Anti-LAIR1 antibodies and uses thereof

Abstract

The invention discloses an anti-LAIR1 antibody and an application thereof. The invention belongs to the technical field of biology, and aims to provide a separated antibody or an antigen binding fragment of the antibody capable of binding to a human leukocyte-associated immunoglobulin-like receptor 1 (LAIR1). The antibody or the antigen binding fragment thereof provided by the invention is combined with human LAIR1 and shows more excellent characteristics.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to anti-LAIR1 antibodies and their uses. Background Art

[0002] Immunotherapy has brought great hope for all mankind to overcome cancer. However, at present, the immunotherapy mainly based on PD-1 / PD-L1 benefits a small number of patients and still fails to meet the clinical needs. Therefore, it is extremely important to find the next immunotherapy target at present in order to start a new era of immunotherapy.

[0003] Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1) is also known as cluster of differentiation 305 (CD305). LAIR1 belongs to the members of the immunoglobulin superfamily and the leukocyte-associated inhibitory receptor family. LAIR will express two proteins, LAIR1 and LAIR2. LAIR1 is a type I transmembrane glycoprotein, which contains an extracellular C2-type lg-like domain and two immunoreceptor tyrosine-based inhibitory motifs (ITIM) in its cytoplasmic tail region. The ligands of LAIR1 include collagen, complement component 1q (C1q), surfactant protein D (SP-D), etc. LAIR2 can inhibit the interaction between LAIR1 and its ligands (including collagen, C1q, SP-D, etc.). Its binding force with ligands is higher than that of LAIR1 and it does not play an immunological functional role. Among them, the affinity of collagen with LAIR2 is about 2 times that with LAIR1, and the affinity of C1q with LAIR2 is about 16 times that with LAIR1.

[0004] LAIR1 exerts a series of immunosuppressive functions in vivo to regulate the functions of immune cells. For example, LAIR1 can inhibit the killing effect of NK cells on target cells. Even under some strong activation signals such as positive activation conditions of CD2, CD16, CD69, CD226, etc., it can still play an obvious inhibitory role. At the same time, LAIR1 is also an inhibitory receptor of T cells, which can inhibit the cytotoxic activity of effector T cells and the proliferation of CD4+ T cells during CD3 cross-linking or antigen stimulation, and down-regulate the production of IL-2 and IFN-γ, and at the same time induce the secretion of TGF-β. LAIR1 can also inhibit CD28 +The killing function of T cells, LAIR1 crosslinking results in reduced calcium mobilization induced by the B cell antigen receptor (BCR), and downregulation of Ig and cytokine production. In addition, LAIR1 can also inhibit cytokine-mediated signaling. This LAIR1-mediated inhibition can occur through protein tyrosine phosphatase 1 / 2 (SHP-1 / 2), or by recruiting Src carboxyl-terminal kinase (Csk) to inactivate Src family kinases.

[0005] In normal human tissues, LAIR1 is highly expressed in the appendix, bone marrow, lymph, and placenta, moderately expressed in the lung, spleen, and tonsils, and hardly expressed in the remaining tissues. LAIR1 is expressed in almost all immune system cells, including NK cells, T cells, B cells, monocytes, dendritic cells, macrophages, etc. Compared with normal tissues, LAIR1 is overexpressed in various tumors, including breast cancer, renal cell carcinoma, hepatocellular carcinoma, pancreatic cancer, melanoma, gastric cancer, thymoma, esophageal cancer, acute myeloid leukemia, etc. High expression of LAIR1 in tumor tissues is associated with poor prognosis of various tumors, including renal cancer, breast cancer, hepatocellular carcinoma, pediatric acute lymphoblastic leukemia (ALL), low-grade glioma, colorectal adenocarcinoma, ovarian serous cystadenocarcinoma, etc. Compared with low expression of LAIR1, the survival rate of patients with high expression of LAIR1 is significantly reduced. The literature shows that LAIR1 may be a potential marker for resistance to anti-PD-1 / PD-L1 therapy in melanoma and lung cancer. Research on the target and immune-related mechanisms shows that in the tumor microenvironment, LAIR1 binds to ligands such as collagen and C1q (collagen and C1q are highly expressed in tumor tissues, and collagen is a diagnostic and prognostic marker for various tumors) to exert immune cell inhibitory functions, including inhibiting the cytotoxic activities mediated by NK cells and T cells, inducing the differentiation of M2 anti-inflammatory macrophages, etc., and then playing a tumor-promoting role. Currently, the drugs under research are all developed based on this mechanism. There are both preclinical descriptive and in vivo validation evidences for new LAIR1 drugs in tumors such as breast cancer, colorectal cancer, and pancreatic cancer. The single-agent release data of LAIR1 is limited, but it has a synergistic effect when combined with PD-1 monoclonal antibody or TGF-β / PD-L1 bispecific antibody. Summary of the Invention

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

[0007] In a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof that has binding specificity for 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, the heavy-chain variable region comprises heavy-chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, the light-chain variable region comprises light-chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, and wherein: The amino acid sequence of the HCDR1 is as shown in SEQ ID NO: 2, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO: 3, the amino acid sequence of the HCDR3 is as shown in SEQ ID NO: 4, the amino acid sequence of the LCDR1 is as shown in SEQ ID NO: 6, the amino acid sequence of the LCDR2 is as shown in SEQ ID NO: 7, and the amino acid sequence of the LCDR3 is as shown in SEQ ID NO: 8.

[0008] In some embodiments, in the above antibody or antigen-binding fragment thereof, the heavy-chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 1, 13, 14, 15, 16, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in any one of SEQ ID NOs: 1, 13, 14, 15, 16, or a sequence having one or more amino acid substitutions (such as conservative substitutions), deletions, or insertions, or any combination thereof, as compared to the amino acid sequence shown in any one of SEQ ID NOs: 1, 13, 14, 15, 16; and / or The light-chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 5, 17, 18, 19, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity to the amino acid sequence shown in any one of SEQ ID NOs: 5, 17, 18, 19, or a sequence having one or more amino acid substitutions (such as conservative substitutions), deletions, or insertions, or any combination thereof, as compared to the amino acid sequence shown in any one of SEQ ID NOs: 5, 17, 18, 19.

[0009] In some embodiments, in any of the above-described antibodies or antigen-binding fragments thereof: 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 2) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13 or consists thereof; 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 consists thereof; or 3) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 14 or consists thereof; 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 consists thereof; or 4) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15 or consists thereof; 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 consists thereof; or 5) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 16 or consists thereof; 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 consists thereof.

[0010] In some embodiments, in any of the above-mentioned antibodies or antigen-binding fragments thereof, the antibody or antigen-binding fragment further comprises a heavy chain constant region, a light chain constant region, an Fc region or a combination thereof.

[0011] In some embodiments, in any of the above-mentioned antibodies or antigen-binding fragments thereof, the light chain constant region is a κ-chain or λ-chain constant region.

[0012] In some embodiments, in any of the above-mentioned antibodies or antigen-binding fragments thereof, the heavy chain constant region is selected from the IgG, IgM, IgA, IgE or IgD classes.

[0013] In some embodiments, in any of the above-mentioned 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 subclasses.

[0014] In some embodiments, in any of the above-mentioned antibodies or antigen-binding fragments thereof, the antibody or antigen-binding fragment 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.

[0015] In some embodiments, in any of the above-mentioned antibodies or antigen-binding fragments thereof, the antibody or antigen-binding fragment is a chimeric antibody or a humanized antibody.

[0016] In some embodiments, in any of the antibodies or antigen-binding fragments thereof described above, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 9; and / or the amino acid sequence of the light chain constant region is as shown in SEQ ID NO: 10. In some embodiments, in any of the antibodies or antigen-binding fragments thereof described above, the antigen-binding fragment is a Fab, Fv or scFv fragment.

[0017] In some embodiments, in any of the antibodies or antigen-binding fragments thereof described above, the antibody or antigen-binding fragment is a monoclonal antibody (including full-length monoclonal antibody), a polyclonal antibody or a multispecific antibody (such as a bispecific antibody).

[0018] In a second aspect, the present invention provides a nucleic acid molecule encoding any of the antibodies or antigen-binding fragments thereof described above; 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, etc.

