Myeloid cell engager antibody targeting ILT7 and CD89, and use thereof

Abstract

The present invention relates to an anti-ILT7 antibody and a myeloid cell engager antibody targeting ILT7 and CD89. Further provided in the present invention are a method for preparing the anti-ILT7 antibody and the myeloid cell engager antibody, and a method for treating or preventing a disease using the antibody.

Description

Myeloid cell engager antibodies targeting ilt7 and cd89 and uses thereof TECHNICAL FIELD

[0001] The present invention relates to anti-ILT7 antibodies and myeloid cell engager antibodies targeting CD89 and ILT7. The invention also provides methods of making the antibodies, and methods of using them to treat or prevent various diseases (e.g., autoimmune diseases, cancers, and infectious diseases). BACKGROUND

[0002] In recent years, bispecific antibodies capable of simultaneously binding two different antigens or antigen epitopes have shown great clinical value, among which several CD3-based bispecific antibodies, i.e., T-cell engagers, have been successfully approved for use in anti-tumor therapy. Such bispecific antibodies achieve the goal of targeted killing of tumor cells by simultaneously binding to an antigen molecule on the surface of tumor cells (Tumor-associated Antigen, TAA) and CD3 on the surface of T cells, thereby redirecting and / or activating cytotoxic T cells (CTLs). However, the clinical application of T-cell engagers is often accompanied by cytokine storm caused by excessive activation of T cells, which leads to serious side effects (Wilke et al., Expert Opin Drug Saf 2017, 16:1191-1202). In addition, compared with traditional monospecific antibodies, bispecific antibody drugs still have many challenges in production and quality control due to their complex structure, which are manifested as follows: (1) the expression level in industrial production is relatively low, and the consistency between batches is difficult to control; (2) the asymmetric structure of bispecific antibodies containing Fc full-length antibody often accompanies problems such as homodimerization of heavy chains and / or mispairing of light chains during expression, thereby producing various dimer byproducts, which are difficult to separate by downstream purification processes.

[0003] CD89, also known as immunoglobulin A (IgA) Fc receptor (FcαRI), is a receptor protein that is expressed in a restricted manner in myeloid cells (including neutrophils, eosinophils, monocytes, and macrophages). Binding and activation of CD89 triggers phagocytosis, trogocytosis, antigen presentation function of myeloid cells, and initiation of production and release of superoxide and various cytokines and immune mediators by myeloid cells (Bakema and Egmond, Mucosal Immunol 2011, 4:612-624).

[0004] Plasmacytoid dendritic cells (pDC) are considered as the main source of type I interferons (IFN-I) (Liu et al., Annu Rev Immunol 2005, 23:275-306), and dysregulated pDCs produce large amounts of IFN-I, which plays a central role in the pathogenesis of systemic lupus erythematosus (SLE) (Crow et al., Arthritis Res Ther 2010, 12(Suppl. 1):S5; Elkon et al., Curr Opin Rheumatol 2012, 24:499-505). Some therapeutic agents targeting pDCs for the treatment of SLE are currently in clinical trials, such as anti-ILT7 antibody VIB7734 and anti-BDCA2 antibody BIIB059.

[0005] In view of this, the present application develops a novel myeloid engager antibody capable of selectively depleting pDCs, which can effectively activate one or more myeloid effector cells (e.g., neutrophils and macrophages) other than T cells to selectively kill pDCs, and is expected to reverse the immune dysfunction of patients with pDC-related diseases (e.g., SLE), delay disease progression, and improve disease symptoms, without triggering cytokine storm, and is relatively easy to obtain high-purity target heterodimeric antibody products in the production process. SUMMARY

[0006] The present application provides an anti-ILT7 antibody and an anti-CD89 antibody, and a myeloid engager antibody targeting ILT7 and CD89 (e.g., an anti-CD89 / ILT7 bispecific antibody) constructed based on the two. In one aspect, the anti-ILT7 antibody of the present application can specifically bind to the juxtamembrane end of the extracellular domain of human ILT7; the anti-CD89 antibody of the present application can specifically recognize and bind to the extracellular Ig-like domain 2 of CD89 without affecting the binding of CD89 to IgA. In another aspect, the myeloid engager antibody (e.g., bispecific antibody) of the present application is a non-symmetrical heterodimeric molecule containing a constant region, which can simultaneously bind and crosslink myeloid cells expressing CD89 and target cells expressing ILT7, thereby activating the CD89 +The myeloid cells mediate directed killing of the target cells by activated myeloid cells. The part of the myeloid cell engager antibody that binds to CD89 is a monovalent antigen binding domain, thus avoiding induction of non-directed activation of myeloid cells in the absence of target cells; and the myeloid cells do not secrete or only secrete limited amounts of cytokines upon activation, thus the risk of the myeloid cell engager antibody directly triggering a cytokine storm is very low. In a further aspect, the present application introduces a series of amino acid mutations in the sequence of the antibody to optimize its Chemistry, Manufacturing, and Controls (CMC) properties, including improving the stability of the molecule, facilitating the formation of heterodimers during expression, and facilitating the separation of the desired heterodimer from the homodimer byproduct by the purification process, solving the technical problems of difficult production, poor stability, and low uniformity and purity of bispecific antibodies.

[0007] In one aspect, the present application provides an isolated anti-ILT7 antibody or antigen binding fragment thereof, which is capable of specifically binding to the extracellular domain of human ILT7 and is capable of specifically binding to pDC expressing ILT7, and has no cross-binding activity to other members of the ILT family, including ILT1, ILT2, ILT3, ILT4, ILT5, ILT6, ILT8, ILT11, LIR6 and LIR8.

[0008] In some embodiments, the anti-ILT7 antibody includes mouse anti-human ILT7 antibody or chimeric and / or humanized antibodies derived therefrom, and optimized antibodies thereof. In some embodiments, the anti-ILT7 antibody or antigen binding fragment thereof is capable of specifically binding to the juxtamembrane end of the extracellular domain of human ILT7, preferably an epitope comprising amino acid residues 420-446 of human ILT7.

[0009] In one aspect, the present application provides an isolated anti-CD89 antibody or antigen binding fragment thereof, which is capable of specifically recognizing and binding to the extracellular Ig-like domain 2 of CD89, and its binding site to CD89 is different from the binding site of CD89 to IgA.

[0010] In some embodiments, the anti-CD89 antibody includes fully human or humanized anti-CD89 antibody or antibodies derived / optimized therefrom (e.g., Mab 14.1 antibody and optimized antibodies thereof as described in WO2002064634). In one embodiment, the fully human or humanized anti-CD89 antibody or antibodies derived / optimized therefrom or antigen binding fragment thereof does not inhibit the binding of IgA to CD89, or does not compete with IgA for binding to CD89.

[0011] In some embodiments, the anti-CD89 antibody or antigen-binding fragment thereof comprises an scFv molecule of anti-CD89, or an scFv-Fc molecule of anti-CD89 fused with an IgG Fc fragment (e.g., a human IgG Fc fragment, such as a human IgG1 Fc fragment), which can be a scFv molecule with no disulfide bond at the VH-VL interface, or a scFv molecule with a disulfide bond at the VH-VL interface to enhance its stability, preferably a scFv molecule with a disulfide bond at the VH-VL interface. The disulfide bond at the VH-VL interface is introduced by introducing at least one cysteine residue in the VH domain and the VL domain of the anti-CD89 scFv molecule, respectively, so that at least one disulfide bond is formed between the VH domain and the VL domain of the scFv molecule, thereby enhancing the stability of the scFv molecule. The scFv molecule with a disulfide bond at the VH-VL interface maintains the binding activity and specificity of the anti-CD89 antibody or antigen-binding fragment thereof to CD89.

[0012] In one aspect, the present application provides a myeloid engager antibody targeting ILT7 and CD89, and an exemplary myeloid engager antibody is an anti-CD89 / ILT7 bispecific antibody comprising a first antigen binding domain and a second antigen binding domain. The first antigen binding domain specifically binds to CD89 on the surface of myeloid cells, preferably a scFv domain with a disulfide bond at the VH-VL interface; and the second antigen binding domain specifically binds to ILT7. The bispecific antibody can crosslink myeloid cells expressing CD89 with target cells expressing ILT7, direct activation of myeloid cells expressing CD89, and effectively mediate activated CD89 + myeloid cells expressing CD89 do not massively secrete cytokines after activation, so the risk of triggering cytokine storm is extremely low.

[0013] Further, the myeloid engager antibody is an asymmetric heterodimeric molecule comprising a constant region comprising a heavy chain constant region and a light chain constant region. The heavy chain constant region comprises native and mutant protein forms of human IgG (including human IgG1, IgG2, IgG3, IgG4) heavy chain constant regions, preferably mutant protein forms. The mutant protein forms of the heavy chain constant region include, but are not limited to, mutations introduced into the human IgG heavy chain constant region to promote heterodimer formation, mutations that facilitate separation of the target heterodimer from homodimer byproducts by purification processes, and / or mutations that reduce or enhance Fc Effector Function of the heavy chain constant region. The light chain constant region is a human lambda or kappa light chain constant region, preferably a human kappa light chain constant region (comprising an amino acid sequence as shown in SEQ ID NO: 166).

[0014] In some embodiments, the first antigen binding domain of the myeloid engager antibody targeting ILT7 and CD89 is selected from an anti-CD89 antibody or antigen binding fragment thereof that is capable of specifically binding to the extracellular domain of CD89, preferably an antibody or antigen binding fragment thereof that specifically binds to human CD89 extracellular Ig-like domain 2; the second antigen binding domain of the myeloid engager antibody is selected from an anti-ILT7 antibody or antigen binding fragment thereof that is capable of specifically binding to the extracellular domain of ILT7 (preferably the juxtamembrane 420-446 amino acid residues of the human ILT7 extracellular domain), which can be an anti-ILT7 antibody or antigen binding fragment thereof of the present application, or other anti-ILT7 antibody or antigen binding fragment thereof (e.g., the VIB7734 antibody or antigen binding fragment thereof described in WO2017156298). In some embodiments, the myeloid engager antibody has no cross- binding activity to other members of the ILT family, including ILT1, ILT2, ILT3, ILT4, ILT5, ILT6, ILT8, ILT11, LIR6, and LIR8. In some embodiments, the myeloid engager antibody mediates myeloid directed killing of pDC by specifically binding to CD89 on the surface of myeloid cells (e.g., neutrophils and macrophages) and ILT7 on the surface of pDC and activating the myeloid cells. In one embodiment, the myeloid engager antibody is capable of significantly inhibiting the production or secretion of type I interferon (IFN-I) and does not elicit cytokine storm.

[0015] In another aspect, the present application relates to isolated nucleic acid molecules (also referred to as “polynucleotides”) encoding the anti-CD89 antibodies or antigen binding fragments thereof, the anti-ILT7 antibodies or antigen binding fragments thereof, and the myeloid engager antibodies targeting ILT7 and CD89, as well as expression vectors comprising the nucleic acid molecules and host cells comprising the nucleic acid molecules or expression vectors. The present application also relates to methods of using the host cells to produce the anti-CD89 antibodies or antigen binding fragments thereof, the anti-ILT7 antibodies or antigen binding fragments thereof, and the myeloid engager antibodies targeting ILT7 and CD89 described herein, comprising culturing the host cells and recovering the antibodies or antigen binding fragments thereof from the culture.

[0016] In another aspect, the present application relates to immunoconjugates, chimeric antigen receptors, engineered T cell receptors, or oncolytic viruses comprising the anti-ILT7 antibodies or antigen binding fragments thereof described herein.

[0017] In another aspect, the present application relates to a pharmaceutical composition comprising a myeloid engager antibody targeting ILT7 and CD89 described herein, or an immunoconjugate, chimeric antigen receptor, engineered T cell receptor, or oncolytic virus comprising the anti-ILT7 antibody or antigen-binding fragment thereof described, and a pharmaceutically acceptable carrier.

[0018] In another aspect, the present application relates to a kit comprising a myeloid engager antibody targeting ILT7 and CD89 described herein, or an immunoconjugate, chimeric antigen receptor, engineered T cell receptor, or oncolytic virus comprising the anti-ILT7 antibody or antigen-binding fragment thereof described, or a pharmaceutical composition described herein, and optionally at least one additional therapeutic agent.

[0019] In another aspect, the present application relates to a method of treating and / or preventing a disorder associated with pDCs, especially dysregulated pDCs, comprising administering to a subject in need thereof an effective amount of a myeloid engager antibody targeting ILT7 and CD89 of the present application, or an immunoconjugate, chimeric antigen receptor, engineered T cell receptor, or oncolytic virus comprising the anti-ILT7 antibody or antigen-binding fragment thereof described, or a pharmaceutical composition or kit described herein. Alternatively, the present application relates to the use of the above myeloid engager antibody, or immunoconjugate, chimeric antigen receptor, engineered T cell receptor, or oncolytic virus comprising the anti-ILT7 antibody or antigen-binding fragment thereof described, or pharmaceutical composition or kit, for the manufacture of a medicament for treating and / or preventing a disorder associated with pDCs. Alternatively, the present application relates to the myeloid engager antibody targeting ILT7 and CD89, or immunoconjugate, chimeric antigen receptor, engineered T cell receptor, or oncolytic virus comprising the anti-ILT7 antibody or antigen-binding fragment thereof described, or pharmaceutical composition or kit, for use in treating and / or preventing a disorder associated with pDCs.

[0020] In some embodiments, the pDC-related disorder includes, but is not limited to, autoimmune diseases (e.g., systemic lupus erythematosus, cutaneous lupus, discoid lupus, lupus nephritis, multiple sclerosis, scleroderma, dermatomyositis, polymyositis, psoriasis, psoriatic arthritis, rheumatoid arthritis, myasthenia gravis, Goodpasture's syndrome, Sjogren's syndrome, systemic sclerosis, inflammatory bowel disease, irritable bowel syndrome, and Type I diabetes), cancers (e.g., Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN), and various solid tumors [including, but not limited to, lung cancer, liver cancer, stomach cancer, kidney cancer, head and neck cancer, nasopharyngeal cancer, cervical cancer, endometrial cancer, myeloid sarcoma, osteosarcoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, neuroblastoma] and hematological tumors [including, but not limited to, leukemia, multiple myeloma, malignant lymphoma, myelodysplastic syndrome]), and disorders associated with pDC tissue accumulation.

[0021] In another aspect, the present application relates to a method of detecting the number of pDCs in a sample (e.g., a biological sample, including serum, tissue, biopsy sample) comprising contacting the sample or tissue with an anti-ILT7 antibody or antigen-binding fragment thereof described herein, or a myeloid engager antibody targeting ILT7 and CD89, and then detecting the binding of the antibody or antigen-binding fragment thereof, or bispecific antibody to pDCs in the sample.

[0022] In another aspect, the present application relates to a method of detecting the level of ILT7 expression in a sample (e.g., a biological sample, including serum, tissue, biopsy sample) comprising contacting the sample or tissue with an anti-ILT7 antibody or antigen-binding fragment thereof described herein, or a myeloid engager antibody targeting ILT7 and CD89, and then detecting the binding of the antibody or antigen-binding fragment thereof, or bispecific antibody to ILT7 in the sample.

[0023] The present application will be further illustrated by the following figures and specific embodiments, other features and advantages of the present application will be apparent from the figures and specific embodiments, and these figures and specific embodiments should not be considered limiting the scope of the present application, and changes readily occurring to those skilled in the art will be included within the spirit and scope of the present application and the appended claims. All references cited in the present application, including published publications, patents and patent applications are incorporated by reference in their entirety. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1. Flow cytometry detection of binding activity of anti-CD89 single-chain antibody with disulfide bond at VH-VL interface to CD89-expressing Jurkat cells, wherein the control antibody is the parent single-chain antibody Mab 14.1-HL1405.

[0025] Figure 2. Binding activity of anti-ILT7 chimeric antibodies to human ILT family protein members (including ILT1, ILT6, ILT7, ILT8, ILT11, ILT2, ILT3, ILT4 and ILT5) was detected by ELISA.

[0026] Figure 3. Binding activity of anti-ILT7 chimeric antibodies to 293T engineered cell lines expressing each protein member of ILT family (including ILT7, ILT1, ILT8, ILT11, ILT2, ILT3, ILT4 and ILT5) was detected by flow cytometry, wherein the control antibody was BM antibody.

[0027] Figure 4. Binding activity of anti-ILT7 humanized antibodies HuA4c-L1H4 and HuA4c-L1H11 to ILT family protein members was detected by flow cytometry and ELISA. Figure 4A shows the binding activity of HuA4c-L1H4 and HuA4c-L1H11 to 293T engineered cell lines expressing each protein member of ILT family (including 293T-ILT7, -ILT1, -ILT8, -ILT11, -LIR6, -ILT2, -ILT3, -ILT4, -ILT5 and -LIR8) by flow cytometry; Figure 4B shows the binding activity of HuA4c-L1H4, HuA4c-L1H11 to ILT7 and ILT6 by ELISA, wherein the control antibody was BM antibody.

[0028] Figure 5. Binding activity of anti-ILT7 humanized antibodies HuA4c-L1H4 and HuA4c-L1H11 to human primary pDC cells was detected by flow cytometry, wherein the control antibody was BM antibody.

[0029] Figure 6. Schematic diagram of anti-CD89 / ILT7 bispecific antibodies. The configuration of exemplary bispecific antibodies is 2+1 scFv-IgG or scFv-Fab-Fc: Fab-Fc asymmetric structure, wherein subunit A is in the form of scFv-Fab-Fc, subunit B is in the form of Fab-Fc, the scFv domain is selected from anti-CD89 single-chain antibody containing disulfide bond at VH-VL interface, the IgG domain or Fab-Fc domain is anti-ILT7 antibody sequence, wherein the light chain variable region of the scFv domain is connected to the heavy chain variable region of the IgG domain or Fab-Fc domain through a polypeptide linker (G4S)2, the antibody heavy chain constant region comprises “knob-into-hole” mutation, “pI mutation” and L234F / L235E / P331S mutation (EU Numbering, referred to as “TM mutation”).

[0030] Figure 7. Biological activity of anti-CD89 / ILT7 bispecific antibodies detected by reporter gene assay, in which target cells are 293T engineered cell line expressing human ILT7 (293T-ILT7) and wild type 293T cell line, respectively.

[0031] Figure 8. Activity of anti-CD89 / ILT7 bispecific antibodies Cy01-L1H4 and Cy09-L1H4 in co-binding ILT7 and CD89 detected by ForteBio, in which control group includes anti-ILT7 humanized antibody HuA4c-L1H4 and anti-CD89 antibody Mab14.1-HL1405.

[0032] Figure 9. Binding activity of anti-CD89 / ILT7 bispecific antibodies Cy01-L1H4 and Cy09-L1H4 to members of ILT family proteins detected by flow cytometry and ELISA. In which, Figure 9A is flow cytometry detecting binding activity of Cy01-L1H4 and Cy09-L1H4 to 293T engineered cell lines expressing members of ILT family proteins (including 293T-ILT7, -ILT1, -ILT8, -ILT11, -LIR6, -ILT2, -ILT3, -ILT4, -ILT5 and -LIR8); Figure 9B is ELISA detecting binding activity of Cy01-L1H4 and Cy09-L1H4 to ILT7 and ILT6, in which control antibody is BM antibody.

[0033] Figure 10. Directed killing effect of anti-CD89 / ILT7 bispecific antibodies Cy01-L1H4 and Cy09-L1H4 mediated by human primary leukocytes on 293T engineered cell line expressing ILT7 and firefly luciferase (293T-ILT7-luc) detected, in which control group is 293T engineered cell line expressing firefly luciferase only (293T-luc).

[0034] Figure 11. Activity of anti-CD89 / ILT7 bispecific antibody Cy09-L1H4 in mediating killing of pDC by myeloid cells in human primary leukocytes indirectly evaluated by detecting expression / secretion level of IFNa. Shown in the figure are representative experimental results selected from one healthy person and one SLE patient (shown as response curve), as well as summary and comparison of the activity of Cy09-L1H4 in inhibiting IFNa production / secretion by pDC cells measured in 16 healthy person peripheral blood samples and 9 SLE patient peripheral blood samples (shown as EC50).

[0035] Figure 12. Detection of whether anti-CD89 / ILT7 bispecific antibody Cy09-L1H4 induces cytokine release from myeloid cells using peripheral blood derived from healthy people and SLE patients, wherein the negative control is blank medium and the positive control is Human CD3 / CD28 T Cell Activator.

[0036] Figure 13. Detection of ADCC activity of anti-CD89 / ILT7 bispecific antibody Cy09-L1H4 using reporter gene method, wherein the positive control antibody is anti-ILT7 antibody HuA4c-L1H4 with wild-type Fc sequence.

[0037] Figure 14. Linear elution of pH and salt concentration for the mixture of anti-CD89 / ILT7 bispecific antibody heterodimeric protein and “pore-pore” homodimeric protein using CEX-HPLC to determine the retention time of each protein elution peak, wherein Buffer A is 20 mM NaH2PO4, 20 mM Citric Acid, pH 5.5; Buffer B is 20 mM NaH2PO4, 20 mM Citric Acid, 200 mM NaCl, pH 8.0; the number marked at the peak tip is the retention time (minute) of the elution peak. DETAILED DESCRIPTION

[0038] DEFINITIONS

[0039] 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 application belongs. For purposes of the present application, the following terms are defined below.

[0040] As used herein, the terms “ILT7”, “ILT7 receptor”, and “ILT7 protein” can be used interchangeably, and the protein is also known as LILRA4 or CD85g. Unless otherwise indicated, “ILT7” as expressed herein refers to any native form of human ILT7, which can have an amino acid sequence as set forth in SEQ ID NO: 161 and / or a full-length ILT7 amino acid sequence as set forth in UniProtKB Accession No. P59901, and can be naturally expressed by cells (including pDC) or cells transfected with an ILT7 gene or cDNA. The term includes naturally occurring ILT7 allelic variants and splice variants, isoforms, homologs, and species homologs. ILT7 can be isolated from a human body, or can be produced by recombinant or synthetic methods.

[0041] The full-length ILT7 protein consists of 499 amino acids, wherein the first 1-23 amino acid residues are signal peptide, the 24th-446th amino acid residues are extracellular domain (ECD), the 447th-467th amino acid residues are transmembrane domain, and the 468th-499th amino acid residues are intracellular domain. The extracellular domain comprises 4 C2-type Ig-like domains, i.e., domain 1 (Ig1, 24th-118th amino acid residues), domain 2 (Ig2, 123rd-213th amino acid residues), domain 3 (Ig3, 224th-313th amino acid residues), and domain 4 (Ig4, 324th-413th amino acid residues). ILT7 is a member of the Immunoglobulin-like Transcript (ILT) or Leukocyte Immunoglobulin-like Receptor (LILR or LIR) gene family. The amino acid sequences of the extracellular domains of the members of the ILT family are highly conserved.

[0042] In the present text, the terms "CD89", "CD89 protein", "CD89 receptor", "FcaRI", or "IgA receptor" can be used interchangeably. CD89 is a glycosylated transmembrane receptor, comprising two extracellular domains, a transmembrane domain and an intracellular domain, which is constitutively expressed on neutrophils, eosinophils, macrophages and most monocytes, and the expression level is affected by inflammatory microenvironment. For example, the expression of CD89 on neutrophils is significantly increased after stimulation by G-CSF or GM-CSF (Weisbart et al., Nature 1988, 332: 647). The extracellular Ig-like domain 1 of CD89 binds IgA1 or IgA2 with moderate affinity (K D ~5 x 10 -7 M) and mediates effector cell functions including ADCC and phagocytosis, as well as release of limited inflammatory mediators and cytokines (Shen, Immunol Res 1992, 11: 273; Morton et al., Crit Rev Immunol 1996, 16: 423).

[0043] In this context, "plasmacytoid dendritic cells (pDCs)" are a special population of dendritic cells present in peripheral blood and secondary lymphoid organs, which participate in the immune response of the organism by secreting type I interferons (IFN-I) and as antigen-presenting cells, and also have the function of mediating the generation of immune tolerance. Although the number of pDCs represents only about 0.1-0.5% of peripheral blood mononuclear cells (PBMCs), they are the main source of IFN-I (Siegal et al., Science 1999, 284: 1835-1837; Liu et al., Annu Rev Immunol 2005, 23: 275-306). IFN-I not only plays an important role in the natural immune response against viruses and tumors, but is also involved in the regulation of the differentiation, survival and homeostasis of immune cells (such as T cells, B cells) and the control of the cell cycle. However, excessive secretion of IFN-I triggers persistent IFN-I signaling, leading to excessive inflammatory response and immune dysfunction (Chen et al., J Autoimmun 2017, 83: 1-11; Picard et al., Autoimmun Rev 2017, 16: 897-902). Studies have found that pDCs are enriched in the skin and other damaged organs (such as the kidney) of patients with various autoimmune diseases (such as dermatomyositis, polymyositis, systemic lupus erythematosus [SLE], cutaneous lupus erythematosus [CLE], psoriasis, alopecia areata and vitiligo), and the interferon activity in these tissues or organs is also high (Bertolotti et al., Pigment Cell Melanoma Res 2014, 27: 398-407; Blomberg et al., Lupus 2001, 10: 484-490; Farkas et al., Am J Pathol 2001, 159: 237-243; Greenberg et al., Arthritis Res Ther 2010, 12: S4). In addition, pDCs can also enable tumor cells to achieve immune escape and promote tumor proliferation by promoting the formation of a tumor microenvironment and mediating immune tolerance in the tumor microenvironment (Li et al., Front Immunol 2017, 8: 1268; Cao et al., Immunol Rev 2010; 234: 163).

