Antibody binding to lilrb1 / 2 or PD1-lilrb1 / 2 and use thereof

Abstract

Provided are an antibody specifically binding to LILRB1 / 2, such as a VHH antibody or a heavy-chain antibody, and a bispecific antibody constructed based thereon, such as a bispecific antibody specifically binding to PD1 and LILRB1 / 2. Further provided are a nucleic acid encoding the antibody, a method for preparing the antibody, and a method for using same and the use thereof, e.g., the therapeutic use.

Description

Antibodies that bind to LILRB1 / 2 or PD1-LILRB1 / 2 and their uses

[0001] The present invention relates to antibodies that specifically bind to LILRB1 / 2, such as VHH antibodies or heavy chain antibodies, and bispecific antibodies constructed based thereon, such as bispecific antibodies that specifically bind to PD1 and LILRB1 / 2. The present invention also relates to nucleic acids encoding the antibodies, methods for preparing the antibodies, and methods and uses thereof, such as therapeutic applications. Background Art

[0002] The leukocyte immunoglobulin-like receptors (LILRs) consist of the inhibitory LILR subfamily B (LILRB1-5) and the activating LILR subfamily A (LILRA1-6). LILRB family receptors are type I transmembrane glycoproteins that contain an Ig-like domain in their extracellular region and an immunoreceptor tyrosine-based inhibitory motif (ITIM) domain in their intracellular region, making them immunosuppressive receptors. LILRB1 is widely expressed on myeloid cells, B cells, NK cell subsets, and T cells, while LILRB2-LILRB5 are primarily expressed on myeloid cells. LILRBs (LILRB1-5) are also highly expressed on cancer stem cells and may directly regulate cancer initiation and recurrence, as well as cancer stem cell activity. Furthermore, LILRBs have diverse biological functions, including regulating inflammatory responses, immune tolerance, cell differentiation, and neural plasticity, and play important roles in a variety of diseases, including inflammatory, autoimmune, infectious, and neurological disorders and malignancies.

[0003] LILRB1 binds to a variety of ligands, including human leukocyte antigen (HLA) class I molecules, human cytomegalovirus UL18 protein, and calcium-binding proteins S100A8 and S100A9. LILRB2 interacts with related ligands within the tumor microenvironment, including HLA-G, ANGPTLs, SEMA4A, and CD1d, enabling myeloid cells to promote tumor growth and enhance tumor immune evasion. LILRB1 and LILRB2 receptors, through interaction with human leukocyte antigen-G (HLA-G), inhibit cytotoxic T cells, NK cells, and B cells, induce T cell anergy, regulate myeloid cells, and promote Tregs. Furthermore, HLA-G expression on antigen-presenting cells, such as MDSCs or DCs, promotes T cell hyporesponsiveness and induces Treg differentiation. The HLA-G / LILRB1 interaction can inhibit the proliferation of neoplastic B cells. Interactions between ligands and LILRBs are considered immune checkpoints in cancer. When stimulated by ligands such as HLA-G on tumor cells, LILRBs inhibit immune activation, thereby indirectly promoting tumor growth. LILRBs are also specifically expressed or upregulated in lung, gastric, breast, and pancreatic cancer cells. Because LILRBs act as both immune checkpoint molecules and tumor-maintaining factors without affecting hematopoiesis and normal development, they are considered a target for cancer therapy.

[0004] PD-1 (Programmed cell death-1) is a receptor that inhibits T cell activation and is an important target for tumor immunotherapy. PD-1 expression remains high on antigen-specific T cells that have been stimulated for a long time, making them less responsive to stimulation. As an inhibitory signaling mechanism, the PD-1 axis controls immune responses. Blocking the PD-1 axis has been shown to have long-lasting effects on various tumors, demonstrating the critical role of PD-1 in blocking anti-tumor immunity.

[0005] The prior art has antibodies against LILRB1&2, such as the LILRB1 antibody disclosed in WO2021028921, and the LILRB2 antibodies disclosed in WO2022087188A1 and WO2021138079A1.

[0006] Furthermore, although the prior art discloses numerous PD1 antibodies such as pembrolizumab, and there is the use of anti-LILRB1 / 2 antibodies in combination with PD1 antibodies, no effective multispecific antibody that specifically binds to PD1 and LILRB1 / 2 simultaneously has been developed.

[0007] Therefore, there is a need for a new antibody against LILRB1&2, as well as a PD1 and LILRB1&2 (LILRB1 / 2) bispecific antibody constructed based on this, which has improved properties. Summary of the Invention

[0008] The present invention constructs an antibody that specifically binds to LILRB1 and LILRB2 (LILRB1 / 2), and then constructs a bispecific antibody that binds to PD1 and LILRB1 / 2 based on the antibody, forming an engager between tumor cells and immune cells such as macrophages, DCs, and NK cells. PD1 antibodies relieve the inhibition of T cells, while LILRB1&2 antibodies activate the activity of immune cells such as macrophages, NK, T, and DCs, thereby improving the immune microenvironment and ultimately forming an immune synergistic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Flow cytometry results of antibody binding to CHOK1-huLILRB1 cells.

[0010] Figure 2 Flow cytometry results of antibody binding to CHOK1-huLILRB1 cells.

[0011] Figure 3 DSF melting peak of humanized antibody.

[0012] Figure 4 Flow cytometry results of antibody binding to CHOK1-huLILRB1 cells.

[0013] Figure 5 shows flow cytometry results of antibody binding to 293T-RheLILRB1 (FHG) cells.

[0014] Figure 6 Flow cytometry results of antibodies blocking the binding of human LILRB1 protein to A375-HLAG cells.

[0015] Figure 7 Flow cytometry results of antibody blocking human LILRB1 protein binding to RPMI-8226 (MHC-Ⅰ) cells.

[0016] FIG8 Flow cytometry results of antibodies blocking the binding of human LILRB2 protein to A375-HLAG cells.

[0017] Figure 9 Flow cytometry results of antibody blocking human LILRB2 protein binding to RPMI-8226 (MHC-Ⅰ) cells.

[0018] FIG10 Flow cytometry results of antibody blocking human LILRB2 protein binding to RPMI-8226 (MHC-Ⅰ) cells.

[0019] Figure 11 Flow cytometry results of antibody-induced NK cell killing of A375-HLAG cells.

[0020] Figure 12 Flow cytometry results of antibody-induced macrophage phagocytosis of RPMI-8226 cells.

[0021] Figure 13 Flow cytometry results of antibody-induced macrophage phagocytosis of RPMI-8226 cells.

[0022] Figure 14 Detection of TNFα release in the supernatant after the antibody interacted with PBMC.

[0023] Figure 15 Detection of TNFα release in the supernatant after the antibody interacted with PBMC.

[0024] Figure 16 Detection of TNFα release in the supernatant after the interaction of antibodies with DCs.

[0025] Figure 17 Detection of TNFα release in the supernatant after the antibody interacted with DC.

[0026] FIG18 Detection of antibodies relieving the inhibition of MDSC on allogeneic T cell proliferation.

[0027] Figure 19 is a schematic diagram of the bispecific molecular structure.

[0028] Figure 20 shows the DSF melting peak of the bispecific antibody.

[0029] Figure 21 Detection of TNFα release in the supernatant after the anti-PD1 / anti-LILRB1 / 2 bispecific antibody interacts with macrophages.

[0030] Figure 22 Changes in surface marker CD163 after macrophages acted with anti-PD1 / anti-LILRB1 / 2 bispecific antibodies.

[0031] Figure 23 shows changes in the surface marker CD86 of macrophages after treatment with anti-PD1 / anti-LILRB1 / 2 bispecific antibodies.

[0032] Figure 24 Detection of IFN-γ release induced by anti-PD1 / anti-LILRB1 / 2 bispecific antibody in mixed lymphoid reaction system.

[0033] Figure 25 Detection of TNFα release induced by anti-PD1 / anti-LILRB1 / 2 bispecific antibody in DC activation.

[0034] Figure 26 Flow cytometric detection of surface CD83 in DCs induced by anti-PD1 / anti-LILRB1 / 2 bispecific antibodies.

[0035] Figure 27 Detection of IFNγ release in the supernatant of the allogeneic DC-T mixed lymphocyte reaction system induced by anti-PD1 / anti-LILRB1 / 2 bispecifics.

[0036] Figure 28 Tumor volume change curve.

[0037] Figure 29 shows the weight change curve of mice.

[0038] Figure 30 shows the proportion of hCD45+ positive cells in mouse peripheral blood.

[0039] Figure 31 Tumor volume change curve.

[0040] Figure 32 shows the weight change curve of mice.

[0041] Detailed Description of the Invention

[0042] 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.

[0043] I. Definition

[0044] 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.

[0045] 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.

[0046] As used herein, the term "and / or" means any one of the alternatives or two or more or all of the alternatives.

[0047] 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.

[0048] 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.

[0049] As used herein, the term "antibody that binds to PD1" or "anti-PD1 antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize PD1 or bind thereto. In one embodiment, the antigen-binding region that binds to PD1 in the anti-PD1 antibody or its antigen-binding fragment or bispecific antibody of the present invention may have a higher binding activity to PD1 or cells expressing PD1. The binding affinity of the antibody to PD1 can be detected by flow cytometry or biofilm optical interference technology. Preferably, in the bispecific antibody molecule of the present invention, the binding affinity of the antigen-binding region that binds to PD1 to human PD1 is between 1-100 nM. In some embodiments, the antigen-binding region that binds to PD1 in the anti-PD1 antibody or its antigen-binding fragment or bispecific antibody of the present invention binds to human PD1 with a higher binding affinity. Any known PD1 antibody is suitable for constructing the bispecific antibody of the present invention.

[0050] 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).

[0051] The term "LILR" refers to leukocyte immunoglobulin-like receptors (LILRs), members of which include inhibitory LILR subfamily B (LILRB1-5) and activating LILR subfamily A (LILRA1-6), for example, human LILRB1 protein under Uniprot Accession No. Q8NHL6, rhesus macaque LILRB1 protein under Uniprot Accession No. F7H3G7, cynomolgus macaque LILRB1 protein under NCBI Sequence No. XP_045236898.1, or human LILRB2 protein under Uniprot Accession No. Q8N423. In one embodiment, the human LILRB1 protein of the present invention comprises or consists of the amino acid sequence set forth in SEQ ID NO: 47, 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 rhesus macaque LILRB1 protein of the invention comprises, or consists of, the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In one embodiment, the cynomolgus macaque LILRB1 protein of the invention comprises, or consists of, the amino acid sequence of SEQ ID NO: 49, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In one embodiment, the human LILRB2 protein of the invention comprises, or consists of, the amino acid sequence of SEQ ID NO: 50, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0052] As used herein, the term "antibody that binds to LILRB1 / 2" or "anti-LILRB1 / 2 antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize or bind to LILRB1 and LILRB2. In one embodiment, the antigen-binding region that binds to LILRB1 / 2 in the anti-LILRB1 / 2 antibody or antigen-binding fragment thereof or bispecific antibody of the present invention may have higher binding activity to LILRB1 and LILRB2 or higher binding activity to cells expressing LILRB1 and / or LILRB2. The binding affinity of the antibody to LILRB1 and LILRB2, respectively, can be detected by flow cytometry or biofilm optical interferometry. In one embodiment, the anti-LILRB1 / 2 antibody of the present invention specifically binds to LILRB1 and LILRB2, such as human or monkey (e.g., rhesus monkey or cynomolgus monkey) LILRB1 and LILRB2. In one embodiment, the antigen-binding region that binds to LILRB1 / 2 in the anti-LILRB1 / 2 antibody or bispecific antibody of the present invention has high affinity binding activity to cells expressing human or monkey (e.g., rhesus monkey or cynomolgus monkey) LILRB1 and / or LILRB2. In one embodiment, the antigen-binding region that binds to LILRB1 / 2 in the anti-LILRB1 / 2 antibody or antigen-binding fragment thereof or bispecific antibody of the present invention has cross-reactivity to human and monkey (e.g., rhesus monkey or cynomolgus monkey) LILRB1 and / or LILRB2, i.e., it can bind to human and monkey (e.g., rhesus monkey or cynomolgus monkey) LILRB1 and / or LILRB2.

[0053] 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 a D265A and / or P329A mutation. 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

[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: 33 or 34;

[0055] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 33 or 34; 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: 33 or 34.

[0057] 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.

[0058] (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: 36;

[0059] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 36; or

[0060] (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 the amino acid sequence selected from SEQ ID NO: 36.

[0061] The term "antibody fragment" includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0062] 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.

[0063] "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 domain and the VL domain 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."

[0064] 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).

[0065] The single domain antibodies or VHHs of the present invention may also be contained in a larger polypeptide / protein. Examples of polypeptides / proteins containing the VHHs of the present invention include, but are not limited to, heavy chain antibodies (HcAbs) or multispecific antibodies.

[0066] The "heavy chain antibody" described in the present invention refers to an antibody without a light chain, for example, it may comprise VH-Fc or VH-CH2-CH3 or VH-hinge region-CH2-CH3 from the N segment to the C segment, or may comprise VH-CH1-CH2-CH3. The heavy chain antibody of the present invention may also encompass homodimers, such as heavy chain dimer antibodies without light chains. The heavy chain antibody may comprise VH from a standard antibody or VH from a single domain antibody. For example, the VH in the heavy chain antibody may be 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 or 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 to an Fc region, such as a human IgG Fc region, such as the Fc region of human IgG1, 2, 3, or 4. When "VHH" is mentioned in the context of a heavy chain antibody or a multispecific antibody, it should be understood that it is part of a multispecific antibody, not a separate molecule.

[0067] 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.

[0068] 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.

[0069] 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 to 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), and the VH / VL pair can be contained in a single polypeptide chain (e.g., in scFv) or in two separated polypeptide chains (e.g., in a Fab heavy chain and a Fab light chain, respectively). In one embodiment, one or more of the antigen binding regions for the bispecific antibody molecule of the present invention can be Fab.

[0070] As used herein, the term "monospecific" antibody refers to an antibody having one or more binding regions, each of which binds to the same epitope of the same antigen. For example, the present invention provides monospecific antibodies against LILRB1 / 2.

[0071] 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 LILRB1 / 2 and PD1.

[0072] 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.

[0073] 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.

[0074] "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.

[0075] The following are the regional ranges of CDRs defined using the Kabat, AbM, Chothia, Contact, and IMGT schemes.

[0076] 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).

[0077] In one embodiment, the HCDRs in the VHH or heavy chain antibodies of the invention are determined according to the IMGT protocol.

[0078] In one embodiment, the HCDRs and LCDRs in the antibodies of the invention are defined according to the Kabat scheme.

[0079] 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" excludes the heavy chain variable region VH and light chain variable region VL of an immunoglobulin, as well as the heavy chain constant region CH1 (without the hinge region) and the light chain constant region CL, but may include the hinge region at the N-terminus of the heavy chain constant region in some cases. In some embodiments, the heavy chain constant region Fc suitable for use in 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 mutations that reduce binding to Fcγ receptors, such as the LALA mutation, the D265A mutation, and / or the P329A mutation, preferably comprising the LALA mutation and the D265A and P329A mutations. In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO: 35, 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, the Fc fragments dimerize to form Fc dimers. In some embodiments, the Fc fragments homodimerize. In some embodiments, the 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.

[0080] 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.

[0081] A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody (e.g., a murine or camelid 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] 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.

[0087] 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, or 30 amino acids. In one embodiment, the connecting peptide comprises the amino acid sequence (GS)n(SEQ ID NO:69), (GGS)n(SEQ ID NO:70), (GSGGS)n(SEQ ID NO:71), (GGGGS)n(SEQ ID NO:72), (GGGS)n(SEQ ID NO:73), and (GGGGS)nG(SEQ ID NO:74), 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, or 10. Useful linkers also include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. In some embodiments, the peptide linker is (GGGGS)n(SEQ ID NO:75), wherein n=1, 2, 3, or 4, for example, the sequence shown in SEQ ID NO:23 or 24.

[0088] 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.

[0089] 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.

[0090] The terms "subject" or "individual" 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 subject is a human.

[0091] The term "treat," ...

[0092] 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, individuals 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 individuals at risk for cancer.

[0093] 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).

[0094] 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.

[0095] "Chemotherapeutic agents" include chemical compounds useful in treating cancer or immune system disorders.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, etc., which is administered together with the active substance.

[0104] 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.

[0105] The term "pharmaceutical combination or combination product" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit. The term "non-fixed combination" means that the active ingredients (e.g., (i) the bispecific antibody of the present invention, and (ii) other therapeutic agent) 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 with prophylactic or therapeutically effective levels in the patient. 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.

[0106] 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.

[0107] "Individual / patient / individual sample" refers to a collection of cells or fluids obtained from a patient or individual. 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 individual'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.

[0108] II. Anti-LILRB1 / 2 Antibodies

[0109] In one aspect, the present invention provides a LILRB1 / 2 antibody having a higher binding affinity to LILRB1 and / or LILRB2. In some embodiments, the LILRB1 / 2 antibody of the present invention is suitable for constructing an antigen-binding region in a multispecific antibody molecule.

