CD3-targeting antibody and use thereof

Abstract

The present invention relates to the field of antibodies, specifically to a CD3-binding molecule or a leukocyte immunoglobulin‑like receptor subfamily B member 4 (LILRB4)-binding molecule, and in particular to an antibody and a fragment thereof that specifically recognize CD3 or LILRB4, and a bispecific antibody constructed on the basis of the antibody and the fragment. Therefore, the present invention also relates to the field of bispecific antibody drugs. The bispecific antibody comprises a domain that binds to human LILRB4 and another domain that binds to CD3. In addition, the present invention also relates to a nucleic acid or a host cell comprising the antibody or the fragment thereof, a drug comprising the antibody or the fragment thereof, and a therapeutic and diagnostic method using the antibody or the fragment thereof, or a use.

Description

An antibody targeting CD3 and its application Technical Field

[0001] This invention belongs to the field of antibodies, specifically relating to a CD3-binding molecule or a LILRB4-binding molecule, particularly antibodies and fragments thereof that specifically recognize CD3 or LILRB4, and bispecific antibodies constructed based thereon. Therefore, this invention also relates to the field of bispecific antibody pharmaceuticals comprising a domain that binds to a member of the human leukocyte immunoglobulin-like receptor subfamily B (LILRB4) and another domain that binds to CD3. Furthermore, this invention relates to nucleic acids or host cells comprising such antibodies or fragments thereof, pharmaceuticals comprising such antibodies or fragments thereof, and methods or uses for treatment and diagnosis employing these antibodies and fragments. Background Technology

[0002] T-cell engagers are a class of bispecific antibodies that can simultaneously bind to CD3 on T cells and tumor-associated antigens (TAAs) on tumor cells (Labrijn, AF, et al. (2019). "Bispecific antibodies: a mechanistic review of the pipeline." Nat Rev Drug Discov 18(8):585-608.)(Ellerman, D. (2019). "Bispecific T-cell engagers: Towards understanding variables influencing the in vitro potency and tumor selectivity and their modulation to enhance their efficacy and safety." Methods 154:102-117.). These bispecific antibodies, by simultaneously binding to T cells and tumor cells, drive the formation of immune synapses between cells, activating T cells to kill tumor cells. Blinatumomab, as the first marketed T-cell linker, has proven the feasibility of this theory. More T-cell linkers are in early development and are widely used to treat hematologic malignancies and solid tumors (Goebeler, ME and RC Bargou (2020). "T cell-engaging therapies-BiTEs and beyond." Nat Rev Clin Oncol 17(7):418-434.).

[0003] Studies have found that LILRB4 is highly expressed in various solid tumors and hematological malignancies. This molecule promotes tumor immune escape by interacting with immune cells in the tumor microenvironment, making it an emerging target for cancer therapy. It is believed to have the potential to serve as a target for T-cell linkers. Specifically, LILRB4 exhibits significant immunomodulatory functions in various solid tumors and hematological malignancies, including acute myeloid leukemia, chronic lymphocytic leukemia, pancreatic cancer, non-small cell lung cancer, colorectal cancer, melanoma, and liver cancer.

[0004] LILRB4 is highly expressed on M4 and M5 cells of the FAB classification of acute myeloid leukemia (AML) and is an AML-specific marker. It supports tumor cell infiltration into tissues and inhibits T cell activity through the ApoE / LILRB4 / SHP-2 / uPAR / Arginase-1 signaling axis in AML cells, and is closely related to the survival of AML patients (Deng M. et al., *Nature* (2018)). AML, a malignant hematologic malignancy, affects people of all ages, but is most common in the elderly, with a median age of diagnosis of 68 years. Two-thirds of patients are over 55 years old, and the five-year survival rate for patients over 60 years old is less than 10%. Current clinical treatments for AML include chemotherapy and hematopoietic stem cell transplantation. Adult AML patients can achieve good results with chemotherapy and hematopoietic stem cell transplantation. However, for elderly AML patients (≥60 years old) or relapsed / refractory AML patients, there is currently a lack of effective treatments, which remains a very challenging problem in clinical treatment, and new therapies are urgently needed.

[0005] Besides AML, LILRB4 is also expressed in chronic myelomonocytic leukemia (CMML). Studies have shown that LILRB4 expression in CMML is significantly increased compared to healthy individuals and patients with myelodysplastic syndromes (MDS) (Leuk Lymphoma. 2020 June; 61(6):1493–1499). CMML is a clonal hematopoietic stem cell / progenitor cell disorder with an inherent risk of transformation into AML (15%–20% within 3–5 years). As a heterogeneous disease, its clinical course and prognosis vary, and there is currently no standard treatment, leaving the clinical field almost entirely unexplored. IO202 is a monoclonal antibody targeting LILRB4, and its application in the treatment of CMML is in Phase 1 clinical trials, showing promising therapeutic benefits, including improved symptoms, reduced transfusion frequency, decreased immature cells and / or monocytes, and remission of thrombocytopenia.

[0006] In recent years, an increasing number of studies have shown that LILRB4 is expressed and plays an important role in multiple myeloma (MM). One report indicated that the expression level of LILRB4 in MM (Western medicine) was as high as 88.96%, and the article also demonstrated that T-cell adaptor drugs (TCEs) targeting LILRB4 had a strong killing effect on MM tumor cell lines. It can be said that LILRB4 is a potential target for MM (Di Meo et al., 2023, Cell Reports Medicine 4). Although there are relatively abundant treatment options for MM, providing patients with choices, MM patients are prone to relapse and drug resistance, and new therapies are still needed for this population.

[0007] Currently, the treatment of AML, CMML, or MM, especially for end-line patients, still requires new therapies that are more likely to provide durable responses to a larger patient population. Currently, there is only one LILRB4-targeting monoclonal antibody in clinical trials, IO202 (CN 115551894 A). Although it has shown some clinical response, its efficacy in AML is poor, with only one partial response (PR) (n=6). As a monoclonal antibody, its efficacy is mainly achieved by blocking LILRB4 / ApoE signaling, indirectly activating T cells. It is dependent on both TCR and MHC, or indirectly activates NK cells through Fc to exert its killing effect. It cannot maximize the use of immune cells to achieve a strong killing effect, therefore its clinical efficacy is poor for refractory hematological malignancies like AML. As mentioned earlier, the T-cell linker, containing an antigen domain that binds to CD3 on T cells and a bispecific antibody that binds to a protein expressed on target cells, can bridge T cells and target cells together, forming an immune synapse to induce targeted lysis of target cells. This mechanism differs from chemotherapy and immunotherapy; it does not rely on the major histocompatibility complex (MHC) and T-cell antigen receptor (TCR) and can work directly and efficiently.

[0008] Therefore, the present invention addresses the need to develop novel anti-CD3 antibodies and antibodies that specifically bind to novel other antigens such as LILRB4, as well as novel bispecific antibodies (e.g., bispecific antibodies that specifically bind to CD3 and other antigens such as LILRB4) such as T-cell adaptors based on these novel CD3 antibodies. Summary of the Invention

[0009] This invention provides a novel anti-CD3 antibody or its antigen-binding fragment.

[0010] The present invention also provides a novel anti-LILRB4 antibody or its antigen-binding fragment.

[0011] This invention also provides a bispecific antibody that can simultaneously target LILRB4 and CD3. The bispecific antibody of this invention is a T-cell adaptor (TCE) targeting LILRB4, which does not depend on TCR and MHC. It can directly bridge T cells and tumor cells, forming an immune synapse, activating T cells, and killing tumor cells, thereby directly maximizing the utilization of immune cells to achieve their potential and thus maximizing efficacy. Therefore, the bispecific antibody of this invention has a stronger killing effect than monoclonal antibodies targeting LILRB4 and will have better clinical efficacy, especially for refractory AML, CMML, or MM.

[0012] The bispecific antibodies of the present invention have one or more of the following characteristics:

[0013] (i) Specific binding to primate CD3, such as human or monkey CD3, such as CD3εγ protein, for example, binding to CD3 (e.g. human CD3) with the following equilibrium dissociation constant (KD), said KD being less than about 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 4 nM or 3 nM, or in the range of about 1 nM, 2 nM to any of the above values, or in the range of any of the above values, for example as detected by Fortebio;

[0014] (ii) Binding primate LILRB4 with high affinity, such as human LILRB4 or monkey LILRB4, for example, with the following equilibrium dissociation constant (K D ) binds to LILRB4 (e.g., human LILRB4), the K D Less than about 20 nM, 15 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM or 0.5 nM, or in the range of about 0.1 nM, 0.2 nM, 0.3 nM or 0.4 nM to any of the above values, or in the range of any of the above values, such as those detected by Fortebio;

[0015] (iii) Specific binding to LILRB4, such as human or monkey LILRB4.

[0016] (iv) Specific binding to Jurkat T cells;

[0017] (v) Specifically binds to peripheral blood T cells;

[0018] (vi) Specific binding to LILRB4-positive cells (e.g., human or monkey cells), such as LILRB4-positive tumor cell lines, such as leukemia cell lines, such as human acute myeloid leukemia tumor cells like human acute monocytic leukemia cells or human myeloid monocytic leukemia cells, such as binding in a LILRB4 expression abundance-dependent manner.

[0019] (vii) In the presence of LILRB4-positive cells, it specifically activates T cells; for example, it can bridge LILRB4-positive target cells and T cells, promote T cell activation, and does not non-specifically activate T cells in the absence of tumor cell lines.

[0020] (viii) Specific killing of LILRB4-positive cells (e.g., human or monkey cells), such as LILRB4-positive tumor cells, such as leukemia cells, such as human acute myeloid leukemia tumor cells like human acute monocytic leukemia cells or human myeloid monocytic leukemia cells, such as binding in a LILRB4 expression abundance-dependent manner.

[0021] (ix) It does not cause nonspecific killing of cells that do not express LILRB4;

[0022] (x) It has a killing effect on normal monocytes and / or no killing effect on normal B cells;

[0023] (xi) Concentration-dependent activation of T cells and / or promotion of T cell secretion of granzymes;

[0024] (xii). Stimulating PBMCs to secrete cytokines (e.g., IL2, IFNγ, TNF-α, or IL-10) at a level lower than that of control antibodies such as Tab1 or Glofitamab in the absence of tumor cells is safe;

[0025] (xiii) In the presence of tumor cells, stimulation of PBMCs to secrete cytokines (e.g., IL2, IFNγ, TNF-α, IL-10 and / or IL-6) is more effective than in the absence of tumor cells.

[0026] (xiv). Combined with multiple myeloma tumor cells;

[0027] (xv) It can kill multiple myeloma tumor cells;

[0028] (xvi) It has an inhibitory effect on LILRB4-positive tumors, such as leukemia, such as acute myeloid leukemia tumors, such as human acute monocytic leukemia tumors or human myeloid monocytic leukemia tumors, such as dose-dependent inhibition, such as superior to the control antibody Tab1;

[0029] (xvii). It has an inhibitory effect on multiple myeloma, for example, it is superior to the control antibody Tab1. Attached Figure Description

[0030] Figure 1: Binding of humanized anti-CD3 antibodies 16H4, 313D9 and 341F5 to Jurkat (1A) and human T cells (1B);

[0031] Figure 2: Binding of humanized anti-CD3 antibodies 16H4, 313D9 and 341F5 to monkey PBMCs;

[0032] Figure 3: Activation of Jurkat NF-AT by humanized anti-CD3 antibodies 16H4, 313D9 and 341F5;

[0033] Figure 4: Binding of the humanized anti-LILRB4 antibody H69L28 to tumor cell lines THP-1 (4A) and MV4-11 (4B);

[0034] Figure 5: Binding of the humanized anti-LILRB4 antibody H69L28 to monkey CHO-K1 Rhesus cells overexpressing LILRB4;

[0035] Figure 6: Binding of the obtained humanized anti-LILRB4 antibody to homology family proteins;

[0036] Figure 7: Binding of anti-LILRB4 monoclonal antibody to three cell types: CHO-K1 (7A), HEK293 (7B), and U937 (7C);

[0037] Figure 8: Structure of the dual antibody CD3×LILRB4, where 8A shows the "1+2" ​​structure (corresponding to clone F5-313D9 or F5-341F5), and 8B shows the 1+1 structure (corresponding to clone F3-16H4).

[0038] Figure 9: Binding of Jurkat to the "1+2" ​​structured dual antibody CD3×LILRB4;

[0039] Figure 10: Binding of the "1+2" ​​structured bispecific antibody CD3×LILRB4 to T cells. 10A shows the experimental batch results with Tab1 as the control and isotype IgG1-LALA as the negative control, and 10B shows the experimental batch results with F5-AMG as the control.

[0040] Figure 11: Binding of the "1+2" ​​bispecific antibody CD3×LILRB4 to AML tumor cell lines THP-1 (11A), MV4-11 (11B), and MOLM13 (11C).

[0041] Figure 12: Activation of Jurkat NF-AT by the "1+2" ​​structured dual antibody CD3×LILRB4;

[0042] Figure 13: Killing effect of the "1+2" ​​structured dual antibody CD3×LILRB4 on AML tumor cell lines THP-1 (13A), MV4-11 (13B) and MOLM13 (13C);

[0043] Figure 14: Killing effect of the "1+2" ​​structured double antibody CD3×LILRB4 on the negative cell line RPMI8226;

[0044] Figure 15: Killing of B cells by the "1+2" ​​structured dual antibody CD3×LILRB4;

[0045] Figure 16: The activation of T cells (16A, 16B) and secretion of granzymes (16C, 16D) by the "1+2" ​​structured bispecific antibody CD3×LILRB4 in the presence of THP-1;

[0046] Figure 17: Secretion of cytokines IL2 (17A), IFN-γ (17B), TNF-α (17C), and IL-10 (17D) by the "1+2" ​​bispecific antibody CD3×LILRB4 in the absence of tumor cells;

[0047] Figure 18: Secretion of cytokines IL-2 (18A), IFN-γ (18B), TNF-α (18C), IL-10 (18D), and IL-6 (18E) by the "1+2" ​​bispecific antibody CD3×LILRB4 in the presence of tumor cells;

[0048] Figure 19: Binding of the "1+2" ​​structured bispecific antibody CD3×LILRB4 to the MM tumor cell line MM1.S;

[0049] Figure 20: Killing effect of the "1+2" ​​structured dual antibody CD3×LILRB4 on the MM tumor cell line MM1.S;

[0050] Figure 21: Efficacy of the "1+2" ​​structured bispecific antibody CD3×LILRB4 in the PBMC-reconstructed B-NDG mouse THP-1-Luc human myeloid leukemia in situ model;

[0051] Figure 22: Efficacy of the "1+2" ​​structured dual anti-CD3×LILRB4 in a PBMC-reconstructed MOLM-13 acute myeloid leukemia subcutaneous xenograft model;

[0052] Figure 23: Efficacy of the "1+2" ​​bispecific antibody CD3×LILRB4 in a subcutaneous xenograft model of MM1.S multiple myeloma reconstructed by PBMC.

[0053] Figure 24: Binding of Jurkat to the "1+1" structured dual antibody CD3×LILRB4;

[0054] Figure 25: Binding of the "1+1" structured bispecific antibody CD3×LILRB4 to T cells;

[0055] Figure 26: Binding of the "1+1" structured bispecific antibody CD3×LILRB4 to AML tumor cell lines THP-1 (26A), MV4-11 (26B), and MOLM13 (26C);

[0056] Figure 27: Activation of Jurkat NF-AT by the "1+1" structured dual antibody CD3×LILRB4;

[0057] Figure 28: Killing effect of the "1+1" structured dual antibody CD3×LILRB4 on AML tumor cell lines THP-1 (28A), MV4-11 (28B), and MOLM13 (28C);

[0058] Figure 29: Killing effect of "1+1" structured double antibody CD3×LILRB4 on negative cell lines;

[0059] Figure 30: Killing of B cells by the "1+1" structured bispecific antibody CD3×LILRB4;

[0060] Figure 31: Activation of T cells by the "1+1" structured bispecific antibody CD3×LILRB4 in the presence of THP-1 (Figures 31A, 31B) and secretion of granzymes (31C, 31D);

[0061] Figure 32: Secretion of cytokines IL-2 (32A), IFN-γ (32B), TNF-α (32C), and IL-10 (32D) by the "1+1" bispecific antibody CD3×LILRB4 in the absence of tumor cells;

[0062] Figure 33: Secretion of cytokines IL-2 (33A), IFN-γ (33B), TNF-α (33C), IL-10 (33D), and IL-6 (33E) by the "1+1" bispecific antibody CD3×LILRB4 in the presence of tumor cells;

[0063] Figure 34: Binding of the "1+1" structured bispecific antibody CD3×LILRB4 to the MM tumor cell line MM1.S;

[0064] Figure 35: Killing effect of the "1+1" structured dual antibody CD3×LILRB4 on the MM tumor cell line MM1.S;

[0065] Figure 36: Efficacy of the "1+1" structured bispecific antibody CD3×LILRB4 in the PBMC-reconstructed B-NDG mouse THP-1-Luc human myeloid leukemia in situ model;

[0066] Figure 37: Efficacy of the "1+1" bispecific antibody CD3×LILRB4 in a PBMC-reconstructed MOLM-13 acute myeloid leukemia subcutaneous xenograft model.

[0067] Invention Details

[0068] Before describing the invention in detail below, it should be understood that the invention is not limited to the specific methodologies, schemes, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0069] I. Definition

[0070] To explain this specification, the following definitions will be used, and terms used in the singular may also include plural forms, where appropriate. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive.

[0071] The term “about” when used in conjunction with a numeric value means to encompass a range of numeric values ​​that have a lower limit of 5% (e.g., 4%, 3%, 2%, or 1%) smaller than the specified numeric value and an upper limit of 5% larger than the specified numeric value.

[0072] As used herein, the term “and / or” means any one of the options or two or more of the options.

[0073] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover combinations of the stated elements, integers, or steps. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region composed of that specific sequence.

[0074] When “first” and “second” are mentioned in this article, it is only to distinguish between two structural domains or two chains, and does not indicate the location of the two structural domains in any way.

[0075] As used herein, the term "CD3" refers to the antigen expressed on T cells as a part of the multimolecular T cell receptor (TCR), namely the T cell adaptor antigen, the T cell surface glycoprotein CD3, which is composed of homodimers or heterodimers formed by two of the following four receptor chains: CD3-ε, CD3-δ, CD3-ζ, and CD3-γ. Human CD3-εn (hCD3ε) contains the amino acid sequence described in UniProtKB / Swiss-Prot: P07766. Human CD3-δ (hCD3δ) contains the amino acid sequence described in UniProtKB / Swiss-Prot: P04234. In some embodiments, the CD3 referred to in this invention refers to CD3 derived from humans or monkeys (e.g., cynomolgus monkeys).

[0076] As used herein, the terms "CD3-binding antibody" or "anti-CD3 antibody" encompass antibodies and their antigen-binding fragments that specifically recognize or bind to a single CD3 subunit (e.g., ε, δ, γ, or ζ), and dimer complexes that specifically recognize two CD3 subunits (e.g., γ / ε, δ / ε, and ζ / ζ CD3 dimers) and their associated antibodies and antigen-binding fragments. The antibodies and antigen-binding fragments of the present invention can bind to soluble CD3, bound CD3, and / or CD3 expressed on the cell surface. Soluble CD3 comprises native CD3 protein and recombinant CD3 protein variants, such as monomeric and dimer CD3 structures lacking a transmembrane region or otherwise not binding to the cell membrane. In one embodiment, the antigen-binding region of the CD3-binding fragment or bispecific antibody of the present invention may have low binding activity to CD3 or CD3-expressing cells (e.g., T cells). The binding affinity of the antibody to CD3 can be detected by flow cytometry or biofilm layer optical interferometry.

[0077] The term "LILRB4" (Leukocyte Immunoglobulin-like Receptor B4, member of the human leukocyte immunoglobulin-like receptor subfamily B), also known as ILT3, is a myeloid immune checkpoint molecule belonging to the B subfamily of the leukocyte immunoglobulin-like receptor (LILR) family. The encoding gene is located on human chromosome 19q13.4 in the leukocyte receptor cluster. LILRB4 contains an IgG-like domain in its extracellular region and an immunoreceptor tyrosine inhibitory motif (ITIM) in its intracellular region, through which it transmits inhibitory signals and participates in the negative regulation of immune cell activation. LILRB4 is mainly expressed on the surface of myeloid cells such as monocytes, macrophages, and dendritic cells, as well as some tumor cells, suppressing innate immunity autonomously and inhibiting T cell activation indirectly. Examples of LILRB4 include, for instance, the human LILRB4 protein at Uniprotocol accession number Q8NHJ6 or the cynomolgus monkey LILRB4 protein at accession number A0A5F8AGT6.

[0078] The terms "full-length antibody" or "complete antibody" are used interchangeably herein and refer to antibody molecules that have the molecular structure of natural immunoglobulins. In the case of a conventional four-chain IgG antibody, a full-length antibody consists of two heavy chains (H) and two light chains (L) linked together by disulfide bonds. In the case of a heavy chain antibody that has only heavy chains and lacks light chains, a full-length antibody consists of two heavy chains (H) linked together by disulfide bonds. For a conventional four-chain IgG antibody, the heavy chain of a full-length antibody typically consists of a heavy chain variable region (abbreviated as VH in this document) and a heavy chain constant region, wherein the heavy chain constant region contains at least three domains CH1, CH2, and CH3. The light chain of a full-length antibody consists of a light chain variable region (abbreviated as VL in this document) and a light chain constant region, wherein the light chain constant region consists of one domain CL. Each heavy chain variable region VH and each light chain variable region consists 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 constant region does not directly participate in antibody-antigen binding but exhibits various effector functions. In some embodiments, the antibody heavy chain constant region HC of the present invention is the heavy chain constant region of IgG1, IgG2, IgG3, or IgG4, preferably the heavy chain constant region of IgG1. In some embodiments, the heavy chain constant region contains an LALA mutation. In some embodiments, the antibody light chain constant region LC of the present invention is a Lambda or Kappa light chain constant region.

[0079] The term "antibody fragment" includes a portion of a complete antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment. An "antigen-binding fragment" of an antibody is a molecule distinct from a full-length antibody, containing a portion of the full-length antibody but capable of binding antigens to the full-length antibody or competing with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment originates) for antigen binding. Antigen-binding fragments can be prepared by recombinant DNA technology or by enzymatic or chemical cleavage of the complete antibody. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabody, single-domain antibody (sdAb), nanobody, and sc(Fv)2. For example, a Fab fragment can be obtained by digesting a full-length antibody with papain. Furthermore, digestion of a complete antibody with pepsin below the disulfide bonds in the hinge region produces F(ab')2, a dimer of Fab', which is a divalent antibody fragment. F(ab')2 can be reduced under neutral conditions by breaking the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into a Fab' monomer. Fab' monomers are essentially Fab fragments with hinge regions. The Fv fragment consists of the VL and VH domains of an antibody single arm. The two domains, VL and VH, of the Fv fragment can be encoded by independent genes, but they can also be produced as a single protein chain using a recombinant approach, linking these two domains with a synthetic linker peptide. In this single protein chain, the VL and VH regions pair to form a single-chain Fv (scFv). sc(Fv)2 is a small antibody in which two VH and two VL regions are linked by a linker to form a single chain.

[0080] The term "Fab fragment" or "Fab" is used interchangeably herein to refer to an immunoglobulin fragment consisting of two polypeptide chains containing an immunoglobulin heavy chain variable domain (VH), a heavy chain constant domain (CH1), a light chain variable domain (VL), and a light chain constant domain (CL). One polypeptide chain contains VH and a constant region selected from CH1 and CL from its N-terminus to its C-terminus, while the other polypeptide chain contains VL and another constant region selected from CL and CH1 from its N-terminus to its C-terminus. The VH and VL domains pair to form an antigen-binding site. In this document, the Fab polypeptide chain containing the heavy chain constant region CH1 is also referred to as the "Fab heavy chain," and correspondingly, the Fab polypeptide chain containing the light chain constant region CL is also referred to as the "Fab light chain."

[0081] The terms “Fc region” or “Fc domain” used herein are used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region. These terms include native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (residues 446-447) of the Fc region may or may not be present. Unless otherwise stated herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0082] The complementarity-determining region (CDR) or CDR is a region within the antibody variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while those located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of many known antibody CDR assignment schemes, including, for example: Chothia (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)) based on antibody three-dimensional structure and CDR loop topology; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., USDapartment of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); and the International ImMunoGeneTics. The database (IMGT) (on the World Wide Web at imgt.cines.fr / ) and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0083] The following is the region range of the CDR defined using the Kabat scheme (http: / / www.bioinf.org.uk / abs / info.html).

[0084] Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" encompasses a CDR sequence determined in any of the foregoing manner. A CDR may also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the exemplary CDRs of this invention). Unless otherwise stated, in this invention, when referring to the position of residues in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it means the numbering position according to 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)).

[0085] In one embodiment, the CDR of the antibody heavy chain variable region is determined according to Kabat. In some embodiments, the CDR of the antibody light chain variable region is determined according to Kabat. In some embodiments, the CDRs of both the heavy chain and light chain variable regions of the antibody are determined according to Kabat.