[0044] In the present context, "a cell expressing ILT7" or "a cell expressing an ILT family member" can be a naturally occurring cell (e.g., a pDC) or a cell produced recombinantly by introducing a nucleic acid encoding an ILT7 or other member of the ILT family protein into a host cell.

[0045] In the present context, "a cell expressing CD89" can be a naturally occurring cell (e.g., a neutrophil, eosinophil, macrophage, monocyte) or a cell produced recombinantly by introducing a nucleic acid encoding CD89 into a host cell.

[0046] In the present context, "bispecific" or "bi-specific" is intended to include any antibody or antigen-binding fragment capable of specifically binding to two different antigens or epitopes of an antigen, comprising two separate antigen-binding domains each having a unique antigen-binding specificity. For example, where one antigen-binding domain binds to a first antigen or epitope of an antigen, the other antigen-binding domain binds to a second antigen different from the first antigen, or a different epitope of the first antigen. The first and second are used only to facilitate differentiation between the different antigens or epitopes bound, unless explicitly stated otherwise, and the use of these terms is not intended to impart a particular order of binding to different antigens or epitopes.

[0047] Monospecific antibody refers to an antibody or antigen-binding fragment having only one binding specificity, i.e., the antigen-binding domain of a monospecific antibody binds to a single epitope of a single antigen. In some embodiments, examples of monospecific antibodies include anti-CD89 monospecific antibodies and anti-ILT7 monospecific antibodies of the present application.

[0048] In the present context, "antigen-binding domain" or "antigen-binding region" or "epitope-binding domain" are used interchangeably to refer to a specific region on an antibody or antigen-binding fragment or derivative thereof that is directly involved in specific interaction with a target antigen, e.g., by binding, steric hindrance, stabilizing / destabilizing, spatial distribution, etc., to reach a dynamic equilibrium. In the present application, "antigen-binding domain" also refers to a specific region on an antibody or antigen-binding fragment or derivative thereof that interacts with a specific epitope on CD89 or ILT7, by binding, steric hindrance, stabilizing / destabilizing, spatial distribution, etc., to reach a dynamic equilibrium between the two.

[0049] An "antibody" refers to a polypeptide or protein that is capable of specifically recognizing and binding an antigen, generally encoded by one or more immunoglobulin genes or fragments thereof. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, some of which can be further divided into subclasses or isotypes, e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. A typical immunoglobulin (e.g., antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminal variable region of each chain defines a site that is specifically involved in binding the antigen. Antibody heavy chains are composed of a heavy chain variable region (VH) and a heavy chain constant region (CH), which in turn is generally composed of three domains, CH1, CH2 and CH3. Antibody light chains are composed of a light chain variable region (VL) and a light chain constant region (CL), which in turn is generally composed of one domain, CL. The pairing of VHand VLtogether creates a single antigen binding site. Endogenous VLis encoded by gene segments V (variable) and J (joining), and endogenous VHis encoded by V, D (diversity), and J. Both VLor VHinclude a Region of Hypervariability, or Complementarity Determining Region (CDR), and a Framework Region (FR). The terms "variable region" or "V region" are used interchangeably to refer to either a heavy chain variable region or a light chain variable region arranged in order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from amino terminus to carboxy terminus. The term "J region" refers to the subsequence of the variable region that encodes the C-terminal portion comprising CDR3 and FR4. The V region or J region can be naturally occurring, recombinant, or synthetic. In this document, antibody light chain variable regions and / or antibody heavy chain variable regions are sometimes collectively referred to as "antibody variable regions," and antibody light chains and / or antibody heavy chains are collectively referred to as "antibody chains." In certain embodiments, the FRs of the antibodies or antigen-binding fragments thereof provided herein can be identical to the human germline sequences, or can be naturally or artificially modified.

[0050] The location of CDRs and FRs can be determined using a variety of definition methods well known in the art, e.g., Kabat, Chothia, IMGT, and Contact (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 1991 Fifth Edition, NIH Publication No. 91-3242; Johnson et al., Nucleic Acids Res 2001, 29:205-206; Chothia & Lesk, J Mol Biol 1987, 196:901-917; Chothia et al., Nature 1989, 342:877-883; Chothia et al., J Mol Biol 1992, 227:799-817; Al-Lazikani et al., J Mol Biol 1997, 273:927-748; Lefranc et al., Nucleic Acids Research 1999, 27:209-212; MacCallum et al., J Mol Biol 1996, 262:732-745). Definitions of antigen binding sites are also described in Ruiz et al., Nucleic Acids Res 2000, 28:219-221; Lefranc, Nucleic Acids Res 2001, 29:207-209; Lefranc, The Immunologist 1999, 7:132-136; Lefranc et al., Dev Comp Immunol 2003, 27:55-77; MacCallum et al., J Mol Biol 1996, 262:732-745; Martin et al., Proc Natl Acad Sci USA 1989, 86:9268-9272; Martin et al., Methods Enzymol 1991, 203:121-153; Sternberg, ed., Protein Structure Prediction, 1996 Oxford University Press, 141-172. Any one of the definition methods is incorporated herein to determine the CDRs in the anti-CD89 antibodies or antigen-binding fragments thereof, the anti-ILT7 antibodies or antigen-binding fragments thereof, or the anti-CD89 / ILT7 bispecific antibodies of the present application, and Table 1 shows the location numbering of the antibody CDR amino acid sequences determined using different definition methods. The exact number of amino acid residues encompassing a particular CDR varies with the sequence of the CDR. One of skill in the art, given the amino acid sequence of an antibody variable region, can determine the CDRs of that antibody by routine methods including, without limitation, the definitions described.

[0051] Table 1. CDRs determined by different definition methods 1 1 The numbering of all CDRs in Table 1 follows the numbering system set forth by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 1991 Fifth Edition, NIH Publication No. 91-3242).

[0052] In addition, Kabat et al. also defined a numbering system for variable region sequences that can be applied to any antibody. One of ordinary skill in the art can apply this "Kabat numbering" system to the variable region sequences of any antibody without reliance on experimental data other than the antibody sequence itself to determine the variable region sequences. Unless otherwise indicated, the numbering of particular amino acid residues in the variable region of the antigen binding domain of the anti-CD89 antibodies, anti-ILT7 antibodies, or anti-CD89 / ILT7 bispecific antibodies of the application is according to the Kabat numbering system.

[0053] Antibodies exist as intact immunoglobulins or as a number of fragments produced by digestion with various peptidases. Although defined in terms of digestion of intact antibodies, fragments of an antibody can be synthesized de novo using recombinant DNA techniques or by chemical cleavage methods. In the present context, the term "antigen-binding fragment" of an antibody (or simply "antibody portion" or "antibody fragment") refers to a portion of an antibody that contains one or more CDRs or any other antibody fragment that is capable of binding to an antigen (e.g., ILT7 or an extracellular domain of ILT7, CD89 or an extracellular domain of CD89), but which does not have the structure of an intact antibody. An antigen-binding fragment can have the same antigenic binding activity as an intact antibody. In certain embodiments, an antigen-binding fragment can contain one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies. Antigen-binding fragments include, without limitation: (1) a "Fab" fragment, a monovalent fragment consisting of the VH, VL, CL, and CHI domains; (2) a "F(ab')2" fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (3) a "Fv" fragment, consisting of the VL and VH domains of a single arm of an antibody, is the minimum antibody fragment that contains a complete antigen-binding site; (4) a "Fd" fragment consisting of the VH and CHI domains; (5) a "single-chain Fv antibody" ("scFv"), "single-chain antibody" or "scFv molecule" refers to an engineered antibody in which the variable domain of the light chain and the variable domain of the heavy chain are directly linked together or linked by a short peptide chain (Huston et al., Proc Natl Acad Sci USA 1988, 85:5879-5883; Bird et al., Science 1988, 242:423-426); further, single-chain antibodies include "linear antibodies", which comprise a pair of tandem Fv segments (VH-CH1-VH-CH1) joined by a linker, together with a complementary light chain polypeptide to form a pair of antigen binding regions (Zapata et al., Protein Eng 1995, 8:1057-1062; US5641870); (6) a "dAb" fragment (Ward et al., Nature 1989, 341 :544-546; WO 90 / 05144A1) comprises a single variable domain, e.g., a VH domain.A single domain antibody (sdAb) is an isolated immunoglobulin domain; (7) A "diabody" is a bivalent, bispecific antibody in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and creating two antigen binding sites (Holliger et al., Proc Natl Acad Sci USA 1993, 90:6444-6448; Poljak et al., Structure 1994, 2:1121-1123; EP 404097; WO 93 / 11161).

[0054] The term "Fc region" or "Fc domain" herein refers to a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region, e.g., the immunoglobulin heavy chain constant region other than the first constant region (CHI). For IgG, the Fc region can comprise immunoglobulin domains CH2 and CH3 and a hinge region between CHI and CH2. The Fc region used herein includes native sequence Fc regions and / or variants of the Fc region and can be part of an anti-CD89 antibody, an anti-ILT7 antibody, or an anti-CD89 / ILT7 bispecific antibody of the application. It is understood that the boundaries of the Fc region can vary, however, the human IgG heavy chain Fc region is usually defined as stretching from an amino-terminal residue at position 226 or 230, to the carboxy-terminal residue at position 447, according to the EU numbering system / conventions, as found in Kabat et al., Sequences of Proteins of Immunological Interest, 1991, 5th Ed., NIH Publication 91-3242.

[0055] "myeloid engager antibody targeting ILT7 and CD89" or "myeloid engager targeting ILT7 and CD89" herein refers to a polypeptide comprising an anti-ILT7 antibody or antigen binding fragment thereof and an anti-CD89 antibody or antigen binding fragment thereof, e.g., an anti-CD89 / ILT7 bispecific antibody.

[0056] The term "anti-CD89 antibody" or "an antibody that specifically binds CD89" refers to any form of antibody or fragment thereof that specifically binds CD89 and encompasses both polyclonal and monoclonal antibodies, as well as biologically functional antibody fragments so long as the fragments are capable of specifically binding CD89. The anti-CD89 antibody of the present application, the anti-CD89 portion of the anti-CD89 / ILT7 bispecific antibody, is preferably an antibody or antibody fragment that specifically binds to the extracellular domain of CD89, more preferably an antibody or antibody fragment that specifically binds to the extracellular Ig-like domain 2 of CD89.

[0057] The term "anti-ILT7 antibody" or "an antibody that specifically binds to ILT7" refers to any form of antibody or fragment thereof that specifically binds to ILT7, and encompasses both polyclonal antibodies and biologically functional antibody fragments so long as the fragments are capable of specifically binding to ILT7. The anti-ILT7 antibody of the present application, the anti-ILT7 antibody portion of the anti-CD89 / ILT7 bispecific antibody, is preferably an antibody or antibody fragment that specifically binds to the extracellular domain of ILT7, and more preferably an antibody or antibody fragment that specifically binds to amino acid residues 420-446 of the extracellular domain of ILT7.

[0058] In the present context, the term "specifically binds" or "binding specificity" or "specific for" or "binds" refers to a binding reaction that is determinative of the presence of a target molecule (e.g., an antigen) in a heterogeneous population of proteins and other biological materials (e.g., a biological sample such as blood, serum, plasma, or a tissue sample), that is, of the target molecule, as opposed to that of other non-target molecules, and is capable of distinguishing between those non-specific or background interactions. For example, an antibody that specifically binds to a target molecule (which can be an antigen) has a greater affinity for the target molecule, a greater avidity for the target molecule, binds more readily, and / or remains bound more tightly to the target molecule than it does to other non-target molecules. Thus, such selectivity can be achieved by designing the immunizing antigen and preparing / screening antibodies capable of distinguishing between binding to members of the ILT family, as desired. Various immunoassay methods can be used to select antibodies specifically immunoreactive with the particular protein, e.g., solid-phase ELISA immunoassays, flow cytometry fluorescence-activated cell sorting (FACS), cellular fluorescence assays, or Surface Plasmon Resonance (SPR) techniques to detect binding of the antibodies or antigen-binding fragments thereof of the present disclosure to a target antigen / protein. Typically, the specific or selective binding of an antibody or binding agent to an antigen will result in a signal at least two times the background signal, more typically, at least 10-100 times the background signal, and most typically, at least 1000 times the background signal, under the assay conditions to be used, and substantially no binding to other antigens / proteins present in the sample. In certain embodiments, the antibody specifically binds to a target antigen / protein with an equilibrium dissociation constant (K D ) of <1 μΜ, <100 nM, <10 nM, <1 nM, or <0.1 nM.

[0059] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts. Monoclonal antibodies display specificity and affinity for a particular epitope. Monoclonal antibodies can be produced by the hybridoma method first described by Kohler et al., Nature 1975, 256:495, or can be made using recombinant DNA methods (see US4816567), or can be isolated from a phage antibody library, e.g., using the techniques described in Clackson et al., Nature 1991, 352:624-628; Marks et al., J Mol Biol 1991, 222:581-597.

[0060] The term "chimeric antibody" as used herein refers to an antibody that contains sequences derived from two different antibodies (e.g., US4816567), typically from different species. For example, a chimeric antibody comprises human and rodent antibody fragments, typically human constant regions and mouse variable regions. Methods for producing chimeric antibodies include conventional recombinant DNA and gene transfection techniques known to those of ordinary skill in the art (e.g., Morrison et al., Proc Natl Acad Sci USA 1984, 81:6851-6855; US5202238 and US5204244).

[0061] The term "humanized antibody or humanized antigen binding fragment" as used herein refers to an antibody or antigen binding fragment that includes CDRs derived from a non-human animal, FR regions derived from a human, and constant regions derived from a human. The humanized antibody optionally further comprises at least a portion of a constant region of a human immunoglobulin. Humanized antibodies or antigen binding fragments are useful as therapeutic agents for administration to humans due to their reduced immunogenicity. In some embodiments, the non-human animal is a mammal such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster. In some embodiments, the humanized antibody or antigen binding fragment has substantially all of the other portions of the antibody except the CDR sequences composed of human sequences. In some embodiments, a humanized antibody can be further modified, improved, and optimized for specificity, antigen binding affinity, and / or activity by substituting (e.g., by back mutation) an amino acid residue in a human immunoglobulin FR region with the corresponding amino acid residue in the antibody of the non-human species. In some embodiments, the FR regions derived from a human can include the same amino acid sequence as in the human antibody from which they are derived, or they can include some amino acid alterations, e.g., no more than 7, 6, 5, 4, 3, 2, or 1 amino acid alterations. In some embodiments, the amino acid alterations can be present only in the heavy chain FR regions, only in the light chain FR regions, or in both chains.

[0062] In the present context, the term "corresponding human germline sequence" refers to an antibody variable region amino acid sequence or subsequence that has a higher amino acid sequence identity to a reference human germline immunoglobulin variable region amino acid sequence than to all other known human germline immunoglobulin variable region amino acid sequences. The corresponding human germline sequence can be an individual framework region, an individual complementarity determining region, a framework region and a complementarity determining region, a variable region, or other combinations comprising variable region sequences or subsequences. Sequence identity can be determined using methods described herein, e.g., using BLAST, ALIGN, or other alignment algorithms known in the art to align two sequences. The corresponding human germline amino acid sequence can have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reference human germline immunoglobulin variable region amino acid sequence.

[0063] The anti-CD89 antibodies, anti-ILT7 antibodies, anti-CD89 / ILT7 bispecific antibodies of the present application can be selected from any one or more of the following forms, including chimeric forms, non-human humanized forms, humanized forms, or fully human forms, as long as the forms are capable of specifically binding to the target molecules (e.g., CD89 and / or ILT7).

[0064] The term "epitope" as used herein refers to a Protein Determinant, a portion of an antigen that is capable of being recognized by and specifically bound to an antibody. Epitopes are generally composed of chemically distinct groups of molecules, such as amino acids or sugar side chains, and are usually found on the surface of a molecule, and often have specific three-dimensional structural characteristics, as well as specific charge characteristics. The portion of the antibody or antigen-binding fragment thereof that recognizes the epitope is called the paratope.

[0065] The term "affinity" or "binding affinity" refers to the intrinsic binding capacity of an interaction between a molecule (e.g., a receptor or an antigen) and its partner (e.g., a ligand or an antibody), i.e., the strength of the sum total of all non-covalent interactions. Unless otherwise stated, "binding affinity" as used herein is used to reflect the intrinsic binding affinity of a one-to-one interaction between members of a binding pair (e.g., a receptor and a ligand or an antigen and an antibody). The affinity of a molecule X for its partner Y can be generally expressed in terms of the equilibrium dissociation constant (K D ), which is the ratio of the dissociation rate constant (k dis or k off ) and the association rate constant (k a or k on ). Affinity can be measured by common methods known in the art, including the methods used in the present application.

[0066] In the present context, the terms "cross-react", "cross-bind", "cross-reactivity" or "cross-reactive" are used interchangeably and refer to the ability of an antibody to have specificity for one antigen and also to react with a second antigen, and is a measure of the relatedness between two different antigens. Thus, an antibody has cross-reactivity if it binds to an antigen other than the target antigen. Cross-reactive epitopes typically contain many amino acid sequences homologous to the target antigen epitope, or similar structural features, and in some cases can bind to the antibody more fittingly than the target antigen epitope. An antibody can be considered to have "high specificity" for an antigenic epitope if it does not bind to other sequences or structures than the target antigen epitope and the cross-reactive epitope.

[0067] In the present context, the term "fusion" or "fused" when used in relation to amino acid sequences (e.g. peptides, polypeptides or proteins) refers to the combination of two or more amino acid sequences into a single amino acid sequence that does not occur in nature by chemical bonding or recombinant means. The fused amino acid sequence can be produced by recombination of two genes encoding the polynucleotide sequences, and can be expressed by methods of introducing the construct containing the recombined polynucleotide into a host cell.

[0068] In the present context, the term "antibody variant" or "antibody variant" refers to an antibody polypeptide sequence containing at least one amino acid mutation in a reference antibody variable region. A variant can be substantially homologous or substantially identical to the unmodified antibody. In some embodiments, there are amino acid mutations in one, two, three, four, five, and / or six CDRs of an anti-CD89 antibody, an anti-ILT7 antibody, or an anti-CD89 / ILT7 bispecific antibody of the present application to improve and optimize the performance of the antibody or antigen binding portion, including but not limited to increasing the degree of humanization, enhancing the binding affinity or binding activity to the target protein / antigen (e.g., CD89 and / or ILT7), increasing the expression level, and / or increasing the stability (e.g., reducing or eliminating the risk of aspartate isomerization and / or asparagine deamidation). In some embodiments, there are amino acid mutations in one, two, three, and / or four FRs of an anti-CD89 antibody, an anti-ILT7 antibody, or an anti-CD89 / ILT7 bispecific antibody of the present application to improve and optimize the performance of the antibody or antigen binding portion, including but not limited to increasing the degree of humanization of the antibody or antigen binding portion, enhancing the binding affinity or binding activity to the target molecule (e.g., CD89 and / or ILT7), increasing the expression level or monomer content, and / or increasing the stability (e.g., reducing or eliminating the risk of aspartate isomerization and / or asparagine deamidation). In some embodiments, there is one or more amino acid mutations in the CDRs and / or FRs of an anti-CD89 antibody, an anti-ILT7 antibody, or an anti-CD89 / ILT7 bispecific antibody of the present application to increase the degree of humanization of the antibody or antigen binding portion, enhance the binding affinity or binding activity to the target molecule (e.g., CD89 and / or ILT7), increase the expression level or monomer content, and / or increase the stability (e.g., reducing or eliminating the risk of aspartate isomerization and / or asparagine deamidation). In some embodiments, the amino acid mutations include amino acid substitutions, deletions, insertions, or any combination thereof.

[0069] wherein the amino acid substitutions include conservative amino acid substitutions and non-conservative amino acid substitutions, conservative amino acid substitutions involve substitution with another amino acid in the same class (e.g., chemically or functionally similar), and non-conservative substitutions involve substitution with an amino acid in a different class (chemically or functionally dissimilar). One of ordinary skill in the art can make conservative or non-conservative amino acid substitutions based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues involved. For example, (i) non-polar (hydrophobic) amino acids include alanine (Ala, A), leucine (Leu, L), isoleucine (Ile, I), valine (Val, V), proline (Pro, P), phenylalanine (Phe, F), tryptophan (Trp, W), and methionine (Met, M); (ii) polar neutral amino acids include glycine (Gly, G), serine (Ser, S), threonine (Thr, T), cysteine (Cys, C), tyrosine (Tyr, Y), asparagine (Asn, N), and glutamine (Gln, Q); (iii) positively charged (basic) amino acids include arginine (Arg, R), lysine (Lys, K), and histidine (His, H); (iv) negatively charged (acidic) amino acids include aspartic acid (Asp, D) and glutamic acid (Glu, E). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein, and non-conservative amino acid substitutions can result in substantial changes in the properties or functions of a protein. Although the site or region into which an amino acid sequence mutation is introduced can be predetermined, the potential changes in protein properties or functions resulting from non-conservative substitutions are unpredictable. In some embodiments, the anti-CD89 antibodies, anti-ILT7 antibodies, or anti-CD89 / ILT7 bispecific antibodies of the present application are unexpectedly and significantly altered in function and performance, such as antigen binding affinity, cell binding activity, molecular stability, etc., by non-conservative amino acid substitutions.

[0070] In the present context, the terms "identical" or "identity" or "percent identity" or "percent sequence identity" in relation to a plurality of polypeptide sequences are used interchangeably and refer to the percentage of amino acid residues in a candidate sequence that have the same amino acid residues as a reference sequence when aligned for maximum sequence comparison, and wherein the number of gaps, if any, is minimized by introducing of appropriate gaps. The conservative substitution of an amino acid residue can or can not be considered as an identical residue. Alignment of sequences for the determination of percent sequence identity can be performed by tools disclosed in the art, such as BLASTp, ClustalW2 (see also Higgins et al., Methods Enzymol 1996, 266:383-402; Larkin et al., Bioinformatics 2007, 23:2947-2948) and ALIGN or Megalign (DNASTAR) software. The tools can be used by the skilled person using the default parameters of the tools or appropriately adjusted parameters according to the needs of the alignment, such as by choosing the appropriate algorithm for sequence alignment.

[0071] By "reducing or eliminating the risk of deamidation" in relation to an amino acid sequence, it is meant a sequence in which an amino acid residue susceptible to deamidation is replaced by an amino acid residue less susceptible or not susceptible to deamidation. Deamidation is a chemical reaction in which the amide function in the side chain of an asparagine or glutamine is removed or converted into another function. Typically, asparagine (Asn) can be converted into aspartic acid or isoaspartic acid, and glutamine (Gln) can be converted into glutamic acid or pyroglutamic acid. Deamidation is prone to occur in an amino acid sequence comprising an Asn-Gly, Asn-Ser or Asn-Thr site, and asparagine is more prone to deamidation than glutamine. Deamidation of asparagine and / or glutamine can alter the structure of the antibody and its stability and / or function (e.g. antibody-antigen binding), and therefore, it is desirable to reduce or eliminate the risk of deamidation. The risk of deamidation at these sites can be reduced or eliminated by predicting the amino acid residues in the variable region of an antibody that are susceptible to deamidation (Sydow et al., PLoS ONE 2014, 9: e100736), and replacing these sites with amino acid residues less susceptible or not susceptible to deamidation.

[0072] In the present context, the term "asparagine isomerization" refers to a sequence comprising an asparagine residue which is prone to isomerization. Isomerization causes a change in the antibody conformation, which in turn changes the antibody surface charge, resulting in antibody charge heterogeneity. Preferably, the antibodies of the present application do not comprise asparagine isomerization sites. Asparagine isomerization is prone to occur on D-G sequences, and isomerization has also been reported to occur on D-H or D-S sequences, resulting in isoasparagine residues, which introduce a linkage into the polypeptide chain, resulting in a decrease in the stability of the polypeptide chain (also known as the isoasparagine effect), and can also result in a decrease in antibody-antigen binding affinity.

[0073] In the present context, the term "isolated" when referring to a protein means that the protein is substantially free of other cellular components with which it is associated in nature, preferably in a homogeneous state, e.g., an isolated protein can be removed from its natural or native environment. An isolated protein can be lyophilized or in an aqueous solution. Typically, its purity and homogeneity can be determined using analytical chemistry techniques (e.g., polyacrylamide gel electrophoresis or high performance liquid chromatography). A protein in an isolated preparation is substantially purified. The term "purified" means that the protein produces essentially one band on a non-reducing gel. In particular, this means that the protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure. For purposes of the present application, a recombinant protein expressed in a host cell is considered isolated in some embodiments, as are native or recombinant proteins that are separated, fractionated, or partially or substantially purified by any of the techniques known to those skilled in the art. In some embodiments, an "isolated antibody" is one which is substantially free of other antibodies of different antigenic specificities (e.g., an isolated antibody which specifically binds CD89 is substantially free of antibodies that specifically bind antigens other than CD89, or an isolated antibody which specifically binds ILT7 is substantially free of antibodies that specifically bind antigens other than ILT7). Moreover, an isolated antibody can be substantially free of other cellular material and / or chemicals. In some embodiments, a recombinant polynucleotide encoding a polypeptide or protein of the present application (e.g., an anti-CD89 antibody, an anti-ILT7 antibody, or an anti-CD89 / ILT7 bispecific antibody) comprised in a vector is considered isolated. Other examples of isolated polynucleotides include a recombinant polynucleotide comprised in a heterologous host cell or a purified (partially or substantially) polynucleotide in solution.