[0019] In a third aspect, the present invention provides a recombinant vector comprising the above nucleic acid molecule.

[0020] In a fourth aspect, the present invention provides a recombinant cell comprising the above nucleic acid molecule and / or the above recombinant vector, or expressing any of the antibodies or antigen-binding fragments thereof described above.

[0021] In a fifth aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof of the present invention, the method comprising culturing a recombinant cell comprising a nucleic acid molecule encoding the antibody or antigen-binding fragment of the present invention under conditions suitable for expressing the antibody. In some embodiments, the method further comprises: recovering the antibody or antigen-binding fragment thereof from the recombinant cell or the culture medium.

[0022] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier.

[0023] In some embodiments, the above 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: checkpoint inhibitors of T cells, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, anti-LAG-3 antibodies, anti-TGF-β antibodies.

[0024] In a seventh aspect, the present invention provides the use of any of the above-mentioned antibodies or their antigen-binding fragments, any of the above-mentioned nucleic acid molecules, any of the above-mentioned recombinant vectors, any of the above-mentioned recombinant cells, and / or any of the above-mentioned pharmaceutical compositions in the preparation of any of the following products: (1) A product for detecting LAIR1; (2) A product for stimulating or enhancing the immune response; (3) A product for preventing and / or treating diseases; Preferably, the diseases are 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, lung cancer.

[0025] The antibody or its antigen-binding fragment provided by the present invention binds to human LAIR1 and exhibits many excellent characteristics, including the following aspects: 1. The antibody or its antigen-binding fragment of the present invention binds to human LAIR1, cynomolgus monkey LAIR1, and primary human CD3 + T cells with high affinity, can regulate the immune response, and indicate that cynomolgus monkeys can be used as relevant species for preclinical studies; 2. The antibody of the present invention does not bind to LAIR2 of the same family. LAIR2 is a soluble protein. Not binding to LAIR2 can precisely target LAIR1 and avoid the reduction of drug efficacy caused by off-target effects, etc.; 3. The antibody of the present invention can block the binding to collagen. Collagen transmits inhibitory signals through LAIR1 (such as in the tumor microenvironment). After the antibody blocks, the immunosuppression can be relieved and the anti-tumor immunity can be enhanced; 4. Can activate primary human CD3 + T cell proliferation and the secretion of IL-2 and IFN-γ, thereby stimulating T cell activation to trigger tumor immune responses; 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 immune responses. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1A Is the binding activity of a murine monoclonal antibody against human LAIR1 to human LAIR1 protein.

[0027] Figure 1B Is the binding activity of a murine monoclonal antibody against human LAIR1 to cynomolgus monkey LAIR1 protein.

[0028] Figure 1C Is the binding activity of a murine monoclonal antibody against human LAIR1 to human LAIR2 protein.

[0029] Figure 2 shows the binding activity of a murine monoclonal antibody against human LAIR1 to a cell line expressing human LAIR1.

[0030] Figure 3 It is the binding activity of a murine monoclonal antibody against human LAIR1 to a cell line expressing cynomolgus monkey LAIR1.

[0031] Figure 4 It is the activity of a murine monoclonal antibody against human LAIR1 to block the interaction between human LAIR1 protein and Collagen type I. Figure 5 It is the binding activity of murine monoclonal antibodies mAb003 and mAb013 against human LAIR1 to the CHOK1 cell line expressing human LAIR1.

[0032] Figure 6 It is the binding activity of murine monoclonal antibodies mAb003 and mAb013 against human LAIR1 to primary human CD3 + T cells.

[0033] Figure 7A It is the activity of murine monoclonal antibodies mAb003 and mAb013 against human LAIR1 to block the interaction between LAIR1 protein and Collagen type I. Figure 7B It is the activity of murine monoclonal antibodies mAb003 and mAb013 against human LAIR1 to block the interaction between LAIR1 protein and Collagen type III. Figure 7C It is the activity of murine monoclonal antibodies mAb003 and mAb013 against human LAIR1 to block the interaction between LAIR1 protein and Collagen type IV. Figure 8 It is the activity of anti-human LAIR1 positive control antibody NGM438 and murine monoclonal antibodies mAb003 and mAb013 against human LAIR1 to activate T cell secretion of IL-2 by anti-CD3 (OKT3) and anti-CD28. Figure 9A shows the binding activity of a chimeric antibody ch75A81E9 against human LAIR1 to a cell line expressing human LAIR1.

[0034] Figure 9B It is the binding activity of a chimeric antibody ch75A81E9 against human LAIR1 to a cell line expressing cynomolgus monkey LAIR1.

[0035] Figure 10A shows the binding activity of a humanized antibody against human LAIR1 to human LAIR1 protein.

[0036] Figure 10B shows the binding activity of a humanized antibody against human LAIR1 to cynomolgus monkey LAIR1 protein.

[0037] Figure 10C shows the binding activity of a humanized antibody against human LAIR1 to primary human CD3 + T cells.

[0038] Figure 11A shows the binding activity of a humanized antibody against human LAIR1 to a cell line expressing human LAIR1.

[0039] Figure 11B shows the binding activity of a humanized antibody against human LAIR1 to a cell line expressing cynomolgus monkey LAIR1.

[0040] Figure 12A shows the activity of a humanized antibody against human LAIR1 in activating the proliferation of primary human CD3 + T cells (donor LP230111009). Figure 12B shows the activity of a humanized antibody against human LAIR1 in activating the proliferation of primary human CD3 + T cells (donor LP230112007). Figure 12C shows the activity of a humanized antibody against human LAIR1 in activating the secretion of IL-2 by primary human CD3 + T cells (donor LP230111009). Figure 12D shows the activity of a humanized antibody against human LAIR1 in activating the secretion of IL-2 by primary human CD3 + T cells (donor LP230112007). Figure 12E shows the activity of a humanized antibody against human LAIR1 in activating the secretion of IFN-γ by primary human CD3 + T cells (donor LP230111009). Figure 12F shows the activity of a humanized antibody against human LAIR1 in activating the secretion of IFN-γ by primary human CD3 + T cells (donor LP230112007). Figure 13 shows the activity of a humanized antibody against human LAIR1 in activating the secretion of CCL4 by collagen-polarized monocyte-derived dendritic cells. Detailed implementation manners The present invention will be further described below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate the present invention rather than to limit the scope of the present invention. Unless otherwise specified, the technical means used in the embodiments are all conventional operations in the art, or the experimental methods recommended by the kit and instrument manufacturers. The reagents and biological materials used in the embodiments can be obtained from commercial sources unless otherwise specified.

[0041] Abbreviations and Definitions Unless otherwise stated, the following terms shall have the meanings set forth below. Other terms or abbreviations have meanings well known in the art.

[0042] As used herein, the term "antibody" generally refers to an immunoglobulin molecule composed 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 the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined 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 is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of one domain, CL. The constant domains do not directly participate in the binding of the antibody to the antigen, but exhibit various effector functions, such as mediating the binding of the immunoglobulin 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 further divided into regions with high variability (called complementarity-determining regions (CDRs)), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen-binding site. The assignment of amino acids to each region or domain 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. "Antibody" refers to any form of an antibody that exhibits the desired biological activity (e.g., inhibiting the binding of a ligand to its receptor or inhibiting ligand-induced receptor signal transduction). Thus, "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, etc.

[0043] "Hypervariable region" refers to the antibody amino acid residues responsible for antigen binding. The hypervariable region contains the following amino acid residues: amino acid residues from the "complementary determining region" or "CDR" defined by sequence alignment. "Framework" residues or "FR" residues are variable domain residues other than the hypervariable region residues defined herein.

[0044] As used herein, the term "complementary 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, those skilled in the art will readily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0045] "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 its structure, the antibody fragment binds to the same antigen recognized by the intact antibody. The term "antigen-binding fragment" includes aptamers, mirror image isomers, and bispecific antibodies. The term "antigen-binding fragment" also includes any synthetic or genetically engineered protein that acts as an antibody by binding to a specific antigen to form a complex.

[0046] "Fab fragment" consists of a light chain and the CH1 and variable regions of a heavy chain. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0047] "Fc region" contains two heavy chain fragments that include the CH2 and CH3 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.

