) at a concentration of 1 μg / mL. The plates were coated overnight at 4°C with 100 μL / well. After washing three times with PBST (containing 0.05% Tween20), 2% BSA was added at 300 μL / well and incubated at 37°C for 1 hour. 1. Add PBST (containing 0.05% Tween20) to the wells and wash three times; prepare 660 μL / well of the mouse monoclonal antibody against human LAIR1 and the positive control antibody (DX26, LA235N297A, Anti-LAIR2 mAb) to a stock solution with a concentration of 66.667 nM using blocking solution; dilute the stock solution with blocking solution at a dilution factor of 3, for a total of 12 steps; add the antibody at 100 μL / well and incubate at 37°C for 1 hour; add PBST (containing 0.05% Tween20) at 200 μL / well and wash three times; add the secondary antibody (Anti-Mouse IgG-HRP (Sigma, Catalog No.: A0168), 1:5000 dilution) to each well at 100 μL / well and incubate at room temperature for 1 hour; add PBST (containing 0.05% Tween20) at 200 μL / well and wash three times; pat dry, add 50 μL The TMB colorimetric solution was used for color development for approximately 15 minutes, and then the reaction was terminated with sulfuric acid. OD450 was read on a microplate reader. Data were analyzed using Graphpad Prism 8.0 software, with the logarithm of the antibody concentration as the x-axis and the corresponding OD450 value as the y-axis. A four-parameter regression model was used to fit the antibody dose-effect curve and calculate the EC50. The results are shown in the figure. Figure 1A 、 Figure 1B 、 Figure 1C and as shown in Table 2.

[0126] Table 2 Binding activity of mouse monoclonal antibodies against human LAIR1 to human LAIR1, cynomolgus monkey LAIR1, and human LAIR2 proteins

[0127]

[0128] The results showed that mAb003, mAb004, mAb012 and mAb013 all had strong binding activity to human and cynomolgus macaque LAIR1 proteins; in addition, except for mAb004 which had weak binding to human LAIR2 protein, the other three mouse monoclonal antibodies against human LAIR1 did not bind to human LAIR2.

[0129] (2) Binding experiments of mouse monoclonal antibodies against human LAIR1 with cell lines endogenously expressing human LAIR1 and cell lines expressing cynomolgus monkey LAIR1

[0130] The concentration of Jurkat T cell line endogenously expressing human LAIR1 (Jiman Biotechnology (Shanghai) Co., Ltd., Catalog No.: GM-C03922) or CHOK1 cell line expressing cynomolgus macaque LAIR1 (CynoLAIR1-CHOK1 (Kangyuan Broad Biotechnology (Beijing) Co., Ltd., Catalog No.: KC-2190)) was adjusted to 1×10 6 Cells were plated at a concentration of 100 μL / well in a 96-well U-bottom plate. After centrifugation, the supernatant was discarded. Mouse monoclonal antibodies against human LAIR1 and positive control antibodies (DX26, LA235 N297A, and NGM438) were diluted to a starting working concentration of 200 nM in FACS buffer (PBS containing 1% FBS). A 1:3 serial dilution was then performed in FACS buffer for a total of 12 concentration points (including the final point containing no antibody). Cells were resuspended by adding 100 μL / well of the serially diluted antibodies to the plate, pipetting to mix thoroughly, and incubating at 4°C for 1 hour. After incubation, the cells were centrifuged and washed three times with FACS buffer. The AlexaFlour-647 labeled anti-mouse secondary antibody (Invitrogen, catalog number: A-31571) was diluted 1:1000 with FACS buffer (PBS solution containing 1% FBS), 100 μL of secondary antibody dilution was added to each well, the cell pellet was resuspended, pipetting and mixing were carried out, and the cells were incubated at 4°C for about 45 minutes. After incubation, the cells were centrifuged and washed three times with FACS buffer. FACS buffer was added to the well plate at 100 μL / well to resuspend the cells. The mean fluorescence intensity (MFI) was read by flow cytometer (BDCelesta), and the experimental data were analyzed by Graphpad Prism 8.0 software. The logarithm of the antibody concentration was used as the x-axis, and the corresponding MFI value was used as the y-axis. A four-parameter regression model was selected to fit the antibody dose-effect curve and calculate the EC50. The results are shown in Figure 2. Figure 2 、 Figure 3 and shown in Table 3.

[0131] Table 3 Binding of anti-human LAIR1 mouse monoclonal antibodies to human LAIR1-expressing cell lines and cynomolgus monkey LAIR1-expressing cell lines

[0132]

[0133] The results showed that mAb003, mAb012, and mAb013 all had good binding to the Jurkat T cell line. In addition, mAb003 and mAb013 had good binding to the cynomolgus monkey CynoLAIR1-CHOK1 cell line.