[0074] In the present context, the term "engineered" includes any manipulation of the polypeptide or protein backbone, or post-translational modification of a naturally occurring or recombinant protein or polypeptide. Engineering includes mutation of the amino acid sequence, modification of glycosylation or side chain groups of individual amino acids, and combinations of these methods.

[0075] As used herein, the term "polypeptide" refers to polymers of amino acids and its equivalents, and is not intended to specify a particular length of the product. Thus, "peptides" and "proteins" are included within the definition of polypeptides. Also included within the definition of polypeptides are "antibodies" as defined herein.

[0076] As used herein, the term "asymmetric" means that the bispecific antibody of the present application cannot be divided into two identical parts. Specifically, certain natural antibodies (e.g., IgG) comprise two identical heavy chains and two identical light chains, wherein the part consisting of one heavy chain and one light chain is mutually symmetric to the part consisting of the other heavy chain and the other light chain. Preferably, the asymmetric bispecific antibody of the present application is a 3-valent heterodimer or heterodimeric bispecific antibody consisting of three different polypeptide chains, wherein the two heavy chains are not identical and the two light chains are identical.

[0077] The term "heterodimer" or "heterodimeric bispecific antibody" refers herein to an asymmetric bispecific antibody consisting of two different self-assembled paired heavy chains and two identical light chains, which comprises two different subunits (e.g., subunit A and B), wherein subunit A comprises two different antigen binding domains and subunit B comprises one antigen binding domain. The heterodimeric bispecific antibody of the present application mainly avoids the heavy chain mispairing problem by self-assembly based on the Fc domains in the two heavy chains. Since the bispecific antibody of the present application has only one kind of light chain, there is no light chain mispairing problem. In some embodiments, exemplary formats of the bispecific antibody of the present application are 2+1 scFv-IgG or scFv-Fab-Fc:Fab-Fc; accordingly, subunit A is scFv-Fab-Fc, subunit B is Fab-Fc, and the heavy chain Fc regions of subunits A and B have different amino acid sequences. The subunits A and B are used for the convenience of distinguishing the two different parts / half antibodies of the bispecific antibody, and the use of these terms is not intended to impart a specific order of the two different parts / half antibodies of the bispecific antibody, unless explicitly so stated.

[0078] Unless otherwise indicated by context, a "derivative" is a polypeptide or fragment thereof having one or more non-conservative or conservative amino acid substitutions relative to the parent antibody or polypeptide; or a polypeptide or fragment thereof modified by covalent attachment of a second molecule, such as by attachment of a heterologous polypeptide, or by glycosylation, acetylation, phosphorylation, etc. The definition of "derivative" can also include polypeptides of one or more amino acid analogs (e.g., unnatural amino acids, etc.), as well as other modifications (both naturally-occurring and non-naturally-occurring) known in the art.

[0079] In the present context, the term "expression vector" or "vector" refers to a vehicle into which a polynucleotide or nucleic acid encoding a protein can be operatively inserted and the protein is expressed. The vector can be used to transform, transduce or transfect a host cell so that the genetic material elements it carries are expressed within the host cell.

[0080] In the present context, the term "host cell" is a cell into which a foreign polynucleotide, nucleic acid and / or vector is introduced. The host cell includes "transformants" and "transformed cells," including the primary transformed cell and progeny derived therefrom, regardless of the number of transfers. The progeny can not be identical to the parent cell both in nucleic acid content and in physical characteristics, but rather can include mutations that arise during the multiplication of the cells. Mutant progeny that have the same function or biological activity as the originally transformed cell are included in the present application, as are screening or selection of such mutant progeny in the initially transformed cell.

[0081] In the present context, the terms "subject," "patient" or "individual" are used interchangeably and include, but are not limited to, a mammal such as a human, a non-human primate (e.g., a monkey), a mouse, a pig, a dog, a cat, a cow, a goat, a rabbit, a rat, a guinea pig, a hamster, a horse, a sheep, or other non-human mammal; a non-mammal such as a non-mammalian vertebrate, e.g., a bird (e.g., a chicken, an emu, or a duck) or a fish; and a non-mammalian invertebrate. In some embodiments, the subject and pharmaceutical compositions of the uses or methods of the present application are for treating (prophylactically and / or therapeutically) a non-human animal.

[0082] In the present context, "Treating," "Treatment" or "Treat" of a disease or condition refers to Alleviating or Alleviation of a disease or condition, reducing the speed of onset or development of a disease or condition, reducing the risk of developing a disease or condition, or delaying the development of symptoms associated with a disease or condition, reducing or terminating symptoms associated with a disease or condition, producing complete or partial reversal of a disease or condition, curing a disease or condition, or a combination thereof.

[0083] The term "therapeutically effective amount," "effective dose" or "effective amount" refers to the amount or concentration of an active ingredient or agent, and the period of time for which it is required, which is necessary to achieve an effective prevention or improvement of symptoms associated with a disease or condition, alleviation or reduction of the severity of a disease or condition, and / or delay or halt of the progression of a disease, or to achieve a therapeutic benefit that is significantly greater than any toxic or deleterious effects caused by the treatment. The therapeutically effective amount of a formulation, antibody or antigen-binding fragment thereof, bispecific antibody or composition of the present application can vary depending on a variety of factors, such as the disease state, the age, sex, and weight of the individual, and the ability of the formulation, antibody or antigen-binding fragment thereof, bispecific antibody or composition to elicit a desired response in the individual.

[0084] In the present context, the term "pharmaceutically acceptable" or "pharmaceutically acceptable" refers to a carrier, vehicle, diluent, adjuvant, and / or salt that is chemically and / or physiologically compatible to the other ingredients used in the formulation and physiologically compatible to the subject.

[0085] The term "about" when used in association with a numerical value means a numerical value that encompasses 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, or in one embodiment a lower limit that is 10% less and an upper limit that is 10% greater, or in another embodiment a lower limit that is 15% less and an upper limit that is 15% greater, or in another embodiment a lower limit that is 20% less and an upper limit that is 20% greater.

[0086] The term "and / or" should be understood to mean either one of the items or any combination of the items in the alternatives.

[0087] As used herein, the terms "comprise" or "comprising" or "contain" or "containing" or "include" or "including" or "have" or "having" or "involve" or "involving" are interchangeable with respect to the inclusion of the elements, integers or steps that are described in the respective clause.

[0088] As used herein, the term "optional" means that the object modified by the term exists or does not exist, e.g. "the kit comprises optionally at least one additional therapeutic agent" means that the kit can or can not comprise at least one additional therapeutic agent.

[0089] As used herein, "some embodiments," "one embodiment," "one specific embodiment," or "a specific embodiment," or combinations thereof, means that a specific feature, structure, or characteristic described is included in at least one embodiment of the application. Therefore, repeated use of the phrases "some embodiments," "one embodiment," "one specific embodiment," or "a specific embodiment," or combinations thereof, does not necessarily refer to the same embodiment. In addition, the described features, structures, or characteristics can be combined in any suitable manner in one or more embodiments.

[0090] The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0091] For purposes of United States patent practice, the patents, patent applications, and other publications recited herein are hereby expressly incorporated by reference for all purposes. These publications are provided solely for their disclosure prior to the filing date of the present application. All statements as to the date or

[0092] Various aspects of the application will be described in further detail in the following sections.

[0093] 1. Anti-ILT7 antibody or antigen-binding fragment thereof

[0094] In one aspect, the present application provides an isolated anti-ILT7 antibody or antigen-binding fragment thereof, which is capable of specifically binding to the extracellular domain of ILT7. Preferably, the anti-ILT7 antibody or antigen-binding fragment thereof is capable of specifically binding to amino acid residues 420-446 of the extracellular domain of human ILT7.

[0095] To obtain an antibody or antigen-binding fragment thereof capable of specifically binding to human ILT7, the present application designs a specific immunizing antigen, which, when used to immunize an animal (e.g., a mouse) alone or in combination with other immunizing antigens, is capable of eliciting a high titer immune response, and through cell fusion and hybridoma screening, an anti-ILT7 antibody or antigen-binding fragment thereof is obtained that specifically binds to human ILT7 only, and has no or little cross-reactivity with other members of the human ILT family, including ILT1, ILT2, ILT3, ILT4, ILT5, ILT6, ILT8, ILT11, LIR6 and LIR8. The immunizing antigen is designed by aligning the amino acid sequences of the extracellular domains of each member of the human ILT family, and selecting a portion that has relatively low degree of amino acid sequence homology between members, and preferably comprises the amino acid sequence of 420-446 of the extracellular domain of human ILT7. The screening of the hybridoma includes using various methods known in the art to determine the binding activity of the supernatant containing the antibody or antigen-binding fragment thereof, or antigen-binding molecule of interest to ILT7 or other members of the ILT family, including but not limited to ELISA, cell fluorescence, flow cytometry and SPR molecular interaction analysis.

[0096] The anti-ILT7 antibody or antigen-binding fragment thereof can specifically recognize the juxtamembrane sequence of the extracellular domain of ILT7, in particular, the amino acid residues 420-446 of the extracellular domain of human ILT7, and the binding epitope is different from the epitope (i.e., Ig1 domain) bound by the reference antibody VIB7734 to human ILT7. The anti-ILT7 antibody or antigen-binding fragment thereof can specifically bind to cells expressing ILT7, including pDC and a cell line generated by introducing nucleic acid encoding ILT7 into 293T cells. The anti-ILT7 antibody or antigen-binding fragment thereof has a high binding affinity to ILT7, with a Kd D value of < 5 x 10 -8 M, preferably < 1 x 10 -8 M, or < 5 x 10 -9 M, more preferably < 5 x 10 -9 M. The anti-ILT7 antibody or antigen-binding fragment thereof has no cross-binding activity to other members of the ILT family, including ILT1, ILT2, ILT3, ILT4, ILT5, ILT6, ILT8, ILT11, LIR6 and LIR8.

[0097] The anti-ILT7 antibody of the present application can also optionally comprise F(ab')2, Fab, Fab', Fv, scFv, scFv-Fc, single domain antibody (sdAb), or have an IgG type. The anti-ILT7 antibody of the present application can be a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody, a monoclonal antibody, a polyclonal antibody, a monospecific antibody, a multispecific antibody (e.g., a bispecific antibody), and fragments thereof, as long as the antibody can specifically recognize and bind to the extracellular domain of ILT7 and has no cross-binding activity to other members of the ILT family. In some embodiments, the anti-ILT7 antibody is selected from mouse anti-human ILT7 antibodies and humanized antibodies and optimized antibodies thereof.

[0098] In another aspect, the present application provides an anti-ILT7 antibody or antigen-binding fragment thereof as shown in Table 2, which can specifically recognize and bind to the amino acid residues 420-446 of the extracellular domain of human ILT7 and has no cross-binding activity to other members of the ILT family.

[0099] The heavy chain variable region CDRs and light chain variable region CDRs of the anti-ILT7 antibody or antigen-binding fragment thereof of the present application are defined by the Kabat numbering system. However, as is well known in the art, the CDR regions can also be defined based on other numbering systems / methods such as Chothia and IMGT, AbM or Contact numbering systems / methods of the heavy chain / light chain variable region sequences, and the CDR regions defined thereby are within the scope of the present application.

[0100] Table 2. CDR regions of anti-ILT7 antibodies, and amino acid sequence ID numbers for heavy chain variable regions and light chain variable regions

[0101] In some embodiments, an anti-ILT7 antibody of the application or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13 and / or a VL comprising an amino acid sequence as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14. In one embodiment, an anti-ILT7 antibody of the application or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO: 1 and / or a VL comprising an amino acid sequence as set forth in SEQ ID NO: 2. In one embodiment, an anti-ILT7 antibody of the application or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO: 3 and / or a VL comprising an amino acid sequence as set forth in SEQ ID NO: 4. In one embodiment, an anti-ILT7 antibody of the application or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO: 5 and / or a VL comprising an amino acid sequence as set forth in SEQ ID NO: 6. In one embodiment, an anti-ILT7 antibody of the application or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO: 7 and / or a VL comprising an amino acid sequence as set forth in SEQ ID NO: 8. In one embodiment, an anti-ILT7 antibody of the application or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO: 9 and / or a VL comprising an amino acid sequence as set forth in SEQ ID NO: 10. In one embodiment, an anti-ILT7 antibody of the application or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO: 11 and / or a VL comprising an amino acid sequence as set forth in SEQ ID NO: 12. In one embodiment, an anti-ILT7 antibody of the application or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO: 13 and / or a VL comprising an amino acid sequence as set forth in SEQ ID NO: 14.

[0102] In some embodiments, the anti-ILT7 antibodies or antigen-binding fragments thereof of the present application comprise: one or more CDRs in a VH comprising an amino acid sequence as set forth in SEQ ID NO: 3 or an amino acid sequence as set forth in SEQ ID NOs: 15, 17, and 25, or a variant thereof; one or more CDRs in a VH comprising an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence as set forth in SEQ ID NOs: 114, 115, and 116, or a variant thereof; one or more CDRs in a VH comprising an amino acid sequence as set forth in SEQ ID NO: 5 or an amino acid sequence as set forth in SEQ ID NOs: 120, 121, and 122, or a variant thereof; one or more CDRs in a VH comprising an amino acid sequence as set forth in SEQ ID NO: 7 or an amino acid sequence as set forth in SEQ ID NOs: 126, 127, and 128, or a variant thereof; one or more CDRs in a VH comprising an amino acid sequence as set forth in SEQ ID NO: 9 or an amino acid sequence as set forth in SEQ ID NOs: 132, 133, and 134, or a variant thereof; one or more CDRs in a VH comprising an amino acid sequence as set forth in SEQ ID NO: 11 or an amino acid sequence as set forth in SEQ ID NOs: 138, 139, and 140, or a variant thereof; and / or one or more CDRs in a VH comprising an amino acid sequence as set forth in SEQ ID NO: 13 or an amino acid sequence as set forth in SEQ ID NOs: 144, 145, and 146, or a variant thereof; variants described above include humanized antibodies or any other variant described in the present application. In some embodiments, the anti-ILT7 antibodies or antigen-binding fragments thereof of the present application comprise one or more CDRs in a VH comprising an amino acid sequence as set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13, or any combination of the CDRs.

[0103] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment thereof of the present application further comprises: one or more CDRs in a VL comprising an amino acid sequence as set forth in SEQ ID NO: 4 or an amino acid sequence as set forth in SEQ ID NOs: 26, 31, and 37, or a variant thereof; one or more CDRs in a VL comprising an amino acid sequence as set forth in SEQ ID NO: 2 or an amino acid sequence as set forth in SEQ ID NOs: 117, 118, and 119, or a variant thereof; one or more CDRs in a VL comprising an amino acid sequence as set forth in SEQ ID NO: 6 or an amino acid sequence as set forth in SEQ ID NOs: 123, 124, and 125, or a variant thereof; one or more CDRs in a VL comprising an amino acid sequence as set forth in SEQ ID NO: 8 or an amino acid sequence as set forth in SEQ ID NOs: 129, 130, and 131, or a variant thereof; one or more CDRs in a VL comprising an amino acid sequence as set forth in SEQ ID NO: 10 or an amino acid sequence as set forth in SEQ ID NOs: 135, 136, and 137, or a variant thereof; one or more CDRs in a VL comprising an amino acid sequence as set forth in SEQ ID NO: 12 or an amino acid sequence as set forth in SEQ ID NOs: 141, 142, and 143, or a variant thereof; and / or one or more CDRs in a VL comprising an amino acid sequence as set forth in SEQ ID NO: 14 or an amino acid sequence as set forth in SEQ ID NOs: 147, 148, and 149, or a variant thereof; variants described above include humanized antibodies or any other variant described in the present application. In some embodiments, the anti-ILT7 antibody or antigen-binding fragment thereof of the present application further comprises one or more CDRs in a VL comprising an amino acid sequence as set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14, or any combination of the CDRs.

[0104] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment thereof of the present application comprises: one or more CDRs of a VH comprising an amino acid sequence as set forth in SEQ ID NO: 3 or an amino acid sequence as set forth in SEQ ID NOs: 15, 17, and 25, or a variant thereof, and one or more CDRs in a VL comprising an amino acid sequence as set forth in SEQ ID NO: 4 or an amino acid sequence as set forth in SEQ ID NOs: 26, 31, and 37, or a variant thereof; one or more CDRs of a VH comprising an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence as set forth in SEQ ID NOs: 114, 115, and 116, or a variant thereof, and one or more CDRs in a VL comprising an amino acid sequence as set forth in SEQ ID NO: 2 or an amino acid sequence as set forth in SEQ ID NOs: 117, 118, and 119, or a variant thereof; one or more CDRs of a VH comprising an amino acid sequence as set forth in SEQ ID NO: 5 or an amino acid sequence as set forth in SEQ ID NOs: 120, 121, and 122, or a variant thereof, and one or more CDRs in a VL comprising an amino acid sequence as set forth in SEQ ID NO: 6 or an amino acid sequence as set forth in SEQ ID NOs: 123, 124, and 125, or a variant thereof; one or more CDRs of a VH comprising an amino acid sequence as set forth in SEQ ID NO: 7 or an amino acid sequence as set forth in SEQ ID NOs: 126, 127, and 128, or a variant thereof, and one or more CDRs in a VL comprising an amino acid sequence as set forth in SEQ ID NO: 8 or an amino acid sequence as set forth in SEQ ID NOs: 129, 130, and 131, or a variant thereof; one or more CDRs of a VH comprising an amino acid sequence as set forth in SEQ ID NO: 9 or an amino acid sequence as set forth in SEQ ID NOs: 132, 133, and 134, or a variant thereof, and one or more CDRs in a VL comprising an amino acid sequence as set forth in SEQ ID NO: 10 or an amino acid sequence as set forth in SEQ ID NOs: 135, 136, and 137, or a variant thereof; one or more CDRs of a VH comprising an amino acid sequence as set forth in SEQ ID NO: 11 or an amino acid sequence as set forth in SEQ ID NOs: 138, 139, and 140, or a variant thereof, and one or more CDRs in a VL comprising an amino acid sequence as set forth in SEQ ID NO: 12 or an amino acid sequence as set forth in SEQ ID NOs: 141, 142, and 143, or a variant thereof;and / or one or more CDRs of a VH comprising an amino acid sequence as set forth in SEQ ID NO: 13 or an amino acid sequence as set forth in SEQ ID NOs: 144, 145, and 146, or a variant thereof, and one or more CDRs of a VL comprising an amino acid sequence as set forth in SEQ ID NO: 14 or an amino acid sequence as set forth in SEQ ID NOs: 147, 148, and 149, or a variant thereof; variants include humanized antibodies or any other variant described herein.

[0105] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment thereof of the present application comprises: a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 114, 15, 120, 126, 132, 138, or 144, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 115, 17, 121, 127, 133, 139, or 145, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 116, 25, 122, 128, 134, 140, or 146; and a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 117, 26, 123, 129, 135, 141, or 147, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 118, 31, 124, 130, 136, 142, or 148, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 119, 37, 125, 131, 137, 143, or 149.

[0106] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment thereof of the present application comprises: a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 15, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 17, a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 25, and a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 26, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 31, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 37.

[0107] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment thereof comprises a HCDR1 having an amino acid sequence of SEQ ID NO: 114, a HCDR2 having an amino acid sequence of SEQ ID NO: 115, a HCDR3 having an amino acid sequence of SEQ ID NO: 116 (the amino acid sequence is YSK), and a LCDR1 having an amino acid sequence of SEQ ID NO: 117, a LCDR2 having an amino acid sequence of SEQ ID NO: 118, a LCDR3 having an amino acid sequence of SEQ ID NO: 119.

[0108] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment thereof comprises a HCDR1 having an amino acid sequence of SEQ ID NO: 120, a HCDR2 having an amino acid sequence of SEQ ID NO: 121, a HCDR3 having an amino acid sequence of SEQ ID NO: 122 (the amino acid sequence is YSN), and a LCDR1 having an amino acid sequence of SEQ ID NO: 123, a LCDR2 having an amino acid sequence of SEQ ID NO: 124, a LCDR3 having an amino acid sequence of SEQ ID NO: 125.

[0109] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment thereof comprises a HCDR1 having an amino acid sequence of SEQ ID NO: 126, a HCDR2 having an amino acid sequence of SEQ ID NO: 127, a HCDR3 having an amino acid sequence of SEQ ID NO: 128 (the amino acid sequence is FAF), and a LCDR1 having an amino acid sequence of SEQ ID NO: 129, a LCDR2 having an amino acid sequence of SEQ ID NO: 130, a LCDR3 having an amino acid sequence of SEQ ID NO: 131.

[0110] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment thereof comprises a HCDR1 having an amino acid sequence of SEQ ID NO: 132, a HCDR2 having an amino acid sequence of SEQ ID NO: 133, a HCDR3 having an amino acid sequence of SEQ ID NO: 134 (the amino acid sequence is YSN), and a LCDR1 having an amino acid sequence of SEQ ID NO: 135, a LCDR2 having an amino acid sequence of SEQ ID NO: 136, a LCDR3 having an amino acid sequence of SEQ ID NO: 137.

[0111] In some embodiments, the anti-ILT7 antibody or its antigen-binding fragment comprises: HCDR1 having the amino acid sequence shown in SEQ ID NO:138, HCDR2 having the amino acid sequence shown in SEQ ID NO:139, HCDR3 having the amino acid sequence shown in SEQ ID NO:140 (amino acid sequence YNY), and LCDR1 having the amino acid sequence shown in SEQ ID NO:141, LCDR2 having the amino acid sequence shown in SEQ ID NO:142, and LCDR3 having the amino acid sequence shown in SEQ ID NO:143.

[0112] In some embodiments, the anti-ILT7 antibody or its antigen-binding fragment comprises: HCDR1 having the amino acid sequence shown in SEQ ID NO:144, HCDR2 having the amino acid sequence shown in SEQ ID NO:145, HCDR3 having the amino acid sequence shown in SEQ ID NO:146 (amino acid sequence YSN), and LCDR1 having the amino acid sequence shown in SEQ ID NO:147, LCDR2 having the amino acid sequence shown in SEQ ID NO:148, and LCDR3 having the amino acid sequence shown in SEQ ID NO:149.

[0113] The binding affinity (K) of the above-mentioned anti-ILT7 murine antibody or its antigen-binding fragment to human ILT7 of the present invention is [missing information]. D Value < 5 × 10 -8 M, preferably, <1×10 -8 M. The antibody or its antigen-binding fragment does not have cross-binding activity with other members of the ILT family (including ILT1, ILT2, ILT3, ILT4, ILT5, ILT6, ILT8, ILT11, LIR6 and LIR8).

[0114] On the other hand, the anti-ILT7 antibody of the present invention or its antigen-binding fragment VH and / or VL can be used as starting materials for engineering to prepare an anti-ILT7 antibody more suitable for human administration. The antibody can be engineered by mutating one or more amino acid residues within one or both variable regions (i.e., VH and / or VL), for example, by mutating one or more CDR regions and / or one or more FR regions.

[0115] In some embodiments, the anti-ILT7 antibody variable regions of the present application are engineered by CDR grafting (see, e.g., Riechmann et al., Nature 1998, 332:323-327; Jones et al., Nature 1986, 321 :522-525; Queen et al., Proc Natl Acad Sci USA 1989, 86:10029-10033. See also US5225539, US5530101, US5585089, US5693762, and US6180370).

[0116] In some embodiments, the present application relates to an isolated anti-ILT7 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequences of HCDR1, HCDR2, and HCDR3 as described herein, and / or a light chain variable region comprising the amino acid sequences of LCDR1, LCDR2, and LCDR3 as described herein. Although the antibodies or antigen-binding fragments thereof comprise the CDR sequences of the antibody VH and VL of the present application, they can comprise different FR sequences.

[0117] Such FR sequences can be obtained from a public database of DNA or from a reference disclosing germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available at www.mrc-cpe.cam.ac.uk / vbase); and in Kabat et al., Sequences of Proteins of Immunological Interest, 1991 Fifth Edition, NIH Publication No. 91-3242; Tomlinson et al., J Mol Biol 1992, 227:776-798; and Cox et al., Eur J Immunol 1994, 24:827-836; the contents of each of which are expressly incorporated herein by reference. As another example, germline DNA sequences of heavy and light chain variable region genes of human antibodies can be found in the IMGT database. For example, the following heavy chain germline sequence found in human immunoglobulins: IGHV1-3*01 can be obtained by IMGT accession number. As another example, the following light chain germline sequence found in human immunoglobulins: IGKV1-NL1*01 can be obtained by IMGT accession number.