[0048] The "Fv region" contains the variable regions from both the heavy and light chains, but lacks the constant regions.

[0049] A "single-chain Fv antibody" (or "scFv antibody") refers to an antibody fragment that contains the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain. Generally, the Fv polypeptide additionally contains a polypeptide linker between the VH and VL domains, which enables the scFv to form the required structure for antigen binding. For a review of scFv, see U.S. Patent No. 6,423,538.

[0050] Those skilled in the art will understand that the classes of antibody heavy chains include gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), and there are also some subclasses (e.g., γ1-γ4). The nature of this chain determines the "class" of the antibody to be IgG, IgM, IgA, IgD, or IgE, respectively. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, have been well characterized and the functional specificities conferred are also known. All immunoglobulin classes are within the scope of the protection disclosed in the present invention. In some embodiments, the immunoglobulin molecule is of the IgG class. IgG typically contains 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. These four chains are linked by disulfide bonds in a "Y" configuration, where the light chains start from the "mouth" of the "Y" and continue through the variable regions surrounding the heavy chains. An antibody in the IgG1 form is a subclass of IgG, and its heavy chain is of the γ1 subtype. In some embodiments, the antibody disclosed in the present invention is IgG1.

[0051] As used herein, the term "heavy chain constant region" includes amino acid sequences derived from immunoglobulin heavy chains. 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 a variant or fragment 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, the polypeptide of the present disclosure comprises a polypeptide chain containing a CH3 domain. Additionally, an antibody for use in the present disclosure may lack at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). As described above, one of ordinary skill in the art will understand that the heavy chain constant region can be modified such that it differs in amino acid sequence from a naturally occurring immunoglobulin molecule.

[0052] An "isolated antibody" is an antibody that is separated from all or some of the components of its natural environment. The contaminating components of its natural environment are substances that would interfere with the diagnostic or therapeutic use of the antibody and can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified to the extent that: (1) more than 95% by weight of the antibody, such as more than 99% by weight as determined by the Lowry method; (2) to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spinning cup sequencer; or (3) is determined to be homogeneous by SDS-PAGE stained with Coomassie blue or silver stain under reducing or non-reducing conditions. Isolated antibodies include antibodies in situ within recombinant cells because at least one component of the antibody's natural environment will be absent. Isolated antibodies are generally prepared by at least one purification step. In some embodiments, the purity of the isolated antibody is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or a range between any two of these values (including the endpoints) or any value therein.

[0053] "Nucleic acid" or "polynucleotide" refers to a polymeric molecule composed of individual nucleotides: adenine (A), cytosine (C), guanine (G), thymine (T) (or uracil (U) in RNA), such as DNA, RNA, or their modifications. Nucleic acid molecules can be natural nucleic acid molecules, synthetic nucleic acid molecules, 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 (such as probes, primers, ESTs, 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.

[0054] "Isolated nucleic acid molecule" refers to 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 distinguishable from the nucleic acid molecule present in its natural cell. However, an isolated nucleic acid molecule includes the nucleic acid molecule contained in a cell that normally expresses an antibody, for example, the chromosomal location of the nucleic acid molecule is different from that of the natural cell.

[0055] The term "identity" as used herein can be evaluated by the naked eye or by computer software (such as the software programs described by Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology). When the positions in the sequences being compared are occupied by the same base or amino acid, the molecules are identical at that position. The identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. A polynucleotide sequence or an amino acid sequence having a certain percentage (such as 90%, 95%, 98%, or 99%) of "sequence identity" with another sequence means that when the sequences are aligned, that percentage of the bases or amino acids in the two sequences being compared are the same.

[0056] "Monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, and each antibody constituting the group is identical. Monoclonal antibodies are highly specific and can target a single antigenic site. In addition, in contrast to conventional (polyclonal) antibody preparations that usually include multiple different antibodies directed against multiple different determinants (epitopes), each monoclonal antibody targets only a single determinant on the antigen.

[0057] The term "chimeric antibody" refers to an antibody in which part of the heavy chain and / or light chain is derived from one source or species, while the remaining part of the heavy chain and / or light chain is derived from a different source or species.

[0058] "Immune cells" include cells of hematopoietic origin that play a role in the immune response. Immune cells include: B lymphocytes, T lymphocytes, natural killer cells, monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0059] As used herein, a sequence "variant" refers to a sequence that differs from the shown sequence at one or more amino acid residues but retains the biological activity of the resulting molecule.

[0060] "Amino acid" refers to an organic compound containing both an amino group and a carboxyl group, such as an α-amino acid, which can be encoded directly or in precursor form by nucleic acids. 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 referred to as "degeneracy of the genetic code". Amino acids include natural amino acids 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).

[0061] "Conservative substitution variants" or "conservative amino acid substitutions" refer to amino acid substitutions known to those skilled in the art, and making such substitutions generally does not change the biological activity of the resulting molecule. Generally, it is recognized by those skilled in the art that a single amino acid substitution in a non-essential region of a polypeptide generally does not change the biological activity. Conservative substitutions can be substitutions of amino acids with chemically similar side chains, 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; 6) acidic side chains: aspartic acid and glutamic acid.

[0062] As used herein, the term "about" refers to a value within an acceptable error range of a specific 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 measuring system). Alternatively, "about" can mean a range of up to ±20%, such as a range of ±10%, ±5% or ±1%. Unless otherwise specified, when a specific value appears in the present application and claims, the meaning of "about" should be assumed to be within the acceptable error range of that specific value.

[0063] When referring to a ligand / receptor, antibody / antigen or other binding pair, "specific" binding refers to determining whether a binding reaction of a protein such as LAIR1 exists in a heterogeneous population of proteins and / or other biological reagents. Thus, under the specified conditions, a particular ligand / antigen binds to a particular receptor / antibody and does not bind to other proteins present in the sample in significant amounts.

[0064] When applied to a polynucleotide, the term "encoding" means that a polynucleotide is called "encoding" a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and / or translated to produce an mRNA of a polypeptide and / or a fragment thereof. The antisense strand is the complement of such a nucleic acid and the coding sequence can be deduced therefrom.

[0065] When "administering" and "treating" are used in reference to an animal, human, subject, cell, tissue, organ or biological fluid, it means 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 the cell with a reagent and contacting the cell with a fluid stream that contacts the cell. "Administering" and "treating" also mean, for example, in vitro and ex vivo treatment of cells by a reagent, diagnostic agent, binding composition or by other cells.

[0066] The term "treatment" includes the amelioration or cessation of a disorder or its symptoms. Treatment includes inhibition, e.g., reducing the overall frequency of onset of a disorder or its symptoms.

[0067] The term "prevention" includes avoiding the initial occurrence of a disorder or its symptoms.

[0068] As used herein, the term "therapeutically effective amount" or "effective amount" refers to an amount of an anti-LAIR1 antibody or an antigen-binding fragment thereof that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective to prevent or slow a disease or disorder to be treated. A therapeutically effective dose further refers to an amount of the antibody or antigen-binding fragment thereof sufficient to result in symptom alleviation, such as treating, curing, preventing, or slowing a related medical condition, or increasing the rate of treatment, cure, prevention, or slowdown of the condition. The effective amount for a particular subject can vary depending on multiple factors, such as the disease to be treated, the overall health of the patient, the method and route of administration, the dose, and the severity of side effects. The effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects. When administering an active ingredient given alone to an individual, the therapeutically effective amount refers to that individual ingredient. When administered in combination, the therapeutically effective amount refers to the combined amount of the active ingredients that produce a therapeutic effect, whether administered jointly, sequentially, or simultaneously.

[0069] Pharmaceutical composition The present invention also provides pharmaceutical compositions. Such compositions comprise an effective dose of an antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

[0070] In some embodiments, the term "pharmaceutically acceptable carrier" refers to a substance that is approved by a governmental regulatory agency or listed in other recognized pharmacopoeias for use in animals, particularly in humans. Additionally, a "pharmaceutically acceptable carrier" will generally be any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation adjuvant.