[0134] (3) Anti-human LAIR1 mouse monoclonal antibody blocks the interaction between human LAIR1 protein and collagen type I

[0135] The coating protein was human collagen type I protein (Abcam, catalog number: ab7533) at a concentration of 2 μg / ml, and the plate was coated with 100 μL / well at 4°C overnight; 2% BSA was added at 150 μL / well and incubated at room temperature for 1 hour; PBST (containing 0.05% Tween20) was added at 200 μL / well and washed three times; mouse monoclonal antibody against human LAIR1 and positive control antibody (DX26, LA235 N297A) were prepared into 210 μL of 400 nM stock solution with blocking solution; the stock solution was diluted with blocking solution at a dilution factor of 3, with a total of 12 gradients; hLAIR1-mFc protein (ACROBiosystems, catalog number: LA1-H5253) was diluted with blocking solution to a concentration of 2 μg / mL, and the antibody solution and hLAIR1-mFc solution were incubated together with equal volumes, and then the mixture was diluted with 100 μL of blocking solution. μL was added to the well plate and incubated at room temperature for 1 hour; PBST (containing 0.05% Tween20) was added at 200 μL / well to wash three times; secondary antibody (Anti-Mouse IgG-HRP (Sigma, Catalog No.: A0168), 1:5000 dilution) was added to each well at 100 μL / well and incubated at room temperature for 1 hour; PBST (containing 0.05% Tween20) was added at 200 μL / well to wash three times; after patting dry, 100 μL TMB color development solution was added, color was developed for about 15 minutes, and then the reaction was terminated with sulfuric acid. OD450 was read on a microplate reader. Graphpad Prism 8.0 software was used to analyze the data, with the logarithm of the antibody concentration as the x-axis and the corresponding OD450 value as the y-axis. A four-parameter regression model was selected to fit the antibody dose-effect curve and calculate IC50. The results are shown in the figure. Figure 4 and as shown in Table 4.

[0136] Table 4 Activity of anti-human LAIR1 mouse monoclonal antibodies in blocking the interaction between human LAIR1 protein and collagen type I

[0137]

[0138] The results showed that mAb003, mAb004, mAb012, and mAb013 all had the activity of blocking the interaction between human LAIR1 protein and Collagentype I.

[0139] (4) mAb003 and mAb013 and primary human CD3 + Binding experiments between T cells and CHOK1 cell lines expressing human LAIR1

[0140] Based on the above experimental results of binding to human LAIR1, cynomolgus monkey LAIR1, human LAIR2, Jurkat T cells, and CynoLAIR1-CHOK1 and blocking human LAIR1 and collagen type I, mAb003 and mAb013 were selected for further characterization.

[0141] Primary human CD3 + T cells (derived from human CD3 + The concentration of T cells (PBMC from TPCS, Catalog No. PB050C) or CHOK1 cell line expressing human LAIR1 (hLAIR1-CHOK1 (Kangyuan Broad Biotechnology (Beijing) Co., Ltd., Catalog No. KC-1370)) was adjusted to 1 × 10 6 Cells / mL, 100 μL / well was plated in a 96-well U-bottom plate, and the supernatant was discarded after centrifugation. Mouse monoclonal antibody against human LAIR1 and negative control antibody (mIgG1) were diluted to a starting working concentration of 200 nM (for hLAIR1-CHOK1 cells) or 100 nM (for primary human CD3 +T cells), and then diluted with FACS buffer at a gradient of 1:3, for a total of 12 concentration points (including the last concentration point without antibody solution). 100 μL / well of the gradient diluted antibody was added to the well plate to resuspend the cells, pipetted to mix, and incubated at 4°C for 1 hour. After incubation, the cells were centrifuged and washed three times with FACS buffer. AlexaFlour-647-labeled anti-mouse secondary antibody (Jackson ImmunoResearch, cat. no. 115-605-071) was diluted 1:1000 with FACS buffer (PBS solution containing 1% FBS), 100 μL of secondary antibody dilution was added to each well, the cell pellet was resuspended, pipetted to mix, and incubated at 4°C for about 45 minutes. After incubation, the cells were centrifuged, washed three times with FACS buffer, and 100 μL / well of FACS buffer was added to the well plate to resuspend the cells. The mean fluorescence intensity (MFI) was read by flow cytometry (BECKMAN COULTER cytoFLEX). The experimental data were analyzed using Graphpad Prism 8.0 software. The logarithm of the antibody concentration was used as the x-axis and the corresponding MFI value was used as the y-axis. A four-parameter regression model was used to fit the antibody dose-effect curve and calculate the EC50. The results are shown in Figure 2. Figure 5 and Figure 6 shown.

