Antibodies binding lilrb4 and CD3 and uses thereof

Abstract

An antibody capable of binding to LILRB4 and a bispecific antibody capable of binding to LILRB4 and CD3 are provided. Nucleic acid molecules encoding the antibodies, expression vectors, host cells and methods for expressing the antibodies are also provided. Also provided are methods of treating solid tumors, multiple myeloma or leukemia, such as acute myelogenous leukemia (AML), using the anti-LILRB4xCD3 double antibodies of the present invention.

Description

Antibodies that bind LILRB4 and CD3 and their uses Technical Field

[0001] The present invention provides an antibody capable of binding to LILRB4 and a bispecific antibody capable of binding to LILRB4 and CD3. Also provided are nucleic acid molecules encoding the antibodies, expression vectors, host cells, and methods for expressing the antibodies. The present invention also relates to methods of using the anti-LILRB4×CD3 bispecific antibodies of the present invention to treat solid tumors, multiple myeloma, or leukemias, such as acute myeloid leukemia (AML). Background Art

[0002] Leukocyte Ig-like receptor B4 (LILRB4), also known as immunoglobulin-like transcript 3 (ILT3 or ILT-3), leukocyte immunoglobulin-like receptor 5 (LIR5 or LIR-5) and CD85k or CD85K, is a type I transmembrane protein that contains two immunoglobulin domains extracellularly and a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) intracellularly, and is involved in the downregulation of immune responses.

[0003] LILRB4 supports tumor cell infiltration into tissues and inhibits T cell activity through a signaling pathway involving APOE, LILRB4, SHP-2, μPAR, and ARG1 in tumor cells (LILRB4 signaling in leukaemia cells mediates T cell suppression and tumor infiltration, Nature (2018) 562: 605-09).

[0004] LILRB4 antibodies can activate dendritic cells, relieve the immunosuppressive effects of MSDCs, and have the effect of treating cancer (WO2019099597A2).

[0005] Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy that is the most common acute leukemia in adults and a common pediatric cancer. It is characterized by the clonal expansion of myeloid cells in peripheral blood, bone marrow (BM), and / or other tissues (NCCN INSIGHTS,Acute Myeloid Leukemia,Version 2.2021). Despite significant advances in understanding AML biology, the five-year survival rate for AML patients is only 20-40% (Expression of Immune Inhibitory Receptor ILT3 in Acute Myeloid Leukemia with Monocytic Differentiation, Cytometry Part B (Clinical Cytometry) 84B:21–29 (2013)). Currently, treatment for acute myeloid leukemia includes intensive cytotoxic chemotherapy, often followed by bone marrow conditioning and stem cell transplantation. Despite treatment, relapse and refractory disease remain a significant problem for adults and children with acute myeloid leukemia, with survival rates for relapsed patients below 15%. Monocytic AML (FAB M5) is a common subtype of AML, accounting for 20% of pediatric AML cases and more than half of infant AML cases. Clinical studies have also shown that monocytic AML has a greater risk of myeloid and extramedullary relapse after stem cell transplantation compared with non-monocytic AML. Therefore, new therapeutic strategies are needed to improve the poor prognosis of patients with relapsed or refractory monocytic AML (A Novel Anti-LILRB4 CAR-T Cell for the Treatment of Monocytic AML. Molecular Therapy 2018, 26(10), 2487–2495.). LILRB4 is expressed on monocytes, macrophages, and dendritic cells, but not on normal CD34+ hematopoietic stem cells. Furthermore, LILRB4 is a tumor-associated antigen that is significantly upregulated in acute monocytic leukemia (FAB M4, M5) and is expressed in leukemic stem cell populations. Data indicate that LILRB4 expression is negatively correlated with AML patient survival.

[0006] Currently under development for the treatment of AML, CD3 bispecific antibodies target antigens such as CD33, CD123, and FLT3. Bispecific antibodies against these antigens can lead to non-targeted elimination of hematopoietic stem cells (HSCs), resulting in bone marrow suppression or bone marrow ablation, and increased susceptibility to infection (A Novel Anti-LILRB4 CAR-T Cell for the Treatment of Monocytic AML. Molecular Therapy 2018, 26(10), 2487–2495.). SGN-33A, which targets CD33, was terminated early in a clinical study in combination with azacitabine / decitabine (NCT02785900) due to side effects, especially a high mortality rate caused by infection (Acute Myeloid Leukemia: Therapeutic Targeting of Stem Cells. Expert Opinion on Therapeutic Targets 2022, 26(6), 547–556.). Therefore, it is particularly important to identify and target AML cells rather than normal hematopoietic stem cells. LILRB4 is expressed on LSCs but not on hematopoietic stem cells and multipotent progenitor cells (MPPs). Therefore, antibody therapy targeting LILRB4 has low bone marrow toxicity and is an ideal antigen target (A Novel Anti-LILRB4CAR-T Cell for the Treatment of Monocytic AML. Molecular Therapy 2018, 26(10), 2487–2495.).

[0007] LILRB4 monoclonal antibody can exert anti-tumor activity by activating immune effector cells (such as macrophages and NK cells). LILRB4×CD3 bispecific antibody can mediate T cell killing of tumor cells expressing LILRB4. CAR-T cells that target and bind to LILRB4 can provide effective anti-tumor capabilities through T cell-mediated cytotoxicity. LILRB4 antibodies bound to toxins can destroy tumor cells. After binding to LILRB4 and being internalized by target tumor cells, therapeutic toxic drugs are internalized and can then exert cytotoxic effects to achieve the purpose of treating tumors (LILRB4, an immune checkpoint on myeloid cells. Blood Science, (2022) 4, 49-56).

[0008] The principle of bispecific antibodies is to simultaneously target two antigens, combining the binding specificities and biological functions of two antibodies into a single molecule: one for tumor-associated surface antigens and the other for surface antigens on effector cells (such as T cells and NK cells). Through this dual specificity, effector cells are used to specifically kill tumor cells (Acute myeloid leukemia targets for bispecific antibodies, Citation: Blood Cancer Journal (2017) 7, e522).

[0009] There is a need to develop antibodies that specifically bind to LILRB4, in particular, to develop bispecific antibodies that specifically bind to CD3 and LILRB4, thereby utilizing the cytotoxic effect generated by T cell activation to kill tumor cells.

[0010] Summary of the Invention

[0011] The present invention provides a novel anti-LILRB4 antibody or an antigen-binding fragment thereof.

[0012] In some embodiments, the anti-LILRB4 antibody of the present invention is a full-length antibody or an antigen-binding fragment thereof, or is a VHH antibody or an antibody comprising a VHH, such as a VHH-Fc.

[0013] The present invention also provides a multispecific binding molecule constructed using the anti-LILRB4 antibody of the present invention, such as a multispecific antibody, such as a bispecific antibody. In some embodiments, the bispecific antibody specifically binds to LILRB4 and CD3.

[0014] Therefore, the present invention provides a LILRB4×CD3 bispecific antibody, which can activate T cells by specifically binding to AML cells that highly express LILRB4, thereby generating a T cell cytotoxic effect to kill tumor cells.

[0015] In some embodiments, the anti-LILRB4 antibodies of the invention specifically bind to LILRB4 (e.g., human LILRB4 and / or monkey (e.g., cynomolgus monkey) LILRB4) with high affinity. In some embodiments, the anti-LILRB4 antibodies specifically bind to cells expressing LILRB4, such as tumor cells.

[0016] In some embodiments, the anti-LILRB4 antibodies or antigen-binding fragments thereof of the present invention can activate dendritic cells, for example, induce them to release cytokines, such as tumor necrosis factor, such as TNF-α.

[0017] In some embodiments, the LILRB4 antibody or antigen-binding fragment thereof of the present invention can promote the differentiation of myeloid cells in PBMCs toward a pro-inflammatory phenotype, such as reducing the release of IL-10 in the PBMC system.

[0018] The anti-CD3 / LILRB4 bispecific antibodies of the present invention have one or more of the following characteristics:

[0019] (1) binding to human and / or monkey (e.g., cynomolgus monkey) LILRB4, for example, binding to LILRB4 expressed on cells, for example, binding to LILRB4 with high affinity;

[0020] (2) binds to human and / or monkey (e.g., cynomolgus monkey) CD3 with moderate affinity;

[0021] (3) The binding affinity for human LILRB4 is stronger than that for human CD3 (e.g., K D The value is two orders of magnitude higher), thus having lower side effects caused by targeting CD3;

[0022] (4) Induce T cell proliferation;

[0023] (5) More specific induction of T cells to kill LILRB4-expressing cells, for example, the ability to specifically induce T cells to kill LILRB4-expressing cells at a lower efficiency-target ratio;

[0024] (6) having good cytokine induction specificity; for example, inducing the release of inflammatory cytokines only in cells expressing LILRB4 (for example, still inducing the release of inflammatory cytokines at a lower efficiency target ratio), for example, interleukins, such as IL-2; for example, interferons, such as IFNγ; and / or tumor necrosis factors, such as TNFα; or

[0025] (7) Effective treatment of LILRB4-related indications, such as solid tumors, multiple myeloma, or leukemia, such as acute myeloid leukemia (AML). BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The preferred embodiments of the present invention described in detail below will be better understood when read in conjunction with the following drawings. For the purpose of illustrating the present invention, the drawings show presently preferred embodiments. However, it should be understood that the present invention is not limited to the precise arrangements and means of the embodiments shown in the drawings.

[0027] Figures 1A-C show schematic diagrams of the structures of three anti-LILRB4×CD3 bispecific antibodies. Figure 1A shows a schematic diagram of the structure of huB1G5-CD3Fab, Figure 1B shows a schematic diagram of the structure of huB1G5-CD3-2VHH, and Figure 1C shows a schematic diagram of the structure of huB1G5-ScFv-2VHH.

[0028] Figures 2A-B show the binding of anti-LILRB4 chimeric antibodies to engineered cells. Figure 2A shows the binding of anti-LILRB4 chimeric antibodies (labeled as Antibody B1A2 and Antibody B1G5) and antibody h193 to engineered cells expressing human / cynomolgus monkey LILRB4 (CHO-K1-huLILRB4 cells and CHO-K1-cynoLILRB4). Figure 2B shows the binding of anti-LILRB4 chimeric antibodies (labeled as Antibody 2A3 and Antibody 1H12) and antibody h193 to cells expressing human / cynomolgus monkey LILRB4 protein (CHO-K1-huLILRB4 cells and CHO-K1-cynoLILRB4).

[0029] FIG3 shows the binding of humanized anti-LILRB4 antibodies (labeled as antibody huB1G5-1, antibody huB1G5-3) and antibody h193 to tumor cells (MV-411) expressing human LILRB4.

[0030] FIG4 shows the results of humanized anti-CD3 antibodies 017-Roche-CD3, DA023AH23L2, and 017-2 activating the Jurkat / NFAT-luc reporter gene system.

[0031] Figures 5A-D show that the anti-LILRB4×CD3 bispecific antibodies (labeled as huB1G5-CD3Fab and huB1G5-CD3-2VHH) can specifically induce T cells to kill HL-60 tumor cells at a lower efficiency target ratio (E:T=1:1) (Figure 5A), releasing inflammatory cytokines TNFα (Figure 5B) and IFNγ (Figure 5C), and the anti-LILRB4×CD3 bispecific antibodies (labeled as huB1G5-CD3-2VHH and huB1G5-ScFv-2VHH) and the bispecific antibody 4ab3-4ab S91A can specifically induce T cells to kill HL-60 tumor cells at a lower efficiency target ratio (E:T=1:1) (Figure 5D).

[0032] Figures 6A-D show the efficacy of LILRB4×CD3 bispecific antibodies in solid tumor animal models. Figure 6A shows the changes in tumor volume after administration of anti-LILRB4×CD3 bispecific antibodies (labeled as huB1G5-CD3Fab and huB1G5-CD3-2VHH) to tumor-bearing mice. Figure 6B shows the changes in mouse body weight after administration of anti-LILRB4×CD3 bispecific antibodies (labeled as huB1G5-CD3Fab and huB1G5-CD3-2VHH) to tumor-bearing mice. Figure 6C shows the changes in tumor volume after administration of anti-LILRB4×CD3 bispecific antibodies (labeled as huB1G5-CD3-2VHH and huB1G5-ScFv-2VHH) to tumor-bearing mice. FIG6D shows the changes in body weight of tumor-bearing mice after administration of anti-LILRB4×CD3 bispecific antibodies (labeled as huB1G5-CD3-2VHH and huB1G5-ScFv-2VHH).

[0033] Figures 7A-C show the changes in mouse survival (7A), changes in tumor fluorescence intensity in mice (7B), and changes in in vivo imaging of mice (7C) after administration of anti-LILRB4×CD3 bispecific antibodies (labeled as huB1G5-CD3-2VHH and huB1G5-ScFv-2VHH).

[0034] Figures 8A-C show the results of mixed lymphocyte reactions using anti-LILRB4×CD3 bispecific antibodies. These results demonstrate that anti-LILRB4×CD3 bispecific antibodies (labeled as huB1G5-CD3-2VHH and huB1G5-ScFv-2VHH) can promote the release of IL-2 (Figure 8A) and IFN-γ (Figure 8B) in a DC-T mixed lymphocyte reaction system, with the amount of release being dose-dependent with antibody concentration. Figure 8C demonstrates that anti-LILRB4×CD3 bispecific antibodies (labeled as huB1G5-CD3-2VHH and huB1G5-ScFv-2VHH) can promote T cell proliferation in a DC-T mixed lymphocyte reaction system, with the amount of cell proliferation being dose-dependent with antibody concentration.

[0035] Figures 9A-C show that humanized anti-LILRB4 antibody (labeled as huB1G5-3) and antibody h193 can significantly reduce the secretion of IL-10 in the system at three tested concentrations compared with two negative controls.

[0036] Detailed Description of the Invention

[0037] It should be understood that the present invention is not limited to the specific methodology, protocols and reagents described herein, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.

[0038] I. Definition

[0039] For the purpose of interpreting this specification, the following definitions will be used, and whenever appropriate, terms used in the singular may also include the plural, and vice versa. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0040] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0041] As used herein, the term "and / or" means any one of the alternatives or two or more or all of the alternatives.

[0042] As used herein, the terms "comprising" or "including" are intended to include the recited 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 indicated, the context of consisting of the recited elements, integers, or steps is also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0043] When "first" and "second" are mentioned herein, it is only to distinguish the two domains or two chains, but does not indicate the positions of the two domains in any way.

[0044] As used herein, the term "CD3" refers to an antigen expressed on T cells as part of the multimolecular T cell receptor (TCR), namely the T cell engaging antigen, the T cell surface glycoprotein CD3, which is composed of a homodimer or heterodimer formed by two of the following four receptor chains: CD3-ε, CD3-δ, CD3-ζ, and CD3-γ. Human CD3-εn (hCD3ε) comprises the amino acid sequence described in UniProtKB / Swiss-Prot: P07766. Human CD3-δ (hCD3δ) comprises the amino acid sequence described in UniProtKB / Swiss-Prot: P04234. In some embodiments, the CD3 described herein refers to CD3 from humans or monkeys (e.g., cynomolgus monkeys).

[0045] As used herein, the term "antibody that binds to CD3" or "anti-CD3 antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize or bind to a single CD3 subunit (e.g., epsilon, delta, gamma, or zeta), as well as antibodies and antigen-binding fragments thereof that specifically recognize or bind to a dimeric complex of two CD3 subunits (e.g., gamma / epsilon, delta / epsilon, and zeta / zeta CD3 dimers) and antibodies and antigen-binding fragments thereof that bind to the same. 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 includes natural CD3 protein and recombinant CD3 protein variants, for example, monomeric and dimeric CD3 structures that lack a transmembrane region or are otherwise not bound to the cell membrane. In one embodiment, the antigen-binding region that binds to CD3 in the anti-CD3 antibody, antigen-binding fragment, or bispecific antibody of the present invention can have low binding activity to CD3 or cells expressing CD3 (e.g., T cells). The binding affinity of the antibody to CD3 can be detected by flow cytometry or biofilm optical interferometry. Preferably, in the bispecific antibody molecule of the present invention, the antigen-binding region that binds to CD3 has a binding affinity for human or monkey (cynomolgus monkey) CD3 of between 1-1000 nM, for example, between 1-100 nM. In some embodiments, the antigen-binding region that binds to CD3 in the anti-CD3 antibody, antigen-binding fragment thereof, or bispecific antibody of the present invention binds to human and / or monkey (e.g., cynomolgus monkey) CD3 with a lower binding affinity, thereby being able to activate human and / or monkey (e.g., cynomolgus monkey) T cells.

[0046] Effector cells include effector T cells (T lymphocytes), such as CD4+ T cells, CD8+ T cells, Th1, Th2 and regulatory T cells (Tregs). Effector cells can also include natural killer cells, macrophages, granulocytes, plasma cells or B cells (lymphocytes).

[0047] Leukocyte immunoglobulin-like receptor subfamily B, member 4 (LILRB4) is a protein encoded by the LILRB4 gene in humans. This gene is a member of the leukocyte immunoglobulin-like receptor (LIR) family and is found in the gene cluster at chromosomal region 19q13.4. The encoded protein belongs to the subfamily B class of LIR receptors, which contain two or four extracellular immunoglobulin domains, a transmembrane domain, and two to four cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). In one embodiment, the human LILRB4 protein of the invention comprises or consists of the amino acid sequence set forth in SEQ ID NO:65, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, the cynomolgus monkey LILRB4 protein of the present invention comprises the amino acid sequence shown in SEQ ID NO:66, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consists of the sequence.

[0048] In one embodiment, the antigen-binding region that binds to LILRB4 in an anti-LILRB4 antibody, or antigen-binding fragment (e.g., Fab), or bispecific antibody of the present invention has high-affinity binding activity for cells expressing human LILRB4. In some embodiments, the binding affinity of the bispecific antibody of the present invention for human LILRB4 is higher than its binding affinity for human CD3, for example, 1-2 orders of magnitude higher. In one embodiment, the binding affinity is determined by flow cytometry. In one embodiment, the antigen-binding region that binds to LILRB4 in an anti-LILRB4 antibody, or antigen-binding fragment (e.g., Fab), or bispecific antibody of the present invention has cross-reactivity to human and monkey (e.g., cynomolgus monkey) LILRB4, i.e., it can bind to human and monkey (e.g., cynomolgus monkey) LILRB4.

[0049] The terms "whole antibody," "full-length antibody," "complete antibody," and "intact antibody" are used interchangeably herein to refer to a naturally occurring glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further divided into hypervariable regions (complementarity determining regions (CDRs) interposed with relatively conserved regions (framework regions (FRs)). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions. In some embodiments, the heavy chain constant region HC of the antibody 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 comprises a LALA mutation. In some embodiments, the heavy chain constant region comprises D265A and P329A mutations. In some embodiments, the heavy chain constant region comprises a LALA mutation and D265A and P329A mutations. In some preferred embodiments, the heavy chain constant region HC of the antibody of the present invention

[0050] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 52 or 57;

[0051] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 52 or 57; or

[0052] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 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 an amino acid sequence selected from SEQ ID NO: 52 or 57.

[0053] In some embodiments, the antibody light chain constant region LC of the present invention is a Lambda or Kappa light chain constant region. In some embodiments, the antibody light chain constant region LC of the present invention is a Lambda or Kappa light chain constant region.

[0054] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 56 or 58;

[0055] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 56 or 58; or

[0056] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 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 an amino acid sequence selected from SEQ ID NO: 56 or 58.

[0057] The term "antibody fragment" includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0058] The term "antigen-binding fragment" is a portion or segment of an intact or complete antibody that has fewer amino acid residues than the intact or complete antibody and that is capable of binding to an antigen or competing with the intact antibody (i.e., the intact antibody from which the antigen-binding fragment is derived) for antigen binding. Antigen-binding fragments can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabodies, and single-domain antibodies (sdAbs). The Fab fragment is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains. For example, Fab fragments can be obtained by digesting an intact antibody with papain. In addition, digesting an intact antibody with pepsin below the disulfide bonds in the hinge region produces F(ab')2, a dimer of Fab', 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. The Fab' monomer is essentially a Fab fragment with a hinge region (for a more detailed description of other antibody fragments, see: Fundamental Immunology, edited by W.E. Paul, Raven Press, NY (1993)). The Fv fragment consists of the VL and VH domains of a single antibody arm. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by independent genes, recombinant methods can be used to connect them via a synthetic linker peptide that enables the two domains to be produced as a single protein chain, in which the VL and VH regions are paired to form a single-chain Fv (scFv). The antibody fragment can be obtained by chemical methods, recombinant DNA methods, or protease digestion.

[0059] "Fab fragment" or "Fab" are used interchangeably herein to refer to an immunoglobulin fragment composed of two polypeptide chains comprising 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), wherein one polypeptide chain comprises, from N-terminus to C-terminus, VH and a constant region selected from CH1 and CL, and the other polypeptide chain comprises, from N-terminus to C-terminus, VL and another constant region selected from CL and CH1, wherein the VH and VL domains pair to form an antigen-binding site. Herein, the Fab chain comprising the heavy chain constant region (CH1) is also referred to as the "Fab heavy chain"; accordingly, the Fab chain comprising the light chain constant region (CL) is also referred to as the "Fab light chain."

[0060] The terms "VHH" or "VHH antibody" are used interchangeably herein and generally refer to an antibody that comprises or consists of only one heavy chain variable region and has antigen binding activity. VHHs generally comprise three CDRs in four highly conserved framework regions and generally have the following structure: FR1-CDR-FR2-CDR2-FR3-CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4; CDR1 to CDR3 refer to complementarity determining regions 1-3. The CDR sequences in the VHH variable region can be determined according to any of the CDR definition schemes described in the "Definitions" section, and preferably the boundaries of the three CDRs in the variable region sequence can be defined by IMGT. VHHs generally include only heavy chain variable domains derived from heavy chain antibodies lacking light chains, also known as nanobodies. The VHHs used in the present invention are preferably from camelids, such as alpacas, or are humanized or sequence-optimized forms thereof (e.g., affinity mature forms to increase binding affinity). In some embodiments, a VHH of the invention is a monovalent, monospecific polypeptide molecule that consists of, or consists essentially of, a single heavy chain variable region (eg, the heavy chain variable region of a heavy chain antibody).