[0086] It should be noted that the boundaries of the CDRs of the variable region of the same antibody may differ based on different assignment schemes. That is, the CDR sequences of the variable region of the same antibody defined under different assignment schemes are different. Therefore, when referring to antibodies defined by the specific CDR sequence of this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from the specific CDR boundaries defined by this invention due to the application of different schemes (e.g., different assignment scheme rules or combinations).

[0087] In this paper, when each domain or fragment is labeled with a superscript, it indicates that the domain or fragment is an antibody or antigen-binding region that specifically binds to the antigen indicated by the superscript. For example, VH CD3 and VL CD3 These represent VH and VL in the antibody or antigen-binding region that specifically binds to CD3, respectively. CD3 scFv, VH, which specifically binds to CD3 LILRB4 and VL LILRB4 These represent VH and VL in the antibody or antigen-binding region that specifically binds to LILRB4, or Fab, respectively. LILRB4 This indicates the Fab fragment that specifically binds to LILRB4.

[0088] The term "chimeric antibody" refers to an antibody molecule in which (a) a constant region or a portion thereof is altered, replaced, or exchanged, thereby linking the antigen-binding site to a different or altered class, effector function, and / or species of constant region, or to a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug), which confers novel properties to the chimeric antibody; or (b) a variable region or a portion thereof is altered, replaced, or exchanged with a variable region having different or altered antigen specificity. For example, a mouse antibody can be modified by replacing its constant region with a constant region derived from human immunoglobulins. Due to the replacement with a human constant region, the chimeric antibody can retain its specificity in recognizing antigens while exhibiting reduced immunogenicity in humans, as compared to the original mouse antibody.

[0089] A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody (such as a mouse monoclonal antibody) while exhibiting low immunogenicity when administered to humans as a therapeutic agent. This can be achieved, for example, by retaining the non-human antigen-binding site and replacing the remaining portion of the antibody with its corresponding human portion (i.e., replacing the non-binding portions of the constant region and variable region with the corresponding portions of the human antibody).

[0090] As used herein, the terms “anti,” “binding,” or “specific binding” mean that the binding interaction is selective for the target or antigen and can be distinguished from unwanted or nonspecific interactions. The ability of a binding site to bind to a specific target or antigen can be determined by flow cytometry or enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as radioimmunoassay (RIA), thin-layer interferometry, MSD assay, or surface plasmon resonance (SPR).

[0091] The term "antigen" refers to a molecule that triggers an immune response. This immune response may involve antibody production or activation of specific immune cells, or both. Those skilled in the art will understand that any macromolecule, including virtually all proteins or peptides, can be used as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA.

[0092] As used herein, the term "antigen-binding region" refers to the portion of a multispecific binding molecule, such as a bispecific binding molecule, that binds to a specific target or antigen. The antigen-binding region can be an antibody or immunoglobulin itself or an antibody fragment. Such an antigen-binding region may or may not have a tertiary structure independent of the remaining portion of the bispecific antibody molecule and may bind to or not bind to its target as a standalone entity. In one embodiment, the antigen-binding region of the bispecific antibody molecule of the present invention comprises a VH / VL pair consisting of a variable region (VL) of the antibody light chain and a variable region (VH) of the antibody heavy chain, said VH / VL pair may be contained in a single polypeptide chain (e.g., in scFv) or in two separate polypeptide chains (e.g., in a Fab heavy chain and a Fab light chain, respectively). In one embodiment, one or more of the antigen-binding regions of the bispecific antibody molecule of the present invention may be Fab. In one embodiment, one or more of the antigen-binding regions of the bispecific antibody molecule of the present invention may be scFv.

[0093] As used herein, the term "monospecific" antibody refers to an antibody having one or more binding regions, each of which binds to the same epitope of the same antigen. For example, the present invention provides monospecific antibodies against CD3 or LILRB4.

[0094] As used herein, the term "multispecific" antibody refers to an antibody having at least two antigen-binding regions, each of which binds to a different epitope of the same antigen or to a different epitope of a different antigen. A multispecific antibody is an antibody that has binding specificity to at least two different antigenic epitopes. In one embodiment, this document provides such a bispecific antibody having binding specificity against a first antigen and a second antigen. For example, the present invention provides a bispecific antibody against CD3 and LILRB4, also referred to as "CD3×LILRB4" or "CD3×LILRB4 bispecific antibody".

[0095] The term "CH1 region" refers to the portion of the antibody heavy chain polypeptide extending from EU position 118 to EU position 216 (EU numbering system). In some cases, such as during Fab construction, the CH1 region may also include a partial hinge region, such as EPKSC. Therefore, in some embodiments, the term "CH1 region" refers to the portion of the antibody heavy chain polypeptide extending from EU position 118 to EU position 220 (EU numbering system). In one embodiment, the CH1 domain comprises or consists of the amino acid sequence of ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC (SEQ ID NO: 31).

[0096] Examples of "effector functions" of immunoglobulins include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen-presenting cell uptake of antigens, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0097] The term "knob-in-hole" mutation, as used herein, refers to the introduction of mutations, respectively, into the first and second Fc peptides, to form a knot and a complementary hole at the interface between the first and second Fc peptides. Knob-in-hole technology is known in the art to modify interfaces between different chains of an antibody molecule to facilitate proper association between the chains. Typically, this technique involves introducing a knot at the interface of one chain and a corresponding hole at the interface of the chain to be paired with it, allowing the knot to be placed within the hole. A preferred interface comprises the CH3 domain of the heavy chain constant domain of one chain and the CH3 domain of the heavy chain constant domain of the chain to be paired with it. The knot can be constructed by replacing a small amino acid side chain from the CH3 domain interface of the heavy chain constant domain of one chain with a larger side chain (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with smaller side chains (e.g., alanine or threonine), compensating holes of the same or similar size as the protrusions are constructed at the interface of the CH3 domain of the heavy chain constant domain of the other chain to be paired. Another alternative interface is the CL domain of the Fab fragment described above, containing the light chain and the CH1 domain of the heavy chain, which promotes proper heterodimerization between the two chains of the Fab fragment by constructing protrusion-hole interactions.

[0098] As used herein, the term "connector" refers to any molecule that enables the direct linking of different domains of a multispecific antibody. Examples of connectors that establish covalent links between different molecular parts include peptide connectors and non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyethylene, or copolymers of PEG and polypropylene glycol. In some embodiments, the connector is a peptide connector, which refers to a short amino acid sequence consisting of amino acids, such as glycine (G) and / or serine (S) and / or threonine residues (T) used alone or in combination, or hinge regions from immunoglobulins, for linking the amino acid sequence of a first part of a multispecific antibody to a second part of the multispecific antibody. For example, a peptide connector can link a first antigen-binding region of a multispecific antibody to a second antigen-binding region. For example, a peptide connector can also link one part of an antibody to another part of an antibody, such as linking Fab to scFv, or linking VH to VL of an antibody. Preferably, the peptide connector has a length sufficient to link two entities in such a way that they maintain their conformation relative to each other without impeding the desired activity. In one embodiment, the linker peptide has a length of 5-50 amino acids, for example, 10, 15, 20, 25, or 30 amino acids. In one embodiment, the linker peptide comprises the amino acid sequences (GKPGS)n(GS)n, (GGS)n, (GSGGS)n, (GGGGS)n, (GGGS)n, and (GGGGS)nG, where n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, or 10. Useful linkers also include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. In some embodiments, the peptide linker is (GGGGS)n, where n = 1, 2, 3, 4, or 5, such as the sequence shown in SEQ ID NO:48; or the peptide linker is (GKPGS)n, where n = 1, 2, 3, 4, or 5, such as the sequence shown in SEQ ID NO:47.

[0099] In some embodiments, the hinge region or a portion thereof of an immunoglobulin may also serve as the linking peptide. In some embodiments, a partial hinge region may be introduced to connect the two parts if neither of them contains a hinge region or not all of its hinge region. For example, if scFv is linked to the Fc region, and the Fc region does not contain all of its hinge region, a partial hinge region may be introduced to connect scFv and the Fc region. In some embodiments, the hinge region may have a mutation, such as a C220S mutation at position C220. Therefore, in some embodiments, the hinge region used to connect the two parts may be a hinge region or a portion thereof of an immunoglobulin (e.g., IgG, such as IgG1, IgG2, IgG3, or IgG4) (e.g., EPKSC, SEQ ID NO:42) or a mutated hinge region or a portion thereof, such as EPKSS (SEQ ID NO:43).

[0100] "Immune conjugates" are antibodies that are conjugated with one or more other substances (such as macromolecules, radioactive elements, cytotoxic agents, etc.).

[0101] "Individual" or "subject" includes mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0102] The term "effective amount" refers to the amount or dose of the antibody, fragment, composition, or combination of the present invention, which, when administered to a patient in a single or multiple doses, produces the desired effect in a patient requiring treatment or prevention. "Therapeutic effective amount" refers to the amount that, at the required dose and for the required duration, effectively achieves the desired therapeutic outcome. "Prophylactic effective amount" refers to the amount that, at the required dose and for the required duration, effectively achieves the desired preventive outcome. Generally, because the prophylactic dose is used in the subject before or at an earlier stage of the disease, the prophylactic effective amount will be less than the therapeutic effective amount.

[0103] The term "host cell" refers to a cell into which exogenous polynucleotides have been introduced, including the progeny of such cells. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their derived progeny, regardless of the number of passages. Host cells can be any type of cell system that can be used to produce the antibody molecules of this invention, including eukaryotic cells, such as mammalian cells, insect cells, and yeast cells; and prokaryotic cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0104] As used herein, the term "label" refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and promotes the detection of the conjugated or fused reagent. The label itself may be detectable (e.g., radioisotope labeling or fluorescent labeling) or, in the case of enzymatic labeling, may catalyze a chemical change in a detectable substrate compound or composition. The term is intended to cover both direct labeling of probes or antibodies by conjugation (i.e., physical linking) to a detectable substance and indirect labeling of probes or antibodies by reaction with another directly labeled reagent.

[0105] "Isolated" antibodies or molecules are those that have been separated from components of their natural environment. In some embodiments, the antibody or molecule is purified to a purity of more than 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0106] The "percentage of identity (%)" for an amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to those in the specific amino acid sequence shown in this specification, after comparing the candidate sequence with the specific amino acid sequence shown herein and, if necessary, introducing vacancies to achieve the maximum percentage of sequence identity, and without considering any conserved substitutions as part of the sequence identity. In some embodiments, the invention contemplates variants of the antibody molecules of the invention that have a considerable degree of identity with respect to the antibody molecules and their sequences specifically disclosed herein, for example, an identity of at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% or higher. These variants may contain conserved changes.

[0107] For polypeptide sequences, "conservative alteration" includes substitutions, deletions, or additions to the polypeptide sequence that do not substantially change the desired functional activity of the polypeptide sequence. For example, a conserved substitution often results in a chemically similar amino acid being replaced. Providing conserved representations of functionally similar amino acids is well known in the art. The following are eight groups of amino acids containing mutually conserved substitutions: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M). In some embodiments, the term "conserved sequence alteration" is used to refer to amino acid modifications that do not significantly affect or alter the target antigen binding characteristics of the antibody molecule or binding protein molecule of the present invention containing the amino acid sequence. For example, conserved modified variants maintain at least 80%, 85%, 90%, 95%, 98%, 99% or higher, such as 100-110% or higher, binding affinity to the target antigen relative to the parent antibody or binding protein.

[0108] The term "therapeutic agent" as used herein encompasses any substance effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (such as immunosuppressants).

[0109] The term "cytotoxic agent" generally refers to substances that kill cells or cause cell death or destruction. "Chemotherapy agents" include chemical compounds that are useful in treating cancer or immune system diseases.

[0110] The term "small molecule drug" refers to low-molecular-weight organic compounds that can regulate biological processes. "Small molecule" is defined as a molecule with a molecular weight less than 10 kDa, typically less than 2 kDa, and preferably less than 1 kDa. As therapeutic agents, small molecules can penetrate cells more easily, are less susceptible to degradation, and are less likely to elicit an immune response than large molecules.

[0111] The term "immunomodulator" as used in this article refers to natural or synthetic active agents or drugs that inhibit or regulate immune responses.

[0112] The term "pharmaceutical excipient" refers to diluents, adjuvants (such as Freund's adjuvants (complete and incomplete)), carriers, or stabilizers that are applied together with the active substance.

[0113] The term "pharmaceutical composition" refers to a composition which is present in a form that allows the biological activity of the active ingredient contained therein to be effective and does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition.

[0114] The terms "drug combination" or "combination product" refer to non-fixed or fixed combinations, including but not limited to kits / reagents and pharmaceutical compositions. The term "non-fixed combination" means that active ingredients (e.g., (i) the molecules of the present invention, and (ii) other therapeutic agents) are administered to a patient simultaneously, without a specific time limit, or sequentially at the same or different time intervals, in separate entities, wherein such administration to the patient provides a preventive or therapeutically effective level. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. Preferably, the dosage and / or time interval of the two or more active agents are selected so that the combined use of the components produces an effect greater than that achieved by using any one component alone in treating a disease or condition. Each component may be in a separate formulation, and the formulations may be the same or different.

[0115] The term "combination therapy" refers to the administration of two or more therapeutic agents or modes of treatment to treat the disease described herein. Such administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule containing active ingredients in a fixed proportion. Alternatively, such administration includes the co-administration of individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids may be reconstituted or diluted to the desired dose prior to administration. Furthermore, such administration includes the sequential administration of each type of therapeutic agent at substantially the same time or at different times. The treatment regimen will provide the beneficial effect of the combination of drugs in treating the condition or symptom described herein.

[0116] When used in this article, "treatment" means to slow down, interrupt, block, alleviate, stop, reduce, or reverse the onset of symptoms, complications, or biochemical indicators of a disease, relieve symptoms, or prevent or inhibit the further development of the disease, condition, or symptom.

[0117] When used in this article, "prevention" includes the suppression of the occurrence or development of a disease or condition or the symptoms of a particular disease or condition.

[0118] The term "expression vector" refers to a vector containing recombinant polynucleotides, which includes an expression control sequence that effectively links the nucleotide sequence to be expressed. Expression vectors contain sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system.

[0119] "Subject / Patient / Individual Sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of a tissue or cell sample can be solid tissue, such as fresh, frozen, and / or preserved organ or tissue samples, biopsy samples, or puncture samples; blood or any blood component; body fluids, such as tears, vitreous fluid, cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; or cells from any stage of pregnancy or development in the subject. Tissue samples may contain compounds that are naturally occurring and do not mix with tissues, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0120] All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Any or all features discussed above and throughout this application may be combined in various embodiments of the invention. Furthermore, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will become apparent from this specification and the accompanying drawings and from the appended claims.

[0121] II. Specific Implementation Plan

[0122] In a first aspect, the present invention relates to an anti-CD3 antibody or a fragment thereof.

[0123] In some embodiments, the anti-CD3 antibody or fragment thereof of the present invention binds to primate CD3 (e.g., human CD3 or monkey CD3, such as CD3εγ protein) with high affinity, for example, with the following equilibrium dissociation constant (K). D K binds to CD3 (e.g., human CD3), D Less than about 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 4 nM or 3 nM, or in the range of about 1 nM, 2 nM to any of the above values, or in the range of any of the above values, such as those detected by Fortebio.

[0124] In some embodiments, the anti-CD3 antibody or fragment thereof of the present invention has one or more of the following properties:

[0125] (i) It exhibits concentration-dependent binding to CD3-positive tumor cell lines and / or to T cells;

[0126] (ii) Combine with monkey PBMCs, such as cynomolgus monkey PBMCs; or

[0127] (iii) It can activate TCR-mediated signaling pathways and activate T cells.

[0128] In some embodiments, the anti-CD3 antibody is a monoclonal antibody. In some embodiments, the anti-CD3 antibody of the present invention is a chimeric antibody or a humanized antibody. In some embodiments, the anti-CD3 antibody of the present invention is a full-length antibody. In one embodiment, the anti-CD3 antibody of the present invention also encompasses its antibody fragment, preferably an antigen-binding fragment, such as antibody fragments selected from Fab, Fab', F(ab')2, Fv, scFv, diabody, or sc(Fv)2.

[0129] In some embodiments, the anti-CD3 antibody of the present invention is suitable for constructing multispecific antibodies, such as bispecific antibodies. In some embodiments, the anti-CD3 antibody molecule is in the form of a bispecific or multispecific antibody molecule. Therefore, when referring to anti-CD3 antibody, it also encompasses multispecific antibodies that specifically bind to CD3, such as bispecific antibodies.

[0130] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises three complementarity-determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, and / or three complementarity-determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3. In some aspects, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region (VH, which may also be designated VH herein). CD3 ), and / or the light chain variable region (VL, which may also be identified as VL in this document). CD3 In some aspects, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL).

[0131] In some embodiments, the heavy chain variable region of the anti-CD3 antibody or its antigen-binding fragment described in this invention...

[0132] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 1, 9, 14, 33, or 35; or

[0133] (ii) Contains or consists of an amino acid sequence selected from or composed of said amino acid sequence; or

[0134] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 1, 9, 14, 33, or 35, preferably, the amino acid alterations do not occur in the CDR region.

[0135] In some embodiments, the light chain variable region of the anti-CD3 antibody or its antigen-binding fragment described in this invention...

[0136] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 2, 10, 15, 32, or 34; or

[0137] (ii) Contains or consists of an amino acid sequence selected from or composed of said amino acid sequence; or

[0138] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO:2, 10, 15, 32, or 34, preferably, the amino acid alterations do not occur in the CDR region.

[0139] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises three complementarity-determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, and / or three complementarity-determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, wherein

[0140] The three complementary determinant regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, are respectively the three complementary determinant regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in SEQ ID NO: 1, 9, 14, 33, or 35, and / or

[0141] The three complementary determination regions (LCDRs) from the light chain variable region, LCDR1, LCDR2 and LCDR3 are the three complementary determination regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO:2, 10, 15, 32 or 34.

[0142] For example, the HCDR and LCDR can be determined according to any CDR determination scheme known in the art, such as Kabat, Chothia, IMGT or AbM or a combination thereof. Preferably, the HCDR and LCDR can be determined according to the Kabat scheme.

[0143] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment described in this invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3, or comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein...

[0144] HCDR1 contains, or is composed of, the amino acid sequence of SEQ ID NO:3 or 11, or contains an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:3 or 11.

[0145] HCDR2 contains, or is composed of, the amino acid sequence of SEQ ID NO:4, 12 or 16, or contains an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:4, 12 or 16.

[0146] HCDR3 contains, or is composed of, the amino acid sequence of SEQ ID NO:5, 13 or 17, or contains an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:5, 13 or 17.

[0147] LCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:6, or contains an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:6.

[0148] LCDR2 contains, or is composed of, the amino acid sequence of SEQ ID NO:7, or contains an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:7; and / or

[0149] LCDR3 contains, or is composed of, the amino acid sequence of SEQ ID NO:8, or contains an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:8.

[0150] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment described in this invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein...

[0151] (i) HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:3; HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:4; HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:5; LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:6; LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:7; and LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:8.

[0152] (ii). HCDR1 contains the amino acid sequence shown in SEQ ID NO:3; HCDR2 contains the amino acid sequence shown in SEQ ID NO:4; HCDR3 contains the amino acid sequence shown in SEQ ID NO:5; LCDR1 contains the amino acid sequence shown in SEQ ID NO:6; LCDR2 contains the amino acid sequence shown in SEQ ID NO:7; and LCDR3 contains the amino acid sequence shown in SEQ ID NO:8.

[0153] (iii) HCDR1 consists of the amino acid sequence shown in SEQ ID NO:3; HCDR2 consists of the amino acid sequence shown in SEQ ID NO:4; HCDR3 consists of the amino acid sequence shown in SEQ ID NO:5; LCDR1 consists of the amino acid sequence shown in SEQ ID NO:6; LCDR2 consists of the amino acid sequence shown in SEQ ID NO:7; and LCDR3 consists of the amino acid sequence shown in SEQ ID NO:8.

[0154] (iv) HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:11; HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:12; HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:13; LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:6; LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:7; and LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:8.

[0155] (v) HCDR1 contains the amino acid sequence shown in SEQ ID NO:11; HCDR2 contains the amino acid sequence shown in SEQ ID NO:12; HCDR3 contains the amino acid sequence shown in SEQ ID NO:13; LCDR1 contains the amino acid sequence shown in SEQ ID NO:6; LCDR2 contains the amino acid sequence shown in SEQ ID NO:7; and LCDR3 contains the amino acid sequence shown in SEQ ID NO:8.

[0156] (vi) HCDR1 consists of the amino acid sequence shown in SEQ ID NO:11; HCDR2 consists of the amino acid sequence shown in SEQ ID NO:12; HCDR3 consists of the amino acid sequence shown in SEQ ID NO:13; LCDR1 consists of the amino acid sequence shown in SEQ ID NO:6; LCDR2 consists of the amino acid sequence shown in SEQ ID NO:7; and LCDR3 consists of the amino acid sequence shown in SEQ ID NO:8.

[0157] (vii) HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:11; HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:16; HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:17; LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:6; LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:7; and LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:8.

[0158] (viii) HCDR1 contains the amino acid sequence shown in SEQ ID NO:11; HCDR2 contains the amino acid sequence shown in SEQ ID NO:16; HCDR3 contains the amino acid sequence shown in SEQ ID NO:17; LCDR1 contains the amino acid sequence shown in SEQ ID NO:6; LCDR2 contains the amino acid sequence shown in SEQ ID NO:7, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:8; or

[0159] (ix). HCDR1 consists of the amino acid sequence shown in SEQ ID NO:11; HCDR2 consists of the amino acid sequence shown in SEQ ID NO:16; HCDR3 consists of the amino acid sequence shown in SEQ ID NO:17; LCDR1 consists of the amino acid sequence shown in SEQ ID NO:6; LCDR2 consists of the amino acid sequence shown in SEQ ID NO:7; and LCDR3 consists of the amino acid sequence shown in SEQ ID NO:8.

[0160] In some specific embodiments of the present invention, the anti-CD3 antibody or its antigen-binding fragment comprises:

[0161] 1) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:1 or 33, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:2 or 32;

[0162] 2) The three complementary determining regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO:9 or 35, and the three complementary determining regions LCDR1, LCDR2, and LCDR3 contained in VL as shown in SEQ ID NO:10 or 34; or

[0163] 3) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:14, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:15;

[0164] For example, the HCDR and LCDR can be determined according to any CDR determination scheme known in the art, such as Kabat, Chothia, IMGT or AbM or a combination thereof. Preferably, the HCDR and LCDR can be determined according to the Kabat scheme.

[0165] In some specific embodiments of the present invention, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as described above, and / or the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as described above. In some specific embodiments of the present invention, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are as described above.

[0166] In some specific embodiments of the present invention, the anti-CD3 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein...

[0167] (i) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:1 or 33, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:2 or 32, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0168] (ii) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0169] (iii) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:33, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:32, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0170] (iv) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9 or 35, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 10 or 34, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0171] (v) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:9, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:10, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0172] (vi) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:35, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:34, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; or

[0173] (vii) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:14, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:15, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0174] In some specific embodiments of the present invention, the anti-CD3 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein...

[0175] (i) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:1 or 33; the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:2 or 32;

[0176] (ii) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:1; the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:2;

[0177] (iii) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:33; the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:32;

[0178] (iv) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO: 9 or 35; the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO: 10 or 34;

[0179] (v) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:9; the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:10;

[0180] (vi) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:35; the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:34; or

[0181] (vii) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:14; the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:15.

[0182] In some specific embodiments of the present invention, the anti-CD3 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein...

[0183] (i). The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:1 or 33; the light chain variable region contains the amino acid sequence shown in SEQ ID NO:2 or 32;

[0184] (ii). 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:2;

[0185] (iii) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:33; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:32;

[0186] (iv). The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9 or 35; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 10 or 34.

[0187] (v) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:9; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:10;

[0188] (vi) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:35; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:34; or

[0189] (vii) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:14; the light chain variable region contains the amino acid sequence shown in SEQ ID NO:15.

[0190] In some specific embodiments of the present invention, the anti-CD3 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein...

[0191] (i) The heavy chain variable region is composed of the amino acid sequence shown in SEQ ID NO:1 or 33; the light chain variable region is composed of the amino acid sequence shown in SEQ ID NO:2 or 32.

[0192] (ii). The heavy chain variable region is composed of the amino acid sequence shown in SEQ ID NO:1; the light chain variable region is composed of the amino acid sequence shown in SEQ ID NO:2;

[0193] (iii) The heavy chain variable region is composed of the amino acid sequence shown in SEQ ID NO:33; the light chain variable region is composed of the amino acid sequence shown in SEQ ID NO:32;

[0194] (iv) The heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 9 or 35; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 10 or 34.

[0195] (v) The heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:9; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:10;

[0196] (vi) The heavy chain variable region is composed of the amino acid sequence shown in SEQ ID NO:35; the light chain variable region is composed of the amino acid sequence shown in SEQ ID NO:34; or

[0197] (vii) The heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:14; the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:15.