[0142] The results showed that both mAb003 and mAb013 could bind to hLAIR1-CHOK1 cells, with an EC50 of 0.52 nM and a maximum MFI of 81176 for mAb003 and 0.67 nM and a maximum MFI of 133985 for mAb013. + For T cells, the EC50 of mAb003 was 0.89 nM, with a maximum MFI of 33233; the EC50 of mAb013 was 0.21 nM, with a maximum MFI of 54996.

[0143] (5) mAb003 and mAb013 block the interaction between human LAIR1 protein and different collagens

[0144] The coating protein was human collagen type I protein (Abcam, Catalog No.: ab7533), collagen type III protein (Abcam, Catalog No.: ab7535), or collagen type IV protein (Abcam, Catalog No.: ab7536) at a concentration of 2 μg / mL, and the plates were coated overnight at 4°C with 100 μL / well. 2% BSA was added at 150 μL / well and incubated at room temperature for 1 hour. PBST (containing 0.05% Tween20) was added at 200 μL / well and washed three times. Mouse monoclonal antibody against human LAIR1, positive control antibody (NGM438), and negative control antibody (mIgG1) were prepared into 990 μL of a 400 nM stock solution using blocking buffer. The stock solution was diluted with blocking buffer by a dilution factor of 3 for a total of 12 steps. hLAIR1-mFc protein (ACROBiosystems, Catalog No.: LA1-H5253) was diluted with blocking buffer to a concentration of 2 After incubating equal volumes of antibody solution and hLAIR1-mFc solution at 4 μg / mL or 4 μg / mL, 100 μL of the mixture was added to the well plate and incubated at 37°C for 1 hour. The wells were washed three times with PBST (containing 0.05% Tween 20) at a rate of 200 μL / well. Secondary antibody (Anti-Mouse IgG-HRP (Sigma, Catalog No. A0168), diluted 1:5000) was added to each well at a rate of 100 μL / well and incubated at room temperature for 1 hour. The wells were washed three times with PBST (containing 0.05% Tween 20) at a rate of 200 μL / well. After patting dry, 100 μL of TMB color development solution was added and color was developed for approximately 15 minutes. The reaction was then terminated with sulfuric acid. OD450 values ​​were read on a microplate reader. The data were analyzed using Graphpad Prism 8.0 software. The logarithm of the antibody concentration was used as the x-axis and the corresponding OD450 value was used as the y-axis. A four-parameter regression model was used to fit the antibody dose-effect curve and calculate the IC50. The results are shown in the figure. Figure 7A 、 Figure 7B 、 Figure 7C and as shown in Table 5.

[0145] Table 5 Activity of anti-human LAIR1 mouse monoclonal antibodies in blocking the interaction between LAIR1 protein and different collagens

[0146]

[0147] The results showed that both mAb003 and mAb013 could block the interaction between human LAIR1 protein and collagen type I, type III, and type IV, and their IC50 values ​​were comparable to that of the positive control NGM438.

[0148] (6) Effects of mAb003 and mAb013 on activation of primary human CD3 + T cell IL-2 secretion activity

[0149] Human collagen type I protein (Abcam, Catalog No. ab7533) and a series of concentration gradients of OKT3 (Biolegend, Catalog No. 317326) were pre-coated in a 96-well plate. 100 μL / well was added to the 96-well plate to make the final concentration of human collagen type I protein coating 5 μg / mL and the starting concentration of OKT3 coating 10 μg / mL. The concentrations were diluted 3-fold with DPBS (Gibco, Catalog No. 14190-144) for a total of 6 concentration points. Incubate overnight at 4°C. On the second day, wash the 96-well plate 3 times with DPBS (Gibco, Catalog No. 14190-144), and aspirate the liquid in the wells for the final time. Human CD3 was isolated from frozen human PBMC (TPCS, Catalog No. PB025C). + T cells, anti-CD28 antibody (Invitrogen, catalog number: 16-0289-85) (concentration of 10 μg / mL) was added to the adjusted T cells (concentration 1e6 / mL), and added to a 96-well plate at 100 μL / well. Then, mouse monoclonal antibody against human LAIR1, positive control antibody (NGM438), and negative control antibody (mIgG1) (all at a concentration of 10 μg / mL) were added at 100 μL / well, with a total volume of 200 μL per well. The above plates were incubated at 5% CO2 and 37°C for 3 days. After 3 days, the culture supernatant was collected, and the relative concentration of IL-2 in the above culture supernatant was detected using the hIL-2 HTRF kit (Cisbio, catalog number: 62HIL02PEH). The results are shown in Figure 8 shown.

[0150] The results showed that mAb013 and the positive control NGM438 could upregulate human CD3 + T cell IL-2 secretion, activation of primary human CD3 + In T cells, mAb013 activity was comparable to the positive control NGM438.