[0071] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle used in treatment in conjunction with an active ingredient. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, 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. Aqueous solutions of saline, glucose, and glycerol can also be used as liquid carriers, particularly for injection solutions. Examples of suitable pharmaceutical carriers are described in E. W. Martin's Remington's Pharmaceutical Sciences, 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 formulation should be suitable for the mode of administration. The formulation can be encapsulated in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0072] In some embodiments, the pharmaceutical compositions of the present invention can be administered by any suitable route known in the art, including but not limited to: oral, nasal, intradermal, subcutaneous, intravenous, intramuscular, intratracheal, intrapleural, intraperitoneal, intraarterial, lymphatic, and / or cerebrospinal.

[0073] In some embodiments, the composition is formulated into a pharmaceutical composition suitable for intravenous injection into humans according to conventional procedures. Compositions for intravenous administration are usually solutions in sterile isotonic aqueous buffers. The pharmaceutical composition may also contain solubilizing agents and local anesthetics such as lidocaine to relieve the pain at the injection site. Generally, the active ingredient is supplied individually or in admixture in unit dosage form, such as in a sealed container (such as an ampoule or sachet) that can indicate the amount of the active agent, in the form of a dry lyophilized powder or an anhydrous concentrate. In the case of administering the composition by infusion, the composition can be dispensed in an infusion bottle containing sterile pharmaceutical-grade water or saline. In the case of administering the composition by injection, an ampoule of sterile water for injection or saline can be used so that the active ingredient can be mixed before administration.

[0074] The antibodies or antigen-binding fragments thereof of the present invention include their salt forms. Pharmaceutically acceptable salts include those derived from anions such as hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those derived from cations such as sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0075] DX26 was purchased from BD Pharmingen™, catalog number: 550810, and it is an anti-LAIR1 antibody.

[0076] LA235 N297A is LA-235 in US2019 / 0338026A1, and it is an anti-LAIR1 antibody.

[0077] Anti-LAIR2 mAb was purchased from Invitrogen, catalog number: MA5-24041.

[0078] NGM438 is Hz47H1.v4 in WO2021 / 262597A2, and it is an anti-human LAIR1 antibody.

[0079] Example 1: Generation of Murine Monoclonal Antibodies Against Human LAIR1 1. Obtaining Hybridomas Mice were immunized with LAIR1 protein, and SP2 / 0 cells were fused. Positive clones were screened by ELISA to obtain hybridomas. The specific method is as follows: Animal Immunization: Human LAIR1 Protein, mIgG2a Tag (ACROBiosystems, Catalog No.: LA1-H5253) was used as the immunogen and diluted with normal saline to 1 mg / mL. Then it was mixed with an equal volume of adjuvant CFA (Sigma, Catalog No.: F5881) or IFA (Sigma, Catalog No.: F5506) (CFA was used for the primary immunization and IFA was used for subsequent immunizations). Female Balb / c and SJL mice (6 - 8 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were immunized with the protein by intraperitoneal injection (50 μg / mouse for the first immunization, 25 μg / mouse for each subsequent immunization). Protein immunization was repeated more than 3 times at 2-week intervals. 14 days after the last immunization, 25 μg of the immunogen was injected intraperitoneally again for booster immunization. 3 days later, the spleens of the mice were taken for cell fusion.

[0080] 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 + ), and cultured in 96-well culture plates with HAT medium (Sigma, Catalog No.: H0262). After 10 days, the hybridoma cell supernatants were screened for antibodies.

[0081] ELISA Screening of Positive Clones: The hybridoma supernatants were added to 96-well flat-bottom detection plates (Corning, Catalog No.: 9018) coated with human LAIR1 protein (Human LAIR1 / CD305 Protein, His Tag (ACROBiosystems, Catalog No.: CD5-H52H1)) at 100 μL per well 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 blotting dry, 100 μL of TMB (Yingchuang Biotech, Catalog No.: EL0009) was added to each well. After 15 minutes of color development, 50 μL of sulfuric acid was added to each well to terminate the reaction. The OD values were read on an enzyme-linked immunosorbent assay reader. The cells in the positive clone culture wells were mixed and aspirated into a centrifuge tube. An appropriate amount of medium was added, mixed well, and a small amount of cells was aspirated for counting. According to the counting results, the hybridoma cells were diluted to 5 cells per mL. 0.2 mL of the diluted cell suspension was added to each well of a 96-well plate. After culturing for one week, the culture supernatants containing single cell colonies were selected and detected again by the above method.

[0082] 2. Preparation of Murine Monoclonal Antibodies Against Human LAIR1 After culturing hybridoma cells in serum-free medium for 10 days, the supernatant was collected, and the murine monoclonal antibody was purified using a Protein A column (Bogelong (Shanghai) Biotechnology Co., Ltd., catalog number: AA0272) to obtain the purified monoclonal antibody. Table 1 shows the murine monoclonal antibody against human LAIR1 and its corresponding hybridoma clone number.

[0083] Table 1 Murine Monoclonal Antibody Against Human LAIR1

[0084] 3. Functional Identification of Murine Monoclonal Antibody Against Human LAIR1 (1) Binding Activity of Murine Monoclonal Antibody Against Human LAIR1 to Human LAIR1, Cynomolgus Monkey LAIR1, and Human LAIR2 Proteins The coating proteins are human LAIR1 protein (Human LAIR1 / CD305 Protein, Fc Tag (ACROBiosystems, catalog number: LA1-H5252)), cynomolgus LAIR1 protein (Recombinant Cynomolgus LAIR1 Fc Chimera Protein, CF (R&D Systems, catalog number: 10226-LR-050)), or human LAIR2 protein (Recombinant Human LAIR2 Fc Chimera Protein, CF (R&D Systems, catalog number: 10166-LR-050)), with a concentration of 1 μg / mL. Coat the plates overnight at 4°C at 100 μL / well; after adding PBST (containing 0.05% Tween20) and washing three times, add 2% BSA at 300 μL / well and incubate at 37°C for 1 hour; add PBST (containing 0.05% Tween20) at 200 μL / well and wash three times; prepare a stock solution of 660 μL with a concentration of 66.667 nM of murine monoclonal antibody against human LAIR1 and positive control antibodies (DX26, LA235N297A, Anti-LAIR2 mAb) using the blocking solution; dilute the stock solution with the blocking solution at a dilution factor of 3, for a total of 12 gradients; add the antibodies 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 number: A0168), diluted 1:5000), add 100 μL / well to each well, and incubate at room temperature for 1 hour; add PBST (containing 0.05% Tween20) at 200 μL / well and wash three times; after patting dry, add 50 μL of TMB chromogenic solution and develop color for about 15 minutes, then terminate the reaction with sulfuric acid. Read the OD450 value with an enzyme-linked immunosorbent assay reader. Analyze the data using Graphpad Prism 8.0 software. Use the logarithm of the antibody concentration as the x-axis and the corresponding OD450 value as the y-axis, select the four-parameter equation regression model, fit the antibody dose-effect curve, and calculate the EC50. The results are as shown in Figure 1A , Figure 1B , Figure 1C and Table 2.

[0085] Table 2 Binding activities of murine monoclonal antibodies against human LAIR1 with human LAIR1, cynomolgus LAIR1, and human LAIR2 proteins

[0086] The results showed that mAb003, mAb004, mAb012, and mAb013 all had strong binding activities with human and cynomolgus monkey LAIR1 proteins; in addition, except for mAb004 having weak binding with human LAIR2 protein, the other three murine monoclonal antibodies against human LAIR1 did not bind to human LAIR2.