[0198] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention further comprises an antibody heavy chain constant region. In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention further comprises an antibody light chain constant region. In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention further comprises both a heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region comprises an Fc region, such as the Fc region as defined herein.

[0199] In some embodiments, the heavy chain constant region is a heavy chain constant region derived from (e.g., human) IgG, such as the heavy chain constant region of (e.g., human) IgG1, IgG2, IgG3, or IgG4, preferably the heavy chain constant region of (e.g., human) IgG1. In some embodiments, the heavy chain constant region of the anti-CD3 antibody of the present invention contains a mutation that reduces binding to the Fcγ receptor, such as the L234A / L235A mutation (also known as the LALA mutation).

[0200] In some implementations, the heavy chain constant region

[0201] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:36;

[0202] (ii) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:37 and containing an L234A / L235A mutation; or

[0203] (iii) Contains or is composed of an amino acid sequence selected from SEQ ID NO: 36 or 37; or

[0204] (iv) An amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 36 or 37.

[0205] In some embodiments, the antibody light chain constant region of the present invention is a light chain constant region derived from (e.g., human) lambda or Kappa light chain constant regions, preferably (human) Kappa light chain constant regions. In some embodiments, the antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment in the form of IgG1 and contains a human Kappa light chain constant region.

[0206] In some implementations, the light chain constant region

[0207] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:49;

[0208] (ii) Containing or consisting of the amino acid sequence of SEQ ID NO:49; or

[0209] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO:49.

[0210] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises an antibody heavy chain (HC). In some embodiments, the heavy chain comprises or consists of antibody heavy chain variable regions and antibody heavy chain constant regions as defined herein. In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises an antibody light chain (LC). In some embodiments, the antibody light chain comprises or consists of antibody light chain variable regions and antibody light chain constant regions as defined herein.

[0211] In some embodiments, the anti-CD3 antibody of the present invention or its antigen-binding fragment comprises the heavy chain and the light chain. In some embodiments, the anti-CD3 antibody of the present invention or its antigen-binding fragment comprises two of the heavy chains and two of the light chains, or is composed of two of the heavy chains and two of the light chains, for example, comprising two identical heavy chains and two identical light chains, or composed of two identical heavy chains and two identical light chains.

[0212] In one embodiment of the invention, the amino acid alterations described herein include substitution, insertion, or deletion of amino acids. Preferably, the amino acid alterations described herein are amino acid substitutions, preferably conservative substitutions. In a preferred embodiment, the amino acid alterations described herein occur in regions outside the CDR (e.g., in the FR). More preferably, the amino acid alterations described herein occur in regions outside the heavy chain variable region and / or outside the light chain variable region. In some embodiments, the substitution is a conservative substitution. A conservative substitution refers to the substitution of one amino acid with another amino acid of the same class, such as the substitution of one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid with another neutral amino acid. In some embodiments, the substitution occurs in the CDR region of the antibody. Typically, the resulting variant has modifications (e.g., improvements) relative to the parent antibody in certain biological properties (e.g., increased affinity) and / or will have substantially retained certain biological properties of the parent antibody. In some embodiments, it may be necessary to produce cysteine-engineered antibodies, such as "thioMAb," wherein one or more residues of the antibody are substituted with cysteine ​​residues. In some embodiments, the antibodies provided herein may be further modified to contain other non-protein moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-diane, poly-1,3,6-triane, ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0213] In some embodiments, the anti-CD3 antibody of the present invention is an antibody in the form of IgG1, IgG2, IgG3, or IgG4.

[0214] In a second aspect, the present invention relates to an anti-LILRB4 antibody or a fragment thereof.

[0215] In some embodiments, the anti-LILRB4 antibody or fragment thereof of the present invention binds to primate LILRB4 (e.g., human LILRB4 or monkey LILRB4, such as LILRB4 of rhesus monkeys or cynomolgus monkeys) with high affinity, for example, with the following equilibrium dissociation constant (K D ) binds to LILRB4 (e.g., human LILRB4), the K DLess than about 20 nM, 15 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM or 0.5 nM, or in the range of about 0.1 nM, 0.2 nM, 0.3 nM or 0.4 nM to any of the above values, or in the range of any of the above values, such as those detected by Fortebio.

[0216] In some embodiments, the anti-LILRB4 antibody or fragment thereof of the present invention has one or more of the following properties:

[0217] (i) Specifically binds to LILRB4-positive cells (e.g., human or monkey cells), such as LILRB4-positive tumor cell lines, such as leukemia cell lines, such as human acute monocytic leukemia cells or human myeloid monocytic leukemia cells.

[0218] (ii) It does not bind to other members of the LILRB4 family, but binds specifically to LILRB4; and / or

[0219] (iii) It does not bind nonspecifically to cell lines that do not express LILRB4.

[0220] In some embodiments, the anti-LILRB4 antibody is a monoclonal antibody. In some embodiments, the anti-LILRB4 antibody of the present invention is a chimeric antibody or a humanized antibody. In some embodiments, the anti-LILRB4 antibody of the present invention is a full-length antibody. In one embodiment, the anti-LILRB4 antibody of the present invention also encompasses its antibody fragment, preferably an antigen-binding fragment, such as antibody fragments selected from: Fab, Fab', F(ab')2, Fv, scFv, diabody antibody, or sc(Fv)2.

[0221] In some embodiments, the anti-LILRB4 antibody of the present invention is suitable for constructing multispecific antibodies, such as bispecific antibodies. In some embodiments, the anti-LILRB4 antibody molecule is in the form of a bispecific or multispecific antibody molecule. Therefore, when referring to anti-LILRB4 antibody, it also encompasses multispecific antibodies, such as bispecific antibodies, that specifically bind to LILRB4.

[0222] In some embodiments, the anti-LILRB4 antibody or its antigen-binding fragment of the present invention comprises three complementarity-determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, and / or three complementarity-determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3. In some aspects, the anti-LILRB4 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region (VH, which may also be designated VH herein). LILRB4 ), and / or the light chain variable region (VL, which may also be identified as VH in this document).LILRB4 In some aspects, the anti-LILRB4 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL).

[0223] In some embodiments, the heavy chain variable region of the anti-LILRB4 antibody or its antigen-binding fragment described in this invention...

[0224] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:18; or

[0225] (ii) Containing or consisting of the amino acid sequence of SEQ ID NO:18; or

[0226] (iii) An amino acid sequence comprising or consisting of the amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO:18, preferably, the amino acid changes do not occur in the CDR region.

[0227] In some embodiments, the light chain variable region of the anti-LILRB4 antibody or its antigen-binding fragment described in this invention...

[0228] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:19; or

[0229] (ii) Containing or consisting of the amino acid sequence of SEQ ID NO:19; or

[0230] (iii) An amino acid sequence comprising or consisting of the amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO:19, preferably, the amino acid changes do not occur in the CDR region.

[0231] In some embodiments, the anti-LILRB4 antibody or its antigen-binding fragment according to the present invention comprises three complementarity-determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, and / or three complementarity-determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, wherein

[0232] The three complementary determinant regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, are respectively the three complementary determinant regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO:18, and / or

[0233] The three complementary determination regions (LCDRs) from the light chain variable region, LCDR1, LCDR2 and LCDR3 are the three complementary determination regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO:19.

[0234] For example, the HCDR and LCDR can be determined according to any CDR determination scheme known in the art, such as Kabat, Chothia, IMGT or AbM or a combination thereof. Preferably, the HCDR and LCDR can be determined according to the Kabat scheme.

[0235] In some embodiments, the anti-LILRB4 antibody or its antigen-binding fragment described in this invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3, or comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein...

[0236] HCDR1 contains, or is composed of, the amino acid sequence of SEQ ID NO:20, or contains an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:20.

[0237] HCDR2 contains the amino acid sequence of SEQ ID NO:21, or is composed of the amino acid sequence, or contains an amino acid sequence that has one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:21.

[0238] HCDR3 contains, or is composed of, the amino acid sequence of SEQ ID NO:22, or contains an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:22.

[0239] LCDR1 contains the amino acid sequence of SEQ ID NO:23, or is composed of the amino acid sequence, or contains an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:6.

[0240] LCDR2 contains, or is composed of, the amino acid sequence of SEQ ID NO:24, or contains an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:7; and / or

[0241] LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:25, or contains an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:8.

[0242] In some embodiments, the anti-LILRB4 antibody or its antigen-binding fragment described in this invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein...

[0243] (i) HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:20; HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:21; HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:22; LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:23; LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:24; and LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:25.

[0244] (ii) HCDR1 contains the amino acid sequence shown in SEQ ID NO:20; HCDR2 contains the amino acid sequence shown in SEQ ID NO:21; HCDR3 contains the amino acid sequence shown in SEQ ID NO:22; LCDR1 contains the amino acid sequence shown in SEQ ID NO:23; LCDR2 contains the amino acid sequence shown in SEQ ID NO:24; and LCDR3 contains the amino acid sequence shown in SEQ ID NO:25; or

[0245] (iii) HCDR1 consists of the amino acid sequence shown in SEQ ID NO:20; HCDR2 consists of the amino acid sequence shown in SEQ ID NO:21; HCDR3 consists of the amino acid sequence shown in SEQ ID NO:22; LCDR1 consists of the amino acid sequence shown in SEQ ID NO:23; LCDR2 consists of the amino acid sequence shown in SEQ ID NO:24; and LCDR3 consists of the amino acid sequence shown in SEQ ID NO:25.

[0246] In some specific embodiments of the present invention, the anti-LILRB4 antibody or its antigen-binding fragment of the present invention comprises the three complementary determinant regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:18, and the three complementary determinant regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:19.

[0247] For example, the HCDR and LCDR can be determined according to any CDR determination scheme known in the art, such as Kabat, Chothia, IMGT or AbM or a combination thereof. Preferably, the HCDR and LCDR can be determined according to the Kabat scheme.

[0248] In some specific embodiments of the present invention, the anti-LILRB4 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as described above, and / or the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as described above. In some specific embodiments of the present invention, the anti-LILRB4 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are as described above.

[0249] In some specific embodiments of the present invention, the anti-LILRB4 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:18, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:19, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0250] In some specific embodiments of the present invention, the anti-LILRB4 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:18; and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:19. In some specific embodiments of the present invention, the anti-LILRB4 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:18; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:19. In some specific embodiments of the present invention, the anti-LILRB4 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is composed of the amino acid sequence shown in SEQ ID NO:18; and the light chain variable region is composed of the amino acid sequence shown in SEQ ID NO:19.

[0251] In some embodiments, the anti-LILRB4 antibody or its antigen-binding fragment of the present invention further comprises an antibody heavy chain constant region. In some embodiments, the anti-LILRB4 antibody or its antigen-binding fragment of the present invention further comprises an antibody light chain constant region. In some embodiments, the anti-LILRB4 antibody or its antigen-binding fragment of the present invention further comprises both a heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region comprises an Fc region, such as the Fc region as defined herein.

[0252] In some embodiments, the heavy chain constant region is a heavy chain constant region derived from (e.g., human) IgG, such as (e.g., human) IgG1, IgG2, IgG3, or IgG4, preferably (e.g., human) IgG1. In some embodiments, the heavy chain constant region of the anti-LILRB4 antibody of the present invention contains a mutation that reduces binding to the Fcγ receptor, such as the L234A / L235A mutation (also known as the LALA mutation).

[0253] In some implementations, the heavy chain constant region

[0254] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:36;

[0255] (ii) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:37 and containing an L234A / L235A mutation; or

[0256] (iii) Contains or is composed of an amino acid sequence selected from SEQ ID NO: 36 or 37; or

[0257] (iv) An amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 36 or 37.

[0258] In some embodiments, the antibody light chain constant region of the present invention is a light chain constant region derived from (e.g., human) lambda or Kappa light chain constant regions, preferably (human) Kappa light chain constant regions. In some embodiments, the antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment in the form of IgG1 and contains a human Kappa light chain constant region.

[0259] In some implementations, the light chain constant region

[0260] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:49;

[0261] (ii) Containing or consisting of the amino acid sequence of SEQ ID NO:49; or

[0262] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO:49.

[0263] In some embodiments, the anti-LILRB4 antibody or its antigen-binding fragment of the present invention comprises an antibody heavy chain (HC). In some embodiments, the heavy chain comprises or consists of antibody heavy chain variable regions and antibody heavy chain constant regions as defined herein. In some embodiments, the anti-LILRB4 antibody or its antigen-binding fragment of the present invention comprises an antibody light chain (LC). In some embodiments, the antibody light chain comprises or consists of antibody light chain variable regions and antibody light chain constant regions as defined herein.

[0264] In some embodiments, the anti-LILRB4 antibody or its antigen-binding fragment of the present invention comprises the heavy chain and the light chain. In some embodiments, the anti-LILRB4 antibody or its antigen-binding fragment of the present invention comprises two of the heavy chains and two of the light chains, or consists of two of the heavy chains and two of the light chains, for example, comprising two identical heavy chains and two identical light chains, or consisting of two identical heavy chains and two identical light chains.

[0265] In one embodiment of the invention, the amino acid alterations described herein include substitution, insertion, or deletion of amino acids. Preferably, the amino acid alterations described herein are amino acid substitutions, preferably conservative substitutions. In a preferred embodiment, the amino acid alterations described herein occur in regions outside the CDR (e.g., in the FR). More preferably, the amino acid alterations described herein occur in regions outside the heavy chain variable region and / or outside the light chain variable region. In some embodiments, the substitution is a conservative substitution. A conservative substitution refers to the substitution of one amino acid with another amino acid of the same class, such as the substitution of one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid with another neutral amino acid. In some embodiments, the substitution occurs in the CDR region of the antibody. Typically, the resulting variant has modifications (e.g., improvements) relative to the parent antibody in certain biological properties (e.g., increased affinity) and / or will have substantially retained certain biological properties of the parent antibody. In some embodiments, it may be necessary to produce cysteine-engineered antibodies, such as "thioMAb," wherein one or more residues of the antibody are substituted with cysteine ​​residues. In some embodiments, the antibodies provided herein may be further modified to contain other non-protein moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-diane, poly-1,3,6-triane, ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0266] In some embodiments, the anti-LILRB4 antibody of the present invention is an antibody in the form of IgG1, IgG2, IgG3, or IgG4.

[0267] In a third aspect, the present invention relates to a multispecific antibody, such as a bispecific antibody, which specifically binds to CD3 and / or LILRB4.

[0268] In some embodiments, the present invention relates to a multispecific antibody, such as a bispecific antibody, that specifically binds to CD3 and one or more other antigens, such as tumor-associated antigens or tumor antigens, wherein the multispecific antibody comprises an antigen-binding region that specifically binds to CD3 and an antigen-binding region that specifically binds to the other antigen. In some embodiments, the other antigen is LILRB4.

[0269] In some embodiments, the present invention relates to a multispecific antibody, such as a bispecific antibody, that specifically binds to LILRB4 and one or more other antigens, such as CD3, wherein the multispecific antibody comprises an antigen-binding region that specifically binds to LILRB4 and an antigen-binding region that specifically binds to the other antigens.

[0270] In some embodiments, the present invention relates to a multispecific antibody that specifically binds to CD3 and LILRB4, and optionally one or more other antigens, such as tumor-associated antigens or tumor antigens, wherein the multispecific antibody comprises an antigen-binding region that specifically binds to CD3 and an antigen-binding region that specifically binds to LILRB4, and optionally one or more antigen-binding regions that specifically bind to other antigens.

[0271] In some embodiments, the present invention relates to a bispecific antibody that specifically binds to CD3 and LILRB4.

[0272] In some specific embodiments, the present invention relates to a bispecific antibody comprising

[0273] The first antigen-binding region and the second antigen-binding region specifically bind CD3, and / or the second antigen-binding region specifically binds LILRB4.

[0274] In some embodiments, the multispecific antibody of the present invention is an IgG-like multispecific antibody. In some embodiments, the bispecific antibody of the present invention is an IgG-like bispecific antibody. As used herein, "IgG-like bispecific antibody" or "IgG-like multispecific antibody" refers to a bispecific antibody or multispecific antibody comprising an Fc dimer. Therefore, in some embodiments, the multispecific antibody of the present invention, such as a bispecific antibody, comprises an Fc dimer. In some embodiments, the Fc dimer comprises a first Fc region and a second Fc region, wherein the first Fc region and the second Fc region may be the same or different.

[0275] In some embodiments, the multispecific antibody of the present invention, such as a bispecific antibody, comprises two antigen-binding regions that specifically bind to LILRB4 and one antigen-binding region that specifically binds to CD3. In some embodiments, the multispecific antibody of the present invention comprises one antigen-binding region that specifically binds to LILRB4 and one antigen-binding region that specifically binds to CD3.

[0276] The present invention describes various components / domains for multispecific antibodies, such as bispecific antibodies. Those skilled in the art will understand that, unless the context explicitly indicates otherwise, any combination of any technical features of any component of a multispecific antibody, such as a bispecific antibody, described herein is within the scope of this invention. Furthermore, those skilled in the art will understand that, unless the context explicitly indicates otherwise, the antibodies of this invention (including antibodies of any form) may comprise any such combination.

[0277] In some embodiments, the first antigen-binding region (the antigen-binding region specifically binding to CD3) of the multispecific antibody, such as a bispecific antibody, suitable for the present invention may comprise, or be composed of, the full-length anti-CD3 antibody or its antigen-binding fragment described in the first aspect of this invention, as long as it can specifically bind to CD3, including but not limited to, for example, a full-length antibody specifically binding to CD3, Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, single-domain antibody such as VHH, dAb (domain antibody), heavy-chain antibody, or linear antibody, etc. In some embodiments, the first antigen-binding region is derived from the anti-CD3 antibody or its antigen-binding fragment described in the first aspect of this invention, for example, the scFv fragment of the anti-CD3 antibody described in the first aspect.

[0278] In some embodiments, the CD3-specific antigen-binding region comprises 1, 2, 3, 4, 5, or 6 CDRs of the anti-CD3 antibody described in the first aspect of this document. In some embodiments, the CD3-specific antigen-binding region comprises the anti-CD3 antibodies HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 described in the first aspect of this document. In some embodiments, the CD3-specific antigen-binding region comprises the heavy chain variable region VH (also referred to as VH) of the anti-CD3 antibody described in the first aspect of this document. CD3 ) and light chain variable region VL (also known as VL) CD3 ), particularly combinations of VH and VL containing anti-CD3 antibodies as described in the first aspect of this document.

[0279] Unless otherwise specified in this article, VH CD3VH refers to the heavy chain variable region contained in an anti-CD3 antibody or its antigen-binding fragment or antigen-binding region that specifically binds to CD3. In this document, unless otherwise specified, VL refers to the variable region (VH). CD3 It refers to the light chain variable region (VL) contained in anti-CD3 antibodies or their antigen-binding fragments or antigen-binding regions that specifically bind to CD3.

[0280] In some embodiments, the first antigen-binding region is an scFv fragment of an antibody that specifically binds to CD3 as described in the first aspect of this document. CD3 ).

[0281] In some embodiments, the scFv fragment comprises a polypeptide chain containing antibody VH and VL domains, wherein the VH and VL are linked (e.g., via a linker) to pair and form an antigen-binding site. In some embodiments, the scFv is in a trans configuration, comprising or consisting of VH, a linker, and VL (VH-linker-VL) from the N-terminus to the C-terminus. In other embodiments, the scFv is in a cis configuration, comprising or consisting of VL, a linker, and VH (VL-linker-VH) from the N-terminus to the C-terminus. In some embodiments, the linker is a peptide linker composed of amino acid residues. Suitable peptide linkers are known to those skilled in the art.

[0282] In some embodiments, the antigen-binding region that specifically binds to CD3 is a CD3-specific scFv CD3 Its N-terminus to C-terminus contains or consists of the following:

[0283] (i)VH CD3 -VL CD3 Where “-” represents a connector or direct connection, preferably a connector, where VH CD3 C-terminus and VL CD3 The N-terminus is connected directly or via a connector; or

[0284] (ii)VL CD3 -VH CD3 Where "-" represents a connector or direct connection, preferably a connector, where VL CD3 C-terminus and VH CD3 The N-terminus is connected directly or via a connector.

[0285] In one embodiment, the linker is 5-50 amino acids long, for example 5-30 amino acids long, such as 15 or 20 amino acids long. In one embodiment, the linker is an amino acid sequence (GKPGS)n, where n = 1, 2, 3, 4 or 5, preferably n = 3 or 4, more preferably n = 4. In one embodiment, the linker comprises or consists of the amino acid sequence shown in SEQ ID NO:45.

[0286] In some implementations, the scFv CD3 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which contain anti-CD3 antibodies as described in the first aspect of this document.

[0287] In some implementations, the VH CD3 It comprises HCDR1, HCDR2, and HCDR3, which are anti-CD3 antibodies as described in the first aspect of this document. In some embodiments, the VH CD3 It is the heavy chain variable region VH of the anti-CD3 antibody described in the first aspect of this document. In some embodiments, the VL... CD3 LCDR1, LCDR2, and LCDR3 comprise anti-CD3 antibodies as described in the first aspect of this document. In some embodiments, the VL CD3 It is the light chain variable region (VL) of the anti-CD3 antibody described in the first aspect of this article.

[0288] In some implementations, the VH CD3 The VL contains HCDR1, HCDR2, and HCDR3, which are anti-CD3 antibodies as described in the first aspect of this document. CD3 LCDR1, LCDR2, and LCDR3 comprise anti-CD3 antibodies as described in the first aspect of this document. In some embodiments, the VH CD3 It is the heavy chain variable region VH of the anti-CD3 antibody described in the first aspect of this document, and the VL CD3 It is the heavy chain variable region VL of the anti-CD3 antibody described in the first aspect of this document. In some embodiments, the VH... CD3 and the VL CD3 These are VH and VL in the combination of anti-CD3 antibodies VH and VL as described in the first aspect of this article.

[0289] In some preferred embodiments, the scFv antigen-binding region is a disulfide-bonded scFv. In some preferred embodiments, the scFv introduces a disulfide bond between VH and VL to increase stability, i.e., by mutating the amino acid at the corresponding site in VH and VL to cysteine. In some preferred embodiments, the disulfide-bonded scFv has a cysteine ​​mutation at amino acid position 44 in VH and a cysteine ​​mutation at amino acid position 100 in VL. In some embodiments, the scFv at the disulfide bond site contains a Q100C mutation in VL and a G44C mutation in VH.

[0290] In some embodiments, the scFv comprises the amino acid sequence shown in SEQ ID NO:52, 53 or 54, or comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or is composed of said amino acid sequence.

[0291] In some embodiments, the second antigen-binding region (the antigen-binding region specifically binding to LILRB4) of the multispecific antibody, such as a bispecific antibody, suitable for use in this invention may comprise, or be composed of, the full-length anti-LILRB4 antibody or its antigen-binding fragment as described in the second aspect of this invention, as long as it can specifically bind to LILRB4, including but not limited to, for example, a full-length antibody specifically binding to LILRB4, Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies such as VHH, dAb (domain antibody), heavy-chain antibodies, or linear antibodies. In some embodiments, the second antigen-binding region is derived from the anti-LILRB4 antibody or its antigen-binding fragment as described in the second aspect of this invention, for example, the Fab of the anti-LILRB4 antibody as described in the second aspect.

[0292] In some embodiments, the antigen-binding region specifically binding to LILRB4 comprises 1, 2, 3, 4, 5, or 6 CDRs of the anti-LILRB4 antibody described in the second aspect of this document. In some embodiments, the antigen-binding region specifically binding to LILRB4 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the anti-LILRB4 antibody described in the second aspect of this document. In some embodiments, the antigen-binding region specifically binding to LILRB4 comprises the heavy chain variable region VH (also referred to as VH) of the anti-LILRB4 antibody described in the second aspect of this document. LILRB4 ) and light chain variable region VL (also known as VL) LILRB4 ), particularly combinations of VH and VL containing anti-LILRB4 antibodies as described in the second aspect of this document.

[0293] Unless otherwise specified in this article, VH LILRB4 This refers to the heavy chain variable region (VH) contained in anti-LILRB4 antibodies or their antigen-binding fragments or antigen-binding regions that specifically bind to LILRB4. In this document, unless otherwise specified, VH... LILRB4 It refers to the light chain variable region (VL) contained in the anti-LILRB4 antibody or its antigen-binding fragment or antigen-binding region that specifically binds to LILRB4.

[0294] In some embodiments, the second antigen-binding region is the Fab fragment of an antibody that specifically binds to LILRB4 as described in the second aspect of this document. LILRB4 ).

[0295] In some embodiments, the Fab fragment consists of two polypeptide chains comprising antibody VH, CH1, VL, and CL domains, wherein VH pairs with VL and CH1 pairs with CL to form an antigen-binding region. In some embodiments, in the Fab, one chain comprises or consists of VH and CH1 from the N-terminus to the C-terminus (i.e., VH-CH1, where the C-terminus of VH is connected to the N-terminus of CH1), and the other chain comprises or consists of VL and CL from the N-terminus to the C-terminus (i.e., VL-CL, where the C-terminus of VL is connected to the N-terminus of CL). In this document, the Fab chain comprising VH-CH1 is also referred to as the Fab heavy chain, and the Fab chain comprising VL-CL is also referred to as the Fab light chain.