[0151] Example 2: Sequencing of mouse monoclonal antibodies against human LAIR1 and functional identification of chimeric antibodies

[0152] 1. Sequencing of mouse monoclonal antibodies against human LAIR1 and preparation of chimeric antibodies

[0153] Based on the results of Example 1, mAb013 (also referred to in the present invention as its corresponding hybridoma clone number 75A81E9) was sequenced, and the heavy chain variable region, light chain variable region and CDR sequences are shown in Table 6.

[0154] Table 6 CDR sequences and variable region sequences of the mouse monoclonal antibody mAb013 against human LAIR1 (determined according to the Kabat protocol)

[0155]

[0156] The light and heavy chain variable regions were separately constructed onto human constant regions (IgG1 / κ, Table 7) to construct the corresponding chimeric antibodies (Table 8), and the sequences were verified by sequencing. Chimeric antibodies are named by appending the prefix "ch" to the corresponding hybridoma clone number. For example, the chimeric antibody obtained in this example using hybridoma clone 75A81E9 was named ch75A81E9 and used for in vitro functional characterization.

[0157] The nucleic acid encoding the chimeric antibody was expressed in Expi293 cells and purified using a Protein A column as follows:

[0158] Expi293 cells expressing chimeric antibodies: One day before transfection, dilute Expi293 cells (Thermo, Cat. No. A14635CN) to a density of 1.5 × 10 6 Cells / mL were cultured in a shaker at 120 rpm at 37°C and 8% CO2. The next day, the viable cell density and survival rate were determined. The cell transfection density should be around 3×10 6cells / mL, with a cell viability greater than 95%. Prepare the PEI / plasmid complex: Mix PEI (1 mg / mL, Polysciences, Catalog No. 24765-1) by inverting. Dilute the ch75A81E9 heavy and light chain plasmids to a concentration of 1 μg / mL in OPM-293CD05 medium (Shanghai Aopmin Biotech Co., Ltd., Catalog No. 81075-001). Gently mix to achieve a 1:1.5 ratio of heavy and light chain plasmids. Dilute the PEI reagent in OPM-293 CD05 medium to a volume of 1 / 20 of the transfection volume. Gently invert to mix. Incubate at room temperature for 5 minutes. Add the diluted PEI reagent to the diluted plasmid and gently invert to mix. Incubate the PEI / plasmid complex at room temperature for 15 minutes, then slowly add the solution dropwise to the transfer shake flask, gently swirling the flask during addition. After transfection, incubate the flask in a shaker at 37°C, 8% CO₂, and 120 rpm. On the second day after transfection (24 hours after transfection), add 10% OPM-293 ProFeed (Shanghai Aopmin Biotech Co., Ltd., Catalog No. F081918) to the flask, gently swirling the flask during addition. Return the flask to the shaker and continue incubation for 5-7 days. Harvest the supernatant.

[0159] Protein A column for antibody purification: Prepare a gravity chromatography column. Open the column cap, place the gasket at the bottom of the column, and tighten. Prepare Protein A medium (Cytiva, Cat. No. 17549801). Calculate the required medium suspension volume based on the target medium volume and the medium suspension ratio: Required medium suspension volume = target medium volume / medium suspension ratio. Vortex the medium thoroughly to ensure complete suspension. Add the medium suspension to the bottom of the gravity chromatography column. Add at least 10 CV of equilibration buffer (PBS) to the column. After equilibration, check the pH at the outlet. If the target pH has not been reached, continue adding equilibration buffer until it reaches the target pH. Slowly add a desired volume of sample to the column. Add at least 10 CV of wash buffer to the column. Slowly add 5 CV of elution buffer (10-50 mM NaAc, pH 3.0-3.5) to the column. Incubate for 3-5 minutes and collect the eluate. Repeat the elution step as needed. Neutralization: Adjust the pH to the target pH with neutralization buffer (1M Tris). Determine the protein concentration using a Nanodrop. Replace the buffer containing the antibody with PBS by ultrafiltration.

[0160] Table 7 Constant region sequences

[0161]

[0162] Table 8 Chimeric antibodies against human LAIR1

[0163]

[0164] 2. Functional characterization of chimeric antibodies against human LAIR1

[0165] Binding assay of anti-human LAIR1 chimeric antibody to cell lines endogenously expressing human LAIR1 and cell lines expressing cynomolgus monkey LAIR1

[0166] The test was performed according to the method (2) in step 3 of Example 1, and the negative control antibody was anti-HEL-Human IgG1 (purchased from Bio-Bio, catalog number: B117901). Figure 9A 、 9B and as shown in Table 9.