[0110] In some embodiments, the anti-LILRB1 / 2 antibodies or antigen-binding fragments thereof of the present invention bind to LILRB1 and / or LILRB2 (e.g., human or monkey (e.g., cynomolgus monkey or rhesus monkey) LILRB1 and / or LILRB2) with higher affinity. In some embodiments, the anti-LILRB1 / 2 antibodies or antigen-binding fragments thereof of the present invention can bind to human LILRB1 / 2 with high affinity, e.g., their K D In some embodiments, the K is less than or equal to about 50 nM, such as less than or equal to about 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, or 0.01 nM, such as less than or equal to about 5 pM, 4 pM, 3 pM, 2 pM, or 1 pM, or therebetween. D The value is greater than or equal to about 0.1 pM.

[0111] In some embodiments, the anti-LILRB1 / 2 antibodies or antigen-binding fragments thereof of the present invention bind to LILRB1 and / or LILRB2 expressed by cells. In some embodiments, the affinity of the anti-LILRB1 / 2 antibodies to LILRB1 and / or LILRB2 expressed by cells is determined by flow cytometry.

[0112] In some embodiments, the anti-LILRB1 / 2 antibodies or antigen-binding fragments thereof of the present invention have good thermal stability, for example, in a DSF assay, Tm1 is greater than 60 degrees Celsius, for example, between about 60-65 degrees Celsius, for example, about 61, 62 or 63 degrees Celsius, for example, between about 62-63 degrees Celsius.

[0113] In some embodiments, the anti-LILRB1 / 2 antibody or antigen-binding fragment thereof of the present invention blocks the binding of human LILRB1 protein or human LILRB2 protein to its ligand, for example, it blocks the binding of human LILRB1 protein to human HLAG protein, and / or blocks the binding of human LILRB1 protein to human MHC-I protein; and / or it blocks the binding of human LILRB2 to human HLAG, and / or it blocks the binding of human LILRB2 to human MHC-I protein.

[0114] In some embodiments, the anti-LILRB1 / 2 antibodies or antigen-binding fragments thereof of the present invention induce immune cells to kill target cells expressing ligands of LILRB1 or LILRB2, such as dendritic cells, macrophages, and NK cells.

[0115] In some embodiments, the anti-LILRB1 / 2 antibodies or antigen-binding fragments thereof of the present invention induce NK cells to kill target cells, such as cells expressing ligands of LILRB1 or 2 (e.g., HLAG proteins). In some embodiments, the anti-LILRB1 / 2 antibodies or antigen-binding fragments thereof of the present invention induce macrophages to phagocytose target cells, such as cells expressing ligands of LILRB1 or 2 (e.g., human MHC-I).

[0116] In some embodiments, the anti-LILRB1 / 2 antibodies or antigen-binding fragments thereof of the present invention activate the release of cytokines from immune cells, such as myeloid cells, macrophages, B cells, NK cells, or T cells, and the cytokines, such as TNFα or IFNγ, are produced in a mixed lymphocyte reaction system. In some embodiments, the anti-LILRB1 / 2 antibodies or antigen-binding fragments thereof of the present invention can promote immune cells (such as macrophages or dendritic cells) to regulate cell markers, such as downregulating the expression of surface CD163 or upregulating the expression of surface CD86 or upregulating the expression of surface CD83.

[0117] In some embodiments, the anti-LILRB1 / 2 antibodies or antigen-binding fragments thereof of the present invention are capable of promoting the differentiation of dendritic cells into mature cells, as indicated by, for example, CD83 expression.

[0118] In some embodiments, the anti-LILRB1 / 2 antibodies or antigen-binding fragments thereof of the present invention relieve MDSCs from suppressing immune cells, such as T cells, such as CD3-positive T cells, for example, promoting the proliferation of such cells.

[0119] Single domain antibodies

[0120] In some embodiments, the anti-LILRB1 / 2 antibodies of the invention are single domain antibodies, particularly VHH antibodies.

[0121] 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 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 individual molecules, single-domain antibodies are also suitable components for constructing multispecific molecules.

[0122] In some embodiments, the anti-LILRB1 / 2 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 by IMGT.

[0123] In some embodiments, the anti-LILRB1 / 2 VHH antibodies of the present invention comprise

[0124] (i) three complementarity determining regions (CDRs) contained in the VH represented by any one of SEQ ID NOs: 1-8, 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-LILRB1 / 2 VHH antibodies of the present invention comprise or consist of a heavy chain variable region comprising

[0128] (i) three complementarity determining regions (CDRs) contained in the VH set forth in any one of SEQ ID NOs: 1-8, or

[0129] (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);

[0130] Preferably, the CDR sequences are defined according to IMGT.

[0131] In some embodiments, the anti-LILRB1 / 2 VHH antibodies of the present invention comprise complementarity determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3. In some embodiments, the anti-LILRB1 / 2 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.

[0132] In some embodiments, the VHH CDR1 comprises or consists of an amino acid sequence selected from SEQ ID NO: 9, 12, 15 or 18, 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: 9, 12, 15 or 18.

[0133] In some embodiments, the VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 10, 13, 16, 19, 21 or 22, 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: 10, 13, 16, 19, 21 or 22.

[0134] In some embodiments, the VHH CDR3 comprises or consists of an amino acid sequence selected from SEQ ID NO: 11, 14, 17 or 20, 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: 11, 14, 17 or 20.

[0135] In one embodiment, the anti-LILRB1 / 2 VHH antibody of the present invention comprises the complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein

[0136] (i) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 9, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 10 or 21, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 11;

[0137] (ii) 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 or 22, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 14;

[0138] (iii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 15, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 16, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 17; or

[0139] (iv) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 18, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 19, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 20.

[0140] In one embodiment, the anti-LILRB1 / 2 VHH antibody of the present invention comprises or consists of a heavy chain variable region, wherein the heavy chain variable region comprises complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein

[0141] (i) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 9, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 10 or 21, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 11;

[0142] (ii) 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 or 22, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 14;

[0143] (iii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 15, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 16, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 17;

[0144] (iv) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 18, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 19, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 20.

[0145] In some embodiments, the anti-LILRB1 / 2 VHH antibodies of the present invention comprise or consist of a heavy chain variable region, wherein the heavy chain variable region

[0146] (i) comprising or consisting of an amino acid sequence that is at least 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: 1-8;

[0147] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 1-8; or

[0148] (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: 1-8, preferably, the amino acid changes do not occur in the CDR regions.

[0149] In some embodiments, the anti-LILRB1 / 2 VHH antibody of the present invention comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 1-8.

[0150] 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.

[0151] 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 3. 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.

[0152] In some embodiments, the humanized single domain antibody of the present invention can be obtained by a method comprising the following steps:

[0153] Determine the CDR loop structure of a parent single domain antibody (e.g., a camelid VHH antibody screened from a phage display library);

[0154] 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;

[0155] 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;

[0156] If necessary, the key amino acids in the FR region are back-mutated to the amino acids corresponding to the VHH antibody to ensure the original affinity, thereby obtaining a humanized anti-LILRB1 / 2 VHH antibody, and the VHH antibody is optionally sequenced.

[0157] In some embodiments, the back mutation site is selected from one or more of the framework (FR).

[0158] In some embodiments, the heavy chain variable region germline gene suitable for humanization of the VHH antibodies of the invention is selected from IGHV3-48*03 or IGHV3-74*01.

[0159] 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., LILRB1 / 2) (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.

[0160] In some embodiments, the present invention provides affinity variants of the VHH polypeptides of the present invention. Preferably, the affinity variants exhibit one or more amino acid changes in the amino acid sequence relative to the parent single domain antibody from which they are derived, wherein the affinity variants have altered binding affinity for the target antigen compared to the parent antibody.

[0161] In some embodiments, the isomerization site in a CDR can be mutated, for example, position 53 in CDR2 can be mutated from aspartic acid to glutamine.

[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-LILRB1 / 2 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 is included between the VHH or its heavy chain variable region and the heavy chain constant region or Fc region, such as an antibody hinge region or a portion thereof contained in the Fc region, such as a hinge region or a portion thereof from IgG (including a native or mutated IgG hinge region or a portion thereof), which can also be considered a connecting peptide.

[0166] 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.

[0167] 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.

[0168] In some embodiments, the heavy chain antibody comprises an Fc portion 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., a 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.

[0169] 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.

[0170] 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.

[0171] In some embodiments, an anti-LILRB1 / 2 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain comprising a heavy chain variable region and an Fc region (eg, an Fc region comprising a hinge region).

[0172] In some embodiments, the anti-LILRB1 / 2 antibodies or antigen-binding fragments thereof of the present invention comprise or consist of a heavy chain comprising or consisting of a heavy chain variable region of a VHH of the present invention and an Fc region, wherein the heavy chain

[0173] (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: 52-59; or

[0174] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 52-59; or

[0175] (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: 52-59, preferably, the amino acid changes do not occur in the CDR regions.

[0176] III. Multispecific Antibodies

[0177] In some embodiments, the anti-LILRB1 / 2 antibodies of the present invention are multispecific antibodies, such as bispecific antibodies or trispecific antibodies. In some embodiments, the anti-LILRB1 / 2 multispecific antibodies of the present invention comprise one binding specificity for LILRB1 / 2 and other binding specificities for one or more molecules (e.g., PD1, PD-L1, or PD-L2).

[0178] Therefore, one aspect of the present invention relates to a bispecific antibody comprising

[0179] a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region specifically binds to LILRB1 / 2, and / or the second antigen-binding region specifically binds to PD1.

[0180] In some embodiments, the first antigen-binding region is from an anti-LILRB1 / 2 antibody described herein, eg, an anti-LILRB1 / 2 VHH.

[0181] In some embodiments, the second antigen-binding region is from an anti-PD1 antibody, such as the anti-PD1 antibody disclosed in WO2008156712A1, such as Pembrolizumab, for example, a Fab fragment of the anti-PD1 antibody.

[0182] The first antigen-binding region of the multispecific antibody suitable for use in the present invention may comprise or consist of the anti-LILRB1 / 2 VHH of the present invention, as long as it can specifically bind to LILRB1 and / or LILRB2.

[0183] The second antigen-binding region of the multispecific antibody suitable for use in the present invention may comprise or consist of an anti-PD1 full-length antibody or an antigen-binding fragment thereof (e.g., the anti-PD1 antibody or antigen-binding fragment thereof disclosed in WO2008156712A1, such as Pembrolizumab or an antigen-binding fragment thereof), as long as it can specifically bind to PD1, including but not limited to, for example, a full-length antibody, single-chain Fv, Fab, Fab', (Fab)2, single-domain antibody, VHH, or heavy chain antibody that specifically binds to PD1.

[0184] In some embodiments, the bispecific antibodies of the present invention are IgG-like bispecific antibodies. "IgG-like bispecific antibodies" as described herein refer to bispecific antibodies or trispecific antibodies comprising an Fc dimer. Therefore, in some embodiments, the bispecific antibodies of the present invention comprise an Fc dimer.

[0185] 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 one antigen and a VHH as an antigen-binding region that specifically binds another antigen. In some embodiments, the IgG-like bispecific antibody comprises a VHH fragment that specifically binds LILRB1 / 2 as a first antigen-binding region and a Fab that specifically binds PD1 as a second antigen-binding region.

[0186] 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 two first antigen-binding regions and two second antigen-binding regions.

[0187] In one embodiment, the bispecific antibody comprises a full-length antibody that specifically binds to one antigen, and two VHH fragments (the same or different) connected at the N-termini or C-termini of its two heavy chains with or without a linker.

[0188] In some embodiments, the bispecific antibodies of the present invention bind to LILRB1 and / or LILRB2 (e.g., human or monkey (e.g., cynomolgus monkey or rhesus monkey) LILRB1 and / or LILRB2) with higher affinity. In some embodiments, the bispecific antibodies of the present invention can bind to human LILRB1 / 2 with high affinity, for example, its K DIn some embodiments, the K is less than or equal to about 50 nM, such as less than or equal to about 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, or therebetween. D In some embodiments, the bispecific antibodies of the present invention also bind to human PD1 protein with high affinity, for example, its K D The value is less than or equal to about 10 nM, such as 1-10 nM, or between 2 nM-10 nM or 5 nM-10 nM, such as less than or equal to about 9 nM, 8 M, 7 nM or 6 nM.

[0189] In some embodiments, the bispecific antibodies of the invention have good thermal stability, for example, in a DSF assay, Tm1 is greater than 60 degrees Celsius, for example, between about 55-65 degrees Celsius, for example, about 59, 60, 61, 62, 63 or 64 degrees Celsius, for example, between about 57-65 degrees Celsius.

[0190] In some embodiments, the bispecific antibodies of the present invention block the binding of human LILRB1 protein or human LILRB2 protein to its ligand, for example, it blocks the binding of human LILRB1 protein to human HLAG protein, and / or blocks the binding of human LILRB1 protein to human MHC-I protein; and / or it blocks the binding of human LILRB2 to human HLAG, and / or it blocks the binding of human LILRB2 to human MHC-I protein.

[0191] In some embodiments, the bispecific antibodies of the present invention induce immune cells to kill target cells expressing the ligand of LILRB1 or LILRB2, such as dendritic cells, macrophages, NK cells, and the like.

[0192] In some embodiments, the bispecific antibodies of the present invention activate the release of immune cell cytokines, such as myeloid cells, macrophages, B cells, NK cells or T cells, and the cytokines are such as TNFα or IFNγ, for example, in a mixed lymphocyte reaction system. In some embodiments, the bispecific antibodies of the present invention can promote immune cells (such as macrophages or dendritic cells) to regulate cell markers, such as downregulating the expression of surface CD163 or upregulating the expression of surface CD86 or upregulating the expression of surface CD83.

[0193] In some embodiments, the bispecific antibodies of the invention are capable of promoting the differentiation of dendritic cells into mature cells, as indicated by, for example, CD83 expression.

[0194] In some embodiments, the bispecific antibodies of the invention promote the proliferation of T cells.

[0195] In some embodiments, the bispecific antibodies of the invention are effective in treating tumors.

[0196] Fab fragments of the multispecific antibodies suitable for use in the present invention

[0197] In some embodiments, the Fab fragment, which is one of the antigen-binding regions of a multispecific antibody, is composed of two polypeptide chains comprising the antibody VH, CH1 (optionally further comprising a hinge region), 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).

[0198] In some embodiments, in the multispecific antibody, the Fab can be fused to the N-terminus of the antibody's Fc domain via the C-terminus of the VH-containing chain. Preferably, the Fab comprises a VH-CH1 chain and a VL-CL chain, and is fused to the antibody Fc domain via the C-terminus of the CH1 of the VH-CH1 chain. 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 via a linker.

[0199] In some embodiments, the CH1 of a Fab may further comprise a partial hinge region, such as 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.

[0200] In some embodiments, the Fab comprised in the multispecific antibody of the invention specifically binds to PD1.

[0201] In some embodiments, the Fab heavy chain comprises VH and CH1 (and optionally comprises part of the hinge region EPKSC at the C-terminus of CH1). In some embodiments, the CH1 is a CH1 from IgG1, IgG2, IgG3 or IgG4, preferably a CH1 from IgG1. In some embodiments, the CH1

[0202] (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: 60;

[0203] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 60; or

[0204] (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: 60.

[0205] In some embodiments, the Fab light chain comprises VL-CL.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.

[0206] Fc dimers suitable for the multispecific antibodies of the present invention

[0207] In one embodiment, the two Fc regions in the multispecific antibody of the present invention dimerize to form a dimeric Fc. Preferably, the two Fc regions heterodimerize to form a heterodimeric Fc.

[0208] 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.

[0209] The Fc region fragments 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., CH2 domain, CH3 domain and 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.

[0210] 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 multispecific 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 an amino acid sequence having at least 90% identity thereto, e.g., 95%, 96%, 97%, 99% or greater identity thereto.

[0211] The Fc region of the antibody or multispecific antibody of the present invention can be mutated to obtain desired properties. Mutations in the Fc region are known in the art.

[0212] 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.

[0213] 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.

[0214] 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: 35 or 61 or 62 or 63, 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: 35 or 61 and comprises L234A / L235A mutations, D265A mutations, and P329A mutations.

[0215] As will be appreciated by those skilled in the art, in order to promote the formation of the multispecific antibodies of the present invention as heterodimers, the Fc regions contained in the multispecific antibodies of the present invention may contain mutations that are conducive to heterodimerization. In one embodiment, mutations are introduced into the CH3 regions of the two Fc regions. Methods for promoting heterodimerization of the Fc regions are known in the art. For example, the CH3 regions of the first Fc region and the CH3 regions of the second Fc region are engineered in a complementary manner so that each CH3 region (or its heavy chain) can no longer homodimerize with itself but is forced to heterodimerize with the other CH3 regions of the complementary engineering transformation (so that the first and second CH3 regions heterodimerize and no homodimer is formed between the two first CH3 regions or the two second CH3 regions). For example, based on the Knob-in-Hole technology, corresponding knob mutations and Hole mutations are introduced into the first monomer Fc region and the second monomer Fc region, respectively. For this technology, see, for example, Merchant, AM, et al. (1998). "An efficient route to human bispecific IgG." Nat Biotechnol 16(7): 677-681.

[0216] Specific binding to the antigen-binding region of PD1

[0217] The anti-PD1 antigen-binding region suitable for constructing the bispecific antibody of the present invention can be derived from any known anti-PD1 antibody, such as the antibodies disclosed in WO2008156712A1 (which is incorporated herein by reference in its entirety), such as Pembrolizumab.

[0218] In some embodiments, the multispecific antibodies of the present invention comprising an antigen-binding region that specifically binds to PD1 bind to PD1 (e.g., human PD1 or monkey PD1, such as cynomolgus monkey PD1) with a desired affinity. In some embodiments, the multispecific antibodies of the present invention comprising an antigen-binding region that specifically binds to PD1 are capable of binding to both human PD1 and monkey PD1, such as cynomolgus monkey PD1. In some embodiments, the affinity of the antibody is determined by thin-layer interferometry or surface plasmon resonance.