[0118] Comparison of antibody amino acid sequences to a compiled protein sequence database can be performed using one of the sequence similarity searching methods known to those skilled in the art, referred to as Gapped BLAST (e.g., Altschul et al., Nucleic Acids Res 1997, 25:3389-3402).

[0119] The FR sequences used for the anti-ILT7 antibodies of the present application are structurally similar (or have a higher degree of homology) to the FR sequences of the murine parent antibodies of the present application. In some embodiments, the CDR1 sequence, the CDR2 sequence, and the CDR3 sequence of the VH or VL are grafted onto an antibody FR sequence that has the same or a higher degree of homology to the germline immunoglobulin gene from which it is derived. In a particular embodiment, the CDR sequences of the VH as set forth in SEQ ID NO: 3, 11, or 13 and / or the CDR sequences of the VL as set forth in SEQ ID NO: 4, 12, or 14 are grafted onto a human IgG FR sequence to obtain a humanized antibody that is capable of retaining the antigen binding properties, e.g., binding affinity and / or binding specificity, similar to the parent antibody comprising a VH as set forth in SEQ ID NO: 3 and a VL as set forth in SEQ ID NO: 4, or comprising a VH as set forth in SEQ ID NO: 11 and a VL as set forth in SEQ ID NO: 12, or comprising a VH as set forth in SEQ ID NO: 13 and a VL as set forth in SEQ ID NO: 14.

[0120] In addition, the VH and VL sequences (or CDR sequences, or full-length heavy and full-length light chain sequences) of other anti-ILT7 antibodies can also be "mixed and matched" with the VH and VL sequences (or CDR sequences, or full-length heavy and full-length light chain sequences) of the anti-ILT7 antibodies of the present application. Preferably, when the VH and VL chains (or CDRs within these chains, or full-length heavy and full-length light chain sequences) are mixed and matched, the VH sequence from a particular VH / VL pair is replaced with a structurally similar VH sequence. Likewise, preferably, the VL sequence from a particular VH / VL pair is replaced with a structurally similar VL sequence. Likewise, a full-length heavy chain sequence from a particular full-length heavy chain / full-length light chain pair should be replaced with a structurally similar full-length heavy chain sequence. Likewise, a full-length light chain sequence from a particular full-length heavy chain / full-length light chain pair should be replaced with a structurally similar full-length light chain sequence.

[0121] Accordingly, in one embodiment, an anti-ILT7 antibody or antigen-binding fragment thereof of the present application comprises: (a) a heavy chain variable region comprising an amino acid sequence listed in Table 2; and (b) a light chain variable region comprising an amino acid sequence listed in Table 2, or a VL of another anti-ILT7 antibody.

[0122] In one embodiment, an anti-ILT7 antibody or antigen-binding fragment thereof of the present application comprises: (a) a heavy chain variable region comprising a VH of another anti-ILT7 antibody; and (b) a light chain variable region comprising an amino acid sequence listed in Table 2.

[0123] In one embodiment, an anti-ILT7 antibody or antigen-binding fragment thereof of the present application comprises: (a) a heavy chain variable region comprising a VH of another anti-ILT7 antibody; and (b) a light chain variable region comprising an amino acid sequence listed in Table 2.

[0124] In one embodiment, an anti-ILT7 antibody or antigen-binding fragment thereof of the present application comprises: (a) a heavy chain variable region comprising a VH of another anti-ILT7 antibody; and (b) a light chain variable region comprising an amino acid sequence listed in Table 2.

[0125] In one embodiment, an anti-ILT7 antibody or antigen-binding fragment thereof of the present application comprises: (a) a heavy chain variable region comprising a VH of another anti-ILT7 antibody; and (b) a light chain variable region comprising an amino acid sequence listed in Table 2.

[0126] In some embodiments, CDR sequences can be grafted onto FR regions that contain one or more mutations as compared to germline sequences. For example, mutating amino acid residues within FR regions can maintain or enhance the antigen binding ability of an antibody (see, e.g., US5530101, US5585089, US5693762, and US6180370). In some embodiments, grafting CDRs of the parent antibodies described herein onto FR regions that have one or more mutations as compared to germline sequences can improve the degree of humanization of the anti-ILT7 antibodies.

[0127] In some specific embodiments, the FR regions of the anti-ILT7 humanized antibodies of the present application have one or more amino acid mutations to improve the degree of humanization of the antibodies. For example, one or more amino acid mutations can be made to the FR regions of the VH having the amino acid sequence set forth in SEQ ID NO: 39, further, one or more amino acid mutations can be made to HFR1, HFR2, and / or HFR3. For example, one or more amino acid mutations can be made to the glutamine residue at position 6, the proline residue at position 9, the valine residue at position 18, and the serine residue at position 28 of HFR1, the arginine residue at position 9 of HFR2 (corresponding to position 44 of the VH having the amino acid sequence set forth in SEQ ID NO: 39), the valine residue at position 2 (corresponding to position 67 of the VH having the amino acid sequence set forth in SEQ ID NO: 39), the leucine residue at position 4 (corresponding to position 69 of the VH having the amino acid sequence set forth in SEQ ID NO: 39), the lysine residue at position 8 (corresponding to position 73 of the VH having the amino acid sequence set forth in SEQ ID NO: 39), the alanine residue at position 10 (corresponding to position 75 of the VH having the amino acid sequence set forth in SEQ ID NO: 39), and the leucine residue at position 17 (corresponding to position 82 of the VH having the amino acid sequence set forth in SEQ ID NO: 39) of HFR3; and / or one or more amino acid mutations can be made to the FR regions of the VL having the amino acid sequence set forth in SEQ ID NO: 60, further, one or more amino acid mutations can be made to LFR2 and / or LFR3, specifically, one or more amino acid mutations can be made to the serine residue at position 9 (corresponding to position 43 of the VL having the amino acid sequence set forth in SEQ ID NO: 60), the valine residue at position 14 (corresponding to position 48 of the VL having the amino acid sequence set forth in SEQ ID NO: 60) of LFR2, and the tyrosine residue at position 15 (corresponding to position 71 of the VL having the amino acid sequence set forth in SEQ ID NO: 60) of LFR3.

[0128] In one embodiment, the anti-ILT7 humanized antibody of the application has one or more amino acid mutations in the HFR region relative to the VH containing the amino acid sequence of SEQ ID NO: 39, including Q6E, P9A or P9G, V18L, S28T, R44G, V67F, L69I, K73T, A75T, and L82M; and / or one or more amino acid mutations in the LFR region (light chain FR region) relative to the VL containing the amino acid sequence of SEQ ID NO: 60, including S43A, V48I, and Y71A or Y71F. The amino acid mutations (in the HFR region and / or the LFR region) can improve the degree of humanization of the antibody.

[0129] In some embodiments, the CDRs of the VH and / or VL of the anti-ILT7 humanized antibody of the application are mutated for amino acids to improve one or more properties of the antibody, including increasing the degree of humanization, improving binding activity or affinity, increasing expression, or altering the germline sequence of the antibody. Site-directed or PCR-induced mutations can be made, and the effects of the mutations on the antibody's binding ability and / or other functional properties can be evaluated using in vitro or in vivo detection methods known in the art. The mutations can be amino acid substitutions, additions, or deletions, preferably amino acid substitutions. In particular, no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid residues within a heavy chain CDR or a light chain CDR are mutated.

[0130] In some embodiments, the anti-ILT7 humanized antibody of the application has no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid mutations within a heavy chain CDR or a light chain CDR.

[0131] In one embodiment, one or more amino acid mutations are made to the CDRs in the VH having the amino acid sequence of SEQ ID NO: 39, and further, one or more amino acid mutations can be made to the HCDR1 and / or HCDR2. For example, one or more amino acid mutations are made to the asparagine residue at position 3 of HCDR1 (corresponding to position 33 of the VH having the amino acid sequence of SEQ ID NO: 39), the asparagine residue at position 1 of HCDR2 (corresponding to position 50 of the VH having the amino acid sequence of SEQ ID NO: 39), the tyrosine residue at position 6 (corresponding to position 54 of the VH having the amino acid sequence of SEQ ID NO: 39), the serine residue at position 9 (corresponding to position 57 of the VH having the amino acid sequence of SEQ ID NO: 39), the serine residue at position 10 (corresponding to position 58 of the VH having the amino acid sequence of SEQ ID NO: 39), and the phenylalanine residue at position 15 (corresponding to position 63 of the VH having the amino acid sequence of SEQ ID NO: 39). Mutations to the asparagine residue at position 3 of HCDR1 (corresponding to position 33 of the VH having the amino acid sequence of SEQ ID NO: 39) include N33A, N33D, N33E, N33G, N33S, or N33Y, preferably N33Y; mutations to the asparagine residue at position 1 of HCDR2 (corresponding to position 50 of the VH having the amino acid sequence of SEQ ID NO: 39) include N50W, N50R, N50G, or N50Y, preferably N50Y; mutations to the tyrosine residue at position 6 (corresponding to position 54 of the VH having the amino acid sequence of SEQ ID NO: 39) include Y54A, Y54F, Y54G, Y54K, Y54S, or Y54T, preferably Y54F; mutations to the serine residue at position 9 (corresponding to position 57 of the VH having the amino acid sequence of SEQ ID NO: 39) include S57A, S57I, S57K, S57N, or S57T, preferably S57T; mutations to the serine residue at position 10 (corresponding to position 58 of the VH having the amino acid sequence of SEQ ID NO: 39) include S58N, S58K, S58D, or S58G, preferably S58D; mutations to the phenylalanine residue at position 15 (corresponding to position 63 of the VH having the amino acid sequence of SEQ ID NO: 39) include F63V.

[0132] In some embodiments, one or more amino acid mutations are made to the HCDR1 and / or HCDR2 of a VH comprising the amino acid sequence set forth in SEQ ID NO: 39. For example, an amino acid mutation is made to the asparagine residue at position 3 of HCDR1 (corresponding to position 33 of a VH comprising the amino acid sequence set forth in SEQ ID NO: 39), and / or the asparagine residue at position 1 of HCDR2 (corresponding to position 50 of a VH comprising the amino acid sequence set forth in SEQ ID NO: 39), the tyrosine residue at position 6 (corresponding to position 54 of a VH comprising the amino acid sequence set forth in SEQ ID NO: 39), the serine residue at position 9 (corresponding to position 57 of a VH comprising the amino acid sequence set forth in SEQ ID NO: 39), the serine residue at position 10 (corresponding to position 58 of a VH comprising the amino acid sequence set forth in SEQ ID NO: 39). Preferably, the amino acid mutation is selected from one or more of N33Y, N50Y, Y54F, S57T, and S58D.

[0133] In one embodiment, one or more amino acid mutations are made to the CDRs in VL having the amino acid sequence set forth in SEQ ID NO: 60, and further, one or more amino acid mutations can be made to LCDR1, LCDR2, and / or LCDR3. For example, one or more amino acid mutations are made to the glycine residue at position 4 (corresponding to position 27 of VL having the amino acid sequence set forth in SEQ ID NO: 60), the asparagine residue at position 5 (corresponding to position 28 of VL having the amino acid sequence set forth in SEQ ID NO: 60), the histidine residue at position 7 (corresponding to position 30 of VL having the amino acid sequence set forth in SEQ ID NO: 60) of LCDR1, the asparagine residue at position 1 (corresponding to position 50 of VL having the amino acid sequence set forth in SEQ ID NO: 60), the lysine residue at position 3 (corresponding to position 52 of VL having the amino acid sequence set forth in SEQ ID NO: 60), the alanine residue at position 6 (corresponding to position 55 of VL having the amino acid sequence set forth in SEQ ID NO: 60), and the glutamic acid residue at position 7 (corresponding to position 56 of VL having the amino acid sequence set forth in SEQ ID NO: 60) of LCDR2, and the histidine residue at position 1 (corresponding to position 89 of VL having the amino acid sequence set forth in SEQ ID NO: 60) and the histidine residue at position 2 (corresponding to position 90 of VL having the amino acid sequence set forth in SEQ ID NO: 60) of LCDR3.Mutations at LCDR1 position 4 (corresponding to VL position 27 with an amino acid sequence set forth in SEQ ID NO: 60) include G27A, G27K, G27N, G27Q, G27R, G27S, or G27T; at position 5 (corresponding to VL position 28 with an amino acid sequence set forth in SEQ ID NO: 60) include N28G, N28I, or N28S; at position 7 (corresponding to VL position 30 with an amino acid sequence set forth in SEQ ID NO: 60) include H30D, H30G, H30K, H30N, H30R, H30S, H30T, or H30Y, preferably H30G, H30N, H30S, or H30Y; at LCDR2 position 1 (corresponding to VL position 50 with an amino acid sequence set forth in SEQ ID NO: 60) include N50A, N50D, N50E, N50G, N50K, N50Q, N50R, N50S, N50W, or N50Y, preferably N50D, N50E, N50G, N50S, or N50Y; at position 3 (corresponding to VL position 52 with an amino acid sequence set forth in SEQ ID NO: 60) include K52D, K52N, K52S, K52T, or K52Y, preferably K52D, K52N, or K52Y; at position 6 (corresponding to VL position 55 with an amino acid sequence set forth in SEQ ID NO: 60) include A55Q or A55E; at position 7 (corresponding to VL position 56 with an amino acid sequence set forth in SEQ ID NO: 60) include E56S or E56T; at LCDR3 position 1 (corresponding to VL position 89 with an amino acid sequence set forth in SEQ ID NO: 60) include H89M, H89L, H89Q, or H89S, preferably H89Q; at position 2 (corresponding to VL position 90 with an amino acid sequence set forth in SEQ ID NO: 60) include H90Q.

[0134] In some embodiments, one or more amino acid mutations are made to the CDRs in VL having the amino acid sequence set forth in SEQ ID NO: 60, and further, one or more amino acid mutations can be made to LCDR1, LCDR2 and / or LCDR3. For example, one or more amino acid mutations are made to the glycine residue at position 4 (corresponding to position 27 of VL having the amino acid sequence set forth in SEQ ID NO: 60), the asparagine residue at position 5 (corresponding to position 28 of VL having the amino acid sequence set forth in SEQ ID NO: 60), the histidine residue at position 7 (corresponding to position 30 of VL having the amino acid sequence set forth in SEQ ID NO: 60) of LCDR1, the lysine residue at position 3 (corresponding to position 52 of VL having the amino acid sequence set forth in SEQ ID NO: 60), the alanine residue at position 6 (corresponding to position 55 of VL having the amino acid sequence set forth in SEQ ID NO: 60), and the glutamic acid residue at position 7 (corresponding to position 56 of VL having the amino acid sequence set forth in SEQ ID NO: 60) of LCDR2, and the histidine residues at positions 1 (corresponding to position 89 of VL having the amino acid sequence set forth in SEQ ID NO: 60) and 2 (corresponding to position 90 of VL having the amino acid sequence set forth in SEQ ID NO: 60) of LCDR3, which can significantly improve the antigen binding affinity of the antibody. Preferably, the amino acid mutations are selected from one or more of G27Q, N28S, H30G, K52N, A55E, E56S, H89Q and H90Q.

[0135] In some embodiments, the present application makes one or more amino acid mutations to the CDRs of the VH and VL of the above-mentioned anti-ILT7 humanized antibody to improve the degree of humanization, antigen binding activity or affinity of the antibody. Specifically, one or more amino acid mutations are made to the asparagine residue at position 3 of HCDR1 (corresponding to position 33 of the VH having the amino acid sequence set forth in SEQ ID NO: 39), the asparagine residue at position 1 of HCDR2 (corresponding to position 50 of the VH having the amino acid sequence set forth in SEQ ID NO: 39), the tyrosine residue at position 6 (corresponding to position 54 of the VH having the amino acid sequence set forth in SEQ ID NO: 39), the serine residue at position 9 (corresponding to position 57 of the VH having the amino acid sequence set forth in SEQ ID NO: 39), the serine residue at position 10 (corresponding to position 58 of the VH having the amino acid sequence set forth in SEQ ID NO: 39) of the VH having the amino acid sequence set forth in SEQ ID NO: 39; and one or more amino acid mutations are made to the glycine residue at position 4 (corresponding to position 27 of the VL having the amino acid sequence set forth in SEQ ID NO: 60), the asparagine residue at position 5 (corresponding to position 28 of the VL having the amino acid sequence set forth in SEQ ID NO: 60), the histidine residue at position 7 (corresponding to position 30 of the VL having the amino acid sequence set forth in SEQ ID NO: 60) of the LCDR1, the lysine residue at position 3 (corresponding to position 52 of the VL having the amino acid sequence set forth in SEQ ID NO: 60), the alanine residue at position 6 (corresponding to position 55 of the VL having the amino acid sequence set forth in SEQ ID NO: 60), and the glutamic acid residue at position 7 (corresponding to position 56 of the VL having the amino acid sequence set forth in SEQ ID NO: 60) of the LCDR2, the histidine residue at position 1 (corresponding to position 89 of the VL having the amino acid sequence set forth in SEQ ID NO: 60) and position 2 (corresponding to position 90 of the VL having the amino acid sequence set forth in SEQ ID NO: 60) of the LCDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 60.

[0136] In some embodiments, one or more amino acid mutations can also be made to the CDRs and FRs of the variable region of the anti-ILT7 humanized antibody to further improve one or more properties of the antibody (e.g., to improve the degree of humanization, to improve the antigen binding affinity).

[0137] In some embodiments, the 2 CDRs and 3 FRs of the VH (amino acid sequence shown as SEQ ID NO: 39) of the above-mentioned anti-ILT7 humanized antibody are subjected to a plurality of amino acid mutations, preferably the mutation sites include one or more of the following amino acid residues: the serine residue at position 28 of HFR1 of the VH comprising the amino acid sequence shown as SEQ ID NO: 39 (corresponding to the 28thposition of the VH comprising the amino acid sequence shown as SEQ ID NO: 39), the asparagine residue at position 3 of HCDR1 (corresponding to the 33rdposition of the VH comprising the amino acid sequence shown as SEQ ID NO: 39), the arginine residue at position 9 of HFR2 (corresponding to the 44thposition of the VH comprising the amino acid sequence shown as SEQ ID NO: 39), the asparagine residue at position 1 of HCDR2 (corresponding to the 50thposition of the VH comprising the amino acid sequence shown as SEQ ID NO: 39), the tyrosine residue at position 6 (corresponding to the 54thposition of the VH comprising the amino acid sequence shown as SEQ ID NO: 39), the serine residue at position 9 (corresponding to the 57thposition of the VH comprising the amino acid sequence shown as SEQ ID NO: 39), the serine residue at position 10 (corresponding to the 58thposition of the VH comprising the amino acid sequence shown as SEQ ID NO: 39), and the alanine residue at position 10 of HFR3 (corresponding to the 75thposition of the VH comprising the amino acid sequence shown as SEQ ID NO: 39). Preferably, the amino acid mutations are selected from one or more of S28T, N33Y, R44G, N50Y, Y54F, S57T, S58D, A75T.

[0138] In some embodiments, the VL of the above-mentioned anti-ILT7 humanized antibody (the amino acid sequence of which is shown as SEQ ID NO: 60) is subjected to a plurality of amino acid mutations in the 3 CDRs and 2 FRs thereof, and preferred mutation sites include: glycine residue at position 4 of LCDR1 (corresponding to position 27 of the VL having the amino acid sequence shown as SEQ ID NO: 60), asparagine residue at position 5 (corresponding to position 28 of the VL having the amino acid sequence shown as SEQ ID NO: 60), histidine residue at position 7 (corresponding to position 30 of the VL having the amino acid sequence shown as SEQ ID NO: 60), serine residue at position 9 of LFR2 (corresponding to position 43 of the VL having the amino acid sequence shown as SEQ ID NO: 60), valine residue at position 14 (corresponding to position 48 of the VL having the amino acid sequence shown as SEQ ID NO: 60), lysine residue at position 3 of LCDR2 (corresponding to position 52 of the VL having the amino acid sequence shown as SEQ ID NO: 60), alanine residue at position 6 (corresponding to position 55 of the VL having the amino acid sequence shown as SEQ ID NO: 60), and glutamic acid residue at position 7 (corresponding to position 56 of the VL having the amino acid sequence shown as SEQ ID NO: 60), tyrosine residue at position 15 of LFR3 (corresponding to position 71 of the VL having the amino acid sequence shown as SEQ ID NO: 60), histidine residues at positions 1 and 2 of LCDR3 (corresponding to positions 89 and 90 of the VL having the amino acid sequence shown as SEQ ID NO: 60). Preferably, the amino acid mutations are selected from one or more of G27Q, N28S, H30G, S43A, V48I, K52N, A55E, E56S, Y71A, H89Q, and H90Q.

[0139] By subjecting the CDRs and FRs of the variable region of the anti-ILT7 humanized antibody of the present application to the above-mentioned amino acid mutations, the antibody not only has a higher degree of humanization, but also maintains antigen binding affinity and binding activity comparable to the parent antibody.

[0140] In other embodiments, the present application provides an isolated anti-ILT7 antibody or antigen binding fragment thereof capable of specifically binding to amino acid residues 420-446 of the extracellular domain of human ILT7, which comprises: (1) HCDR1 having the amino acid sequence of GYX 15 MI(X 15 = N or Y; shown as SEQ ID NO: 150), preferably, X 15 = Y; (2) HCDR2 having the amino acid sequence of X 16IDPFX 17 GGX 18 X 19 YNQKX 20 KG(X 16 =N, W or Y; X 17 =Y or F; X 18 =S, A, I, K, N or T; X 19 =S, N, K, D or G; X 20 =F or V; as represented by SEQ ID NO: 151) HCDR2, preferably X 16 =Y, X 17 =F, X 18 =T, X 19 =D, X 20 =F; (3) HCDR3 having an amino acid sequence as represented by SEQ ID NO: 25; (4) LCDR1 having an amino acid sequence of RASX 21 X 22 IX 23 NYLA(X 21 =G, A, K, N, Q, R, S or T; X 22 =N, G, I or S; X 23 =H, G, N, S or Y; as represented by SEQ ID NO: 152) HCDR2, preferably X 21 =Q, X 22 =S, X 23 =G; (5) LCDR2 having an amino acid sequence of X 24 AX 25 TLX 26 X 27 (X 24 =N, D, E, G, S or Y; X 25 =K, D, N or Y; X 26 =A, Q or E; X 27 =E, T or S; as represented by SEQ ID NO: 153) HCDR2, preferably X 24 =N, X 25 =S or N, X 26 =E, X 27 =S; (6) LCDR3 having an amino acid sequence of X 28 X 29 YYSTPLT(X 28 =H or Q; X 29 =H or Q; as represented by SEQ ID NO: 154) HCDR2, preferably X 28 =Q, X 29= Q, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively. The anti-ILT7 antibody or antigen-binding fragment thereof has a higher degree of humanization and maintains antigen binding affinity and binding activity comparable to the parent antibody.

[0141] In some specific embodiments, the anti-ILT7 antibody or antigen-binding fragment thereof comprises: a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 15 or 16, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, or 24, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 25; and a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 26, 27, 28, 29, or 30, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 31, 32, 33, 34, 35, or 36, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 37 or 38.

[0142] In some specific embodiments, the anti-ILT7 antibody or antigen-binding fragment thereof comprises: a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 15 or 16, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 17, 18, 19, 20, or 21, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 25; and a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 26, 27, 28, or 29, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 31, 33, or 35, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 37 or 38.

[0143] In some specific embodiments, the anti-ILT7 antibody or antigen-binding fragment thereof comprises: a HCDR1 having an amino acid sequence of SEQ ID NO: 16, a HCDR2 having an amino acid sequence of SEQ ID NO: 23 or 24 (preferably SEQ ID NO: 24), and a HCDR3 having an amino acid sequence of SEQ ID NO: 25; and a LCDR1 having an amino acid sequence of SEQ ID NO: 30, a LCDR2 having an amino acid sequence of SEQ ID NO: 36, and a LCDR3 having an amino acid sequence of SEQ ID NO: 38.

[0144] In one specific embodiment, the anti-ILT7 antibody or antigen-binding fragment thereof of the present application comprises:

[0145] (1) a HCDR1 having an amino acid sequence of SEQ ID NO: 15, a HCDR2 having an amino acid sequence of SEQ ID NO: 17, a HCDR3 having an amino acid sequence of SEQ ID NO: 25, and a LCDR1 having an amino acid sequence of SEQ ID NO: 26, 27, 28, or 29, a LCDR2 having an amino acid sequence of SEQ ID NO: 31, a LCDR3 having an amino acid sequence of SEQ ID NO: 37, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or

[0146] (2) a HCDR1 having an amino acid sequence of SEQ ID NO: 15, a HCDR2 having an amino acid sequence of SEQ ID NO: 17, a HCDR3 having an amino acid sequence of SEQ ID NO: 25, and a LCDR1 having an amino acid sequence of SEQ ID NO: 26, a LCDR2 having an amino acid sequence of SEQ ID NO: 32, 33, 34, or 35, a LCDR3 having an amino acid sequence of SEQ ID NO: 37, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or

[0147] (3) HCDR1 having an amino acid sequence of SEQ ID NO: 15, HCDR2 having an amino acid sequence of SEQ ID NO: 18, 19, 20, 21, or 22, HCDR3 having an amino acid sequence of SEQ ID NO: 25, and LCDR1 having an amino acid sequence of SEQ ID NO: 26, LCDR2 having an amino acid sequence of SEQ ID NO: 31, LCDR3 having an amino acid sequence of SEQ ID NO: 37, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or

[0148] (4) HCDR1 having an amino acid sequence of SEQ ID NO: 15, HCDR2 having an amino acid sequence of SEQ ID NO: 17, HCDR3 having an amino acid sequence of SEQ ID NO: 25, and LCDR1 having an amino acid sequence of SEQ ID NO: 26, LCDR2 having an amino acid sequence of SEQ ID NO: 31, LCDR3 having an amino acid sequence of SEQ ID NO: 38, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or

[0149] (5) a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 16, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 17, a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 25, and a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 26, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 31, a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 37, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or

[0150] (6) a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 16, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 23 or 24, a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 25, and a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 30, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 36, a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 38, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0151] In one embodiment, the anti-ILT7 antibody or antigen-binding fragment thereof of the present application comprises a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 16, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 23 or 24, a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 25, and a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 30, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 36, a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 38, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively. Preferably, the anti-ILT7 antibody or antigen-binding fragment thereof comprises a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 16, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 24, a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 25, and a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 30, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 36, a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 38, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively. The anti-ILT7 antibody or antigen-binding fragment thereof has a binding affinity (KD) of 5 x 10-7M or less to human ILT7. D )<5 x 10 -9 M.