[0061] The single-domain antibodies or VHHs of the present invention may also be contained in larger polypeptides / proteins. Examples of polypeptides / proteins containing the VHHs of the present invention include, but are not limited to, heavy chain antibodies (HcAbs) or bispecific antibodies or fusion proteins. The "heavy chain antibody" of the present invention refers to an antibody without a light chain, for example, from the N segment to the C segment, it may contain VH-Fc or VH-CH2-CH3 or VH-hinge region-CH2-CH3, or it may contain VH-CH1-CH2-CH3. The heavy chain antibodies of the present invention may also encompass homodimers, such as heavy chain dimer antibodies without light chains. The heavy chain antibody may contain a VH from a standard antibody or a VH from a single-domain antibody. For example, the VH in the heavy chain antibody may be a VHH. In some embodiments, the heavy chain antibody of the present invention may be a heavy chain antibody having a framework region and / or a heavy chain constant region derived from a camelid (llama, camel, especially alpaca), a humanized form thereof, a sequence-optimized form thereof (affinity matured form), or a fragment thereof (e.g., a fragment comprising at least a portion of the constant region). The heavy chain antibodies of the present invention also encompass antibodies formed by fusing a heavy chain variable region or VHH with an Fc region (e.g., a human IgG Fc region, such as a human IgG1 or IgG4 Fc region). When "VHH" is mentioned in the context of a heavy chain antibody, a bispecific antibody, or a fusion protein, it should be understood that it is part of a bispecific antibody, not as a separate molecule.

[0062] The term "target" refers to the object to which a binding molecule is directed. A target can be an antigen, a ligand, or a receptor.

[0063] The term "antigen" refers to a molecule that elicits an immune response. This immune response may involve the production of antibodies or the activation of specific immune cells, or both. The skilled artisan will appreciate that any macromolecule, including essentially all proteins or peptides, can serve as an antigen. Additionally, antigens can be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to the portion of an antigen that specifically interacts with an antibody molecule.

[0064] As used herein, the term "target binding region" refers to a multispecific binding molecule, such as a portion of a bispecific binding molecule that binds to a specific target or antigen. The target binding region can be, for example, an antibody or immunoglobulin itself or an antibody fragment. This target binding region may or may not have a tertiary structure independent of the remainder of the bispecific antibody molecule and may bind or not bind to its target as a separate entity. The target binding region can also be a receptor or ligand, or a domain of a receptor that is capable of binding to a ligand. In the case of multispecific antibodies or bispecific antibodies, the "target binding region" is also referred to as an "antigen binding region." In one embodiment, the antigen binding region for the bispecific antibody molecule of the present invention comprises a VH / VL pair consisting of an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), the VH / VL pair being comprised of a single polypeptide chain (e.g., in an scFv) or being comprised of two separated polypeptide chains (e.g., in a Fab heavy chain and a Fab light chain, respectively). In one embodiment, the antigen binding region for the bispecific antibody molecule of the present invention can be a Fab. In one embodiment, the antigen binding region of the bispecific antibody molecule used in the invention may comprise only VH, eg, be derived from VHH, eg, be VHH.

[0065] When referring to "the target binding region is derived from an antibody", it means that the binding domain constituting the target binding region is or is derived from the binding domain of the antibody that specifically binds to the antigen, for example, the heavy chain variable region and / or light chain variable region of the target binding region is or is derived from the heavy chain variable region and / or light chain variable region of the antibody, or one, two, three, four, five or six CDRs of the target binding region are CDRs of the antibody.

[0066] The term "derived from" means that the fragment in the target binding region is substantially identical to the fragment of the antibody from which it is derived, but has a mutation, such as a substitution, deletion, or addition, at one or more sites. In a specific embodiment, the mutation is not in the CDR of the antibody. In a specific embodiment, the mutation is not in the variable region of the antibody.

[0067] 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 a monospecific antibody against LILRB4.

[0068] 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 different epitopes of different antigens. A multispecific antibody is an antibody that has binding specificity for at least two different antigenic epitopes. In one embodiment, provided herein are bispecific antibodies that have binding specificity for a first antigen and a second antigen. For example, the present invention provides bispecific antibodies against LILRB4 and CD3.

[0069] The term "multispecific binding molecule" refers to a multispecific binding molecule that is at least bispecific, e.g., a bispecific binding molecule, i.e., the molecule comprises at least a first target binding region and a second target binding region, wherein the first target binding region binds one target and the second target binding region binds another target. Thus, the multispecific binding molecules according to the present invention comprise specificities for at least two different targets. The molecules according to the present invention also encompass multispecific molecules comprising multiple target binding regions, such as trispecific binding molecules. In some embodiments, where the binding molecule is an antibody, the target is an antigen. In some embodiments, the bispecific binding molecules of the present invention are bispecific antibodies.

[0070] As used herein, the terms "antibody," "multispecific binding molecule," and "multispecific antibody" are not mutually exclusive when referred to herein.

[0071] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable regions of the heavy and light chains of natural antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions.

[0072] "Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms structurally determined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs 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 a number of well-known antibody CDR assignment schemes, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (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)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics 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.

[0073] The following are the regional ranges of CDRs defined using the Kabat, AbM, Chothia, Contact, and IMGT schemes.

[0074] Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways. CDRs can also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the exemplary CDRs of the present invention). Unless otherwise indicated, in the present invention, when referring to residue positions in an antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the numbering position of the Kabat numbering system according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0075] In some embodiments, the HCDRs in the VHH antibodies of the invention are determined according to the IMGT protocol.

[0076] In some embodiments, the HCDRs and / or LCDRs in the antibodies of the invention are determined according to the Kabat scheme.

[0077] The term "Fc domain" or "Fc region" or "Fc fragment" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" comprises two or three constant domains, namely a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in a native antibody, an immunoglobulin Fc domain comprises the second and third constant domains (CH2 domain and CH3 domain) of two heavy chains derived from IgG, IgA, and IgD classes of antibodies; or the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of two heavy chains derived from IgM and IgE classes of antibodies. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or heavy chain constant region is according to the EU numbering system (also known as the EU index) as described in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) (https: / / pubmed.ncbi.nlm.nih.gov / 5257969 / ), see also http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html. As used herein, the term "Fc domain" or "Fc region" or "Fc fragment" does not include the heavy chain variable region VH and light chain variable region VL of an immunoglobulin, as well as the heavy chain constant region CH1 and light chain constant region CL, but in some cases may include the hinge region at the N-terminus of the heavy chain constant region, such as EPKSS or EPKSC. In some embodiments, the heavy chain constant region Fc suitable for the present invention is derived from an antibody heavy chain constant region, such as the constant region of human IgG1, IgG2, IgG3 or IgG4, preferably the constant region of IgG1. In some embodiments, the Fc region comprises a mutation that reduces binding to Fcγ receptors, such as a LALA mutation, a D265A mutation and / or a P329A mutation, preferably comprising a LALA mutation and D265A and P329A mutations. In some embodiments, Fc comprises an amino acid sequence as shown in any one of SEQ ID NOs: 53-55 or SEQ ID NO: 68, or comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consists of said amino acid sequence. In some embodiments of the present invention, Fc fragments dimerize to form Fc dimers. In some embodiments, Fc fragments heterodimerize to form Fc heterodimers. In the case where the Fc fragment heterodimerizes into a heterodimer, the Fc fragment may comprise a mutation for heterodimerization, such as a knob into hole mutation.

[0078] The term "CH1 region" refers to the portion of an antibody heavy chain polypeptide extending from EU position 118 to EU position 216 (EU numbering system). In some cases, for example, when constructing a Fab, the CH1 region may also include a portion of the hinge region, such as EPKSC. Therefore, in some embodiments, the term "CH1" region refers to the portion of an antibody heavy chain polypeptide extending from EU position 118 to EU position 220 (EU numbering system). In one embodiment, the CH1 domain comprises the amino acid sequence of ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO: 51). In some embodiments, CH1 may also include a portion of the hinge region, such as EPKSC or EPKSS. For example, in the Fab fragment of an antibody, CH1 may include the hinge region EPKSC.

[0079] 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 cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B-cell receptor), and B-cell activation.

[0080] The term "chimeric antibody" is an antibody molecule in which (a) the constant region or a portion thereof is changed, replaced or exchanged so that the antigen binding site is connected to a constant region of a different or altered class, effector function and / or species or a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug) that imparts new properties to the chimeric antibody; or (b) the variable region or a portion thereof is changed, replaced or exchanged with a variable region having a different or altered antigenic specificity. For example, a mouse antibody can be modified by replacing its constant region with a constant region from a human immunoglobulin. Due to the replacement with a human constant region, the chimeric antibody can retain its specificity in recognizing the antigen while having reduced immunogenicity in humans as compared to the original mouse antibody.

[0081] A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody (e.g., a camelid VHH antibody) while being less immunogenic 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 portions of the antibody with their human counterparts (i.e., replacing portions of the variable region that do not participate in binding with corresponding portions of a human antibody).

[0082] As used herein, the terms "anti," "binding," or "specific binding" mean that the binding is selective for the target or antigen and can be distinguished from unwanted or non-specific 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 by radioimmunoassay (RIA) or thin-layer interferometry or MSD assays or surface plasmon resonance (SPR).

[0083] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K D ) indicates that the dissociation constant is the dissociation rate constant and the association rate constant (K dis and K on ). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.

[0084] "Percent identity (%)" of an amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the specific amino acid sequence set forth in this specification, after aligning the candidate sequence with the specific amino acid sequence set forth in this specification and introducing gaps, if necessary, to achieve the maximum percentage identity, and not considering any conservative substitutions as part of the sequence identity. In some embodiments, the present invention contemplates variants of the antibody molecules of the present invention that have a substantial degree of identity, e.g., at least 80%, 85%, 90%, 95%, 97%, 98% or 99% or more, relative to the antibody molecules and sequences thereof specifically disclosed herein. The variants may comprise conservative changes or be conservatively modified variants.

[0085] For polypeptide sequences, "conservative changes" include replacements, deletions or additions to the polypeptide sequence, but do not substantially change the desired functional activity of the polypeptide sequence. For example, conservative substitutions often result in a certain amino acid being replaced with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following lists 8 groups of amino acids containing mutually conservative replacements: 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 "conservative sequence change" is used to refer to amino acid modifications that do not significantly affect or change the target antigen binding characteristics of the antibody molecule or binding protein molecule of the present invention containing the amino acid sequence. For example, a conservatively modified variant retains at least 80%, 85%, 90%, 95%, 98%, 99% or more, such as 100-110% or more, binding affinity for the antigen of interest relative to the parent antibody or binding protein.

[0086] "Knob-in-hole" mutations or "knob-in-knob" mutations or "knob-in-hole" mutations are used herein to refer to the introduction of mutations into a first Fc polypeptide and a second Fc polypeptide, respectively, using the "knob-in-knob" technique to form a protrusion ("knob") and a complementary cavity ("hole") at the interface of the first Fc polypeptide and at the interface of the second Fc polypeptide. It is known in the art that the "knob-in-hole" technique can modify the interface between different chains of an antibody molecule to promote the correct association of the chains of the antibody molecule. Typically, the technique involves introducing a "protrusion / knob" at the interface of one chain and a corresponding "hole / knob" at the interface of the other chain to be paired with it, so that the protrusion can be placed in the cavity. 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 other chain to be paired with it. The protrusion can be constructed by replacing small amino acid side chains from the interface of the CH3 domain of the heavy chain constant domain of one chain with larger side chains (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with smaller side chains (e.g., alanine or threonine), a compensatory cavity of the same or similar size as the protrusion is constructed at the interface of the CH3 domain of the heavy chain constant domain of the other chain to be paired. Another optional interface is the CL domain of the light chain and the CH1 domain of the heavy chain of the Fab fragment described above, which promotes correct heterodimerization between the two chains of the Fab fragment by establishing a protrusion-cavity interaction.

[0087] "Single-chain variable fragment" or "scFv" is used herein to refer to a single-chain antibody fragment comprising a heavy chain variable domain VH and a light chain variable domain VL connected by a linker, wherein VH and VL pair to form an antigen binding site.

[0088] In this article, the antibody constant region or antibody constant domain, including CH1, CL and Fc domains and CH2, CH3 and optional CH4 domains constituting the Fc domain, can be selected according to the intended function of the antibody molecule. For example, the constant region can be an IgA, IgD, IgE, IgG or IgM region, in particular an immunoglobulin constant domain of human IgG, for example, a constant domain of human IgG1, IgG2, IgG3 or IgG4, preferably a constant domain of human IgG1. The immunoglobulin constant region can have a native sequence or a variant sequence.

[0089] As used herein, the term "joint" refers to any molecule that enables the direct connection of the different parts of a bispecific binding molecule. The example of a covalently linked joint between different molecular parts includes a peptide joint and a non-protein polymer, including but not limited to a copolymer of polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene or polyethylene glycol, polypropylene glycol. In some embodiments, the joint is a peptide joint (also referred to as a "connecting peptide"), 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 a hinge region from an immunoglobulin, for connecting the amino acid sequence of the first part of the binding molecule to the second part of the binding molecule. For example, a peptide joint can connect the first target binding region of the binding molecule to the second target binding region. For example, a peptide joint can also connect a part of an antibody to another part of the antibody, such as connecting a light chain variable region to a heavy chain variable region. Preferably, the peptide joint has a length that is sufficient to connect two entities in a manner such that they maintain their conformations relative to each other so as not to hinder the desired activity. In one embodiment, the connecting peptide has a length of 5-50 amino acids, for example, 10, 15, 20, 25, 30 amino acids. In one embodiment, the connecting peptide comprises the amino acid sequence (GS)n(SEQ ID NO:80), (GGS)n(SEQ ID NO:81), (GSGGS)n(SEQ ID NO:82), (GGGGS)n(SEQ ID NO:83), (GGGS)n(SEQ ID NO:84) and (GGGGS)nG(SEQ ID NO:85), wherein 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, 10. Useful linkers also include glycine-alanine polymers, alanine-serine polymers and other flexible linkers. In some embodiments, the peptide linker is (GGGGS)n(SEQ ID NO:86), wherein n=1, 2, 3, 4 or 5, preferably, n=3 or 4, for example, the sequence shown in SEQ ID NO:60 or 70.

[0090] In yet another embodiment, the connecting peptide is a hinge region or a portion of a hinge region from an immunoglobulin, including a native hinge region or a portion thereof, or a mutated hinge region or a portion thereof. In one embodiment, the connecting peptide is, for example, a hinge region or a portion thereof (e.g., EPKSC (SEQ ID NO: 67)) of an immunoglobulin (e.g., IgG, e.g., IgG1, IgG2, IgG3, or IgG4) or a mutated hinge region or a portion thereof, e.g., EPKSS (SEQ ID NO: 59). Alternatively, a computer program can be used to simulate the three-dimensional structure of proteins and peptides, or by phage display methods, to rationally design suitable flexible connecting peptides.

[0091] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom. Host cells are any type of cell system that can be used to produce the antibody molecules of the present invention, including eukaryotic cells, e.g., mammalian cells, insect cells, yeast cells; and prokaryotic cells, e.g., E. coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.

[0092] The term "vector" when used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is attached. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which it has been introduced. The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences that are operatively linked to the nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) into which recombinant polynucleotides are incorporated.

[0093] The terms "individual" or "subject" are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual is a human.

[0094] The term "treat," ...

[0095] The term "prevent" includes the inhibition of the development or progression of a disease or condition or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a preventative regimen. Generally, in the context of cancer, the term "prevent" refers to the administration of a drug before the development of signs or symptoms of cancer, particularly in a subject at risk for cancer.

[0096] 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 (eg, immunosuppressants).

[0097] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction.

[0098] "Chemotherapeutic agents" include chemical compounds useful in treating cancer or immune system disorders.

[0099] The term "small molecule drug" refers to low molecular weight organic compounds that are capable of regulating biological processes. "Small molecules" are defined as molecules with a molecular weight of less than 10 kD, typically less than 2 kD, and preferably less than 10 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimics. As therapeutic agents, small molecules can be more cell-permeable, less susceptible to degradation, and less prone to eliciting an immune response than macromolecules.

[0100] As used herein, the term "immunomodulator" refers to a natural or synthetic agent or drug that inhibits or modulates an immune response. The immune response can be a humoral response or a cellular response. Immunomodulators include immunosuppressants. In some embodiments, the immunomodulators of the present invention include immune checkpoint inhibitors or immune checkpoint agonists.

[0101] The term "effective amount" refers to that amount or dosage of an antibody or fragment or composition or combination of the present invention which, after single or multiple doses, produces the desired effect in a patient in need of treatment or prevention.

[0102] A "therapeutically effective amount" is an amount effective to achieve the desired therapeutic outcome at the required dosage and for the required period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or composition or combination are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter or improves a measurable parameter by at least about 40%, even more preferably by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100%, relative to an untreated subject.

[0103] A "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result, at the required dosage and for the required period of time. Typically, a prophylactic amount will be less than a therapeutically effective amount because a prophylactic dose is used in a subject prior to or at an earlier stage of disease.

[0104] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when referred to herein. The term "tumor" encompasses both solid tumors and hematologic tumors.

[0105] The term "anti-tumor effect" or "tumor inhibitory effect" or "tumor suppressive effect" refers to a biological effect that can be demonstrated by various means, including but not limited to, for example, a decrease in tumor volume, a decrease in tumor cell number, a decrease in tumor cell proliferation, or a decrease in tumor cell survival.

[0106] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.

[0107] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0108] The term "drug combination or combination product" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit, a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) an antibody or bispecific antibody of the present invention, and (ii) other therapeutic agents) are administered to a patient simultaneously, without specific time restrictions, or at the same or different time intervals, in a separate entity, wherein such administration provides two or more active agents at a prophylactic or therapeutically effective level in the patient's body. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. The dosage and / or time interval of the two or more active agents are preferably selected so that the combined use of the parts can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in the form of a separate formulation, which can be the same or different.

[0109] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiotherapy or surgery) to treat diseases described herein. This administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed ratio of active ingredients. Alternatively, this administration includes co-administration of each active ingredient in a variety of or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids can be reconstituted or diluted to the desired dose before administration. In addition, this administration also includes using each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the therapeutic regimen will provide the beneficial effects of the drug combination in treating disorders or conditions described herein.

[0110] "Subject / patient / individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample can be solid tissue, such as from fresh, frozen and / or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood component; body fluids, such as tears, vitreous humor, cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time during the subject's pregnancy or development. In some embodiments, the tissue sample is tumor tissue. The tissue sample may contain compounds that are not naturally mixed with tissues in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0111] II. Anti-LILRB4 Antibodies

[0112] In one aspect, the present invention provides a LILRB4 antibody having a higher binding affinity to LILRB4. In some embodiments, the LILRB4 antibody of the present invention is suitable for constructing an antigen-binding region in a bispecific antibody molecule.

[0113] In some embodiments, the anti-LILRB4 antibodies or antigen-binding fragments thereof of the present invention bind to LILRB4 (e.g., human LILRB4 or monkey LILRB4, such as cynomolgus monkey LILRB4) with high affinity. In some embodiments, the anti-LILRB4 antibodies or antigen-binding fragments thereof of the present invention are capable of binding to both human LILRB4 and monkey LILRB4, such as cynomolgus monkey LILRB4.

[0114] In some embodiments, the anti-LILRB4 antibodies or antigen-binding fragments thereof of the present invention bind to LILRB4 expressed by cells. In some embodiments, the affinity of the anti-LILRB4 antibodies to LILRB4 expressed by cells is determined by flow cytometry.

[0115] Single domain antibodies

[0116] In some embodiments, the anti-LILRB4 antibodies of the invention are single domain antibodies, particularly VHH antibodies.

[0117] Single-domain antibodies, or VHH antibodies, have a molecular weight approximately one-tenth that of a human IgG molecule and a physical diameter of only a few nanometers. Due to their small molecular size, single-domain monoclonal antibodies offer advantages over conventional four-chain antibodies: high stability and solubility, as well as the ability to recognize hidden antigenic sites. Furthermore, single-domain antibodies are cheaper to prepare than conventional four-chain antibodies. In addition to their use as standalone molecules, single-domain antibodies are also suitable components for constructing multispecific molecules.

[0118] In some embodiments, the anti-LILRB4 single-domain antibody of the present invention is a VHH antibody comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region generally has the following structure: FR1-VHH CDR1-FR2-VHH CDR2-FR3-VHH CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4; VHH CDR1 to VHH CDR3 refer to complementarity determining regions 1 to 3. The CDR sequences in the VHH variable region can be determined according to any of the CDR definition schemes described in the "Definitions" section, and preferably, the boundaries of the three CDRs in the VHH sequence can be defined using IMGT.

[0119] In some embodiments, the anti-LILRB4 VHH antibody of the present invention comprises

[0120] (i) three complementarity determining regions (CDRs) contained in the VH represented by any one of SEQ ID NOs: 11, 16, 21, 25, 30, 32, 36, 39 and 42, or

[0121] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three CDR regions relative to the sequence of (i);

[0122] Preferably, the CDR sequences are defined according to IMGT.

[0123] In some embodiments, the anti-LILRB4 VHH antibodies of the present invention comprise or consist of a heavy chain variable region comprising

[0124] (i) three complementarity determining regions (CDRs) contained in the VH represented by any one of SEQ ID NOs: 11, 16, 21, 25, 30, 32, 36, 39 and 42, or

[0125] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three CDR regions relative to the sequence of (i);

[0126] Preferably, the CDR sequences are defined according to IMGT.

[0127] In some embodiments, the anti-LILRB4 VHH antibodies of the present invention comprise complementarity determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3. In some embodiments, the anti-LILRB4 VHH antibodies of the present invention comprise or consist of a heavy chain variable region comprising complementarity determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3.

[0128] In some embodiments, the VHH CDR1 comprises or consists of an amino acid sequence selected from SEQ ID NO: 12, 17, 26 or 75, or the VHH CDR1 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from SEQ ID NO: 12, 17, 26 or 75.

[0129] In some embodiments, the VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 13, 18, 22, 27, 33 or 37, or the VHH CDR2 comprises 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: 13, 18, 22, 27, 33 or 37.

[0130] In some embodiments, the VHH CDR3 comprises or consists of an amino acid sequence selected from SEQ ID NO: 14, 19, 23, 28, 34 or 40, or the VHH CDR3 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from SEQ ID NO: 14, 19, 23, 28, 34 or 40.

[0131] In one embodiment, the anti-LILRB4 VHH antibody of the present invention comprises the complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein

[0132] (i) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 12, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 13, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 14; or

[0133] (ii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 17, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 18, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 19;

[0134] (iii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 75, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 22, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 23;

[0135] (iv) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 26, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 27, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 28;

[0136] (v) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 17, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 33 or 37, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 34 or 40;

[0137] (vi) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 17, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 33 or 37, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 34; or

[0138] (vii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 17, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 33 or 37, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 40.