[0219] In some embodiments, the multispecific antibody of the present invention comprising an antigen-binding region that specifically binds to PD1 has an equilibrium dissociation constant (K D ) binds to human PD1 or monkey PD1, such as cynomolgus monkey PD1. In some embodiments, the multispecific antibody of the present invention comprising an antigen binding region that specifically binds to PD1, such as its K D Less than or equal to about 10 nM, such as with a K between about 1-10 nM, or 2 nM-10 nM or 5 nM-10 nM (e.g., less than or equal to about 9 nM, 8 M, 7 nM or 6 nM) D Binds to human PD1, such as cynomolgus monkey PD1.

[0220] In some embodiments, the multispecific antibodies of the present invention comprising an antigen-binding region that specifically binds to PD1 bind to PD1 expressed in cells.

[0221] In some embodiments, the antigen-binding region of the present invention that specifically binds to PD1 comprises three complementarity determining regions (HCDRs) from the heavy chain variable region of the antibody disclosed in WO2008156712A1, such as pembrolizumab, namely HCDR1, HCDR2, and HCDR3.

[0222] In some embodiments, the antigen-binding region that specifically binds to PD1 of the present invention comprises three complementarity-determining regions (LCDRs) from the light chain variable region of the antibody disclosed in WO2008156712A1, such as pembrolizumab, LCDR1, LCDR2, and LCDR3.

[0223] In some embodiments, the antigen-binding region of the present invention that specifically binds to PD1 comprises three complementarity determining regions (HCDRs) from the heavy chain variable region and three complementarity determining regions (LCDRs) from the light chain variable region of the antibody disclosed in WO2008156712A1, such as pembrolizumab.

[0224] In some aspects, the antigen binding region that specifically binds to PD1 of the present invention comprises an antibody disclosed in WO2008156712A1, such as the heavy chain variable region (VH) of Pembrolizumab. In some aspects, the antigen binding region that specifically binds to PD1 of the present invention comprises an antibody disclosed in WO2008156712A1, such as the light chain variable region (VH) of Pembrolizumab. In some aspects, the antigen binding region that specifically binds to PD1 of the present invention comprises an antibody disclosed in WO2008156712A1, such as the heavy chain variable region (VH) and light chain variable region (VL) of Pembrolizumab. In some embodiments, the heavy chain variable region comprises 3 complementary determining regions (CDRs) from the heavy chain variable region of the antibody disclosed in WO2008156712A1, such as Pembrolizumab, HCDR1, HCDR2 and HCDR3. In some embodiments, the light chain variable region comprises three complementarity determining regions (CDRs), LCDR1, LCDR2, and LCDR3, from the light chain variable region of an antibody disclosed in WO2008156712A1, such as pembrolizumab.

[0225] In some embodiments, the antigen-binding region that specifically binds to PD1 of the present invention further comprises an antibody heavy chain constant region HC. In some embodiments, the antigen-binding region that specifically binds to PD1 of the present invention further comprises an antibody light chain constant region LC. In some embodiments, the antigen-binding region that specifically binds to PD1 of the present invention further comprises a heavy chain constant region HC and a light chain constant region LC.

[0226] In some embodiments, the heavy chain variable region VH of the antigen binding region that specifically binds to PD1 of the present invention is

[0227] (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: 25; or

[0228] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 25; or

[0229] (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: 25, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0230] In some embodiments, the antigen-binding region light chain variable region VL of the present invention that specifically binds to PD1

[0231] (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: 29; or

[0232] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 29; or

[0233] (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: 29, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0234] In some embodiments, the three complementary determining regions (HCDRs) of the antigen binding region of the present invention that specifically bind to PD1 are from the heavy chain variable region, HCDR1, HCDR2 and HCDR3.

[0235] (i) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 25, or

[0236] (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 (i),

[0237] Preferably, the HCDRs are defined according to Kabat.

[0238] In some embodiments, the three complementary determining regions (LCDRs) of the antigen binding region of the present invention that specifically bind to PD1 are from the light chain variable region, LCDR1, LCDR2 and LCDR3.

[0239] (i) three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 29, or

[0240] (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 LCDR regions relative to the sequence of (i);

[0241] Preferably, the LCDR is determined according to Kabat.

[0242] In some embodiments, in the antigen binding region of the present invention that specifically binds to PD1,

[0243] HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 26; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 27; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 28; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 30; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 31; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 32.

[0244] In some specific embodiments of the present invention, the antigen binding region that specifically binds to PD1 of the present invention comprises VH and VL, wherein

[0245] The VH comprises the amino acid sequence of SEQ ID NO:25, 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:29, 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.

[0246] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to PD1 of the present invention comprises three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO: 25, and three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in VL as shown in SEQ ID NO: 29.

[0247] In some specific embodiments of the present invention, the antigen binding region that specifically binds to PD1 of the present invention comprises: HCDR1 as shown in SEQ ID NO:26, HCDR2 as shown in SEQ ID NO:27, HCDR3 as shown in SEQ ID NO:28; LCDR1 as shown in SEQ ID NO:30, LCDR2 as shown in SEQ ID NO:31 and LCDR3 as shown in SEQ ID NO:32.

[0248] 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.

[0249] In some embodiments, the antigen-binding region of the present invention that specifically binds to PD1 comprises a heavy chain constant region or a fragment thereof (e.g., CH1) that is an IgG1, IgG2, IgG3, or IgG4 constant region or a fragment thereof. In some embodiments, the antigen-binding region of the present invention that specifically binds to PD1 comprises a light chain constant region or a fragment thereof that is a kappa or lambda light chain constant region or a fragment thereof, such as a kappa light chain constant region or a fragment thereof.

[0250] In one embodiment, the antigen-binding region that specifically binds to PD1 of the present invention is a fragment (e.g., an antigen-binding fragment) of an anti-PD1 antibody (e.g., an antibody disclosed in WO2008156712A1, such as Pembrolizumab), preferably selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, single-domain antibody such as VHH, dAb (domain antibody) or linear antibody. In one embodiment, the antigen-binding region that specifically binds to PD1 of the present invention is an antibody disclosed in WO2008156712A1, such as Fab of Pembrolizumab. In one embodiment, the antigen-binding region that specifically binds to PD1 of the present invention is a Fab comprising the heavy chain variable region VH and the light chain variable region VL described in this section.

[0251] In some embodiments, the Fab heavy chain comprises VH and CH1 (and optionally a portion of the hinge region EPKSC at the C-terminus of CH1), wherein VH is the VH of an anti-PD1 antibody.

[0252] In some embodiments, the Fab light chain comprises VL-CL, wherein VL is the VL of an anti-PD1 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.

[0253] In some embodiments, the Fab heavy chain

[0254] (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: 64;

[0255] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 64; or

[0256] (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: 64.

[0257] In some embodiments, the Fab light chain

[0258] (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: 38;

[0259] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 38; or

[0260] (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: 38.

[0261] Exemplary bispecific antibody molecules

[0262] In some preferred embodiments, the present invention provides a bispecific antibody comprising an antigen-binding region that specifically binds to LILRB1 / 2 and an antigen-binding region that specifically binds to PD1, and optionally an Fc region.

[0263] 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 VHH fragment that specifically binds to LILRB1 / 2, and the second antigen-binding region is a Fab fragment that specifically binds to PD1. In some embodiments, the bispecific antibody of the present invention comprises two first antigen-binding regions and two second antigen-binding regions, for example, wherein the two first antigen-binding regions may be the same or different, and / or the two second antigen-binding regions may be the same or different.

[0264] Thus, in some embodiments, the present invention relates to a bispecific antibody that is an IgG-like bispecific antibody comprising a heavy chain and a light chain, wherein

[0265] The heavy chain contains from N-terminus to C-terminus

[0266] a Fab heavy chain that specifically binds to PD-1, an Fc region, and a VHH fragment that specifically binds to LILRB1 / 2, which is connected to the C-terminus of the Fc region via a linker or not (for example, the N-terminus of the VHH fragment is connected to the C-terminus of the Fc region via a linker or not), or

[0267] A VHH fragment that specifically binds to LILRB1 / 2, a Fab heavy chain that specifically binds to PD-1 (e.g., the N-terminus of the Fab heavy chain is connected to the C-terminus of the VHH fragment via or without a linker), and an Fc region;

[0268] and a light chain comprising a Fab light chain that specifically binds to PD1.

[0269] In some embodiments, the Fc region includes or does not include a hinge region.

[0270] In some embodiments, the present invention relates to a bispecific antibody, which is an IgG-like bispecific antibody comprising a heavy chain and a light chain, wherein

[0271] The heavy chain comprises a VH that specifically binds to the antigen-binding region of PD-1, a heavy chain constant region connected to the C-terminus thereof, and a VHH that specifically binds to LILRB1 / 2 connected to the C-terminus of the heavy chain constant region via or without a linker (connected to the N-terminus of the VHH); or the heavy chain comprises a VH that specifically binds to the antigen-binding region of PD-1, a heavy chain constant region connected to the C-terminus thereof, and a VHH that specifically binds to LILRB1 / 2 connected to the N-terminus of the VH via or without a linker (connected to the C-terminus of the VHH);

[0272] The light chain comprises a VL that specifically binds to the antigen binding region of PD-1 and a light chain constant region connected to the C-terminus thereof.

[0273] In some embodiments, the present invention relates to a bispecific antibody comprising a full-length antibody that specifically binds to PD1 and a VHH fragment that specifically binds to LILRB1 / 2, linked to the N-terminus or C-terminus of its heavy chain via or without a linker.

[0274] In some embodiments, the connection is direct connection or connection through a linker or connecting peptide or hinge region.In some embodiments, the connecting peptide is selected from (GGGGS)n, wherein n=1, 2, 3 or 4.

[0275] In some embodiments, the bispecific antibody comprises two heavy chains or two light chains, for example, two heavy chains and two light chains. In some embodiments, the two heavy chains may be identical or different. In some embodiments, the two light chains may be identical or different. In some embodiments, the bispecific antibody comprises two identical heavy chains and two identical light chains, or consists of two identical heavy chains and two identical light chains.

[0276] In some embodiments, the VHHs that specifically bind to LILRB1 / 2 can be the same or different, for example, bind to different LILRB1 / 2 epitopes, or bind to the same LILRB1 / 2 epitope but differ in sequence.

[0277] In some embodiments, the Fabs that specifically bind to PD1 can be the same or different, for example, bind to different PD1 epitopes, or bind to the same PD1 epitope but differ in sequence.

[0278] In some embodiments, the two heavy chains may be identical or different in the constant region or Fc region, e.g., differ in sequence. In some embodiments, the two light chains may be identical or different in the constant region, e.g., differ in sequence.

[0279] In some embodiments, the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 37 or 39, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and / or

[0280] The light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:38, 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.

[0281] IV. Nucleic Acids Encoding Antibodies and Host Cells Containing Them

[0282] In one aspect, the invention provides a nucleic acid encoding any of the above anti-LILRB1 / 2 antibodies or bispecific antibodies, or either chain thereof.

[0283] For example, the nucleic acids of the present invention include nucleic acids encoding an amino acid sequence selected from any one of SEQ ID NOs: 1-8, 37, 39 and 52-59, or nucleic acids encoding 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: 1-8, 37, 39 and 52-59.

[0284] 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.

[0285] In one aspect, the present invention provides nucleic acids encoding any of the above antibodies or any antibody chains. When expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid can exhibit human and / or monkey (e.g., cynomolgus monkey) LILRB1 and / or LILRB2 antigen binding ability.

[0286] In another aspect, the present invention provides a nucleic acid encoding any of the above bispecific antibodies. When expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid can exhibit binding ability to human PD1 and human or monkey (e.g., rhesus monkey or cynomolgus monkey) LILRB1 and / or LILRB2 antigens.

[0287] In one embodiment, the nucleic acids encoding each chain of the antibody or bispecific antibody of the present invention can be in the same vector or in different vectors. In another embodiment, the nucleic acids encoding each chain of the antibody or bispecific antibody of the present invention can be introduced into the same or different host cells for expression. Therefore, in some embodiments, the production method of the antibody or bispecific antibody of the present invention comprises the steps of culturing a host cell containing the nucleic acids encoding each chain under conditions suitable for expressing each chain of the molecule to produce the antibody or bispecific antibody of the present invention.

[0288] 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 eukaryotic expression vector is, for example, a pcDNA vector, such as pcDNA3.1.

[0289] 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-LILRB1 / 2 antibody or 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.

[0290] In some embodiments, 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. Mammalian host cell lines suitable for producing antibodies are known in the art.

[0291] V. Production and Purification of the Anti-LILRB1 / 2 Antibody or Bispecific Antibody of the Invention

[0292] In one embodiment, a method for preparing an anti-LILRB1 / 2 antibody or bispecific antibody of the present invention is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the anti-LILRB1 / 2 antibody or 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 expression of the anti-LILRB1 / 2 antibody or bispecific antibody or its chains, as provided above, and optionally recovering the anti-LILRB1 / 2 antibody or bispecific antibody from the host cell (or host cell culture medium).

[0293] The polynucleotide encoding the polypeptide chains of the anti-LILRB1 / 2 antibodies or 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 purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection or other conventional techniques.

[0294] Anti-LILRB1 / 2 antibodies or 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.

[0295] The purity of the antibody molecules of the present 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.

[0296] VI. Assays for Anti-LILRB1 / 2 Antibodies or Bispecific Antibodies

[0297] The anti-LILRB1 / 2 antibodies or 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.

[0298] In one aspect, the anti-LILRB1 / 2 antibody or bispecific antibody of the present invention is tested for its target (e.g., antigen, such as 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 PD1 and / or LILRB1 / 2 (or PD1 and / or LILRB1 / 2 expressed on a cell) can be determined using methods known in the art, and exemplary methods are disclosed herein. In some embodiments, radioimmunoassay (RIA) or thin-layer interferometry (BLI) or electrochemiluminescence (ECL) or surface plasmon resonance (SPR) or flow cytometry (FACS) is used for measurement.

[0299] The present invention also provides assays for identifying the physicochemical properties of anti-LILRB1 / 2 antibodies or bispecific antibodies, such as purity or thermal stability assays, such as those described in the Examples, such as DSC or DSF.

[0300] The present invention also provides assays for identifying the biological activity of an anti-LILRB1 / 2 antibody or bispecific antibody. The biological activity is selected from the properties of the anti-LILRB1 / 2 antibody or bispecific antibody of the present invention, for example, by the methods described in the Examples.

[0301] In some embodiments, the measuring of biological activity comprises measuring one or more activities of the anti-LILRB1 / 2 antibody or bispecific antibody selected from the following, for example, as measured by the assay described in Example 3 or Example 4:

[0302] Blocking the binding of human LILRB1 protein or human LILRB2 protein to its ligand;

[0303] Inducing immune cells to kill target cells expressing LILRB1 or LILRB2 ligands;

[0304] Inducing NK cells to kill target cells, such as cells expressing ligands for LILRB1 or 2 (such as HLAG proteins);

[0305] Inducing macrophages to phagocytose target cells, such as cells expressing ligands for LILRB1 or 2 (e.g., human MHC-I);

[0306] Activating the release of cytokines from immune cells, such as myeloid cells, macrophages, B cells, NK cells, or T cells, such as TNFα or IFNγ, for example, in a mixed lymphocyte reaction system;

[0307] Promoting immune cells (such as macrophages or dendritic cells) to regulate cell markers, such as downregulating the expression of surface CD163 or upregulating the expression of surface CD86 or upregulating the expression of surface CD83;

[0308] Promotes differentiation of dendritic cells into mature cells, as indicated by CD83 expression;

[0309] Relieving the inhibition of MDSC on immune cells, such as T cells, such as CD3-positive T cells, such as promoting the proliferation of the cells;

[0310] Promote T cell proliferation;

[0311] or tumor therapeutic activity.

[0312] 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.

[0313] VII. Immunoconjugates, Pharmaceutical Compositions, Pharmaceutical Combination Products, and Kits of Anti-LILRB1 / 2 Antibodies or Bispecific Antibodies of the Invention

[0314] In some embodiments, the present invention provides an immunoconjugate comprising any of the anti-LILRB1 / 2 antibodies or bispecific antibodies described herein. Preferably, the immunoconjugate comprises one or more other therapeutic agents (e.g., cytotoxins or small molecule compounds) or markers. In some embodiments, the immunoconjugate is an antibody-drug-conjugate (ADC).

[0315] In some embodiments, the present invention provides a composition, a medicament, or a formulation comprising any of the anti-LILRB1 / 2 antibodies or bispecific antibodies described herein, preferably a composition is a pharmaceutical composition.

[0316] In one embodiment, a composition, e.g., a pharmaceutical composition, comprises an anti-LILRB1 / 2 antibody or bispecific antibody of the invention in combination with one or more additional therapeutic agents.

[0317] The composition, medicament 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. As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic agents and absorption delaying agents, etc.

[0318] 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.

[0319] 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.

[0320] Medicaments or formulations comprising the anti-LILRB1 / 2 antibodies or bispecific antibodies described herein can be prepared by mixing the anti-LILRB1 / 2 antibodies or bispecific antibodies of the invention having the desired purity with one or more optional pharmaceutical excipients, for example, in the form of a lyophilized formulation or aqueous solution.

[0321] 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.