[0296] In some embodiments, the antigen-binding region that specifically binds to LILRB4 is a Fabry-Perot protein that specifically binds to LILRB4. LILRB4 It contains Fab heavy chains and Fab light chains, or is composed of Fab heavy chains and Fab light chains, as shown below:

[0297] Fab heavy chain: from N end to C end: VH LILRB4 -CH1, where VH LILRB4 The C-terminus of CH1 is directly connected to the N-terminus of CH1;

[0298] Fab Light Chain: From N-end to C-end: VL LILRB4 -CL, where VL LILRB4 The C-terminus of CL is directly connected to the N-terminus of CL;

[0299] Where “-” represents a direct connection; CH1 is the first structural domain CH1 of the heavy chain constant region, and CL is the light chain constant region.

[0300] In some implementations, the Fab LILRB4HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, which contain anti-LILRB4 antibodies as described in the second aspect of this document.

[0301] In some implementations, the VH LILRB4 The HCDR1, HCDR2, and HCDR3 comprise the anti-LILRB4 antibody as described in the second aspect of this document. In some embodiments, the VH LILRB4 It is the heavy chain variable region VH of the anti-LILRB4 antibody described in the second aspect of this document. In some embodiments, the VH... LILRB4 LCDR1, LCDR2, and LCDR3 comprise anti-CD3 antibodies as described in the second aspect of this document. In some embodiments, the VL LILRB4 It is the light chain variable region VL of the anti-LILRB4 antibody described in the second aspect of this article.

[0302] In some implementations, the VH LILRB4 Containing HCDR1, HCDR2, and HCDR3, as described in the second aspect of this document, and the VL LILRB4 LCDR1, LCDR2, and LCDR3 comprise the anti-LILRB4 antibody as described in the second aspect of this document. In some embodiments, the VH LILRB4 It is the heavy chain variable region VH of the anti-LILRB4 antibody described in the second aspect of this document, and the VL LILRB4 It is the heavy chain variable region VL of the anti-LILRB4 antibody described in the second aspect of this document. In some embodiments, the VH LILRB4 and the VL LILRB4 These are VH and VL in the combination of VH and VL of the anti-LILRB4 antibody as described in the second aspect of this document.

[0303] In some embodiments, CH1 is derived from IgG, such as human IgG, like IgG1, IgG2, IgG3, or IgG4, preferably CH1 of human IgG1. In some embodiments, CH1

[0304] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:31; or

[0305] (ii) Contains or consists of the amino acid sequence of SEQ ID NO:31.

[0306] In some embodiments, the CL is a light chain constant region derived from the antibody kappa or lambda light chain (e.g., human kappa or lambda light chain), preferably from the light chain constant region of the kappa light chain, such as the human kappa light chain. In some embodiments, the CL is a light chain constant region of the anti-LILRB4 antibody as described in the second aspect herein. In some embodiments, the Fab light chain is the light chain of the anti-LILRB4 antibody as described in the second aspect herein.

[0307] In some implementations, the CL

[0308] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:49; or

[0309] (ii) Contains or consists of the amino acid sequence of SEQ ID NO:49.

[0310] In some embodiments, the multispecific antibody, such as a bispecific antibody, further comprises two Fc regions (a first Fc region and a second Fc region), which are capable of dimerizing to form a dimer Fc. Preferably, the two Fc regions heterodimerize to form a heterodimer Fc.

[0311] In some implementations, the first and second Fc regions are identical. In other implementations, the first and second Fc regions are different, and they pair up and heterodimerize.

[0312] The Fc region is the C-terminal constant domain of an immunoglobulin, interacting with cell surface Fc receptors and some proteins of the complement system. An immunoglobulin Fc region typically contains two or three heavy-chain constant domains (designated CH2, CH3, and CH4) and a hinge region, and usually exists in a dimerized form. The two chains in a dimerized Fc region can be linked by disulfide bonds within the hinge region. The immunoglobulin Fc region used in the multispecific antibody of this invention can be derived from any immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region contains at least the immunoglobulin CH2 and CH3 domains. In some embodiments, the immunoglobulin Fc region also contains a complete or partial hinge region. In some embodiments, the immunoglobulin Fc region, from the N-terminus to the C-terminus, comprises, or consists of, the complete or partial hinge region of an immunoglobulin, the CH2 domain, and the CH3 domain. In this article, unless otherwise specified, the Fc region refers to the Fc region that contains a partial hinge region at the N-terminus (corresponding to the hinge region of IgG1 Fc region E2221-P230 (EU number)).

[0313] In some embodiments, the Fc region is derived from IgG, such as human IgG, such as IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is a (human) IgG Fc region, such as human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc. In some embodiments, the Fc region comprises the amino acid sequence shown in SEQ ID NO:38, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity with said amino acid sequence, or is composed of said amino acid sequence.

[0314] The immunoglobulin Fc region of the multispecific antibody used in this invention can be the natural Fc region sequence. Alternatively, the Fc region can contain mutations relative to the natural Fc sequence. Mutations include substitutions, insertions, and / or deletions.

[0315] When the multispecific antibody of the present invention contains an asymmetric double-stranded structure, the Fc region preferably contains a KiH mutation that promotes the correct heterodimerization of the antibody polypeptide chain.

[0316] For example, to promote heterodimerization, a Knob-into-Hole (KiH) mutation can be introduced into the CH3 domain. This technique is described in, for example, US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9,617-621 (1996) and Carter, J Immunol Meth 248,7-15 (2001). In this case, one Fc strand is designed to contain a large protruding residue (i.e., Knob), while the other Fc strand is designed to contain a complementary pocket (i.e., Hole). Suitable positions for the KiH mutation are known in the art. For example, a common mutation combination is to mutate the T366 position of one strand to Y (T366Y) and the Y407 position of the other strand to T (Y407T). This complementary mutation allows the two strands to bind specifically, forming a stable heterodimer. Another common combination is to achieve a similar heterodimerization effect by mutating T366 to W and Y407 to A. Furthermore, exemplary KiH mutations also include combinations of Knob mutation T366W and Hole mutations T366S, L368A, and Y407V. In some embodiments, the Fc regions may also contain cysteine ​​residue substitutions to obtain non-natural disulfide bond linkages. In some embodiments, one Fc region contains S354C or E356C, and the other Fc region contains Y349C.

[0317] In one specific implementation, the Fc region containing the Knob mutation contains the amino acid substitution T366W, and the Fc region containing the hole mutation contains the amino acid substitutions T366S, L368A, and Y407V (numbered according to the EU index).

[0318] In one specific implementation, the Fc region containing the Knob mutation contains amino acid substitutions S354C and T366W, and the Fc region containing the hole mutation contains amino acid substitutions Y349C, T366S, L368A and Y407V (numbered according to the EU index).

[0319] In addition, H435R and Y436F mutations can be further included in the Fc region containing the Hole mutation to prevent the obtained multispecific antibody from binding to Protein A, thus facilitating purification.

[0320] In one specific implementation, the Fc region containing the Knob mutation contains amino acid substitutions S354C and T366W, and the Fc region containing the hole mutation contains amino acid substitutions Y349C, T366S, L368A and Y407V, as well as H435R and Y436F mutations (numbered according to the EU index).

[0321] In some embodiments, depending on the specific application of the antibody or antibody-based molecule, the Fc region may also contain mutations that alter effector function. For example, where effector function is not required, the Fc region may contain mutations that reduce or eliminate effector function. In some embodiments, preferably, the Fc region contains mutations that reduce or eliminate the interaction between the Fc region and the Fcγ receptor, such as the LALA mutation (L234A / L235A) where lysine (L) at positions 234 and 235 of the Fc region is replaced with alanine (A), to reduce Fcγ receptor-mediated off-target cytotoxicity. Alternatively or additionally, mutations may be introduced into the Fc region to increase binding to FcRn and / or remove protease sites, and / or introduce amino acid modifications that can be used for coupling with active molecules. Therefore, in one specific embodiment, the multispecific antibody of the present invention comprises an Fc region containing a mutation that reduces or eliminates the Fc region and the Fcγ receptor, such as the LALA mutation. For example, the Fc region containing the LALA mutation contains an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:39, such as containing or consisting of the amino acid sequence shown in SEQ ID NO:39.

[0322] Alternatively, for antibody production purposes, the Fc region may be mutated, for example, by removing or replacing amino acids that may undergo post-translational modifications (e.g., glycosylation), to provide improved drugability and developability of therapeutic antibodies.

[0323] Therefore, in one specific embodiment, the multispecific antibody of the present invention comprises two heterodimerized Fc regions, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence shown in SEQ ID NO:40 and comprises the mutations Y349C, T366S, L368A, and Y407V, as well as the mutations H435R and Y436F, and optionally the mutations L234A / L235A; and the other Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence shown in SEQ ID NO:41 and comprises the mutations S354C and T366W, and optionally the mutations L234A / L235A.

[0324] Therefore, in one specific embodiment, the multispecific antibody of the present invention comprises two heterodimerized Fc regions, wherein one Fc region polypeptide comprises or is composed of the amino acid sequence shown in SEQ ID NO:40, and the other Fc region comprises or is composed of the amino acid sequence shown in SEQ ID NO:41.

[0325] The multispecific antibodies of the present invention, such as bispecific antibodies, can have any structure comprising an antigen-binding region that specifically binds to CD3, and / or an antigen-binding region that specifically binds to LILRB4, and / or an Fc region, as long as they can function as multispecific antibodies. Various suitable forms of multispecific antibodies, such as bispecific antibodies, are known in the art.

[0326] In some embodiments, the multispecific antibody of the present invention is an IgG-like bispecific antibody comprising a Fab fragment as an antigen-binding region specifically binding to one antigen and an scFv as an antigen-binding region specifically binding to another antigen. In some embodiments, the IgG-like bispecific antibody comprises, as described herein, an scFv specifically binding to CD3 as a first antigen-binding region, and comprises, as described herein, a Fab fragment specifically binding to anti-LILRB4 as a second antigen-binding region, and optionally, an Fc region or Fc dimer as described herein. In one embodiment, the bispecific antibody may comprise one or more first antigen-binding regions. In one embodiment, the bispecific antibody may comprise one or more second antigen-binding regions.

[0327] In some implementations, the bispecific antibody may comprise one or two second antigen-binding regions and a first antigen-binding region.

[0328] In some embodiments, the scFv can be linked to the N-terminus of the Fc domain of the antibody via the C-terminus of the chain. In some embodiments, in the bispecific antibody, the scFv is linked at the N-terminus of the chain to the C-terminus of another antigen-binding region (e.g., the Fab heavy chain), and at the C-terminus of the chain to the N-terminus of the antibody's Fc domain.

[0329] In some embodiments, in the bispecific antibody, the scFv can be linked to the N-terminus of the Fc domain of the antibody via the C-terminus of the VH chain. In some embodiments, in the bispecific antibody, the scFv is linked at the C-terminus of another antigen-binding region (e.g., the Fab heavy chain) at the N-terminus of the VL chain, and at the N-terminus of the Fc domain of the antibody at the C-terminus of the VH chain. In some embodiments, in the bispecific antibody, the scFv can be linked to the N-terminus of the Fc domain of the antibody via the C-terminus of the VL chain. In some embodiments, in the bispecific antibody, the scFv is linked at the C-terminus of another antigen-binding region (e.g., the Fab heavy chain) at the N-terminus of the VH chain, and at the N-terminus of the Fc domain of the antibody at the C-terminus of the VL chain.

[0330] In some embodiments, the scFv is connected to the Fc region via a partial hinge region, for example via EPKSC (SEQ ID NO:42) or EPKSS (SEQ ID NO:43). In some embodiments, the scFv is connected to a second antigen-binding region, such as Fab, or Fab heavy chain, via a linker, which can be any peptide linker, such as any peptide linker as described herein. In one embodiment, the linker is an amino acid sequence (GGGGS)n, where n = 1, 2, 3, 4, or 5, preferably n = 2. In one embodiment, the linker comprises or consists of the amino acid sequence shown in SEQ ID NO:46.

[0331] In some embodiments, in the bispecific antibody, the Fab heavy chain variable region can be linked to the N-terminus of the Fc domain via the C-terminus of CH1, or to the N-terminus of a first antigen-binding region such as scFv.

[0332] In some specific embodiments, the bispecific antibody of the present invention comprises a first antigen-binding region, a second antigen-binding region, and an Fc dimer, wherein the first antigen-binding region is the scFv fragment specifically binding to CD3 as described herein, and the second antigen-binding region is the Fab fragment specifically binding to anti-LILRB4 as described herein. In some embodiments, the bispecific antibody of the present invention comprises one first antigen-binding region and one or two second antigen-binding regions, for example, wherein the two second antigen-binding regions may be the same or different.

[0333] Therefore, in some specific embodiments, the bispecific antibody is an IgG-like bispecific antibody comprising one or two anti-LILRB4 Fabs described herein, an anti-CD3 antibody scFv and an Fc heterodimer described herein, wherein the C-terminus of the heavy chain of the Fab is linked to the N-terminus of the first Fc region (preferably directly linked), and the C-terminus of the scFv is linked to the N-terminus of the second Fc region (preferably via a partial hinge region such as EPKSS or EPKSC, more preferably EPKSS).

[0334] Optionally, the N-end of the scFv is also connected to the C-end of another Fab heavy chain (preferably via a connector such as (GGGGS)n where n = an integer from 1 to 10, such as 1, 2, 3, 4 or 5);

[0335] The first Fc region and the second Fc region are preferably different, as described herein.

[0336] In some embodiments, if the bispecific antibody comprises Fabs of two anti-LILRB4 antibodies... LILRB4 The two Fabs can be the same or different, for example, combining different LILRB4 epitopes, or combining the same LILRB4 epitopes but different in sequence; preferably, the two Fabs are the same.

[0337] In some embodiments, the bispecific antibody of the present invention comprises or consists of the following chains:

[0338] First chain, from the N-terminus to the C-terminus: VH LILRB4 -CH1- First Fc region;

[0339] First light chain, from the N-terminus to the C-terminus: VL LILRB4 -CL; and

[0340] The second chain, from the N-terminus to the C-terminus: VL CD3 -Connector 1-VH CD3 -Partial hinge area-Second Fc area;

[0341] Where “-” represents a direct connection;

[0342] VH LILRB4 It is the VH, VL anti-LILRB4 antibody as described in the second aspect of this article. LILRB4 It is the VL of the anti-LILRB4 antibody as described in the second aspect of this document, and CH1 and CL are CH1 and CL as defined herein, and VH LILRB4 -CH1 and VL LILRB4 -CL constitutes the Fab fragment of the anti-LILRB4 antibody as defined in this article. LILRB4 ;

[0343] VH CD3 It is the anti-CD3 antibody VH, VL as described in the first aspect of this article. CD3 It is the VL of the anti-CD3 antibody as described in the first aspect of this document, and VL CD3 -Connector 1-VH CD3 The scFv fragments that constitute anti-CD3 antibodies as defined in this article CD3 ;

[0344] Optionally, the connector 1 is as shown in SEQ ID NO:45; and a portion of the hinge area is as shown in SEQ ID NO:42 or 43, preferably as shown in SEQ ID NO:43;

[0345] Optionally, the first Fc region and the second Fc region are different. For example, the first Fc region contains Y349C, T366S, L368A and Y407V, as well as H435R and Y436F mutations and L234A / L235A mutations. Specifically, it can be the sequence shown in SEQ ID NO: 40. The second Fc region contains S354C and T366W and L234A / L235A mutations. Specifically, it can be the sequence shown in SEQ ID NO: 41.

[0346] In some embodiments, the bispecific antibody of the present invention comprises or consists of the following chains:

[0347] First chain, from the N-terminus to the C-terminus: VH LILRB4 -CH1- First Fc region;

[0348] First light chain, from the N-terminus to the C-terminus: VL LILRB4 -CL; and

[0349] The second chain, from the N-terminus to the C-terminus: VH LILRB4 -CH1-Connector 2-VL CD3 -Connector 1-VH CD3 -Partial hinge area-Second Fc;

[0350] The second light chain, from the N-terminus to the C-terminus: VL LILRB4 -CL;

[0351] Where “-” represents a direct connection;

[0352] VH LILRB4 It is the VH, VL anti-LILRB4 antibody as described in the second aspect of this article. LILRB4 It is the VL of the anti-LILRB4 antibody as described in the second aspect of this document, and CH1 and CL are CH1 and CL as defined herein, and VH LILRB4 -CH1 and VLLILRB4 -CL constitutes the Fab fragment of the anti-LILRB4 antibody as defined in this article. LILRB4 Preferably, VH in the first heavy chain and the second heavy chain LILRB4 The same, and / or VL in the first and second light chains LILRB4 The same; more preferably, VH in the first and second heavy chains. LILRB4 -CH1 is the same and / or the first light chain and the second light chain are the same;

[0353] VH CD3 It is the anti-CD3 antibody VH, VL as described in the first aspect of this article. CD3 It is the VL of the anti-CD3 antibody as described in the first aspect of this document, and VL CD3 -Connector 1-VH CD3 The scFv fragments that constitute anti-CD3 antibodies as defined in this article CD3 ;

[0354] Optionally, connector 1 is as shown in SEQ ID NO:45, connector 2 is as shown in SEQ ID NO:46, and / or a portion of the hinge area is as shown in SEQ ID NO:42 or 43, preferably as shown in SEQ ID NO:43;

[0355] Optionally, the first Fc region and the second Fc region are different. For example, the first Fc region contains Y349C, T366S, L368A and Y407V, as well as H435R and Y436F mutations and L234A / L235A mutations, and the second Fc region contains S354C and T366W and L234A / L235A mutations.

[0356] In some embodiments, the bispecific antibody of the present invention comprises or consists of the following chains:

[0357] The first heavy chain comprises the amino acid sequence shown in SEQ ID NO:26, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with said amino acid sequence, or is composed of said sequence;

[0358] The second chain comprises the amino acid sequence shown in SEQ ID NO:27, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with said amino acid sequence, or is composed of said sequence; and / or

[0359] The first light chain comprises the amino acid sequence shown in SEQ ID NO:28, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence, or is composed of the sequence.

[0360] In some embodiments, the bispecific antibody of the present invention comprises or consists of the following chains:

[0361] The first chain comprises or consists of the amino acid sequence shown in SEQ ID NO:26;

[0362] The second chain comprises or consists of the amino acid sequence shown in SEQ ID NO:27; and

[0363] The first light chain comprises or consists of the amino acid sequence shown in SEQ ID NO:28.

[0364] In some embodiments, the bispecific antibody of the present invention comprises or consists of the following chains:

[0365] The first heavy chain comprises the amino acid sequence shown in SEQ ID NO:26, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with said amino acid sequence, or is composed of said sequence;

[0366] The second chain comprises the amino acid sequence shown in SEQ ID NO:29 or 30, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with said amino acid sequence, or is composed of said sequence;

[0367] A first light chain comprising the amino acid sequence shown in SEQ ID NO:28, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with said amino acid sequence, or consisting of said sequence, and / or

[0368] The second light chain comprises the amino acid sequence shown in SEQ ID NO:28, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence, or is composed of the sequence.

[0369] In some embodiments, the bispecific antibody of the present invention comprises or consists of the following chains:

[0370] The first chain comprises or consists of the amino acid sequence shown in SEQ ID NO:26;

[0371] The second chain comprises or consists of the amino acid sequence shown in SEQ ID NO:29 or 30;

[0372] A first light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:28; and

[0373] The second light chain comprises or consists of the amino acid sequence shown in SEQ ID NO:28.

[0374] Those skilled in the art will understand that, unless the context clearly indicates otherwise, any combination of any technical features of any component of the multispecific antibody, such as a bispecific antibody, described herein is within the scope of this invention. Furthermore, those skilled in the art will understand that, unless the context clearly indicates otherwise, the antibodies of this invention (including antibodies of any form) may contain any such combination of features.

[0375] In a fourth aspect, the present invention provides a nucleic acid encoding any antibody molecule described herein or any strand thereof.

[0376] For example, the nucleic acid of the present invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NO: 1, 2, 9, 10, 14, 15, 18, 19, 26-30, and 32-35, or a nucleic acid encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from any one of SEQ ID NO: 1, 2, 9, 10, 14, 15, 18, 19, 26-30, and 32-35. As will be apparent to those skilled in the art, due to codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences. The nucleic acid sequence encoding the molecule of the present invention can be generated using methods well known in the art, such as de novo solid-phase DNA synthesis or by PCR amplification.

[0377] In one aspect, the present invention provides nucleic acids encoding any antibody molecule or fragment thereof or any strand thereof described herein. When expressed from a suitable expression vector, the polypeptide encoded by said nucleic acid is capable of exhibiting human (and / or monkey, e.g., cynomolgus monkey) CD3 antigen binding ability. For example, in some embodiments, the nucleic acid encoding the variable region of the heavy chain and / or light chain is operatively linked in a reading frame to a nucleic acid encoding the constant region of the heavy chain and / or light chain, thereby producing nucleic acids encoding said antibody heavy chain and / or light chain when expressed from a suitable expression vector.

[0378] To facilitate production and purification, a secretory signal peptide and / or a tag peptide that facilitates purification can be fused to the N-terminus of the heavy chain and / or light chain of the antibody.

[0379] In a fifth aspect, the present invention provides one or more vectors comprising the nucleic acid described in the fourth aspect.

[0380] In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, granules, λ phages, or yeast artificial chromosomes (YACs). In one embodiment, the vector is a pCDNA vector, such as pCDNA3.1.

[0381] In a sixth aspect, the present invention provides a host cell comprising the nucleic acid described in the fourth aspect or comprising the vector described in the fifth aspect.

[0382] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells (e.g., CHO-S or CHO-K) or 293 cells (e.g., 293F or HEK293 cells)) or other cells suitable for preparing antibodies or fragments thereof. In one embodiment, the host cell is prokaryotic, such as bacteria, e.g., Escherichia coli.

[0383] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing antibodies or fragments thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antibodies. For example, fungal and yeast strains whose glycosylation pathways have been “humanized” result in the production of antibodies with partial or complete human glycosylation patterns. Host cells suitable for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines modified to be suitable for suspension growth can be used. Other examples of useful mammalian host cell lines include the monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney lines (HEK293, 293F, or 293T cells), etc. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including CHO-S cells or CHO-K, etc.; and myeloma cell lines such as Y0, NSO, and Sp2 / 0. Mammalian host cell lines suitable for antibody production are known in the art.

[0384] In a seventh aspect, the present invention provides a method for preparing any antibody molecule or fragment thereof (preferably an antigen-binding fragment) described herein.

[0385] In some embodiments, the method includes culturing the host cells under conditions suitable for expressing nucleic acids encoding the antibody or a fragment thereof (preferably an antigen-binding fragment) or any one or both strands thereof, and optionally isolating the antibody or a fragment thereof (preferably an antigen-binding fragment). In one embodiment, the method further includes recovering any antibody molecule or a fragment thereof (preferably an antigen-binding fragment) described herein from the host cells.

[0386] Polynucleotides encoding the polypeptide chain of the present invention can be inserted into one or more vectors for further cloning and / or expression in host cells. Expression vectors can be constructed using methods well known to those skilled in the art. Once an expression vector containing one or more nucleic acid molecules of the present invention has been prepared for expression, the expression vector can be transfected or introduced into suitable host cells. Various techniques can be used to achieve this, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection, or other conventional techniques.

[0387] The antibodies prepared as described herein can be purified using known existing techniques such as high-performance liquid chromatography, ion-exchange chromatography, gel electrophoresis, affinity chromatography (e.g., Protein A), size exclusion chromatography, etc. The actual conditions used to purify a specific protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, which are obvious to those skilled in the art.

[0388] The purity of the antibody molecules of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc.

[0389] Any antibody molecule or fragment thereof described herein can be identified, screened, or characterized for its physical / chemical properties and / or biological activity using a variety of assays known in the art. Exemplary methods are described in the embodiments of this invention.

[0390] In an eighth aspect, the present invention provides an immunoconjugate comprising any antibody molecule or fragment thereof (preferably an antigen-binding fragment) provided herein and other substances, such as therapeutic agents or labels.

[0391] In some embodiments, the therapeutic agent may be a therapeutic agent suitable for forming an immune conjugate with any antibody molecule or fragment thereof provided herein. In some embodiments, the therapeutic agent is selected from chemotherapeutic agents, cytotoxic agents, or small molecule drugs.

[0392] In a ninth aspect, the present invention provides compositions, such as pharmaceutical compositions.

[0393] In some embodiments, the composition comprises any antibody molecule or fragment thereof (preferably an antigen-binding fragment) or immunoconjugate provided herein. In some embodiments, the pharmaceutical composition is a pharmaceutical formulation. The invention also includes compositions comprising polynucleotides encoding any antibody molecule or fragment thereof (preferably an antigen-binding fragment) provided herein (including pharmaceutical compositions such as pharmaceutical formulations).

[0394] In one embodiment, the composition further comprises a pharmaceutical excipient. As used herein, "pharmaceutical excipient" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents. For information on the use and applications of pharmaceutical excipients, see also "Handbook of Pharmaceutical Excipients," 8th edition, R.C. Rowe, P.J. Seskey, and S.C. Wen, Pharmaceutical Press, London, Chicago.