[0167] Table 9 Binding of anti-human LAIR1 chimeric antibodies to cell lines expressing human LAIR1 and cell lines expressing cynomolgus monkey LAIR1

[0168]

[0169] The results showed that the anti-human LAIR1 chimeric antibody ch75A81E9 had strong binding activity to Jurkat T cell line and cynomolgus monkey CynoLAIR1-CHOK1 cell line, similar to the positive control antibody NGM438.

[0170] Example 3: Humanization of anti-human LAIR1 antibodies and expression and purification of humanized antibodies

[0171] Using Kabat numbering to identify CDRs, the human germline gene with the highest homology to the mouse sequence was selected as the acceptor framework, and the mouse CDRs were transplanted into the human framework. Based on the importance of amino acids, backmutations were performed, remutating key amino acids in the transplanted framework region to their corresponding mouse counterparts. Several variants were designed for each heavy and light chain.

[0172] 1. Humanization

[0173] The sequence of the murine antibody mAb013 (i.e., 75A81E9) was analyzed and aligned with the human germline genome according to Kabat. The framework region sequences of IGKV1-12*01 were identified as the humanized light chain framework sequence, while the framework region sequences of IGHV3-73*01 were identified as the humanized heavy chain framework sequence. Through CDR-grafting (CDRs were determined using the Kabat notation), the heavy and light chain CDRs were juxtaposed onto the selected humanized framework sequences. Simultaneously, backmutations were designed at key sites within the framework regions to generate several humanized antibody variable regions, as shown in the humanized sequences in Table 10. The heavy and light chain variable region sequences were combined with the human IgG1 constant region (Kabat, human IGHG1*01) and human kappa constant region, respectively, to generate humanized antibodies. The specific heavy and light chain variable region sequence combinations obtained for the humanized antibody 75A81E9 are shown in Table 11.

[0174] Table 10 Humanized sequences of anti-LAIR1 antibody 75A81E9

[0175]

[0176] Table 11 Heavy chain variable region sequences and light chain variable region sequences corresponding to the constructed humanized antibodies

[0177]

[0178] 2. Expression and purification of humanized anti-human LAIR1 antibody

[0179] The light and heavy chain variable regions of the humanized antibodies listed in Table 10 were constructed onto human constant regions (hIgG1 / κ, Table 7) according to the combinations listed in Table 11. Gene synthesis was performed and sequencing confirmed consistency with the designed sequences. Expression in Expi293 cells was performed according to the method in Example 2 and purification was performed using a Protein A column.

[0180] 3. Functional characterization of humanized anti-human LAIR1 antibodies

[0181] (1) Binding activity of anti-human LAIR1 humanized antibody to human LAIR1 and cynomolgus monkey LAIR1 protein

[0182] The coating proteins were human LAIR1 protein (Human LAIR1 / CD305 Protein, His Tag (ACROBiosystems, Catalog No.: CD5-H52H1) and cynomolgus monkey LAIR1 protein (Shanghai Hongcheng Pharmaceutical Co., Ltd., Catalog No.: TP1106) at a concentration of 1 μg / mL, and 100 μL / well was used to coat the plate overnight at 4°C; 2% BSA was added at 150 μL / well and incubated at room temperature for 1 hour; PBST (containing 0.05% Tween20) was added at 200 μL / well and washed three times; anti-human LAIR1 humanized antibody, chimeric antibody ch75A81E9, and negative control antibody anti-HEL-Human IgG1 (purchased from Baiying Bio, Catalog No.: B117901) were prepared into 900 μL of a stock solution with a concentration of 33.333 nM using blocking solution; the stock solution was diluted with blocking solution at a dilution factor of 3, for a total of 8 gradients; Antibody was added at 200 μL / well and incubated at room temperature for 1 hour. PBST (containing 0.05% Tween 20) was added and washed three times at 200 μL / well. Secondary antibody (goat anti-human Fab-HRP (Sigma, Catalog No. A0293-1ML) for coated cynomolgus macaque LAIR1 protein or goat anti-human IgG Fc-HRP (Sigma, Catalog No. A0170-1ML) for coated human LAIR1 protein was added at a dilution of 1:5000) was added to each well at 100 μL / well and incubated at room temperature for 1 hour. PBST (containing 0.05% Tween 20) was added and washed three times at 200 μL / well. After patting dry, 100 μL of TMB color development solution was added and color was developed for approximately 15 minutes, and then the reaction was terminated with sulfuric acid. OD450 was read on a microplate reader. Graphpad Prism was used. The data were analyzed using the 8.0 software. The logarithm of the antibody concentration was used as the x-axis and the corresponding OD450 value was used as the y-axis. A four-parameter regression model was used to fit the antibody dose-effect curve and calculate the EC50. The results are shown in the figure. Figure 10A 、 Figure 10B and as shown in Table 12.