[0322] The present invention also provides a pharmaceutical combination or a pharmaceutical combination product comprising the anti-PD1 antibody or anti-LILRB1 / 2 antibody or bispecific antibody of the present invention, and one or more other therapeutic agents.

[0323] The present invention also provides a complete kit comprising the drug combination, for example, the complete kit comprises in the same package:

[0324] - a first container containing a pharmaceutical composition comprising the anti-LILRB1 / 2 antibody or bispecific antibody of the present invention;

[0325] - A second container containing a pharmaceutical composition comprising an additional therapeutic agent.

[0326] 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 or an immunoactivator).

[0327] VIII. Uses of anti-LILRB1 / 2 antibodies or bispecific antibodies and methods of using the same

[0328] In one aspect, the present invention relates to an anti-LILRB1 / 2 antibody or bispecific antibody of the invention (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, combination products, etc. comprising the same) for use in therapy.

[0329] In some embodiments, the present invention relates to the anti-LILRB1 / 2 antibodies or bispecific antibodies of the present invention (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, combination products, etc. comprising the same) for use as medicaments.

[0330] In another aspect, the present invention provides a method for preventing or treating a disease in an individual, comprising administering to the individual an anti-LILRB1 / 2 antibody or 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 an individual, comprising administering to the individual a bispecific antibody of the present invention, or an immunoconjugate, composition, or drug or formulation comprising the same.

[0331] In some embodiments, the disease is a LILRB1 / 2-associated disease, such as a disease in which LILRB1 / 2 is abnormally expressed (e.g., overexpressed). 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 tumor may be a solid tumor or a blood tumor, such as a malignant solid tumor or a blood tumor, such as cancer. In some embodiments, the cancer is melanoma, lung cancer, gastric cancer, breast cancer, or pancreatic cancer.

[0332] In some embodiments, the disease treatment would benefit from activation of the PD1 signaling pathway and / or activation of T cells.

[0333] In some embodiments, the cancer is a LILRB1 or LILRB2 or LILRB1 and LILRB2 positive cancer. In some embodiments, the cancer is characterized by elevated LILRB1 or LILRB2 or LILRB1 and LILRB2 protein levels and / or nucleic acid levels in the patient (e.g., in the patient's cancer tissue or cells) (e.g., compared to the LILRB1 or LILRB2 or LILRB1 and LILRB2 protein levels and / or nucleic acid levels in the same tissue of a healthy individual, or compared to the LILRB1 or LILRB2 or LILRB1 and LILRB2 protein levels and / or nucleic acid levels in adjacent healthy tissue of the patient). ), for example, a cancer in which tumor cells have elevated LILRB1 or LILRB2 or both protein levels and / or nucleic acid levels (for example, compared to LILRB1 or LILRB2 or both protein levels and / or nucleic acid levels in cells of the same tissue of a healthy individual, or compared to LILRB1 or LILRB2 or both protein levels and / or nucleic acid levels in healthy cells of the same tissue or cells of adjacent healthy tissue of the patient).

[0334] In some embodiments, the cancer is a PD1 (or PD-L1 or PD-L2) positive cancer. In some embodiments, the cancer is characterized by elevated protein levels and / or nucleic acid levels of PD1 (or PD-L1 or PD-L2) in the patient (e.g., in the patient's cancer tissue or cells) (e.g., compared to the protein levels and / or nucleic acid levels of PD1 (or PD-L1 or PD-L2) in the same tissue of a healthy individual, or compared to the protein levels and / or nucleic acid levels of PD1 (or PD-L1 or PD-L2) in adjacent healthy tissues of the patient), for example, the tumor cells of the cancer have elevated protein levels and / or nucleic acid levels of PD1 (or PD-L1 or PD-L2) (e.g., compared to the protein levels and / or nucleic acid levels of PD1 (or PD-L1 or PD-L2) in cells of the same tissue of a healthy individual, or compared to the protein levels and / or nucleic acid levels of PD1 (or PD-L1 or PD-L2) in healthy cells of the same tissue of the patient or cells of adjacent healthy tissue).

[0335] In some embodiments, the cancer is positive for both PD1 (or PD-L1 or PD-L2) and LILRB1 or LILRB2 or both.

[0336] The anti-LILRB1 / 2 antibodies or 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, intrapulmonary administration and intranasal 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.

[0337] For the prevention or treatment of disease, the appropriate dosage of the anti-LILRB1 / 2 antibodies or bispecific antibodies of the 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 treatment, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient as a single treatment or over a series of treatments.

[0338] In other aspects, the present invention provides the anti-LILRB1 / 2 antibodies or bispecific antibodies of the present invention, or immunoconjugates or compositions or combination products comprising the same, for use as described herein, such as for preventing or treating the relevant diseases or disorders mentioned herein.

[0339] In other aspects, the present invention provides use of the anti-LILRB1 / 2 antibody or bispecific antibody of the present invention, or an immunoconjugate or composition or combination product comprising the same, in the production or preparation of a medicament for the uses described herein, e.g., for preventing or treating the relevant diseases or conditions mentioned herein.

[0340] In some embodiments, the anti-LILRB1 / 2 antibody or 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.

[0341] In some embodiments, the treatment modality is, for example, surgery or radiation therapy.

[0342] 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).

[0343] IX. Diagnosis and Testing

[0344] In one aspect, the present invention also relates to methods for diagnosis and detection (eg, for diagnostic or non-diagnostic purposes) of anti-LILRB1 / 2 antibodies or bispecific antibodies and compositions for diagnosis and detection comprising the same.

[0345] In certain embodiments, the anti-LILRB1 / 2 antibodies provided herein can be used to detect the presence of LILRB1 or LILRB2 or LILRB1 and LILRB2 in a biological sample. In certain embodiments, the bispecific antibodies provided herein can be used to detect the presence of PD1 and / or LILRB1 or LILRB2 or LILRB1 and LILRB2 in a biological sample.

[0346] 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 blood, serum, or plasma.

[0347] In certain embodiments, the method comprises contacting a biological sample with an anti-LILRB1 / 2 antibody or bispecific antibody as described herein under conditions that allow binding to LILRB1 or LILRB2 or LILRB1 and LILRB2, and detecting whether a complex is formed between the anti-LILRB1 / 2 antibody or bispecific antibody and LILRB1 or LILRB2 or LILRB1 and LILRB2. The formation of a complex indicates the presence of LILRB1 / 2. The method can be an in vitro or in vivo method.

[0348] In certain embodiments, labeled anti-LILRB1 / 2 antibodies or bispecific antibodies are 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 indirectly detectable moieties, 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.

[0349] In some embodiments provided herein, the sample is obtained prior to treatment with an anti-LILRB1 / 2 antibody or 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.

[0350] In some embodiments, LILRB1 or LILRB2 or both are detected prior to treatment, eg, prior to initiation of treatment or prior to a treatment after a treatment interval.

[0351] X. Specific Implementation Plan

[0352] In one aspect, the present invention relates to the following embodiments:

[0353] 1. A VHH antibody that specifically binds to LILRB1 / 2, comprising

[0354] The three complementarity determining regions (CDRs) contained in the VHH shown in any one of SEQ ID NOs: 1-8,

[0355] Preferably, the CDR sequences are defined according to IMGT.

[0356] 2. The VHH antibody of embodiment 1, comprising the complementarity determining regions (CDRs) VHH CDR1, VHH CDR2 and VHH CDR3, wherein

[0357] (i) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 9, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 10 or 21, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 11;

[0358] (ii) 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 or 22, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 14;

[0359] (iii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 15, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 16, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 17; or

[0360] (iv) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 18, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 19, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 20.

[0361] 3. The VHH antibody of embodiment 1, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region

[0362] (i) comprising or consisting of an amino acid sequence that is at least 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: 1-8;

[0363] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 1-8; or

[0364] (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: 1-8, preferably, the amino acid changes do not occur in the CDR regions.

[0365] 4. A heavy chain antibody that specifically binds to LILRB1 / 2, comprising the VHH antibody of any one of embodiments 1-3.

[0366] 5. The heavy chain antibody of embodiment 4, comprising the VHH antibody of any one of embodiments 1-3 linked to an antibody constant region or Fc region, preferably, the antibody constant region or Fc region is derived from human IgG1, human IgG2, human IgG3 or human IgG4.

[0367] 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, optionally comprising L234A / L235A mutations, D265A mutations and P329A mutations, preferably, the Fc region

[0368] (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: 35 or 61 or 62 or 63; or

[0369] (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 35 or 61 or 62 or 63; or

[0370] (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: 35 or 61 or 62 or 63.

[0371] 7. The heavy chain antibody according to embodiment 4, wherein

[0372] (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: 52-59; or

[0373] (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 52-59; or

[0374] (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: 52-59, preferably, the amino acid changes do not occur in the CDR regions.

[0375] 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 humanized antibody.

[0376] 9. A multispecific antibody comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region specifically binds to LILRB1 / 2 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. Preferably, the multispecific antibody is a bispecific antibody.

[0377] 10. The multispecific antibody of embodiment 9, wherein the second antigen binding region specifically binds to PD1.

[0378] 11. The multispecific antibody of embodiment 10, wherein the second 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

[0379] (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: 25, and LCDR1, LCDR2, and LCDR3 are the three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the VL set forth in SEQ ID NO: 29; or

[0380] (ii) HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 26; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 27; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 28; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 30; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 31; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 32.

[0381] 12. The multispecific antibody of embodiment 11, wherein the second antigen-binding region comprises VH and VL, wherein

[0382] The VH comprises or consists of the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and / or

[0383] The VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 29, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0384] 13. The multispecific antibody according to any one of embodiments 10-12, wherein the second antigen binding region is a Fab that specifically binds to PD1.

[0385] 14. The multispecific antibody according to embodiment 13, wherein the Fab comprises VH and CH1 of the second antigen binding region, wherein the CH1 is CH1 from IgG1, IgG2, IgG3 or IgG4, preferably CH1 from IgG1.

[0386] 15. The multispecific antibody of embodiment 14, wherein the CH1

[0387] (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: 60; or

[0388] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 60.

[0389] 16. The multispecific antibody of embodiment 15, wherein the Fab heavy chain of the second antigen binding region

[0390] (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: 64;

[0391] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 64; or

[0392] (iii) an amino acid sequence comprising or consisting 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 an amino acid sequence selected from SEQ ID NO: 64;

[0393] and / or the second antigen binding region of the Fab light chain

[0394] (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: 38;

[0395] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 38; or

[0396] (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: 38.

[0397] 17. The multispecific antibody of embodiment 10, wherein the second antigen binding region comprises

[0398] (1) Three complementarity-determining regions (HCDRs) from the heavy chain variable region and three complementarity-determining regions (LCDRs) from the light chain variable region of pembrolizumab;

[0399] (2) the heavy chain variable region (VH) and light chain variable region (VL) of pembrolizumab; or

[0400] (3) Fab of Pembrolizumab.

[0401] 18. The multispecific antibody of any one of embodiments 9-17, wherein the first antigen binding region is the VHH of any one of embodiments 1-3 and 8.

[0402] 19. The multispecific antibody of any one of embodiments 9-18, wherein the multispecific antibody is an IgG-like bispecific antibody comprising an Fc dimer, wherein the two Fc regions constituting the Fc dimer are identical, and optionally, the Fc region further comprises a mutation that reduces binding to Fcγ receptors, for example, one or more of the L234A / L235A mutation, the D265A mutation, or the P329A mutation; for example, the L234A / L235A mutation, the D265A mutation, and the P329A mutation.

[0403] 20. The bispecific antibody of embodiment 19, wherein the Fc region

[0404] (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: 35 or 61 or 62 or 63; or

[0405] (ii) comprising or consisting of the amino acid sequence shown in SEQ ID NO: 35 or 61 or 62 or 63; or

[0406] (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: 35 or 61 or 62 or 63.

[0407] 21. The multispecific antibody of any one of embodiments 9-20, which is a bispecific antibody and comprises a first antigen binding region, a second antigen binding region and an Fc dimer, wherein the first antigen binding region is a VHH that specifically binds to LILRB1 / 2, such as the VHH of any one of embodiments 1-3 and 8, and the second antigen binding region is a Fab fragment that specifically binds to PD1.

[0408] 22. The multispecific antibody of embodiment 21, wherein the bispecific antibody comprises two first antigen-binding regions and two second antigen-binding regions, for example, the two first antigen-binding regions may be the same or different, or / and the two second antigen-binding regions may be the same or different.

[0409] 23. The multispecific antibody of embodiment 21 or 22, comprising a heavy chain and a light chain, wherein

[0410] The heavy chain contains from N-terminus to C-terminus

[0411] a Fab heavy chain that specifically binds to PD-1, an Fc region, and a VHH fragment that specifically binds to LILRB1 / 2, which is connected to the C-terminus of the Fc region via a linker or not (for example, the N-terminus of the VHH fragment is connected to the C-terminus of the Fc region via a linker or not), or

[0412] A VHH fragment that specifically binds to LILRB1 / 2, a Fab heavy chain that specifically binds to PD-1 (e.g., the N-terminus of the Fab heavy chain is connected to the C-terminus of the VHH fragment via or without a linker), and an Fc region;

[0413] and a light chain comprising a Fab light chain that specifically binds to PD1.

[0414] 24. The multispecific antibody of embodiment 21 or 22, comprising a heavy chain and a light chain, wherein

[0415] The heavy chain comprises a VH that specifically binds to the antigen-binding region of PD-1, a heavy chain constant region connected to the C-terminus thereof, and a VHH that specifically binds to LILRB1 / 2 connected to the C-terminus of the heavy chain constant region via or without a linker (connected to the N-terminus of the VHH); or the heavy chain comprises a VH that specifically binds to the antigen-binding region of PD-1, a heavy chain constant region connected to the C-terminus thereof, and a VHH that specifically binds to LILRB1 / 2 connected to the N-terminus of the VH via or without a linker (connected to the C-terminus of the VHH);

[0416] The light chain comprises a VL that specifically binds to the antigen binding region of PD-1 and a light chain constant region connected to the C-terminus thereof.

[0417] 25. The multispecific antibody of embodiment 23 or 24, comprising two heavy chains or two light chains, for example comprising two heavy chains and two light chains, wherein the two heavy chains are identical or different, or the two light chains are identical or different.

[0418] 26. The multispecific antibody of any one of embodiments 23-25, wherein

[0419] The heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 37 or 39, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and / or

[0420] The light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:38, 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.

[0421] 27. The multispecific antibody of any one of embodiments 9-20, which is a bispecific antibody and comprises a full-length antibody that specifically binds to PD1 and a VHH of any one of embodiments 1-3 and 8 connected to the N-terminus or C-terminus of its heavy chain via or without a linker.

[0422] 28. The multispecific antibody of embodiment 27, wherein the full-length antibody that specifically binds to PD1 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as defined in embodiment 7; or comprises VH and VL as defined in embodiment 8, or comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3; or VH and VL of an antibody disclosed in 156712A1, such as pembrolizumab.

[0423] 29. 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 multispecific antibody of any one of embodiments 9-28, or consisting of said nucleic acid sequence.

[0424] 30. An expression vector comprising the nucleic acid molecule of embodiment 29.

[0425] 31. A host cell comprising the nucleic acid molecule of embodiment 29 or the expression vector of embodiment 30. 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.

[0426] 32. 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 multispecific antibody of any one of embodiments 9-28, the method comprising culturing a host cell comprising the nucleic acid molecule of embodiment 29 or the expression vector of embodiment 30 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).

[0427] 33. 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 multispecific antibody of any one of embodiments 9-28.

[0428] 34. 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 multispecific antibody of any one of embodiments 9-28, or the immunoconjugate of embodiment 33, and optionally a pharmaceutically acceptable excipient.

[0429] 35. 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 multispecific antibody of any one of embodiments 9-28, or the immunoconjugate of embodiment 33, and one or more other therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators).

[0430] 36. A method for preventing or treating a tumor, such as cancer, in an individual, comprising administering to the individual 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 9-28, or the immunoconjugate of embodiment 33, or the pharmaceutical composition or drug or formulation of embodiment 34; or the pharmaceutical combination product of embodiment 35.

[0431] 37. The method of embodiment 36, wherein the tumor cells of the tumor have elevated protein levels and / or nucleic acid levels of LILRB1 / 2 (e.g., elevated expression), and optionally elevated protein levels and / or nucleic acid levels of PD1 (e.g., elevated expression).

[0432] 38. The method of embodiment 36 or 37, wherein the tumor is a solid tumor or a blood tumor, such as a malignant solid tumor or a blood tumor, such as a cancer, such as the cancer is melanoma, lung cancer, gastric cancer, breast cancer or pancreatic cancer.

[0433] 39. The method of any one of embodiments 36-38, wherein the method further comprises administering the drug in combination with other therapies, such as treatment modalities (e.g., surgery or radiotherapy) and / or other therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators).

[0434] 40. A method for detecting the presence of LILRB1 / 2 in a biological sample, comprising:

[0435] (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 multispecific antibody of any one of embodiments 9-28 under conditions that allow binding thereof to LILRB1 / 2,

[0436] (ii) detecting whether a complex is formed between the antibody or bispecific antibody and LILRB1 / 2,

[0437] The formation of the complex indicates the presence of LILRB1 / 2. Example

[0438] Example 1. Generation of anti-human LILRB1 / 2 antibodies

[0439] 1.1 Animal immunization

[0440] Two healthy adult Alpaca alpacas were immunized with human LILRB1 (Uniprot sequence number: Q8NHL6, SEQ ID NO: 47). The human LILRB1 protein used was the LILRB1 extracellular domain with a His tag (Sino-Biological, Cat. No. 16014-H08H). The alpacas were immunized four times, every two weeks (Table 1). Blood was collected 7 days after the third and fourth immunizations, and the alpaca immune sera were analyzed by FACS using human LILRB1-his protein and rhesus monkey LILRB1-his protein (F7H3G7) (KACTUS, Cat. No. LIL-CM1B1), respectively. Serum from the NB313-B alpaca after four immunizations was used for PBMC isolation and phage library construction.