[0152] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment thereof of the present application further comprises a heavy chain variable region (VH) having the HCDR1, HCDR2, and HCDR3, and a light chain variable region (VL) having the LCDR1, LCDR2, and LCDR3.

[0153] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment thereof of the present application comprises a VH and a VL comprising an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) identical to the VH amino acid sequence as set forth in SEQ ID NO: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 and the VL amino acid sequence as set forth in SEQ ID NO: 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72, respectively. In some embodiments, the amino acid sequence that is at least 80% identical compared to the amino acid sequences set forth in SEQ ID NOs: 39-59 and 60-72 has differences in amino acids that are primarily in or all in the FRs. In some preferred embodiments, the anti-ILT7 antibody or antigen-binding fragment thereof of the present application comprises a VH and a VL comprising an amino acid sequence that is at least 80% identical to the VH amino acid sequence as set forth in SEQ ID NO: 39, 43, 44, 45, 46, 47, 48, 49, 52, 53, or 54 and the VL amino acid sequence as set forth in SEQ ID NO: 60, 61, 62, 63, 64, 65, 67, 69, 70, or 71, respectively. In some preferred embodiments, the anti-ILT7 antibody or antigen-binding fragment thereof of the present application comprises a VH and a VL comprising an amino acid sequence that is at least 80% identical to the VH amino acid sequence as set forth in SEQ ID NO: 58 or 59 and the VL amino acid sequence as set forth in SEQ ID NO: 72, respectively.

[0154] In one embodiment, the anti-ILT7 antibody or antigen-binding fragment thereof of the present application comprises: (1) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a VHcomprising the amino acid sequence of SEQ ID NO: 39 and a VLcomprising the amino acid sequence of SEQ ID NO: 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or 71, respectively; or (2) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a VHcomprising the amino acid sequence of SEQ ID NO: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 and a VLcomprising the amino acid sequence of SEQ ID NO: 60, respectively; or (3) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a VHcomprising the amino acid sequence of SEQ ID NO: 56, 57, 58, or 59 and a VLcomprising the amino acid sequence of SEQ ID NO: 72, respectively. Preferably, the anti-ILT7 antibody or antigen-binding fragment thereof of the present application comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a VHcomprising the amino acid sequence of SEQ ID NO: 58 or 59 and a VLcomprising the amino acid sequence of SEQ ID NO: 72, respectively; more preferably, the anti-ILT7 antibody or antigen-binding fragment thereof of the present application comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a VHcomprising the amino acid sequence of SEQ ID NO: 58 and a VLcomprising the amino acid sequence of SEQ ID NO: 72, respectively.

[0155] The anti-ILT7 humanized antibody or antigen-binding fragment thereof of the present application can specifically bind to human ILT7 without cross-binding activity to other members of the human ILT family, including ILT1, ILT2, ILT3, ILT4, ILT5, ILT6, ILT8, ILT11, LIR6 and LIR8; the anti-ILT7 humanized antibody or antigen-binding fragment thereof has a high degree of humanization and a high binding affinity to human ILT7 (K D <5 x 10 -9 M); the anti-ILT7 humanized antibody or antigen-binding fragment thereof can bind to human primary pDC cells.

[0156] 2. Anti-CD89 antibodies and antigen-binding fragments thereof

[0157] In one aspect, the present application provides an isolated anti-CD89 antibody or antigen-binding fragment thereof that can specifically recognize and bind to CD89 extracellular Ig-like domain 2 without blocking the binding of IgA to CD89. The anti-CD89 antibody or antigen-binding fragment thereof of the present application does not significantly activate myeloid cells expressing CD89 in monovalent binding state.

[0158] The anti-CD89 antibody of the present application can also optionally comprise a Fab, Fab', F(ab')2, Fv, scFv, scFv-Fc, single domain antibody (sdAb), or have an IgG type. The anti-CD89 antibody of the present application can be a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody, can be a monoclonal antibody, a polyclonal antibody, a monospecific antibody, a bispecific antibody, a multispecific antibody, and an antibody fragment, as long as the antibody can specifically recognize and bind to CD89 extracellular Ig-like domain 2. In some embodiments, the anti-CD89 antibody is selected from a fully human anti-CD89 antibody or an optimized antibody or antibody fragment thereof.

[0159] In some embodiments, the anti-CD89 antibody or antigen-binding fragment thereof comprises an anti-CD89 scFv molecule, or an anti-CD89 scFv-Fc molecule fused with an IgG Fc fragment. The scFv molecule comprises one heavy chain variable region (VH) and one light chain variable region (VL), wherein each variable region or a portion thereof is derived from the same antibody or different antibodies. The VH-VL interface of the scFv molecule can contain or not contain a disulfide bond. The scFv molecule can be constructed in the order of VH-linker-VL, or VL-linker-VH.

[0160] Generally, scFv molecules without disulfide bond at the VH-VL interface are less stable and more prone to aggregation (Worn and Pluckthun, J Mol Biol 2001, 305:989-1010; Rothlisberger et al., J Mol Biol 2005, 347:773-789). The present application enhances the stability of the scFv molecules, e.g., reduces the occurrence of aggregation, by introducing disulfide bond between the VH and VL domains.

[0161] "Introduced" or "introducing" as used herein refers to a mutation, including substitution, insertion or addition, of an amino acid residue originally or naturally present in a parent polypeptide, e.g., an anti-CD89 scFv molecule of the present application. For example, an amino acid residue originally or naturally present at a particular position in a parent polypeptide, e.g., an anti-CD89 scFv molecule of the present application, is referred to as an "original" or "native" amino acid residue, and the corresponding introduced mutation is a cysteine residue different from the original / native amino acid residue at the same position in the parent polypeptide.

[0162] In some embodiments, the VH-VL interface of the scFv molecule contains a disulfide bond to cross-link the FR region of the VH domain and the FR region of the VL domain, or the CDR region of the VH and the CDR region of the VL, or the CDR region of the VH and the FR region of the VL in the scFv molecule. For example, the disulfide bond is to cross-link the FR2 of the VH and the FR4 of the VL, or to cross-link the FR4 of the VH and the FR2 of the VL, or to cross-link the CDR3 of the VH and the CDR3 of the VL, or to cross-link the CDR2 of the VH and the CDR3 of the VL, or to cross-link the CDR3 of the VH and the CDR1 of the VL, or to cross-link the CDR3 of the VH and the FR2 of the VL. Preferably, the disulfide bond is to cross-link the FR2 of the VH and the FR4 of the VL, or to cross-link the CDR2 of the VH and the CDR3 of the VL, or to cross-link the CDR3 of the VH and the FR2 or CDR3 of the VL, or to cross-link the FR4 of the VH and the FR2 of the VL. In particular embodiments, the disulfide bond contained in the VH-VL interface of the anti-CD89 scFv molecule is formed by introducing at least one cysteine residue at positions 44, 45, 60, 100, 100a, 101, 103, 105 and 106 of the VH domain, and positions 34, 43, 46, 91, 95, 96, 98, 100 and 101 of the VL domain, respectively, in the anti-CD89 scFv molecule, wherein the amino acid residue positions of the VH domain and the VL domain are according to the Kabat numbering. The formation of the disulfide bond can be determined by art-known analytical methods, e.g., by mass spectrometry.

[0163] To mutate the amino acid residues at specific sites in the VH and VL domains of the anti-CD89 scFv molecules to cysteine residues, the coding sequences of the VH and VL domains can be manipulated, for example, the codons encoding the native amino acids are replaced with codons encoding cysteine.

[0164] In one embodiment, a cysteine residue is introduced into the VH and VL domains of the anti-CD89 scFv molecules of the application by way of an amino acid mutation, preferably the amino acid mutation is an amino acid substitution, insertion or addition, for example, a non-cysteine residue can be substituted with a cysteine residue, or a cysteine residue or a polypeptide fragment comprising a cysteine residue is inserted or added at a position adjacent to the position of the non-cysteine residue, the position of substitution or insertion or addition can be determined as such that after the substitution of the non-cysteine residue with a cysteine residue or the insertion or addition of a cysteine residue or a polypeptide fragment comprising a cysteine residue, a disulfide bond can be formed at the interface of the VH and VL domains of the anti-CD89 scFv molecules of the application. To this end, a number of factors need to be considered, for example, the spatial distance between the pair of cysteine residues intended to form a disulfide bond needs to be close enough, and / or the substitution or insertion or addition does not significantly alter the antigen binding property of the anti-CD89 scFv molecules.

[0165] The distance and angle between two amino acid residues to be mutated can be determined by methods known in the art, including but not limited to, distance mapping by photodetection, computer modeling, NMR spectroscopy or X-ray crystallography. In some embodiments, the crystal structure of a target protein (e.g., the VH and VL domains of an anti-CD89 antibody or antigen-binding fragment thereof, which can be a scFv) can be obtained from a public database (e.g., the PDB database) or elucidated using methods such as X-ray crystallography, and then computer software is used to determine the distance and angle between amino acid residues based on the protein crystal structure data. Further, it is desirable to identify amino acid residues in the target protein that are important for stabilizing the VH-VL interface, which can be located anywhere on the VH-VL interface. In some embodiments, the site for introducing a first cysteine residue in the VH domain and the site for introducing a second cysteine residue in the VL domain of the anti-CD89 scFv molecule are able to pair correctly, thereby enabling a stable disulfide bond to form at the VH-VL interface to improve the stability (e.g., thermal stability) of the target protein and reduce the risk of mispairing of the target protein during production. In one embodiment, the sites that are able to pair correctly are located at the edge of the VH-VL interface, and the pairing amino acid residues are in close proximity, e.g., the spatial distance between the pairing amino acid residues is less than 10 angstroms, preferably about 5 angstroms. Once the pairing sites for introducing cysteine residues or the pairing amino acid residues to be mutated are determined, one skilled in the art can mutate the amino acid codons to be mutated to cysteine codons by methods known in the art (e.g., PCR mutagenesis, site-directed mutagenesis or cassette mutagenesis).

[0166] Exemplary introduced cysteine residues are cysteine mutations that substitute an original / natural amino acid residue in the VH domain and / or VL domain, which include combinations of: VH44-VL100 (see, e.g., Reiter et al., Biochemistry 1994, 33:5451-5459; Reiter et al., J Biol Chem 1994, 269:18327-18331; Rajagopal et al., Protein Engineering 1997, 10:1453-1459), VH44-VL101, VH45-VL98, VH60-VL95, VH100-VL91, VH100-VL96, VH101-VL46, VH103-VL43, VH105-VL43 (see, e.g., Brinkmann et al., Proc Natl Acad Sci USA 1993, 90:7538-7542; Jung et al., Proteins 1994, 19:35-47), VH106-VL43, VH100a-VL34, and VH100a-VL91; preferably, the pairing positions of introduced cysteine residues in VH and VL, respectively, include VH44-VL100, VH44-VL101, VH60-VL95, VH100-VL91, VH101-VL46, VH103-VL43, and VH105-VL43.

[0167] The present application introduces cysteine residues in the VH domain and VL domain of an anti-CD89 scFv molecule, respectively, to construct a scFv molecule with a disulfide bond in the VH-VL interface. The effects of the introduction of the disulfide bond are reflected in various aspects, for example, the scFv molecule with a disulfide bond in the VH-VL interface has a higher proportion of monomer components in expression, has better thermal stability, can be constructed into an antigen-binding molecule (e.g., scFv-Fc) with a longer half-life, can be fused with a polypeptide that binds to other target antigens to construct a bispecific or multispecific antigen-binding molecule, and / or can be expressed and assembled into a monospecific or multispecific antigen-binding molecule with specific biological activity.

[0168] In some embodiments, the introduction of a disulfide bond in the VH-VL interface of an anti-CD89 scFv molecule can increase the thermal stability of the molecule. Specifically, compared with an anti-CD89 scFv without a disulfide bond in the VH-VL interface, the anti-CD89 scFv with a disulfide bond in the VH-VL interface has a Tm aggValues increase by about 3°C or more. Thermal stability can be assessed using Differential Scanning Calorimetry (DSC) or Differential Scanning Fluorimetry (DSF). Exemplary disulfide bond containing scFv molecules with increased thermal stability include VH and VL containing cysteine residues in the pairing positions of VH44-VL100, VH44-VL101, VH60-VL95, VH100-VL91, VH101-VL46, VH103-VL43, and VH105-VL43, preferably VH44-VL100.

[0169] In some embodiments, the VH-VL interface of the disulfide bond containing scFv exhibits the ability to maintain a relatively high level of monomer content under a range of test conditions.

[0170] In another aspect, the anti-CD89 antibodies or antigen-binding fragments thereof of the present application include a VH and a VL. In some embodiments, the antibodies of the present application are derived from an anti-CD89 antibody including a VH containing the amino acid sequence of SEQ ID NO: 90 and a VL containing the amino acid sequence of SEQ ID NO: 104 (i.e., the parent antibody Mab 14.1, see WO2002064634, which is incorporated herein by reference), and are capable of specifically recognizing and binding to CD89 extracellular Ig-like domain 2 without blocking IgA binding to CD89.

[0171] In some embodiments, the anti-CD89 antibodies of the present application are derived from an antibody including a VH containing the amino acid sequence of SEQ ID NO: 90:

[0172] In some embodiments, the anti-CD89 antibodies of the present application are derived from an antibody including a VL containing the amino acid sequence of SEQ ID NO: 104:

[0173] In some embodiments, the anti-CD89 antibodies of the present application comprise one or more CDRs in a VH comprising an amino acid sequence as set forth in SEQ ID NO: 90 or an amino acid sequence as set forth in SEQ ID NOs: 73, 74, and 80, or a variant thereof, including a humanized antibody or any other variant described herein. In some embodiments, the anti-CD89 antibodies comprise a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 73, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 74, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 80.

[0174] In some embodiments, the anti-CD89 antibodies of the present application further comprise one or more CDRs in a VL comprising an amino acid sequence as set forth in SEQ ID NO: 104 or an amino acid sequence as set forth in SEQ ID NOs: 83, 84, and 86, or a variant thereof, including a humanized antibody or any other variant described herein. In some embodiments, the anti-CD89 antibodies comprise a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 83, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 84, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 86.

[0175] In some specific embodiments, the anti-CD89 antibodies comprise a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 73, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 74, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 80, and a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 83, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 84, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 86.

[0176] In another aspect, the VH and / or VL of the anti-CD89 antibodies of the present application can be used as starting material for engineering to make antibodies described herein, i.e., optimized antibodies of the parent antibody Mab 14.1, that are more suitable for administration to humans.

[0177] In some embodiments, the anti-CD89 optimized antibodies of the present application comprise antibodies or antigen-binding fragments thereof having a VH and / or VL that has been modified as compared to the VH (amino acid sequence as set forth in SEQ ID NO: 90) and / or VL (amino acid sequence as set forth in SEQ ID NO: 104) of the parent antibody Mab 14.1. Modifications introduced in the parent antibody include introducing amino acid mutations (including amino acid substitutions, deletions, insertions, or any combination thereof) in the heavy chain variable region CDRs, light chain variable region CDRs, and / or FRs of the parent antibody to increase the degree of humanization of the antibody and / or reduce or eliminate the risk of aspartate isomerization and / or asparagine deamidation.

[0178] In some specific embodiments, the anti-CD89 optimized antibodies of the present application have one or more amino acid mutations in the FR regions of the parent antibody Mab 14.1 to increase the degree of humanization of the parent antibody. For example, the FR regions of the VH of Mab 14.1 (amino acid sequence as set forth in SEQ ID NO: 90) are subjected to one or more amino acid mutations, further, amino acid mutations can be introduced in HFR2 and / or HFR3, for example, mutating the aspartic acid residue at position 11 of HFR2 (corresponding to position 46 of the VH having the amino acid sequence as set forth in SEQ ID NO: 90), and / or mutating the valine residue at position 31 of HFR3 (corresponding to position 93 of the VH having the amino acid sequence as set forth in SEQ ID NO: 90).

[0179] In one specific embodiment, the anti-CD89 optimized antibodies of the present application have one or more amino acid mutations in the heavy chain FR regions (HFRs) of the VH of the parent antibody Mab 14.1 (amino acid sequence as set forth in SEQ ID NO: 90), including D46E and / or V93A, which mutations are capable of significantly increasing the degree of humanization of the parent antibody Mab 14.1.

[0180] In some embodiments, the anti-CD89 optimized antibodies of the application have one or more amino acid mutations in the CDR regions (including heavy chain variable region CDRs and / or light chain variable region CDRs) of the parent antibody Mab 14.1 that increase the degree of humanization of the antibody while retaining the antigen binding affinity of the parent antibody. For example, one or more amino acid mutations are made to the CDRs of the VH (amino acid sequence set forth in SEQ ID NO: 90) and / or VL (amino acid sequence set forth in SEQ ID NO: 104) of Mab 14.1, such as an amino acid mutation at position 59 (F59) of the VH comprising the amino acid sequence set forth in SEQ ID NO: 90, and / or at position 56 (G56) of the VL comprising the amino acid sequence set forth in SEQ ID NO: 104; such as an amino acid mutation in the VH amino acid sequence selected from F59Y, and / or an amino acid mutation in the VL amino acid sequence selected from G56S. Further, one or more amino acid mutations can be introduced in HCDR2 and / or LCDR2, such as a mutation at the phenylalanine residue at position 11 of HCDR2 (corresponding to position 59 of the VH comprising the amino acid sequence set forth in SEQ ID NO: 90), and / or at the glycine residue at position 7 of LCDR2 (corresponding to position 56 of the VL comprising the amino acid sequence set forth in SEQ ID NO: 104). The mutation at the phenylalanine residue at position 11 of HCDR2 (corresponding to position 59 of the VH amino acid sequence set forth in SEQ ID NO: 90) comprises F59Y; the mutation at the glycine residue at position 7 of LCDR2 (corresponding to position 56 of the VL comprising the amino acid sequence set forth in SEQ ID NO: 104) comprises G56A, G56S, or G56T, preferably G56S.

[0181] In some embodiments, the anti-CD89 optimized antibodies of the application have one or more amino acid mutations in the CDR regions (including heavy chain variable region CDRs and / or light chain variable region CDRs) of the parent antibody Mab 14.1 that reduce the potential for aspartate isomerization and / or asparagine deamidation. For example, no more than 1, 2, 3, 4, or 5 amino acid mutations, which can be amino acid substitutions, additions, or deletions, preferably amino acid substitutions, are introduced in the heavy chain variable region CDRs or light chain variable region CDRs of the parent antibody Mab 14.1.

[0182] In one embodiment, one or more amino acid mutations are made in the CDR regions of the VH of Mab 14.1 (amino acid sequence shown as SEQ ID NO: 90) to reduce or eliminate the risk of aspartate isomerization of the antibody. In preferred embodiments, the anti-CD89 optimized antibodies of the application do not contain aspartate isomerization sites, which can be eliminated by introducing one or more amino acid mutations in HCDR2 of Mab 14.1, for example, 1, 2, 3, 4, or 5 mutations can be made in the aspartate residue at position 4 (corresponding to position 52a of the VH containing the amino acid sequence shown as SEQ ID NO: 90), the aspartate residue at position 5 (corresponding to position 53 of the VH containing the amino acid sequence shown as SEQ ID NO: 90), the glycine residue at position 6 (corresponding to position 54 of the VH containing the amino acid sequence shown as SEQ ID NO: 90), the aspartate residue at position 13 (corresponding to position 61 of the VH containing the amino acid sequence shown as SEQ ID NO: 90), and / or the serine residue at position 14 (corresponding to position 62 of the VH containing the amino acid sequence shown as SEQ ID NO: 90) of HCDR2 to reduce or eliminate the risk of aspartate isomerization of the parental antibody Mab 14.1 amino acid sequence. Among them, the mutation of the aspartate residue at position 4 (corresponding to position 52a of the VH containing the amino acid sequence shown as SEQ ID NO: 90) of HCDR2 includes D52aG, D52aA, D52aY, or D52aT; the mutation of the aspartate residue at position 5 (corresponding to position 53 of the VH containing the amino acid sequence shown as SEQ ID NO: 90) includes D53H, D53I, D53T, D53Y, D53S, or D53G; the mutation of the glycine residue at position 6 (corresponding to position 54 of the VH containing the amino acid sequence shown as SEQ ID NO: 90) includes G54D, G54A, G54S, or G54T; the mutation of the aspartate residue at position 13 (corresponding to position 61 of the VH containing the amino acid sequence shown as SEQ ID NO: 90) includes D61E, D61A, or D61Q; the mutation of the serine residue at position 14 (corresponding to position 62 of the VH containing the amino acid sequence shown as SEQ ID NO: 90) includes S62K, S62N, S62P, or S62R.

[0183] In some embodiments, one or more amino acid mutations are made to the CDR2 region in the VH of Mab 14.1 (amino acid sequence set forth as SEQ ID NO: 90) to reduce or eliminate the risk of aspartate isomerization for the antibody, for example, mutating the aspartate residue at position 5 (corresponding to position 53 of the VH having the amino acid sequence set forth as SEQ ID NO: 90) and / or the serine residue at position 14 (corresponding to position 62 of the VH having the amino acid sequence set forth as SEQ ID NO: 90) of HCDR2 of Mab 14.1; for example, at least one amino acid mutation selected from D53H, D53I, D53T, D53Y, S62K, S62N, S62P, or S62R.

[0184] In one specific embodiment, one or more amino acid mutations are made to the CDR regions in the VL of Mab 14.1 (amino acid sequence set forth as SEQ ID NO: 104) to reduce or eliminate the risk of deamidation for the antibody. For example, the asparagine residue at position 4 (corresponding to position 92 of the VL having the amino acid sequence set forth as SEQ ID NO: 104) and / or the serine residue at position 5 (corresponding to position 93 of the VL having the amino acid sequence set forth as SEQ ID NO: 104) of LCDR3 can be mutated. Mutations to the asparagine residue at position 4 (corresponding to position 92 of the VL having the amino acid sequence set forth as SEQ ID NO: 104) of LCDR3 include N92A, N92G, N92H, N92L, N92S, N92T, or N92Y; mutations to the serine residue at position 5 (corresponding to position 93 of the VL having the amino acid sequence set forth as SEQ ID NO: 104) include S93D, S93H, S93N, S93Q, S93R, or S93T.

[0185] In some embodiments, multiple amino acid mutations can also be made to the CDR regions (including the heavy chain variable region CDRs and / or the light chain variable region CDRs) and FR regions of Mab 14.1 to further improve one or more properties of the antibody, for example, to increase the degree of humanization, to increase the expression level or monomer purity, and to enhance stability, etc.

[0186] In some embodiments, one CDR region and one or two FR regions of the VH of Mab 14.1 (amino acid sequence shown as SEQ ID NO: 90) are subjected to a plurality of amino acid mutations at positions including: aspartic acid residue at position 5 of HCDR2 (corresponding to position 53 of VH having the amino acid sequence shown as SEQ ID NO: 90), phenylalanine residue at position 11 (corresponding to position 59 of VH having the amino acid sequence shown as SEQ ID NO: 90), serine residue at position 14 (corresponding to position 62 of VH having the amino acid sequence shown as SEQ ID NO: 90), and aspartic acid residue at position 11 of HFR2 (corresponding to position 46 of VH having the amino acid sequence shown as SEQ ID NO: 90), and / or valine residue at position 31 of HFR3 (corresponding to position 93 of VH having the amino acid sequence shown as SEQ ID NO: 90); for example, the amino acid mutations are selected from D53H, D53I, D53T, D53Y, F59Y, S62K, S62N, S62P, S62R, D46E, and / or V93A, preferably D53H, F59Y, S62K, D46E, and / or V93A.

[0187] In some embodiments, two CDR regions of the VL of Mab 14.1 (amino acid sequence shown as SEQ ID NO: 104) are subjected to a plurality of amino acid mutations at positions including: glycine residue at position 7 of LCDR2 (corresponding to position 56 of VL having the amino acid sequence shown as SEQ ID NO: 104), and / or asparagine residue at position 4 of LCDR3 (corresponding to position 92 of VL having the amino acid sequence shown as SEQ ID NO: 104); for example, the amino acid mutations are selected from G56A, G56S, G56T, N92A, N92G, N92H, N92L, N92S, N92T, and / or N92Y, preferably G56S and / or N92Y.