[0139] In one embodiment, an anti-LILRB4 VHH antibody of the invention comprises or consists of a heavy chain variable region, wherein said heavy chain variable region comprises complementarity determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, wherein

[0140] (i) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 12, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 13, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 14; or

[0141] (ii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 17, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 18, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 19;

[0142] (iii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 75, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 22, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 23;

[0143] (iv) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 26, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 27, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 28;

[0144] (v) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 17, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 33 or 37, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 34 or 40;

[0145] (vi) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 17, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 33 or 37, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 34; or

[0146] (vii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 17, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 33 or 37, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 40.

[0147] In some embodiments, the anti-LILRB4 VHH antibodies of the present invention comprise or consist of a heavy chain variable region, wherein the heavy chain variable region

[0148] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 11, 16, 21, 25, 30, 32, 36, 39 and 42; or

[0149] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 11, 16, 21, 25, 30, 32, 36, 39 and 42; or

[0150] (iii) an amino acid sequence comprising 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 shown in any one of SEQ ID NOs: 11, 16, 21, 25, 30, 32, 36, 39, and 42, preferably, the amino acid changes do not occur in the CDR regions.

[0151] In some embodiments, the anti-LILRB4 VHH antibody of the present invention comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 11, 16, 21, 25, 30, 32, 36, 39, and 42.

[0152] In some embodiments, the VHH antibodies of the present invention comprise CDR amino acid sequences and / or framework (FR) amino acid sequences derived from camelid heavy chain antibodies produced by immunizing camelids (e.g., alpacas). In some embodiments, the VHH monoclonal antibodies of the present invention derived from camelid heavy chain antibodies can be engineered, for example, to comprise framework region sequences derived from human amino acid sequences (i.e., human antibodies) or other non-camelid mammalian species. In one embodiment, to further improve the properties (e.g., affinity) of the engineered antibodies, camelid amino acid residues located at corresponding positions in the parent camelid antibody can be introduced into the engineered antibodies at one or more positions (e.g., framework regions) by back mutation.

[0153] In one embodiment, the VHH antibody of the present invention is a humanized antibody. Humanization can be achieved by replacing one or more amino acid residues, especially framework region sequences, in a non-human natural VHH sequence (e.g., a VHH sequence from camelids or alpacas immunization) with residues at corresponding positions in the heavy chain VH of a conventional human antibody. Methods for humanizing VHHs are well known in the art, such as the method described in Example 5. Typically, humanizing substitutions are performed in a manner that maintains the favorable binding properties of single-domain antibodies. Tests for determining the biological properties of humanized single-domain antibodies, such as binding affinity, are well known in the art to determine and select suitable humanized residue mutations or combinations of mutations.

[0154] In some embodiments, the humanized single domain antibody of the present invention can be obtained by a method comprising the following steps:

[0155] Determine the CDR loop structure of a parent single domain antibody (e.g., a camelid VHH antibody screened from a phage display library);

[0156] Find the closest homologous sequence for each V / J region in the human germline sequence database as a template, for example, by aligning the IMGT human antibody heavy chain variable region germline gene database (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi) and selecting a heavy chain variable region germline gene with high homology to the VHH antibody as a template;

[0157] The CDRs of the VHH antibody are respectively transplanted into the corresponding selected human templates to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CR3-FR4. Preferably, the framework sequence used for replacement has structural similarity with the framework sequence of the antibody to be humanized, for example, having a sequence identity of at least 80%, 85%, 90%, or 95%, 96%, 97%, 98%, or 99% or more;

[0158] If necessary, key amino acids in the FR region are backmutated to amino acids corresponding to the VHH antibody to ensure the original affinity, thereby obtaining a humanized anti-LILRB4 VHH antibody, and the VHH antibody is optionally sequenced.

[0159] In some embodiments, the back mutation site is selected from one or more of the framework (FR).

[0160] In some embodiments, the heavy chain variable region germline gene suitable for humanization of the VHH antibodies of the present invention is IGHV3-7*01.

[0161] In some embodiments, the present invention also provides functional variants of single domain antibodies of the present invention (particularly VHH antibodies). The functional variants can be introduced into the encoding nucleic acid sequence of the exemplary single domain antibody of the present invention by methods well known in the present invention, such as by random or site-directed mutagenesis, for example, by introducing CDR sequences and / or FR sequences, and then screening (for example, by phage display library screening) variants that maintain the desired properties to obtain functional variants. Typically, functional variants maintain significant sequence identity with the parent single domain antibody (or VHH). Preferably, the functional variant maintains the desired biological properties of the parent single domain antibody (or VHH), for example, relative to the biological activity of the parent, the variant has a comparable (for example, at least 50%, 60%, 70%, 80%, preferably more than 90%) biological activity, or improved biological activity (for example, 110-150% or higher). The desired biological properties include, for example, but are not limited to, binding affinity to the target antigen (e.g., LILRB4) (as measured by a KD value), activity in blocking binding of the target antigen to a receptor (e.g., as measured by an IC50 value), activity in activating T cells in in vitro or in vivo experiments (e.g., as measured by released cytokines), and inhibition of tumor growth / survival in in vitro or in vivo experiments.

[0162] Heavy chain antibodies

[0163] In another aspect of the present invention, the present invention also provides a heavy chain antibody comprising the heavy chain variable region of the VHH antibody of the present invention.

[0164] In some embodiments, a single domain antibody or VHH of the present invention (e.g., a camel-derived VHH or a humanized form thereof) can be linked to a constant region of a human antibody or a portion thereof, such as an Fc region, to produce a heavy chain antibody comprising a VHH-constant region or VHH-CH1-Fc or VHH-Fc. In one embodiment, the heavy chain antibody comprises a VHH antibody of the present invention and an Fc region at its C-terminus. In some embodiments, VHH and Fc are linked by a hinge region or a portion thereof, such as a hinge region from IgG (e.g., a hinge region of IgG1, 2, 3, or 4) or a portion thereof.

[0165] In some embodiments, the anti-LILRB4 heavy chain antibodies of the present invention comprise a VHH as defined herein, or a heavy chain variable region thereof, and a heavy chain constant region or an Fc region of a heavy chain constant region. In some embodiments, a connecting peptide, such as an antibody hinge region or a portion thereof, such as a hinge region or a portion thereof from IgG (including a native or mutated IgG hinge region or a portion thereof), is included between the VHH or its heavy chain variable region and the heavy chain constant region or Fc region.

[0166] In some embodiments, the connecting peptide is a hinge region or portion thereof from human IgG1, 2, 3 or 4, including a native or mutated hinge region or portion thereof, such as a hinge region from human IgG1, for example, the connecting peptide is EPKSS (SEQ ID NO: 59) or EPKSC (SEQ ID NO: 67).

[0167] In one embodiment, the heavy chain antibody comprises an Fc region or portion thereof from a camelid (e.g., an alpaca). In one embodiment, the heavy chain antibody is produced and isolated by immunizing the camelid, e.g., an alpaca. Various methods are known in the art for immunizing camelids and isolating the resulting VHH antibodies or heavy chain antibodies against an antigen of interest.

[0168] In some embodiments, the heavy chain antibody comprises a constant region from a human or non-human primate (eg, cynomolgus monkey) antibody, such as a constant region from human IgG1, human IgG2, human IgG3, or human IgG4.

[0169] In some embodiments, the heavy chain antibody comprises an Fc region or portion thereof from a human or non-human primate (e.g., cynomolgus monkey). In yet another embodiment, the heavy chain antibody comprises a human IgG Fc region, e.g., human IgG1, human IgG2, human IgG3, or human IgG4 Fc region, preferably a human IgG1 or human IgG4 Fc region, such as a human IgG1 Fc region.

[0170] In one embodiment, the heavy chain antibody according to the present invention can dimerize with another polypeptide chain (e.g., the same or different heavy chain antibody) comprising an Fc region through the Fc region. Therefore, in one embodiment, the present invention also provides a homologous or heterologous multimeric protein comprising a heavy chain antibody of the present invention. In a preferred embodiment, the protein preferably comprises a heavy chain antibody formed by pairing two identical heavy chain antibody chains.

[0171] In some embodiments, the anti-LILRB4 heavy chain antibodies of the present invention are chimeric antibodies. In some embodiments, the anti-LILRB4 antibodies of the present invention are humanized antibodies.

[0172] The Fc region of the present invention may be mutated to obtain desired properties. Mutations to the Fc region are known in the art and are defined below.

[0173] In some embodiments, the anti-LILRB4 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain comprising a heavy chain variable region, an Fc region, and a connecting peptide connecting the heavy chain variable region and the Fc region. Preferably, the connecting peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59 or 67.

[0174] In some embodiments, the anti-LILRB4 antibody or antigen-binding fragment thereof of the present invention comprises or consists of a heavy chain comprising or consisting of a heavy chain variable region of a VHH of the present invention, a connecting peptide, and an Fc region, wherein the heavy chain

[0175] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 15, 20, 24, 29, 31, 35, 38, 41 or 43; or

[0176] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NO: 15, 20, 24, 29, 31, 35, 38, 41 or 43; or

[0177] (iii) an amino acid sequence comprising 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 shown in any one of SEQ ID NOs: 15, 20, 24, 29, 31, 35, 38, 41, or 43, preferably, the amino acid changes do not occur in the CDR regions.

[0178] III. Humanized CD3 Antibody

[0179] On the one hand, the present invention provides a humanized CD3 antibody, which has better binding affinity to CD3 and is therefore more suitable for constructing the antigen binding region in a bispecific antibody molecule.

[0180] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention bind to CD3 (e.g., human CD3 or monkey CD3, such as cynomolgus CD3) with a desired affinity. In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention are capable of binding to both human CD3 and monkey CD3, such as cynomolgus CD3. In some embodiments, the affinity of the antibody is determined by thin-layer interferometry or surface plasmon resonance.

[0181] In some embodiments, the anti-CD3 antibodies of the present invention have an equilibrium dissociation constant (K D ) binds to human CD3 or monkey CD3, such as cynomolgus monkey CD3, said K D In some embodiments, the anti-CD3 antibodies of the present invention have a K of about 10nM-100nM, or 20nM-100nM or 50nM-100nM. DBinds to monkey CD3, such as cynomolgus monkey CD3. In some embodiments, the anti-CD3 antibodies of the invention have a K between about 100-1000 nM (e.g., about 200, 300, 400, or 500-1000 nM). D Binds to human CD3.

[0182] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention bind to CD3 on the surface of effector cells. In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention are capable of activating effector cells. In some embodiments, the effector cells are T cells. In some embodiments, the binding is detected by flow cytometry. In some embodiments, the activation effect of the CD3 antibody is detected using a reporter gene detection system (e.g., the Jurkat / NFAT-luc reporter gene system).

[0183] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention are capable of activating effector cells to induce killing of tumor cells.

[0184] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention comprise three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3.

[0185] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention comprise three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0186] In some embodiments, an anti-CD3 antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (HCDRs) from a heavy chain variable region and three complementarity determining regions (LCDRs) from a light chain variable region.

[0187] In some aspects, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH). In some aspects, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention comprise a light chain variable region (VH). In some aspects, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises three complementary determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementary determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0188] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody heavy chain constant region HC. In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody light chain constant region LC. In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention further comprise a heavy chain constant region HC and a light chain constant region LC.

[0189] In some embodiments, the heavy chain variable region VH of the anti-CD3 antibody of the present invention is

[0190] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 3; or

[0191] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 3; or

[0192] (iii) an amino acid sequence comprising 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: 3, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0193] In some embodiments, the light chain variable region VL of the anti-CD3 antibody of the present invention is

[0194] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 4; or

[0195] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 4; or

[0196] (iii) an amino acid sequence comprising 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: 4, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0197] In some embodiments, the three complementarity determining regions (HCDRs) from the heavy chain variable region of the anti-CD3 antibody of the present invention, HCDR1, HCDR2 and HCDR3 are

[0198] (i) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 3, or

[0199] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in the three HCDR regions relative to the sequence of any one of (i),

[0200] Preferably, the HCDRs are defined according to Kabat.

[0201] In some embodiments, the three complementarity determining regions (LCDRs), LCDR1, LCDR2, and LCDR3, from the light chain variable region of the anti-CD3 antibody of the invention are

[0202] (i) three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 4, or

[0203] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in the three LCDR regions relative to the sequence of any one of (i);

[0204] Preferably, the LCDR is determined according to Kabat.

[0205] In some embodiments, in the anti-CD3 antibodies of the present invention,

[0206] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 5; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 6; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 7; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 8; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 9; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 10.

[0207] In some specific embodiments of the present invention, the anti-CD3 antibody or antigen-binding fragment thereof comprises VH and VL, wherein

[0208] The VH comprises the amino acid sequence of SEQ ID NO:3, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of said amino acid sequence, and the VL comprises the amino acid sequence of SEQ ID NO:4, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of said amino acid sequence.

[0209] In some specific embodiments of the present invention, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:3, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:4.

[0210] In some specific embodiments of the present invention, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises: HCDR1 as shown in SEQ ID NO: 5, HCDR2 as shown in SEQ ID NO: 6, HCDR3 as shown in SEQ ID NO: 7; LCDR1 as shown in SEQ ID NO: 8, LCDR2 as shown in SEQ ID NO: 9 and LCDR3 as shown in SEQ ID NO: 10.

[0211] In one embodiment of the present invention, the amino acid changes described herein include amino acid substitutions, insertions, or deletions. In preferred embodiments, the amino acid changes described herein occur in regions outside the CDRs (e.g., in the FRs). More preferably, the amino acid changes described herein occur in regions outside the heavy chain variable region and / or outside the light chain variable region. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

[0212] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the present invention have one or more of the following properties:

[0213] (i) exhibiting the same or similar binding affinity and / or specificity for CD3 as the antibodies of the present invention;

[0214] (ii) inhibiting (e.g., competitively inhibiting) the binding of an antibody of the invention to CD3;

[0215] (iii) binds to the same or overlapping epitope as an antibody of the invention;

[0216] (iv) competing with the antibodies of the present invention for binding to CD3;

[0217] (v) possess one or more biological properties of an antibody of the invention.

[0218] In some embodiments, the anti-CD3 antibodies of the present invention are IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, or IgG4 antibodies, for example, IgG1 antibodies. In some embodiments, the heavy chain constant region of the anti-CD3 antibodies of the present invention is an IgG1, IgG2, IgG3, or IgG4 constant region. In some embodiments, the light chain constant region of the anti-CD3 antibodies of the present invention is a kappa or lambda light chain constant region, for example, a lambda light chain constant region.

[0219] In some embodiments, the anti-CD3 antibody is a monoclonal antibody.

[0220] In some embodiments, the anti-CD3 antibody is humanized.

[0221] In one embodiment, the anti-CD3 antibodies of the present invention also encompass antibody fragments thereof (e.g., antigen-binding fragments), preferably antibody fragments selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies such as VHH, dAb (domain antibody), or linear antibodies.

[0222] In one embodiment, the anti-CD3 antibody fragment of the present invention is a Fab comprising VH and VL as well as CH1 and CL as described herein. In one embodiment, the anti-CD3 antibody fragment suitable for constructing the bispecific antibody of the present invention is a Fab.

[0223] In one embodiment, the Fab comprises the heavy chain variable region VH and light chain variable region VL described in this section.

[0224] In some embodiments, the Fab heavy chain comprises VH and CH1 (and optionally part of the hinge region EPKSS or EPKSC at the C-terminus of CH1), wherein VH is the VH of an anti-CD3 antibody.

[0225] In some embodiments, the Fab light chain comprises VL-CL, wherein VL is the VL of an anti-CD3 antibody. In some embodiments, the CL is a light chain constant region from an antibody kappa or lambda light chain, preferably a light chain constant region from a kappa light chain.

[0226] In some embodiments, the Fab heavy chain

[0227] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 87;

[0228] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 87; or

[0229] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 87.

[0230] In some embodiments, the Fab light chain

[0231] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 2;

[0232] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 2; or

[0233] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 2.

[0234] In one embodiment, the anti-CD3 antibody fragment of the invention is a scFv comprising VH and VL as described herein, and optionally a connecting peptide, wherein for example the connecting peptide is (GGGGS) n , wherein n=1, 2, 3, 4 or 5, such as n=3 or 4, such as the linker set forth in SEQ ID NO: 70. In some embodiments, the scFv comprises the amino acid sequence set forth in SEQ ID NO: 70, or comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto (preferably having 1-6 identical CDRs thereto). In one embodiment, the anti-CD3 antibody fragment suitable for constructing the bispecific antibody of the present invention is a scFv.

[0235] IV. Multispecific Antibodies

[0236] In some embodiments, an anti-LILRB4 antibody of the invention is a multispecific antibody, eg, a bispecific antibody, comprising, eg, one binding specificity for LILRB4 and other binding specificities for one or more molecules (eg, CD3).

[0237] Therefore, one aspect of the present invention relates to a bispecific antibody comprising

[0238] a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region specifically binds to CD3, and / or the second antigen-binding region specifically binds to LILRB4.

[0239] In some embodiments, the first antigen-binding region is from an anti-CD3 antibody, such as SP34 antibody, or a humanized antibody thereof, such as an anti-CD3 humanized antibody described herein, such as a Fab fragment or scFv fragment of an anti-CD3 antibody.

[0240] In some embodiments, the second antigen-binding region is from an anti-LILRB4 antibody or antigen-binding fragment thereof described herein, eg, an anti-LILRB4 VHH.

[0241] The first antigen-binding region of the bispecific antibody suitable for use in the present invention may comprise or consist of an anti-CD3 full-length antibody or an antigen-binding fragment thereof, as long as it can specifically bind to CD3, including but not limited to, for example, a full-length antibody, single-chain Fv (scFv), Fab, Fab', (Fab)2, single-domain antibody, VHH or heavy chain antibody that specifically binds to CD3.

[0242] The second antigen-binding region of the bispecific antibody suitable for use in the present invention may comprise or consist of an anti-LILRB4 full-length antibody or an antigen-binding fragment thereof, as long as it can specifically bind to CD3, including but not limited to, for example, a full-length antibody, single-chain Fv (scFv), Fab, Fab', (Fab)2, single-domain antibody, etc. that specifically binds to LILRB4.

[0243] The second antigen-binding region of the bispecific antibody suitable for use in the present invention may comprise or consist of the anti-LILRB4 VHH or heavy chain antibody of the present invention, as long as it can specifically bind to LILRB4, including but not limited to, for example, a VHH or heavy chain antibody that specifically binds to LILRB4.

[0244] In some embodiments, the multispecific antibodies of the present invention, such as bispecific antibodies, are IgG-like bispecific antibodies. As used herein, "IgG-like bispecific antibodies" refer to bispecific antibodies that comprise an Fc dimer. Thus, in some embodiments, the bispecific antibodies of the present invention comprise an Fc dimer.

[0245] In some embodiments, the bispecific antibodies of the present invention are IgG-like bispecific antibodies, which comprise a Fab fragment as an antigen-binding region that specifically binds to one antigen and a VHH as an antigen-binding region that specifically binds to another antigen. In some embodiments, the IgG-like bispecific antibody comprises a Fab that specifically binds to CD3 as a first antigen-binding region and a VHH that specifically binds to LILRB4 as a second antigen-binding region.

[0246] In some embodiments, the bispecific antibodies of the present invention are IgG-like bispecific antibodies, which comprise an scFv fragment as an antigen-binding region that specifically binds one antigen and a VHH as an antigen-binding region that specifically binds another antigen. In some embodiments, the IgG-like bispecific antibody comprises an scFv that specifically binds CD3 as a first antigen-binding region and a VHH that specifically binds LILRB4 as a second antigen-binding region.

[0247] In some embodiments, the first antigen-binding region that specifically binds to CD3 is selected from scFv or Fab, and / or the second antigen-binding region that specifically binds to LILRB4 is a VHH.

[0248] 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. In one embodiment, the bispecific antibody comprises one first antigen-binding region and one or more (e.g., two) second antigen-binding regions.

[0249] Fab fragments suitable for use in the bispecific antibodies of the present invention

[0250] In some embodiments, the Fab fragment as one of the bispecific antibody antigen-binding regions is composed of two polypeptide chains comprising the antibody VH, CH1, VL and CL domains, wherein the VH is paired with the VL and the CH1 is paired with the CL to form an antigen-binding region. In some embodiments, in Fab, one chain comprises VH and CH1 from N-terminus to C-terminus (i.e., VH-CH1), and the other chain comprises VL and CL from N-terminus to C-terminus (i.e., VL-CL).

[0251] In some embodiments, in the bispecific antibody, Fab can be fused to the N-terminus of the Fc domain of the antibody through the C-terminus of the chain comprising VH, wherein the Fc domain may or may not include a hinge region (e.g., EPKSS or EPKSC). Preferably, the Fab comprises a VH-CH1 chain and a VL-CL chain, and is fused to the antibody Fc domain through the C-terminus of the CH1 of the VH-CH1 chain, wherein the Fc domain may or may not include a hinge region (e.g., EPKSS or EPKSC). Herein, the Fab chain connected to the Fc dimer is also referred to as the Fab heavy chain, and the Fab chain not connected to the Fc dimer is also referred to as the Fab light chain. In some embodiments, the fusion is direct fusion, or fusion through a linker such as a connecting peptide.

[0252] In some embodiments, the CH1 of a Fab may further comprise a partial hinge region, such as EPKSS or EPKSC, to facilitate formation of a stable structure, for example, to facilitate the preparation of multispecific antibodies. When the Fc fused to the Fab heavy chain does not comprise this partial hinge region, or when the Fab heavy chain is fused to a non-Fc domain, the C-terminus of the CH1 may comprise this partial hinge region to facilitate formation of a stable structure. When the Fc fused to the Fab heavy chain comprises this partial hinge region, the C-terminus of the CH1 of the Fab heavy chain may not comprise this partial hinge region.

[0253] In some embodiments, the Fab fragment contained in the bispecific antibody of the present invention specifically binds to CD3. In some embodiments, the Fab fragment contained in the bispecific antibody of the present invention is derived from the anti-CD3 antibody of the present invention, which comprises the heavy chain variable region VH and the light chain variable region VL of the anti-CD3 antibody of the present invention.

[0254] In some embodiments, the Fab heavy chain comprises VH and CH1 (and optionally comprises part of the hinge region EPKSS or EPKSC at the C-terminus of CH1), wherein VH is the VH of the anti-CD3 antibody of the present invention. In some embodiments, the CH1 is CH1 from IgG1, IgG2, IgG3 or IgG4, preferably CH1 from IgG1. In some embodiments, the CH1

[0255] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 51;

[0256] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 51; or

[0257] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 51.

[0258] In some embodiments, the Fab light chain comprises VL-CL, wherein VL is the VL of an anti-CD3 antibody of the present invention. In some embodiments, the CL is a light chain constant region from a kappa or lambda light chain of an antibody, preferably a light chain constant region from a kappa light chain. In some embodiments, the CL is a light chain constant region of an anti-CD3 antibody of the present invention. In some embodiments, the Fab light chain is a light chain of an anti-CD3 antibody of the present invention.