[0441] Table 1. Alpaca immunization schedule

[0442] 1.2 Phage Library

[0443] Using phage display technology, antibody genes from PBMCs (PBMCs) obtained from NB313-B alpacas after quadruple immunization were cloned into a phage display vector to construct an antibody library. Peripheral blood was isolated from NB313-B alpacas, and total RNA was extracted from PBMCs using the RNAprep pure Cell Kit (TIANGEN, Cat# DP430). RNA was reverse-transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo, K1622). Single-domain antibody VHH genes were amplified using the cDNA as a PCR template, using a high-fidelity polymerase. The VHH gene fragments were inserted into a phage display vector and transformed into competent Escherichia coli. TG1 bacteria transformed with the correct antibody plasmid were amplified, ultimately constructing an antibody gene library containing VHH gene fragments with a library capacity of 1.29E+09.

[0444] 1.3 Phage panning

[0445] Using phage display technology, phage libraries were screened on solid phase with human LILRB1-his protein in the first round and monkey LILRB1-his protein in the second round to obtain VHH antibodies that bind to both human and monkey LILRB1.

[0446] After two rounds of screening, the eluted phage was diluted and used to infect TG1 cells in the logarithmic phase. The cells were then plated on 2YT (50 μg / mL carbenicillin + 2% glucose) plates and incubated overnight at 37°C. The next day, a single colony was picked from the plate and plated in a deep-well plate containing 400 μL of 2YT (50 μg / mL carbenicillin) liquid medium. The culture was continued at 37°C, 220 rpm, until the logarithmic phase was reached. 1 mM IPTG was added, and the cells were induced at 30°C overnight. The next day, the plates were centrifuged at 500g for 5 minutes, and the supernatant was collected for ELISA analysis.

[0447] Human LILRB1-his was diluted to a concentration of 4 μg / mL using PBS buffer at pH 7.4, added to a 96-well ELISA plate at a volume of 50 μL / well, and placed at 4°C overnight. The next day, after discarding the liquid, 200 μL / well of 1% skim milk blocking solution diluted with PBS was added, and the plates were incubated in a 37°C incubator for 1 hour for blocking. After blocking, the blocking buffer was discarded and the plate was washed twice with PBST buffer (pH 7.4 PBS containing 0.005% Tween-20). 50 μL / well of supernatant was added and the plate was incubated at 37°C for 1 hour. The reaction solution was discarded and the plate was washed twice with PBST. 50 μL / well of diluted HRP-conjugated mouse anti-HA tag secondary antibody (Sino Biological, Cat. No. 100028-MM10) was added and incubated at 37°C for 1 hour. The plate was washed twice with PBST and 50 μL / well of TMB was added for 5 minutes of color development. The reaction was terminated by adding 50 μL / well of 1 M H₂SO₄. The absorbance was read at 450 nm using a microplate reader to count the number of clones binding to human LILRB1 and monkey LILRB1. Candidate clones were induced to express antibodies, and the supernatants were collected for ELISA analysis.

[0448] Positive clones that bind to human LILRB1 and monkey LILRB1 were selected for sequencing and identification. After screening, four antibody clones were finally selected for further analysis and research. The amino acid sequences of the VHH variable regions of the generated antibodies and the EU or Kabat variable region sequences of the antibodies are shown in Table 2.

[0449] Table 2: Variable region amino acid sequences and CDRs of camel-derived anti-LILRB1 / 2 VHH antibodies

[0450] Example 2: Characterization of LILRB1 / 2 antibodies obtained by phage display technology

[0451] 2.1 Synthesis, expression and purification of camel-derived anti-LILRB1 / 2 VHH antibodies and control antibodies

[0452] The DNA sequences of the four camel-derived VHH antibodies in Table 2 were codon-optimized and gene-synthesized by Universal Biosystems (Anhui) Co., Ltd. The genes encoding the VHH antibodies were inserted into an expression vector containing a gene encoding the human IgG1 mut heavy chain constant region Fc (mutation sites C220S, L234A, L235A, D265A, P329A, SEQ ID NO: 35).

[0453] The anti-LILRB1 control antibody 15G8 sequence (heavy chain: SEQ ID NO: 40; light chain: SEQ ID NO: 41) was derived from patent WO2021028921, the anti-LILRB2 control antibody IO-108 sequence (heavy chain: SEQ ID NO: 42; light chain: SEQ ID NO: 43) was derived from patent WO2022087188A1, and the anti-LILRB2 control antibody MK-4830 sequence (heavy chain: SEQ ID NO: 44; light chain: SEQ ID NO: 45) was derived from patent WO2021138079A1. The genes encoding the anti-LILRB1 and anti-LILRB2 control antibodies were respectively inserted into expression vectors to obtain plasmids encoding anti-LILRB1 or anti-LILRB2 antibodies; the anti-PD1 control antibody was pembrolizumab (heavy chain: SEQ ID NO: 46; light chain: SEQ ID NO: 47). NO:38), INN sequence number is 9798, and the gene encoding the anti-PD-1 control antibody is inserted into the expression vector pcDNA3.1.

[0454] Use ExpiCHO according to the manufacturer's product manual. TM The camel-derived anti-LILRB1 / 2 VHH plasmid and control antibody plasmid obtained above were transfected into ExpiCHO-S cells to express anti-LILRB1 / 2, anti-LILRB1, anti-LILRB2, or anti-PD1 antibodies, respectively. The cells were cultured for 10-12 days after transfection. When the cell viability dropped to 60% to 70%, the supernatant was collected and the antibody expressed in the supernatant was purified using the MabSelect Sure Protein A affinity chromatography system (GE healthcare). The purified antibody was concentrated, sterile filtered, and the purity of the antibody protein was detected by SDS-PAGE and molecular exclusion.

[0455] 2.2 Physicochemical Analysis of Camel-derived Anti-LILRB1 / 2 VHH Antibodies and Control Antibodies

[0456] The purity of the obtained camel-derived anti-LILRB1 / 2 VHH antibody was confirmed by size exclusion chromatography. Specifically, 100 mM sodium phosphate + 100 mM Na2SO4 (pH 7.0) was used as the running buffer, and 20 μg of sample was injected onto a TSK G3000SWXL column. The column was run for 30 minutes. The collected effluent was measured using an Agilent 1220 HPLC, and the data were analyzed using OpenLAB software.

[0457] Table 3: Purity of anti-LILRB1 / 2 VHH antibodies and control antibodies

[0458] 2.3 Binding of camel-derived anti-LILRB1 / 2 VHH antibodies to human LILRB1 protein expressed on the cell surface

[0459] The human LILRB1 protein (Q8NHL6) gene sequence was codon-optimized and synthesized by General Biosystems (Anhui) Co., Ltd. and cloned into a stable expression vector. The gene encoding the human LILRB1 protein was transfected into CHO-K1 cells (purchased from the American Type Culture Collection (ATCC), Catalog No. CCL-61) using a Bio-Rad electroporator (BIO-RAD, Model: Gene Pul Ser X Cell™). The transfected cells were selected with 4 μg / mL puromycin (Gibco, Catalog No. A1113802) and plated to obtain engineered CHO-K1 cells that overexpress human LILRB1, designated CHOK1-huLILRB1. These cells were cultured in complete DME / F12 (Hyclone, Catalog No. SH30261.01) medium supplemented with 10% fetal bovine serum (Gibco, Catalog No. 10099-141) and 4 μg / mL puromycin.

[0460] When CHOK1-huLILRB1 cells were pressure-cultured to 80-90% confluence, they were digested with 0.25% trypsin (Gibco, Catalog No. 25200072) to obtain a single-cell suspension. The suspension was centrifuged at 400 × g at room temperature, and the culture medium was discarded. The cell pellet was washed once with PBS (Hyclone, Catalog No. SH30256.01) and centrifuged, and the supernatant was discarded. The cell pellet was resuspended in a serial dilution of the camel-derived anti-LILRB1 / 2 VHH antibody obtained in the present invention and the control antibody 15G8 analog (i.e., a synthetic 15G8 control antibody, with an initial concentration of 25 μg / mL and a 4-fold serial dilution to 6.25 μg / mL, 1.563 μg / mL, 0.391 μg / mL, 0.0977 μg / mL, 0.0244 μg / mL, 0.0061 μg / mL, and 0.00153 μg / mL, for a total of 8 points) and incubated at 4°C for 30 minutes. After washing the cells once with PBS, the cell pellet was resuspended in 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). The cells were incubated at 4°C in the dark for 30 minutes. After washing the cells twice with PBS and resuspending them, the fluorescence signal in the PE channel was detected using a flow cytometer (Beckman, CytoFlex). The results are shown in Figures 1 and 2.

[0461] The results shown in Figures 1 and 2 show that the camel-derived anti-LILRB1 / 2 VHH antibody can bind to CHOK1-huLILRB1 engineered cells expressing human LILRB1 protein, and the binding ability of the camel-derived anti-LILRB1 / 2 VHH antibody is slightly weaker than that of the control antibody 15G8 analog.

[0462] Example 3: Humanization and characterization of camel-derived anti-LILRB1 / 2 antibodies

[0463] 3.1 Humanization of camel-derived anti-LILRB1 / 2 antibodies

[0464] Camel-derived antibodies VH1 and VH15 were humanized, and VHH antibodies were humanized using CDR grafting technology. In short: the above VH1 and VH15 sequences were searched and compared in the IMGT database. The sequence of VH1 was searched and compared in the IMGT database, and the human germline gene sequence IGHV3-48*03 with high homology to the VH1 variable region was obtained as the VHH variable region humanization framework. The CDRs of the VH1 variable region were transplanted into the corresponding humanized framework to form a humanized anti-VH1 antibody variable region. The sequence of VH15 was searched and compared in the IMGT database, and the human germline gene sequence IGHV3-74*01 with high homology to the VH15 variable region was obtained as the VHH variable region humanization framework. The CDRs of the VH15 variable region were transplanted into the corresponding humanized framework to form a humanized anti-VH15 antibody variable region. To maintain the affinity of the camelid LILRB1 / 2 antibody, the variable regions of the obtained humanized antibodies were backmutated. The isomerization site at position H53 in the CDR2 region of the VH1 antibody was mutated from aspartic acid to glutamine, and the isomerization site at position H53 in the CDR2 region of the VH15 antibody was mutated from aspartic acid to glutamine.

[0465] The humanized VHH sequence was thus obtained. Table 4 lists the amino acid sequence of the humanized antibody VHH and the corresponding mutation sites.

[0466] Table 4 Humanized antibody VHH amino acid sequence

[0467] The variable region amino acid sequence was sent to Universal Biosystems (Anhui) Co., Ltd. for codon optimization and gene synthesis. The genes encoding the VHH region of the antibody were sequentially inserted into an expression vector containing the gene encoding the human IgG1 mut heavy chain constant region Fc (SEQ ID NO: 35) to obtain a plasmid expressing the full-length heavy chain of the anti-LILRB1 / 2 humanized antibody.

[0468] 3.2 Expression and purification of humanized anti-LILRB1 / 2 antibodies

[0469] The VHH-encoding plasmids were transfected into ExpiCHO-S cells to express humanized anti-LILRB1 / 2 antibodies and purified accordingly (see Example 2.1 for specific methods). The purified antibodies were concentrated, sterile filtered, and protein purity was determined by SDS-PAGE and size exclusion chromatography (SEC).

[0470] 3.3 Physicochemical Analysis of Humanized Anti-LILRB1 / 2 Antibodies

[0471] The purity of the obtained humanized anti-LILRB1 / 2 antibody was confirmed by size exclusion chromatography. Specifically, 20 μg of sample was injected onto a TSK G3000SWXL column using 100 mM sodium phosphate + 100 mM Na2SO4 (pH 7.0) as the running buffer. The column was run for 30 minutes. The collected effluent was measured using an Agilent 1220 HPLC, and the data were analyzed using OpenLAB software.

[0472] Table 5 Purity of humanized antibodies

[0473] 3.4 Thermal Stability Analysis of Humanized Anti-LILRB1 / 2 Antibodies (DSF)

[0474] Differential scanning fluorimetry (DSF) is used to assess the thermal stability of proteins by measuring the amount of fluorescent dye bound to structurally altered proteins as the temperature is increased. Specifically, it is used to measure the thermal transition midpoint (Tm), an indicator of the relative stability of a protein in a liquid.

[0475] Antibodies VH1-2, VH1-D53E, VH15-2, and VH15-D53E were diluted to 5 μM in PBS buffer. A fluorescent dye (SYPRO Orange, Sigma, S5692) was diluted 150-fold and thoroughly mixed with the antibodies. The samples were then transferred to a 96-well plate and assayed in a quantitative fluorescence PCR instrument (Bio-Rad, CFX96 touch). The sample volume per well was 25 μl. The temperature range was 25–95°C, with a ramp rate of 1°C / min. The results were analyzed using Bio-Rad CFX Manager automated analysis software. The DSF assay results are shown in Table 6 and Figure 3. As shown in Table 6, the Tm values ​​for VH1-2, VH1-D53E, VH15-2, and VH15-D53E were 62.2°C, 63.0°C, 62.2°C, and 62.3°C, respectively (Tm1, the first minimum temperature, corresponds to the Tm of the CH2 domain of the Fc fragment).

[0476] Table 6 Thermal stability of humanized antibodies

[0477] 3.5 Affinity Binding of Humanized Anti-LILRB1 / 2 Antibodies to Human LILRB1 Protein, Monkey LILRB1 Protein, and Human LILRB2 Protein

[0478] In this experiment, the binding affinity of humanized anti-LILRB1 / 2 antibodies to human LILRB1 protein (Sino Biological, Catalog No. 16014-H08H), human LILRB2 protein (Sino Biological, Catalog No. 14132-H08H), rhesus monkey LILRB1 protein (Kactus Biosystems, Catalog No. LIL-CM1B1), and cynomolgus monkey LILRB1 protein (ACRO Biosystems, Catalog No. CDJ-C52H3) was detected using the ForteBio Octet RED96e according to the manufacturer's instructions.

[0479] Briefly, an AHC sensor (ForteBio, Catalog No. 18-5060) was placed in running buffer (1X PBS, Cytiva, Catalog No. SH30256.01, containing 0.02% Tween 20, 0.1% BSA, pH 7.0) and pre-equilibrated at room temperature for 10 minutes. Kinetic experiments were performed in a 96-well plate according to the following steps:

[0480] a) Equilibrate the baseline with running buffer for 180 s;

[0481] b) Add each antibody diluted in running buffer to a final concentration of 5 μg / mL and solidify for 200 s;

[0482] c) Equilibrate the baseline with running buffer for 180 s;

[0483] d) Add the following concentrations of human LILRB1 protein, human LILRB2 protein, rhesus monkey LILRB1 protein, and cynomolgus monkey LILRB1 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 180 s.

[0484] 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;

[0485] Table 7 summarizes the binding affinities of the humanized anti-LILRB1 / 2 antibodies of the present invention to human LILRB1 protein, monkey LILRB1 protein and human LILRB2 protein, respectively.

[0486] Table 7 Binding affinity of humanized anti-LILRB1 / 2 antibodies to human LILRB1 protein, monkey LILRB1 protein and human LILRB2 protein

[0487] Table 7 shows that the humanized anti-LILRB1 / 2 antibodies of the present invention can bind to human LILRB1 protein, monkey LILRB1 protein and human LILRB2 protein with high affinity.

[0488] 3.6 Binding of Humanized Anti-LILRB1 / 2 VHH Antibodies to Human LILRB1 Protein or Monkey LILRB1 Protein Expressed on the Cell Surface

[0489] The gene sequence of rhesus monkey LILRB1 protein (F7H3G7) was codon optimized and synthesized by Universal Biosystems (Anhui) Co., Ltd. and cloned into a stable expression vector. TM The gene sequence encoding the rhesus macaque LILRB1 protein was transfected into 293T cells (purchased from the Cell Bank of the Committee for Type Culture Collection of the Chinese Academy of Sciences, Catalog No. SCSP-502) using 2000 transfection reagent (Invitrogen, Catalog No. 11668019). The transfected cells were subjected to pressure selection with 0.3 μg / mL puromycin for over one week to obtain engineered 293T cells that highly express rhesus macaque LILRB1, designated 293T-RheLILRB1(FGH). These cells were cultured in complete DMEM (Gibco, Catalog No. 11995065) supplemented with 10% fetal bovine serum and 0.3 μg / mL puromycin.