[0087] (2) Binding experiments of murine monoclonal antibodies against human LAIR1 with cell lines endogenously expressing human LAIR1 and cell lines expressing cynomolgus monkey LAIR1 Adjust the concentration of Jurkat T cell line (GemGenBio Technology (Shanghai) Co., Ltd., product number: GM-C03922) endogenously expressing human LAIR1 or CHOK1 cell line (CynoLAIR1-CHOK1 (Kangyuan Botech Co., Ltd., product number: KC-2190)) expressing cynomolgus monkey LAIR1 to 1×10 6 cells / mL with FACS buffer (PBS solution containing 1% FBS), place 100 μL per well in a 96-well U-bottom plate, discard the supernatant after centrifugation. Dilute the murine monoclonal antibodies against human LAIR1 and positive control antibodies (DX26, LA235 N297A, NGM438) to the initial working concentration of 200 nM with FACS buffer (PBS solution containing 1% FBS), and then perform a 1:3 gradient dilution with FACS buffer, with a total of 12 concentration points (including the last concentration point without antibody solution). Add the gradient-diluted antibodies to the plate at 100 μL per well to resuspend the cells, pipette and mix well, and incubate at 4°C for 1 hour. After incubation, the cells are centrifuged and washed three times with FACS buffer. Dilute the AlexaFlour-647-labeled anti-mouse secondary antibody (Invitrogen, product number: A-31571) 1:1000 with FACS buffer (PBS solution containing 1% FBS), add 100 μL of the secondary antibody dilution to each well, resuspend the cell pellet, pipette and mix well, and incubate at 4°C for about 45 minutes. After incubation, the cells are centrifuged, washed three times with FACS buffer, and then resuspend the cells in the plate with FACS buffer at 100 μL per well. Read the mean fluorescence intensity (Median Fluorescence Intensity, MFI) with a flow cytometer (BD Celesta), analyze the experimental data using Graphpad Prism 8.0 software, use the logarithm of the antibody concentration as the x-axis and the corresponding MFI value as the y-axis, select a four-parameter equation regression model, fit the antibody dose-effect curve, and calculate the EC50. The results are as Figure 2 、 Figure 3 shown in Table 3.

[0088] Table 3 Binding of Murine Monoclonal Antibodies Against Human LAIR1 to Cell Lines Expressing Human LAIR1 and Cell Lines Expressing Cynomolgus Monkey LAIR1

[0089] 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.

[0090] (3) Activity of Murine Monoclonal Antibodies Against Human LAIR1 in Blocking the Interaction between Human LAIR1 Protein and Collagen type I The protein coated on the plate was human Collagen type I protein (Abcam, catalog number: ab7533), with a concentration of 2 μg / ml. It was coated on the plate at 4°C overnight at 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; the murine monoclonal antibodies against human LAIR1 and positive control antibodies (DX26, LA235 N297A) were prepared into a mother liquor with a concentration of 400 nM and a volume of 210 μL using the blocking solution; the mother liquor was diluted with the blocking solution at a dilution factor of 3, for a total of 12 gradients; the hLAIR1-mFc protein (ACROBiosystems, catalog number: LA1-H5253) was diluted with the blocking solution to a concentration of 2 μg / mL. After the antibody solution and the hLAIR1-mFc solution were incubated in equal volumes, 100 μL of the mixture was added to the wells and incubated at room temperature for 1 hour; PBST (containing 0.05% Tween20) was added at 200 μL / well and washed three times; the secondary antibody (Anti-Mouse IgG-HRP (Sigma, catalog number: A0168), diluted 1:5000) was added at 100 μL / well to each well and incubated at room temperature for 1 hour; PBST (containing 0.05% Tween20) was added at 200 μL / well and washed three times; after patting dry, 100 μL of TMB chromogenic solution was added and developed for about 15 minutes, and then the reaction was terminated with sulfuric acid. The OD450 was read using an enzyme-linked immunosorbent assay reader. The data was 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 equation regression model was selected to fit the antibody dose-effect curve and calculate the IC50. The results are as Figure 4 and shown in Table 4.

[0091] Table 4 Activity of murine monoclonal antibodies against human LAIR1 in blocking the interaction between human LAIR1 protein and Collagen type I

[0092] The results showed that mAb003, mAb004, mAb012, and mAb013 all had the activity to block the interaction between human LAIR1 protein and Collagen type I.

[0093] (4) Binding experiments of mAb003 and mAb013 with primary human CD3 + T cells and CHOK1 cell line expressing human LAIR1 Based on the above experimental results of binding to human LAIR1, cynomolgus monkey LAIR1, human LAIR2, Jurkat T cells, CynoLAIR1-CHOK1 and blocking the interaction between human LAIR1 and Collagen type I, mAb003 and mAb013 were selected for further characterization.

[0094] Adjust the concentration of primary human CD3 + T cells (derived from human CD3 + T cells isolated from PBMC, PBMC from TPCS, product number: PB050C) or CHOK1 cell line expressing human LAIR1 (hLAIR1-CHOK1 (Kangyuan Bchuang Biotechnology (Beijing) Co., Ltd., product number: KC-1370)) to 1×10 6 cells / mL, place 100 μL / well in a 96-well U-bottom plate, discard the supernatant after centrifugation. Dilute murine monoclonal antibodies against human LAIR1 and negative control antibody (mIgG1) with FACS buffer (PBS solution containing 1% FBS) to the initial working concentration of 200 nM (for hLAIR1-CHOK1 cells) or 100 nM (for primary human CD3 +T cells), and then serially diluted 1:3 with FACS buffer to a total of 12 concentration points (including the last concentration point of the antibody-free solution). Add 100 μL of the serially diluted antibody per well to the plate to resuspend the cells, pipette to mix well, and incubate at 4°C for 1 hour. After incubation, the cells were centrifuged and washed three times with FACS buffer. Dilute the AlexaFlour-647-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch, catalog number: 115-605-071) 1:1000 with FACS buffer (PBS solution containing 1% FBS), add 100 μL of the secondary antibody dilution per well, resuspend the cell pellet, pipette to mix well, and incubate at 4°C for approximately 45 minutes. After incubation, the cells were centrifuged and washed three times with FACS buffer, and then resuspended with 100 μL of FACS buffer per well. The mean fluorescence intensity (Median Fluorescence Intensity, MFI) was read using a flow cytometer (BECKMAN COULTER cytoFLEX). The experimental data were analyzed using Graphpad Prism 8.0 software. With the logarithm of the antibody concentration on the x-axis and the corresponding MFI value on the y-axis, a four-parameter equation regression model was selected to fit the antibody dose-response curve, and the EC50 was calculated. The results are shown as Figure 5 and Figure 6 shown.

[0095] The results showed that both mAb003 and mAb013 could bind to hLAIR1-CHOK1 cells. The EC50 of mAb003 was 0.52 nM, and the highest MFI was 81176; the EC50 of mAb013 was 0.67 nM, and the highest MFI was 133985. Both mAb003 and mAb013 could bind to primary human CD3 + T cells. The EC50 of mAb003 was 0.89 nM, and the highest MFI was 33233; the EC50 of mAb013 was 0.21 nM, and the highest MFI was 54996.

[0096] (5) Activity of mAb003 and mAb013 in blocking the interaction between human LAIR1 protein and different Collagens The plate coating proteins were human Collagen type I protein (Abcam, catalog number: ab7533), Collagen type III protein (Abcam, catalog number: ab7535), or Collagen type IV protein (Abcam, catalog number: ab7536), with a concentration of 2 μg / mL. The plates were coated overnight at 4°C at 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; a mouse monoclonal antibody against human LAIR1, a positive control antibody (NGM438), and a negative control antibody (mIgG1) were prepared with a blocking solution to a mother liquor with a concentration of 400 nM in 990 μL; the mother liquor was diluted with the blocking solution at a dilution factor of 3, with a total of 12 gradients; the hLAIR1-mFc protein (ACROBiosystems, catalog number: LA1-H5253) was diluted with the blocking solution to a concentration of 2 μg / mL or 4 μg / mL. After the antibody solution and the hLAIR1-mFc solution were incubated in equal volumes, 100 μL of the mixture was added to each well and incubated at 37°C for 1 hour; PBST (containing 0.05% Tween20) was added at 200 μL / well and washed three times; a secondary antibody (Anti-Mouse IgG-HRP (Sigma, catalog number: A0168), diluted 1:5000) was added at 100 μL / well to each well and incubated at room temperature for 1 hour; PBST (containing 0.05% Tween20) was added at 200 μL / well and washed three times; after blotting dry, 100 μL of TMB chromogenic solution was added and developed for about 15 minutes, and then the reaction was terminated with sulfuric acid. The OD450 was read with an enzyme-linked immunosorbent assay reader. The data were analyzed using Graphpad Prism 8.0 software. With the logarithm of the antibody concentration on the x-axis and the corresponding OD450 value on the y-axis, a four-parameter equation regression model was selected to fit the antibody dose-effect curve and calculate the IC50. The results are as Figure 7A , Figure 7B , Figure 7C and Table 5 show.