[0183] The results showed that, except for the humanized antibody 75A81E9-hzVH4 hzVL2, the binding ability of all humanized antibodies to human LAIR1 protein and crab-eating macaque LAIR1 protein was comparable to that of the chimeric antibody ch75A81E9.

[0184] (2) Anti-LAIR1 humanized antibody and primary human CD3 + T cell binding assay

[0185] Primary human CD3+ The concentration of T cells (Allcells, catalog number: FPB009-1F-C-10M) was adjusted to 1×10 6 100 μL / well of the diluted antibody was plated in a 96-well U-bottom plate at 100 cells / mL. After centrifugation, the supernatant was discarded. Anti-human LAIR1 humanized antibody, chimeric antibody ch75A81E9, and negative control antibody anti-HEL-Human IgG1 (Bio-Invitrogen, Catalog No. B117901) were diluted to a starting working concentration of 100 nM in FACS buffer (PBS containing 1% FBS). A 1:5 serial dilution was then performed in FACS buffer, resulting in eight antibody concentrations. 100 μL / well of the serially diluted antibody was added to the plate to resuspend the cells, pipetted to mix, and incubated at 4°C for 1 hour. After incubation, the cells were centrifuged and washed three times with FACS buffer. AlexaFlour-647-conjugated anti-human secondary antibody (Invitrogen, Catalog No. A-21445) was diluted 1:1000 in FACS buffer (PBS containing 1% FBS). 100 μL of the secondary antibody dilution was added to each well, and the cell pellet was resuspended, mixed by pipetting, and incubated at 4°C for approximately 45 minutes. After incubation, the cells were centrifuged and washed three times with FACS buffer. T cells were labeled with Brilliant Violet 421™ anti-human CD3 Antibody (Biolegend, Catalog No. 317344), mixed by pipetting, and incubated at 4°C for approximately 30 minutes. After incubation, the cells were centrifuged and washed three times with FACS buffer. 100 μL of FACS buffer was added to the plate to resuspend the cells. The mean fluorescence intensity (MFI) was read by flow cytometry (BD Cellesta) and the experimental data were analyzed using Graphpad Prism 8.0 software. The logarithm of the antibody concentration was used as the x-axis and the corresponding MFI value was used as the y-axis. A four-parameter regression model was used to fit the antibody dose-effect curve and calculate the EC50. Figure 10C and as shown in Table 12.

[0186] The results showed that except for the humanized antibody 75A81E9-hzVH4 hzVL2, all humanized antibodies had high affinity with primary human CD3 + The T cell binding abilities of these antibodies were comparable to those of the chimeric antibody ch75A81E9.

[0187] Table 12 Anti-LAIR1 humanized antibody binding activity

[0188]

[0189] (3) Based on the above-mentioned ability to bind to human LAIR1 protein, cynomolgus monkey LAIR1 protein, primary human CD3 + Based on the T cell binding ability, the five best humanized antibodies 75A81E9-hzVH2 hzVL1, 75A81E9-hzVH3 hzVL1, 75A81E9-hzVH4 hzVL1, 75A81E9-hzVH3 hzVL3, and 75A81E9-hzVH4 hzVL3 were selected for further characterization.

[0190] i. Binding to cell lines endogenously expressing human LAIR1 and cynomolgus monkey LAIR1

[0191] The concentration of Jurkat T cells endogenously expressing human LAIR1 (Jiman Biotechnology (Shanghai) Co., Ltd., Catalog No. GM-C03922) or CHOK1 cell line expressing cynomolgus macaque LAIR1 (CynoLAIR1-CHOK1 (Kangyuan Broad Biotechnology (Beijing) Co., Ltd., Catalog No. KC-2190)) was adjusted to 1×10 6100 μL / well of a 96-well U-bottom plate was plated at 40 cells / mL. After centrifugation, the supernatant was discarded. Five optimal humanized anti-human LAIR1 antibodies, the chimeric antibody ch75A81E9, a positive control antibody (NGM438), and a negative control antibody, anti-HEL-Human IgG1 (Bio-Invitrogen, Catalog No. B117901), were diluted in FACS buffer (PBS containing 1% FBS) to a starting working concentration of 20 nM (for Jurkat T cells endogenously expressing human LAIR1) or 100 nM (for the CHOK1 cell line expressing cynomolgus macaque LAIR1). These were then serially diluted 1:5 in FACS buffer to a total of seven antibody concentration points (for Jurkat T cells endogenously expressing human LAIR1) or eight antibody concentration points (for the CHOK1 cell line expressing cynomolgus macaque LAIR1). Serially diluted antibodies were added to the plate at 100 μL / well to resuspend the cells, pipette to mix, and incubate at 4°C for 1 hour. After incubation, the cells were centrifuged and washed three times with FACS buffer. AlexaFlour-647-conjugated anti-human secondary antibody (Invitrogen, Cat. No. A-21445) was diluted 1:1000 in FACS buffer (PBS containing 1% FBS). 100 μL of the secondary antibody dilution was added to each well, and the cell pellet was resuspended, pipette to mix, and incubate at 4°C for approximately 45 minutes. After incubation, the cells were centrifuged, washed three times with FACS buffer, and then 100 μL / well of FACS buffer was added to resuspend the cells. The mean fluorescence intensity (MFI) was read by flow cytometry (BD Cellesta) and the experimental data were analyzed using Graphpad Prism 8.0 software. The logarithm of the antibody concentration was used as the x-axis and the corresponding MFI value was used as the y-axis. A four-parameter regression model was used to fit the antibody dose-effect curve and calculate the EC50. Figure 11A 、 Figure 11B and as shown in Table 13.