[0490] CHOK1-huLILRB1 and 293T-RheLILRB1 (FGH) cells were pressure cultured to 80-90% confluence and then digested with 0.25% trypsin to obtain a single-cell suspension. The suspension was centrifuged at 400×g at room temperature, and the culture medium was discarded. The cell pellet was washed once with PBS and centrifuged, and the supernatant was discarded. The cell pellet was resuspended in a gradient dilution of the humanized anti-LILRB1 / 2 VHH antibody obtained by the present invention and the control antibody 15G8 analog (i.e., the synthetic control antibody 15G8, with an initial concentration of 25 μg / mL and a 4-fold gradient dilution to 6.25 μg / mL, 1.563 μg / mL, 0.391 μg / mL, 0.0977 μg / mL, 0.0244 μg / mL, 0.0061 μg / mL, and 0.00153 μg / mL, a total of 8 points) and incubated at 4°C for 30 minutes. After washing the cells once with PBS, the cell pellet was resuspended in a 1:200 dilution of the fluorescent secondary antibody, R-PE-conjugated AffiniPure Goat Anti-Human IgG, FcγFragment Specific. The cells were incubated at 4°C in the dark for 30 minutes. After washing the cells twice with PBS and resuspending them, the fluorescence signal in the PE channel was detected by flow cytometry. The results are shown in Figures 4 and 5.

[0491] The results shown in Figure 4 show that the humanized anti-LILRB1 / 2 VHH antibody can bind to CHOK1-huLILRB1 engineered cells expressing human LILRB1 protein, and the binding ability of the test antibody is slightly weaker than that of the control antibody 15G8 analog.

[0492] The results shown in Figure 5 show that the humanized anti-LILRB1 / 2 VHH antibody can bind to 293T-RheLILRB1 (FGH) engineered cells expressing rhesus monkey LILRB1 protein, and the binding ability of the tested antibody is significantly better than that of the control antibody 15G8 analog.

[0493] 3.7 Humanized anti-LILRB1 / 2 VHH antibodies block the binding of human LILRB1 or human LILRB2 protein to ligands

[0494] The human HLA-G gene sequence (Uniprot sequence number: P17693, SEQ ID NO: 51) was codon-optimized and gene-synthesized by Universal Biosystems (Anhui) Co., Ltd. and cloned into a stable expression vector. TMA375 cells (purchased from the Cell Bank of the Chinese Academy of Sciences, Catalog No. TCHu155) were transfected with the gene sequence encoding the human HLA-G protein using 2000 transfection reagent. After virus infection, the cells were subjected to pressure selection with 0.5 μg / mL puromycin and limited dilution monoclonal screening to obtain A375 cells that highly express the human HLA-G protein, designated A375-HLAG. These cells were cultured in complete DMEM medium supplemented with 10% fetal bovine serum and 0.5 μg / mL puromycin. RPMI-8226 cells (purchased from the Cell Bank of the Chinese Academy of Sciences, Catalog No. TCHu234) naturally express MHC-I and were cultured in complete 1640 (Gibco, Catalog No. 22400089) medium supplemented with 10% fetal bovine serum.

[0495] When A375-HLAG cells were cultured under pressure to a confluence of 80-90%, they were digested with 0.25% trypsin to obtain a single cell suspension. RPMI-8226 cells were cultured to a density of 0.5-2×10 6 The humanized anti-LILRB1 / 2 VHH antibody and the positive control antibody 15G8 analog obtained by the present invention were gradiently diluted with complete culture medium (initial concentration was 1050nM, 3-fold gradient down to 350nM, 116.7nM, 38.89nM, 12.963nM, 4.321nM, 1.4403nM and 0.4801nM, a total of 8 points) to obtain an antibody gradient dilution solution. The human LILRB1-mFc (SEQ ID NO: 65) protein was diluted to 15μg / mL with complete culture medium to obtain a protein dilution solution.

[0496] Equal volumes of antibody gradient dilution and protein dilution (50 μL-50 μL) were gently mixed and incubated at room temperature for 20 minutes. The two single cell suspensions were centrifuged at 400 × g at room temperature and the culture medium was discarded. The cell pellet was washed once with complete culture medium and centrifuged and the supernatant was discarded. The cell pellet was resuspended in complete culture medium to a density of 2 × 10 6 Cells were plated with the antibody and protein mixture at a concentration of 50 μL / well, gently mixed, and incubated at 4°C for 30 minutes. The cells were washed once with complete culture medium and resuspended in a 1:200 dilution of the fluorescent secondary antibody, R-PE-conjugated AffiniPure Goat Anti-Mouse IgG (subclasses 1+2a+2b+3), FcγFragment Specific (Jackson ImmunoResearch, Cat. No. 115-115-164). The cell pellet was incubated at 4°C in the dark for 30 minutes. The cells were washed twice with PBS and resuspended. Fluorescence signals in the PE channel were detected by flow cytometry. The results are shown in Figures 6 and 7.

[0497] The results shown in Figures 6 and 7 show that the humanized anti-LILRB1 / 2 VHH antibody can block the binding of human LILRB1 protein to A375-HLAG engineered cells expressing human HLAG protein, and can also block the binding of human LILRB1 protein to RPMI-8226 cells expressing human MHC-I protein. The blocking ability of the tested antibody is comparable to or better than that of the control antibody 15G8 or the control antibody 15G8 analog.

[0498] When A375-HLAG cells were cultured under pressure to a confluence of 80-90%, they were digested with 0.25% trypsin to obtain a single cell suspension. RPMI-8226 cells were cultured to a density of 0.5-2×10 6 The humanized anti-LILRB1 / 2 VHH antibody obtained in the present invention and the positive control antibodies IO-108analog and MK-4830analog and the negative control antibodies (IgG4 control, Ultra-LEAF TM Purified Human IgG4 Isotype Ctrl Recombinant Antibody (Biolegend, Cat. No. 403702) or IgG1 mut control, Human IgG1, kappa Isotype Control (Sino Biological, Cat. No. HG1K) were serially diluted in complete medium (initial concentration: 6000 nM, followed by a three-fold dilution series to 8 concentrations: 2000 nM, 666.7 nM, 222.2 nM, 74.07 nM, 24.69 nM, 8.23 ​​nM, and 2.74 nM). A dilution series was prepared. Human LILRB2-mFc protein was diluted to 150 μg / mL in complete medium to prepare a protein dilution series. Equal volumes (50 μL to 50 μL) of the antibody serial dilution series and protein dilution series were gently mixed and incubated at room temperature for 20 minutes. The two single cell suspensions were centrifuged at 400 × g at room temperature and the culture medium was discarded. The cell pellets were washed once with complete culture medium and centrifuged and the supernatant was discarded. The cell pellets were resuspended in complete culture medium to a density of 2 × 10 6Cells were plated at 50 μL / well in the antibody and protein mixture, gently mixed, and incubated at 4°C for 30 minutes. Wash the cells once with complete culture medium, then resuspend the cell pellet in a 1:200 dilution of the fluorescent secondary antibody, R-PE-conjugated AffiniPure Goat Anti-Mouse IgG (subclasses 1+2a+2b+3), FcγFragment Specific. Incubate at 4°C in the dark for 30 minutes. Wash the cells twice with PBS, then resuspend them. Finally, analyze the fluorescence signal in the PE channel using a flow cytometer. The results are shown in Figures 8-10.

[0499] The results shown in Figures 8, 9 and 10 show that the humanized anti-LILRB1 / 2 VHH antibody can block the binding of human LILRB2 protein to A375-HLAG engineered cells expressing human HLAG protein, and can also block the binding of human LILRB2 protein to RPMI-8226 cells expressing human MHC-I protein. The blocking ability of the tested antibodies is comparable to that of the control antibodies IO-108analog and MK-4830analog.

[0500] 3.8 Humanized anti-LILRB1 / 2 VHH antibody induces NK cells to kill target cells

[0501] Take out the frozen human PBMC (purchased from Shanghai Rubai Biotechnology Co., Ltd., product number: PBMNC050C, batch number: 2204148361) from the liquid nitrogen tank and thaw it quickly in a 37°C water bath. Centrifuge to obtain the cell pellet. TM Pure NK cells were isolated using human NK cell isolation reagent (Stem, Catalog No. 17955) and activated overnight in 1640 complete medium supplemented with 10% fetal bovine serum and 200 IU / mL human IL-2 (Jiangsu Jinsili Pharmaceutical Co., Ltd., Catalog No. NA). The next day, NK cells were collected and centrifuged at 400 × g for 5 minutes at room temperature, and the supernatant was discarded. The cell pellet was resuspended in 1640 complete medium supplemented with 10% fetal bovine serum and 100 IU / mL human IL-2. The cell count was adjusted to a density of 4.5 × 10 4 / 50μL / well was inoculated into an ultra-low adsorption 96-well U-bottom plate (Corning, Cat. No. CLS7007). The humanized anti-LILRB1 / 2VHH antibody obtained in the present invention, the positive control antibody 15G8 analog, and the negative control antibody (IgG1 control) were gradiently diluted with 1640 complete medium containing 10% fetal bovine serum and 100IU / mL human IL-2 (the initial concentration was 400nM, and the 5-fold gradient was diluted downward to 80nM, 16nM, 3.2nM, 0.64nM, 0.128nM, 0.0256nM and 0.00512nM, a total of 8 points), and 50μL / well was inoculated. When A375-HLAG cells were pressure-cultured to a confluence of 80-90%, they were digested with 0.25% trypsin to obtain a single cell suspension. CTV (CellTrace TM A375-HLAG cells were stained and labeled using the Violet Cell Proliferation Kit (Invitrogen, Cat. No. C34557). After labeling, the cells were centrifuged and the supernatant discarded. The cell pellet was resuspended in 1640 complete medium containing 10% fetal bovine serum and 100 IU / mL human IL-2. The cell count was adjusted and the cell density was adjusted to 1.5 × 10 4 Inoculate 100 μL / well. Gently mix and incubate in a 37°C incubator for 4 hours. After incubation, add propidium iodide (PI, Beyotime Biotechnology Co., Ltd., Catalog No. ST511) to a final concentration of 1:500. Incubate at room temperature for 5 minutes, and then measure the apoptosis rate of A375-HLAG cells by flow cytometry.

[0502] The results shown in Figure 11 show that the humanized anti-LILRB1 / 2 VHH antibody can induce human NK cells to kill A375-HLAG engineered cells expressing human HLAG protein, and the NK cell killing ability of the tested antibody is comparable to or better than that of the positive control antibody 15G8.

[0503] 3.9 Humanized anti-LILRB1 / 2 VHH antibody induces macrophage phagocytosis of RPMI-8226

[0504] Cryopreserved human PBMCs (purchased from Shanghai Rubai Biotechnology Co., Ltd., Catalog No. PBMNC050C, Batch No. 2104250211; or purchased from Shanghai Aoneng Biotechnology Co., Ltd., Catalog No. FPB004F-C, Batch No. LP220624012) were removed from a liquid nitrogen tank and rapidly thawed in a 37°C water bath. Cell pellets were obtained by centrifugation. Pure monocytes were isolated using a human total monocyte isolation kit (Miltenyi Biotec, Catalog No. 130-096-537) according to the manufacturer's instructions. Monocytes were cultured for 9 days in 1640 complete medium (factor-containing complete medium) supplemented with 10% fetal bovine serum and 100 ng / mL recombinant human M-CSF (PeproTech, Catalog No. 300-25-250 μg). Fresh factor-containing complete medium was replaced every 3 days to induce monocyte-to-macrophage differentiation. The successfully induced macrophages were digested with 0.25% trypsin-EDTA to obtain a single cell suspension, centrifuged at 400 × g for 5 minutes at room temperature, and the supernatant was discarded. The cell pellet was resuspended in PBS, counted, and the cell density was adjusted to 2 × 10 6 / mL. According to the instructions, add the live cell stain CytoTell Red 650 (Xi'an Baiying Biotechnology Co., Ltd., catalog number: 22255) to label and stain the cells. After washing the labeled macrophages with complete culture medium to remove the dye, resuspend them to 5×10 5 / mL, 100 μL / well was inoculated into an ultra-low adsorption 96-well U-bottom plate.

[0505] The humanized anti-LILRB1 / 2 VHH antibody obtained by the present invention and the positive control antibody 15G8 analog and the combined anti-human CD47 monoclonal antibody (Magrolimab, heavy chain: SEQ ID NO: 66; light chain: SEQ ID NO: 67) were serially diluted with 1640 complete medium containing 10% fetal bovine serum (initial concentration was 600 nM or 40 μg / mL, 10-fold gradient down to 60 nM, 6 nM and 0.6 nM or 4 μg / mL, 0.4 μg / mL and 0.04 μg / mL for a total of 4 points), 50 μL / well was inoculated and mixed evenly with macrophages, and the culture plate was incubated in a 37°C incubator for 30 minutes. The target cells were cultured in RPMI-8226 to a density of 0.5-2×10 6 When the cell suspension reached 2 × 10 / mL, the cells were dispersed evenly by gentle blowing to obtain a single cell suspension. The suspension was centrifuged at 400 × g for 5 minutes at room temperature, and the supernatant was discarded. The cell pellet was resuspended in PBS, and the cell density was counted and adjusted to 2 × 10 6 / mL. CFSE (BioTracker 488 Green CSFE Cell Proliferation Kit, Sigma, Catalog No.: SCT110) was added to the cells according to the instructions. The labeled target cells were washed with complete culture medium to remove the dye and resuspended to 5×10 5 / mL or 4×10 5 / mL, inoculate 50 μL / well. After gentle mixing, incubate in a 37°C incubator for 4 hours. Flow cytometry was used to determine the proportion of cells positive for both CFSE and APC channels. The results are shown in Figures 12 and 13.

[0506] The results shown in Figures 12 and 13 show that the humanized anti-LILRB1 / 2 VHH antibody combined with Magrolimab can promote human macrophages to phagocytose RPMI-8226 cells expressing human MHC-I. The test antibody combined with Magrolimab analog is equivalent to or better than the positive control antibody combined with Magrolimab analog (i.e., synthetic Magrolimab antibody) in promoting macrophage phagocytosis of RPMI-8226, and both are significantly better than Magrolimab analog alone.

[0507] 3.10 Humanized anti-LILRB1 / 2 VHH antibody induces cytokine release after activation of PBMC cells

[0508] Frozen human PBMCs (purchased from Shanghai Rubai Biotechnology Co., Ltd., catalog number: PBMNC050C, batch number: 2105030018 or 2204080023) were taken out of the liquid nitrogen tank and thawed rapidly in a 37°C water bath. Cell pellets were obtained by centrifugation. PBMCs were resuspended in 1640 complete medium containing 10% fetal bovine serum and the density was adjusted to 1.5×10 5 100 μL / well was inoculated into a 96-well plate (Corning, Cat. No. 3599). CD3 monoclonal antibody (OKT3, Invitrogen, Cat. No. 16-0037-85) was diluted to 40 ng / mL with complete culture medium and loaded at 50 μL / well to activate PBMC overnight. Alternatively, CD3 monoclonal antibody was diluted to 0.3 μg / mL with PBS and loaded at 50 μL / well into a 96-well plate. After standing overnight at 4°C, the antibody dilution was discarded and the CD3 antibody was coated onto the 96-well plate. Then, 1.5×10 5 PBMCs were seeded into 96-well plates at 150 μL / well.

[0509] The humanized anti-LILRB1 / 2 VHH antibody obtained in the present invention, as well as the positive control antibodies 15G8 analog, MK-4830 analog, and IO-108 analog, and negative control antibodies (IgG4 control, IgG1 control) were diluted in a gradient with complete medium containing 1640 (initial concentration: 400 nM or 40 μg / mL, followed by a 10-fold gradient dilution to three concentration points of 40 nM and 4 nM or two concentration points of 4 μg / mL). 50 μL / well was inoculated and mixed evenly with PBMC cells. The culture plate was incubated in a 37°C incubator for 3 days. The TNFα content in the supernatant was assayed according to the instructions of the Human TNFα Detection Kit @ HTRF (Cisbio, Catalog No.: 62HTNFAPEH).

[0510] The results shown in Figures 14 and 15 demonstrate that the humanized anti-LILRB1 / 2 VHH antibody can activate PBMCs to release TNFα. The level of TNFα release promoted by the tested antibody in PBMCs was comparable to that of the positive antibodies MK-4830 analog and IO-108 analog, and significantly better than that of the positive antibody 15G8 analog and the negative control antibody.

[0511] 3.11 Humanized anti-LILRB1 / 2 VHH antibody induces cytokine release after activation of DC cells

[0512] Frozen human PBMCs (purchased from Shanghai Rubai Biotechnology Co., Ltd., catalog number: PBMNC050C, batch number: 2202103579 or 2204060906) were removed from a liquid nitrogen tank 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 number: 130-096-537) according to the manufacturer's instructions. Monocytes were cultured for 4 days in 1640 complete medium supplemented with 10% fetal bovine serum, 100 ng / mL recombinant human GM-CSF (PeproTech, catalog number: 00-03-100UG), and 10 ng / mL recombinant human IL-4 (PeproTech, catalog number: 200-04-100UG) to induce monocyte differentiation into immature DCs (imDCs). Induced imDCs were collected and centrifuged at 400 × g for 5 minutes at room temperature. The supernatant was discarded, and the cell pellet was resuspended in complete medium. The cell count was counted and the cell density was adjusted to 2 × 10 5 / mL, according to 2×10 4 / 100 μL / well was inoculated into a 96-well flat-bottom plate.

[0513] The humanized anti-LILRB1 / 2 VHH antibody obtained in the present invention, the positive control antibodies 15G8 analog and IO-108 analog, and the negative control antibodies (IgG4 control, IgG1 control) were serially diluted with 1640 complete medium (initial concentration: 400 nM, 10-fold gradient dilution to 40 nM and 4 nM concentrations), and 50 μL / well was inoculated. Lipopolysaccharide (LPS, Sigma, Cat. No. LPS25) was diluted to 400 ng / mL with 1640 complete medium, and 50 μL / well was inoculated. After the antibody and LPS were evenly mixed with the imDC cells, the culture plate was incubated in a 37°C incubator for 2 days. The TNFα content of the supernatant was determined according to the instructions of the Human TNFα Detection Kit @ HTRF.