[0097] Table 5 Activity of mouse monoclonal antibodies against human LAIR1 in blocking the interaction of LAIR1 protein with different Collagens

[0098] The results showed that both mAb003 and mAb013 could block the interaction of human LAIR1 protein with Collagen type I, type III, and type IV, and their IC50 was comparable to that of the positive control NGM438.

[0099] (6) The activities of mAb003 and mAb013 on activating primary human CD3 + T cells to secrete IL-2 Pre-coat human Collagen type I protein (Abcam, catalog number: ab7533) and a series of concentration gradients of OKT3 (Biolegend, catalog number: 317326) in a 96-well plate in advance, and add them to the 96-well plate at 100 μL / well, so that the final coating concentration of human Collagentype I protein is 5 μg / mL, and the starting concentration of the final coating concentration of OKT3 is 10 μg / mL. Dilute them with DPBS (Gibco, catalog number: 14190-144) at a 3-fold concentration gradient, with 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 number: 14190-144), and finally aspirate the liquid in the wells completely. Isolate human CD3 + T cells from cryopreserved human PBMC (TPCS, catalog number: PB025C), add anti-CD28 antibody (Invitrogen, catalog number: 16-0289-85) (concentration: 10 μg / mL) to the adjusted T cells (concentration 1e6 / mL), and add them to the 96-well plate at 100 μL / well. Then add murine monoclonal antibodies against human LAIR1, positive control antibody (NGM438), and negative control antibody (mIgG1) (concentrations are all 10 μg / mL) to the 96-well plate at 100 μL / well, with a total volume of 200 μL per well. Incubate the above plate under the conditions of 5% CO2 and 37°C for 3 days. After 3 days, collect the culture supernatant, and detect the relative concentration of IL-2 in the above culture supernatant using the hIL-2 HTRF kit (Cisbio, catalog number: 62HIL02PEH). The results are as Figure 8 shown.

[0100] The results show that both mAb013 and the positive control NGM438 can upregulate the secretion of IL-2 by human CD3 + T cells and activate primary human CD3 + T cells, and the activity of mAb013 is comparable to that of the positive control NGM438.

[0101] Example 2: Sequencing of murine monoclonal antibodies against human LAIR1 and identification of the functions of chimeric antibodies 1. Sequencing of murine monoclonal antibodies against human LAIR1 and preparation of chimeric antibodies According to the results of Example 1, sequence mAb013 (which can also be represented by its corresponding hybridoma clone number 75A81E9 in the present invention). The heavy chain variable region, light chain variable region, and CDR sequences are shown in Table 6.

[0102] CDR sequences and variable region sequences of murine monoclonal antibody mAb013 against human LAIR1 (determined according to the Kabat scheme)

[0103] The light and heavy chain variable regions were respectively constructed onto human constant regions (IgG1 / K, Table 7) to construct the corresponding chimeric antibodies (Table 8), and the sequences were verified by sequencing. The chimeric antibodies were named by adding the prefix "ch" to the corresponding hybridoma clone number. For example, the chimeric antibody obtained through this example using hybridoma clone 75A81E9 was named ch75A81E9 and was used for in vitro functional identification.

[0104] The corresponding nucleic acids encoding the chimeric antibodies were expressed using Expi293 cells and purified using a Protein A column. The specific method is as follows: Expression of chimeric antibodies in Expi293 cells: One day before transfection, dilute the density of Expi293 cells (Thermo, catalog number: A14635CN) to 1.5×10 6 cells / mL and culture them in a shaker at 37°C and 8% CO2 at 120 rpm. The next day, measure the viable cell density and survival rate. The cell transfection density should be 3×10 6 cells / mL and the cell viability should be greater than 95%. Preparation of the PEI / plasmid complex: Invert and mix PEI (1 mg / mL, Polysciences, catalog number: 24765-1). Dilute the ch75A81E9 heavy chain plasmid and light chain plasmid with OPM-293CD05 medium (Shanghai OPMI Biotechnology Co., Ltd., catalog number: 81075-001). The plasmid concentration is 1 μg / mL, and the volume of the medium diluting the plasmid is 1 / 20 of the transfection volume. Gently mix. The ratio of the plasmids expressing the heavy and light chains is 1:1.5. Dilute the PEI reagent with OPM-293 CD05 medium. The volume of the medium diluting the PEI is 1 / 20 of the transfection volume. Gently invert and mix, and incubate at room temperature for 5 minutes. Add the diluted PEI reagent to the diluted plasmid and gently invert and mix. Incubate the PEI / plasmid complex at room temperature for 15 minutes, and then slowly add the solution dropwise to the transfer shake flask while gently rotating the shake flask during the addition. After transfection, place the shake flask in a shaker at 37°C and 8% CO2 at 120 rpm for culture. On the second day after transfection (24 hours after transfection), add 10% OPM-293 ProFeed (Shanghai OPMI Biotechnology Co., Ltd., catalog number: F081918) to the shake flask while gently rotating the shake flask, and then return the shake flask to the shaker for continued culture for 5-7 days, and harvest the supernatant.

[0105] Protein A Column Purification of Antibodies: Prepare a gravity chromatography column. Open the upper cover of the gravity chromatography column, place the gasket at the bottom of the gravity column, and press it firmly. Prepare the packing Protein A (Cytiva, catalog number: 17549801). Accurately calculate the volume of the packing suspension required based on the target packing volume and the suspension ratio of the packing. The volume of the packing suspension required = target packing volume / suspension ratio of the packing. Vortex the packing thoroughly to ensure complete suspension. Add the packing suspension to the bottom of the gravity chromatography column. Add at least 10 CV of the equilibration buffer PBS to the gravity chromatography column. After equilibration, measure the pH at the outlet. If it does not reach the target pH, continue to add the equilibration buffer until the target pH is reached. Slowly add a certain volume of the sample to the gravity chromatography column. Add at least 10 CV of the washing buffer to the gravity chromatography column. Slowly add 5 CV of the elution buffer (10 - 50 mM NaAc, pH 3.0 - pH 3.5) to the gravity chromatography column and incubate for 3 - 5 minutes to collect the eluate. Repeat the elution step as needed. Neutralization: Adjust the pH to the target pH with the neutralization buffer (1M Tris). Measure the protein concentration using Nanodrop. Replace the buffer for storing the antibody with PBS by ultrafiltration buffer exchange.

[0106] Table 7 Constant Region Sequences

[0107] Table 8 Chimeric Antibodies Against Human LAIR1

[0108] 2. Functional Identification of Chimeric Antibodies Against Human LAIR1 Binding Assays of Chimeric Antibodies Against Human LAIR1 to Cell Lines Endogenously Expressing Human LAIR1 and Cell Lines Expressing Cynomolgus Monkey LAIR1 Detect according to the method in (2) of step 3 in Example 1. The negative control antibody is anti-HEL-HumanIgG1 (purchased from Bio Ying, catalog number: B117901). The results are as Figure 9A 、 9B and shown in Table 9.

[0109] Table 9 Binding of Chimeric Antibodies Against Human LAIR1 to Cell Lines Expressing Human LAIR1 and Cell Lines Expressing Cynomolgus Monkey LAIR1

[0110] The results showed that the chimeric antibody ch75A81E9 against human LAIR1 was similar to the positive control antibody NGM438 and had strong binding activity to the Jurkat T cell line and the cynomolgus monkey CynoLAIR1-CHOK1 cell line.

[0111] Example 3: Humanization of Anti-human LAIR1 Antibody and Expression and Purification of the Humanized Antibody The CDRs were determined using the Kabat numbering system. The human germline gene with the highest homology to the murine sequence was selected as the acceptor framework, and the CDRs of the murine sequence were transplanted into the human framework. According to the importance of amino acids, back mutations were performed, that is, some key amino acids in the framework region after transplantation were reverted to the corresponding murine amino acids, and several variants were designed for the heavy and light chains respectively.