[0259] scFv suitable for the bispecific antibodies of the present invention

[0260] In some embodiments, the scFv fragment as one of the binding regions of a bispecific antibody is composed of a polypeptide chain comprising the VH and VL domains of an antibody, wherein the VH and VL are connected (e.g., via a linker) to pair to form an antigen binding site. In some embodiments, the scFv is in a trans configuration and comprises, from N-terminus to C-terminus: VH, a linker, and VL (VH-linker-VL). In other embodiments, the scFv is in a cis configuration and comprises, from N-terminus to C-terminus: VL, a linker, and VH (VL-linker-VH). In some embodiments, the linker is a peptide linker consisting of amino acid residues. Suitable peptide linkers are known to those skilled in the art. In one embodiment, the linker is 5-50 amino acids in length, e.g., 5-30 amino acids in length, e.g., 15 amino acids or 20 amino acids in length. In one embodiment, the linker comprises the amino acid sequence (G4S)n, wherein n=1, 2, 3, 4 or 5, preferably, n=3 or 4, more preferably, n=3.

[0261] In some preferred embodiments, the scFv antigen binding region contained in the antibody molecule of the invention is a disulfide-stabilized scFv.

[0262] In some embodiments, in the bispecific antibody, the scFv can be fused 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 fused to the C-terminus of another antigen binding region (e.g., a Fab heavy chain) at the N-terminus of the chain and to the N-terminus of the Fc domain of the antibody at the C-terminus of the chain.

[0263] In some embodiments, in the bispecific antibody, the scFv can be fused to the N-terminus of the Fc domain of the antibody via the C-terminus of the chain comprising VL. In some embodiments, in the bispecific antibody, the scFv is fused to the C-terminus of another antigen binding region (e.g., a Fab heavy chain) at the N-terminus of the VH chain and to the N-terminus of the Fc domain of the antibody at the C-terminus of the VL chain.

[0264] In some embodiments, in the bispecific antibody, the scFv can be fused to the N-terminus of the Fc domain of the antibody via the C-terminus of the chain comprising VH. In some embodiments, in the bispecific antibody, the scFv is fused to the C-terminus of another antigen binding region (e.g., a Fab heavy chain) at the N-terminus of the VL chain and to the N-terminus of the Fc domain of the antibody at the C-terminus of the VH chain.

[0265] In some embodiments, the scFv contained in the bispecific antibody of the present invention specifically binds to CD3. In some embodiments, the scFv contained in the bispecific antibody of the present invention is derived from the anti-CD3 antibody of the present invention, which comprises the heavy chain variable region VH and the light chain variable region VL of the anti-CD3 antibody of the present invention.

[0266] Fc dimers suitable for the bispecific antibodies of the present invention

[0267] In one embodiment, the two Fc regions in the bispecific antibody of the present invention dimerize to form a dimeric Fc. Preferably, the two Fc regions heterodimerize to form a heterodimeric Fc.

[0268] In some embodiments, the first and second Fc regions are identical. In other embodiments, the first and second Fc regions are different and pair and heterodimerize.

[0269] The Fc region fragment suitable for antibody molecules of the present invention can be any antibody Fc region. The Fc region can include native sequence Fc regions and variant Fc regions. The native sequence Fc domain covers naturally occurring various immunoglobulin Fc sequences, such as various Ig subtypes and the Fc regions of their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520.). For example, the Fc region of an antibody of the present invention can include two or three constant domains, i.e., a CH2 domain, a CH3 domain, and an optional CH4 domain. In some embodiments, the antibody Fc region can also have an IgG hinge region or a partial IgG hinge region at the N-terminus, such as an IgG1 hinge region or a partial IgG1 hinge region. Mutations can be contained in the hinge region. In some embodiments, the hinge region can be EPKSS or EPKSC.

[0270] Preferably, the Fc region of an antibody of the present invention comprises, from N-terminus to C-terminus, CH2-CH3, or, from N-terminus to C-terminus, hinge region-CH2-CH3. In some embodiments, the Fc region suitable for use in an antibody or bispecific antibody of the present invention is a human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, human IgG3, or human IgG4 Fc. In one embodiment, the Fc region is derived from a human IgG1 Fc, e.g., comprising or consisting of the amino acid sequence of SEQ ID NO: 55 or 68, or an amino acid sequence having at least 90% identity thereto, e.g., 95%, 96%, 97%, 99% or greater identity thereto.

[0271] The Fc region of the antibody or bispecific antibody of the present invention can be mutated to obtain desired properties. Mutations in the Fc region are known in the art.

[0272] In one embodiment, the Fc region is modified with respect to the properties of the effector function of the Fc region (e.g., the complement activation function of the Fc region). In one embodiment, the effector function has been reduced or eliminated relative to a wild-type Fc region. In one embodiment, the effector function is reduced or eliminated by a method selected from the group consisting of: using an Fc isotype that naturally has reduced or eliminated effector function, and Fc region modification.

[0273] In a preferred embodiment, the Fc region has reduced effector function mediated by the Fc region, such as reduced or abolished ADCC or ADCP or CDC effector function, eg, comprises a mutation that achieves the above function.

[0274] As will be appreciated by those skilled in the art, according to the intended use of the antibody molecule of the present invention, the antibody molecule of the present invention may also include modifications in the Fc domain that alter the binding affinity to one or more Fc receptors. In one embodiment, the Fc receptor is an Fcγ receptor, particularly a human Fcγ receptor. In some embodiments, the Fc region comprises a mutation that reduces binding to the Fcγ receptor. For example, in some embodiments, the Fc region used for the present invention has one or more of the L234A / L235A mutation, the D265A mutation, or the P329A mutation that reduces binding to the Fcγ receptor. In some embodiments, the Fc region used for the present invention has a L234A / L235A mutation, the D265A mutation, and the P329A mutation that reduces binding to the Fcγ receptor. In another preferred embodiment, the Fc fragment may have a mutation that results in increased serum half-life, such as a mutation that improves binding of the Fc fragment to FcRn. In some embodiments, the Fc region comprising a mutation that reduces binding to an Fcγ receptor comprises or consists of the amino acid sequence of SEQ ID NO: 53 or 54, or an amino acid sequence having at least 90% identity thereto, e.g., 95%, 96%, 97%, 99% or more identity. In some embodiments, the Fc region comprises an amino acid sequence having at least 90% identity, e.g., 95%, 96%, 97%, 99% or more identity to SEQ ID NO: 53 or 54 and comprises an L234A / L235A mutation, a D265A mutation, and a P329A mutation.

[0275] As will be appreciated by those skilled in the art, in order to promote the formation of the bispecific antibodies of the present invention as heterodimers, the Fc regions comprised by the bispecific antibodies of the present invention may comprise mutations that are conducive to heterodimerization. In one embodiment, mutations are introduced into the CH3 regions of the two Fc regions.

[0276] Methods for promoting heterodimerization of Fc regions are known in the art. For example, the CH3 region of a first Fc region and the CH3 region of a second Fc region are engineered in a complementary manner such that each CH3 region (or a heavy chain comprising it) is no longer able to homodimerize with itself but is forced to heterodimerize with the other complementary engineered CH3 region (such that the first and second CH3 regions heterodimerize and homodimers are not formed between the two first CH3 regions or the two second CH3 regions).

[0277] Preferably, corresponding knob mutations and hole mutations are introduced into the first monomer Fc region and the second monomer Fc region, respectively, based on the Knob-in-Hole technique. For this technique, see, for example, Merchant, AM, et al. (1998). "An efficient route to human bispecific IgG." Nat Biotechnol 16(7):677-681.

[0278] In a specific embodiment, in the CH3 region of one Fc region, the threonine residue at position 366 is replaced with a tryptophan residue (T366W) (knob mutation); and in the CH3 region of another Fc region, the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (hole mutation), optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the EU index).

[0279] In a further embodiment, in the CH3 region of one Fc region, the knob mutation comprises or consists of substitution of the threonine residue at position 366 with a tryptophan residue (T366W) and substitution of the serine residue at position 354 with a cysteine ​​residue (S354C) or substitution of the glutamic acid residue at position 356 with a cysteine ​​residue (E356C) (in particular, substitution of the serine residue at position 354 with a cysteine ​​residue); and in the CH3 region of another Fc region, the hole mutation comprises or consists of substitution of the tyrosine residue at position 407 with a valine residue (Y407V), optionally substitution of the threonine residue at position 366 with a serine residue (T366S) and substitution of the leucine residue at position 368 with an alanine residue (L368A) (numbering according to the EU index), optionally substitution of the tyrosine residue at position 349 with a cysteine ​​residue (Y349C) (numbering according to the EU index).

[0280] In a specific embodiment, one Fc region comprises amino acid substitutions S354C and T366W (knob mutation), and the other Fc region comprises amino acid substitutions Y349C, T366S, L368A, and Y407V (hole mutation) (numbering is according to the EU index).

[0281] Thus, in a specific embodiment, the bispecific antibody of the present invention comprises two Fc regions that are heterodimerized, wherein

[0282] a) one Fc-region polypeptide comprises the mutation T366W and the other Fc-region polypeptide comprises the mutations T366S, L368A and Y407V, or

[0283] b) one Fc-region polypeptide comprises the mutations T366W and Y349C and the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and S354C, or

[0284] c) one Fc-region polypeptide comprises the mutations T366W and S354C, and the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V, and Y349C;

[0285] Optionally, the Fc region further comprises a mutation that reduces binding to an Fcγ receptor, eg, one or more of a L234A / L235A mutation, a D265A mutation, or a P329A mutation; eg, a L234A / L235A mutation, a D265A mutation, and a P329A mutation.

[0286] In some embodiments, the Fc region further comprises other mutations that facilitate purification of the heterodimer.

[0287] In a specific embodiment, the two Fc regions of the bispecific antibody of the invention are heterodimerized, wherein

[0288] The first Fc region comprises a knob mutation comprising or consisting of an amino acid sequence of SEQ ID NO: 49 or 50 or 73, or an amino acid sequence at least 90% identical thereto, e.g., 95%, 96%, 97%, 99% or more identical thereto; in some embodiments, the Fc region comprises an amino acid sequence at least 90% identical, e.g., 95%, 96%, 97%, 99% or more identical to SEQ ID NO: 49 or 50 or 73 and comprises a knob mutation (e.g., S354C and T366W); in some embodiments, the Fc region comprises or does not comprise a hinge region EPKSS or EPKSC;

[0289] The second Fc region comprises a hole mutation, which comprises or consists of an amino acid sequence having at least 90% identity, e.g., 95%, 96%, 97%, 99% or higher identity, to an amino acid sequence of SEQ ID NO: 47 or 48 or 74. In some embodiments, the Fc region comprises an amino acid sequence having at least 90% identity, e.g., 95%, 96%, 97%, 99% or higher identity, to SEQ ID NO: 47 or 48 or 74 and comprises hole mutations (T366S, L368A, Y407V, and Y349C); in some embodiments, the Fc region comprises or does not comprise a hinge region EPKSS or EPKSC.

[0290] Exemplary bispecific antibody molecules

[0291] In some preferred embodiments, the present invention provides a bispecific antibody comprising a VHH that specifically binds to LILRB4 and an antigen binding region that specifically binds to CD3, and optionally an Fc region.

[0292] In some embodiments, the present invention provides a bispecific antibody comprising a first antigen-binding region, a second antigen-binding region, and an Fc dimer, wherein the first antigen-binding region is a Fab fragment or scFv that specifically binds to CD3, and the second antigen-binding region is a VHH that specifically binds to LILRB4.

[0293] In some embodiments, the bispecific antibodies of the present invention comprise a first antigen-binding region and a second antigen-binding region. In some embodiments, the bispecific antibodies of the present invention comprise a first antigen-binding region and two second antigen-binding regions. In some embodiments, the two second antigen-binding regions are the same or different, for example, two different antigen-binding regions bind to different antigenic epitopes or have different sequences.

[0294] In some embodiments, the present invention relates to a bispecific antibody that is an IgG-like bispecific antibody comprising at least one anti-LILRB4 VHH, a Fab fragment of an anti-CD3 antibody, and an Fc heterodimer. In some embodiments, the bispecific antibody comprises one anti-LILRB4 VHH. In some embodiments, the bispecific antibody comprises two anti-LILRB4 VHHs. In some embodiments, the two VHHs are the same or different, for example, two different VHHs bind to different anti-LILRB4 epitopes or have different sequences.

[0295] In some embodiments, in the bispecific antibody,

[0296] The Fab comprises VH-CH1 and VL-CL, and the VHH comprises a heavy chain variable region.

[0297] wherein the C-terminus of CH1 of the Fab is fused to the CH2 or hinge region of the first Fc region (eg, comprising a knob mutation) to form a heavy chain,

[0298] The one VHH is fused to a second Fc region (e.g., comprising a hole mutation) (e.g., the C-terminus of the VHH is fused to the CH2 or hinge region of the second Fc region) to constitute another heavy chain;

[0299] and

[0300] The VL-CL of the Fab constitutes the light chain;

[0301] Optionally, the additional VHH (eg, another VHH) is fused to the N-terminus of the VH of the Fab fragment.

[0302] In some embodiments, the present invention relates to a bispecific antibody that is an IgG-like bispecific antibody comprising at least one anti-LILRB4 VHH, an anti-CD3 antibody scFv, and an Fc heterodimer. In some embodiments, the bispecific antibody comprises one anti-LILRB4 VHH. In some embodiments, the bispecific antibody comprises two anti-LILRB4 VHHs. In some embodiments, the two VHHs are the same or different, for example, two different VHHs bind to different anti-LILRB4 epitopes or have different sequences.

[0303] In some embodiments, in the bispecific antibody,

[0304] The VHH comprises or consists of a heavy chain variable region, and the scFv comprises VH and VL.

[0305] wherein the C-terminus of the scFv is fused to the CH2 or hinge region of the first Fc region (eg, comprising a knob mutation),

[0306] The one VHH is fused to a second Fc region (e.g., comprising a hole mutation) (e.g., the C-terminus of the VHH is fused to the CH2 or hinge region of the second Fc region);

[0307] Optionally, the additional VHH (eg, another VHH) is fused to the N-terminus of the scFv.

[0308] In some embodiments, the scFv comprises VH and VL in order from N-terminus to C-terminus.

[0309] In some embodiments, the fusion is direct fusion or fusion through a linker or connecting peptide. In some embodiments, the VH and VL in the scFv are connected by a linker or connecting peptide. In some embodiments, the linker or connecting peptide is selected from (GGGGS)n, wherein n=1, 2, 3 or 4. In some embodiments, the linker or connecting peptide is GGGGS. In one embodiment, the linker (e.g., the linker contained in the scFv) comprises or consists of the amino acid sequence shown in SEQ ID NO:70. In some embodiments, the Fc region includes or does not include a hinge region. In some embodiments, the hinge region is selected from EPKSS (SEQ ID NO:59) or EPKSC (SEQ ID NO:67).

[0310] Therefore, in some embodiments, the present invention relates to a bispecific antibody, which is an IgG-like bispecific antibody, comprising an anti-LILRB4 VHH, an anti-CD3 antibody Fab fragment and an Fc heterodimer, wherein

[0311] The Fab fragment comprises VH-CH1 and VL-CL, wherein the VHH comprises a heavy chain variable region,

[0312] wherein the C-terminus of CH1 of the Fab fragment is fused to the CH2 or hinge region of the first Fc region (e.g., comprising a knob mutation) to constitute the first heavy chain;

[0313] The VHH is fused to a second Fc region (e.g., comprising a hole mutation) to form a second heavy chain (e.g., the C-terminus of the VHH is fused to the CH2 or hinge region of the second Fc region);

[0314] and

[0315] The VL-CL of the Fab fragment constitutes the light chain.

[0316] In one embodiment, the present invention relates to a bispecific antibody, which is an IgG-like bispecific antibody comprising an anti-LILRB4 VHH, an anti-CD3 antibody Fab fragment and an Fc heterodimer, wherein

[0317] The Fab fragment comprises VH-CH1 and VL-CL, wherein the VHH comprises a heavy chain variable region,

[0318] wherein the C-terminus of CH1 of the Fab fragment is fused to the CH2 or hinge region of the first Fc region comprising a knob mutation, constituting the first heavy chain;

[0319] The C-terminus of the VHH is fused to a second Fc region containing a hole mutation to form a second heavy chain;

[0320] and

[0321] The VL-CL of the Fab fragment constitutes the light chain.

[0322] In some embodiments, the first heavy chain comprises, or consists of, the amino acid sequence set forth in SEQ ID NO:44, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to said amino acid sequence;

[0323] The second heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 45, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; and / or

[0324] The light chain comprises or consists of the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence.

[0325] Therefore, in some embodiments, the present invention relates to a bispecific antibody, which is an IgG-like bispecific antibody, comprising two anti-LILRB4 VHHs, an anti-CD3 antibody Fab fragment and an Fc heterodimer, wherein

[0326] The Fab fragment comprises VH-CH1 and VL-CL, wherein the VHH comprises a heavy chain variable region,

[0327] wherein the C-terminus of the first VHH is fused to the N-terminus of the VH of the Fab fragment and the C-terminus of the CH1 of the Fab fragment is fused to the CH2 or hinge region of the Fc region (e.g., comprising a knob mutation), constituting a first heavy chain;

[0328] The second VHH is fused to the Fc region (e.g., comprising a hole mutation) to form the second heavy chain (e.g., the C-terminus of the VHH is fused to the CH2 or hinge region of the second Fc region);

[0329] and

[0330] The VL-CL of the Fab fragment constitutes the light chain.

[0331] In one embodiment, the present invention relates to a bispecific antibody, which is an IgG-like bispecific antibody comprising two anti-LILRB4 VHHs, an anti-CD3 antibody Fab fragment and an Fc heterodimer, wherein

[0332] The Fab fragment comprises VH-CH1 and VL-CL, wherein the VHH comprises a heavy chain variable region,

[0333] wherein the C-terminus of the first VHH is fused to the N-terminus of the VH of the Fab fragment and the C-terminus of the CH1 of the Fab fragment is fused to the CH2 or hinge region of the Fc region comprising a knob mutation, constituting a first heavy chain;

[0334] The C-terminus of the second VHH was fused to the Fc region containing the hole mutation to form the second heavy chain;

[0335] and

[0336] The VL-CL of the Fab fragment constitutes the light chain.

[0337] In some embodiments, the first VHH and the second VHH may be the same. In some embodiments, the first VHH and the second VHH may be different, for example, the first VHH and the second VHH may be different humanized antibodies of the same camelid VHH antibody, or the first VHH and the second VHH may be different humanized antibodies derived from different camelid antibodies.

[0338] In some embodiments, the first heavy chain comprises, or consists of, the amino acid sequence set forth in SEQ ID NO:46, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to said amino acid sequence;

[0339] The second heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 45, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; and / or

[0340] The light chain comprises or consists of the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence.

[0341] Therefore, in some embodiments, the present invention relates to a bispecific antibody, which is an IgG-like bispecific antibody, comprising two anti-LILRB4 VHHs, an anti-CD3 antibody scFv and an Fc heterodimer, wherein

[0342] The scFv comprises VH and VL from N-terminus to C-terminus (wherein VH and VL are connected by a linker or connecting peptide, for example, the linker or connecting peptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 70), the VHH comprises a heavy chain variable region,

[0343] wherein the C-terminus of the first VHH is fused to the N-terminus of the VH of the scFv and the C-terminus of the VL of the scFv is fused to the CH2 or hinge region of the Fc region (e.g., comprising a knob mutation), constituting the first heavy chain; and

[0344] The second VHH is fused to the Fc region (eg, comprising a hole mutation) to form the second heavy chain (eg, the C-terminus of the VHH is fused to the CH2 or hinge region of the second Fc region).

[0345] In one embodiment, the present invention relates to a bispecific antibody, which is an IgG-like bispecific antibody comprising two anti-LILRB4 VHHs, an anti-CD3 antibody scFv and an Fc heterodimer, wherein

[0346] The scFv comprises VH and VL from N-terminus to C-terminus (wherein VH and VL are connected by a linker or connecting peptide, for example, the linker or connecting peptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 70), the VHH comprises a heavy chain variable region,

[0347] wherein the C-terminus of the first VHH is fused to the N-terminus of the VH of the scFv and the C-terminus of the VL of the scFv is fused to the CH2 or hinge region of the Fc region comprising a knob mutation, constituting the first heavy chain; and

[0348] The C-terminus of the second VHH was fused to the Fc region containing a hole mutation to form the second heavy chain.

[0349] In some embodiments, the first VHH and the second VHH may be the same. In some embodiments, the first VHH and the second VHH may be different, for example, the first VHH and the second VHH may be different humanized antibodies of the same camelid VHH antibody, or the first VHH and the second VHH may be different humanized antibodies derived from different camelid antibodies.

[0350] In some embodiments, the first heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:72, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; and / or

[0351] The second heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:45, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence.

[0352] V. Nucleic acids encoding antibodies and host cells containing the same

[0353] In one aspect, the present invention provides a nucleic acid encoding any of the above anti-LILRB4 antibodies or antigen-binding fragments thereof or multispecific antibodies such as bispecific antibodies or any one chain thereof.

[0354] 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 11, 15, 16, 20, 21, 24, 25, 29, 30, 31, 32, 35, 36, 38, 39, 41, 42, 43, 44, 45, 46 or 72.

[0355] As will be appreciated by those skilled in the art, because of codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by a variety of nucleic acid sequences. Nucleic acid sequences encoding molecules of the present invention can be produced by methods well known in the art, for example, by de novo solid phase DNA synthesis, or by PCR amplification.

[0356] In one aspect, the present invention provides nucleic acids encoding any of the above antibodies or antigen-binding fragments thereof or any antibody chains. When expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid is capable of exhibiting human and / or monkey (e.g., cynomolgus monkey) LILRB4 antigen binding ability. In one embodiment, the nucleic acids encoding the various chains of the antibody may be in the same vector or in different vectors. In yet another embodiment, the nucleic acids encoding the various chains of the antibody may be introduced into the same or different host cells for expression. Therefore, in some embodiments, the method for producing the antibodies of the present invention comprises the steps of culturing host cells containing nucleic acids encoding the various chains under conditions suitable for expressing the various chains of the molecule to produce the antibodies of the present invention.