[0514] The results shown in Figures 16 and 17 demonstrate that humanized anti-LILRB1 / 2 VHH antibodies can promote the transformation of imDCs into mature DCs and the release of TNFα. The levels of TNFα release promoted by the tested antibodies in DC maturation were comparable to those of the positive antibody 10-108 analog and significantly better than those of the positive antibody 15G8 analog and the negative control antibody.

[0515] 3.12 Humanized anti-LILRB1 / 2 VHH antibody relieves MDSC cells from CD3 + Inhibition of T cell proliferation

[0516] Cryopreserved human PBMCs (purchased from Shanghai Rubai Biotechnology Co., Ltd., Catalog No. PBMNC050C, Batch No. 230111008 or LP220623007) 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 according to the manufacturer's instructions and cultured for 14 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 100 ng / mL recombinant human IL-6 (Recombinant Proteins, Catalog No. UA040052). The medium was replaced every four days with fresh complete medium containing GM-CSF and IL-6 to induce monocyte differentiation into MDSCs.

[0517] Take out the frozen human PBMC (purchased from Shanghai Rubai Biotechnology Co., Ltd., product number: PBMNC050C, batch number: 2301310011) from the liquid nitrogen tank and thaw it quickly in a 37℃ water bath. Centrifuge to obtain the cell pellet. TMPure T cells were isolated using human T cell sorting reagents. T cells were stained and labeled using the Cell Trace Violet Cell Proliferation Kit (Invitrogen, Cat. No. C34557) according to the instructions. T cells were stained with CTV and resuspended in 1640 complete medium containing 10% fetal bovine serum to a total cell density of 1×106 / mL. 5 / 100 μL / well was inoculated into a 96-well U-bottom plate and mixed evenly with the induced MDSCs obtained above (the effector-target ratio was 1:1).

[0518] The humanized anti-LILRB1 / 2 VHH antibody obtained by the present invention and the positive control antibodies MK4830 analog and IO-108 analog were diluted to 400nM with 1640 complete medium and inoculated at 50μL / well. Enced T cell activation (Kinsher, Catalog No.: L00899-1) was diluted to 0.5ul / well with 1640 complete medium and inoculated at 50μL / well. An empty control group (No drug) without any antibody and a control group (CD3 only) without MDSC and proliferated under CD3 / CD28 stimulation were also set up. After the antibody, T cell activation, MDSC and T cells were mixed evenly, the culture plate was incubated in a 37°C incubator for 3 days. The proportion of cells with CTV low was detected by flow cytometry.

[0519] The results shown in Figure 18 demonstrate that humanized anti-LILRB1 / 2 VHH antibodies can relieve MDSC inhibition of T cell proliferation. The ability of the tested antibodies to relieve MDSC inhibition of T cells was superior to that of the positive antibody MK-4830 analog and comparable to that of the positive antibody IO-108 analog.

[0520] Example 4: Anti-PD1-LILRB1 / 2 bispecific antibody

[0521] 4.1 Construction of bispecific antibodies

[0522] The present invention constructs bispecific antibodies with a symmetrical structure as shown in Figure 19, wherein the anti-LILRB1 / 2 portion of each bispecific antibody is derived from the above-mentioned humanized LILRB1 / 2 antibody, the anti-PD1 portion is derived from the pembrolizumab antibody (INN Number: 9798), and the bispecific antibody constant region is a human IgG1 mut heavy chain constant region gene (mutation sites L234A, L235A, D265A, P329A). This type of bispecific antibody is also referred to herein as an "anti-PD1 / LILRB1 / 2 bispecific antibody" or "anti-PD1 / anti-LILRB1 / 2 bispecific antibody", and sometimes simply referred to as a "bispecific antibody molecule, bispecific antibody". The anti-LILRB1 / 2 portion of the anti-PD1 / LILRB1 / 2 bispecific antibody targets LILRB1 or 2, as well as cells expressing LILRB1 / 2, while the anti-PD1 portion targets PD1 and cells expressing PD1, blocking the PD1-PD L1 signaling pathway and relieving T cell inhibition. The dual antibody binds to LILRB1 / 2 and PD1 on tumor cells simultaneously, blocking both signaling pathways and achieving synergistic activation.

[0523] The present application uses a standard construction method to obtain two anti-PD1 / LILRB1 / 2 bispecific antibodies with the structure shown in Figure 19, and their amino acid sequences are listed in Table 10 below. The heavy chains of the bispecific antibodies are named according to the source sequence in order from N-terminus to C-terminus. For example, VH1DE-Key-1mut indicates that the sequence includes, from N segment to C-terminus, VH1-D53E VHH, Pembrolizumab heavy chain variable region and constant region (with mutations L234A, L235A, D265A, P329A); Key-1mut-VH1DE indicates that the sequence includes, from N segment to C-terminus, Pembrolizumab heavy chain variable region, constant region (with mutations L234A, L235A, D265A, P329A) and VH1-D53E VHH.

[0524] Each bispecific antibody molecule was constructed as follows:

[0525] VH1DE-Key-1mut (or Key-1mut-VH1DE): The heavy chain nucleotide sequence encoding the humanized anti-LILRB1 / 2 antibody VH1-D53E and the heavy chain nucleotide sequence encoding the anti-PD1 antibody are cloned into a vector with a constant region (IgG1 mut) to express the VH1DE-Key-1mut (or Key-1mut-VH1DE) molecule.

[0526] Pembrolizumab-L: The nucleotide sequence encoding the anti-PD1 antibody light chain was cloned into the vector and expressed to obtain the Pembrolizumab-L molecule.

[0527] According to the specific composition shown in Table 8, the above-mentioned expression vectors were combined accordingly and expressed under appropriate conditions to obtain the bispecific antibody shown in Figure 19.

[0528] The steps for construction, expression, purification, and preliminary analysis of the bispecific antibody were the same as those in Example 2.1.

[0529] Table 8 Composition of anti-PD1 / LILRB1 / 2 bispecific antibodies

[0530] 4.2 Bispecific Antibody Thermal Stability Analysis (DSF)

[0531] Differential scanning fluorimetry (DSF) is used to assess the thermal stability of proteins by measuring the amount of fluorescent dye bound to structurally altered proteins as the temperature is increased. Specifically, it is used to measure the thermal transition midpoint (Tm), an indicator of the relative stability of a protein in a liquid.

[0532] Antibodies Bi-PL-6 and Bi-PL-9 were diluted to 5 μM in PBS buffer. The fluorescent dye (SYPRO Orange, Sigma, S5692) was diluted 150-fold and thoroughly mixed with the antibodies. The samples were then transferred to a 96-well plate and assayed in a fluorescent quantitative PCR instrument (Bio-Rad, CFX96 touch). The sample volume per well was 25 μl. The temperature range was 25-95°C, with a heating rate of 1°C / min. The results were analyzed using Bio-Rad CFX Manager automated analysis software. The DSF test results are shown in Table 9 and Figure 20. As shown in Table 9, the Tm1 values ​​of the Bi-PL-6 and Bi-PL-9 antibodies were 58.8°C and 64.7°C, respectively.

[0533] Table 9 Thermal stability of bispecific antibodies

[0534] 4.3 Affinity Binding of Bispecific Antibodies to Human LILRB1, LILRB2, or PD1 Proteins

[0535] In this experiment, the binding affinity of the bispecific antibody to human LILRB1 protein (Sino Biological, Catalog No. 16014-H08H), human LILRB2 protein (Sino Biological, Catalog No. 14132-H08H), and human PD-1 protein (ACRO Biosystems, Catalog No. PD1-H522a) was detected using the ForteBio Octet RED96e according to the manufacturer's instructions.

[0536] Briefly, an AHC sensor (ForteBio, Catalog No. 18-5060) was placed in running buffer (1X PBS, Cytiva, Catalog No. SH30256.01, containing 0.02% Tween 20, 0.1% BSA, pH 7.0) and pre-equilibrated at room temperature for 10 minutes. Kinetic experiments were performed in a 96-well plate according to the following steps:

[0537] a) Equilibrate the baseline with running buffer for 180 s;

[0538] b) Add each antibody diluted in running buffer to a final concentration of 5 μg / mL and solidify for 200 s;

[0539] c) Equilibrate the baseline with running buffer for 180 s;

[0540] d) Add the following concentrations of human LILRB1 protein, human LILRB2 protein, and human PD-1 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 180 s.

[0541] e) Regeneration solution (0.01 M Gly-HCl, pH 1.5) was used for regeneration for 30 seconds. The experimental data were fitted and calculated using a 1:1 binding model using Fortebio Data Analysis software.

[0542] Table 10 summarizes the binding affinity of the bispecific antibodies to human LILRB1 protein, human LILRB2 protein, or human PD1 protein.

[0543] Table 10 Binding affinity of bispecific antibodies to human LILRB1 protein, human LILRB2 protein or human PD1 protein

[0544] Table 10 shows that the bispecific antibody of the present invention can specifically bind to human LILRB1 protein, human LILRB2 protein or human PD1 protein, and has a higher affinity for human LILRB2 protein than for human LILRB1 protein. The bispecific antibody and pembrolizumab analog have comparable affinity for PD-1 protein.

[0545] 4.4 Anti-PD1 / anti-LILRB1 / 2 bispecific antibodies induce cytokine release and cell marker expression after macrophage activation

[0546] Frozen human PBMCs (purchased from Shanghai Rubai Biotechnology Co., Ltd., product number: PBMNC050C, batch: 2206301324) were taken out of the liquid nitrogen tank and quickly thawed in a 37°C water bath, and the cell pellet was obtained by centrifugation. According to the instructions, pure monocytes were isolated using a human total monocyte isolation kit and cultured with 1640 complete medium containing 10% fetal bovine serum and 100 ng / mL recombinant human M-CSF (complete medium containing factors) for 7 days. Fresh complete medium containing factors was replaced every 3 days to induce monocytes to differentiate into macrophages. Successfully induced macrophages were digested with 0.25% trypsin-EDTA to obtain a single cell suspension, centrifuged at 400×g for 5 minutes at room temperature, and the supernatant was discarded. The cell pellet was resuspended in complete medium, counted, and the cell density was adjusted to 5×10 5 / mL, according to 5×10 4 / 100 μL / well was inoculated into a 96-well flat-bottom plate.

[0547] The PD1-LILRB1 / 2 bifunctional antibody, humanized anti-LILRB1 / 2 VHH antibody, and negative control antibody obtained in the present invention were gradiently diluted with 1640 complete medium containing 10% fetal bovine serum (initial concentration was 600 nM, 20-fold gradient down to 30 nM and 1.5 nM for a total of 3 points) to obtain an antibody dilution solution, and 25 μL / well was inoculated. LPS was diluted to 60 ng / mL with 1640 complete medium and 25 μL / well was inoculated. The antibody dilution solution, LPS, and macrophages were mixed evenly, and the culture plate was incubated in a 37°C incubator for 2 days. The TNFα content of the supernatant was detected according to the instructions of the Human TNFα Detection Kit @ HTRF (Cisbio, Catalog No.: 62HIFNGPEH). Macrophages were digested with 0.25% lysate to single cells and collected. They were then incubated with directly fluorescently labeled anti-human CD163 (APC anti-human CD163 Antibody, Biolegend, Catalog No. 333610) and anti-human CD86 (FITC anti-human CD86 Antibody, Biolegend, Catalog No. 374204) antibodies diluted according to the manufacturer's instructions. Macrophage surface expression of CD163 and CD86 was assessed by flow cytometry. The results are shown in Figures 21, 22, and 23.

[0548] The results shown in Figure 21 show that the PD1-LILRB1 / 2 bifunctional antibody and the humanized anti-LILRB1 / 2 VHH antibody can promote the release of TNFα by macrophages. The test antibodies promoted the release of TNFα by macrophages at a level significantly better than the negative control antibody.

[0549] The results shown in Figure 22 show that the PD1-LILRB1 / 2 bifunctional antibody and the humanized anti-LILRB1 / 2 VHH antibody can promote the downregulation of surface CD163 expression on macrophages. The test antibodies promoted the downregulation of surface CD163 expression on macrophages at a significantly better level than the negative control antibody.

[0550] The results shown in Figure 23 show that the PD1-LILRB1 / 2 bifunctional antibody and the humanized anti-LILRB1 / 2 VHH antibody can promote the upregulation of surface CD86 expression on macrophages. At high concentrations, the test antibodies promoted the upregulation of surface CD86 expression on macrophages significantly better than the negative control antibody.

[0551] 4.5 Anti-PD1 / anti-LILRB1 / 2 bispecific antibodies activate macrophages-MLR

[0552] Frozen human PBMCs (purchased from Shanghai Rubai Biotechnology Co., Ltd., catalog number: PBMNC050C, batch number: 2210104295) were removed from the liquid nitrogen tank and rapidly thawed in a 37°C water bath. Cell pellets were obtained by centrifugation. Pure monocytes were isolated using a human total monocyte isolation kit according to the instructions and resuspended in 1640 complete medium containing 10% fetal bovine serum. The cells were counted and the cell density was adjusted to 5×10 5 / mL, according to 5×10 4 / 100 μL / well was inoculated into a 96-well flat-bottom plate.

[0553] The PD1-LILRB1 / 2 bifunctional antibody, humanized anti-LILRB1 / 2 VHH antibody, anti-PD-1 positive antibody (Pembrolizumab analog, expressed by Nanjing Weilizhibo Biotechnology Co., Ltd.) and negative control antibody obtained in the present invention were gradiently diluted to 20nM with 1640 complete medium and inoculated at 50μL / well. Human recombinant M-CSF was gradiently diluted to 400ng / mL with 1640 complete medium and inoculated at 50μL / well. After mixing evenly, the mixture was incubated in a 37°C incubator for 7 days. After 7 days, 150μL / well of the supernatant was aspirated and discarded to obtain induced macrophages.

[0554] The same human PBMCs 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 human T cell separation reagent (Stem, Cat. No. 17951) and resuspended in 1640 complete medium containing 10% fetal bovine serum to a cell density of 1×10 6 / mL, according to 5×10 4 / 50μL / well was inoculated into a 96-well flat-bottom plate and mixed evenly with the above-induced macrophages.

[0555] The PD1-LILRB1 / 2 bifunctional antibody, humanized anti-LILRB1 / 2 VHH antibody, anti-PD-1 positive antibody and negative control antibody (IgG1 control) obtained by the present invention were gradiently diluted to 15nM with 1640 complete medium and inoculated at 50μL / well. A 1:1 mixture of humanized anti-LILRB1 / 2 VHH antibody + anti-PD-1 positive antibody was inoculated at 50μL / well. The OKT-3 antibody was gradiently diluted to 40ng / mL with 1640 complete medium and inoculated at 50μL / well, with a final concentration of 10ng / ml. The antibody to be tested and the OKT-3 antibody were mixed evenly and incubated in a 37°C incubator for 5 days. The IFNγ content of the supernatant was detected according to the instructions of the Human IFNγ Detection Kit @HTRF (Cisbio, Catalog No.: 62HIFNGPEH).

[0556] The results shown in Figure 24 show that the PD1-LILRB1 / 2 bifunctional antibody can promote the secretion of IFNγ in the macrophage-T cell mixed system at a concentration of 5 nM, and the factor secretion level is significantly higher than that of the negative control antibody and the humanized anti-LILRB1 / 2 VHH antibody and anti-PD-1 positive antibody used alone or in combination.

[0557] 4.6 Anti-PD1 / anti-LILRB1 / 2 bispecific antibodies induce cytokine release and cell marker expression after DC activation

[0558] Frozen human PBMCs (purchased from Shanghai Rubai Biotechnology Co., Ltd., catalog number: PBMNC050C, batch number: 2210104295) were removed from the liquid nitrogen tank and rapidly thawed in a 37°C water bath. Cell pellets were obtained by centrifugation. Pure monocytes were isolated using a human total monocyte isolation kit according to the instructions. The cells were cultured for 4 days in 1640 complete medium containing 10% fetal bovine serum, 100 ng / mL recombinant human GM-CSF, and 10 ng / mL recombinant human IL-4 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 resuspended in complete medium, counted, and the cell density was adjusted to 2 × 10 5 / mL, according to 2×10 4 / 100 μL / well was inoculated into a 96-well flat-bottom plate.

[0559] The PD1-LILRB1 / 2 bifunctional antibody, humanized anti-LILRB1 / 2 VHH antibody, positive control antibody, and negative control antibody obtained in the present invention were diluted with 1640 complete medium (initial concentration was 400nM, 20-fold gradient down to three concentration points of 20nM and 1nM), and 50μL / well was inoculated. LPS was diluted to 400ng / mL with 1640 complete medium and 50μL / well was inoculated. After the antibodies and LPS were evenly mixed with the imDC cells, the culture plate was incubated in a 37°C incubator for 2 days to induce the imDC into mature DC cells. The TNFα content of the supernatant was detected according to the instructions of the Human TNFα Detection Kit @ HTRF. The results are shown in Figure 25. The mature DC cells were collected and labeled with a directly labeled fluorescent antibody against human CD83 (PE anti-human CD83 Antibody, Biolegend, Cat. No.: 305322) diluted according to the instructions. The expression of CD83 on the DC surface was detected by flow cytometry. The results are shown in Figure 26.