[0192] Table 13 Anti-LAIR1 humanized antibody binding activity to cells

[0193]

[0194] The results showed that the anti-human LAIR1 humanized antibody had strong binding activity to human Jurkat T cells and cynomolgus monkey CynoLAIR1-CHOK1 cell line, similar to the positive control antibody NGM438.

[0195] ii. Activate CD3 + T cell experiments

[0196] In a 96-well plate, pre-coat human collagen type I (Millipore, Catalog No. CC050) and a series of OKT3 concentration gradients (Biolegend, Catalog No. 317326) simultaneously. Add 100 μL / well to the 96-well plate, so that the final concentration of human collagen type I protein coating is 5 μg / mL and the starting concentration of OKT3 coating is 15 μg / mL. Dilute the concentration three-fold with DPBS (Gibco, Catalog No. 14190-144) for a total of 5 concentration points. Incubate overnight at 4°C. On the second day, wash the 96-well plate three times with DPBS (Gibco, Catalog No. 14190-144), aspirating the liquid in the wells for the final time. Rapidly thaw human CD3 in a 37°C water bath. + T cells (Allcells, Catalog No. FPB009-1F-C-10M, Donor 1: LP230111009, Donor 2: LP230112007) were adjusted to a concentration of 1e6 / mL and plated into a 96-well plate at 100 μL / well. Five optimal humanized LAIR1 antibodies, the chimeric antibody ch75A81E9, a positive control antibody (NGM438), and a negative control antibody, anti-HEL-Human IgG1 (Bio-Innovation, Catalog No. B117901), were then added at 100 μL / well, for a total volume of 200 μL per well. The cells were incubated at 37°C with 5% CO2 for 5 days. After 5 days, the culture supernatant was collected and the concentrations of IL-2 and IFN-γ in the culture supernatant were measured using the hIL-2 HTRF kit (Cisbio, Catalog No.: 62HIL02PEH) and the hIFNgamma HTRF kit (Cisbio, Catalog No.: 62HIFNG PEH). The number of viable cells was also measured using the Cell Titer Turbo 2.0 (Adamas Life, Catalog No.: RA-GL11). The results are shown in Figure 2. Figure 12A (Donor 1, cell proliferation assay), Figure 12B (Donor 2, cell proliferation assay), Figure 12C (Donor 1, human IL-2 detection), Figure 12D (Donor 2, human IL-2 detection), Figure 12E (Donor 1, human IFN-γ detection) and Figure 12F (Donor 2, human IFN-γ detection) as shown.

[0197] The results showed that both the humanized antibody and the positive control NGM438 could activate primary human CD3 +T cell proliferation and IL-2 and IFN-γ secretion, among which 75A81E9-hzVH3 hzVL3 activity was comparable to or better than the positive control NGM438.

[0198] iii. Activity assay for CCL4 secretion by collagen-polarized monocyte-derived dendritic cells

[0199] Human monocytes were isolated from human PBMCs (Allcells, Catalog No. FPB004F-C) using a human monocyte kit (Stemcell, Catalog No. 19059). Monocytes were then induced into DCs in 6-well plates with recombinant human GM-CSF (Peprotech, Catalog No. 300-03) and IL-4 (Peprotech, Catalog No. 200-04). 96-well plates were pre-coated with human collagen type I (Millipore, Catalog No. CC050) at 5 μg / mL. On the second day, the 96-well plates were washed three times with DPBS (Gibco, Catalog No. 14190-144), and the liquid was aspirated after the final wash. The induced DCs were harvested, adjusted to a concentration of 5e5 / mL, and plated at 100 μL per well. Then, 100 μL of the optimal five humanized LAIR1 antibodies, a positive control antibody (NGM438), and anti-HEL-Human IgG1 (from Bio-Bio, Catalog No. B117901) were added to each well, for a total volume of 200 μL per well. The cells were incubated at 37°C with 5% CO2 for 3 days. After 3 days, the culture supernatant was collected and the CCL4 concentration in the culture supernatant was measured using a CCL4 HTRF kit (Cisbio, Catalog No. 62HCCL4PEG). The results are shown in Figure 2. Figure 13 shown.