[0357] In another aspect, the present invention provides nucleic acids encoding any of the above bispecific antibodies. When expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid is capable of exhibiting binding ability to human or monkey (e.g., cynomolgus monkey) CD3 and human or monkey (e.g., cynomolgus monkey) LILRB4 antigens. In one embodiment, the nucleic acids encoding each chain of the bispecific antibody can be in the same vector or in different vectors. In another embodiment, the nucleic acids encoding each chain of the bispecific antibody can be introduced into the same or different host cells for expression. Therefore, in some embodiments, the method for producing the bispecific antibody of the present invention comprises the steps of culturing a host cell containing nucleic acids encoding each chain under conditions suitable for expressing each chain of the molecule to produce the bispecific antibody of the present invention.

[0358] In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). In one embodiment, the vector is, for example, a pcDNA vector, such as pcDNA3.1.

[0359] In one embodiment, a host cell comprising the nucleic acid or the vector is provided, for example, a vector for cloning or expressing an anti-LILRB4 antibody or antigen-binding fragment thereof or a bispecific antibody. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells such as CHO cells (e.g., CHO-S, such as ExpiCHO-S) 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, for example, a bacterium, such as Escherichia coli.

[0360] 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 antibody-encoding vectors. For example, fungal and yeast strains whose glycosylation pathways have been "humanized" result in the production of antibodies with partially or fully 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 are monkey kidney CV1 lines (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 DHFR-CHO cells, CHO-S cells, ExpiCHO, etc.; and myeloma cell lines such as Y0, NS0, and Sp2 / 0. Suitable mammalian host cell lines for producing antibodies are known in the art.

[0361] VI. Production and Purification of Anti-LILRB4 Antibodies or Antigen-Binding Fragments or Bispecific Antibodies of the Invention

[0362] In one embodiment, a method for preparing an anti-LILRB4 antibody or an antigen-binding fragment thereof or a multispecific antibody such as a bispecific antibody of the present invention is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the anti-LILRB4 antibody or its antigen-binding fragment or multispecific antibody such as a bispecific antibody (e.g., any one polypeptide chain and / or multiple polypeptide chains) or an expression vector comprising the nucleic acid under conditions suitable for the expression of the anti-LILRB4 antibody or its antigen-binding fragment or multispecific antibody such as a bispecific antibody or its chains, as provided above, and optionally recovering the anti-LILRB4 antibody or its antigen-binding fragment or multispecific antibody such as a bispecific antibody from the host cell (or host cell culture medium).

[0363] The polynucleotide encoding the polypeptide chains of the anti-LILRB4 antibodies or antigen-binding fragments thereof or multispecific antibodies, such as bispecific antibodies, of the present invention can be inserted into one or more vectors for further cloning and / or expression in host cells. Methods well known to those skilled in the art can be used to construct expression vectors. Once an expression vector comprising one or more nucleic acid molecules of the present invention has been prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection, or other conventional techniques.

[0364] Anti-LILRB4 antibodies or antigen-binding fragments thereof, or multispecific antibodies such as bispecific antibodies prepared as described herein can be purified by known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these will be apparent to those skilled in the art.

[0365] The purity of the molecules of the invention can be determined by any of a number of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

[0366] VII. Assays for Anti-LILRB4 Antibodies or Antigen-Binding Fragments or Multispecific Antibodies

[0367] The anti-LILRB4 antibodies or antigen-binding fragments thereof or multispecific antibodies, such as bispecific antibodies, provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by various assays known in the art.

[0368] In one aspect, an anti-LILRB4 antibody or antigen-binding fragment thereof or a multispecific antibody, such as a bispecific antibody, of the present invention is tested for target (e.g., antigen, e.g., free antigen or antigen expressed on a cell) binding activity, for example, by known methods such as thin-layer interferometry, ELISA, flow cytometry, etc. Binding to CD3 and / or LILRB4 (or CD3 and / or LILRB4 expressed on a cell) can be determined using methods known in the art, exemplary methods of which are disclosed herein. In some embodiments, radioimmunoassay (RIA), thin-layer interferometry (BLI), electrochemiluminescence (ECL), surface plasmon resonance (SPR), or flow cytometry (FACS) is used for measurement.

[0369] The present invention also provides assays for identifying the biological activity of an anti-LILRB4 antibody, or antigen-binding fragment thereof, or a multispecific antibody, such as a bispecific antibody. The biological activity is selected from the properties of the anti-LILRB4 antibody, or antigen-binding fragment thereof, or a multispecific antibody, such as a bispecific antibody, of the present invention.

[0370] For example, the binding activity of the antibody molecules of the present invention to cells expressing CD3 and / or LILRB4 can be determined by methods known in the art, such as fluorescent reporter molecules and flow cytometry, or the exemplary methods disclosed in the Examples herein, for example, the binding of the antibody molecules of the present invention to CD3 and / or LILRB4 expressed on cells can be determined by the methods shown in Examples 3, 4 or 5.

[0371] For example, the activation activity of the antibody molecules of the present invention on T cells or the activation of T cell proliferation can be determined by methods known in the art, such as T cell activation test systems, such as Jurkat / NFAT-luc (luciferase) reporter gene systems or primary T cell-dependent cytotoxicity test systems, for example, by implementing the methods shown in 4 or 6 or 9, by detecting the CD3 signaling pathway in T cells or by detecting the release of cytokines (e.g., interleukins such as IL2; interferons such as IFNγ; tumor necrosis factors such as TNFα) after activation of T cells. For example, the inhibitory activity or structural safety of the antibody molecules of the present invention on tumors can be determined by methods known in the art, such as tumor inhibition experiments on mouse tumor models.

[0372] Cells used in any of the above in vitro assays are primary cells or cell lines, including cells that naturally express or overexpress CD3 (e.g., human or monkey (e.g., cynomolgus macaque)) or LILRB4 (e.g., human or monkey (e.g., cynomolgus macaque) LILRB4), or cells engineered to express CD3 or LILRB4, such as CHO cells or CHO-K1 cells, MV-411, HL-60, or MOLM-13.

[0373] It will be appreciated that any of the above assays can be performed using a combination of the antibodies of the invention and additional active agents.

[0374] VIII. Immunoconjugates, pharmaceutical compositions, pharmaceutical combination products, and kits of the anti-LILRB4 antibodies or bispecific antibodies of the present invention

[0375] In some embodiments, the present invention provides an immunoconjugate comprising any of the anti-LILRB4 antibodies or antigen-binding fragments thereof described herein, or a multispecific antibody such as a bispecific antibody. Preferably, the immunoconjugate comprises one or more other therapeutic agents (e.g., cytotoxins or small molecule compounds) or a label.

[0376] In some embodiments, the present invention provides a composition, a medicament, or a formulation comprising any anti-LILRB4 antibody or antigen-binding fragment thereof or a multispecific antibody such as a bispecific antibody described herein, preferably a composition is a pharmaceutical composition.

[0377] In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition, e.g., a pharmaceutical composition, comprises an anti-LILRB4 antibody or antigen-binding fragment thereof or a multispecific antibody such as a bispecific antibody of the present invention in combination with one or more other therapeutic agents.

[0378] The composition, medicine or preparation of the present invention may further comprise suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.

[0379] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0380] For the use of pharmaceutical excipients and their applications, see also "Handbook of Pharmaceutical Excipients", 8th edition, RC Rowe, PJ Eskey and S C Owen, Pharmaceutical Press, London, Chicago.

[0381] The compositions, medicaments or preparations of the present invention can be in various forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injections or eye drops), powders or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

[0382] A medicament or formulation comprising the anti-LILRB4 antibody or antigen-binding fragment thereof or multispecific antibody such as a bispecific antibody described herein can be prepared by mixing an anti-LILRB4 antibody or antigen-binding fragment thereof or multispecific antibody such as a bispecific antibody of the present invention having a desired degree of purity with one or more optional pharmaceutical excipients, for example, in the form of a lyophilized formulation or an aqueous solution.

[0383] The compositions or medicaments or formulations of the present invention may also contain more than one active ingredient as required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to also provide other therapeutic agents.

[0384] The present invention also provides a pharmaceutical combination or pharmaceutical combination product comprising an anti-LILRB4 antibody or antigen-binding fragment thereof or a multispecific antibody such as a bispecific antibody of the present invention, and one or more other therapeutic agents.

[0385] The present invention also provides a complete kit comprising the drug combination, for example, the complete kit comprises in the same package:

[0386] - a first container containing a pharmaceutical composition comprising an anti-LILRB4 antibody or antigen-binding fragment thereof or a multispecific antibody such as a bispecific antibody of the present invention;

[0387] - A second container containing a pharmaceutical composition comprising an additional therapeutic agent.

[0388] In some embodiments, the additional therapeutic agent is, for example, a chemotherapeutic agent, a cytokine, a cytotoxic agent, an additional antibody, a small molecule drug, or an immunomodulatory agent (eg, an immunosuppressant).

[0389] IX. Uses of anti-LILRB4 antibodies or antigen-binding fragments thereof or multispecific antibodies and methods of using the same

[0390] In one aspect, the present invention provides a method for preventing or treating a disease in a subject, comprising administering to the subject an anti-LILRB4 antibody, or an antigen-binding fragment thereof, or a multispecific antibody, such as a bispecific antibody, of the present invention, or an immunoconjugate, composition, or drug or formulation comprising the same. In some embodiments, the present invention provides a method for specifically activating T cells in a subject, comprising administering to the subject an anti-LILRB4 antibody, or an antigen-binding fragment thereof, or a multispecific antibody, such as a bispecific antibody, of the present invention, or an immunoconjugate, composition, or drug or formulation comprising the same.

[0391] In some embodiments, the disease is, for example, a tumor, such as cancer. The cancer may be in the early, middle, or late stages or may be metastatic. In some embodiments, the cancer may be a solid tumor or a hematologic tumor. In some embodiments, the cancer is a hematologic tumor, such as a solid tumor, multiple myeloma, or leukemia, such as acute myeloid leukemia (AML), such as monocytic AML and non-monocytic AML, such as human myelomonocytic leukemia cells, human promyelocytic acute leukemia, or human acute myeloid leukemia.

[0392] In some embodiments, the disease treatment would benefit from activation of the CD3 signaling pathway and / or activation of T cells.

[0393] In some embodiments, the cancer is a cancer characterized by having (e.g., expressing the LILRB4 protein or comprising the nucleic acid compared to the same tissue of a healthy individual without the LILRB4 protein or nucleic acid or compared to an adjacent healthy tissue of the patient) or having elevated protein and / or nucleic acid levels of LILRB4 (e.g., elevated expression, e.g., compared to the protein and / or nucleic acid levels of LILRB4 in the same tissue of a healthy individual or compared to the protein and / or nucleic acid levels of LILRB4 in an adjacent healthy tissue of the patient) in the patient (e.g., in cancer tissue of the patient), e.g., having (e.g., expressing the LILRB4 protein or comprising the nucleic acid compared to cells of the same tissue of a healthy individual without the LILRB4 protein or nucleic acid or compared to cells of an adjacent healthy tissue of the patient) or having elevated protein and / or nucleic acid levels of LILRB4 (e.g., elevated expression, e.g., compared to the protein and / or nucleic acid levels of LILRB4 in cells of the same tissue of a healthy individual or compared to the protein and / or nucleic acid levels of LILRB4 in healthy cells of the same tissue or adjacent healthy tissue of the patient) in tumor cells of the cancer.

[0394] The anti-LILRB4 antibodies or antigen-binding fragments thereof or multispecific antibodies such as bispecific antibodies of the present invention (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, combination products, etc. comprising the same) can be administered by any suitable method, including parenteral administration, and, if desired for local treatment, intralesional administration. Parenteral injection or infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous injection or infusion. Depending to some extent on whether the medication is short-term or long-term, the medication can be administered by any suitable route, such as by injection, such as intravenous or subcutaneous injection. Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

[0395] For the prevention or treatment of disease, the appropriate dosage of the anti-LILRB4 antibodies, or antigen-binding fragments thereof, or multispecific antibodies, such as bispecific antibodies, of the present invention (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, combination products, etc. comprising the same) (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the administration is for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The molecules are suitably administered to the patient as a single treatment or over a series of treatments. In other aspects, the present invention provides for the use of the anti-LILRB4 antibodies, or antigen-binding fragments thereof, or multispecific antibodies, such as bispecific antibodies, or immunoconjugates, compositions, or combination products comprising the same, of the present invention in the manufacture or preparation of a medicament for the uses described herein, e.g., for the prevention or treatment of the relevant diseases or conditions mentioned herein.

[0396] In some embodiments, an anti-LILRB4 antibody or antigen-binding fragment thereof or a multispecific antibody such as a bispecific antibody (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, etc. comprising the same) can also be administered in combination with one or more other therapies, such as treatment modalities and / or other therapeutic agents, for the uses described herein, e.g., for preventing and / or treating the relevant diseases or conditions mentioned herein.

[0397] In some embodiments, the treatment modality is, for example, radiation therapy.

[0398] In some embodiments, the additional therapeutic agent is, for example, a chemotherapeutic agent, a cytokine, a cytotoxic agent, an additional antibody, a small molecule drug, or an immunomodulatory agent (eg, an immunosuppressant).

[0399] X. Diagnosis and testing

[0400] In one aspect, the present invention also relates to methods for diagnosis and detection (e.g., for diagnostic or non-diagnostic purposes) of anti-LILRB4 antibodies or antigen-binding fragments thereof or multispecific antibodies such as bispecific antibodies and compositions for diagnosis and detection comprising the same.

[0401] In certain embodiments, the anti-LILRB4 antibodies or antigen-binding fragments thereof or multispecific antibodies such as bispecific antibodies provided herein can be used to detect the presence of LILRB4 in a biological sample. In certain embodiments, the anti-bispecific antibodies provided herein can be used to detect the presence of CD3 and / or LILRB4 in a biological sample.

[0402] The term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods can involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays, PCR-techniques (e.g., RT-PCR). In certain embodiments, the biological sample is a body fluid, such as bone marrow, blood, serum, or plasma.

[0403] In certain embodiments, the method comprises contacting a biological sample with an anti-LILRB4 antibody, or an antigen-binding fragment thereof, or a multispecific antibody, such as a bispecific antibody, as described herein, under conditions that allow binding to LILRB4, and detecting whether a complex is formed between the anti-LILRB4 antibody, or an antigen-binding fragment thereof, or a multispecific antibody, such as a bispecific antibody, and LILRB4. The formation of a complex indicates the presence of LILRB4. The method can be an in vitro or in vivo method.

[0404] In certain embodiments, a labeled anti-LILRB4 antibody or antigen-binding fragment thereof or multispecific antibody, such as a bispecific antibody, is provided. Labels include, but are not limited to, directly detectable labels or moieties (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as moieties that are indirectly detected, such as enzymes or ligands, e.g., via an enzymatic reaction or molecular interaction. In some embodiments, the label is, for example, a marker such as biotin or hFc.

[0405] In some embodiments provided herein, the sample is obtained prior to treatment with an anti-LILRB4 antibody, or antigen-binding fragment thereof, or a multispecific antibody, such as a bispecific antibody, of the invention. In some embodiments, the sample is obtained prior to treatment with another therapy. In some embodiments, the sample is obtained during treatment with another therapy, or after treatment with another therapy.

[0406] In some embodiments, LILRB4 is detected prior to treatment, eg, prior to initiation of treatment or prior to a treatment after a treatment interval.

[0407] XI. Specific Implementation Plan

[0408] In some aspects, the present invention relates to the following specific embodiments:

[0409] 1. A VHH antibody that specifically binds to LILRB4, comprising

[0410] three complementarity determining regions (CDRs) contained in the VHH represented by any one of SEQ ID NOs: 11, 16, 21, 25, 30, 32, 36, 39 and 42,

[0411] Preferably, the CDR sequences are defined according to IMGT.

[0412] 2. The VHH antibody of embodiment 1, comprising the complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein

[0413] (i) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 12, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 13, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 14; or

[0414] (ii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 17, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 18, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 19;

[0415] (iii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 75, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 22, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 23;

[0416] (iv) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 26, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 27, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 28;

[0417] (v) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 17, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 33 or 37, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 34 or 40;

[0418] (vi) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 17, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 33 or 37, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 34; or

[0419] (vii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 17, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 33 or 37, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 40.

[0420] 3. The VHH antibody of embodiment 1, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region

[0421] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 11, 16, 21, 25, 30, 32, 36, 39 and 42; or

[0422] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 11, 16, 21, 25, 30, 32, 36, 39 and 42; or

[0423] (iii) an amino acid sequence comprising 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 shown in any one of SEQ ID NOs: 11, 16, 21, 25, 30, 32, 36, 39, and 42, preferably, the amino acid changes do not occur in the CDR regions.

[0424] 4. A heavy chain antibody that specifically binds to LILRB4, comprising the VHH antibody of any one of embodiments 1-3.

[0425] 5. The heavy chain antibody of embodiment 4, comprising the VHH antibody of any one of embodiments 1-3 connected to an antibody constant region or Fc region, preferably, the antibody constant region or Fc region is from human IgG1, human IgG2, human IgG3 or human IgG4, optionally, the VHH antibody is connected to the Fc region via a hinge region or a portion thereof, preferably, the amino acid sequence of the hinge region portion is EPKSS (SEQ ID NO: 59) or EPKSC (SEQ ID NO: 67).

[0426] 6. The heavy chain antibody of embodiment 4, comprising the VHH antibody of any one of embodiments 1-3 linked to an antibody Fc region, wherein the Fc region is an Fc region from human IgG1, IgG2, IgG3 or IgG4, preferably, the Fc region

[0427] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 55 or 68; or

[0428] (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO: 55 or 68; or

[0429] (iii) an amino acid sequence comprising 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 shown in SEQ ID NO: 55 or 68.

[0430] 7. The heavy chain antibody according to embodiment 4, wherein

[0431] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 15, 20, 24, 29, 31, 35, 38, 41 or 43; or

[0432] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NO: 15, 20, 24, 29, 31, 35, 38, 41 or 43; or

[0433] (iii) an amino acid sequence comprising 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 shown in any one of SEQ ID NOs: 15, 20, 24, 29, 31, 35, 38, 41, or 43, preferably, the amino acid changes do not occur in the CDR regions.

[0434] 8. The VHH antibody of any one of embodiments 1-3, or the heavy chain antibody of any one of embodiments 4-7, wherein the antibody is a chimeric antibody or a humanized antibody.

[0435] 9. A bispecific antibody comprising a first antigen-binding region and a second antigen-binding region, wherein the second antigen-binding region specifically binds to LILRB4, and comprises the VHH antibody of any one of embodiments 1-3 and 8, or the heavy chain antibody of any one of embodiments 4-8.

[0436] 10. The bispecific antibody of embodiment 9, wherein the first antigen-binding region specifically binds to CD3.

[0437] 11. The bispecific antibody of embodiment 10, wherein the first antigen-binding region comprises a VH and a VL, wherein the VH comprises three complementarity-determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, and the VL comprises three complementarity-determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, wherein

[0438] (i) HCDR1, HCDR2 and HCDR3 are the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH set forth in SEQ ID NO: 3, and LCDR1, LCDR2 and LCDR3 are the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL set forth in any one of SEQ ID NO: 4; or

[0439] (ii) HCDR1 consists of the amino acid sequence set forth in SEQ ID NO:5, HCDR2 consists of the amino acid sequence set forth in SEQ ID NO:6, HCDR3 consists of the amino acid sequence set forth in SEQ ID NO:7, LCDR1 consists of the amino acid sequence set forth in SEQ ID NO:8, LCDR2 consists of the amino acid sequence set forth in SEQ ID NO:9, and LCDR3 consists of the amino acid sequence set forth in SEQ ID NO:10.

[0440] 12. The bispecific antibody of embodiment 11, wherein the first antigen-binding region comprises VH and VL, wherein

[0441] The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto,

[0442] and / or the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0443] 13. The bispecific antibody according to any one of embodiments 10-12, wherein the first antigen binding region is a Fab that specifically binds to CD3.

[0444] 14. The bispecific antibody of embodiment 13, wherein the Fab comprises CH1, wherein the CH1 is CH1 from IgG1, IgG2, IgG3 or IgG4, preferably CH1 from IgG1.

[0445] 15. The bispecific antibody of embodiment 14, wherein the CH1

[0446] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 51; or

[0447] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 51.

[0448] 16. The bispecific antibody of any one of embodiments 10-12, wherein the first antigen binding region is a scFv that specifically binds to CD3, and the scFv comprises VH and VL as defined in embodiment 11 or 12, optionally, the VH and VL are connected via a linker, and the linker, for example, comprises the amino acid sequence (G4S)n, wherein n=1, 2, 3, 4 or 5, preferably, n=3 or 4, more preferably, n=3.

[0449] 17. The bispecific antibody of any one of embodiments 9-16, wherein the second antigen binding region is the VHH of any one of embodiments 1-3 and 8.

[0450] 18. The bispecific antibody of any one of embodiments 9-17, 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.

[0451] 19. The bispecific antibody of embodiment 18, wherein the two Fc regions are different, preferably, corresponding knob mutations and hole mutations are introduced into the two Fc regions, respectively.

[0452] 20. The bispecific antibody of embodiment 19, wherein

[0453] a) one Fc-region polypeptide comprises the knob mutation T366W and the other Fc-region polypeptide comprises the hole mutations T366S, L368A and Y407V, or

[0454] b) one Fc-region polypeptide comprises the knob mutations T366W and Y349C, and the other Fc-region polypeptide comprises the hole mutations T366S, L368A, Y407V, and S354C, or

[0455] c) one Fc-region polypeptide comprises knob mutations T366W and S354C, and the other Fc-region polypeptide comprises hole mutations T366S, L368A, Y407V, and Y349C;

[0456] Optionally, the Fc region further comprises a mutation that reduces binding to an Fcγ receptor, eg, one or more of a L234A / L235A mutation, a D265A mutation, or a P329A mutation; eg, a L234A / L235A mutation, a D265A mutation, and a P329A mutation.

[0457] 21. The bispecific antibody of any one of embodiments 18-20, wherein one or both of the Fc regions comprise a hinge region, e.g., EPKSS or EPKSC.

[0458] 22. The bispecific antibody of embodiment 21, wherein

[0459] (i) Fc region containing knob mutation

[0460] a) comprises or consists of the amino acid sequence of SEQ ID NO: 49 or 50 or 73; or

[0461] b) comprising or consisting of an amino acid sequence that is at least 90% identical, such as 95%, 96%, 97%, 99% or more identical to SEQ ID NO: 49 or 50 or 73 and comprises knob mutations (such as S354C and T366W); and / or

[0462] (ii) Fc region containing hole mutation

[0463] a) comprises or consists of the amino acid sequence of SEQ ID NO: 47 or 48 or 74; or

[0464] b) comprising or consisting of an amino acid sequence that is at least 90% identical, e.g., 95%, 96%, 97%, 99% or more identical to SEQ ID NO: 47 or 48 or 74 and comprises hole mutations (e.g., Y349C, T366S, L368A and Y407V).