[0112] 1. Humanization The sequence of the murine antibody mAb013 (i.e., 75A81E9) was analyzed and compared with the Kabat human germline genes. The framework region sequence of IGKV1-12*01 was determined as the humanized framework sequence for the light chain, and the framework region sequence of IGHV3-73*01 was determined as the humanized framework sequence for the heavy chain. By CDR-grafting (the CDRs were determined using the Kabat coding method), the CDRs of the heavy and light chains were respectively juxtaposed to the selected humanized framework sequences. At the same time, back mutation designs were carried out on the key sites in the framework region to obtain several humanized antibody variable regions, such as the humanized sequences in Table 10. The variable region sequences of the heavy and light chains were respectively combined with the human IgG1 constant region (Kabat, human IGHG1*01) and the human kappa constant region to obtain the humanized antibody. The sequence combinations of the heavy and light chain variable regions of the specifically obtained 75A81E9 humanized antibody are shown in Table 11.

[0113] Table 10 Humanized Sequences of Anti-LAIR1 Antibody 75A81E9

[0114] Table 11 Heavy Chain Variable Region Sequences and Light Chain Variable Region Sequences Corresponding to the Constructed Humanized Antibodies

[0115] 2. Expression and Purification of Anti-human LAIR1 Humanized Antibody The variable regions of the light and heavy chains of the humanized antibody in Table 10 were respectively constructed onto the human constant regions (hIgG1 / K, Table 7) according to the combinations in Table 11, gene synthesis was carried out, and sequencing confirmed that it was consistent with the designed sequence. Expression was carried out in Expi293 cells according to the method in Example 2, and purification was carried out using a Protein A column.

[0116] 3. Functional Identification of Anti-human LAIR1 Humanized Antibody (1) Binding Activity of Anti-human LAIR1 Humanized Antibody to Human LAIR1 and Cynomolgus Monkey LAIR1 Proteins The plate coating proteins were Human LAIR1 Protein, His Tag (ACROBiosystems, product number: CD5-H52H1) and cynomolgus monkey LAIR1 Protein (Shanghai Hongcheng Pharmaceutical Co., Ltd., product number: TP1106), with a concentration of 1 μg / mL. They were used to coat the plates at 4°C overnight at 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; the humanized antibody against human LAIR1, chimeric antibody ch75A81E9, and negative control antibody anti-HEL-Human IgG1 (purchased from Bio Ying, product number: B117901) were prepared into a mother liquor with a concentration of 33.333 nM in 900 μL using the blocking solution; the mother liquor was diluted with the blocking solution at a dilution factor of 3, with a total of 8 gradients; the antibodies were added at 100 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; the secondary antibody (goat anti-human Fab-HRP (Sigma, product number: A0293-1ML) was added for the cynomolgus monkey LAIR1 protein-coated plates or goat anti-human IgG Fc-HRP (Sigma, product number: A0170-1ML), diluted 1:5000) was added at 100 μL / well to each well and incubated at room temperature for 1 hour; PBST (containing 0.05% Tween20) was added at 200 μL / well and washed three times; after patting dry, 100 μL of TMB chromogenic solution was added and developed for about 15 minutes, and then the reaction was terminated with sulfuric acid. The OD450 was read using an enzyme-linked immunosorbent assay reader. The data was 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 equation regression model was selected to fit the antibody dose-effect curve and calculate the EC50. The results are as Figure 10A , Figure 10B and shown in Table 12.

[0117] The results showed that, except for the humanized antibody 75A81E9-hzVH4 hzVL2, the binding abilities of all humanized antibodies to human LAIR1 protein and cynomolgus monkey LAIR1 protein were comparable to those of the chimeric antibody ch75A81E9. (2) Binding experiment of anti-LAIR1 humanized antibody to primary human CD3 + T cell Primary human CD3 was diluted with FACS buffer (PBS solution containing 1% FBS) +The concentration of T cells (Allcells, catalog number: FPB009-1F-C-10M) was adjusted to 1×10 6 cells / mL. They were placed in a 96-well U-bottom plate at 100 μL / well, and the supernatant was discarded after centrifugation. The anti-human LAIR1 humanized antibody, chimeric antibody ch75A81E9, and negative control antibody anti-HEL-Human IgG1 (from Bioyiteng, catalog number: B117901) were diluted to the initial working concentration of 100 nM with FACS buffer (PBS solution containing 1% FBS), and then serially diluted 1:5 with FACS buffer for a total of 8 antibody concentration points. The serially diluted antibodies were added to the wells at 100 μL / well to resuspend the cells, and the cells were pipetted and mixed well, and then incubated at 4°C for 1 hour. After incubation, the cells were centrifuged and washed three times with FACS buffer. The AlexaFlour-647-labeled anti-human secondary antibody (Invitrogen, catalog number: A-21445) was diluted 1:1000 with FACS buffer (PBS solution containing 1% FBS), and 100 μL of the secondary antibody dilution was added to each well to resuspend the cell pellet, and the cells were pipetted and mixed well, and then incubated at 4°C for about 45 minutes. After incubation, the cells were centrifuged and washed three times with FACS buffer, and then the T cells were labeled with Brilliant Violet 421™ anti-human CD3 Antibody (Biolegend, catalog number: 317344), pipetted and mixed well, and then incubated at 4°C for about 30 minutes. After incubation, the cells were centrifuged and washed three times with FACS buffer, and then FACS buffer was added to the wells at 100 μL / well to resuspend the cells. The mean fluorescence intensity (Median Fluorescence Intensity, MFI) was read using a flow cytometer (BD Celesta), 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 equation regression model was selected to fit the antibody dose-response curve, and the EC50 was calculated. The results are shown in Figure 10C and Table 12.

[0118] The results showed that, except for the humanized antibody 75A81E9-hzVH4 hzVL2, the binding ability of all humanized antibodies to primary human CD3 + T cells was comparable to that of the chimeric antibody ch75A81E9. Table 12 Binding activity of anti-LAIR1 humanized antibodies

[0119] (3) Based on the above-mentioned capabilities of human LAIR1 protein, binding ability with cynomolgus monkey LAIR1 protein, and binding ability with primary human CD3 + T cells, select the top 5 humanized antibodies 75A81E9-hzVH2 hzVL1, 75A81E9-hzVH3 hzVL1, 75A81E9-hzVH4 hzVL1, 75A81E9-hzVH3 hzVL3, 75A81E9-hzVH4 hzVL3 for further characterization.

[0120] i. Binding to endogenous human LAIR1-expressing cell lines and cynomolgus monkey LAIR1-expressing cell lines Adjust the concentration of Jurkat T cells (Genomeditech Co., Ltd., Shanghai, China, Catalog No.: GM-C03922) that endogenously express human LAIR1 or the CHOK1 cell line that expresses cynomolgus monkey LAIR1 (CynoLAIR1-CHOK1 (Kangyuan Botech Co., Ltd., Beijing, China, Catalog No.: KC-2190)) to 1×10 with FACS buffer (PBS solution containing 1% FBS). 6cells / mL, placed in a 96-well U-bottom plate at 100 μL / well, and the supernatant was discarded after centrifugation. The optimal 5 anti-human LAIR1 humanized antibodies, chimeric antibody ch75A81E9, positive control antibody (NGM438), and negative control antibody anti-HEL-Human IgG1 (from Bio Ying, catalog number: B117901) were diluted to the initial working concentration of 20 nM (for Jurkat T cells endogenously expressing human LAIR1) or 100 nM (for CHOK1 cell line expressing cynomolgus monkey LAIR1) with FACS buffer (PBS solution containing 1% FBS), and then serially diluted 1:5 with FACS buffer, with a total of 7 antibody concentration points (for Jurkat T cells endogenously expressing human LAIR1) or 8 antibody concentration points (for CHOK1 cell line expressing cynomolgus monkey LAIR1). The serially diluted antibodies were added to the plate at 100 μL / well to resuspend the cells, pipetted to mix well, and incubated at 4°C for 1 hour. After incubation, the cells were centrifuged and washed three times with FACS buffer. The AlexaFlour-647-labeled anti-human secondary antibody (Invitrogen, catalog number: A-21445) was diluted 1:1000 with FACS buffer (PBS solution containing 1% FBS), 100 μL of the secondary antibody dilution was added to each well, the cell pellet was resuspended, pipetted to mix well, and incubated at 4°C for approximately 45 minutes. After incubation, the cells were centrifuged and washed three times with FACS buffer, and then resuspended with FACS buffer at 100 μL / well. The mean fluorescence intensity (Median Fluorescence Intensity, MFI) was read using a flow cytometer (BD Celesta), and the experimental data were analyzed using Graphpad Prism 8.0 software. With the logarithm of the antibody concentration as the x-axis and the corresponding MFI value as the y-axis, a four-parameter equation regression model was selected to fit the antibody dose-response curve, and the EC50 was calculated. The results are as Figure 11A , Figure 11B and shown in Table 13.