[0395] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusionable solutions), powders or suspensions, liposomes, and suppositories. Preferred forms depend on the intended administration method and therapeutic use.

[0396] Pharmaceutical formulations containing antibodies described herein can be prepared by mixing any antibody molecule or fragment thereof (preferably an antigen-binding fragment) or immunoconjugate having the desired purity provided herein with one or more optional pharmaceutical excipients.

[0397] The pharmaceutical compositions or formulations of the present invention may also comprise more than one active ingredient, said active ingredient being required for a specific indication to be treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, it is desirable to also provide other therapeutic agents, such as chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immunosuppressants). The active ingredients are suitably combined in amounts effective for the intended use.

[0398] In a tenth aspect, the present invention provides a pharmaceutical combination and a medicine box.

[0399] In some embodiments, the present invention also provides pharmaceutical combinations or combination products comprising any antibody molecule or fragment thereof (preferably an antigen-binding fragment) or immune conjugate provided herein, and one or more other therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, vaccines, small molecule drugs, or immunomodulators (e.g., immunosuppressants).

[0400] Another object of the present invention is to provide a kit containing the drug combination of the present invention.

[0401] In an eleventh aspect, the present invention provides uses and methods for applying any antibody molecule or fragment thereof (preferably an antigen-binding fragment), immunoconjugate, pharmaceutical composition, or combination of pharmaceuticals described herein.

[0402] The expression of LILRB4 on the surface of tumor cells in various diseases, such as acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML), and multiple myeloma (MM), provides a basis for targeting these tumor types. Furthermore, CD3×LILRB4 has the potential to target any tumor type that expresses LILRB4.

[0403] In some embodiments, the present invention provides a method for treating LILRB4-related disease in an individual, comprising administering to a subject any antibody molecule or fragment thereof (preferably an antigen-binding fragment) or immunoconjugate or pharmaceutical composition or combination thereof described herein.

[0404] In some embodiments, the present invention provides any antibody molecule or fragment thereof (preferably an antigen-binding fragment) or immunoconjugate or pharmaceutical composition or combination thereof described herein for the treatment of LILRB4-related diseases.

[0405] In some embodiments, the present invention provides the use of any antibody molecule or fragment thereof (preferably an antigen-binding fragment) or immunoconjugate or pharmaceutical composition or combination thereof described herein for the treatment of LILRB4-related diseases.

[0406] In some embodiments, the present invention provides the use of any antibody molecule or fragment thereof (preferably an antigen-binding fragment) or immunoconjugate or pharmaceutical composition or combination described herein in the preparation of a medicament for the treatment of LILRB4-related disease.

[0407] In some implementations, the LILRB4-related diseases described herein include tumors, such as cancer. The cancer can be in its early, intermediate, or late stages, or it can be metastatic. In some implementations, the cancer can be a solid tumor or a hematologic malignancy.

[0408] In some implementations, the tumor is a LILRB4-positive tumor.

[0409] In some implementations, the tumor or cancer is a hematologic malignancy, such as lymphoma, leukemia, or myelodysplastic syndrome, such as multiple myeloma, such as acute myeloid leukemia (AML), such as human acute monocytic leukemia or human myeloid monocytic leukemia, such as chronic myeloid leukemia, such as chronic myelomonocytic leukemia (CMML).

[0410] In one embodiment, the LILRB4-positive tumor refers to a tumor in which LILRB4 is expressed (e.g., highly, moderately, or poorly expressed) in an individual's tumor tissue, such as blood or tumor cells. In another embodiment, the tumor is defined as having elevated levels of LILRB4 protein (e.g., expression) or LILRB4 nucleic acid compared to adjacent normal tissue or normal cells (e.g., normal cells in tissue) of an individual or the same tissue or cells of a healthy individual.

[0411] In some embodiments, any antibody molecule or fragment thereof (preferably an antigen-binding fragment) or immunoconjugate or pharmaceutical composition or combination thereof described herein may also be administered in combination with one or more other therapies, such as modes of treatment and / or other therapeutic agents.

[0412] In some implementations, the treatment modality includes surgery, radiation therapy (e.g., external beam therapy, which involves three-dimensional conformal radiation therapy in which the irradiation area is designed), local irradiation (e.g., irradiation directed at a pre-selected target or organ), or focused irradiation. In some implementations, the therapeutic agent is selected from chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, vaccines, small molecule drugs, or immunomodulators (e.g., immunosuppressants).

[0413] Such combination therapies encompass combined administration (e.g., two or more therapeutic agents contained in the same formulation or separate formulations) and separate administration, in which case the antibody of the present invention may be administered before, simultaneously with, and / or after the administration of other therapeutic agents and / or pharmaceuticals.

[0414] Any antibody molecule or fragment thereof (preferably an antigen-binding fragment), immunoconjugate, or pharmaceutical composition or combination thereof described herein may be administered by any suitable method, including parenteral administration, and, if necessary, intralesional administration. Parenteral injection or infusion includes intravenous injection or infusion.

[0415] For the prevention or treatment of disease, the appropriate dose (when used alone or in combination with one or more other therapeutic agents) of any antibody molecule or fragment thereof (preferably an antigen-binding fragment), immunoconjugate, or pharmaceutical composition or combination thereof described herein will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether it is administered for prophylactic or therapeutic purposes, prior treatment, the patient's clinical history and response to the antibody, the mode of administration, the bioavailability characteristics of the administered formulation, the chosen dosing regimen, the use of any concomitant therapies, and the judgment of the attending physician. The antibody is appropriately administered to the patient as a single treatment or after a series of treatments.

[0416] In a twelfth aspect, the invention also relates to methods for diagnosis and detection using the anti-LILRB4 antibody or multispecific antibody described herein, and compositions for diagnosis and detection comprising the same.

[0417] In some implementations, the anti-LILRB4 antibody described herein, or its antigen-binding fragment or multispecific antibody, can be used to detect the presence of LILRB4 in biological samples.

[0418] When used herein, the term "detection" includes both quantitative and qualitative detection. Exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads with antibody molecules, ELISA assays, and PCR techniques (e.g., RT-PCR). In some embodiments, the biological sample is blood, serum, or other liquid samples of biological origin. In some embodiments, the biological sample comprises cells or tissues. In some embodiments, the biological sample is derived from tumor tissue or cancer tissue.

[0419] In one implementation, an anti-LILRB4 antibody or its antigen-binding fragment or multispecific antibody described herein is provided for use in diagnostic or detection methods.

[0420] In another aspect, a method for detecting the presence of LILRB4 in a biological sample is provided. In some embodiments, the method comprises detecting the presence of the LILRB4 protein in the biological sample. In some embodiments, LILRB4 is human LILRB4 or monkey LILRB4. In some embodiments, the method comprises contacting the biological sample with an anti-LILRB4 antibody or its antigen-binding fragment or multispecific antibody as described herein under conditions that allow it to bind to LILRB4, and detecting whether a complex is formed between the anti-LILRB4 antibody or its antigen-binding fragment or multispecific antibody described herein and LILRB4. The formation of the complex indicates the presence of LILRB4. This method may be an in vitro or in vivo method. In one embodiment, the anti-LILRB4 antibody or its antigen-binding fragment or multispecific antibody described herein is used to select a subject suitable for treatment using any antibody molecule or its fragment (preferably an antigen-binding fragment) or immunoconjugate or pharmaceutical composition or combination of drugs described herein, for example, where LILRB4 is a biomarker for selecting the subject.

[0421] In some embodiments, a method for detecting LILRB4 in a sample is provided, the method comprising:

[0422] (a) Contact the sample with the anti-LILRB4 antibody or its antigen-binding fragment or multispecific antibody described herein; and

[0423] (b) Detecting the formation of a complex between an anti-LILRB4 antibody or its antigen-binding fragment or a multispecific antibody and LILRB4; optionally, the antibody or its antigen-binding fragment is detectably labeled.

[0424] In some embodiments, a labeled anti-LILRB4 antibody or its antigen-binding fragment or multispecific antibody is provided. Labeling includes, but is not limited to, labels or portions that are directly detected (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and portions that are indirectly detected, such as enzymes or ligands, for example, through enzymatic reactions or molecular interactions.

[0425] In some embodiments provided herein, the sample is obtained prior to treatment with any antibody molecule or fragment thereof (preferably an antigen-binding fragment), immunoconjugate, or pharmaceutical composition or combination thereof described herein. In some embodiments, the sample is obtained prior to other therapies. In some embodiments, the sample is obtained during or after treatment with other therapies.

[0426] In some implementations, LILRB4 is detected before treatment, for example, before starting treatment or before a treatment after a treatment interval.

[0427] In some embodiments, a method for treating the disease of the present invention is provided, the method comprising: testing a subject (e.g., a sample) for the presence of LLRRB4, thereby determining a LLRRB4 value; comparing the LLRRB4 value with a control value (e.g., a value in a healthy individual or normal tissue); and if the LLRRB4 value is greater than the control value, administering to the subject a therapeutically effective amount of any antibody molecule or fragment thereof (preferably an antigen-binding fragment) or immunoconjugate or pharmaceutical composition or combination thereof, optionally in combination with one or more other therapies, thereby treating the disease.

[0428] In some embodiments, a method for treating the disease of the present invention is provided, the method comprising: testing a subject (e.g., a sample) for the presence of LILRB4, thereby determining LILRB4; comparing the LILRB4 value with a control value (e.g., a value in a normal individual); and if the LILRB4 value is greater than the control value, administering to the subject a therapeutically effective amount of any antibody molecule or fragment thereof (preferably an antigen-binding fragment) or immunoconjugate or pharmaceutical composition or combination thereof, optionally in combination with one or more other therapies, thereby treating the disease.

[0429] These and other aspects and embodiments of the invention are described in the accompanying drawings (briefly described below) and the following detailed description of the invention, and are exemplified in the following embodiments. Any or all features discussed above and throughout this application may be combined in various embodiments of the invention. The following embodiments further illustrate the invention; however, it should be understood that the embodiments are described in an illustrative rather than limiting manner, and various modifications can be made by those skilled in the art. Example

[0430] Example 1: Immunization, screening, and preparation of anti-CD3 antibodies

[0431] Antibodies against CD3 were obtained by immunizing SJL mice (6-8 weeks old) with human CD3εγ protein (Rich Chemicals) and cynomolgus monkey CD3εγ protein (Acro, CDG-C52W6) or T cells as immunogens. The binding activity of hybridoma cell supernatant to human and cynomolgus monkey CD3εγ proteins was detected by ELISA. ELISA was performed using standard protocols.

[0432] In summary, human and monkey CD3εγ protein was coated at a concentration of 1 μg / ml onto 96-well plates (Corning 9018) and incubated overnight at 4°C. The plates were washed three times with PBS containing 0.05% Tween 20. Subsequently, the plates were blocked with PBS containing 1% BSA for 2 hours. After washing three times with PBS containing 0.05% Tween 20, 50 μL of hybridoma supernatant was added, and the plates were incubated at 37°C for 1 hour. The plates were washed three times with PBS containing 0.05% Tween 20. HRP-conjugated goat anti-mouse IgG-Fc antibody (Sigma, A0168) was added for detection. The assay was performed at room temperature using TMB (InnoReagents, EL0009) substrate and measured at 450 nm using a microplate reader.

[0433] To measure the binding affinity of hybridoma supernatant to CD3 on the cell surface, flow cytometry was used. Approximately 3-5 x 10⁻⁶ cells were used. 5 Cells were incubated with hybridoma cell supernatant at 4°C for 1 hour. They were washed three times with PBS (FACS buffer) containing 2% FBS, then resuspended in 100 μL (1:1000 with FACS buffer) of donkey anti-mouse IgG (H+L) Alexa Fluor 488 (INVITROGEN, A21202) and incubated at 4°C for 30 minutes. Cells were washed three times with FACS buffer, centrifuged, and resuspended for flow cytometry analysis. Specific anti-CD3 clones 16H4, 313D9, and 341F5 were identified to obtain humanized anti-CD3 antibodies, numbered 16H4, 313D9, and 341F5, respectively.

[0434] The VH and VL sequences of antibodies 16H4, 313D9, and 341F5, as well as their specific CDRs, are shown in the sequence information. The heavy chain constant region of the monoclonal antibody is SEQ ID NO:36, and the light chain constant region is SEQ ID NO:49.

[0435] The preparation process of CD3 monoclonal antibody is as follows: Human HEK293 cells are cultured in F17 medium supplemented with Glutsmax-I and F-68, and the cell concentration is adjusted to 2-3 x 10⁻⁶ cells / mL. 6 HEK293 cell solution was obtained by mixing [amount] ml of the culture medium. The coding genes for the heavy chain (VH+ heavy chain constant region) and light chain (VL+ light chain constant region) of the monoclonal antibody were constructed into the PTT5 vector. The PTT5 vector (synthesized by Suzhou Genewiz) containing the coding genes for the CD3 domain was added to opti-MEM (Gibco) to obtain solution A. Transfection reagent PEI (Polysciences, 24885-2) was added to opti-MEM medium to obtain solution B. Solutions A and B were then mixed to obtain the transfection mixture, which was added to the HEK293 cell solution after 20 minutes. After culturing at 37°C and 5% CO2 for 1 day, 20% TNI (Organotechnie, 19553) was added, and the cells were transferred to 37°C and 5% CO2 for 7 days. The culture supernatant was harvested and purified using a Protein A purification column (GE) to obtain the CD3 monoclonal antibody.

[0436] Example 2: Characterization of humanized anti-CD3 antibody

[0437] Example 2.1 Affinity of humanized anti-CD3 antibodies 16H4, 313D9, and 341F5 to human and monkey CD3εγ protein.

[0438] Antibody affinity was determined using the anti-human antibody capture method on a Fortebio (BLITZ pro1.1.0.28) instrument. During the assay, the capture antibody (AHC) bioprobe of the Fc fragment of the anti-human antibody was immersed in 1x KB buffer (PBS + 0.2% Tween 20 + 0.1% BSA) for 10 min. 200 μl of diluted antibody sample was loaded onto the AHC bioprobe at a working concentration of 10 μg / mL, and then equilibrated in KB buffer for 100 s. The AHC probe was then further subjected to a binding reaction with serially diluted (100 nM starting, 2-fold dilution) human CD3εγ protein or monkey CD3εγ (purchased from ACRO biosystem) for 600 s. Afterward, the AHC probe was transferred to KB buffer for dissociation for 600 s. After the experiment, the blank control response value was subtracted, and the antigen-antibody binding kinetic constant was calculated using 1:1 Langmuir binding mode fitting software. The instrument's built-in software was then used for curve processing and fitting. The results are shown in Table 1. 16H4, 313D9 and 341F5 can all bind to human CD3εγ protein and monkey CD3εγ.

[0439] Table 1: Affinity of anti-CD3 humanized antibodies 16H4, 313D9, and 341F5 to human CD3εγ protein and monkey CD3εγ.

[0440] Example 2.2 Binding of humanized anti-CD3 antibodies 16H4, 313D9, and 341F5 to the CD3-positive tumor cell line Jurkat and to human T cells.

[0441] Prepare anti-CD3 antibodies at different concentration gradients (starting at 200 nM, triple-dilution, 12 spots (including 0 spot)) using FACS buffer. Add 100 μL to each well of a 96-well plate. (2 x 10⁻⁶) 6100 μL of Jurkat cells / ml were added to a 96-well plate with neutralizing antibody and incubated at 4°C for 0.5 h. After washing three times with PBS (FACS buffer) containing 2% FBS, the cells were centrifuged and resuspended in 100 μL (1:1000 with FACS buffer) of goat anti-human IgG (Jackson Immune Research, 109-135-098) and incubated at 4°C for 30 min. The cells were washed three times with FACS buffer, resuspended in FACS buffer, and used for flow cytometry analysis. T cell binding was performed using the same method as Jurkat, where T cells were isolated from PBMC (purchased from Ruibai Biotechnology) using a T cell isolation kit (STEMCELL, Cat#17951). The specific results are shown in Figures 1A and 1B, which show that 16H4, 313D9, and 341F5 exhibited concentration-dependent binding to both Jurkat and human T cells.

[0442] Example 2.3 Binding of humanized anti-CD3 antibodies 16H4, 313D9, and 341F5 to monkey PBMCs

[0443] Prepare anti-CD3 antibodies at different concentration gradients using FACS buffer (starting concentration 200 nM, triple dilution, 12 spots (including 0 spot)). Add 100 μL to each well of a 96-well plate. 6 100 μL of neutralizing antibody was added to 96-well plates of cynomolgus monkey PBMCs (Rubai Biotechnology) and incubated at 4°C for 0.5 h. Cells were washed three times with PBS (FACS buffer) containing 2% FBS, centrifuged, and then resuspended in 100 μL (1:1000 with FACS buffer) of goat anti-human IgG (Jackson Immune Research, 109-135-098) and incubated at 4°C for 30 min. Cells were washed three times with FACS buffer, resuspended in FACS buffer, and analyzed by flow cytometry. The results are shown in Figure 2. 16H4, 313D9, and 341F5 all bound to monkey PBMCs, indicating that these antibodies can also bind to monkey cells at the cellular level.

[0444] Example 2.4 Activation of the Jurkat NF-AT reporter gene by humanized anti-CD3 antibodies 16H4, 313D9, and 341F5

[0445] 1x10 5Jurkat NF-AT (Cobioer, CBP74025) cells were added to 96-well white plates, centrifuged, and the supernatant was discarded. 100 μL of antibody or negative control IgG1 LALA (Abinvivo, B109802) at different concentrations (starting at 200 nM, triple-diluted, 12 spots including 0) were added. The plates were incubated at 37°C for 6-12 hours. Then, 100 μL / well of luciferase substrate (Gen Biz, DD1201-03) was added to the plates, and the plates were incubated at room temperature for 5 minutes. The results were detected using a multi-functional microplate reader, as shown in Figure 3. 16H4, 313D9, and 341F5 all activated Jurkat NF-AT, indicating that 16H4, 313D9, and 341F5 are CD3 antibodies with activation function.

[0446] Example 3: Characterization of anti-LILRB4 monoclonal antibody

[0447] The antibody (referred to as WT in this paper) was obtained by immunizing SJL mice (6-8 weeks old) with human LILRB4 protein (Acro, LI4-H52H7) and cynomolgus monkey LILRB4 protein (Acro, CDK-C5227). Affinity maturation and modification were then performed on the resulting antibody to obtain a humanized anti-LILRB4 antibody, H69L28. The heavy chain constant region of this monoclonal antibody is SEQ ID NO:36, and the light chain constant region is SEQ ID NO:49.

[0448] The sequence of the H69L28 antibody is shown in the sequence information. Its preparation is as follows: Human HEK293 cells were cultured in F17 medium supplemented with Glutsmax-I and F-68, and the cell concentration was adjusted to 2-3 x 10⁻⁶ cells / mL. 6 HEK293 cell solution was obtained by mixing [amount] ml. The coding genes for the heavy chain (heavy chain variable region + heavy chain constant region) and light chain (light chain variable region + light chain constant region) of the monoclonal antibody were constructed into the PTT5 vector, respectively. The PTT5 vector (synthesized by Suzhou Genewiz) containing the two coding genes for the LILRB4 domain was added to Opti-MEM (Gibco) to obtain solution A. Transfection reagent PEI (Polysciences, 24885-2) was added to Opti-MEM medium to obtain solution B. Solutions A and B were then mixed to obtain the transfection mixture, which was added to the HEK293 cell solution after 20 minutes. After culturing at 37°C and 5% CO2 for 1 day, 20% TNI (Organotechnie, 19553) was added and the mixture was transferred to 37°C and 5% CO2 for 7 days. The culture supernatant was then harvested and purified using a Protein A purification column (GE) to obtain the antibody LILRB4 monoclonal antibody H69L28.

[0449] Example 3.1 Affinity of anti-LILRB4 monoclonal antibody to human and monkey LILRB4 protein

[0450] Antibody affinity was determined using the anti-human antibody capture method on a Fortebio (BLITZ pro1.1.0.28) instrument. During the assay, the capture antibody (AHC) bioprobe of the Fc fragment of the anti-human antibody was immersed in 1x KB buffer (PBS + 0.2% Tween 20 + 0.1% BSA) for 10 min. 200 μl of diluted antibody sample was loaded onto the AHC bioprobe at a working concentration of 10 μg / mL, and then equilibrated in KB buffer for 100 s. The AHC probe was then further subjected to a binding reaction with serially diluted (100 nM starting, 2-fold dilution) human LILRB4 protein or monkey LILRB4 protein (purchased from ACRO biosystems) for 600 s. Afterward, the AHC probe was transferred to KB buffer for dissociation for 600 s. After the experiment, the blank control response value was subtracted, and the antigen-antibody binding kinetic constant was calculated using software to fit a 1:1 Langmuir binding pattern. The instrument's built-in software was then used for curve processing and fitting. The results are shown in Table 2. H69L28 can bind to both human LILRB4 protein and monkey LILRB4.

[0451] Table 2: Affinity of anti-LILRB4 humanized antibody H69L28 to human and monkey LILRB4 proteins

[0452] Example 3.2 Binding of anti-LILRB4 monoclonal antibody to human tumor cell lines

[0453] To evaluate the binding ability of LILRB4 monoclonal antibody to LILRB4 on the cell surface, flow cytometry was performed using LILRB4-positive human tumor cell lines THP-1 (human acute monocytic leukemia cells, donated by the Chinese Academy of Sciences) and MV4-11 (human myeloid monocytic leukemia cells, donated by the Chinese Academy of Sciences). Approximately 2 x 10⁻⁶ cells were used. 5Cells in each well were incubated with different concentrations of antibody (starting at 30 μg / ml, triple-diluted, 12 spots including 0) at 4°C for 0.5 h. Cells were washed three times with PBS (FACS buffer) containing 2% FBS, then resuspended in 100 μL (1:1000 with FACS buffer) of goat anti-human IgG (Jackson Immune Research, 109-135-098) and incubated at 4°C for 30 min. Cells were washed three times with FACS buffer, resuspended in FACS buffer, and used for flow cytometry analysis. The results are shown in Figure 4. The figure shows that the affinity-modified anti-LILRB4 antibody H69L28 exhibited binding to THP-1 and MV4-11 at a rate comparable to or stronger than that of WT (i.e., the unmodified antibody, information in the table below).

[0454] Table 3 Antibody sequences before affinity modification

[0455] Example 3.3 Binding of anti-LILRB4 monoclonal antibody to monkey overexpression of CHO-K1 Rhesus LILRB4

[0456] To evaluate the binding ability of the LILRB4 monoclonal antibody to LILRB4 on the surface of monkey cells, flow cytometry was performed using the LILRB4-overexpressing tumor cell line CHO-K1 Rhesus LILRB4 (internal construction, obtained by constructing rhesus monkey LILRB4 (Uniprot#A0A5F8AGT6) into the plvx-IRES-puro vector, followed by transfection into the CHO-K1 cell line for selection). Approximately 2 x 10-1 5 Cells were incubated with different concentrations of antibody (starting at 30 μg / ml, triple-diluted, 12 spots including 0) at 4°C for 0.5 h. Cells were washed three times with PBS (FACS buffer) containing 2% FBS, then resuspended in 100 μL (1:1000 with FACS buffer) of goat anti-human IgG (Jackson Immune Research, 109-135-098) and incubated at 4°C for 30 min. Cells were washed three times with FACS buffer, resuspended in FACS buffer, and used for flow cytometry analysis. The results are shown in Figure 5. The anti-LILRB4 antibody H69L28 showed comparable binding to WT (i.e., the unmodified antibody) in monkey CHO-K1 Rhesus LILRB4 cell lines.

[0457] Example 3.4 Binding of anti-LILRB4 monoclonal antibody to homology family proteins

[0458] To evaluate the binding of the LILRB4 monoclonal antibody to other members of the family, ELISA was used to detect the binding of the anti-LILRB4 antibody to the following family proteins: LILRA1 (Sino Biological, 17220-H08H), LILRA2 (Sino Biological, 30010-H08H), LILRA3 (Sino Biological, 13549-H08H), LILRA4 (Acro, LI4-H5243), LILRA5 (Acro, LI5-H52H3), LILRA6 (Acro, LI6-H52H3), LILRB1 (Sino Biological, 16014-H08H), LILRB2 (Acro, LI2-H5220), LILRB3 (Sino Biological, 11978-H08H), and LILRB5 (Acro, CDC-H5220).

[0459] ELSIA was performed using a standard protocol. In short, each protein in the family was coated at a concentration of 1 μg / ml onto a 96-well plate (Corning 9018) and incubated overnight at 4°C. The plates were washed three times with PBS containing 0.05% Tween 20. Subsequently, the plates were blocked with PBS containing 1% BSA for 2 hours. After washing three times with PBS containing 0.05% Tween 20, 30 μg / ml anti-LILRB4 antibody was added, and the plates were incubated at 37°C for 1 hour. The plates were washed three times with PBS containing 0.05% Tween 20. Detection was performed using HRP-conjugated goat anti-mouse IgG-Fc antibody (Jackosn ImmunoResearch, 109-035-098). The assay was performed at room temperature using TMB (Thermo Scientific, 34028) substrate for color development and measured at 450 nm using a microplate reader. The specific results are shown in Figure 6. It can be seen that the anti-LILRB4 antibody H69L28 does not bind to other members of the LILRB4 family, but only specifically binds to LILRB4.