[0465] 23. The bispecific antibody of any one of embodiments 9-22, comprising a first antigen-binding region, a second antigen-binding region and an Fc dimer, wherein the first antigen-binding region is a Fab fragment that specifically binds to CD3, and the second antigen-binding region is a VHH that specifically binds to LILRB4, such as the VHH of any one of embodiments 1-3 and 8. For example, the bispecific antibody comprises one first antigen-binding region, and 1 or 2 second antigen-binding regions.

[0466] 24. The bispecific antibody of embodiment 23, comprising an anti-LILRB4 VHH, an anti-CD3 antibody Fab fragment and an Fc heterodimer, wherein

[0467] The Fab fragment comprises VH-CH1 and VL-CL, wherein the VHH comprises a heavy chain variable region,

[0468] wherein the C-terminus of CH1 of the Fab fragment is fused to the CH2 or hinge region of the first Fc region (e.g., an Fc region comprising a Knob mutation) to constitute the first heavy chain;

[0469] The VHH is fused to the CH2 or hinge region of a second Fc region (e.g., an Fc region comprising a Hole mutation) to form a second heavy chain;

[0470] and

[0471] The VL-CL of the Fab fragment constitutes the light chain.

[0472] 25. The bispecific antibody of embodiment 24, wherein the

[0473] The first heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:44, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence;

[0474] The second heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 45, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; and / or

[0475] The light chain comprises or consists of the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence.

[0476] 26. The bispecific antibody of embodiment 23, comprising two anti-LILRB4 VHHs, a Fab fragment of an anti-CD3 antibody and an Fc heterodimer, wherein

[0477] The Fab fragment comprises VH-CH1 and VL-CL, wherein the VHH comprises a heavy chain variable region,

[0478] wherein the C-terminus of the first VHH fragment is fused to the N-terminus of the VH of the Fab fragment and the C-terminus of the CH1 of the Fab fragment is fused to the CH2 or hinge region of the Fc region (e.g., comprising a knob mutation), constituting a first heavy chain;

[0479] The second VHH is fused to the CH2 or hinge region of the Fc region (e.g., comprising a hole mutation) to form the second heavy chain;

[0480] and

[0481] The VL-CL of the Fab fragment constitutes the light chain.

[0482] 27. The bispecific antibody of embodiment 26, wherein the first VHH and the second VHH are the same or different.

[0483] 28. The bispecific antibody of embodiment 26 or 27, wherein the fusion of the first VHH to the Fab is via a connecting peptide, such as (GGGGS)n, wherein n=1, 2, 3 or 4.

[0484] 29. The bispecific antibody of embodiment 26, wherein the

[0485] The first heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:46, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence;

[0486] The second heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 45, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; and / or

[0487] The light chain comprises or consists of the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence.

[0488] 30. The bispecific antibody of embodiment 23, comprising two anti-LILRB4 VHHs, an anti-CD3 antibody scFv and an Fc heterodimer, wherein

[0489] The scFv comprises VH and VL in sequence from N-terminus to C-terminus, wherein the VHH comprises a heavy chain variable region,

[0490] 31. The bispecific antibody of embodiment 30, wherein the VH and VL of the scFv are connected by a linker or a connecting peptide, for example, the linker or connecting peptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 70.

[0491] 32. The bispecific antibody of embodiment 30 or 31, wherein the first VHH and the second VHH are the same or different.

[0492] 33. The bispecific antibody of any one of embodiments 30-32, wherein the fusion of the first VHH to the scFv is via a connecting peptide, such as (GGGGS)n, wherein n=1, 2, 3 or 4.

[0493] 34. The bispecific antibody of embodiment 30, wherein the

[0494] The first heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 72, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; and / or

[0495] The second heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:45, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence.

[0496] 35. A multispecific antibody comprising the VHH antibody of any one of embodiments 1-3 and 8, or the heavy chain antibody of any one of embodiments 4-8, eg, the multispecific antibody is a bispecific antibody.

[0497] 36. A nucleic acid molecule encoding the VHH antibody of any one of embodiments 1-3 and 8, or the heavy chain antibody of any one of embodiments 4-8, or any chain of the bispecific antibody of any one of embodiments 9-34, or any chain of the multispecific antibody of embodiment 35, or consisting of said nucleic acid sequence.

[0498] 37. An expression vector comprising the nucleic acid molecule of embodiment 36.

[0499] 38. A host cell comprising the nucleic acid molecule of embodiment 36 or the expression vector of embodiment 37. Preferably, the host cell is prokaryotic or eukaryotic, such as 293 cells or CHO cells, such as 293F cells or 293T cells or CHO-S cells.

[0500] 39. A method for preparing the VHH antibody of any one of embodiments 1-3 and 8, or the heavy chain antibody of any one of embodiments 4-8, or the bispecific antibody of any one of embodiments 9-34, or the multispecific antibody of embodiment 35, the method comprising culturing a host cell comprising the nucleic acid molecule of embodiment 36 or the expression vector of embodiment 37 under conditions suitable for expression of the chains of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0501] 40. An immunoconjugate comprising the VHH antibody of any one of embodiments 1-3 and 8, or the heavy chain antibody of any one of embodiments 4-8, or the bispecific antibody of any one of embodiments 9-34, or the multispecific antibody of embodiment 35.

[0502] 41. A pharmaceutical composition, medicament or formulation comprising the VHH antibody of any one of embodiments 1-3 and 8, or the heavy chain antibody of any one of embodiments 4-8, or the bispecific antibody of any one of embodiments 9-34, or the multispecific antibody of embodiment 35, or the immunoconjugate of embodiment 40, and optionally a pharmaceutically acceptable excipient.

[0503] 42. A pharmaceutical combination product comprising the VHH antibody of any one of embodiments 1-3 and 8, or the heavy chain antibody of any one of embodiments 4-8, or the bispecific antibody of any one of embodiments 9-34, or the multispecific antibody of embodiment 35, or the immunoconjugate of embodiment 40, and one or more other therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators).

[0504] 43. A method for preventing or treating cancer in a subject, comprising administering to the subject an effective amount of the VHH antibody of any one of embodiments 1-3 and 8, or the heavy chain antibody of any one of embodiments 4-8, or the bispecific antibody of any one of embodiments 9-34, or the multispecific antibody of embodiment 35, or the immunoconjugate of embodiment 40, or the pharmaceutical composition or drug or formulation of embodiment 41; or the pharmaceutical combination product of embodiment 42.

[0505] 44. The method of embodiment 43, wherein the cancer has or has elevated protein and / or nucleic acid levels (e.g., elevated expression) of LILRB4 in tumor cells.

[0506] 45. The method of embodiment 43 or 44, wherein the cancer is a solid tumor or a blood tumor, such as a solid tumor, multiple myeloma or a leukemia, such as acute myeloid leukemia (AML), such as monocytic AML and non-monocytic AML, such as human myelomonocytic leukemia cells, human promyelocytic acute leukemia or human acute myeloid leukemia.

[0507] 46. ​​The method of any one of embodiments 43-45, wherein the method further comprises administering the drug in combination with other therapies, such as treatment modalities (e.g., radiotherapy) and / or other therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators).

[0508] 47. A method for detecting the presence of LILRB4 in a biological sample, comprising

[0509] (i) contacting a biological sample with the VHH antibody of any one of embodiments 1-3 and 8, or the heavy chain antibody of any one of embodiments 4-8, or the bispecific antibody of any one of embodiments 9-34, or the multispecific antibody of embodiment 35 under conditions that allow binding thereof to LILRB4,

[0510] (ii) detecting whether a complex is formed between the antibody or bispecific antibody and LILRB4,

[0511] The formation of the complex indicates the presence of LILRB4. Example

[0512] Example 1: Immunization to generate LILRB4 antibodies

[0513] The anti-human LILRB4 antibody of the present invention can be prepared by the following method. However, the preparation method of the anti-human LILRB4 antibody of the present invention is not limited to this method, and can also be prepared by other methods known in the art.

[0514] 1.1 Alpaca immune library construction and screening

[0515] 1.1.1 Animal immunization

[0516] Two alpacas (Chengdu Apak Biotechnology Co., Ltd.) were immunized with human LILRB4-his recombinant protein (ACRO, Catalog No. LI4-H52H7). Four 21-day immunizations were administered to the alpacas (Table 1). Venous blood was collected from the alpacas 10 days after the second and third booster immunizations. Mouse immune sera were assayed by ELISA using human LILRB4-his recombinant protein. One alpaca was selected for blood collection and library establishment.

[0517] Table 1. Alpaca immunization schedule

[0518] 1.1.2 Construction and screening of alpaca immune library

[0519] Total RNA was extracted from alpaca peripheral blood using the Trizol method and reverse transcribed using the PrimeScript II 1st Strand cDNA Synthesis Kit (Takara, Cat. No. 6210A) to obtain cDNA. Nested PCR amplification of the cDNA was performed to recover a 750 bp product containing the nanobody, which was then purified by a second round of PCR amplification. The VHHs were then ligated into phage vectors using enzyme digestion and ligation, followed by electroporation, transformation, and plate plating. The next day, the library capacity was determined to be 2 × 10e8 CFU, with an insertion rate of 100%. After two rounds of solid-phase selection with human LILRB4 recombinant protein, positive clones were identified by sequencing. The partial amino acid sequences obtained are shown in Table 2.

[0520] Table 2. Amino acid sequences of camel-derived VHHs of anti-LILRB4 antibodies

[0521] Example 2: Characterization of anti-LILRB4 antibodies

[0522] 2.1 Synthesis and expression of anti-LILRB4 chimeric antibodies

[0523] The amino acid sequences of the camel-derived antibodies VHH in Table 2 were codon-optimized and gene-synthesized by Universal Biosystems (Anhui) Co., Ltd. The variable regions encoding each VHH antibody were added to the hinge region (SEQ ID NO: 59) and then inserted into the expression vector pcDNA3.1(+) containing the human Fc constant region (SEQ ID NO: 55) to obtain plasmids encoding each antibody.

[0524] The specific chimeric antibody sequences obtained are as follows:

[0525] Table 3. Amino acid sequences of anti-LILRB4 chimeric antibodies

[0526] Plasmids encoding anti-LILRB4 antibodies were transfected into ExpiCHO-S cells to express camelid chimeric antibodies. h193 (a fully human monoclonal antibody against LILRB4) (heavy chain amino acid sequence: SEQ ID NO: 61, light chain amino acid sequence: SEQ ID NO: 62) was used as a control antibody, the sequences of which are from PCT Publication No. WO2020056077A1. Similarly, control antibody h193 was expressed and purified accordingly.

[0527] Specifically, use ExpiCHO according to the manufacturer's product manual. TM ExpiCHO-S cells were transfected with a plasmid encoding an anti-LILRB4 chimeric antibody using an expression system (ThermoFisher, Cat# A29133), and a control antibody, h193, was similarly expressed. Cells were cultured for 10-12 days after transfection. When cell viability dropped to 60% to 70%, the supernatant was collected and the expressed and secreted antibody in the supernatant was purified using a MabSelect Sure Protein A affinity chromatography system (GE healthcare). The purified antibody was concentrated and sterile filtered, and the purity of the antibody protein was tested by SDS-PAGE and size exclusion. The results showed that the antibody purity was >90% and was ready for use in the next step of the experiment.

[0528] 2.2 Anti-LILRB4 chimeric antibodies bind to human and cynomolgus monkey LILRB4-engineered cells

[0529] Cell binding experiments were performed to determine whether the anti-LILRB4 chimeric antibody of the present invention could bind to engineered cells expressing human or cynomolgus monkey LILRB4 protein.

[0530] The human LILRB4 sequence (Q8NHJ6, SEQ ID NO: 65) was transfected into CHO-K1 cells (ATCC, catalog number: CCL-61) by electroporation. TM ), and then screened with 24 μg / mL puromycin (Gibco, Catalog No.: A1113802) to obtain CHO-K1 cells that highly express human LILRB4 (CHO-K1-huLILRB4).

[0531] The sequence encoding cynomolgus monkey LILRB4 (XP_015297198.1, SEQ ID NO: 66) was transfected into CHO-K1 cells (ATCC, catalog number: CCL-61) by electroporation. TM ) or 293T cells (Cell Bank of Type Culture Collection of the Chinese Academy of Sciences, GNHu17), and then screened with 24 μg / mL (for CHO-K1 cells) or 0.3 μg / mL (for 293T cells) puromycin to obtain CHO-K1 cells (CHO-K1-cynoLILRB4) and 293T cells (293T-cynoLILRB4) that highly express cynomolgus macaque LILRB4.

[0532] Adherent CHO-K1-huLILRB4, CHO-K1-cynoLILRB4, and 293T-cynoLILRB4 cell lines were enzymatically digested to obtain single-cell suspensions. The suspensions were centrifuged at 300 × g for 4 minutes at room temperature, and the culture medium was discarded. The resulting cell pellets were resuspended and washed once with PBS buffer, and the supernatant was discarded to obtain another cell pellet. The cell pellets were resuspended in a serial dilution series of anti-LILRB4 chimeric antibodies (initial concentration: 20 μg / mL, 4-fold serial dilutions, totaling 8 concentrations) and incubated at 4°C for 30 minutes. The cells were washed once with PBS, and then a 1:200 dilution of the fluorescent secondary antibody, R-PE-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific (Jackson ImmunoResearch, Cat# 109-116-098), was added and incubated at 4°C in the dark for 30 minutes. The cells were washed twice with PBS and resuspended. Finally, the fluorescence signal was detected by flow cytometry in two channels. The results are shown in Figures 2A and 2B.

[0533] Figure 2A shows that both anti-LILRB4 chimeric antibodies (labeled as antibody B1A2 and antibody B1G5) and antibody h193 can bind to engineered cells expressing human / cynomolgus monkey LILRB4. Figure 2B shows that both chimeric anti-LILRB4 antibodies (labeled as 2A3 and antibody 1H12) and antibody h193 can bind to cells expressing human / cynomolgus monkey LILRB4, and the binding activity is comparable to that of the control antibody h193.

[0534] Example 3: Humanization and characterization of anti-LILRB4 antibodies

[0535] 3.1 Humanization of alpaca-derived anti-LILRB4 antibody

[0536] Antibody B1G5 obtained in Example 2.1 was humanized. Specifically, the sequence of antibody B1G5 was searched and aligned in the IMGT database. The human germline gene sequence IGHV3-7*01, which showed high homology to the variable region of antibody B1G5, was obtained as the humanized framework for the heavy chain variable region. The CDRs in the heavy chain variable region of antibody B1G5 were transplanted into the corresponding humanized framework to form the humanized antibody B1G5. To maintain the affinity of the humanized antibody for LILRB4, the obtained humanized antibody framework region was backmutated.

[0537] Table 4. Heavy chain and variable region sequences of humanized antibodies

[0538] 3.2 Expression and purification of humanized anti-LILRB4 antibody

[0539] Similar to Example 2.1, plasmids encoding full-length anti-LILRB4 humanized antibodies were transfected into ExpiCHO-S cells to express each anti-LILRB4 humanized antibody, and the corresponding anti-LILRB4 humanized antibodies were purified as described in Example 2.1. The purified anti-LILRB4 humanized antibodies were concentrated and sterile filtered, and the purity of the anti-LILRB4 humanized antibodies was determined by SDS-PAGE and size exclusion chromatography (SEC).

[0540] 3.3 Physicochemical Analysis of Humanized Anti-LILRB4 Antibodies

[0541] For the obtained humanized anti-LILRB4 antibodies, the purity of each antibody was confirmed by molecular exclusion chromatography. Specifically, 100mM sodium phosphate + 100mM Na2SO4 (pH 7.0) was used as the running buffer, and 20μg of each humanized anti-LILRB4 antibody sample in Table 4 was injected into the TSK G3000SWXL column. The column was run for 30 minutes. The collected effluent was measured using an Agilent 1220HPLC, and the data was analyzed using OpenLAB software. The results showed that the purity of each humanized anti-LILRB4 antibody was >90% and could be used for the next experiment.

[0542] 3.4 Binding of humanized anti-LILRB4 antibodies to tumor cell lines

[0543] Cell binding experiments were performed to determine whether the anti-LILRB4 humanized antibody of the present invention could bind to tumor cells expressing LILRB4.

[0544] MV-4-11 (human myelomonocytic leukemia, Cell Bank, Chinese Academy of Sciences, Cat# SCSP-5031) cells were harvested and mixed by pipetting to obtain a single-cell suspension. The suspension was centrifuged at 300 × g for 4 minutes at room temperature, and the culture medium was discarded. The resulting cell pellet was washed once with PBS. The cells were then resuspended in a serial dilution series of humanized anti-LILRB4 antibodies (initial concentration: 200 nM, 4-fold serial dilutions, 8 concentrations total) and incubated at 4°C for 30 minutes. The cells were washed once with PBS, and then a 1:200 dilution of the fluorescent secondary antibody, R-PE-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific (Jackson ImmunoResearch, Cat# 109-116-098), was added and incubated at 4°C in the dark for 30 minutes. The cells were washed twice with PBS and resuspended. Finally, the fluorescence signal in two channels was detected by flow cytometry. The results are shown in Figure 3.

[0545] FIG3 shows that both humanized anti-LILRB4 antibodies (labeled as antibody huB1G5-1 and antibody huB1G5-3) and antibody h193 can bind to tumor cells expressing LILRB4 and the binding activity is comparable to that of the control antibody h193.

[0546] Example 4: Preparation and characterization of humanized CD3 antibodies

[0547] 4.1 Preparation for CD3 Antibody Humanization

[0548] The SP34 antibody is a murine antibody that specifically binds to the CD3 epsilon subunit (CD3ε) and has cross-species activity with humans and monkeys (Pessano, S., et al. (1985). "The T3 / T cell receptor complex: antigenic distinction between the two 20-kd T3 (T3-delta and T3-epsilon) subunits." EMBO J 4(2):337-344.). In this example, the SP34 antibody was used as a candidate molecule for humanization.

[0549] Mouse anti-SP34 was humanized using CDR grafting technology. Briefly, the SP34 VH and VL sequences were searched and aligned in the IMGT database to obtain the human germline gene sequences IGHV3-73*01 and IGLV7-46*01, which showed high homology to the SP34 heavy and light chain variable regions, respectively. These sequences served as frameworks for the humanized antibody variable regions. The CDRs of mouse anti-SP34 were then grafted onto the corresponding humanized antibody heavy and light chain variable region frameworks, respectively, to form the humanized anti-CD3 antibody variable regions. To maintain the affinity of the mouse SP34 antibody, the resulting humanized antibody variable regions were backmutated.

[0550] In this way, the humanized heavy chain variable region sequence (hu34H1) and light chain variable region sequence (hu34L2) of the SP34 antibody were obtained, and then the SP34 antibody humanized antibody was obtained. The specific sequences are shown in Table 5.

[0551] Table 5. Sequences of humanized anti-CD3 antibodies (CDR sequences all use Kabat encoding)

[0552] The sequence of the humanized control antibody (017-Roche-CD3) is from patent US2016 / 0075785A1, and its heavy chain amino acid sequence is SEQ ID NO: 63, and its light chain is SEQ ID NO: 64. For details, please refer to the sequence listing.

[0553] The variable region amino acid sequences of Table 5 and the control antibody 017-Roche-CD3 were sent to General Biology System (Anhui) Co., Ltd. for codon optimization and gene synthesis. The genes encoding the VH and VL regions of the 017-Roche-CD3 antibody were sequentially inserted into the expression vector pcDNA3.1(+) containing the gene encoding the human IgG1 heavy chain constant region (amino acid sequence SEQ ID NO: 52) and the lambda light chain constant region (amino acid sequence SEQ ID NO: 56) to obtain a plasmid expressing the full-length heavy chain hu34H1 (SEQ ID NO: 1) of the anti-CD3 humanized antibody 017-2 and a plasmid encoding the full-length light chain hu34L2 (SEQ ID NO: 2). The synthesized VH region (hu34H1) and VL region (hu34L2) were simultaneously linked via a (G4S)3 linker peptide (SEQ ID NO: 70) to form the single-chain antibody 23L2 (23L2ScFv) (SEQ ID NO: 69). This was then inserted into the expression vector pcDNA3.1(+) containing the gene encoding the human IgG1 heavy chain constant region Fc region (amino acid sequence SEQ ID NO: 53), generating a plasmid expressing DA023AH23L2 (scFv-Fc) (SEQ ID NO: 71). The heavy chain constant regions used all contained mutations at the L234A, L235A, D265A, and P329A (according to Eu encoding) sites to eliminate effector function.

[0554] The single-chain antibody (23L2) was designated 23L2ScFv, and its amino acid sequence is shown in SEQ ID NO: 69. A synthetic anti-CD3 single-chain antibody containing a constant Fc domain was designated DA023AH23L2 (VH-connector-VL-Fc), and its sequence is shown in SEQ ID NO: 71, wherein the amino acid sequence of VH-connector-VL is SEQ ID NO: 69, the amino acid sequence of the connector is SEQ ID NO: 70, and the amino acid sequence of the Fc region is SEQ ID NO: 53.

[0555] Use ExpiCHO according to the manufacturer's product manual. TMThe plasmids obtained above were co-transfected into ExpiCHO-S cells expressing control antibodies 017-Roche-CD3, 017-2, and DA023AH23L2 using an expression system (ThermoFisher, Cat#A29133). Cells were cultured for 10-12 days after transfection. When cell viability dropped to 60% to 70%, the supernatant was collected and the antibodies expressed in the supernatant were purified using the MabSelect Sure Protein A affinity chromatography system (GE healthcare). The purified antibodies were concentrated and sterile filtered, and the purity of the antibody protein was determined by SDS-PAGE and size exclusion. The results showed that the antibody purity met the requirements and was suitable for use in the next step of the experiment.

[0556] 4.2 Detection of the activation effect of humanized CD3 antibodies

[0557] The activation of CD3 antibodies was detected using the Jurkat / NFAT-luc (luciferase) reporter gene system. Following the manufacturer's instructions, the reporter gene element in NFAT-luc2P (plasmid: Promega, Cat#E8481) was transfected into Jurkat (Cell Bank of the Chinese Academy of Sciences, Cat#SCSP-513) cells by electroporation using Gene Pul Ser X Cell™ (BIO-RAD). NFAT-luc2P polyclonal cell lines were then selected with hygromycin B (Invitrogen, Cat#10687010). Stable and highly expressing NFAT-luc2P monoclonal cell lines, Jurkat / NFAT-luc, were obtained by limiting dilution of the polyclonal cells.