[0188] By subjecting the CDR regions and FR regions of the parent antibody Mab 14.1 to the above-described amino acid mutations, the optimized antibodies not only have an improved degree of humanization, reduced risk of aspartate isomerization and / or asparagine deamidation, but also maintain antigen binding activity comparable to the parent antibody.

[0189] In another aspect, the amino acid sequence numbering of the heavy chain CDR regions, light chain CDR regions, and heavy chain variable region and light chain variable region of the anti-CD89 antibodies of the present application (including the parent antibody Mab 14.1 and the optimized antibodies thereof) are summarized in Table 3, wherein the heavy chain variable region CDRs and light chain variable region CDRs are defined by the Kabat numbering system.

[0190] Table 3. Amino acid sequence ID numbers for CDR regions of anti-CD89 antibodies, and heavy chain variable region and light chain variable region

[0191] In some embodiments, the present application provides an isolated anti-CD89 antibody or antigen-binding fragment thereof, comprising: (1) a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 73; (2) a HCDR2 having an amino acid sequence of VISX1X2X3RNKYX4AX5X6VKG (X1= D, G, A, Y or T; X2= D, H, I, T, Y, S or G; X3= G, A, D, S or T; X4= F or Y; X5= D, E, A or Q; X6= S, K, N, P or R; as set forth in SEQ ID NO: 155), preferably, X1= D, X2= H, X3= G, X4= Y, X5= D, X6= K; (3) a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 80; (4) a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 83; (5) a LCDR2 having an amino acid sequence of GASSLEX7 (X7= G, A, S or T; as set forth in SEQ ID NO: 156), preferably, X7= S; (6) a LCDR3 having an amino acid sequence of QQFX8X9YPFT (X8= N, A, G, H, L, S, T or Y; X9= S, D, H, N, Q, R or T; as set forth in SEQ ID NO: 157), preferably, X8= Y, X9= S, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively.

[0192] In some specific embodiments, the anti-CD89 antibody or antigen-binding fragment thereof of the present application comprises: a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 73, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 74, 75, 76, 77, or 78, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 80, and a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 83, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 84 or 85, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 86 or 87.

[0193] In one specific embodiment, the anti-CD89 antibody or antigen-binding fragment thereof of the present application comprises:

[0194] (1) a HCDR1 having an amino acid sequence of SEQ ID NO: 73, a HCDR2 having an amino acid sequence of SEQ ID NO: 74, 75, 76, or 77, a HCDR3 having an amino acid sequence of SEQ ID NO: 80; and a LCDR1 having an amino acid sequence of SEQ ID NO: 83, a LCDR2 having an amino acid sequence of SEQ ID NO: 84, a LCDR3 having an amino acid sequence of SEQ ID NO: 86, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or

[0195] (2) a HCDR1 having an amino acid sequence of SEQ ID NO: 73, a HCDR2 having an amino acid sequence of SEQ ID NO: 74, a HCDR3 having an amino acid sequence of SEQ ID NO: 80; and a LCDR1 having an amino acid sequence of SEQ ID NO: 83, a LCDR2 having an amino acid sequence of SEQ ID NO: 85, a LCDR3 having an amino acid sequence of SEQ ID NO: 86, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or

[0196] (3) a HCDR1 having an amino acid sequence of SEQ ID NO:73, a HCDR2 having an amino acid sequence of SEQ ID NO:74, a HCDR3 having an amino acid sequence of SEQ ID NO:80; and a LCDR1 having an amino acid sequence of SEQ ID NO:83, a LCDR2 having an amino acid sequence of SEQ ID NO:84, a LCDR3 having an amino acid sequence of SEQ ID NO:87, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or

[0197] (4) a HCDR1 having an amino acid sequence of SEQ ID NO:73, a HCDR2 having an amino acid sequence of SEQ ID NO:78, a HCDR3 having an amino acid sequence of SEQ ID NO:80; and a LCDR1 having an amino acid sequence of SEQ ID NO:83, a LCDR2 having an amino acid sequence of SEQ ID NO:85, a LCDR3 having an amino acid sequence of SEQ ID NO:87, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0198] Preferably, the anti-CD89 antibody or antigen-binding fragment thereof comprises: a HCDR1 having an amino acid sequence of SEQ ID NO: 73, a HCDR2 having an amino acid sequence of SEQ ID NO: 78, a HCDR3 having an amino acid sequence of SEQ ID NO: 80; and a LCDR1 having an amino acid sequence of SEQ ID NO: 83, a LCDR2 having an amino acid sequence of SEQ ID NO: 85, a LCDR3 having an amino acid sequence of SEQ ID NO: 87, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0199] In some embodiments, the anti-CD89 antibody or antigen-binding fragment thereof of the present application further comprises a heavy chain variable region (VH) having the HCDR1, HCDR2, and HCDR3, and a light chain variable region (VL) having the LCDR1, LCDR2, and LCDR3.

[0200] In some embodiments, the anti-CD89 antibody or antigen-binding fragment thereof of the present application comprises: (1) a VH comprising an amino acid sequence of SEQ ID NO: 90, 91, 92, 93, 94, 95, 96, or 97, or an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical thereto, and (2) a VL comprising an amino acid sequence of SEQ ID NO: 104, 105, 106, or 107, or an amino acid sequence that is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical thereto. Preferably, the differences in amino acids between the amino acid sequence of SEQ ID NO: 90, 91, 92, 93, 94, 95, 96, or 97, and an amino acid sequence that is at least 80% identical thereto are primarily or entirely in the FR regions of the heavy chain of the anti-CD89 antibody; the differences in amino acids between the amino acid sequence of SEQ ID NO: 104, 105, 106, or 107, and an amino acid sequence that is at least 80% identical thereto are primarily or entirely in the FR regions of the light chain of the anti-CD89 antibody.

[0201] In some embodiments, the anti-CD89 antibody or antigen-binding fragment thereof of the present application comprises:

[0202] (1) have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence of a VH that has an amino acid sequence of SEQ ID NO: 90 and a VL that has an amino acid sequence of SEQ ID NO: 104, 105, or 106, respectively; or

[0203] (2) have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence of a VH that has an amino acid sequence of SEQ ID NO: 91, 92, 93, 94, or 95 and a VL that has an amino acid sequence of SEQ ID NO: 104, respectively; or

[0204] (3) have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence of a VH that has an amino acid sequence of SEQ ID NO: 96 or 97 and a VL that has an amino acid sequence of SEQ ID NO: 107, respectively.

[0205] Preferably, amino acid differences between an amino acid sequence of SEQ ID NO: 90, 91, 92, 93, 94, 95, 96, or 97 and an amino acid sequence having at least 85% identity thereto are located primarily or entirely in the FR regions of the heavy chain of the anti-CD89 antibody; and amino acid differences between an amino acid sequence of SEQ ID NO: 104, 105, 106, or 107 and an amino acid sequence having at least 85% identity thereto are located primarily or entirely in the FR regions of the light chain of the anti-CD89 antibody.

[0206] In one embodiment, the anti-CD89 antibody or antigen-binding fragment thereof of the present application comprises: an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH comprising the amino acid sequence of SEQ ID NO: 96 or 97 and the VL comprising the amino acid sequence of SEQ ID NO: 107, respectively. Preferably, the antibody or antigen-binding fragment thereof comprises: an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH comprising the amino acid sequence of SEQ ID NO: 97 and the VL comprising the amino acid sequence of SEQ ID NO: 107, respectively. Preferably, the differences in amino acids between the VH comprising the amino acid sequence of SEQ ID NO: 96 or 97 and an amino acid sequence at least 85% identical thereto are mainly or entirely located in the FR regions of the heavy chain of the anti-CD89 antibody; the differences in amino acids between the VL comprising the amino acid sequence of SEQ ID NO: 107 and an amino acid sequence at least 85% identical thereto are mainly or entirely located in the FR regions of the light chain of the anti-CD89 antibody.

[0207] In another aspect, the anti-CD89 antibody or antigen-binding fragment thereof of the present application comprises a scFv molecule with a disulfide bond at the VH-VL interface, the VH and VL of the scFv molecule comprising one or more cysteine mutations, respectively, whereby the VH-VL interface of the scFv molecule comprises one or more pairs of disulfide bonds.

[0208] In some embodiments, the VH-VL interface of an anti-CD89 scFv molecule of the application comprises two or more introduced cysteine residues to form the disulfide bond(s). In some particular embodiments, the VH of the VH-VL interface containing disulfide bond(s) of the scFv molecule has one cysteine mutation introduced at position 44, 45, 60, 100, 100a, 101, 103, 105, or 106 relative to the VH of the parent anti-CD89 scFv, and the VL of the VH-VL interface containing disulfide bond(s) of the scFv molecule has another cysteine mutation introduced at position 34, 43, 46, 91, 95, 96, 98, 100, or 101 relative to the VL of the parent anti-CD89 scFv. The VH and VL of the parent anti-CD89 scFv not containing disulfide bond(s) are selected from the VH and VL of an anti-CD89 antibody or antigen-binding fragment thereof of the application, and the VH and VL of the parent anti-CD89 scFv not containing disulfide bond(s) include: (1) a HCDR1 having an amino acid sequence of SEQ ID NO: 73; (2) a HCDR2 having an amino acid sequence of VISX1X2X3RNKYX4AX5X6VKG (X1= D, G, A, Y, or T; X2= D, H, I, T, Y, S, or G; X3= G, A, D, S, or T; X4= F or Y; X5= D, E, A, or Q; X6= S, K, N, P, or R; as set forth in SEQ ID NO: 155); (3) a HCDR3 having an amino acid sequence of SEQ ID NO: 80; (4) a LCDR1 having an amino acid sequence of SEQ ID NO: 83; (5) a LCDR2 having an amino acid sequence of GASSLEX7 (X7= G, A, S, or T; as set forth in SEQ ID NO: 156); (6) a LCDR3 having an amino acid sequence of QQFX8X9YPFT (X8= N, A, G, H, L, S, T, or Y; X9= S, D, H, N, Q, R, or T; as set forth in SEQ ID NO: 157), or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0209] In one embodiment, the disulfide bond comprised in the VH-VL interface of the anti-CD89 scFv molecule of the application is formed by pairing a first cysteine residue and a second cysteine residue introduced in the VH and VL, respectively, of a parent anti-CD89 scFv molecule which VH-VL interface does not comprise a disulfide bond, the pairing of said first and second cysteine residues being selected from one or more of the following combinations of paired amino acid mutations:

[0210] (1) an amino acid mutation G44C at position 44 of the VH of said parent anti-CD89 scFv molecule to introduce a first cysteine residue; and an amino acid mutation P100C or G101C at position 100 or 101 of the VL of said parent anti-CD89 scFv molecule to introduce a second cysteine residue; and / or

[0211] (2) an amino acid mutation A12C at position 12 of HCDR2, i.e. at the amino acid sequence VISX1X2X3RNKYX4AX5X6VKG (X1= D, G, A, Y or T; X2= D, H, I, T, Y, S or G; X3= G, A, D, S or T; X4= F or Y; X5= D, E, A or Q; X6= S, K, N, P or R; as shown in SEQ ID NO: 155) of the VH of said parent anti-CD89 scFv molecule to introduce a first cysteine residue; and an amino acid mutation P7C at position 7 of LCDR3, i.e. at the amino acid sequence QQFX7X8YPFT (X7= N, A, G, H, L, S, T or Y; X8= S, D, H, N, Q, R or T; as shown in SEQ ID NO: 157) of the VL of said parent anti-CD89 scFv molecule to introduce a second cysteine residue; and / or

[0212] (3) an amino acid mutation S6C at position 6 of HCDR3, i.e. at the amino acid sequence as shown in SEQ ID NO: 80 of the VH of said parent anti-CD89 scFv molecule to introduce a first cysteine residue; and an amino acid mutation F3C at position 3 of LCDR3, i.e. at the amino acid sequence QQFX8X9YPFT (X8= N, A, G, H, L, S, T or Y; X9= S, D, H, N, Q, R or T; as shown in SEQ ID NO: 157) of the VL of said parent anti-CD89 scFv molecule to introduce a second cysteine residue; and / or

[0213] (4) an amino acid mutation D9C at position 101 of the VH of the parent anti-CD89 scFv molecule, i.e., at position 9 of HCDR3 having the amino acid sequence set forth in SEQ ID NO: 80, to introduce a first cysteine residue; and an amino acid mutation K46C at position 46 of the VL of the parent anti-CD89 scFv molecule to introduce a second cysteine residue; and / or

[0214] (5) an amino acid mutation W103C or Q105C at position 103 or 105 of the VH of the parent anti-CD89 scFv molecule to introduce a first cysteine residue; and an amino acid mutation A43C at position 43 of the VL of the parent anti-CD89 scFv molecule to introduce a second cysteine residue.

[0215] In other embodiments, the anti-CD89 scFv molecules of the application having a VH-VL interface containing a disulfide bond comprise a HCDR1, a HCDR2, a HCDR3 of a heavy chain variable region and a LCDR1, a LCDR2, a LCDR3 of a light chain variable region, wherein the HCDR2, the HCDR3 and / or the LCDR3 comprise one or more cysteine residue mutations. The heavy chain variable region and the light chain variable region comprise: (1) a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 73; (2) a HCDR2 having an amino acid sequence of VISX1X2X3RNKYX4X 10 X5X6VKG (X1= D, G, A, Y or T; X2= D, H, I, T, Y, S or G; X3= G, A, D, S or T; X4= F or Y; X5= D, E, A or Q; X6= S, K, N, P or R; X 10 = A or C; as set forth in SEQ ID NO: 158), preferably X1= D, X2= H, X3= G, X4= Y, X5= D, X6= K; (3) a HCDR3 having an amino acid sequence of EGYSGX 11 WFX 12 Y (X 11 = S or C, X 12 = D or C; as set forth in SEQ ID NO: 159); (4) a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 83; (5) a LCDR2 having an amino acid sequence of GASSLEX7 (X7= G, A, S or T; as set forth in SEQ ID NO: 156), preferably X7= S; (6) a LCDR3 having an amino acid sequence of QQX 13 X8X9YX 14 FT (X8= N, A, G, H, L, S, T or Y; X9= S, D, H, N, Q, R or T; X 13 = F or C; X14 = P or C; the HCDR3 of SEQ ID NO: 160), preferably X8= Y, X9= S, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0216] In some specific embodiments, the anti-CD89 scFv molecule with a disulfide bond in the VH-VL interface comprises: a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 73, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 78 or 79, a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 80, 81, or 82, a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 83, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 85, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 87, 88, or 89.

[0217] In one specific embodiment, the VH-VL interface of the anti-CD89 scFv molecules of the present application comprising a disulfide bond comprises: (1) a HCDR1 having an amino acid sequence of SEQ ID NO: 73, a HCDR2 having an amino acid sequence of SEQ ID NO: 78, a HCDR3 having an amino acid sequence of SEQ ID NO: 80 or 82, and a LCDR1 having an amino acid sequence of SEQ ID NO: 83, a LCDR2 having an amino acid sequence of SEQ ID NO: 85, a LCDR3 having an amino acid sequence of SEQ ID NO: 87, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively; (2) a HCDR1 having an amino acid sequence of SEQ ID NO: 73, a HCDR2 having an amino acid sequence of SEQ ID NO: 79, a HCDR3 having an amino acid sequence of SEQ ID NO: 80, and a LCDR1 having an amino acid sequence of SEQ ID NO: 83, a LCDR2 having an amino acid sequence of SEQ ID NO: 85, a LCDR3 having an amino acid sequence of SEQ ID NO: 88, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively; or (3) a HCDR1 having an amino acid sequence of SEQ ID NO: 73, a HCDR2 having an amino acid sequence of SEQ ID NO: 78, a HCDR3 having an amino acid sequence of SEQ ID NO: 81, and a LCDR1 having an amino acid sequence of SEQ ID NO: 83, a LCDR2 having an amino acid sequence of SEQ ID NO: 85, a LCDR3 having an amino acid sequence of SEQ ID NO: 89, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively.

[0218] In some embodiments, the VH-VL interface containing a disulfide bond of the anti-CD89 scFv molecules of the application further comprises a heavy chain variable region having the HCDR1, HCDR2, and HCDR3 and a light chain variable region having the LCDR1, LCDR2, and LCDR3.

[0219] In some specific embodiments, the VH-VL interface containing a disulfide bond of the anti-CD89 scFv molecules of the application comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO: 98, 99, 100, 101, 102, or 103, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 108, 109, 110, 111, 112, or 113, or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the amino acid sequence of the VH and VL, respectively. In some preferred embodiments, the VH-VL interface containing a disulfide bond of the anti-CD89 scFv comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO: 98, 99, or 102, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 108, 109, or 112, or an amino acid sequence having at least 80% identity to the amino acid sequence of the VH and VL, respectively.

[0220] In one embodiment, the VH-VL interface disulfide bond containing anti-CD89 scFv molecules of the application comprise: (1) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a VH comprising the amino acid sequence of SEQ ID NO: 98 and a VL comprising the amino acid sequence of SEQ ID NO: 108 or 112, respectively; or (2) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a VH comprising the amino acid sequence of SEQ ID NO: 99 or 102 and a VL comprising the amino acid sequence of SEQ ID NO: 109, respectively; or (3) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a VH comprising the amino acid sequence of SEQ ID NO: 100 and a VL comprising the amino acid sequence of SEQ ID NO: 110, respectively; or (4) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a VH comprising the amino acid sequence of SEQ ID NO: 101 and a VL comprising the amino acid sequence of SEQ ID NO: 111, respectively; or (5) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a VH comprising the amino acid sequence of SEQ ID NO: 103 and a VL comprising the amino acid sequence of SEQ ID NO: 113, respectively.

[0221] In some embodiments, the VH-VL interface disulfide bond containing anti-CD89 scFv molecules of the application comprise a linker that fuses or operably links the VH and VL of the VH-VL interface disulfide bond containing anti-CD89 scFv molecules, said linker being of low immunogenicity, preferably a flexible peptide as linker, for example a linker comprising glycine, or glycine and serine.

[0222] As used herein, the term "linker" refers to a structure that links two compounds, such as two polypeptide molecules (including but not limited to unmodified or modified amino acids or amino acid sequences). The linker can be composed of 1 or more linking molecules, or can include a linking molecule and at least one spacer molecule intended to separate the linking molecule and the compound by a specific distance.

[0223] The term "operably linked" refers to the linkage of amino acid sequences, peptides or proteins with different functional properties, such as the linkage of a VH domain to a VL domain by a linker described herein, or the linkage of a scFv to an Fc, or the linkage of a scFv to a Fab or IgG domain.

[0224] In some embodiments, the anti-CD89 scFv molecule is constructed in the order of VH-linker-VL. The linker comprises the amino acid sequence of (GGGGS)n, wherein n is an integer from 1 to 3, preferably n is 3. In one embodiment, the linker comprises the amino acid sequence as set forth in SEQ ID NO: 167. n (i.e., (G4S)n, wherein n is an integer from 1 to 3, preferably n is 2. In one embodiment, the linker comprises the amino acid sequence as set forth in SEQ ID NO: 168. n ) wherein n is an integer from 1 to 3, preferably n is 3. In one embodiment, the linker comprises the amino acid sequence as set forth in SEQ ID NO: 167.

[0225] In some embodiments, the anti-CD89 antibody of the present application comprises an anti-CD89 scFv-Fc fusion protein, which is constructed by fusing or operably linking an anti-CD89 scFv molecule with a disulfide bond-containing interface of VH-VL to an IgG Fc (such as human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc) via a linker. The linker comprises the amino acid sequence of (G4S)n, wherein n is an integer from 1 to 3, preferably n is 2. In one embodiment, the linker comprises the amino acid sequence as set forth in SEQ ID NO: 168. n ) wherein n is an integer from 1 to 3, preferably n is 2. In one embodiment, the linker comprises the amino acid sequence as set forth in SEQ ID NO: 168.

[0226] The anti-CD89 antibody or antigen-binding fragment thereof of the present application has a scFv form, in particular, the anti-CD89 antibody or antigen-binding fragment thereof is an anti-CD89 scFv molecule with a disulfide bond-containing interface of VH-VL, which significantly improves the stability of the antibody molecule while maintaining the binding specificity and binding activity of the parent anti-CD89 scFv molecule.

[0227] 3. Myeloid engager antibodies targeting ILT7 and CD89

[0228] In one aspect, the present application provides a myeloid engager antibody targeting ILT7 and CD89, exemplary myeloid engager antibody is an anti-CD89 / ILT7 bispecific antibody, which is constructed based on an anti-CD89 monospecific antibody or antigen binding fragment thereof and an anti-ILT7 monospecific antibody or antigen binding fragment thereof described in the present application, comprising a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain specifically binds to CD89 and the second antigen binding domain specifically binds to amino acid residues 420-446 of the extracellular domain of human ILT7.

[0229] The formats of the anti-CD89 / ILT7 bispecific antibodies of the present application include, but are not limited to, BiTE or Diabody-based bispecific formats, (scFv)2-Fc, scFv-Fab, scFv-IgG or IgG-scFv fusion proteins, DVD-Ig, Quadroma, Knob-into-hole, Common Light Chain, CrossMab, CrossFab, SEEDbody, Leucine Zipper, Duobody, IgG1 / IgG2, Dual-acting Fab (DAF)-IgG, and Mab 2 The bispecific formats (see, e.g., Klein et al., mAbs 2012, 4:653-663 and references cited therein) can also be symmetric or asymmetric bispecific formats.

[0230] The anti-CD89 / ILT7 bispecific antibody adopts a structure of asymmetric heterodimer 2+1 scFv-IgG or scFv-Fab-Fc:Fab-Fc, which comprises two different subunits (A and B), each of which comprises a constant region comprising a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region comprises a native or mutant protein form of a human IgG heavy chain constant region, and also comprises a polypeptide truncated form containing a hinge region that promotes dimer formation. In certain embodiments, the Fc region comprises antibody CH2 and CH3 domains. The fusion protein comprising the Fc portion can be purified by a Protein A or Protein G affinity chromatography column, and can extend the plasma / serum half-life. The preferred Fc region is derived from human IgG, including IgG1, IgG2, IgG3 and IgG4. In this context, the position of a specific amino acid residue of the Fc region is determined according to the EU numbering system, for example, positions 99-330 of the human IgG1 heavy chain constant region amino acid sequence shown in SEQ ID NO: 163. The light chain constant region is a human lambda or kappa light chain constant region, preferably a human kappa light chain constant region (comprising an amino acid sequence shown in SEQ ID NO: 166). In some embodiments, subunit A is a bivalent scFv-Fab-Fc form, and subunit B is a monovalent Fab-Fc form; the scFv portion specifically binds to the CD89 extracellular Ig-like domain 2, which can or can not contain a disulfide bond between the VH domain and the VL domain of the scFv; the Fab-Fc portion specifically binds to the amino acid residues 420-446 of the extracellular domain of ILT7. The anti-CD89 / ILT7 bispecific antibody of the present application can specifically bind to ILT7, retaining the antigen binding affinity (K D value < 5 x 10 -9 M) or binding activity of the corresponding anti-ILT7 antibody or antigen binding fragment thereof, and the bispecific antibody has no cross-binding activity with other members of the ILT family, including ILT1, ILT2, ILT3, ILT4, ILT5, ILT6, ILT8, ILT11, LIR6 and LIR8. At the same time, the anti-CD89 / ILT7 bispecific antibody also retains the antigen binding affinity (K D value < 5 x 10 -8 M, < 1 x 10 -9 M, or < 5 x 10 -10 M) or binding activity of the corresponding anti-CD89 antibody or antigen binding fragment thereof.

[0231] The anti-CD89 / ILT7 bispecific antibody is a non-symmetrical trivalent molecule, i.e., the bispecific antibody binds to CD89 on the surface of myeloid cells in a monovalent manner (i.e., one bispecific antibody molecule binds to one CD89 molecule), and binds to ILT7 on the surface of target cells (e.g., pDC) in a bivalent manner (i.e., one bispecific antibody molecule can bind to one or two ILT7 molecules), and thus the anti-CD89 / ILT7 bispecific antibody can cross-link CD89-expressing myeloid cells to ILT7-expressing target cells (e.g., pDC), thereby activating the CD89-expressing myeloid cells and mediating their directed killing of ILT7-expressing target cells (e.g., pDC), so as to significantly inhibit the secretion of relevant cytokines (e.g., IFN-I, such as IFN-α, secreted by pDC). Since the activation of myeloid cells must be achieved through cell cross-linking, the bispecific antibody can only activate CD89-expressing myeloid cells and further mediate the effector function of myeloid cells (e.g., ADCC / ADCP effect) in the presence of ILT7-expressing target cells (e.g., pDC), i.e., killing / clearance of target cells. The CD89-expressing myeloid cells include neutrophils, eosinophils, macrophages, and monocytes. Thus, the anti-CD89 / ILT7 bispecific antibody of the present application effectively avoids the non-directed activation and killing reaction of myeloid cells in the absence of target cells. In some embodiments, it is found that the anti-CD89 / ILT7 bispecific antibody can effectively activate and mediate the killing of ILT7-expressing target cells (e.g., pDC cells, 293T engineered cells expressing ILT7) by primary human neutrophils, but has no killing effect on cells that do not express ILT7 (e.g., wild-type 293T cells).