[0460] Example 3.5 Non-specific binding of anti-LILRB4 monoclonal antibody to CHO-K1 and HEK293, and binding to the tumor cell line U937 that does not express LILRB4.

[0461] To evaluate the specificity of the LILRB4 monoclonal antibody, flow cytometry was performed using CHO-K1 (Cobioer#CBP60296), HEK293 (ATCC#CRL-1573), and the LILRB4-non-expressing tumor cell line U937 (Cobioer#CBP60277). Approximately 2 x 10⁻⁶ cells were used. 5Cells in each well were incubated at 4°C for 0.5 h with different concentrations (starting at 30 μg / ml, triple-diluted, 12 spots including 0) of antibody H69L28, WT (i.e., pre-affinity maturation antibody), and negative control IgG1 LALA. Cells were washed three times with PBS (FACS buffer) containing 2% FBS, then resuspended in 100 μL (1:1000 with FACS buffer) of goat anti-human IgG (Jackson Immune Research, 109-135-098) and incubated at 4°C for 30 min. Cells were washed three times with FACS buffer, resuspended in FACS buffer, and analyzed by flow cytometry. The results are shown in Figure 7. Antibody H69L28 did not specifically bind to HEK293, CHO-K1, or U937 cells.

[0462] Example 4: Design and preparation of CD3×LILRB4 bispecific antibody

[0463] To obtain a promising bispecific antibody molecule, different bispecific antibody configurations were designed in this example. Figure 8 shows a schematic diagram of the bispecific antibody CD3×LILRB4 structure. CD3 adopts a monovalent configuration of scFv (with a 44C mutation in the heavy chain variable region VH and a 100C mutation in the light chain variable region VL to form interchain disulfide bonds, reducing oligomerization and improving scFv stability). LILRB4 adopts a monovalent (i.e., "1+1" structure, small figure B) or bivalent (i.e., "1+2" ​​structure, small figure A) Fab configuration, with the Fc region being IgG1. The Fc region contains L234A and L235A mutations, and two Fc regions contain Knob mutations (S354C and T366W mutations) and Hole mutations (Y349C+T366S+L368A+Y407V), respectively. Furthermore, the Fc region containing Hole mutations also contains purification-related H435R and Y436F mutations to remove Hole homodimers. The specific sequences of the constructed bispecific antibodies F5-313D9, F5-341F5, and F3-16H4 are shown in the sequence information. F5-313D9 and F5-341F5 have one CD3 binding site and two LILRB4 binding sites (i.e., a "1+2" ​​structure, small figure A), while F3-16H4 has one CD3 binding site and one LILRB4 binding site (i.e., a "1+1" structure, small figure B).

[0464] This embodiment demonstrates the production of bispecific antibodies. The specific experimental procedures are as follows: Human HEK293 cells were cultured in F17 medium supplemented with Glutsmax-I and F-68, and the cell concentration was adjusted to 2-3 x 10⁻⁶ cells / mL. 6HEK293 cell suspension was obtained by mixing [amount] ml. The encoding genes for each strand of the bispecific antibody were constructed into the PTT5 vector. The PTT5 vector (synthesized by Suzhou Genewiz) containing the three-strand encoding genes for the anti-CD3 and anti-LILRB4 domains was added to Opti-MEM (Gibco) to obtain solution A. PEI (Polysciences, 24885-2) transfection reagent was added to Opti-MEM medium to obtain solution B. Solutions A and B were then mixed to obtain the transfection mixture. After 20 minutes, the entire transfection mixture was added to the HEK293 cell suspension. After culturing at 37°C and 5% CO2 for 1 day, 20% TNI (Organotechnie, 19553) was added as feed and the mixture was transferred to 37°C and 5% CO2 for 7 days. The culture supernatant was then harvested, and bispecific antibodies CD3×LILRB4, namely F5-313D9, F5-341F5 and F3-16H4, were obtained using Protein A purification column (GE) and size exclusion chromatography (SEC).

[0465] Example 5: Identification of the binding of the CD3×LILRB4 bispecific antibody "1+2" ​​structure to human and monkey CD3εγ and LILRB4 proteins.

[0466] The binding ability of the anti-CD3 and anti-LILRB4 domains in the "1+2" ​​structure of the CD3×LILRB4 bispecific antibody to their respective antigens was determined by detecting affinity. The specific method can be referred to Example 2.1. The binding ability of the bispecific antibody to CD3 protein and LILRB4 protein was determined by the strength of affinity. The specific results are shown in Tables 4 and 5, indicating that the CD3×LILRB4 bispecific antibodies F5-313D9 and F5-341F5 of the present invention can bind to human and monkey CD3εγ protein and LILRB4 protein.

[0467] Table 4: Affinity of the "1+2" ​​structured bispecific antibody CD3×LILRB4 to human CD3εγ protein and monkey CD3εγ protein.

[0468] Table 5: Affinity of the "1+2" ​​structured bispecific antibody CD3×LILRB4 to human and monkey LILRB4 proteins.

[0469] Example 6: Identification of the binding ability of the CD3×LILRB4 bispecific antibody "1+2" ​​structure to Jurkat T cells

[0470] This embodiment uses flow cytometry to detect the binding characteristics of the CD3×LILRB4 bispecific antibody, and the signal strength after the addition of the bispecific antibody is used to determine the binding characteristics between the bispecific antibody and Jurkat T. The specific experimental procedures are as follows:

[0471] Bispecific antibodies CD3×LILRB4 with different concentration gradients (starting concentration 100 nM, three-fold dilution, 12 spots (including 0 spot)) were prepared using FACS buffer. Tab1 was a positive control bispecific antibody (the bispecific antibody in CN 115551894 A, whose chain sequence can be found in SEQ ID NO: 98, 96, 100, 105, prepared using the method described in Example 4 of this document), and IgG1-LALA was a negative control.

[0472] Add 100 μL to a 96-well plate, and take 2 x 10⁻⁶ μL of the solution. 6 100 μL of / ml Jurkat cells were added to a 96-well plate with neutralizing antibody and incubated at 4°C for 0.5 h. After washing three times with PBS (FACS buffer) containing 2% FBS, the cells were centrifuged and resuspended in 100 μL (1:1000 with FACS buffer) of goat anti-human IgG (Jackson Immune Research, 109-135-098) and incubated at 4°C for 30 min. The cells were then washed three times with FACS buffer, resuspended in FACS buffer, and used for flow cytometry analysis.

[0473] The experimental results are shown in Figure 9. F5-313D9 and F5-341F5 exhibited weak binding to Jurkat.

[0474] Example 7: Identification of the binding ability of the CD3×LILRB4 bispecific antibody "1+2" ​​structure to human peripheral blood T cells.

[0475] This embodiment uses flow cytometry to detect the binding characteristics of the CD3×LILRB4 bispecific antibody, and the signal strength after the addition of the bispecific antibody is used to determine the binding characteristics between the bispecific antibody and human peripheral blood T cells. The specific experimental procedures are as follows:

[0476] T cells were isolated from human peripheral blood mononuclear cells (Rubai Biotechnology) using a T cell isolation kit (stemcell) and diluted with FACS buffer to a concentration of 2 x 10⁻⁶. 6 / ml, take 100μL and add it to a 96-well plate, then add different concentration gradients (starting concentration 1000nM, three-fold dilution, 12 spots (including 0 spot)) of CD3×LILRB4 bispecific antibody and positive control Tab1 (bispecific antibody in CN 115551894 A, the chain sequence of which can be found in SEQ ID NO: 98, 96, 100, 105, prepared as described in Example 4 of this document) and negative control IgG1 LALA (specific sequence is shown in the sequence information table, prepared in a similar manner to the bispecific antibody, see Example 4) 100μL to the 96-well plate, and incubate at 4°C for 0.5 hours. Wash three times with PBS (FACS buffer) containing 2% FBS, centrifuge and discard the supernatant, resuspend in 100μL (1:1000 with FACS buffer) of goat anti-human IgG (Jackson immune research, 109-135-098), and incubate at 4°C for 30 minutes. Cells were washed three times with FACS buffer, resuspended in FACS buffer, and used for flow cytometry analysis.

[0477] The specific experimental results are shown in small figure A in Figure 10. F5-313D9 and F5-341F5 showed weak binding to peripheral blood T cells.

[0478] Example 8: Identification of the binding ability of the CD3×LILRB4 bispecific antibody "1+2" ​​structure to human peripheral blood T cells.

[0479] In this embodiment, flow cytometry was used to detect the differences in binding characteristics of bispecific antibodies with the same "1+2" ​​structure but containing different CD3 binding domains to T cells. The strength of the signal after the addition of bispecific antibodies was used to determine the binding characteristics of bispecific antibodies with the same structure but containing different CD3 binding domains to human peripheral blood T cells.

[0480] The specific experimental procedures are the same as in Example 7.

[0481] The experimental results are shown in Figure 10B, indicating that the bispecific antibodies of the present invention have the same structure but contain different CD3 binding domains, which have different binding abilities to human peripheral blood T cells. F5-AMG (the CD3 binding part uses the CD3 antibody sequence from AMG757, US2020 / 0332002 A1, the bispecific antibody structure and LILRB4 binding domain are the same as F5-313D9 and F5-341F5) showed strong binding to peripheral blood T cells, while F5-313D9 and F5-341F5 showed weak binding to peripheral blood T cells.

[0482] Example 9: Identification of the binding ability of the CD3×LILRB4 bispecific antibody “1+2” structure to human acute myeloid leukemia tumor cell lines THP-1 (LILRB4 highly expressed), MV4-11 (LILRB4 moderately expressed), and MOLM13 (CBP60678, a human myeloid monocytic leukemia cell line, with low LILRB4 expression).

[0483] This embodiment uses flow cytometry to detect the binding characteristics of the CD3×LILRB4 bispecific antibody, and the signal strength after the addition of the bispecific antibody is used to determine the binding characteristics between the bispecific antibody and human acute myeloid leukemia tumor cell lines. The specific experimental procedures are as follows:

[0484] THP-1 was diluted to 2x10 using FACS buffer. 6 / ml, add 100μL to a 96-well plate, then add 100μL each of the CD3×LILRB4 bispecific antibody and the negative control IgG1-LALA at different concentration gradients (starting concentration 100nM, three-fold dilution, 12 spots (including 0 spot)) to the 96-well plate, and incubate at 4°C for 0.5 hours. Wash three times with PBS (FACS buffer) containing 2% FBS, centrifuge and discard the supernatant, resuspend in 100μL (1:1000 with FACS buffer) of goat anti-human IgG (Jackson Immune Research, 109-135-098), and incubate at 4°C for 30 minutes. Wash three times with FACS buffer, resuspend in FACS buffer, and use for flow cytometry analysis. The method used for MV-11 and MOLM13 is the same as that used for THP-1 cell lines.

[0485] The experimental results are shown in Figure 11, indicating that the "1+2" ​​structured bispecific antibody CD3×LILRB4 in this invention exhibits binding ability on human acute myeloid leukemia tumor cell lines THP-1, MV4-11 and MOLM13, and the binding is abundance-dependent.

[0486] Example 10: Identification of the CD3×LILRB4 bispecific antibody “1+2” structure promoting Jurkat NF-AT reporter gene activation

[0487] This embodiment utilizes the Jurkat-NFAT reporter system method to identify the ability of the CD3×LILRB4 bispecific antibody to crosslink LILRB4 on the surface of target cells and CD3 on the surface of effector cells, thereby activating T cells. The intensity of the relative chemiluminescence signal (RLU) is used to determine the ability of the bispecific antibody to activate T cells by bridging LILRB4-positive target cells and T cells. The specific experimental procedures are as follows:

[0488] a. Dilute MV4-11 cells 1x10 using complete culture medium (RPIM1640 + 10% FBS). 5 / well, add 25 μL of cells to a 96-well white plate, or add 25 μL of complete culture medium to a 96-well white plate;

[0489] b. Dilute Jurkat NF-AT cells 1x10 with complete culture medium. 5 / well, take 25ul of cells and add them to a 96-well white plate;

[0490] c. Dilute CD3×LILRB4 antibody, positive control Tab1 and negative control IgG1-LALA to different concentrations using complete culture medium, and add 50 μL to a 96-well white plate.

[0491] d. Place the above white board in a 37℃, 5% CO2 incubator and incubate for 6-12 hours;

[0492] e. Add 100 μL of luciferase substrate per well to the plate, incubate at room temperature for 5 minutes, and then read the plate using a multi-functional microplate reader.

[0493] The specific results are shown in Figure 12, which further demonstrates that the "1+2" ​​structured bispecific antibody CD3×LIRB4 of the present invention can bridge LILRB4-positive target cells and Jurkat T cells, promote Jurkat T cell activation, and does not non-specifically activate Jurkat T cells in the absence of tumor cell lines.

[0494] Example 11: Identification of the killing ability of the CD3×LILRB4 bispecific antibody "1+2" ​​structure against human acute myeloid leukemia tumor cell lines THP-1 (LILRB4 highly expressed), MV4-11 (LILRB4 moderately expressed), and MOLM13 (LILRB4 low expressed).

[0495] This embodiment utilizes a T-cell-dependent cytotoxicity assay (TDCC) to identify the killing ability of the CD3×LILRB4 bispecific antibody against tumor cells, and determines the killing level of the bispecific antibody against tumor cell lines based on the tumor cell mortality rate after bispecific antibody administration. The specific experimental procedures are as follows:

[0496] a. According to CellTrace TM Invitrogen Erythrocyte Proliferation Kit TM According to the instructions (C34564), the tumor cell line THP-1 was labeled and stained.

[0497] b. After staining, dilute THP-1 cells to 8x10⁸ mcg using complete culture medium. 5 / ml, add 50ul of cells to a 96 plate, i.e., 4x10 4 / THP-1 tumor cells

[0498] c. Dilute human peripheral blood mononuclear cells (PBMCs) with complete culture medium to 4 x 10⁻⁶. 6 / ml, add 50ul of cells to a 96 plate, i.e., 2x10 5 / pore PBMC (effector cell PBMC: target cell = 5:1)

[0499] d. Dilute the CD3×LILRB4 antibody and the positive control Tab1 to different concentrations using complete culture medium (starting concentration 13.3 nM, five-fold dilution, 15 spots (including 0 spot)), and add 100 μL to a 96-well plate.

[0500] e. Place the above 96-well plate in a 37°C, 5% CO2 incubator and incubate for 24 hours.

[0501] f. Collect samples using LIVE / DEAD. TM Invitrogen staining kit for fixing purple dead cells TM Cells were stained with L34955 to detect the number of dead cells (BV421 positive by flow cytometry) and positive tumor cell populations (APC positive by flow cytometry). The tumor cell killing efficiency was then calculated using the formula below. Cells without antibody were used as a blank control.

[0502] The methods used for g.MV4-11, MOLM13, and THP-1 cell lines are consistent.

[0503] Kill efficiency (%) = Dead cells (BV421 positive by flow cytometry) / Positive tumor cell population (APC positive by flow cytometry) x 100%

[0504] The specific experimental results are shown in Figure 13 and Table 6: This demonstrates that the bispecific antibody CD3×LILRB4 with the “1+2” structure in this invention exhibits killing function against human acute myeloid leukemia tumor cell lines THP-1, MV4-11 and MOLM13, and the killing is abundance-dependent.

[0505] Table 6: EC50 of bispecific antibody-mediated killing effect on AML tumor cell lines

[0506] Example 12: Identification of the killing effect of the CD3×LILRB4 bispecific antibody "1+2" ​​structure on the LILRB4 negative cell line RPMI8226

[0507] This embodiment utilizes a T-cell-dependent cytotoxicity assay (TDCC) to identify the killing ability of the aforementioned CD3×LILRB4 bispecific antibody and the positive control Tab1 against tumor cells not expressing LILRB4, and to determine the non-specific killing ability of the bispecific antibody based on the tumor cell mortality rate after administration of the bispecific antibody. The killing method is consistent with that in Example 11 above. The blank control refers to the background killing effect without the drug.

[0508] The experimental results are shown in Figure 14: This indicates that the bispecific antibody CD3×LILRB4 in this invention has no killing function on the human LILRB4 negative cell line RPMI8226, which means that the "1+2" ​​structure bispecific antibody CD3×LILRB4 in this invention does not have non-specific killing effect.

[0509] Example 13: Identification of the autocytic effect of the CD3×LILRB4 bispecific antibody "1+2" ​​structure on normal B cells

[0510] This embodiment utilizes T-cell autocytic activity to identify the killing ability of the aforementioned CD3×LILRB4 bispecific antibody against normal B cells. The killing level of the bispecific antibody against normal B cells is determined based on the tumor cell mortality rate after administration of the bispecific antibody. The specific experimental procedures are as follows:

[0511] PBMCs were resuspended in complete culture medium containing 50 ng / ml IL-2 to a final volume of 2 × 10⁻⁶. 6 / ml, add 100μl to a 96-well plate, and prepare CD3×LILRB4 bispecific antibody and Tab1 at different concentration gradients (starting concentration 33.3nM, 5-fold dilution, 19 spots (including 0 spot)). Add 100μl of each to the 96-well plate and incubate at 37°C for 48 hours. At the end of incubation, wash the cells and incubate at room temperature for 10 minutes with 2μL of Fc receptor blocker (BD, 564765). Then, use anti-CD14 flow cytometry antibody (Biolegend, 301804) and anti-CD19 flow cytometry antibody (Biolegend, 392504) and LIVE / DEAD. TM (Invitrogen TM (L34955) was incubated on ice for 30 minutes. B cells were identified as CD19 positive cells.

[0512] The experimental results are shown in Figure 15, indicating that the "1+2" ​​structure bispecific antibody CD3×LILRB4 in this invention has no killing effect on normal B cells.

[0513] Example 14: Identification of the CD3×LILRB4 bispecific antibody “1+2” structure promoting T cell activation and granzyme secretion

[0514] This embodiment uses flow cytometry to identify the activation of human primary T cells and granzyme secretion by the aforementioned CD3×LILRB4 bispecific antibody. The specific experimental procedures are as follows:

[0515] a. Dilute THP-1 cells to 8 x 10⁸ mcg using complete culture medium. 5 / ml, add 50ul of cells to a 96 plate, i.e., 4x10 4 / THP-1 tumor cells

[0516] b. Dilute human peripheral blood mononuclear cells (PBMCs, Ruibai Biotechnology) with complete culture medium to 4 x 10⁻⁶. 6 / ml, add 50ul of cells to a 96 plate, i.e., 2x10 5 / pore PBMC (effector cell PBMC: target cell = 5:1)

[0517] c. Dilute the CD3×LILRB4 antibody and the positive control Tab1 to different concentrations using complete culture medium (starting concentration 13.3 nM, five-fold dilution, 15 spots (including 0 spot)), and add 100 μL to a 96-well plate.

[0518] d. Place the above 96-well plate in a 37°C, 5% CO2 incubator and incubate for 24-48 hours;

[0519] e. Collect samples and perform flow cytometry staining (using flow cytometry antibodies against anti-human CD4 (BD,555346) / anti-human CD8 (BD,555635) / anti-human CD69 (biolegand,310926) / anti-human granzyme B (BD,563388) / live / dead (Thermofish L34976)) to detect the activation of T cells and the level of granzyme secretion.

[0520] The specific experimental results are shown in Figures 16A-D: This demonstrates that the "1+2" ​​structured bispecific antibody CD3×LILRB4 in this invention can activate T cells and secrete granzymes in a concentration-dependent manner.

[0521] Example 15: Cytokine secretion of the CD3×LILRB4 bispecific antibody "1+2" ​​structure in the absence of tumor cells.

[0522] This embodiment utilizes HTRF detection to identify the cytokine secretion of a "1+2" ​​structured CD3×LILRB4 bispecific antibody in the absence of tumor cells. The specific experimental procedures are as follows:

[0523] a. Dilute human peripheral blood mononuclear cells (PBMCs) to 2 x 10⁻⁶ with complete culture medium. 6 / ml, add 100uL of cells to a 96 plate, i.e., 2x10 5 / hole PBMC;

[0524] b. Dilute CD3×LILRB4, positive control (Tab1 and Glofitamab), and negative control IgG1-LALA to different concentrations using complete culture medium (starting concentration 200 nM, three-fold dilution, 15 spots (including 0 spot)), and add 100 μL to a 96-well plate.

[0525] c. Place the above 96-well plate in a 37°C, 5% CO2 incubator and incubate for 24-48 hours.

[0526] d. Collect the supernatant and use HTRF to detect the secretion of cytokines IL-2 (Revvity, 62HIL02PET) / IFN-r (Revvity, 62HIFNGPET) / IL-6 (Revvity, 62HIL06PET) / IL-10 (Revvity, 62HIL10PET) / TNAF-a (Revvity, 62HTNFAPET).

[0527] The specific experimental results are shown in Figure 17: This demonstrates that the "1+2" ​​structured bispecific antibody CD3×LILRB4 in this invention exhibits lower cytokine secretion than Tab1 and the marketed product Glofitamab (Roche's CD3×CD20, prepared as described in Example 4), demonstrating higher safety. IL-6 cytokine was not detected.

[0528] Example 16: Cytokine secretion of the CD3×LILRB4 bispecific antibody "1+2" ​​structure in the presence of tumor cells.

[0529] This embodiment utilizes HTRF detection to identify the cytokine secretion of the aforementioned CD3×LILRB4 bispecific antibody in the presence of tumor cells. The specific experimental procedures are as follows:

[0530] a. Dilute THP-1 cells to 8 x 10⁸ mcg using complete culture medium. 5 / ml, take 50uL of cells and add them to a 96 plate, i.e., 4x10 4 / THP-1 tumor cells

[0531] b. Dilute human peripheral blood mononuclear cells (PBMCs) to 4 x 10⁶ / ml with complete culture medium, and add 50 μL of cells to a 96 plate, i.e., 2 x 10⁶ cells / ml. 5 / pore PBMC (effector cell PBMC: target cell = 5:1)

[0532] c. Dilute the CD3×LILRB4 antibody and the positive control Tab1 to different concentrations using complete culture medium (starting concentration 13.3 nM, five-fold dilution, 15 spots (including 0 spot)), and add 10 μL to a 96-well plate.

[0533] d. Place the above 96-well plate in a 37°C, 5% CO2 incubator and incubate for 24-48 hours;

[0534] e. Collect the supernatant and use HTRF to detect the secretion of cytokines IL-2 (kit: Revvity, 62HIL02PET) / IFN-r (kit: Revvity, 62HIFNGPET) / IL-6 (kit: Revvity, 62HIL06PET) / IL-10 (kit: Revvity, 62HIL10PET) / TNAF-a (kit: Revvity, 62HTNFAPET).

[0535] The specific experimental results are shown in Figures 18A-E: This shows that the "1+2" ​​structure bispecific antibodies CD3×LILRB4F5-313D9 and F5-341F5 in this invention exhibited cytokine secretion comparable to the positive control Tab1.

[0536] Example 17: Identification of the binding ability of the CD3×LILRB4 bispecific antibody "1+2" ​​structure to the human multiple myeloma cell line MM1.S

[0537] This embodiment uses flow cytometry to detect the binding characteristics of the CD3×LILRB4 bispecific antibody, and the signal strength after the addition of the bispecific antibody is used to determine the binding characteristics between the bispecific antibody and MM1.S. The specific experimental procedures are as follows:

[0538] MM1.S tumor cells were diluted to 2 x 10⁻⁶ using FACS buffer. 6 / mL, add 100μL to a 96-well plate, then add 100μL each of the following concentration gradients (starting at 22nM, tripled, 10 spots including 0) of CD3×LILRB4 bispecific antibody, positive control Tab1, and negative control IgG1 LALA to the 96-well plate, and incubate at 4°C for 0.5 hours. Wash three times with PBS (FACS buffer) containing 2% FBS, centrifuge and discard the supernatant, resuspend in 100μL (1:1000 with FACS buffer) of goat anti-human IgG (Jackson Immune Research, 109-135-098), and incubate at 4°C for 30 minutes. Wash three times with FACS buffer, resuspend in FACS buffer, and use for flow cytometry analysis.

[0539] The experimental results are shown in Figure 19, demonstrating that the "1+2" ​​structured bispecific antibody CD3×LILRB4 in this invention exhibits binding ability to the multiple myeloma tumor cell line MM1.S.

[0540] Example 18: Identification of the killing ability of the CD3×LILRB4 bispecific antibody "1+2" ​​structure against the human multiple myeloma cell line MM1.S

[0541] This embodiment utilizes a T-cell-dependent cytotoxicity assay (TDCC) to identify the killing ability of the aforementioned CD3×LILRB4 bispecific antibody against tumor cells, and determines the killing level of the bispecific antibody against tumor cell lines based on the tumor cell mortality rate after bispecific antibody administration. The specific experimental procedures are as follows:

[0542] a. According to CellTrace TM Invitrogen Erythrocyte Proliferation Kit TM According to the instructions (C34564), the tumor cell line MM1S was labeled and stained.