[0558] The cells were collected, centrifuged and the supernatant was removed. The cells were resuspended in assay buffer (99% 1640 medium + 1% FBS) to a cell density of 5 × 10 5 / mL. Cells were seeded in a 384-well plate (Corning, Cat#3570) at 40 μL per well. A serial dilution of humanized anti-CD3 antibodies DA023AH23L2, 017-2, and 017-Roche-CD3 was then added (starting at a final concentration of 20 nM, with a three-fold dilution series in eight steps) at 10 μL per well. The 384-well plate was placed in a 37°C incubator. After 6 hours, 30 μL of One-Glo (Promega, Cat#E6130) reagent was added to each well. Luminescence was measured using a multi-function microplate reader (Tecan Infinite F200PRO). Luciferase expression directly reflects the activation effect of the CD3 antibodies. The results, shown in Figure 4, demonstrate activation by 017-2 and DA023AH23L2.

[0559] Example 5 Preparation and affinity detection of anti-LILRB4×CD3 bispecific antibody

[0560] 5.1 Bispecific Antibody Construction

[0561] The present invention constructs three bispecific antibodies with different asymmetric structures as shown in FIG1 , wherein the anti-LILRB4 portion of each bispecific antibody is derived from the above-mentioned humanized huB1G5-3 antibody, and the anti-CD3 portion is derived from the above-mentioned humanized CD3 antibody. The constant regions of the bispecific antibodies all contain a knob-in-hole structure (KIH) (Merchant, AM, et al. (1998). "An efficient route to human bispecific IgG." Nat Biotechnol 16(7):677-681.). Such bispecific antibodies are also referred to herein as "anti-CD3 / LILRB4 bispecific antibodies" or "anti-CD3 / anti-LILRB4 bispecific antibodies," and sometimes referred to simply as "bispecific antibody molecules, bispecific antibodies." The anti-LILRB4 portion of the anti-CD3 / LILRB4 bispecific antibody targets cells expressing LILRB4, while the anti-CD3 portion activates T cells. The bispecific antibody simultaneously binds to LILRB4 on tumor cells and CD3 on T cells, which helps to target tumor cells through activated T cells.

[0562] This application uses standard construction methods to obtain three anti-CD3 / LILRB4 bispecific antibodies with the structures shown in Figure 1:

[0563] CD3Fab heavy chain-knob: The sequence encoding the heavy chain variable region of the anti-CD3 humanized monoclonal antibody was cloned into the pcDNA3.1(+) vector containing the IgG1 heavy chain constant region containing the Fc (knob) to express the CD3Fab heavy chain-knob molecule.

[0564] huB1G5-hole: The variable region sequence of the anti-LILRB4 humanized antibody huB1G5-3 was cloned into the pcDNA3.1(+) vector containing Fc(hole) to express the huB1G5-hole molecule.

[0565] huB1G5-CD3Fab-knob: Using overlapping PCR, the sequences encoding the huB1G5-3 variable region and the heavy chain variable region of the humanized anti-CD3 monoclonal antibody were concatenated (with a linker GGGGS between the two coding fragments). The concatenated sequences were then cloned into the pcDNA3.1(+) vector containing the IgG1 heavy chain constant region with the Fc(knob) to express the huB1G5-CD3Fab heavy chain-knob molecule.

[0566] CD3Fab light chain: The variable region encoding the anti-CD3 humanized mAb light chain was cloned into the light chain constant region.

[0567] CD3ScFv heavy chain-knob: The nucleotide sequence encoding the anti-CD3 single-chain antibody 23L2 was cloned into the pcDNA3.1(+) vector with Fc(knob) to express the 23L2ScFv-knob molecule.

[0568] huB1G5-Hu34ScFv-knob: Using overlapping PCR, the sequences encoding the huB1G5-3 variable region and the anti-CD3 humanized ScFv were concatenated (with a linker GGGGS between the two coding fragments). The concatenated sequences were then cloned into the pcDNA3.1(+) vector containing the IgG1 heavy chain constant region containing the Fc (knob) to express the huB1G5-CD3Fab heavy chain-knob molecule.

[0569] According to the specific composition shown in Table 6, the above-mentioned expression vectors were combined accordingly and expressed under appropriate conditions to obtain the bispecific antibody in Figure 1.

[0570] The steps for construction, expression, purification, and preliminary analysis of the bispecific antibody were the same as those in Example 2.1.

[0571] Table 6. Humanized bispecific antibodies

[0572] The obtained heavy chain and light chain expression vectors (see Table 6) were co-transfected into ExpiCHO-S cells, and the bispecific antibody protein was expressed under appropriate conditions. The expression, purification, and preliminary analysis steps were the same as in Example 2.1.

[0573] In addition, the amino acid sequence of the synthetic control antibody 4ab3-4ab S91A (CN 115551894 A) (SEQ ID NOs: 76, 77, 78, and 79) was codon-optimized and gene-synthesized by Universal Biosystems (Anhui) Co., Ltd. The expression and purification methods were as described in CN 115551894 A.

[0574] 5.2 Affinity Detection of Bispecific Antibodies to Human / Cynomolgus Monkey LILRB4 and CD3

[0575] In this experiment, the binding affinity of huB1G5-CD3Fab, huB1G5-CD3-2VHH, and huB1G5-ScFv-2VHH bispecific antibodies to human LILRB4 protein (ACRO Biosystems, Catalog No.: LI4-H52H7), cynomolgus macaque LILRB4 protein (ACRO Biosystems, Catalog No.: CDK-C5227), human CD3 protein (Sino Biological, Catalog No.: CT038-H2508H), or cynomolgus macaque CD3 protein (ACRO Biosystems, Catalog No.: CDD-C52W4) was detected using ForteBio Octet RED96e according to the manufacturer's instructions.

[0576] Briefly, an AHC sensor (ForteBio, Catalog No. 18-5060) was placed in running buffer (1X PBS, XiGene, Catalog No. XG3650, containing 0.02% Tween 20, 0.1% BSA, pH 7.0) and pre-equilibrated at room temperature for 10 min.

[0577] The LILRB4 kinetic assay was performed in a 96-well plate format as follows:

[0578] a) Equilibrate the baseline with running buffer for 180 s;

[0579] b) adding each bispecific antibody diluted in running buffer to a final concentration of 5 μg / mL and solidifying for 200 s;

[0580] c) Equilibrate the baseline with running buffer for 300 s;

[0581] d) Add the following concentrations of human LILRB4 protein or cynomolgus monkey LILRB4 protein diluted in running buffer to each well: 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, and 1.56 nM. Bind for 200 s and dissociate for 600 s.

[0582] e) Regeneration solution (0.01 M Gly-HCl, pH 1.5) for 30 seconds. The experimental data were fitted and calculated using the 1:1 binding model using Fortebio Data Analysis software;

[0583] The CD3 kinetics assay was performed in a 96-well plate format using the following steps:

[0584] a) Equilibrate the baseline with running buffer for 300 s;

[0585] b) adding each bispecific antibody diluted in running buffer to a final concentration of 5 μg / mL and solidifying for 200 s;

[0586] c) Equilibrate the baseline with running buffer for 200 s;

[0587] d) Add the following concentrations of human CD3 protein or cynomolgus CD3 protein diluted in running buffer to each well: 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.13 nM. Bind for 120 s and dissociate for 400 s.

[0588] e) Regeneration solution (0.01 M Gly-HCl, pH 1.5) for 30 seconds. The experimental data were fitted and calculated using the 1:1 binding model using Fortebio Data Analysis software;

[0589] Table 7 summarizes the binding affinities of huB1G5-CD3Fab, huB1G5-CD3-2VHH, and huB1G5-ScFv-2VHH bispecific antibodies to human LILRB4 protein, cynomolgus monkey LILRB4 protein, human CD3 protein, or cynomolgus monkey CD3 protein.

[0590] Table 7. Binding affinity of bispecific antibodies to LILRB4 and CD3 proteins

[0591] Table 7 shows that the bispecific antibodies of the present invention can specifically bind to human LILRB4 protein, cynomolgus monkey LILRB4 protein, human CD3 protein or cynomolgus monkey CD3 protein. The K values ​​of the bispecific antibodies huB1G5-CD3Fab, huB1G5-CD3-2VHH and huB1G5-ScFv-2VHH against human LILRB4 protein are 0. D The K value of human CD3 protein D The anti-LILRB4×CD3 bispecific antibodies huB1G5-CD3Fab, huB1G5-CD3-2VHH, and huB1G5-ScFv-2VHH target human LILRB4 with high affinity, effectively reducing the side effects of targeting human CD3.

[0592] Example 6 Cytotoxicity of anti-LILRB4×CD3 bispecific antibody against tumor cells

[0593] To detect the activity of anti-LILRB4×CD3 bispecific antibodies in mediating T cell-mediated cytotoxicity against tumor cells, we established a primary T cell-dependent cytotoxicity assay system using HL-60 (a human promyelocytic acute leukemia cell line, source: Chinese Academy of Sciences, TCHu 23) cells endogenously expressing human LILRB4 as target cells and T cells isolated from human peripheral blood mononuclear cells (PBMCs) as effector cells.

[0594] 6.1 Cytotoxicity of huB1G5-CD3Fab and huB1G5-CD3-2VHH Bispecific Antibodies Against Tumor Cells

[0595] PBMC cells (Shanghai Aoneng Biotechnology Co., Ltd.) were revived and the cell density was adjusted to 1-2 × 10 cells / mL using 1640+10% FBS complete medium. 6 / mL, and activated overnight with the addition of IL-2 (National Medicine Standard No.: S10970058) at a final concentration of 100 IU / mL. The next morning, PBMC cell suspensions were collected and all T cells were isolated using the T cell isolation kit (Stemcell, Cat#17951RF). After centrifugation at 400 g for 5 min, cells were resuspended in MEM-α (Gibco, Cat#41061-029) assay buffer containing 1% FBS (Gibco, Cat#10099-141) and 100 IU / mL IL-2. The cells were counted and the T cell density was adjusted to 2 × 10 5 / mL, 100 μL per well (i.e., 20,000 cells per well) was plated into a 96-well plate (Corning, Cat#3599). Cultured target HL-60 cells were collected by centrifugation at 200g for 5 minutes, the supernatant was discarded, the cells were resuspended in PBS, centrifuged at 200g for 5 minutes, the supernatant was discarded, the cells were resuspended in PBS and counted, and CellTrace was added at a dilution of 1:2000. TM Violet cell proliferation reagent dye (Thermo, C34557) was placed at 37°C for 15 minutes. Five volumes of complete medium were added to terminate the staining. The cells were centrifuged at 200 g for 5 minutes, the supernatant was discarded, and 3 mL of complete medium was added to wash the cells. The cells were resuspended in assay buffer, counted, and the target cell density was adjusted to 2.67 × 10 5100 μL of the test antibody was plated into the 96-well plate (Effector:Target = 1:1) at 75 μL per well (i.e., 20,000 cells per well). The test antibody was prepared at 8x the final assay concentration, with the highest assay concentration being 1 nM. A 5-fold serial dilution was performed to obtain a total of 9 concentration points, with 25 μL added to each well. Minimal killing (target cells plus assay buffer) and natural killer (target cells plus effector cells) controls were also included. The final total volume was 200 μL, and the cells were incubated in a cell culture incubator for a further 22 hours. After incubation, the plates were removed and centrifuged at 2000 rpm for 3 minutes to allow all cells to settle to the bottom of the plate. 100 μL of the supernatant was carefully aspirated into a 96-well V-bottom plate (NEST, 701211). TNFα (PerkinElmer, 62HTNFAPEH) and IFNγ (PerkinElmer, 62HIFNGPEH) cytokine assays were performed according to the cytokine assay kit instructions. The remaining 100 μL of cell suspension was collected into a new 96-well V-bottom plate and centrifuged at 400 g for 3 min. The supernatant was discarded and 100 μL of PBS containing PI dead-alive dye (Biyuntian, ST511) was added to each well to resuspend the cell pellet. After incubation at room temperature for 5 min, the death percentage of target cells was detected by flow cytometry.

[0596] The results, shown in Figures 5A-C, demonstrate that the anti-LILRB4×CD3 bispecific antibodies (labeled as huB1G5-CD3-2VHH and huB1G5-CD3Fab) can still specifically induce T cell killing of HL-60 tumor cells at a lower efficacy-target ratio, with huB1G5-CD3-2VHH exhibiting superior killing activity compared to huB1G5-CD3Fab. Detection of inflammatory cytokine TNFα and IFNγ release was consistent with the killing effect.

[0597] 6.2 Cytotoxicity of huB1G5-CD3-2VHH and huB1G5-ScFv-2VHH Bispecific Antibodies Against Tumor Cells

[0598] PBMC cells (Shanghai Aoneng Biotechnology Co., Ltd.) were revived and the cell density was adjusted to 1-2 × 10 cells / mL using 1640+10% FBS complete medium. 6 / mL, add IL-2 at a final concentration of 100 IU / mL and activate overnight. The next morning, collect PBMC cell suspension, and sort all T cells using the T cell isolation kit (Stemcell, Cat#17951RF). Centrifuge at 400g for 5 minutes, discard the supernatant, resuspend in PBS, centrifuge at 200g for 5 minutes, discard the supernatant, resuspend in PBS and count, and add CellTrace at a dilution of 1:2000. TMViolet cell proliferation reagent dye (Thermo, C34557) was added and placed at 37°C for 15 min. Five volumes of complete medium were added to terminate the staining, and the cells were centrifuged at 200 g for 5 min. 3 mL of complete medium was added to wash the cells again. The cells were resuspended in RPMI 1640 medium (Gibco, Cat#10099-141) containing 10% FBS (Gibco, Cat#10099-141) and 100 IU / mL IL-2 assay buffer. The cells were counted and the T cell density was adjusted to 2 × 10 5 / ml, 100 μL per well (i.e., 20,000 cells per well) was plated into a 96-well plate (Corning, Cat#3599). Cultured target HL-60 cells were collected and centrifuged at 200g for 5 minutes, the supernatant was discarded, and the cells were resuspended in PBS. Centrifuged at 200g for 5 minutes, the supernatant was discarded, and the cells were resuspended in PBS and counted. CytoTell red 650 cell proliferation reagent dye (AAT Bioquest, 22255) was added at a dilution of 1:2000, and the cells were placed at 37°C for 15 minutes. Five volumes of complete medium were added to terminate the staining, and the cells were centrifuged at 200g for 5 minutes. The supernatant was discarded, and 3 mL of complete medium was added to wash the cells once. The cells were resuspended in assay buffer, counted, and the target cell density was adjusted to 2.67×10 5 / ml, plate 75 μL per well (i.e., 20,000 cells per well) into the aforementioned 96-well plate (Effector:Target = 1:1). Prepare the test antibody at 8x the final assay concentration, with the highest assay concentration being 1 nM. Perform a 5-fold serial dilution to obtain a total of 9 concentration points, with 25 μL added to each well. Minimal killing (target cells plus assay buffer) and natural killer (target cells plus effector cells) controls were also included. The final total volume was 200 μL, and the cells were incubated in a cell culture incubator for a further 21 hours. Collect 100 μL of the cell suspension into a new 96-well V-bottom plate, centrifuge at 400 g for 3 minutes, discard the supernatant, and resuspend the cell pellet in 100 μL of PBS containing PI anti-viability dye (Beyotime, ST511) per well. After incubation at room temperature for 5 minutes, the number of remaining viable target cells was determined by flow cytometry, and the percentage of target cell lysis was calculated.

[0599] The results are shown in Figure 5D, which show that the anti-LILRB4×CD3 bispecific antibody (labeled as huB1G5-CD3-2VHH and huB1G5-ScFv-2VHH) and the control antibody 4ab3-4ab S91A can still specifically induce T cells to kill HL-60 tumor cells at a lower efficiency target ratio, and the killing effect of huB1G5-CD3-2VHH and huB1G5-ScFv-2VHH is stronger than that of the control antibody.

[0600] Example 7 In vivo efficacy of anti-LILRB4×CD3 bispecific antibody in the hPBMC-B-NDG mouse MOLM-13 tumor model

[0601] The in vivo antitumor effect of the anti-LILRB4×CD3 bispecific antibody was investigated in the hPBMC-B-NDG mouse MOLM-13 tumor model.

[0602] 7.1 In vivo efficacy of huB1G5-CD3Fab and huB1G5-CD3-2VHH bispecific antibodies

[0603] Severely immunodeficient B-NDG mice were purchased from Biocytogen (Beijing) Pharmaceuticals Co., Ltd.; human peripheral blood mononuclear cells (PBMCs) were purchased from Shanghai Aoneng Biotechnology Co., Ltd.; and human acute myeloid leukemia MOLM-13 cells were purchased from Nanjing Kebai Biotechnology Co., Ltd. Cells were cultured in RPMI 1640 medium supplemented with 20% inactivated fetal bovine serum in a 37°C, 5% CO2 incubator.

[0604] Thirty severely immunodeficient B-NDG mice were inoculated with 5×10 6 Human peripheral blood mononuclear cells / 0.2 mL RPMI 1640 / mouse, 5 × 10 cells were implanted subcutaneously in the right anterior region of the mouse 7 days later. 6 MOLM-13 cells / 0.1 mL RPMI 1640 / mouse. When the average tumor volume reached 182.5 mm 3 Around 20 mice were selected based on tumor volume and PBMC reconstitution and randomly assigned to three experimental groups of 5 mice each. On days 0, 3, and 7 after grouping, mice were intraperitoneally administered with blank PBS, huB1G5-CD3Fab bispecific antibody (0.2 mg / kg), and huB1G5-CD3-2VHH bispecific antibody (0.2 mg / kg), respectively. Tumor volume was monitored twice weekly by vernier caliper measurement starting from the day of grouping and dosing.

[0605] Treatment with the anti-LILRB4×CD3 bispecific antibodies huB1G5-CD3Fab and huB1G5-CD3-2VHH resulted in significant inhibition of tumor growth compared to the PBS group, with TGIs of 49.54% and 96.12%, respectively. The results are shown in FIG6A and Table 8 below.

[0606] Table 8. Inhibition of MOLM-13 tumor growth by the test article Note: a. Mean ± standard error; b. TGI = (1 - change in tumor volume of the treatment group / change in tumor volume of the control group) × 100%; c. Statistical analysis (one-way ANOVA) was performed on the 9th day after administration of the treatment group and the PBS control group. *P < 0.05, ***P < 0.001.

[0607] Throughout the experiment, the animals were active and ate well during the dosing period. Animals in groups G1-G3 all showed some weight gain, indicating good tolerance to the test article. Weight changes for all animals are shown in Figure 6B and Table 9.

[0608] Table 9. Effects of the test articles on the body weight of mice Note: a. Mean ± standard error; b. Body weights of the drug-treated group and the PBS control group were statistically analyzed on the 9th day of administration (One-way ANOVA).

[0609] 7.2 In vivo efficacy of huB1G5-CD3-2VHH and huB1G5-ScFv-2VHH bispecific antibodies

[0610] Severely immunodeficient B-NDG mice were purchased from Biocytogen (Beijing) Pharmaceuticals Co., Ltd.; human peripheral blood mononuclear cells (PBMCs) were purchased from Miaoshun (Shanghai) Biotechnology Co., Ltd.; and human acute myeloid leukemia MOLM-13 cells were purchased from Nanjing Kebai Biotechnology Co., Ltd. Cells were cultured in RPMI 1640 medium supplemented with 20% inactivated fetal bovine serum in a 37°C, 5% CO2 incubator.

[0611] 38 severely immunodeficient B-NDG mice were inoculated with 5×10 6 Human peripheral blood mononuclear cells / 0.2 mL RPMI 1640 / mouse, 5 × 10 cells were implanted subcutaneously in the right anterior region of the mouse 7 days later. 6 The MOLM-13 cells / 0.1mL RPMI 1640 / mice were used. When the average tumor volume reached about 166.64mm3, 25 suitable mice were selected to be enrolled according to the mouse tumor volume and PBMC reconstruction (hCD45% accounting), and were randomly assigned to 5 experimental groups, 5 mice in each group. On the 0th, 3rd and 6th day after grouping, blank control PBS, huB1G5-ScFv-2VHH (0.2 or 2mg / kg) bispecific antibody and huB1G5-CD3-2VHH (0.2 or 2mg / kg) bispecific antibody were administered to the mouse peritoneum respectively. Tumor volume was monitored by vernier caliper measurement twice a week during the experiment.

[0612] Treatment with the anti-LILRB4×CD3 bispecific antibodies huB1G5-ScFv-2VHH (0.2 and 2 mg / kg) and huB1G5-CD3-2VHH (0.2 and 2 mg / kg) resulted in significant inhibition of tumor growth compared to the PBS group, with TGIs of 72.0% and 63.3% for huB1G5-ScFv-2VHH and 54.2% and 87.6% for huB1G5-CD3-2VHH (0.2 and 2 mg / kg), respectively. The results are shown in Figure 6C and Table 10 below.

[0613] Table 10. Inhibition of MOLM-13 tumor growth by the test article Note: a. Mean ± standard error; b. TGI = (1 - change in tumor volume in the treatment group / change in tumor volume in the control group) × 100%; c. Statistical analysis (one-way ANOVA) of the tumor volumes of the treatment groups and the PBS control group on day 9 of group administration was performed, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0614] Throughout the experiment, the animals were active and fed well during the dosing period. Animals in groups G1-G4 all experienced some weight gain, while animals in group G5 showed no significant weight loss, indicating that the animals tolerated the test product well. Weight changes for all animals are shown in Figure 6D and Table 11.

[0615] Table 11. Effects of the test articles on the body weight of mice Note: a. Mean ± standard error; b. Body weights of the drug-treated group and the PBS control group were statistically analyzed on the 9th day of administration (One-way ANOVA).

[0616] Example 8 In vivo efficacy of bispecific antibodies in the MOLM-13-luc hematological tumor model

[0617] The in vivo antitumor effect of the anti-LILRB4×CD3 bispecific antibody was investigated in the MOLM-13-luc hematologic tumor model in hPBMC-B-NDG mice.

[0618] Severely immunodeficient B-NDG mice were purchased from Biocytogen (Beijing) Pharmaceuticals Co., Ltd.; human peripheral blood mononuclear cells (PBMCs) were purchased from Miaoshun (Shanghai) Biotechnology Co., Ltd.; and human acute myeloid leukemia MOLM-13-luc cells were purchased from Nanjing Kebai Biotechnology Co., Ltd. Cells were cultured in RPMI 1640 medium supplemented with 20% inactivated fetal bovine serum and 1 μg / ml puromycin in a 37°C, 5% CO2 incubator.