[0200] The results showed that both the humanized antibody and the positive control NGM438 could activate collagen-polarized monocyte-derived dendritic cells to secrete CCL4, and the activity of 75A81E9-hzVH4 hzVL1 was comparable to or better than that of the positive control NGM438.

Claims

1. An antibody or antigen-binding fragment thereof having binding specificity to human leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, the light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, and wherein: The amino acid sequence of the HCDR1 is shown in SEQ ID NO: 2, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 3, the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 4, the amino acid sequence of the LCDR1 is shown in SEQ ID NO: 6, the amino acid sequence of the LCDR2 is shown in SEQ ID NO: 7, and the amino acid sequence of the LCDR3 is shown in SEQ ID NO:

8.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein: The heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1, 13, 14, 15, and 16, or an amino acid sequence that is at least 90% identical to an amino acid sequence as shown in any one of SEQ ID NOs: 1, 13, 14, 15, and 16, or a sequence having one or more amino acid substitutions, deletions, or insertions, or any combination thereof, compared to an amino acid sequence as shown in any one of SEQ ID NOs: 1, 13, 14, 15, and 16; and / or The light chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 5, 17, 18, and 19, or an amino acid sequence that is at least 90% identical to the amino acid sequence as shown in any one of SEQ ID NOs: 5, 17, 18, and 19, or a sequence having one or more amino acid substitutions, deletions, or insertions, or any combination thereof, compared to the amino acid sequence as shown in any one of SEQ ID NOs: 5, 17, 18, and 19.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein: 1) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 5; or 2) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19; or 3) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 14; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19; or 4) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19; or 5) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO:

19. 4 . The antibody or antigen-binding fragment thereof according to claim 1 , further comprising a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof. The antibody or antigen-binding fragment thereof according to claim 4 , wherein the light chain constant region is a kappa chain or a lambda chain constant region.

6. The antibody or antigen-binding fragment thereof according to claim 4, wherein the heavy chain constant region is selected from the IgG, IgM, IgA, IgE or IgD class. 7 . The antibody or antigen-binding fragment thereof according to claim 6 , wherein the heavy chain constant region is a heavy chain constant region selected from the IgG1, IgG2, IgG3 or IgG4 subclass. 8 . The antibody or antigen-binding fragment thereof according to claim 4 , wherein the antibody or antigen-binding fragment thereof further comprises a human IgG1 heavy chain constant region or a variant thereof, and / or a human κ light chain constant region or a variant thereof.

9. The antibody or antigen-binding fragment thereof according to claim 8, wherein the human IgG1 heavy chain constant region is shown in SEQ ID NO: 9, and the human κ light chain constant region is shown in SEQ ID NO:

10. 10 . The antibody or antigen-binding fragment thereof according to claim 9 , wherein the antibody or antigen-binding fragment thereof is a chimeric antibody or a humanized antibody.

11. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.

12. A recombinant vector comprising the nucleic acid molecule according to claim 11.

13. A recombinant cell comprising the nucleic acid molecule of claim 11 and / or the recombinant vector of claim 12, or expressing the antibody or antigen-binding fragment thereof of any one of claims 1 to 10.

14. A method for preparing an antibody or antigen-binding fragment thereof that binds to LAIR1, the method comprising culturing a recombinant cell comprising a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 10 under conditions suitable for expression of the antibody, optionally further comprising recovering the antibody or antigen-binding fragment thereof from the recombinant cell or culture medium.

15. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, and a pharmaceutically acceptable carrier.

16. The pharmaceutical composition of claim 15, wherein the pharmaceutical composition further comprises a second therapeutic agent.

17. The pharmaceutical composition of claim 16, wherein the second therapeutic agent is a checkpoint inhibitor.

18. The pharmaceutical composition according to claim 17, wherein the checkpoint inhibitor is selected from one or more of the following: a T cell checkpoint inhibitor, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, and an anti-TGF-β antibody.

19. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the nucleic acid molecule according to claim 11, the recombinant vector according to claim 12, the recombinant cell according to claim 13, and / or the pharmaceutical composition according to any one of claims 15 to 18 in the preparation of any of the following products: (1) Testing of LAIR1 products; (2) Products for treating diseases, The disease is selected from one or more of the following: breast cancer, renal cell carcinoma, hepatocellular carcinoma, colorectal cancer, pancreatic cancer, melanoma, gastric cancer, thymoma, esophageal cancer, acute myeloid leukemia, and lung cancer.

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