[0232] Since the anti-CD89 / ILT7 bispecific antibody can direct the clearance of ILT7-expressing pDC cells, it can significantly inhibit the production of IFN-I and other pDC-derived proinflammatory mediators, thereby possibly effectively improving the symptoms of pDC-related diseases, for example, improving the symptoms of SLE patients (including the degree of skin damage and flare rate). In some embodiments, the anti-CD89 / ILT7 bispecific antibody can effectively mediate the killing of ILT7-expressing target cells (e.g., pDC cells) by CD89-expressing myeloid cells (e.g., neutrophils and macrophages), and effectively inhibit the secretion of IFN-α by pDC cells activated by TLR9 agonists (e.g., CpG oligodeoxynucleotides).

[0233] Since the immune effector cells activated by the anti-CD89 / ILT7 bispecific antibody do not include T cells, it does not trigger the release of a large amount of cytokines, thereby having a very low risk of directly triggering a cytokine storm. Thus, the anti-CD89 / ILT7 bispecific antibody of the present application has higher safety in clinical application. In some embodiments, by detecting whether the anti-CD89 / ILT7 bispecific antibody induces a large amount of cytokine release from myeloid cells by peripheral blood from healthy people and SLE patients, the results show that the bispecific antibody does not induce a large amount of cytokine production or secretion from myeloid cells.

[0234] Since the asymmetric heterodimeric bispecific antibody is prone to heavy chain and / or light chain mispairing (e.g., heavy chain / heavy chain mispairing, light chain / light chain mispairing) during production preparation, in order to facilitate the formation of heterodimers and / or facilitate the separation of heterodimers and homodimers by downstream purification processes, the heavy chain constant region of the bispecific antibody is subjected to "knobs and holes" mutations (see, e.g., WO1996027011; Ridgway et al., Protein Engineering 1996, 9:617-621; Atwell et al., J Mol Biol 1997, 270:26-35) and / or "pI mutations", so that the bispecific antibody is prone to form a higher proportion of heterodimers, and its by-products (such as homodimers) are prone to be effectively separated by downstream purification processes, ensuring the uniformity and purity of the final product.

[0235] The "knobs and holes" mutations are generated by substituting an amino acid residue with a larger side chain volume for an amino acid residue with a smaller side chain volume in the CH3 domain of one heavy chain constant region of the bispecific antibody to form a knob, while substituting an amino acid residue with a smaller side chain volume for an amino acid residue with a larger side chain volume in the CH3 domain of the other heavy chain constant region to form a hole, so that the knob is configured in the hole to facilitate the pairing of heterodimeric protein / heterodimeric bispecific antibody heavy chains to form heterodimers. Among them, the amino acid residue with a larger side chain volume is selected from arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W), and the amino acid residue with a smaller side chain volume is selected from alanine (A), serine (S), threonine (T) and valine (V).

[0236] The "pi mutation" is the introduction of an amino acid mutation in the constant region of one or both heavy chains of the bispecific antibody that differs in charge properties from the original amino acid residue, such that each heavy chain has a different pi value, thereby facilitating separation of the heterodimer (A-B) and homodimers (A-A and B-B) having different pi values by ion exchange chromatography. In some embodiments, an amino acid mutation having different charge properties can be introduced into the heavy chain constant region of one (A or B) or both subunits (A and B), whereby the pi of one subunit (e.g., subunit A) is changed, or the pi of both subunits is changed. The pi mutation can be made, for example, by substitution of a positively or negatively charged amino acid residue with an amino acid residue of opposite charge (e.g., aspartic acid or glutamic acid is substituted for lysine or arginine), or substitution of a neutral amino acid residue with a charged amino acid residue (e.g., glycine is substituted for glutamic acid). Following the pi mutation, separation of the heterodimer (A-B) from the homodimers (A-A and B-B) by ion exchange chromatography can be achieved.

[0237] Due to the introduction of "knob and hole" mutations in the heavy chain CH3 region of the bispecific antibody, the resulting homodimers are mainly "hole-hole" dimers. To prevent the formation of bivalent anti-CD89 homodimeric antibodies (e.g., "hole-hole" homodimers of anti-CD89), the present application introduces a "knob" mutation in the Fc region of subunit A (scFv-Fab-Fc) containing the anti-CD89 scFv and a "hole" mutation in the Fc region of subunit B (Fab-Fc). Further, to enable efficient separation of the heterodimer from the "hole-hole" homodimer by ion exchange chromatography, a "pl mutation" can also be introduced in one or both of the heavy chain constant regions of the bispecific antibody, such that the pi difference between the two subunits (A and B) is significantly increased, thereby increasing the pi difference between the heterodimer (A-B) and the homodimer ("hole-hole" or B-B). Preferably, different "pl mutations" are introduced in the two heavy chain constant regions of the bispecific antibody in order to achieve the goal of efficient separation of the homodimer by ion exchange chromatography. In addition, the introduction of a "pl mutation" in the heavy chain constant region of the heterodimer bispecific antibody is also beneficial for quality control of the bispecific antibody, e.g., the residual homodimer in the final heterodimer product can be effectively monitored using high performance liquid ion exchange chromatography. The specific site for the introduction of the "pl mutation" depends on various factors, including the location in the domain of the heterodimer bispecific antibody, functional role, and potential immunogenicity, etc. The "pl mutation" does not have a significant impact on the biological properties of the bispecific antibody. In some embodiments, the introduction of a "pl mutation" in the heavy chain constant region of the anti-CD89 / ILT7 bispecific antibody results in an increased pi difference between the heterodimer and the homodimer, which can be efficiently separated by ion exchange chromatography, and thus the purity of the monomer (i.e., the heterodimer) of the bispecific antibody obtained after isolation and purification is extremely high (up to nearly 100%), and the content of the homodimer is extremely low (not detected).

[0238] By introducing L234F / L235E / P331S mutations (EU Numbering) in the Fc region, the anti-CD89 / ILT7 bispecific antibody has a significantly reduced Fc receptor binding affinity, and thus the Fc effector function (including ADCC and CDC activity) of the bispecific antibody has been substantially eliminated, and other types of immune cells (e.g., T cells, NK cells) cannot be activated. Thus, the risk of toxic side effects caused by antibody Fc effector function is greatly reduced.

[0239] The anti-CD89 / ILT7 bispecific antibody has the characteristics of high stability (e.g., thermal stability) and high monomer content (about 98% or more). In some embodiments, repeated freeze-thaw experiments (e.g., 5 times) and high-temperature accelerated degradation experiments (e.g., placed at 37°C for 7 days) show that the anti-CD89 / ILT7 bispecific antibody produces less than 1% of new aggregates.

[0240] In another aspect, the first and second antigen binding domains of the anti-CD89 / ILT7 bispecific antibody of the present application can be derived from the anti-CD89 antibody or antigen binding fragment thereof and the anti-ILT7 antibody or antigen binding fragment thereof described in the present application, and can also be derived from other anti-CD89 antibody or antigen binding fragment thereof and / or anti-ILT7 antibody or antigen binding fragment thereof known at present (e.g., VIB7734 antibody or antigen binding fragment thereof).

[0241] The exemplary anti-CD89 / ILT7 bispecific antibody of the present application can be constructed based on the anti-CD89 antibody or antigen binding fragment thereof that specifically binds to the extracellular Ig-like domain 2 of CD89 shown in Table 3 and the anti-ILT7 antibody or antigen binding fragment thereof that specifically binds to the extracellular domain of ILT7 shown in Table 2. The exemplary anti-CD89 / ILT7 bispecific antibody of the present application contains two different antigen binding domains, wherein the first antigen binding domain comprises any of at least one CDR region and / or any of the variable regions of the anti-CD89 antibody or antigen binding fragment thereof shown in Table 3, and the second antigen binding domain comprises any of at least one CDR region and / or any of the variable regions of the anti-ILT7 antibody or antigen binding fragment thereof shown in Table 2.

[0242] In some embodiments, the first or second antigen binding domain of the anti-CD89 / ILT7 bispecific antibody comprises the heavy chain variable region CDRs and / or the light chain variable region CDRs of an anti-CD89 antibody or antigen-binding fragment thereof or an anti-ILT7 antibody or antigen-binding fragment thereof, the heavy chain variable region CDRs of the first antigen binding domain comprise the amino acid sequences of any one, two, or three of the heavy chain variable region CDRs of an anti-CD89 antibody or antigen-binding fragment thereof listed in Table 3, or amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively, preferably the heavy chain variable region CDRs of the first antigen binding domain comprise the amino acid sequences of any one, two, or three of the heavy chain variable region CDRs of a VH-VL interface disulfide containing anti-CD89 scFv molecule listed in Table 3, or amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively; the light chain variable region CDRs of the first antigen binding domain comprise the amino acid sequences of any one, two, or three of the light chain variable region CDRs of an anti-CD89 antibody or antigen-binding fragment thereof listed in Table 3, or amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively, preferably the light chain variable region CDRs of the first antigen binding domain comprise the amino acid sequences of any one, two, or three of the light chain variable region CDRs of a VH-VL interface disulfide containing anti-CD89 scFv molecule listed in Table 3, or amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively.the heavy chain variable region CDRs of any one, two, or three of the anti-ILT7 antibodies or antigen binding fragments thereof listed in Table 2, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of HCDR1, HCDR2, and HCDR3, respectively; the light chain variable region CDRs of the second antigen binding domain comprise the amino acid sequences of any one, two, or three of the light chain variable region CDRs of the anti-ILT7 antibodies or antigen binding fragments thereof listed in Table 3, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of LCDR1, LCDR2, and LCDR3, respectively.

[0243] In some embodiments, the first antigen binding domain of the anti-CD89 / ILT7 bispecific antibody comprises the heavy chain variable region CDRs and the light chain variable region CDRs of the VHand VLinterface disulfide-containing anti-CD89 scFv molecules listed in Table 3. Specifically, the first antigen binding domain of the bispecific antibody comprises: an HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 73, an HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 78 or 79, an HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 80, 81, or 82, and an LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 83, an LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 85, and an LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 87, 88, or 89, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0244] In one embodiment, the first antigen binding domain of the anti-CD89 / ILT7 bispecific antibody comprises a HCDR1 having an amino acid sequence of SEQ ID NO: 73, a HCDR2 having an amino acid sequence of SEQ ID NO: 78, a HCDR3 having an amino acid sequence of SEQ ID NO: 80 or 81, a LCDR1 having an amino acid sequence of SEQ ID NO: 83, a LCDR2 having an amino acid sequence of SEQ ID NO: 85, and a LCDR3 having an amino acid sequence of SEQ ID NO: 87 or 89, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively. In one embodiment, the first antigen binding domain of the anti-CD89 / ILT7 bispecific antibody comprises a HCDR1 having an amino acid sequence of SEQ ID NO: 73, a HCDR2 having an amino acid sequence of SEQ ID NO: 78, a HCDR3 having an amino acid sequence of SEQ ID NO: 80, a LCDR1 having an amino acid sequence of SEQ ID NO: 83, a LCDR2 having an amino acid sequence of SEQ ID NO: 85, and a LCDR3 having an amino acid sequence of SEQ ID NO: 87.

[0245] In some embodiments, the second antigen binding domain of the anti-CD89 / ILT7 bispecific antibody comprises a HCDR1 having an amino acid sequence of SEQ ID NO: 16, a HCDR2 having an amino acid sequence of SEQ ID NO: 23 or 24, a HCDR3 having an amino acid sequence of SEQ ID NO: 25, a LCDR1 having an amino acid sequence of SEQ ID NO: 30, a LCDR2 having an amino acid sequence of SEQ ID NO: 36, and a LCDR3 having an amino acid sequence of SEQ ID NO: 38, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0246] In one embodiment, the second antigen binding domain of the anti-CD89 / ILT7 bispecific antibody comprises a HCDR1 having an amino acid sequence of SEQ ID NO: 16, a HCDR2 having an amino acid sequence of SEQ ID NO: 24, a HCDR3 having an amino acid sequence of SEQ ID NO: 25, a LCDR1 having an amino acid sequence of SEQ ID NO: 30, a LCDR2 having an amino acid sequence of SEQ ID NO: 36, and a LCDR3 having an amino acid sequence of SEQ ID NO: 38, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0247] In some embodiments, the first or second antigen binding domain of the anti-CD89 / ILT7 bispecific antibody further comprises a heavy chain variable region and / or a light chain variable region, the heavy chain variable region of the first antigen binding domain comprises any of the VH amino acid sequences of the anti-CD89 antibodies or antigen binding fragments thereof listed in Table 3, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; the light chain variable region of the first antigen binding domain comprises any of the VL amino acid sequences of the anti-CD89 antibodies or antigen binding fragments thereof listed in Table 3, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; the heavy chain variable region of the second antigen binding domain comprises any of the VH amino acid sequences of the anti-ILT7 antibodies or antigen binding fragments thereof listed in Table 2, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; the light chain variable region of the second antigen binding domain comprises any of the VL amino acid sequences of the anti-ILT7 antibodies or antigen binding fragments thereof listed in Table 2, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto.

[0248] In some embodiments, the first antigen binding domain of the anti-CD89 / ILT7 bispecific antibody comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO: 97, 98, 99, 100, 101, 102, or 103, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 107, 108, 109, 110, 111, 112, or 113, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the VH and VL, respectively.

[0249] In one embodiment, the first antigen binding domain of the anti-CD89 / ILT7 bispecific antibody comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO: 98 or 101, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 108, 111, or 112, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the VH and VL, respectively. In one embodiment, the first antigen binding domain of the anti-CD89 / ILT7 bispecific antibody comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO: 98, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 108 or 112.

[0250] In some embodiments, the second antigen domain of the anti-CD89 / ILT7 bispecific antibody comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO: 56, 57, 58, or 59, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 72, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the VH and VL, respectively.

[0251] In one embodiment, the second antigen binding domain of the anti-CD89 / ILT7 bispecific antibody comprises a VH comprising an amino acid sequence of SEQ ID NO: 58 or 59, and a VL comprising an amino acid sequence of SEQ ID NO: 72, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the VH and VL, respectively.

[0252] In another aspect, the exemplary anti-CD89 / ILT7 bispecific antibody of the present application is a non-symmetrical heterodimer comprising a constant region, adopting a 2+1 scFv-IgG or scFv-Fab-Fc:Fab-Fc structure, i.e., the bispecific antibody is a trivalent molecule comprising a subunit A and a subunit B, wherein the subunit A is in the form of scFv-Fab-Fc and the subunit B is in the form of Fab-Fc; the scFv of the bispecific antibody as the first antigen binding domain and the Fab-Fc as the second antigen binding domain, whereby the subunit A comprises the first antigen binding domain, a linker, and the second antigen binding domain, and the subunit B comprises the second antigen binding domain; the scFv domain is fused to or operably linked to the Fab-Fc domain via a linker sequence, which not only has low immunogenicity, but also ensures the stability of the bispecific antibody, preferably a flexible peptide as the linker.

[0253] In some embodiments, the first antigen binding domain of the anti-CD89 / ILT7 bispecific antibody specifically binds to CD89 extracellular Ig-like domain 2, and is a scFv domain, which is a scFv molecule with disulfide bond in the VH-VL interface, and the first antigen binding domain is derived from the heavy chain variable region CDRs and light chain variable region CDRs, and / or the heavy chain variable region VH and light chain variable region VL of the anti-CD89 antibody or antigen binding fragment thereof shown in Table 3, wherein the anti-CD89 antibody or antigen binding fragment thereof is a scFv molecule with disulfide bond in the VH-VL interface; the second antigen binding domain of the anti-CD89 / ILT7 bispecific antibody specifically binds to ILT7 extracellular domain 420-446 amino acid residues, and is a Fab-Fc domain, and the second antigen binding domain is derived from the heavy chain variable region CDRs and light chain variable region CDRs, and / or the heavy chain variable region VH and light chain variable region VL of the anti-ILT7 antibody or antigen binding fragment thereof shown in Table 2; the VL domain C-terminus in the scFv domain is fused to or operably linked to the VH domain N-terminus of the Fab-Fc via a linker to construct a trivalent bispecific antibody.

[0254] In some embodiments, the scFv domain is connected to the Fab-Fc domain via a linker, the linker sequence is (G4S) n , n is an integer greater than 0, such as 1-3, preferably the linker sequence is (G4S)2.

[0255] In one embodiment, the anti-CD89 / ILT7 bispecific antibody comprises first and second antigen binding domains, wherein the first antigen binding domain comprises the VH and VL of an anti-CD89 antibody or antigen binding fragment thereof as shown in Table 3, which is a scFv molecule with disulfide bond in the VH-VL interface; and the second antigen binding domain comprises the VH and VL of an anti-ILT7 antibody or antigen binding fragment thereof as shown in Table 2. The first antigen binding domain is a scFv domain, and the VL domain in the scFv domain is C-terminally connected to the N-terminus of the VH domain of the Fab-Fc via a linker [e.g. (G4S) n , n is an integer greater than 0, such as 1-3; preferably (G4S)2]; and the second antigen binding domain is a Fab-Fc domain.

[0256] In some embodiments, the anti-CD89 / ILT7 bispecific antibody of the present application comprises a constant region, which comprises an antibody heavy chain constant region and a light chain constant region. In some embodiments, the heavy chain constant region comprises an engineered human IgG heavy chain constant region, which is engineered to be able to self-assemble into correct heavy chain / heavy chain pairing, the engineering comprises “knobs-into-holes” mutation and / or “pI mutation”, and can further comprise amino acid mutations in the Fc region of the heavy chain to alter its effector functions. Further, the light chain constant region is a human lambda or kappa light chain constant region, preferably a human kappa light chain constant region (comprising the amino acid sequence as shown in SEQ ID NO: 166).

[0257] In some embodiments, the heavy chain CH3 region of the anti-CD89 / ILT7 bispecific antibody comprises “knobs-into-holes” mutations to facilitate the formation of heterodimers. Specifically, the heavy chain CH3 region of subunit A (scFv-Fab-Fc) comprises “knobs” mutations, which comprise T366W; and the heavy chain CH3 region of subunit B (Fab-Fc) comprises “holes” mutations, which comprise T366S, L368A and Y407V.

[0258] In some embodiments, the heavy chain Fc region of the anti-CD89 / ILT7 bispecific antibody further introduces "pI mutations" to facilitate removal of homodimers by downstream purification processes. In order to minimize the immunogenicity risk caused by "pI mutations", the selection of the anti-CD89 / ILT7 bispecific antibody heavy chain constant region pI mutation sites of the present application is based on the isotype differences among human IgG subtypes (i.e., IgG1, IgG2, IgG3 and IgG4). Specifically, the mutations can be selected by aligning the amino acid sequences of human IgG1, IgG2, IgG3 and IgG4 constant regions, mutating the neutral or basic amino acid residues in the human IgG1 constant region to acidic or neutral amino acid residues at the corresponding sites of other human IgG subtypes to reduce the pI value; and / or mutating the neutral or acidic amino acid residues in the human IgG1 constant region to basic or neutral amino acid residues to increase the pI value. For example, one or more of the mutations shown in Table 4 can be selected to change the heavy chain pI value. In some embodiments, the two heavy chain constant regions of the anti-CD89 / ILT7 bispecific antibody of the present application have different types of "pI mutations", for example, a mutation that reduces the pI value is introduced in one heavy chain constant region, and a mutation that increases the pI value is introduced in the other heavy chain constant region, thereby significantly increasing the pI value difference between the heterodimer and homodimer of the bispecific antibody of the present application, facilitating purification separation. The pI value can be calculated theoretically or determined experimentally. In some embodiments, further mutations that increase the pI value are introduced on the basis of "knobs" mutations to one heavy chain constant region of the bispecific antibody, the "pI mutations" include amino acid mutations Q196K and / or N276K; and further mutations that reduce the pI value are introduced on the basis of "holes" mutations to the other heavy chain constant region of the bispecific antibody, the "pI mutations" include one or more of amino acid mutations G137E, G138S, N203D, K274Q and Q419E. In one embodiment, the theoretical pI value of the heterodimer (A-B) of the heavy chain constant region of the bispecific antibody containing "knobs and holes" mutations and "pI mutations" differs from the theoretical pI value of the formed homodimer (B-B, i.e., "hole-hole") by at least 0.5 pH units, preferably the difference is more than 0.8 pH units. In one embodiment, the heterodimer protein of the bispecific antibody and the "hole-hole" homodimer can be effectively separated by ion exchange chromatography (e.g., high performance liquid cation exchange chromatography), i.e., the heterodimer protein and homodimer show two completely separated elution peaks on the chromatogram, and the residual homodimer by-product in the final product of the heterodimer protein can also be effectively monitored.

[0259] Table 4. Amino acid residues of antibody heavy chain hlgGl constant region pi mutation sites

[0260] One function of the antibody Fc region is to generate "effector functions" with the immune system when the antibody binds its target molecule, including generation of antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cellular cytotoxicity (CDC). The Fc region mediates ADCC and ADCP through binding to Fc receptors on the surface of immune cells, including monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, NK cells, and T cells (Raghavan et al., Annu Rev Cell Dev Biol 1996, 12: 181-220; Ghetie et al., Annu Rev Immunol 2000, 18: 739-766; Ravetch et al., Annu Rev Immunol 2001, 19: 275-290), and mediates CDC through binding to proteins of the complement system, such as Clq (Ward et al., Ther Immunol 1995, 2: 77-94).

[0261] In some specific embodiments, the anti-CD89 / ILT7 bispecific antibodies of the present application comprise an engineered IgG Fc region to reduce its effector functions. Exemplary Fc molecules with reduced effector functions include Fc molecules with the following amino acid substitutions:

[0262] N297A or N297Q (IgGl)

[0263] S267E / L328F (IgGl)

[0264] L234A / L235A (IgGl)

[0265] L234F / L235E / P331S (IgGl)

[0266] C220S / C226S / C229S / P238S (IgGl)

[0267] C226S / C229S / E233P / L234V / L235A (IgGl)

[0268] V234A / G237A (IgG2)

[0269] H268Q / V309L / A330S / A331S (IgG2)

[0270] L235A / G237A / E318A (IgG4)

[0271] A preferred engineered Fc region is a human IgGl Fc with L234F / L235E / P331S (EU numbering system) amino acid substitutions, which can reduce Fc binding to one or more FcyRs and Clq (Oganesyan et al., Acta Crystallogr D Biol Crystallogr 2008, 64:700-704; US5624821; US6194551). The FcyR protein family includes: FcyRI (also known as CD64), including isoforms FcyRIa, FcyRIb, and FcyRIc; FcyRII (also known as CD32), including isoforms FcyRIIa, FcyRIIb, and FcyRIIc; and FcyRIII (also known as CD16), including isoforms FcyRIIIa and FcyRIIIb (Jefferis et al., Immunol Lett 2002, 82:57-65). Of these, FcyRI, FcyRIIa, FcyRIIc, and FcyRIIIa can induce ADCC, endocytosis, phagocytosis, and / or cytokine release. Binding properties include, but are not limited to, binding specificity, binding affinity (K D ), and off- and on-rates (k dis and k a ), respectively), any one or more of which can be analyzed by one of skill in the art to determine whether an engineered Fc region has altered ADCC, ADCP, and / or CDC activity.

[0272] In some embodiments, both heavy chain constant regions of the anti-CD89 / ILT7 bispecific antibody comprise Fc effector function-reducing mutations comprising human IgGl Fc with L234F / L235E / P331S (EU numbering system, referred to as TM mutations) amino acid substitutions, which have reduced binding affinity to one or more FcyRs (e.g., FcyRI, FcyRIIa [131H], FcyRIIb, FcyRIIIa [158F], and FcyRIIIa [158V]). In some embodiments, introducing the L234F / L235E / P331S mutations in both heavy chain constant regions of the anti-CD89 / ILT7 bispecific antibody results in the bispecific antibody substantially not binding to FcyRI, FcyRIIa (131H), FcyRIIb, FcyRIIIa (158F), and / or FcyRIIIa (158V). In some embodiments, introducing the L234F / L235E / P331S mutations in both heavy chain constant regions of the anti-CD89 / ILT7 bispecific antibody results in the bispecific antibody substantially not binding to Clq. In some embodiments, introducing the L234F / L235E / P331S mutations in both heavy chain constant regions of the anti-CD89 / ILT7 bispecific antibody does not affect the binding affinity of the bispecific antibody to FcRn. In some embodiments, introducing the L234F / L235E / P331S mutations in both heavy chain constant regions of the anti-CD89 / ILT7 bispecific antibody significantly reduces the ADCC activity of the bispecific antibody. In one embodiment, the bispecific antibody has significantly reduced or abolished ADCC activity compared to an antibody that does not contain the amino acid mutations in the Fc region. In one embodiment, the bispecific antibody exhibits reduced ADCC activity to the extent that it is not detectable.