[0543] b. After staining, dilute MM1S cells to 8 x 10⁸ mcg using complete culture medium. 5 / ml, add 50ul of cells to a 96 plate, i.e., 4x10 4 / pore THP-1 tumor cells.

[0544] c. Dilute human peripheral blood mononuclear cells (PBMCs, Ruibai Biotechnology) with complete culture medium to 4 x 10⁶ / ml, and add 50 μL of cells to a 96 plate, i.e., 2 x 10⁶ cells / ml. 5 / well PBMC (effect cell PBMC: target cell = 5:1).

[0545] d. Dilute the CD3×LILRB4 antibody and the positive control Tab1 to different concentrations using complete culture medium (starting concentration 3.3 nM, five-fold dilution, 12 spots (including 0 spot)), and add 100 μL to a 96-well plate.

[0546] e. Place the above 96-well plate in a 37°C, 5% CO2 incubator and incubate for 24 hours.

[0547] f. Collect samples using LIVE / DEAD. TM Invitrogen staining kit for fixing purple dead cells TM Cells were stained with L34955 (flow cytometry showed BV421 positive) to detect the number of dead cells (flow cytometry showed APC positive) and positive tumor cell populations (flow cytometry showed APC positive). The tumor cell killing efficiency was then calculated using the formula below. Cells without antibody were used as a blank control.

[0548] Kill efficiency (%) = Dead cells (BV421 positive by flow cytometry) / Positive tumor cell population (APC positive by flow cytometry) x 100%

[0549] The specific experimental results are shown in Figure 20, demonstrating that the bispecific antibody CD3×LILRB4 in this invention exhibits killing function against the multiple myeloma tumor cell line MM1.S.

[0550] Example 19: Identification of the in vivo tumor growth inhibition of the CD3×LILRB4 bispecific antibody "1+2" ​​structure against THP-1, a human acute myeloid leukemia cell line with high LILRB4 expression.

[0551] This embodiment utilizes PBMC-reconstructed B-NDG mice to establish an orthotopic model of THP-1-Luc human myeloid leukemia to detect the in vivo efficacy of the CD3×LILRB4 bispecific antibody. The intracellular biological efficacy of the CD3×LILRB4 bispecific antibody is determined based on its inhibitory effect on mouse tumor growth. Specific experimental procedures are as follows:

[0552] PBMCs (Biocytok, batch number: P121080703C, 5×10⁻⁶) resuspended in serum-free RPMI 1640 medium 6 (0.1 mL / animal) and THP-1-Luc cells (2 × 10⁻⁶) 6 The mixture (0.1 mL / mouse) was injected via the tail vein into 125 B-NDG mice (Biocytok).

[0553] On day 4 post-vaccination, imaging signal values ​​were measured using a small animal imaging system. Based on the imaging signal values ​​and animal weight, 77 mice were randomly selected and enrolled into 11 groups of 7 mice each: G1: PBS, G2: Tab1 (1 mg / kg), G3: F3-16H4 (0.01 mg / kg), G4: F3-16H4 (0.1 mg / kg), G5: F3-16H4 (1 mg / kg), G6: F5-341F5 (0.01 mg / kg), G7: F5-341F5 (0.1 mg / kg), G8: F5-341F5 (1 mg / kg), G9: F5-313D9 (0.01 mg / kg), G10: F5-313D9 (0.1 mg / kg), and G11: F5-313D9 (1 mg / kg).

[0554] Administering began on the day of grouping, with all administration routes being tail vein injection. Administered twice weekly for seven consecutive weeks, ending the experiment five days after the last administration. Tumor imaging signal intensity and body weight were measured twice weekly during the administration and observation period, and the measurements were recorded.

[0555] The specific experimental results are shown in Figure 21, demonstrating that the "1+2" ​​structured bispecific antibody CD3×LILRB4 in this invention exhibited a significant inhibitory effect on mouse tumors in the orthotopic model of THP-1-Luc human myeloid leukemia established in PBMC-reconstructed B-NDG mice. Specifically, both F5-341F5 and F5-313D9 showed dose-dependent tumor inhibition. The 1+2 bispecific antibodies F5-341F5 and F5-313D9 showed tumor inhibition comparable to Tab1 at 1 mg / kg. Specifically, test sample F5-341F5 at test doses of 0.1 mg / kg and 1 mg / kg, and F5-313D9 at test doses of 0.01 mg / kg, 0.1 mg / kg, and 1 mg / kg, all significantly inhibited the growth of THP-1-Luc human myeloid leukemia cells in PBMC-reconstructed B-NDG mice.

[0556] Example 20: Identification of the in vivo tumor growth inhibition of the CD3×LILRB4 bispecific antibody "1+2" ​​structure in the human acute myeloid leukemia LILRB4 low-expressing tumor cell line MOLM13.

[0557] This embodiment utilizes the PPBMC immune system-derived MOLM-13 acute myeloid leukemia subcutaneous xenograft model to further detect the in vivo efficacy of the CD3×LILRB4 bispecific antibody, and determines the intracellular biological efficacy of the CD3×LILRB4 bispecific antibody based on the inhibitory strength of the bispecific antibody on mouse tumor growth.

[0558] The specific experimental procedure was as follows: MOLM13 human acute myeloid leukemia tumor cells were subcutaneously transplanted into the right anterior flank of male NCG mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.). The tumors were allowed to grow to an average size of 58 mm². 3 At that time, mice were randomly divided into eight groups of seven mice each, based on tumor volume. The groups were: PBS (0 mg / kg antibody), Tab1 (2 mg / kg), F3-16H4 (0.5 mg / kg), F3-16H4 (2 mg / kg), F5-341F5 (0.5 mg / kg), F5-341F5 (2 mg / kg), F5-313D9 (0.5 mg / kg), and F5-313D9 (2 mg / kg). All drugs were administered intravenously twice a week for a total of three times. After grouping, the tumor volume and body weight of the mice were measured three times a week.

[0559] The specific experimental results are shown in Figure 22: This demonstrates that the "1+2" ​​structured bispecific antibody CD3×LILRB4 in this invention exhibited a significant inhibitory effect on mouse tumors in the MOLM13 human myeloid leukemia subcutaneous model established in PBMC-reconstructed B-NDG mice. Treatment with low and high doses of F5-313D9 (0.5 mg / kg and 2 mg / kg), high dose of F5-341F5 (2 mg / kg), and Tab1 (2 mg / kg) all significantly inhibited tumor growth. Both the low and high dose groups of F5-341F5 and F5-313D9 showed a dose-dependent tumor-suppressive effect. Furthermore, F5-313D9 (2 mg / kg) showed significantly stronger efficacy compared to Tab1.

[0560] Example 21: Identification of the in vivo tumor growth inhibition of human multiple myeloma MM1.S by the CD3×LILRB4 bispecific antibody "1+2" ​​structure.

[0561] This embodiment utilizes the PPBMC immune system-derived MM1S subcutaneous xenograft model of multiple myeloma to further detect the in vivo efficacy of the CD3×LILRB4 bispecific antibody, and determines the intracellular biological efficacy of the CD3×LILRB4 bispecific antibody based on the inhibitory strength of the bispecific antibody on the growth of mouse tumors.

[0562] The specific experimental procedure was as follows: Human myeloma tumor cells MM1S were subcutaneously transplanted into the right anterior flank of male NCG mice. The tumors were allowed to grow to an average size of 53 mm². 3At approximately 10:00 AM, mice were randomly assigned to three groups of seven mice each, based on tumor volume: PBS, F5-313D9 (2 mg / kg), and F5-341F5 (2 mg / kg). All drugs were administered intravenously twice weekly for a total of five weeks. Tumor volume and body weight were measured twice weekly after grouping.

[0563] The specific experimental results are shown in Figure 23: This demonstrates that the "1+2" ​​structured bispecific antibody CD3×LILRB4 in this invention exhibited a significant inhibitory effect on mouse tumors in the subcutaneous model of MM1S multiple myeloma established in PBMC-reconstructed NCG mice. Treatment with the test drug F5-313D9 (2 mg / kg) significantly inhibited tumor growth. In this experiment, treatment with F5-313D9 (2 mg / kg) showed a significant advantage over F5-341F5 (2 mg / kg).

[0564] Example 22: Identification of the binding of the CD3×LILRB4 bispecific antibody “1+1” structure to human and monkey CD3εγ and LILRB4 proteins.

[0565] The binding ability of the CD3×LILRB4 bispecific antibody "1+1" structure to the CD3 and LILRB4 domains was determined by detecting affinity. The specific method can be referred to Example 2.1. The binding ability of the antibody to CD3 and LILRB4 proteins was determined by the strength of affinity. The specific results are shown in Tables 7 and 8, indicating that the "1+1" structure CD3×LILRB4 bispecific antibody F3-16H4 of the present invention can bind to human and monkey CD3εγ protein and LILRB4 protein.

[0566] Table 7: Affinity of the bispecific antibody CD3×LILRB4 to human CD3εγ protein and monkey CD3εγ protein

[0567] Table 8: Affinity of the bispecific antibody CD3×LILRB4 to human and monkey LILRB4 proteins

[0568] Example 23: Identification of the binding ability of the CD3×LILRB4 bispecific antibody "1+1" structure to Jurkat T cells

[0569] This embodiment uses flow cytometry to detect the binding characteristics of the CD3×LILRB4 bispecific antibody, and the signal strength after the addition of the bispecific antibody is used to determine the binding characteristics between the bispecific antibody and Jurkat T. The specific experimental procedures are as follows:

[0570] Bispecific antibodies CD3×LILRB4 with different concentration gradients (starting concentration 100 nM, three-fold dilution, 12 spots (including 0 spot)) were prepared using FACS buffer. Tab1 was a positive control bispecific antibody (the bispecific antibody in CN 115551894 A, whose chain sequence can be found in SEQ ID NO: 98, 96, 100, 105, prepared using the method described in Example 4 of this document), and IgG1-LALA was a negative control.

[0571] Add 100 μL to a 96-well plate, and take 2 x 10⁻⁶ μL of the solution. 6 100 μL of / ml Jurkat cells were added to a 96-well plate with neutralizing antibody and incubated at 4°C for 0.5 h. After washing three times with PBS (FACS buffer) containing 2% FBS, the cells were centrifuged and resuspended in 100 μL (1:1000 with FACS buffer) of goat anti-human IgG (Jackson Immune Research, 109-135-098) and incubated at 4°C for 30 min. The cells were then washed three times with FACS buffer, resuspended in FACS buffer, and used for flow cytometry analysis.

[0572] The experimental results are shown in Figure 24, indicating that the bispecific antibody F3-16H4 with the "1+1" structure in this invention exhibits a relatively strong binding to Jurkat.

[0573] Example 24: Identification of the binding ability of the CD3×LILRB4 bispecific antibody "1+1" structure to human peripheral blood T cells.

[0574] This embodiment uses flow cytometry to detect the binding characteristics of the CD3×LILRB4 bispecific antibody F3-16H4, and the signal strength after the addition of the bispecific antibody is used to determine the binding characteristics between the bispecific antibody and human peripheral blood T cells. The specific experimental procedures are as follows:

[0575] T cells were isolated from human peripheral blood mononuclear cells (Rubai Biotechnology) using a T cell isolation kit (stemcell) and diluted with FACS buffer to a concentration of 2 x 10⁻⁶. 6 / ml, take 100μL and add it to a 96-well plate, then add different concentration gradients (starting concentration 1000nM, three-fold dilution, 12 spots (including 0 spot)) of CD3×LILRB4 bispecific antibody and positive control Tab1 (bispecific antibody in CN 115551894 A, the chain sequence of which can be found in SEQ ID NO: 98, 96, 100, 105, prepared as described in Example 4 of this document) and negative control IgG1 LALA (specific sequence is shown in the sequence information table, prepared in a similar manner to the bispecific antibody, see Example 4) 100μL to the 96-well plate, and incubate at 4°C for 0.5 hours. Wash three times with PBS (FACS buffer) containing 2% FBS, centrifuge and discard the supernatant, resuspend in 100μL (1:1000 with FACS buffer) of goat anti-human IgG (Jackson immune research, 109-135-098), and incubate at 4°C for 30 minutes. Cells were washed three times with FACS buffer, resuspended in FACS buffer, and used for flow cytometry analysis.

[0576] The experimental results are shown in Figure 25. F3-16H4 showed strong binding to peripheral blood T cells.

[0577] Example 25: Identification of the binding ability of the CD3×LILRB4 bispecific antibody “1+1” structure to human acute myeloid leukemia tumor cell lines THP-1 (LILRB4 highly expressed), MV4-11 (LILRB4 moderately expressed), and MOLM13 (CBP60678, a human myeloid monocytic leukemia cell line, with low LILRB4 expression).

[0578] This embodiment uses flow cytometry to detect the binding characteristics of the CD3×LILRB4 bispecific antibody F3-16H4, and the signal strength after the addition of the bispecific antibody is used to determine the binding characteristics between the bispecific antibody and human acute myeloid leukemia tumor cell lines. The specific experimental procedures are as follows:

[0579] THP-1 was diluted to 2x10 using FACS buffer. 6 / ml, add 100μL to a 96-well plate, then add 100μL each of the CD3×LILRB4 bispecific antibody and the negative control IgG1-LALA at different concentration gradients (starting concentration 100nM, three-fold dilution, 12 spots (including 0 spot)) to the 96-well plate, and incubate at 4°C for 0.5 hours. Wash three times with PBS (FACS buffer) containing 2% FBS, centrifuge and discard the supernatant, resuspend in 100μL (1:1000 with FACS buffer) of goat anti-human IgG (Jackson Immune Research, 109-135-098), and incubate at 4°C for 30 minutes. Wash three times with FACS buffer, resuspend in FACS buffer, and use for flow cytometry analysis. The method used for MV-11 and MOLM13 is the same as that used for THP-1 cell lines.

[0580] The experimental results are shown in Figure 26, indicating that the bispecific antibody F3-16H4 with the "1+1" structure in this invention exhibits binding ability on human acute myeloid leukemia tumor cell lines THP-1, MV4-11 and MOLM13, and the binding is abundance-dependent.

[0581] Example 26: Identification of the CD3×LILRB4 bispecific antibody “1+1” structure promoting Jurkat NF-AT reporter gene activation

[0582] This embodiment utilizes the Jurkat-NFAT reporter system method to identify the ability of the CD3×LILRB4 bispecific antibody F3-16H4 to crosslink LILRB4 on the surface of target cells and CD3 on the surface of effector cells, thereby activating T cells. The intensity of the relative chemiluminescence signal (RLU) is used to determine the ability of the bispecific antibody to activate T cells by bridging LILRB4-positive target cells and T cells. The specific experimental procedures are as follows:

[0583] a. Dilute MV4-11 cells 1x10 using complete culture medium (RPIM1640 + 10% FBS). 5 / well, add 25 μL of cells to a 96-well white plate, or add 25 μL of complete culture medium to a 96-well white plate;

[0584] b. Dilute Jurkat NF-AT cells 1x10 with complete culture medium. 5 / well, take 25ul of cells and add them to a 96-well white plate;

[0585] c. Dilute CD3×LILRB4 antibody, positive control Tab1 and negative control IgG1-LALA to different concentrations using complete culture medium, and add 50 μL to a 96-well white plate.

[0586] d. Place the above white board in a 37℃, 5% CO2 incubator and incubate for 6-12 hours;

[0587] e. Add 100 μL of luciferase substrate per well to the plate, incubate at room temperature for 5 minutes, and then read the plate using a multi-functional microplate reader.

[0588] The specific results are shown in Figure 27, which further demonstrates that the bispecific antibody F3-16H4 with the "1+1" structure of the present invention can bridge LILRB4 positive target cells and Jurkat T cells, promote Jurkat T cell activation, and does not nonspecifically activate Jurkat T cells in the absence of tumor cell lines.

[0589] Example 27. Identification of the killing ability of the CD3×LILRB4 bispecific antibody "1+1" structure against human acute myeloid leukemia tumor cell lines THP-1 (LILRB4 highly expressed), MV4-11 (LILRB4 moderately expressed), and MOLM13 (LILRB4 low expressed).

[0590] This embodiment utilizes a T-cell-dependent cytotoxicity assay (TDCC) to identify the cytotoxic ability of the CD3×LILRB4 bispecific antibody F3-16H4 against tumor cells, and determines the cytotoxic level of the bispecific antibody against tumor cell lines based on the tumor cell mortality rate after bispecific antibody administration. The specific experimental procedures are as follows:

[0591] a. According to CellTrace TM Invitrogen Erythrocyte Proliferation Kit TM According to the instructions (C34564), the tumor cell line THP-1 was labeled and stained.

[0592] b. After staining, dilute THP-1 cells to 8x10⁸ mcg using complete culture medium. 5 / ml, add 50ul of cells to a 96 plate, i.e., 4x10 4 / THP-1 tumor cells

[0593] c. Dilute human peripheral blood mononuclear cells (PBMCs) with complete culture medium to 4 x 10⁻⁶. 6 / ml, add 50ul of cells to a 96 plate, i.e., 2x10 5 / pore PBMC (effector cell PBMC: target cell = 5:1)

[0594] d. Dilute the CD3×LILRB4 antibody and the positive control Tab1 to different concentrations using complete culture medium (starting concentration 13.3 nM, five-fold dilution, 15 spots (including 0 spot)), and add 100 μL to a 96-well plate.

[0595] e. Place the above 96-well plate in a 37°C, 5% CO2 incubator and incubate for 24 hours.

[0596] f. Collect samples using LIVE / DEAD. TM Invitrogen staining kit for fixing purple dead cells TM Cells were stained with L34955 to detect the number of dead cells (BV421 positive by flow cytometry) and positive tumor cell populations (APC positive by flow cytometry). The tumor cell killing efficiency was then calculated using the formula below. Cells without antibody were used as a blank control.

[0597] The methods used for g.MV4-11, MOLM13, and THP-1 cell lines are consistent.

[0598] Kill efficiency (%) = Dead cells (BV421 positive by flow cytometry) / Positive tumor cell population (APC positive by flow cytometry) x 100%

[0599] The specific experimental results are shown in Figure 28 and Table 9: This demonstrates that the bispecific antibody F3-16H4 with the "1+1" structure in this invention exhibits killing function against human acute myeloid leukemia tumor cell lines THP-1, MV4-11 and MOLM13, and the killing is abundance-dependent.

[0600] Table 9: EC50 of bispecific antibody F3-16H4 in killing AML tumor cell lines

[0601] Example 28: Identification of the killing effect of the CD3×LILRB4 bispecific antibody "1+1" structure on the LILRB4 negative cell line RPMI8226

[0602] This embodiment utilizes a T-cell-dependent cytotoxicity assay (TDCC) to identify the killing ability of the "1+1" structured CD3×LILRB4 bispecific antibody against tumor cells that do not express LILRB4, and assesses the non-specific killing ability of the bispecific antibody based on the tumor cell mortality rate after bispecific antibody administration. The killing method is consistent with that in Example 27.

[0603] The experimental results are shown in Figure 29: This indicates that the "1+1" structure bispecific antibody F3-16H4 in this invention has no killing function on the human LILRB4 negative cell line RPMI8226, which means that the bispecific antibody CD3×LILRB4 in this invention does not have non-specific killing effect.

[0604] Example 29: Identification of the autocytic effect of the CD3×LILRB4 bispecific antibody "1+1" structure on normal B cells

[0605] This embodiment utilizes T-cell autocytic activity to identify the cytotoxic ability of the CD3×LILRB4 bispecific antibody F3-16H4 against normal B cells. The cytotoxic level of the bispecific antibody against normal B cells is determined based on the tumor cell mortality rate after administration of the bispecific antibody. The specific experimental procedures are as follows:

[0606] PBMCs were resuspended in complete culture medium containing 50 ng / ml IL-2 to a final volume of 2 × 10⁻⁶. 6 / ml, add 100μl to a 96-well plate, and prepare CD3×LILRB4 bispecific antibody and Tab1 at different concentration gradients (starting concentration 33.3nM, 5-fold dilution, 19 spots (including 0 spot)). Add 100μl of each to the 96-well plate and incubate at 37°C for 48 hours. At the end of incubation, wash the cells and incubate at room temperature for 10 minutes with 2μL of Fc receptor blocker (BD, 564765). Then, use anti-CD14 flow cytometry antibody (Biolegend, 301804) and anti-CD19 flow cytometry antibody (Biolegend, 392504) and LIVE / DEAD. TM (Invitrogen TM (L34955) was incubated on ice for 30 minutes. B cells were identified as CD19 positive cells.

[0607] The specific experimental results are shown in Figure 30: The "1+1" structure bispecific antibody F3-16H4 in this invention has no killing effect on normal B cells.

[0608] Example 30: Identification of the effect of the CD3×LILRB4 bispecific antibody “1+1” structure on T cell activation and granzyme secretion

[0609] This embodiment uses flow cytometry to identify the activation of human primary T cells and granzyme secretion by the CD3×LILRB4 bispecific antibody F3-16H4. The specific experimental procedures are as follows:

[0610] a. Dilute THP-1 cells to 8 x 10⁸ mcg using complete culture medium. 5 / ml, add 50ul of cells to a 96 plate, i.e., 4x10 4 / THP-1 tumor cells

[0611] b. Dilute human peripheral blood mononuclear cells (PBMCs, Ruibai Biotechnology) with complete culture medium to 4 x 10⁻⁶. 6 / ml, add 50ul of cells to a 96 plate, i.e., 2x10 5 / pore PBMC (effector cell PBMC: target cell = 5:1)

[0612] c. Dilute the CD3×LILRB4 antibody and the positive control Tab1 to different concentrations using complete culture medium (starting concentration 13.3 nM, five-fold dilution, 15 spots (including 0 spot)), and add 100 μL to a 96-well plate.

[0613] d. Place the above 96-well plate in a 37°C, 5% CO2 incubator and incubate for 24-48 hours;

[0614] e. Collect samples and perform flow cytometry staining (using flow cytometry antibodies against anti-human CD4 (BD,555346) / anti-human CD8 (BD,555635) / anti-human CD69 (biolegand,310926) / anti-human granzyme B (BD,563388) / live / dead (Thermofish L34976)) to detect the activation of T cells and the level of granzyme secretion.

[0615] The specific experimental results are shown in Figures 31A-D, demonstrating that the "1+1" structure bispecific antibody F3-16H4 in this invention can activate T cells and secrete granzymes in a concentration-dependent manner.

[0616] Example 31: Cytokine secretion of the CD3×LILRB4 bispecific antibody "1+1" structure in the absence of tumor cells

[0617] This embodiment utilizes HTRF detection to identify cytokine secretion by the CD3×LILRB4 bispecific antibody F3-16H4 in the absence of tumor cells. The specific experimental procedures are as follows:

[0618] a. Dilute human peripheral blood mononuclear cells (PBMCs) to 2 x 10⁻⁶ with complete culture medium. 6 / ml, add 100uL of cells to a 96 plate, i.e., 2x10 5 / hole PBMC;

[0619] b. Dilute CD3×LILRB4, positive control (Tab1 and Glofitamab), and negative control IgG1-LALA to different concentrations using complete culture medium (starting concentration 200 nM, three-fold dilution, 15 spots (including 0 spot)), and add 100 μL to a 96-well plate.

[0620] c. Place the above 96-well plate in a 37°C, 5% CO2 incubator and incubate for 24-48 hours.

[0621] d. Collect the supernatant and use HTRF to detect the secretion of cytokines IL-2 (Revvity, 62HIL02PET) / IFN-r (Revvity, 62HIFNGPET) / IL-6 (Revvity, 62HIL06PET) / IL-10 (Revvity, 62HIL10PET) / TNAF-a (Revvity, 62HTNFAPET).

[0622] The specific experimental results are shown in Figure 32, indicating that the "1+1" structured bispecific antibody CD3×LILRB4 in this invention exhibits lower cytokine secretion and demonstrates higher safety. IL-6 cytokine was not detected.

[0623] Example 32: Cytokine secretion of the CD3×LILRB4 bispecific antibody "1+1" structure in the presence of tumor cells.

[0624] This embodiment utilizes HTRF detection to identify cytokine secretion by the CD3×LILRB4 bispecific antibody F3-16H4 in the presence of tumor cells. The specific experimental procedures are as follows:

[0625] a. Dilute THP-1 cells to 8 x 10⁸ mcg using complete culture medium. 5 / ml, take 50uL of cells and add them to a 96 plate, i.e., 4x10 4 / THP-1 tumor cells

[0626] b. Dilute human peripheral blood mononuclear cells (PBMCs) to 4 x 10⁶ / ml with complete culture medium, and add 50 μL of cells to a 96 plate, i.e., 2 x 10⁶ cells / ml. 5 / pore PBMC (effector cell PBMC: target cell = 5:1)

[0627] c. Dilute the CD3×LILRB4 antibody and the positive control Tab1 to different concentrations using complete culture medium (starting concentration 13.3 nM, five-fold dilution, 15 spots (including 0 spot)), and add 10 μL to a 96-well plate.

[0628] d. Place the above 96-well plate in a 37°C, 5% CO2 incubator and incubate for 24-48 hours;

[0629] e. Collect the supernatant and use HTRF to detect the secretion of cytokines IL-2 (kit: Revvity, 62HIL02PET) / IFN-r (kit: Revvity, 62HIFNGPET) / IL-6 (kit: Revvity, 62HIL06PET) / IL-10 (kit: Revvity, 62HIL10PET) / TNAF-a (kit: Revvity, 62HTNFAPET).