[0121] Table 13 Binding activity of anti-LAIR1 humanized antibodies to cells

[0122] The results showed that the anti-human LAIR1 humanized antibodies were similar to the positive control antibody NGM438 and had strong binding activity to human Jurkat T cells and cynomolgus monkey CynoLAIR1-CHOK1 cell lines.

[0123] ii. Activation of CD3 + T cell experiment Pre-coat human Collagen type I (Millipore, catalog number: CC050) and a series of concentration gradients of OKT3 (Biolegend, catalog number: 317326) in a 96-well plate in advance, add 100 μL per well to the 96-well plate, so that the final coating concentration of human Collagentype I protein is 5 μg / mL, and the starting concentration of the final coating concentration of OKT3 is 15 μg / mL. Dilute it with 3-fold concentration gradient of DPBS (Gibco, catalog number: 14190-144), with a total of 5 concentration points. Incubate overnight at 4°C. On the second day, wash the 96-well plate 3 times with DPBS (Gibco, catalog number: 14190-144), and finally aspirate the liquid in the wells completely. Quickly thaw human CD3 + T (Allcells, catalog number: FPB009-1F-C-10M, donor 1: LP230111009, donor 2: LP230112007) in a 37°C water bath, adjust the T cell concentration to 1e6 / mL and add 100 μL per well to the 96-well plate. Then add 100 μL per well of the optimal 5 LAIR1 humanized antibodies, chimeric antibody ch75A81E9, positive control antibody (NGM438), and negative control antibody anti-HEL-HumanIgG1 (from Bio Ying Biotechnology, catalog number: B117901). The total volume per well is 200 μL. Incubate for 5 days under the conditions of 5% CO2 and 37°C. After 5 days, collect the culture supernatant, and use the hIL-2 HTRF kit (Cisbio, catalog number: 62HIL02PEH) and hIFNgamma HTRF kit (Cisbio, catalog number: 62HIFNG PEH) to detect the concentrations of IL-2 and IFN-γ in the above culture supernatant. At the same time, use Cell Titer Turbo 2.0 to detect the number of live cells (Adamas life, catalog number: RA-GL11). The results are as Figure 12A (Donor 1, cell proliferation detection), Figure 12B (Donor 2, cell proliferation detection), 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) shown.

[0124] The results show that both the humanized antibody and the positive control NGM438 can activate primary human CD3 +T cell proliferation and the secretion of IL-2 and IFN-γ, where the activity of 75A81E9-hzVH3 hzVL3 is equivalent to or better than that of the positive control NGM438. iii. Experiment on the activity of collagen-polarized monocyte-derived dendritic cells to secrete CCL4 First, human monocytes were isolated from human PBMC (Allcells, catalog number: FPB004F-C) using a human monocyte kit (Stemcell, catalog number: 19059). The monocytes were induced into DCs in a 6-well plate under the action of recombinant human GM-CSF (Peprotech, catalog number: 300-03) and IL-4 (Peprotech, catalog number: 200-04). Human Collagen type I (Millipore, catalog number: CC050), 5 μg / mL, was pre-coated in a 96-well plate the day before. On the second day, the 96-well plate was washed 3 times with DPBS (Gibco, catalog number: 14190-144), and the liquid in the wells was aspirated clean for the last time. The induced DCs were collected, and the DC cell concentration was adjusted to 5e5 / mL. 100 μL was added to each well of the 96-well plate. Then, 100 μL of each of the top 5 LAIR1 humanized antibodies, the positive control antibody (NGM438), and anti-HEL-Human IgG1 (from Bio Ying, catalog number: B117901) was added to each well, and the total volume per well was 200 μL. They were co-incubated for 3 days at 5% CO2 and 37 °C. After 3 days, the culture supernatant was collected, and the concentration of CCL4 in the above culture supernatant was detected using a CCL4 HTRF kit (Cisbio, catalog number: 62HCCL4PEG). The results are as Figure 13 shown.

[0125] The results showed that both the humanized antibody and the positive control NGM438 could activate collagen-polarized monocyte-derived dendritic cells to secrete CCL4, where the activity of 75A81E9-hzVH4 hzVL1 was equivalent to or better than that of the positive control NGM438.

Claims

1. An antibody or antigen-binding fragment thereof that has binding specificity for 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, the heavy-chain variable region comprises heavy-chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, the light-chain variable region comprises light-chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, and wherein: The amino acid sequence of the HCDR1 is as shown in SEQ ID NO: 2, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO: 3, the amino acid sequence of the HCDR3 is as shown in SEQ ID NO: 4, the amino acid sequence of the LCDR1 is as shown in SEQ ID NO: 6, the amino acid sequence of the LCDR2 is as shown in SEQ ID NO: 7, and the amino acid sequence of the LCDR3 is as 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 the amino acid sequence shown in any one of SEQ ID NO: 1, 13, 14, 15, 16, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 1, 13, 14, 15, 16, or a sequence having one or more amino acid substitutions, deletions, or insertions or any combination thereof as compared with the amino acid sequence shown in any one of SEQ ID NO: 1, 13, 14, 15, 16; and / or The light-chain variable region comprises the amino acid sequence shown in any one of SEQ ID NO: 5, 17, 18, 19, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 5, 17, 18, 19, or a sequence having one or more amino acid substitutions, deletions, or insertions or any combination thereof as compared with the amino acid sequence shown in any one of SEQ ID NO: 5, 17, 18, 19.

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

4. The antibody or antigen-binding fragment thereof according to any one of claims 1-3, which further comprises a heavy chain constant region, a light chain constant region, an Fc region or a combination thereof; Preferably, the light chain constant region is a κ chain or λ chain constant region; Preferably, the heavy chain constant region is selected from the IgG, IgM, IgA, IgE or IgD classes, and more preferably, the heavy chain constant region is a heavy chain constant region selected from the IgG1, IgG2, IgG3 or IgG4 subclasses; More preferably, the antibody or antigen-binding fragment thereof further comprises a human IgG1 heavy chain constant region or a variant thereof, preferably as shown in SEQ ID NO: 9, and / or a human κ light chain constant region or a variant thereof, preferably as shown in SEQ ID NO: 10; Preferably, the antibody or antigen-binding fragment thereof is a chimeric antibody or a humanized antibody.

5. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-4.

6. A recombinant vector comprising the nucleic acid molecule according to claim 5.

7. A recombinant cell comprising the nucleic acid molecule according to claim 5 and / or the recombinant vector according to claim 6, or expressing the antibody or antigen-binding fragment thereof according to any one of claims 1-4.

8. 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 according to any one of claims 1-4 under conditions suitable for expressing the antibody, and optionally the method further comprises recovering the antibody or antigen-binding fragment thereof from the recombinant cell or the culture medium.

9. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-4, and a pharmaceutically acceptable carrier; Preferably, the pharmaceutical composition further comprises a second therapeutic agent; Preferably, the second therapeutic agent is a checkpoint inhibitor; Preferably, the checkpoint inhibitor is selected from one or more of the following: checkpoint inhibitors of T cells, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody, anti-TGF-β antibody.

10. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-4, the nucleic acid molecule according to claim 5, the recombinant vector according to claim 6, the recombinant cell according to claim 7 and / or the pharmaceutical composition according to claim 9 in the preparation of a product as shown in any of the following: (1) A product for detecting LAIR1; (2) A product for stimulating or enhancing immune response; (3) A product for preventing and / or treating diseases; Preferably, the diseases are 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, lung cancer.

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