[0619] Sixty severely immunodeficient B-NDG mice were inoculated with 5×10 6 Human peripheral blood mononuclear cells / 0.2 mL RPMI 1640 / mouse, 2 × 10 cells were inoculated into the tail vein of the mouse 7 days later. 6 MOLM-13-luc cells / 0.2mL RPMI 1640 / mice. According to the mouse in vivo imaging result and PBMC reconstruction (hCD45% accounting), 40 suitable mice were selected to enter the group and randomly assigned to 8 experimental groups, 5 mice in each group. On the 0th, 3rd, 7th, 10th and 14th day after grouping, blank control PBS, 4ab3-4ab S91A (0.2mg / kg) bispecific antibody, huB1G5-ScFv-2VHH (0.02, 0.2 or 2mg / kg) bispecific antibody and huB1G5-CD3-2VHH (0.02, 0.2 or 2mg / kg) bispecific antibody were administered to the mouse peritoneum respectively. Animal status was observed every day during the experiment and animals were subjected to abdomen-upward in vivo imaging at Day8 and Day13.

[0620] Treatment with the anti-LILRB4×CD3 bispecific antibody 4ab3-4ab S91A (0.2 mg / kg), huB1G5-ScFv-2VHH (0.02, 0.2, and 2 mg / kg), and huB1G5-CD3-2VHH (0.02, 0.2, and 2 mg / kg) prolonged the survival of the animals compared to the PBS group. The results are shown in Figure 7A and Table 12 below.

[0621] Table 12. Effects of the test article on animal survival

[0622] Compared to the PBS group, the anti-LILRB4×CD3 bispecific antibody 4ab3-4ab S91A (0.2 mg / kg), huB1G5-ScFv-2VHH (0.02, 0.2, or 2 mg / kg), and huB1G5-CD3-2VHH (0.02, 0.2, or 2 mg / kg) groups all significantly reduced the fluorescence signal. Fluorescence signal profiles of all animals during in vivo imaging are shown in Figures 7B and 7C and Table 13.

[0623] Table 13. Effects of the test articles on the fluorescence signals of in vivo imaging in mice Note: a. Mean ± standard error; b. Statistical analysis (One-way ANOVA) of the fluorescence signals of the drug-treated group and the PBS control group was performed on the 13th day after group administration.

[0624] Example 9 Bispecific Antibody MLR Function

[0625] The release of IL-2 and IFN-γ and the proliferation of T cells in the mixed lymphoid system were detected to determine whether the anti-LILRB4×CD3 bispecific antibody of the present invention could activate T cells.

[0626] Cryopreserved human PBMCs (purchased from Shanghai Aoneng Biotechnology Co., Ltd., Catalog No. PBMNC050C, Batch No. 2108040345) were removed from liquid nitrogen and rapidly thawed in a 37°C water bath. Cell pellets were then centrifuged to obtain pure monocytes. Purified monocytes were isolated using a human total monocyte isolation kit (Miltenyi Biotec, Catalog No. 130-096-537) according to the manufacturer's instructions. These monocytes were cultured for 5 days in 1640 complete medium supplemented with 10% fetal bovine serum, 100 ng / mL recombinant human GM-CSF (PeproTech, Catalog No. 00-03-100UG), and 10 ng / mL recombinant human IL-4 (PeproTech, Catalog No. 200-04-100UG) to induce monocyte differentiation into imDCs. The induced imDCs were collected and centrifuged at 400 × g for 5 minutes at room temperature to discard the supernatant. The cell pellet was cultured in complete medium containing 100 ng / mL lipopolysaccharide (LPS, Sigma, Cat. No. LPS25) for 2 days to promote DC maturation. The harvested DCs were resuspended in complete medium, counted, and the cell density was adjusted to 2 × 10 5 / mL, calculated as 1×10 4 / 50μL / well was inoculated into 96-well U-bottom plates (Corning, Cat. No. 3799). Human PBMCs (purchased from Shanghai Rubai Biotechnology Co., Ltd., Cat. No. PBMNC050C, Batch No. 2301310011) were taken out of the liquid nitrogen tank and thawed quickly in a 37°C water bath. Cell pellets were obtained by centrifugation. TM Pure T cells were isolated using a human T cell isolation kit (Stem, Catalog No. 17951). T cells were stained with CFSE (BioTracker 488 Green CSFE Cell Proliferation Kit, Sigma, Catalog No. C34557) and resuspended in 1640 complete medium containing 10% fetal bovine serum at a cell density of 1 × 10 6 / mL, calculated as 1×10 5 / 100 μL / well was inoculated into a 96-well U-bottom plate and mixed evenly with the induced DCs (Effector cell: Target cell = 10:1).

[0627] The anti-LILRB4×CD3 bispecific antibody of the present invention was diluted with 1640 complete medium in a gradient manner (initial concentration was 1000 nM, 5-fold gradient dilution was performed for a total of 8 concentration points), and 50 μL / well was inoculated. After mixing evenly, the mixture was incubated at 37°C. The IL-2 content of the supernatant after 2 days of incubation was detected according to the instructions of the Human IL2 Detection Kit @ HTRF (Cisbio, Catalog No.: 62HIL02PEH). The IFNγ content of the supernatant after 5 days of incubation was detected according to the instructions of the Human IFNγ Detection Kit @ HTRF (Cisbio, Catalog No.: 62HIFNGPEH). The cell pellet was collected, and the change in the number of CFSE-positive cells was detected by absolute flow cytometry.

[0628] The results shown in Figure 8C show that the anti-LILRB4×CD3 bispecific antibodies of the present invention (labeled as huB1G5-CD3-2VHH and huB1G5-ScFv-2VHH) can promote the proliferation of T cells in the DC-T mixed lymphocyte reaction system, and the amount of cell proliferation is dose-dependent with the antibody concentration. There is no significant difference between the two bispecific antibodies of the present invention. The results shown in Figures 8A and 8B show that the anti-LILRB4×CD3 bispecific antibodies of the present invention (labeled as huB1G5-CD3-2VHH and huB1G5-ScFv-2VHH) can promote the release of IL-2 and IFN-γ in the DC-T mixed lymphocyte reaction system, and the amount of release is dose-dependent with the antibody concentration.

[0629] Example 10 Anti-LILRB4 Antibody Reduces IL-10 Release in PBMC

[0630] The release level of IL-10 in the PBMC system was detected to determine whether the anti-LILRB4 humanized antibody of the present invention can promote the differentiation of myeloid cells in PBMC into a pro-inflammatory phenotype.

[0631] Frozen human PBMCs (purchased from Shanghai Saili & Xuanfeng Biotechnology Co., Ltd., catalog number: HFB-HP050B, batch number: W20210078) were taken out of the liquid nitrogen tank and thawed quickly in a 37°C water bath. Cell pellets were obtained by centrifugation. The cells were resuspended in 1640 (Gibco, catalog number: 22400089) complete medium containing 10% fetal bovine serum (Gibco, catalog number: 10099-141) and then sampled and counted. The number of cells was calculated as 1.5×10 5100 μL was inoculated into the experimental wells of a 96-well culture plate (Corning, Catalog No. 3599). CD3 monoclonal antibody (OKT3, Invitrogen, Catalog No. 16-0037-85) was diluted in complete culture medium and added at 50 μL / well to a final concentration of 10 ng / mL. Activation was performed at 37°C for 6 hours. The anti-human LILRB4 humanized antibody of the present invention, the control antibody h193, and a negative control antibody (IgG4 control, IgG1 control purchased from SinoBiological, Catalog No. HG1K) were diluted in complete culture medium to 400, 40, and 4 nM, respectively. 50 μL / well was added to the sample, with final concentrations of 100, 10, and 1 nM. After mixing, the culture plate was incubated in a 37°C incubator for 3 days. The IL-10 content in the supernatant was detected using an IL-10 detection kit (R&D, Catalog No. DY217B).

[0632] The results shown in Figures 9A, 9B, and 9C demonstrate that the humanized anti-human LILRB4 antibody (labeled huB1G5-3) and antibody h193 of the present invention significantly reduced IL-10 secretion in the system at all three tested concentrations compared to two negative controls. This indicates that the humanized anti-human LILRB4 antibody of the present invention can promote the differentiation of myeloid cells in PBMCs toward a pro-inflammatory phenotype.

[0633] 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 can be combined in various embodiments of the present invention. In addition, the materials, methods and examples described herein are only illustrative and are not intended to be restrictive. Other features, purposes and advantages of the present invention will become apparent from this specification and accompanying drawings and from the appended claims.

[0634] Sequence information:

Claims

1. A VHH antibody that specifically binds to LILRB4, comprising three complementarity determining regions (CDRs) contained in the VHH represented by any one of SEQ ID NOs: 11, 16, 21, 25, 30, 32, 36, 39 and 42, Preferably, the CDR sequences are according to the IMGT definition.

2. The VHH antibody of claim 1, comprising complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein (i) VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 12, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 13, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 14; or (ii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 17, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 18, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 19; (iii) VHH CDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:75, VHH CDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:22, and VHH CDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:23; (iv) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO:26, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO:27, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO:28; (v) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 17, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 33 or 37, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 34 or 40; (vi) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 17, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 33 or 37, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 34; or (vii) VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 17, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 33 or 37, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:

40.

3. The VHH antibody of claim 1, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 11, 16, 21, 25, 30, 32, 36, 39 and 42; or (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 11, 16, 21, 25, 30, 32, 36, 39 and 42; or (iii) comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes compared to the amino acid sequence shown in any one of SEQ ID NOs: 11, 16, 21, 25, 30, 32, 36, 39 and 42. Preferably, the amino acid changes do not occur in the CDR regions.

4. A heavy chain antibody that specifically binds to LILRB4, comprising the VHH antibody of any one of claims 1 to 3.

5. The heavy chain antibody of claim 4, comprising the VHH antibody of any one of claims 1 to 3 connected to an antibody constant region or Fc region, preferably, the antibody constant region or Fc region is from human IgG1, human IgG2, human IgG3 or human IgG4, optionally, the VHH antibody is connected to the Fc region via a hinge region or a portion thereof, preferably, the amino acid sequence of the hinge region portion is EPKSS (SEQ ID NO: 59) or EPKSC (SEQ ID NO: 67).

6. The heavy chain antibody of claim 4, comprising the VHH antibody of any one of claims 1 to 3 linked to an antibody Fc region, wherein the Fc region is an Fc region from human IgG1, IgG2, IgG3 or IgG4, preferably, the Fc region (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:55 or 68; or (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO: 55 or 68; or (iii) comprises an 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 shown in SEQ ID NO: 55 or 68.

7. The heavy chain antibody of claim 4, (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NO: 15, 20, 24, 29, 31, 35, 38, 41 or 43; or (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NO: 15, 20, 24, 29, 31, 35, 38, 41 or 43; or (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in any one of SEQ ID NOs: 15, 20, 24, 29, 31, 35, 38, 41 or 43, preferably, the amino acid changes do not occur in the CDR regions.

8. The VHH antibody of any one of claims 1 to 3, or the heavy chain antibody of any one of claims 4 to 7, wherein the antibody is a chimeric antibody or a humanized antibody.

9. A bispecific antibody comprising a first antigen binding region and a second antigen binding region, wherein the second antigen binding region specifically binds to LILRB4, and comprises the VHH antibody of any one of claims 1 to 3 and 8, or the heavy chain antibody of any one of claims 4 to 8.

10. The bispecific antibody of claim 9, wherein the first antigen binding region specifically binds CD3.

11. The bispecific antibody of claim 10, wherein the first antigen-binding region comprises a VH and a VL, wherein the VH comprises three complementarity determining regions (HCDRs) from a heavy chain variable region, HCDR1, HCDR2, and HCDR3, and the VL comprises three complementarity determining regions (LCDRs) from a light chain variable region, LCDR1, LCDR2, and LCDR3, wherein (i) HCDR1, HCDR2 and HCDR3 are the three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH shown in SEQ ID NO: 3, and LCDR1, LCDR2 and LCDR3 are any one of SEQ ID NO: 4 The three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL of ; or (ii) HCDR1 consists of the amino acid sequence shown in SEQ ID NO:5, HCDR2 consists of the amino acid sequence shown in SEQ ID NO:6, HCDR3 consists of the amino acid sequence shown in SEQ ID NO:7, LCDR1 consists of the amino acid sequence shown in SEQ ID NO:8, LCDR2 consists of the amino acid sequence shown in SEQ ID NO:9, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO:

10.

12. The bispecific antibody of claim 11, wherein the first antigen-binding region comprises VH and VL, wherein The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, And / or the VL contains the amino acid sequence shown in SEQ ID NO:4, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, or consists of the amino acid sequence.

13. The bispecific antibody of any one of claims 10 to 12, wherein the first antigen binding region is a Fab that specifically binds to CD3. The bispecific antibody of claim 13 , wherein the Fab comprises CH1, wherein the CH1 is CH1 derived from IgG1, IgG2, IgG3 or IgG4, preferably CH1 derived from IgG1.

15. The bispecific antibody of claim 14, wherein the CH1 (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 51; or (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO:

51.

16. The bispecific antibody of any one of claims 10-12, wherein the first antigen binding region is a scFv that specifically binds to CD3, the scFv comprising VH and VL as defined in claim 11 or 12, optionally, the VH and VL are connected via a linker, the linker for example comprising the amino acid sequence (G4S)n, wherein n=1, 2, 3, 4 or 5, preferably, n=3 or 4, more preferably, n=3.

17. The bispecific antibody of any one of claims 9-16, wherein the second antigen binding region is the VHH of any one of claims 1-3 and 8. 18 . The bispecific antibody of claim 9 , wherein the bispecific antibody is an IgG-like bispecific antibody comprising an Fc dimer, wherein two Fc regions constituting the Fc dimer are identical or different.

19. The bispecific antibody of claim 18, wherein the two Fc regions are different, and preferably, corresponding knob mutations and hole mutations are introduced into the two Fc regions, respectively.

20. The bispecific antibody of claim 19, wherein a) one Fc-region polypeptide comprises the knob mutation T366W and the other Fc-region polypeptide comprises the hole mutations T366S, L368A and Y407V, or b) one Fc-region polypeptide comprises the knob mutations T366W and Y349C and the other Fc-region polypeptide comprises the hole mutations T366S, L368A, Y407V and S354C, or c) one Fc-region polypeptide comprises knob mutations T366W and S354C, and the other Fc-region polypeptide comprises hole mutations T366S, L368A, Y407V and Y349C; Optionally, the Fc region further comprises a mutation that reduces binding to an Fcγ receptor, for example, one or more of a L234A / L235A mutation, a D265A mutation, or a P329A mutation; for example, a L234A / L235A mutation, a D265A mutation, and a P329A mutation.

21. The bispecific antibody of any one of claims 18-20, wherein one or both of the Fc regions comprise a hinge region, such as EPKSS or EPKSC.

22. The bispecific antibody of claim 21, wherein (i) Fc region containing knob mutation a) comprises or consists of the amino acid sequence SEQ ID NO: 49 or 50 or 73; or b) comprising or consisting of an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or more identity to SEQ ID NO: 49 or 50 or 73 and comprising knob mutations (such as S354C and T366W); and / or (ii) Fc region containing hole mutation a) comprises or consists of the amino acid sequence SEQ ID NO: 47 or 48 or 74; or b) comprising or consisting of an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or more identity to SEQ ID NO: 47 or 48 or 74 and comprising hole mutations (such as Y349C, T366S, L368A and Y407V).

23. The bispecific antibody of any one of claims 9 to 22, comprising a first antigen binding region, a second antigen binding region and an Fc dimer, wherein the first antigen binding region is a Fab fragment that specifically binds to CD3, and the second antigen binding region is a VHH that specifically binds to LILRB4, such as the VHH of any one of claims 1 to 3 and 8, for example, the bispecific antibody comprises one first antigen binding region, and 1 or 2 second antigen binding regions.

24. The bispecific antibody of claim 23, comprising an anti-LILRB4 VHH, an anti-CD3 antibody Fab fragment and an Fc heterodimer, wherein The Fab fragment comprises VH-CH1 and VL-CL, wherein the VHH comprises a heavy chain variable region, wherein the C-terminus of CH1 of the Fab fragment is fused to the CH2 or hinge region of the first Fc region (e.g., an Fc region comprising a Knob mutation) to constitute a first heavy chain; The VHH is fused to the CH2 or hinge region of a second Fc region (e.g., an Fc region comprising a Hole mutation) to form a second heavy chain; and The VL-CL of the Fab fragment constitutes the light chain.

25. The bispecific antibody of claim 24, wherein the The first heavy chain comprises, or consists of, the amino acid sequence set forth in SEQ ID NO:44, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; The second heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:45, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; and / or The light chain comprises the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence, or consists of the sequence.

26. The bispecific antibody of claim 23, comprising two anti-LILRB4 VHHs, a Fab fragment of an anti-CD3 antibody and an Fc heterodimer, wherein The Fab fragment comprises VH-CH1 and VL-CL, wherein the VHH comprises a heavy chain variable region, wherein the C-terminus of the first VHH fragment is fused to the N-terminus of the VH of the Fab fragment and the C-terminus of the CH1 of the Fab fragment is fused to the CH2 or hinge region of the Fc region (e.g., comprising a knob mutation), constituting a first heavy chain; The second VHH is fused to the CH2 or hinge region of the Fc region (e.g., comprising a hole mutation) to form the second heavy chain; and The VL-CL of the Fab fragment constitutes the light chain. The bispecific antibody of claim 26 , wherein the first VHH and the second VHH are the same or different.

28. The bispecific antibody of claim 26 or 27, wherein the fusion of the first VHH to the Fab is via a connecting peptide, such as (GGGGS)n, wherein n=1, 2, 3 or 4.

29. The bispecific antibody of claim 26, wherein the The first heavy chain comprises, or consists of, the amino acid sequence set forth in SEQ ID NO:46, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; The second heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:45, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; and / or The light chain comprises the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence, or consists of the sequence.

30. The bispecific antibody of claim 23, comprising two anti-LILRB4 VHHs, an anti-CD3 antibody scFv and an Fc heterodimer, wherein The scFv comprises VH and VL in sequence from the N-terminus to the C-terminus, wherein the VHH comprises a heavy chain variable region, wherein the C-terminus of the first VHH is fused to the N-terminus of the VH of the scFv and the C-terminus of the VL of the scFv is fused to the CH2 or hinge region of the Fc region (e.g., comprising a knob mutation), constituting a first heavy chain; and The second VHH is fused to the Fc region (eg, comprising a hole mutation) to form the second heavy chain (eg, the C-terminus of the VHH is fused to the CH2 or hinge region of the second Fc region).

31. The bispecific antibody of claim 30, wherein the VH and VL of the scFv are connected via a linker or a connecting peptide, for example, the linker or the connecting peptide comprises or consists of the amino acid sequence shown in SEQ ID NO:

70.

32. The bispecific antibody of claim 30 or 31, wherein the first VHH and the second VHH are the same or different.

33. The bispecific antibody of any one of claims 30-32, wherein the fusion of the first VHH to the scFv is via a connecting peptide, such as (GGGGS)n, wherein n=1, 2, 3 or 4.

34. The bispecific antibody of claim 30, wherein the The first heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:72, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence; and / or The second heavy chain comprises the amino acid sequence shown in SEQ ID NO:45, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence, or consists of the sequence.

35. A multispecific antibody comprising the VHH antibody of any one of claims 1-3 and 8, or the heavy chain antibody of any one of claims 4-8, eg, the multispecific antibody is a bispecific antibody.

36. A nucleic acid molecule encoding the VHH antibody of any one of claims 1 to 3 and 8, or the heavy chain antibody of any one of claims 4 to 8, or any chain of the bispecific antibody of any one of claims 9 to 34, or any chain of the multispecific antibody of claim 35, or consisting of said nucleic acid sequence.

37. An expression vector comprising the nucleic acid molecule of claim 36.

38. A host cell comprising the nucleic acid molecule of claim 36 or the expression vector of claim 37, preferably, the host cell is prokaryotic or eukaryotic, such as 293 cells or CHO cells, such as 293F cells or 293T cells or CHO-S cells.

39. A method for preparing the VHH antibody of any one of claims 1-3 and 8, or the heavy chain antibody of any one of claims 4-8, or the bispecific antibody of any one of claims 9-34, or the multispecific antibody of claim 35, the method comprising culturing a host cell comprising the nucleic acid molecule of claim 36 or the expression vector of claim 37 under conditions suitable for chain expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

40. An immunoconjugate comprising the VHH antibody of any one of claims 1-3 and 8, or the heavy chain antibody of any one of claims 4-8, or the bispecific antibody of any one of claims 9-34, or the multispecific antibody of claim 35.

41. A pharmaceutical composition or medicament or formulation comprising the VHH antibody of any one of claims 1-3 and 8, or the heavy chain antibody of any one of claims 4-8, or the bispecific antibody of any one of claims 9-34, or the multispecific antibody of claim 35, or the immunoconjugate of claim 40 and optionally a pharmaceutically acceptable excipient.

42. A pharmaceutical combination product comprising a VHH antibody according to any one of claims 1 to 3 and 8, or a heavy chain antibody according to any one of claims 4 to 8, or a bispecific antibody according to any one of claims 9 to 34, or a multispecific antibody according to claim 35, or an immunoconjugate according to claim 40, and one or more other therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators).

43. A method for preventing or treating cancer in a subject, comprising administering to the subject an effective amount of the VHH antibody of any one of claims 1-3 and 8, or the heavy chain antibody of any one of claims 4-8, or the bispecific antibody of any one of claims 9-34, or the multispecific antibody of claim 35, or the immunoconjugate of claim 40, or the pharmaceutical composition or medicament or formulation of claim 41; or the pharmaceutical combination product of claim 42.

44. The method of claim 43, wherein the cancer has or has elevated protein levels and / or nucleic acid levels (eg, elevated expression) of LILRB4 in tumor cells.

45. The method of claim 43 or 44, wherein the cancer is a solid tumor or a hematological tumor, such as a solid tumor, multiple myeloma or a leukemia, such as acute myeloid leukemia (AML), such as monocytic AML and non-monocytic AML, such as human myelomonocytic leukemia cells, human promyelocytic acute leukemia or human acute myeloid leukemia.

46. ​​The method of any one of claims 43-45, wherein the method further comprises administering in combination with other therapies such as treatment modalities (e.g., radiation therapy) and / or other therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators).

47. A method for detecting the presence of LILRB4 in a biological sample, comprising (i) contacting a biological sample with the VHH antibody of any one of claims 1 to 3 and 8, or the heavy chain antibody of any one of claims 4 to 8, or the bispecific antibody of any one of claims 9 to 34, or the multispecific antibody of claim 35 under conditions that allow binding thereof to LILRB4, (ii) detecting whether a complex is formed between the antibody or bispecific antibody and LILRB4, The formation of the complex indicates the presence of LILRB4.

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