[0560] The results shown in Figure 25 show that the PD1-LILRB1 / 2 bifunctional antibody and the humanized anti-LILRB1 / 2 VHH antibody can promote the transformation of imDCs into mature DCs and release TNFα. The tested antibodies promoted DC maturation and released TNFα at levels significantly better than the negative control antibody.

[0561] The results shown in Figure 26 show that the PD1-LILRB1 / 2 bifunctional antibody and the humanized anti-LILRB1 / 2 VHH antibody can promote the transformation of imDCs into mature DCs and upregulate surface CD83 expression. The level of CD83 upregulation promoted by the tested antibodies on imDCs was significantly better than that of the negative control antibody.

[0562] 4.7 Anti-PD1 / anti-LILRB1 / 2 bispecific antibodies activate DC cells-MLR

[0563] The frozen human PBMC (purchased from Shanghai Rubai Biotechnology Co., Ltd., product number: PBMNC050C, batch: 2210104295) was taken out of the liquid nitrogen tank and quickly thawed in a 37°C water bath, and the cell pellet was obtained by centrifugation. According to the instructions, pure monocytes were isolated using a human total monocyte isolation kit, and cultured for 5 days with 1640 complete medium containing 10% fetal bovine serum, 100 ng / mL recombinant human GM-CSF, and 10 ng / mL recombinant human IL-4 to induce monocyte differentiation into imDC. The induced imDC was collected, centrifuged at 400 × g for 5 minutes at room temperature, and the supernatant was discarded. The cell pellet was cultured with 1640 complete medium containing 10% fetal bovine serum and 100 ng / mL LPS for 2 days to promote DC maturation. The harvested DC was resuspended in complete medium, counted, and the cell density was adjusted to 2×10 5 / mL, calculated as 1×10 4 / 50μL / well to inoculate 96-well U-bottom plates.

[0564] Take out the frozen human PBMC (purchased from Shanghai Rubai Biotechnology Co., Ltd., product number: PBMNC050C, batch number: 2203010234) from the liquid nitrogen tank and thaw it quickly in a 37°C water bath. Centrifuge to obtain the cell pellet. TM Pure T cells were isolated using human T cell sorting reagent. T cells were stained with CFSE 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 (the effector-target ratio was 10:1).

[0565] The PD1-LILRB1 / 2 bifunctional antibody, humanized anti-LILRB1 / 2 VHH antibody, and negative control antibody obtained in the present invention were inoculated with a 1640 complete medium gradient (initial concentration of 400 nM) at 50 μL / well. The humanized anti-LILRB1 / 2 VHH antibody + positive control antibody mixture was inoculated at 50 μL / well. The system volume was 200 μL / well. After mixing evenly, the mixture was incubated at 37°C for 5 days. The IFNγ content of the supernatant was determined according to the instructions of the Human IFNγ Detection Kit @ HTRF.

[0566] The results shown in Figure 27 demonstrate that the PD1-LILRB1 / 2 bifunctional antibodies Bi-PL-6 and Bi-PL-9 can promote IFNγ secretion in a DC-T mixed lymphocyte reaction system at a concentration of 100 nM. The test antibodies promoted factor secretion levels significantly better than the negative control antibody, and Bi-PL-6 and Bi-PL-9 IFNγ secretion was superior to that of pembrolizumab analogs and the combination of pembrolizumab analogs and VH1-D53E.

[0567] 4.8 Efficacy of anti-PD1 / anti-LILRB1 / 2 bispecific antibodies in a mixed A375-HLAG cell macrophage / PBMC subcutaneous humanized xenograft model

[0568] The anti-tumor effect of the anti-PD1 / anti-LILRB1 / 2 bispecific antibody was studied in a mixed Macrophage / PBMC subcutaneous humanized xenograft tumor model of A375-HLAG cells in NCG mice.

[0569] Eighteen NCG mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0570] A375 primary cells were purchased from the Cell Bank of the Chinese Academy of Sciences, and A375-HLAG cells were obtained as described in Example 3.7. The cells were cultured in a 37°C, 5% CO2 incubator in DMEM containing 10% FBS and 0.5 μg / mL Puromycin.

[0571] PBMCs were purchased from Shanghai Rubai Biotechnology Co., Ltd.

[0572] Similar to the above example 4.5, PBMCs were sorted for monocytes and cultured in vitro for 6 days to induce macrophages. At the same time, the remaining PBMCs after sorting were co-cultured with A375-HLAG cells treated with mitomycin (5 μg / mL for 2 hours, and then replaced with normal culture medium). After 6 days, the macrophages, the co-cultured PBMCs, and the freshly digested A375-HLAG cells were mixed in a ratio of (3:5:50) and inoculated subcutaneously on the right side of NCG mice. When the average tumor volume reached 100 mm 3 At approximately 14 days, the mice were randomly divided into three experimental groups based on tumor volume and body weight, with 6 mice in each group. On days 0, 3, 7, 10, 13, and 17 after grouping, blank control (PBS) (G1), Pembrolizumab (10 mg / kg) (G2), and Bi-PL-6 (11.8 mg / kg) (G3) were intraperitoneally administered to the mice, respectively. The maximum long axis (L) and maximum wide axis (W) of the tumor in the tumor-bearing mice were measured twice a week with a vernier caliper during the experimental period, and the tumor volume was calculated according to the following formula: V = L × W 2 / 2.

[0573] Treatment with the anti-PD1 / anti-LILRB1 / 2 bispecific antibody Bi-PL-6 resulted in significant tumor growth inhibition compared to the PBS group. On day 21 of dosing, 11.8 mg / kg Bi-PL-6 demonstrated a 100.76% tumor growth inhibition rate (TGI rate) compared to the equivalent molar dose of 10 mg / kg pembrolizumab analog. The results are shown in Figure 28.

[0574] Throughout the experiment, the animals were active and ate well during the dosing period, and the weights of animals in groups G1-G3 remained stable, indicating that the animals tolerated the test product well. The weight changes of all animals are shown in Figure 29.

[0575] The proportion of hCD45+ positive cells in the peripheral blood of mice was measured on days 13, 24, and 31 after administration. It was found that treatment with the anti-PD1 / anti-LILRB1 / 2 bispecific antibody Bi-PL-6 significantly increased the number of hCD45+ positive cells compared to the PBS group, thereby exerting an anti-tumor effect. The results are shown in Figure 30.

[0576] 4.9 Efficacy of anti-PD1 / anti-LILRB1 / 2 bispecific antibodies in the A375-HLA G cell CD34 reconstitution mouse model

[0577] The anti-tumor effect of the anti-PD1 / anti-LILRB1 / 2 bispecific antibody was investigated in the CD34 reconstructed mouse A375-HLAG tumor model.

[0578] Twenty-six HSC-NCG-M (IN) mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

[0579] A375 primary cells were purchased from the Cell Bank of the Chinese Academy of Sciences, and A375-HLAG cells were obtained as described in Example 3.7. The cells were cultured in a 37°C, 5% CO2 incubator in DMEM containing 10% FBS and 0.5 μg / mL Puromycin.

[0580] 5×10 6 A375-HLAG cells / 0.1 mL serum-free DMEM / 0.1 mL Matrigel matrix / mouse were used to obtain the CD34 reconstructed mouse A375-HLAG tumor model. When the average tumor volume reached 150 mm 3 At about 14 days, 18 suitable mice were selected according to the tumor volume and body weight of the mice and randomly divided into 3 experimental groups, with 6 mice in each group. On days 0, 3, 6, 10, 13 and 17 after grouping, blank control (PBS) (G1), Pembrolizumab analog (10 mg / kg) (G2), and Bi-PL-6 (11.8 mg / kg) (G3) were intraperitoneally administered to the mice. The maximum long axis (L) and maximum wide axis (W) of the tumor in the tumor-bearing mice were measured twice a week with a vernier caliper during the experiment, and the tumor volume was calculated according to the following formula: V = L × W 2 / 2.

[0581] Treatment with the anti-PD1 / anti-LILRB1 / 2 bispecific antibody Bi-PL-6 resulted in significant tumor growth inhibition compared to the PBS group. On day 19 of dosing, 11.8 mg / kg Bi-PL-6 demonstrated a 50.99% tumor growth inhibition rate (TGI rate) compared to the equivalent molar dose of 10 mg / kg 006-Pembrolizumab analog. The results are shown in Figure 31.

[0582] Throughout the experiment, the animals were active and ate well during the dosing period, and the weights of animals in groups G1-G3 remained stable, indicating that the animals tolerated the test product well. The weight changes of all animals are shown in Figure 32.

[0583] Sequence Listing

Claims

1. A VHH antibody that specifically binds to LILRB1 / 2, comprising The three complementarity determining regions (CDRs) contained in the VHH shown in any one of SEQ ID NOs: 1-8, Preferably, the CDR sequences are defined according to IMGT.

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 of SEQ ID NO: 9, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 10 or 21, VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 11; (ii) 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 or 22, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 14; (iii) VHH CDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 15, VHH CDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 16, and VHH CDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 17; or (iv) VHH CDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 18, VHH CDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 19, and VHH CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:

20.

3. The VHH antibody of claim 1, comprising or consisting of a heavy chain variable region, wherein Heavy chain variable region (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from any one of SEQ ID NOs: 1-8; (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 1-8; 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: 1-8, preferably, the amino acid changes do not occur in the CDR region.

4. A heavy chain antibody that specifically binds to LILRB1 / 2, comprising the VHH antibody according to 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 linked 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.

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, optionally comprising a L234A / L235A mutation, a D265A mutation and a P329A mutation, 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:35 or 61 or 62 or 63; or (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO: 35 or 61 or 62 or 63; 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: 35 or 61 or 62 or 63.

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 NOs: 52-59; or (ii) comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 52-59; 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: 52-59, and 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 humanized antibody.

9. A multispecific antibody comprising a first antigen binding region and a second antigen binding region, wherein the first antigen binding region specifically binds to LILRB1 / 2, and comprises the VHH antibody of any one of claims 1-3 and 8, or the heavy chain antibody of any one of claims 4-8. Preferably, the multispecific antibody is a bispecific antibody.

10. The multispecific antibody of claim 9, wherein the second antigen binding region specifically binds to PD1.

11. The multispecific antibody of claim 10, wherein the second 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 complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 25, and LCDR1, LCDR2 and LCDR3 are the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 29; or (ii) HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:26; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:27; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:28; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:30; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:31; and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:

32.

12. The multispecific antibody of claim 11, wherein the second antigen binding region comprises VH and VL, wherein The VH comprises or consists of the amino acid sequence of SEQ ID NO: 25, 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:29 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 multispecific antibody of any one of claims 10 to 12, wherein the second antigen-binding region is a Fab that specifically binds to PD1. The multispecific antibody of claim 13 , wherein the Fab comprises VH and CH1 of the second antigen-binding region, wherein the CH1 is CH1 derived from IgG1, IgG2, IgG3 or IgG4, preferably CH1 derived from IgG1.

15. The multispecific 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: 60; or (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO:

60.

16. The multispecific antibody of claim 15, wherein the Fab heavy chain of the second antigen binding region (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: 64; (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 64; or (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 an amino acid sequence selected from SEQ ID NO: 64; and / or the second antigen binding region of the Fab light chain (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: 38; (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 38; or (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:

38.

17. The multispecific antibody of claim 10, wherein the second antigen binding region comprises (1) Three complementarity determining regions (HCDRs) from the heavy chain variable region and three complementarity determining regions (LCDRs) from the light chain variable region of pembrolizumab; (2) the heavy chain variable region (VH) and light chain variable region (VL) of pembrolizumab; or (3) Fab of pembrolizumab.

18. The multispecific antibody of any one of claims 9-17, wherein the first antigen binding region is the VHH of any one of claims 1-3 and 8.

19. The multispecific antibody of any one of claims 9-18, wherein the multispecific antibody is an IgG-like bispecific antibody comprising an Fc dimer, wherein the two Fc regions constituting the Fc dimer are identical, and optionally, the Fc region further comprises a mutation that reduces binding to an Fcγ receptor, e.g., one or more of a L234A / L235A mutation, a D265A mutation, or a P329A mutation; e.g., a L234A / L235A mutation, a D265A mutation, and a P329A mutation.

20. The bispecific antibody of claim 19, wherein 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:35 or 61 or 62 or 63; or (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO: 35 or 61 or 62 or 63; 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: 35 or 61 or 62 or 63.

21. The multispecific antibody of any one of claims 9-20, which is a bispecific antibody and comprises a first antigen binding region, a second antigen binding region and an Fc dimer, wherein the first antigen binding region is a VHH that specifically binds to LILRB1 / 2, such as the VHH of any one of claims 1-3 and 8, and the second antigen binding region is a Fab fragment that specifically binds to PD1.

22. The multispecific antibody of claim 21, wherein the bispecific antibody comprises two first antigen-binding regions and two second antigen-binding regions, for example, wherein the two first antigen-binding regions may be the same or different, or / and the two second antigen-binding regions may be the same or different.

23. The multispecific antibody of claim 21 or 22, comprising a heavy chain and a light chain, wherein The heavy chain from N-terminus to C-terminus contains A Fab heavy chain that specifically binds to PD-1, an Fc region, and a VHH fragment that specifically binds to LILRB1 / 2, which is connected to the C-terminus of the Fc region via a linker or not (for example, the N-terminus of the VHH fragment is connected to the C-terminus of the Fc region via a linker or not), or A VHH fragment that specifically binds to LILRB1 / 2, a Fab heavy chain that specifically binds to PD-1 (e.g., the N-terminus of the Fab heavy chain is connected to the C-terminus of the VHH fragment via or without a linker), and an Fc region; And the light chain comprises a Fab light chain that specifically binds to PD1.

24. The multispecific antibody of claim 21 or 22, comprising a heavy chain and a light chain, wherein The heavy chain comprises a VH that specifically binds to the antigen binding region of PD-1 and a heavy chain constant region connected to the C-terminus thereof, and a VHH that specifically binds to LILRB1 / 2 connected to the C-terminus of the heavy chain constant region via or without a linker (connected to the N-terminus of the VHH); or the heavy chain comprises a VH that specifically binds to the antigen binding region of PD-1 and a heavy chain constant region connected to the C-terminus thereof, and a VHH that specifically binds to LILRB1 / 2 connected to the N-terminus of the VH via or without a linker (connected to the C-terminus of the VHH); The light chain comprises a VL that specifically binds to the antigen binding region of PD-1 and a light chain constant region connected to the C-terminus thereof.

25. The multispecific antibody of claim 23 or 24, comprising two heavy chains or two light chains, such as comprising two heavy chains and two light chains, wherein the two heavy chains are identical or different, or the two light chains are identical or different.

26. The multispecific antibody of any one of claims 23-25, wherein The heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 37 or 39, 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:38, 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.

27. The multispecific antibody of any one of claims 9 to 20, which is a bispecific antibody and comprises a full-length antibody that specifically binds to PD1, and the VHH of any one of claims 1 to 3 and 8 connected to the N-terminus or C-terminus of its heavy chain via or without a linker.

28. The multispecific antibody of claim 27, wherein the full-length antibody that specifically binds to PD1 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as defined in claim 7; or comprises VH and VL as defined in claim 8, or comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3; or VH and VL of an antibody disclosed in 156712A1, such as pembrolizumab.

29. 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 one chain of the multispecific antibody of any one of claims 9 to 28, or consisting of said nucleic acid sequence.

30. An expression vector comprising the nucleic acid molecule of claim 29.

31. A host cell comprising the nucleic acid molecule of claim 29 or the expression vector of claim 30, 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.

32. 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 multispecific antibody of any one of claims 9-28, the method comprising culturing a host cell comprising the nucleic acid molecule of claim 29 or the expression vector of claim 30 under conditions suitable for chain expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

33. 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 multispecific antibody of any one of claims 9-28.

34. A pharmaceutical composition or medicament or formulation comprising 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 multi-specific antibody of any one of claims 9 to 28, or the immunoconjugate of claim 33, and optionally a pharmaceutically acceptable excipient.

35. A pharmaceutical combination product 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 multispecific antibody of any one of claims 9-28, or the immunoconjugate of claim 33, and one or more other therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators).

36. A method for preventing or treating a tumor, such as cancer, in an individual, comprising administering to the individual 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 9-28, or the immunoconjugate of claim 33, or the pharmaceutical composition or medicament or formulation of claim 34; or the pharmaceutical combination product of claim 35.

37. The method of claim 36, wherein the tumor cells of the tumor have elevated protein and / or nucleic acid levels (e.g., elevated expression) of LILRB1 / 2, and optionally elevated protein and / or nucleic acid levels (e.g., elevated expression) of PD1.

38. The method of claim 36 or 37, wherein the tumor is a solid tumor or a blood tumor, such as a malignant solid tumor or a blood tumor, such as a cancer, such as the cancer is melanoma, lung cancer, gastric cancer, breast cancer or pancreatic cancer.

39. The method of any one of claims 36-38, wherein the method further comprises administering in combination with other therapies such as treatment modalities (e.g., surgery or radiation therapy) and / or other therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators).

40. A method for detecting the presence of LILRB1 / 2 in a biological sample, comprising (i) contacting a biological sample with 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 multispecific antibody according to any one of claims 9 to 28 under conditions that allow binding thereof to LILRB1 / 2, (ii) detecting whether a complex is formed between the antibody or bispecific antibody and LILRB1 / 2, The formation of the complex indicates the presence of LILRB1 / 2.

Images