[0630] The specific experimental results are shown in Figures 33A-E, demonstrating that the "1+1" structured bispecific antibody F3-16H4 in this invention can promote the secretion of cytokines.

[0631] Example 33: Identification of the binding ability of the CD3×LILRB4 bispecific antibody "1+1" structure to the human multiple myeloma cell line MM1.S

[0632] This embodiment uses flow cytometry to detect the binding characteristics of the CD3×LILRB4 bispecific antibody F3-16H4, and the signal strength after the addition of the bispecific antibody is used to determine the binding characteristics between the bispecific antibody and MM1.S. The specific experimental procedures are as follows:

[0633] MM1.S tumor cells were diluted to 2 x 10⁻⁶ using FACS buffer. 6 / mL, add 100μL to a 96-well plate, then add 100μL each of the following concentration gradients (starting at 22nM, tripled, 10 spots including 0) of CD3×LILRB4 bispecific antibody, positive control Tab1, and negative control IgG1 LALA to the 96-well plate and incubate at 4°C for 0.5 hours. Wash three times with PBS (FACS buffer) containing 2% FBS, centrifuge and discard the supernatant, resuspend in 100μL (1:1000 with FACS buffer) of goat anti-human IgG (Jackson Immune Research, 109-135-098), and incubate at 4°C for 30 minutes. Wash three times with FACS buffer, resuspend in FACS buffer, and use for flow cytometry analysis.

[0634] The experimental results are shown in Figure 34, demonstrating that the "1+1" structure bispecific antibody F3-16H4 in this invention exhibits binding ability to the multiple myeloma tumor cell line MM1.S.

[0635] Example 34: Identification of the killing ability of the CD3×LILRB4 bispecific antibody "1+1" structure against the human multiple myeloma cell line MM1.S

[0636] This embodiment utilizes a T-cell-dependent cytotoxicity assay (TDCC) to identify the cytotoxic ability of the CD3×LILRB4 bispecific antibody F3-16H4 against tumor cells, and determines the cytotoxic level of the bispecific antibody against tumor cell lines based on the tumor cell mortality rate after bispecific antibody administration. The specific experimental procedures are as follows:

[0637] a. According to CellTrace TM Invitrogen Erythrocyte Proliferation Kit TM According to the instructions (C34564), the tumor cell line MM1S was labeled and stained.

[0638] b. After staining, dilute MM1S cells to 8 x 10⁸ mcg using complete culture medium. 5 / ml, add 50ul of cells to a 96 plate, i.e., 4x10 4 / pore THP-1 tumor cells.

[0639] c. Dilute human peripheral blood mononuclear cells (PBMCs, Ruibai Biotechnology) with complete culture medium to 4 x 10⁶ / ml, and add 50 μL of cells to a 96 plate, i.e., 2 x 10⁶ cells / ml. 5 / well PBMC (effect cell PBMC: target cell = 5:1).

[0640] d. Dilute the CD3×LILRB4 antibody and the positive control Tab1 to different concentrations using complete culture medium (starting concentration 3.3 nM, five-fold dilution, 12 spots (including 0 spot)), and add 100 μL to a 96-well plate.

[0641] e. Place the above 96-well plate in a 37°C, 5% CO2 incubator and incubate for 24 hours.

[0642] f. Collect samples using LIVE / DEAD. TM Invitrogen staining kit for fixing purple dead cells TM Cells were stained with L34955 (flow cytometry showed BV421 positive) to detect the number of dead cells (flow cytometry showed APC positive) and positive tumor cell populations (flow cytometry showed APC positive). The tumor cell killing efficiency was then calculated using the formula below. Cells without antibody were used as a blank control.

[0643] Kill efficiency (%) = Dead cells (BV421 positive by flow cytometry) / Positive tumor cell population (APC positive by flow cytometry) x 100%

[0644] The specific experimental results are shown in Figure 35, which demonstrate that the "1+1" structure bispecific antibody F3-16H4 in this invention exhibits killing function against the multiple myeloma tumor cell line MM1.S.

[0645] Example 35: Identification of the in vivo tumor growth inhibition of THP-1, a human acute myeloid leukemia cell line with high LILRB4 expression, by the CD3×LILRB4 bispecific antibody "1+1" structure.

[0646] This embodiment utilizes PBMC-reconstructed B-NDG mice to establish an orthotopic model of THP-1-Luc human myeloid leukemia to detect the in vivo efficacy of the CD3×LILRB4 bispecific antibody. The intracellular biological efficacy of the CD3×LILRB4 bispecific antibody is determined based on its inhibitory effect on mouse tumor growth. Specific experimental procedures are as follows:

[0647] PBMCs (Biocytok, batch number: P121080703C, 5×10⁻⁶) resuspended in serum-free RPMI 1640 medium 6 (0.1 mL / animal) and THP-1-Luc cells (2 × 10⁻⁶) 6 The mixture (0.1 mL / mouse) was injected via the tail vein into 125 B-NDG mice (Biocytok).

[0648] On day 4 post-vaccination, imaging signal values ​​were measured using a small animal imaging system. Based on the imaging signal values ​​and animal weight, 77 mice were randomly selected and enrolled into 11 groups of 7 mice each: G1: PBS, G2: Tab1 (1 mg / kg), G3: F3-16H4 (0.01 mg / kg), G4: F3-16H4 (0.1 mg / kg), G5: F3-16H4 (1 mg / kg), G6: F5-341F5 (0.01 mg / kg), G7: F5-341F5 (0.1 mg / kg), G8: F5-341F5 (1 mg / kg), G9: F5-313D9 (0.01 mg / kg), G10: F5-313D9 (0.1 mg / kg), and G11: F5-313D9 (1 mg / kg).

[0649] Administering began on the day of grouping, with all administration routes being tail vein injection. Administered twice weekly for seven consecutive weeks, ending the experiment five days after the last administration. Tumor imaging signal intensity and body weight were measured twice weekly during the administration and observation period, and the measurements were recorded.

[0650] The specific experimental results are shown in Figure 36: This shows that the F3-16H4 of the CD3×LILRB4 bispecific antibody "1+1" structure in this invention has a significant inhibitory effect on mouse tumors in the THP-1-Luc human myeloid leukemia orthotopic model established in PBMC-reconstructed B-NDG mice, and exhibits a dose-dependent effect.

[0651] Example 36: Identification of the in vivo tumor growth inhibition of the CD3×LILRB4 bispecific antibody "1+1" structure in the human acute myeloid leukemia LILRB4 low-expressing tumor cell line MOLM13.

[0652] This embodiment utilizes the PPBMC immune system-derived MOLM-13 acute myeloid leukemia subcutaneous xenograft model to further detect the in vivo efficacy of the CD3×LILRB4 bispecific antibody, and determines the intracellular biological efficacy of the CD3×LILRB4 bispecific antibody based on the inhibitory strength of the bispecific antibody on mouse tumor growth.

[0653] The specific experimental procedure was as follows: MOLM13 human acute myeloid leukemia tumor cells were subcutaneously transplanted into the right anterior flank of male NCG mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.). The tumors were allowed to grow to an average size of 58 mm². 3 At that time, mice were randomly divided into eight groups of seven mice each, based on tumor volume. The groups were: PBS (0 mg / kg antibody), Tab1 (2 mg / kg), F3-16H4 (0.5 mg / kg), F3-16H4 (2 mg / kg), F5-341F5 (0.5 mg / kg), F5-341F5 (2 mg / kg), F5-313D9 (0.5 mg / kg), and F5-313D9 (2 mg / kg). All drugs were administered intravenously twice a week for a total of three times. After grouping, the tumor volume and body weight of the mice were measured three times a week.

[0654] The specific experimental results are shown in Figure 37: This demonstrates that the "1+1" structure bispecific antibody F3-16H4 in this invention exhibits an inhibitory effect on mouse tumors in the MOLM13 human myeloid leukemia subcutaneous model established in PBMC-reconstructed B-NDG mice.

[0655] Sequence information

Claims

1. An anti-CD3 antibody or its antigen-binding fragment, comprising three CDRs of the heavy chain variable region VH, HCDR1, HCDR2, and HCDR3, and three CDRs of the light chain variable region VL, LCDR1, LCDR2, and LCDR3, wherein... 1) The three complementary determining regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO:9 or 35, and the three complementary determining regions LCDR1, LCDR2, and LCDR3 contained in VL as shown in SEQ ID NO:10 or 34; or 2) The three complementary determining regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO:14, and the three complementary determining regions LCDR1, LCDR2, and LCDR3 contained in VL as shown in SEQ ID NO:15; or 3) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:1 or 33, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:2 or 32.

2. An anti-CD3 antibody or its antigen-binding fragment, comprising HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3, wherein... (i) HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:11; HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:12; HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:13; LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:6; LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:7; and LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:

8. (ii) HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:11; HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:16; HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:17; LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:6; LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:7; and LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:8; or (iii) HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:3; HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:4; HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:5; LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:6; LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:7; and LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:

8.

3. The antibody or antigen-binding fragment of claim 1 or 2, comprising a heavy chain variable region VH, wherein the heavy chain variable region (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 9, 14, 1, 33, or 35; or (ii) Contains or consists of an amino acid sequence selected from or composed of said amino acid sequence, SEQ ID NO: 9, 14, 1, 33 or 35.

4. The antibody or antigen-binding fragment of any one of claims 1-3, comprising a light chain variable region VL, wherein the light chain variable region (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 10, 15, 2, 32, or 34; or (ii) Contains or consists of an amino acid sequence selected from or composed of said amino acid sequence, SEQ ID NO: 10, 15, 2, 32 or 34.

5. The antibody or antigen-binding fragment thereof of any one of claims 1-4, comprising a heavy chain variable region VH and a light chain variable region VL, wherein... (i) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 9 or 35, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 10 or 34, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; (ii) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:9, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:10, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; (iii) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:35, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:34, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; (iv) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:14, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:15, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; (v) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:1 or 33, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:2 or 32, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; (vi) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; or (vii) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:33, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:32, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

6. An anti-CD3 antibody or its antigen-binding fragment, comprising a heavy chain variable region VH and a light chain variable region VL, wherein... (i) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO: 9 or 35; the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO: 10 or 34; (ii). The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:9; the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:10; (iii) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:35; the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:34; (iv) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:14; the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:15; (v) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:1 or 33; the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:2 or 32; (vi) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:1; the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:2; or (vii) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:33; the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:

32.

7. An anti-LILRB4 antibody or its antigen-binding fragment, comprising three CDRs, HCDR1, HCDR2 and HCDR3, of the heavy chain variable region VH, and three CDRs, LCDR1, LCDR2 and LCDR3, of the light chain variable region VL, wherein HCDR1, HCDR2 and HCDR3 are the three complementary determinant regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:18; and LCDR1, LCDR2 and LCDR3 are the three complementary determinant regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:

19.

8. An anti-LILRB4 antibody or its antigen-binding fragment, comprising HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:20; HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:21; HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:22; LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:23; LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:24; and LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:

25.

9. The antibody of claim 7 or 8 or its antigen-binding fragment, comprising a heavy chain variable region VH, wherein the heavy chain variable region (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:18; or (ii) Contains or consists of the amino acid sequence of SEQ ID NO:

18.

10. The antibody or antigen-binding fragment thereof of any one of claims 7-9, comprising a light chain variable region VL, wherein the light chain variable region (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:19; or (ii) Contains or consists of the amino acid sequence of SEQ ID NO:

19.

11. An antibody or antigen-binding fragment thereof according to any one of claims 7-10, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH contains the amino acid sequence shown in SEQ ID NO:18 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO:19 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

12. An anti-LILRB4 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH contains or is composed of the amino acid sequence shown in SEQ ID NO:18, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:

19.

13. The antibody or antigen-binding fragment of any one of claims 1-12, further comprising a heavy chain constant region HC, for example, the antibody heavy chain constant region HC is a heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably a heavy chain constant region of IgG1, optionally, the heavy chain constant region further comprising a mutation that reduces binding to the Fcγ receptor, such as the L234A / L235A mutation; For example, the heavy chain constant region (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO:36; (ii) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO:37 and containing an L234A / L235A mutation; or (iii) Contains or consists of an amino acid sequence selected from or composed of said amino acid sequence.

14. The antibody or antigen-binding fragment of any one of claims 1-13, comprising a light chain constant region, for example, said light chain constant region being a lambda or kappa light chain constant region, for example, said light chain constant region (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:49; (ii) Contains or consists of the amino acid sequence of SEQ ID NO:

49.

15. The antibody or antigen-binding fragment thereof of any one of claims 1-14, wherein the antibody is a chimeric antibody, a humanized antibody, or a monoclonal antibody.

16. The antibody or antigen-binding fragment thereof of any one of claims 1-15, wherein the antigen-binding fragment is an antibody fragment selected from: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, dAb (domain antibody) or heavy-chain antibody.

17. The antibody or antigen-binding fragment thereof of any one of claims 1-16, wherein the antibody is a bispecific antibody or a multispecific antibody.

18. A bispecific antibody comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region specifically binds to CD3, and the second antigen-binding region specifically binds to LILRB4. in, The first antigen-binding region comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as defined in claim 1 or 2, or The first antigen binding region comprises a VH CD3 and a VL CD3 wherein said VH CD3 comprises HCDR1, HCDR2, HCDR3 as defined in claim 1 or 2 and said VL CD3 comprises LCDR1, LCDR2 and LCDR3 as defined in claim 1 or 2; or The VH CD3 and VL CD3 are VH and VL as defined in any one of claims 3-6.

19. The bispecific antibody of claim 18, wherein the first antigen binding region is a scFv of an anti-CD3 antibody as defined in any one of claims 1-6 and claims 13-16 CD3 .

20. The bispecific antibody of claim 19, wherein the scFv comprises a linker, the linker comprising, for example, an amino acid sequence (GKPGS)n, wherein n = 1, 2, 3, 4 or 5, preferably n = 3 or 4, more preferably n = 4.

21. The bispecific antibody of claim 19 or 20, wherein the scFv comprises or consists of, from N- to C-terminus: VL CD3 , a linker, and VH CD3 (VL CD3 - linker - VH CD3 ).

22. The bispecific antibody of any one of claims 18-21, wherein the second antigen-binding region comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as defined in claim 7 or 8; or The second antigen-binding region contains VH LILRB4 and VL LILRB4 ,in The VH LILRB4 Includes HCDR1, HCDR2, HCDR3 as defined in claim 7 or 8, and the VL LILRB4 Includes LCDR1, LCDR2, and LCDR3 as defined in claim 7 or 8; or The VH LILRB4 and VL LILRB4 VH and VL are as defined in any one of claims 9-12.

23. The bispecific antibody of any one of claims 18-22, wherein the second antigen-binding region is the Fab region of the anti-LILRB4 antibody as defined in any one of claims 7-16. LILRB4 .

24. The bispecific antibody of claim 23, wherein the Fab LILRB4 Includes Fab heavy chain VH LILRB4 -Heavy chain constant region CH1 and Fab light chain VL LILRB4 - Light chain constant region CL, wherein the CH1 is CH1 from IgG1, IgG2, IgG3 or IgG4, preferably CH1 from IgG1, for example CH1 (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:31; or (ii) Contains or consists of the amino acid sequence of SEQ ID NO:

31.

25. The bispecific antibody of claim 24, wherein the CL is a lambda or kappa light chain constant region, for example, the light chain constant region. (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:49; (ii) Containing or consisting of the amino acid sequence of SEQ ID NO:

49.

26. The bispecific antibody of any one of claims 18-25, wherein the bispecific antibody is an IgG-like bispecific antibody comprising an Fc dimer, wherein the two Fc regions constituting the Fc dimer are the same or different.

27. The bispecific antibody of claim 26, wherein the Fc region is a human IgG Fc, for example, human IgG1 Fc, human IgG2 Fc, human IgG3 Fc or human IgG4 Fc, wherein the Fc region comprises the amino acid sequence shown in any one of SEQ ID NO:38, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher identity with the amino acid sequence, or is composed of the amino acid sequence.

28. The bispecific antibody of claim 26 or 27, wherein one or both of the Fc regions comprise a mutation that reduces or eliminates the relationship between the Fc region and the Fcγ receptor, such as the LALA mutation. For example, the Fc region comprising the LALA mutation comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:39, such as comprising or consisting of the amino acid sequence shown in SEQ ID NO:

39.

29. The bispecific antibody according to any one of claims 26-28, wherein the two Fc regions are different, preferably, corresponding knob mutations and Hole mutations are introduced into the first monomeric Fc region and the second monomeric Fc region, respectively. For example, the Fc region containing the Knob mutation contains amino acid substitutions S354C and T366W, and the Fc region containing the Hole mutation contains amino acid substitutions Y349C, T366S, L368A, and Y407V (numbered according to the EU index); or the Fc region containing the Knob mutation contains amino acid substitutions S354C and T366W, and the Fc region containing the Hole mutation contains amino acid substitutions Y349C, T366S, L368A, and Y407V, as well as H435R and Y436F mutations (numbered according to the EU index).

30. The bispecific antibody according to any one of claims 18-29, comprising one or two anti-LILRB4 antibodies in a Fab group. LILRB4 A scFv with an anti-CD3 antibody CD3 and Fc heterodimer, wherein one of the Fab LILRB4 The C-terminus of the heavy chain is connected to the N-terminus of the first Fc region (preferably directly connected), and scFv CD3 The C end is connected to the N end of the second Fc region (preferably via a partial hinge region such as EPKSS or EPKSC, more preferably EPKSS); Optionally, the N-terminus of the scFv is also connected to another Fab. LILRB4 The C-end of the heavy chain is connected (preferably via a connector such as (GGGGS)n where n = an integer from 1 to 10, for example 1, 2, 3, 4 or 5).

31. The bispecific antibody of claim 30, comprising or consisting of the following chains: First chain, from the N-terminus to the C-terminus: VH LILRB4 -CH1- First Fc region; First light chain, from the N-terminus to the C-terminus: VL LILRB4 -CL; and The second chain, from the N-terminus to the C-terminus: VL CD3 -Connector 1-VH CD3 -Partial hinge area-Second Fc area; Where "-" represents a direct connection; VH LILRB4 and VL LILRB4 As defined in claim 22, and VH LILRB4 -CH1 and VL LILRB4 -CL constitutes the Fab as defined in any one of claims 22-25 LILRB4 ; VH CD3 and VL CD3 As defined in claim 18, and VL CD3 -Connector 1-VH CD3 scFv constituting an anti-CD3 antibody as defined in any one of claims 19-21 CD3 ;and The Fc region is defined as described in any one of claims 26-29.

32. The bispecific antibody of claim 31, wherein a portion of the hinge region is as shown in SEQ ID NO:42 or 43, preferably as shown in SEQ ID NO:

43.

33. The bispecific antibody of claim 31 or 32, comprising or consisting of the following chains: The first heavy chain comprises the amino acid sequence shown in SEQ ID NO:26, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with said amino acid sequence, or is composed of said sequence; The second chain comprises the amino acid sequence shown in SEQ ID NO:27, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with said amino acid sequence, or is composed of said sequence; and The first light chain comprises the amino acid sequence shown in SEQ ID NO:28, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence, or is composed of the sequence.

34. The bispecific antibody according to any one of claims 31-33, comprising or consisting of the following chains: The first chain comprises or consists of the amino acid sequence shown in SEQ ID NO:26; The second chain comprises or consists of the amino acid sequence shown in SEQ ID NO:27; and The first light chain comprises or consists of the amino acid sequence shown in SEQ ID NO:

28.

35. The bispecific antibody of claim 30, comprising or consisting of the following chains: First chain, from the N-terminus to the C-terminus: VH LILRB4 -CH1- First Fc region; First light chain, from the N-terminus to the C-terminus: VL LILRB4 -CL; and The second chain, from the N-terminus to the C-terminus: VH LILRB4 -CH1-Connector 2-VL CD3 -Connector 1-VH CD3 -Partial hinge area-Second Fc; The second light chain, from the N-terminus to the C-terminus: VL LILRB4 -CL; Where "-" represents a direct connection; VH LILRB4 and VL LILRB4 As defined in claim 22, and VH LILRB4 -CH1 and VL LILRB4 -CL constitutes the Fab as defined in any one of claims 22-25 LILRB4 Preferably, VH in the first heavy chain and the second heavy chain LILRB4 The same, and / or VL in the first and second light chains LILRB4 The same; more preferably, VH in the first and second heavy chains. LILRB4 -CH1 is the same and / or the first light chain and the second light chain are the same; VH CD3 and VL CD3 As defined in claim 18, and VL CD3 -Connector 1-VH CD3 scFv constituting an anti-CD3 antibody as defined in any one of claims 19-21 CD3 ;and The Fc region is defined as described in any one of claims 26-29.

36. The bispecific antibody of claim 35, wherein the linker 2 is as shown in SEQ ID NO:46 and / or a portion of the hinge region is as shown in SEQ ID NO:42 or 43, preferably as shown in SEQ ID NO:

43.

37. The bispecific antibody of claim 35 or 36, comprising or consisting of the following chains: The first heavy chain comprises the amino acid sequence shown in SEQ ID NO:26, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with said amino acid sequence, or is composed of said sequence; The second chain comprises the amino acid sequence shown in SEQ ID NO:29 or 30, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with said amino acid sequence, or is composed of said sequence; and The first light chain comprises the amino acid sequence shown in SEQ ID NO:28, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence, or is composed of the sequence.

38. The bispecific antibody according to any one of claims 35-37, comprising or consisting of the following chains: The first chain comprises or consists of the amino acid sequence shown in SEQ ID NO:26; The second chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 29 or 30; and The first light chain comprises or consists of the amino acid sequence shown in SEQ ID NO:

28.

39. A nucleic acid molecule encoding any chain of the antibody or antigen-binding fragment thereof as claimed in any one of claims 1-17, or any chain of the bispecific antibody as claimed in any one of claims 18-38, or composed of the nucleic acid sequence.

40. An expression vector comprising the nucleic acid molecule of claim 39, preferably, the expression vector being pTT5.

41. A host cell comprising the nucleic acid molecule of claim 39 or the expression vector of claim 40, preferably, the host cell being prokaryotic or eukaryotic, such as 293 cells or CHO cells.

42. A method for preparing the antibody or antigen-binding fragment thereof of any one of claims 1-17 or the bispecific antibody of any one of claims 18-38, the method comprising culturing a host cell containing the nucleic acid molecule of claim 39 or the expression vector of claim 40 or the host cell of claim 41 under conditions suitable for chain expression of the antibody, and optionally recovering the antibody or antigen-binding fragment thereof or bispecific antibody from the host cell (or host cell culture medium).

43. An immunoconjugate comprising the antibody of any one of claims 1-17 or an antigen-binding fragment thereof, or the bispecific antibody of any one of claims 18-38.

44. A pharmaceutical composition comprising the antibody of any one of claims 1-17 or an antigen-binding fragment thereof, or the bispecific antibody of any one of claims 18-38, or the immunoconjugate of claim 43, and optionally a pharmaceutical excipient.

45. A pharmaceutical combination comprising the antibody of any one of claims 1-17 or an antigen-binding fragment thereof, or the bispecific antibody of any one of claims 18-38, or the immunoconjugate of claim 43, and one or more other therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators).

46. ​​A method for preventing or treating tumors in a subject, comprising administering to the subject an effective amount of the antibody of any one of claims 1-17 or an antigen-binding fragment thereof, or a bispecific antibody of any one of claims 18-38, or an immunoconjugate of claim 43, or a pharmaceutical composition of claim 44; or a pharmaceutical composition of claim 45.

47. The method of claim 46, wherein the tumor is a LILRB4-positive cancer, for example, the tumor is characterized by elevated LILRB4 protein and / or nucleic acid levels in the tumor cells of the tumor (e.g., compared to LILRB4 protein and / or nucleic acid levels in cells of the same tissue of a healthy individual, or compared to LILRB4 protein and / or nucleic acid levels in healthy cells of the same tissue of the patient or in cells of adjacent healthy tissue).

48. The method of claim 46 or 47, wherein the cancer is a solid tumor or a hematologic malignancy, such as a hematologic malignancy, such as lymphoma, leukemia, or a myelodysplastic syndrome, such as multiple myeloma, such as acute myeloid leukemia (AML), such as human acute monocytic leukemia or human myeloid monocytic leukemia, such as chronic myeloid leukemia, such as chronic myelomonocytic leukemia (CMML).

49. The method of any one of claims 46-48, wherein the method further comprises administration in combination with other therapies such as treatment modalities (e.g., surgery or radiotherapy) and / or other therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators).

50. A method for detecting the presence of LILRB4 in biological samples, comprising: (i) Contacting a biological sample with the antibody or antigen-binding fragment thereof of any one of claims 7-17 or the bispecific antibody of any one of claims 18-38 under conditions that allow it to bind to LILRB4. (ii) Detect whether a complex is formed between the antibody or its antigen-binding fragment or bispecific antibody and LILRB4. The formation of the complex indicates the presence of LILRB4.

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