[0273] In another aspect, the anti-CD89 / ILT7 bispecific antibody of the present application comprises: a first polypeptide chain, a second polypeptide chain, and two identical third polypeptide chains, wherein the first polypeptide chain comprises, from N-terminus to C-terminus, an anti-CD89 scFv molecule, a heavy chain variable region of an anti-ILT7 antibody, and a first heavy chain constant region, the second polypeptide chain comprises, from N-terminus to C-terminus, the heavy chain variable region of the anti-ILT7 antibody and a second heavy chain constant region, and the third polypeptide chain comprises, from N-terminus to C-terminus, a light chain variable region and a light chain constant region of the anti-ILT7 antibody.

[0274] In some embodiments, the anti-CD89 scFv molecule can or can not contain a disulfide bond between the VH and VL domains thereof, preferably it contains a disulfide bond. In some embodiments, the anti-CD89 scFv molecule comprises a HCDR1 having an amino acid sequence of SEQ ID NO: 73, a HCDR2 having an amino acid sequence of SEQ ID NO: 78, and a HCDR3 having an amino acid sequence of SEQ ID NO: 80 or 81 (preferably SEQ ID NO: 80), and a LCDR1 having an amino acid sequence of SEQ ID NO: 83, a LCDR2 having an amino acid sequence of SEQ ID NO: 85, and a LCDR3 having an amino acid sequence of SEQ ID NO: 87 or 89 (preferably SEQ ID NO: 87), or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the HCDR1, HCDR2 and HCDR3, LCDR1, LCDR2 and LCDR3, respectively.

[0275] In some embodiments, the anti-CD89 scFv molecule comprises a VH comprising an amino acid sequence of SEQ ID NO: 98 or 101, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; and a VL comprising an amino acid sequence of SEQ ID NO: 108, 111, or 112, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto.

[0276] In some embodiments, the anti-CD89 scFv molecule further comprises a linker for linking the VH and VL domains thereof. In some embodiments, the linker comprises (G4S) n , wherein n is an integer from 1 to 3, preferably n is 3. In some embodiments, the linker comprises an amino acid sequence of SEQ ID NO: 167.

[0277] In some embodiments, the heavy chain variable region of the anti-ILT7 antibody comprises a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 16, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 24, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 25, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the HCDR1, HCDR2, and HCDR3, respectively.

[0278] In some embodiments, the heavy chain variable region of the anti-ILT7 antibody comprises an amino acid sequence as set forth in SEQ ID NO: 58 or 59.

[0279] In some embodiments, the first heavy chain constant region and the second heavy chain constant region can be the same or different. In some embodiments, the first heavy chain constant region is a native or mutein form of a human IgGl heavy chain constant region. In some embodiments, the first heavy chain constant region is a mutein form of a human IgGl heavy chain constant region having the mutations that increase the pi value, the TM mutations, and the "knobs" mutations. In some embodiments, the first heavy chain constant region has an amino acid sequence as set forth in SEQ ID NO: 165.

[0280] In some embodiments, the first polypeptide chain further comprises a linker for linking the anti-CD89 scFv molecule and the anti-ILT7 antibody. In some embodiments, the linker comprises (G4S)n, wherein n is an integer from 1 to 3, preferably n is 2. In some embodiments, the linker comprises an amino acid sequence as set forth in SEQ ID NO: 168. n

[0281] In some embodiments, the second heavy chain constant region is a native or mutein form of a human IgGl heavy chain constant region. In some embodiments, the second heavy chain constant region is a mutein form of a human IgGl heavy chain constant region having the mutations that decrease the pi value, the TM mutations, and the "pocket" mutations. In some embodiments, the second heavy chain constant region has an amino acid sequence as set forth in SEQ ID NO: 164.

[0282] ​In some embodiments, the light chain variable region of the anti-ILT7 antibody comprises a LCDR1 having an amino acid sequence of SEQ ID NO: 30, a LCDR2 having an amino acid sequence of SEQ ID NO: 36, and a LCDR3 having an amino acid sequence of SEQ ID NO: 38, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the LCDR1, LCDR2, and LCDR3, respectively.

[0283] In some embodiments, the light chain variable region of the anti-ILT7 antibody comprises an amino acid sequence of SEQ ID NO: 72.

[0284] In some embodiments, the light chain constant region is selected from a human kappa light chain constant region or a human lambda light chain constant region, preferably a human kappa light chain constant region (having an amino acid sequence of SEQ ID NO: 166).

[0285] In another aspect, the anti-CD89 / ILT7 bispecific antibody of the present application comprises two different heavy chains (i.e., referred to as "first and second polypeptide chains") and two identical light chains (i.e., referred to as "third polypeptide chain"), wherein the first polypeptide chain and the third polypeptide chain constitute subunit A of the bispecific antibody; the second polypeptide chain and the third polypeptide chain constitute subunit B of the bispecific antibody. The first polypeptide chain comprises VH1-L1-VL1-L2-VH2-CH a , wherein VH1-L1-VL1 represents a first antigen binding domain that specifically binds to extracellular Ig-like domain 2 of CD89, preferably a VH-VL interface containing disulfide-bonded anti-CD89 scFv molecule, VH1 represents a heavy chain variable region of the first antigen binding domain, L1 represents a linker (such as (G4S) n , wherein n is an integer from 1 to 3), VL1 represents a light chain variable region of the first antigen binding domain, L2 represents a linker connecting the first antigen binding domain and the second antigen binding domain (such as (G4S) n , wherein n is an integer from 1 to 3), VH2 represents a heavy chain variable region of a second antigen binding domain that specifically binds to an epitope of amino acid residues 420-446 of extracellular domain of ILT7, CH a represents a heavy chain constant region of subunit A; the second polypeptide chain comprises VH2-CH b , wherein CH brepresents a heavy chain constant region of a subunit B; the third polypeptide chain comprises VL2-CL, wherein VL2 represents a light chain variable region of a second antigen binding domain that specifically binds to an epitope of amino acid residues 420-446 of an ILT7 extracellular domain, and CL represents an IgG light chain constant region.

[0286] In some embodiments, the VH1 comprises: a HCDR1 having an amino acid sequence of SEQ ID NO: 73, a HCDR2 having an amino acid sequence of SEQ ID NO: 78, and a HCDR3 having an amino acid sequence of SEQ ID NO: 80 or 81 (preferably, SEQ ID NO: 80), or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the HCDR1, HCDR2, and HCDR3, respectively; the L1 comprises a linker sequence of SEQ ID NO: 167; the VL1 comprises: a LCDR1 having an amino acid sequence of SEQ ID NO: 83, a LCDR2 having an amino acid sequence of SEQ ID NO: 85, and a LCDR3 having an amino acid sequence of SEQ ID NO: 87 or 89 (preferably, SEQ ID NO: 87), or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the LCDR1, LCDR2, and LCDR3, respectively; the L2 comprises a linker sequence of SEQ ID NO: 168; the VH2 comprises: a HCDR1 having an amino acid sequence of SEQ ID NO: 16, a HCDR2 having an amino acid sequence of SEQ ID NO: 24, and a HCDR3 having an amino acid sequence of SEQ ID NO: 25, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the HCDR1, HCDR2, and HCDR3, respectively; the CH a comprises an engineered heavy chain constant region comprising a human IgGl heavy chain constant region with a "knobs-into-holes" mutation, a mutation to increase the pi value, and a mutation to decrease Fc effector function, the amino acid sequence of which is set forth in SEQ ID NO: 165.

[0287] In some embodiments, the VH1 comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO: 98 or 101, or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; the VL1 comprises a VL comprising an amino acid sequence as set forth in SEQ ID NO: 108, 111, or 112, or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; and the VH2 comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO: 58 or 59, or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto.

[0288] In some embodiments, the VH1 comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO: 98, or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto; and the VL1 comprises a VL comprising an amino acid sequence as set forth in SEQ ID NO: 108 or 112, or an amino acid sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto.

[0289] In some embodiments, the CH b comprises an engineered heavy chain constant region, including a human IgGl heavy chain constant region with a "pocket" mutation, a mutation to lower the pi value, and a mutation to reduce Fc effector function, the amino acid sequence of which is set forth in SEQ ID NO: 164.

[0290] In some embodiments, the VL2 comprises a LCDR1 having an amino acid sequence of SEQ ID NO: 30, a LCDR2 having an amino acid sequence of SEQ ID NO: 36, and a LCDR3 having an amino acid sequence of SEQ ID NO: 38, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the LCDR1, the LCDR2 and the LCDR3, respectively. The CL is selected from a human kappa constant region or a human lambda constant region, preferably a human kappa constant region (e.g., an amino acid sequence of SEQ ID NO: 166).

[0291] In some specific embodiments, the VL2 comprises a VL comprising an amino acid sequence of SEQ ID NO: 72, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto.

[0292] In some specific embodiments, the anti-CD89 / ILT7 bispecific antibody of the present application comprises:

[0293] (1) a first polypeptide chain, the first polypeptide chain being VH1-L1-VL1-L2-VH2-CH a , the VH1 comprising a HCDR1 having an amino acid sequence of SEQ ID NO: 73, a HCDR2 having an amino acid sequence of SEQ ID NO: 78, and a HCDR3 having an amino acid sequence of SEQ ID NO: 80 or 81, L1 comprising a linker having an amino acid sequence of SEQ ID NO: 167, the VL1 comprising a LCDR1 having an amino acid sequence of SEQ ID NO: 83, a LCDR2 having an amino acid sequence of SEQ ID NO: 85, and a LCDR3 having an amino acid sequence of SEQ ID NO: 87 or 89, L2 comprising a linker having an amino acid sequence of SEQ ID NO: 168, the VH2 comprising a HCDR1 having an amino acid sequence of SEQ ID NO: 16, a HCDR2 having an amino acid sequence of SEQ ID NO: 24, and a HCDR3 having an amino acid sequence of SEQ ID NO: 25, the CH a comprising an amino acid sequence of SEQ ID NO: 165;

[0294] (2) one second polypeptide chain, the second polypeptide chain being VH2-CH b , the VH2 comprising a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 16, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 24, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 25, the CH b comprising an amino acid sequence as set forth in SEQ ID NO: 164;

[0295] (3) two third polypeptide chains, the third polypeptide chain being VL2-CL, the VL2 comprising a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 30, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 36, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 38, the CL comprising an amino acid sequence as set forth in SEQ ID NO: 166.

[0296] In some embodiments, the anti-CD89 / ILT7 bispecific antibodies of the present application comprise:

[0297] (1) one first polypeptide chain comprising: a VH1 comprising an amino acid sequence as set forth in SEQ ID NO: 98 or 101, an L1 comprising an amino acid sequence as set forth in SEQ ID NO: 167, a VL1 comprising an amino acid sequence as set forth in SEQ ID NO: 108, 111, or 112, an L2 comprising an amino acid sequence as set forth in SEQ ID NO: 168, a VH2 comprising an amino acid sequence as set forth in SEQ ID NO: 58 or 59, a CH a ;

[0298] (2) one second polypeptide chain comprising: a VH2 comprising an amino acid sequence as set forth in SEQ ID NO: 58 or 59, a CH b ;

[0299] (3) two third polypeptide chains comprising: a VL2 comprising an amino acid sequence as set forth in SEQ ID NO: 72, a CL comprising an amino acid sequence as set forth in SEQ ID NO: 166.

[0300] In some embodiments, the first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 169, 172, 173, 175, or 176, the second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 170 or 174, and the third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 171.

[0301] In some embodiments, the first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 169 or 172, the second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 170, and the third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 171; or the first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 173, 175, or 176, the second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 174, and the third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 171.

[0302] 4. Methods of producing antibodies of the application

[0303] 4.1 Polynucleotides, vectors, and host cells

[0304] In one aspect, the present application provides a nucleic acid encoding an anti-CD89 antibody or antigen-binding fragment thereof, an anti-ILT7 antibody or antigen-binding fragment thereof, and an anti-CD89 / ILT7 bispecific antibody. The present application also includes polynucleotide variants encoding the amino acid sequences described herein.

[0305] A nucleotide sequence corresponding to an amino acid sequence described herein, which is useful as a probe or primer for nucleic acid isolation or for providing a searchable database can be obtained by back translation of the amino acid sequence. Polymerase chain reaction (PCR) can be used to isolate and amplify DNA sequences encoding an anti-CD89 antibody or antigen-binding fragment thereof, an anti-ILT7 antibody or antigen-binding fragment thereof, or an anti-CD89 / ILT7 bispecific antibody described herein. Oligonucleotides defining the desired ends of the DNA fragment assembly are used as 5' and 3' primers. The oligonucleotides can additionally contain recognition sites for restriction endonucleases to facilitate insertion of the amplified DNA fragment assembly into an expression vector. PCR techniques are described in Saiki et al., Science 1988, 239: 487-491; Wu et al., eds., Recombinant DNA Methodology, 1989 Academic Press, pp. 189-196; Innis et al., eds., PCR Protocols: A Guide to Methods and Applications, 1990 Academic Press.

[0306] Nucleic acid molecules of the present application include single- and double- stranded forms of DNA and RNA, as well as the corresponding complementary sequences, including isolated nucleic acid molecules, preferably DNA or RNA that has been isolated at least once in a substantially pure form and in an amount or concentration that permits the component nucleotide sequences to be identified, manipulated, and recovered by standard biochemical methods (e.g., methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 1989 2nd ed., Cold Spring Harbor Laboratory). Preferably, such sequences are provided and / or constructed in the form of open reading frames interrupted by internal non-translated sequences or introns that are not normally found in eukaryotic genes. Sequences of non-translated DNA can be present 5' or 3' to the open reading frame, where the sequences do not interfere with manipulation or expression of the coding region.

[0307] The anti-CD89 antibodies or antigen-binding fragments thereof, anti-ILT7 antibodies or antigen-binding fragments thereof, or anti-CD89 / ILT7 bispecific antibodies described herein can be prepared by mutagenizing nucleotides at specific sites in the DNA encoding the anti-CD89 antibodies or antigen-binding fragments thereof, anti-ILT7 antibodies or antigen-binding fragments thereof, or anti-CD89 / ILT7 bispecific antibodies using PCR or other techniques known to those of ordinary skill in the art to produce DNA encoding variants, followed by expression of the recombinant DNA in cell culture as outlined herein. In addition, the anti-CD89 antibodies or antigen-binding fragments thereof, anti-ILT7 antibodies or antigen-binding fragments thereof, or anti-CD89 / ILT7 bispecific antibodies can also be prepared using established techniques for in vitro synthesis.

[0308] As is known to those of skill in the art, due to the degeneracy of the genetic code, the anti-CD89 antibodies or antigen-binding fragments thereof, anti-ILT7 antibodies or antigen-binding fragments thereof, or anti-CD89 / ILT7 bispecific antibodies described herein can be encoded by a very large number of nucleic acids, each of which is within the scope of the present application and can be made using standard techniques. Thus, given the particular amino acid sequence identified, one of skill in the art can make many different nucleic acids by simply modifying the coding sequence of each, in a way that does not change the amino acid sequence of the anti-CD89 antibodies or antigen-binding fragments thereof, anti-ILT7 antibodies or antigen-binding fragments thereof, or anti-CD89 / ILT7 bispecific antibodies of the present application.

[0309] In another aspect, the present application also provides expression vectors comprising nucleic acids encoding the anti-CD89 antibodies or antigen-binding fragments thereof, anti-ILT7 antibodies or antigen-binding fragments thereof, or anti-CD89 / ILT7 bispecific antibodies herein.

[0310] A nucleic acid encoding an anti-CD89 antibody or antigen-binding fragment thereof, an anti-ILT7 antibody or antigen-binding fragment thereof, or an anti-CD89 / ILT7 bispecific antibody of the present application can be cloned into a suitable vector for introduction into a host cell for expression of the protein of interest. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. The nucleic acid encoding the protein of interest in the vector is operably linked to a promoter.

[0311] As used herein, the term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcriptional regulatory factor binding sites) and another nucleic acid sequence, and thus that the control sequence controls transcription and / or translation of the other nucleic acid sequence.

[0312] Suitable vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 -derived artificial chromosomes (PAC), bacteriophages (such as lambda phage or M13 phage), and animal viruses, etc. The animal virus species used as vectors are retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). The vectors can contain a variety of expression-controlling elements, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vectors can contain a replication initiation site. The vectors can also include components that assist in their entry into cells, including but not limited to viral particles, liposomes, or protein coats.

[0313] In another aspect, the present application also provides a host cell comprising a nucleic acid or an expression vector encoding an anti-CD89 antibody or antigen-binding fragment thereof, an anti-ILT7 antibody or antigen-binding fragment thereof, or an anti-CD89 / ILT7 bispecific antibody of the present application.

[0314] The host cell can be a eukaryotic cell, for example, a mammalian host cell including, but not limited to, an SV40 transformed monkey kidney cell line CV1 line (COS-7, ATCC, CRL-1651), a human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J Gen Virol 1977, 36:59-74), baby hamster kidney cells (BHK-21, ATCC, CCL-10), Chinese hamster ovary cells / DHFR (CHO, Urlaub et al., Proc Natl Acad Sci USA 1980, 77:4216-4220), mouse Sertoli cells (TM4, Mather, Biol Reprod 1980, 23:243-251), monkey kidney cells (CV1, ATCC, CCL-70), African green monkey kidney cells (VERO-76, ATCC, CRL-1587), human cervical carcinoma cells (HELA, ATCC, CCL-2), canine kidney cells (MDCK, ATCC, CCL-34), buffalo rat liver cells (BRL 3A, ATCC, CRL-1442), human lung cells (W138, ATCC, CCL-75), human hepatoma line (Hep G2, ATCC, HB-8065), mouse mammary tumor (MMT 060562, ATCC, CCL-51), TRI cells (Mather et al., Ann NY Acad Sci 1982, 383:44-68), MRC 5 cells, or FS4 cells.

[0315] 4.2 Generation of Anti-ILT7 Antibodies of the Invention

[0316] The anti-ILT7 antibodies provided by the present application can be murine antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies. The method for preparing the monoclonal antibodies of the present application that specifically bind to ILT7 can be any method known in the art (e.g., hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc.).

[0317] In some embodiments, a polypeptide of any fragment of ILT7 amino acid sequence can be selected as an antigen for immunizing mice to obtain mouse mature B cells, and then mouse myeloma cells and mouse B cells are fused in vitro by hybridoma technology (Kozbor et al., J Immunol 1984, 133:3001; Schook, ed., Monoclonal Antibody Production Techniques and Applications, pp. 51-63, 1987 1st ed., Marcel Dekker; Boerner et al., J Immunol 1991, 147:86) to screen hybridoma cells expressing the target antibody, and then subclone to screen monoclonal hybridoma cell strains expressing the target antibody, thereby obtaining a monoclonal antibody against ILT7.

[0318] The present application has screened a series of antigens for immunizing mice, including polypeptides and cells. In some embodiments, the polypeptides include ILT7 extracellular domain full-length polypeptide or partial polypeptide thereof. In order to obtain an antibody specifically binding to ILT7 without cross-binding activity to other members of the ILT family, the present application has found that the amino acid sequence of 420-446 near the membrane of the ILT7 extracellular domain has the lowest homology to other members of the ILT family by aligning the amino acid sequences of the extracellular domains of each member of the human ILT family, and therefore the present application can select this partial polypeptide (such as the amino acid sequence of 420-446 shown in SEQ ID NO: 161) in the ILT7 extracellular domain as the antigen for immunizing mice. The ILT7 extracellular domain full-length polypeptide or partial polypeptide used as an antigen herein can be obtained by genetically engineering the gene encoding the ILT7 extracellular domain full-length or partial polypeptide to express in host cells, or by in vitro polypeptide synthesis. In some embodiments, the present application also selects a cell strain expressing ILT7 as an immunizing antigen, which can be a naturally occurring cell expressing ILT7 or an engineered cell strain. Preferably, the 293T engineered cell strain expressing ILT7.

[0319] In some embodiments, the present application selects one or more antigens for immunizing mice, and preferably a plurality of antigens (including ILT7 extracellular domain 420-446 amino acid sequence polypeptide, ILT7 extracellular domain full-length polypeptide, and 293T engineered cell strain expressing ILT7) for alternately immunizing mice to obtain the best immunization effect. In some specific embodiments, the present application uses methods for measuring antibody titer, including but not limited to ELISA, FACS detection method.

[0320] In some embodiments, the present application provides a method for obtaining hybridoma cells by cell fusion, which includes but is not limited to the HAT (hypoxanthine-aminopterin-thymidine) selection method (Kohler et al., Nature 1975, 256:495-497; Milstein et al., Nature 1977, 266:550-552). The HAT selection method is to fuse a HGPRT-deficient myeloma cell line with mouse B cells, and then to obtain the target hybridoma cells by screening the cells that survive in the culture medium supplemented with aminopterin.

[0321] In some embodiments, the method for cloning hybridoma cells includes but is not limited to the methods known in the art such as methyl cellulose, soft agarose, and limited dilution method (see, for example, Mishell et al., Selected Methods in Cellular Immunology, 1980 First Edition, WH Freeman & Co), preferably the limited dilution method. In some specific embodiments, after screening the hybridoma cells obtained by the above steps, the limited dilution method is used for 2-4 times of subcloning, and the clones stably expressing the target antibody are selected as the hybridoma cell lines for producing anti-ILT7 monoclonal antibodies.

[0322] In some embodiments, the present application uses various methods to detect and screen the hybridoma cell lines for producing the target antibody, which include but are not limited to cell fluorescence detection method, FACS detection method, and ELISA detection method.

[0323] 5. Preparation method

[0324] The present application provides a method for preparing the anti-CD89 antibody or antigen-binding fragment thereof, the anti-ILT7 antibody or antigen-binding fragment thereof, or the anti-CD89 / ILT7 bispecific antibody of the present application using the host cell.

[0325] The method includes transfecting the nucleic acid or expression vector encoding the anti-CD89 antibody or antigen-binding fragment thereof, the anti-ILT7 antibody or antigen-binding fragment thereof, or the anti-CD89 / ILT7 bispecific antibody of the present application into a host cell, and culturing the host cell in a culture medium for a period of time to express the anti-CD89 antibody or antigen-binding fragment thereof, the anti-ILT7 antibody or antigen-binding fragment thereof, or the anti-CD89 / ILT7 bispecific antibody of the present application. Without limitation, commercially available culture media can be used as the culture medium of the present application.

[0326] Preferably, the expressed anti-CD89 antibody or antigen-binding fragment thereof, anti- ILT7 antibody or antigen-binding fragment thereof, or anti-CD89 / ILT7 bispecific antibody can be secreted into the culture medium in which the host cells are grown. The antibody can be recovered from the culture medium using conventional protein purification methods, such as, for example, centrifugation, filtration, or chromatography; affinity chromatography can also be used to purify the antibody; and other purification techniques can also be employed, such as, for example, anion or cation exchange chromatography, hydrophobic interaction chromatography, and hydroxyapatite chromatography.

[0327] 6. An immunoconjugate, chimeric antigen receptor, engineered T cell receptor, or oncolytic virus

[0328] In one aspect, the present application provides an immunoconjugate comprising an anti- ILT7 antibody or antigen-binding fragment thereof herein. The anti-ILT7 antibody or antigen-binding fragment thereof of the present application can be conjugated to a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC). In an ADC, the linker conjugating the antibody and the therapeutic agent can be a cleavable linker (such as a peptidyl linker, a disulfide bond, or a hydrazone linker), or a non-cleavable linker. The ADC can be prepared using methods described in US7087600, US6989452, US7129261, WO02 / 096910, WO07 / 038658, WO07 / 051081, WO07 / 059404, WO08 / 083312, WO08 / 103693, US20060024317, US20060004081, and US20060247295, the disclosures of which are incorporated herein by reference. The antibody described herein can also be conjugated to a radioisotope (e.g., radioactive iodine) to form a cytotoxic radiopharmaceutical.

[0329] In another aspect, the present application also provides a chimeric antigen receptor, engineered T cell receptor, or oncolytic virus comprising an anti-ILT7 antibody or antigen-binding fragment thereof herein.

[0330] 7. A pharmaceutical composition

[0331] The present application provides a pharmaceutical composition comprising an anti- CD89 / ILT7 bispecific antibody described herein, or an immunoconjugate, chimeric antigen receptor, engineered T cell receptor, or oncolytic virus comprising an anti-ILT7 antibody or antigen-binding fragment thereof described herein, and a pharmaceutically acceptable carrier.

[0332] The pharmaceutical compositions can comprise any kind of pharmaceutically acceptable carrier. Carriers that can be used include excipients, surfactants, thickening or emulsifying agents, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coating agents, disintegrants, lubricants, sweeteners, preservatives, isotonic agents or combinations thereof. The selection and use of appropriate carriers is taught, for example, in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 2003, 20thEdition (Lippincott Williams & Wilkins), the disclosure of which is incorporated herein by reference.

[0333] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or topical administration. For example, "parenteral" administration as used herein refers to modes of administration other than enteral and topical administration, including, but not limited to, injection or infusion either subcutaneously, intracutaneously, intraarterially, intrathecally, intracapsularly, intraorbital, intracardiac, intradermally, intraperitoneally, transtracheally, subcutaneously, subcutaneously, intraarticularly, subcapsularly, subarachnoidly, intraspinally, epidurally and sternal. Alternatively, the antibodies described herein can also be administered by non-parenteral routes (e.g., topical, epidermal or mucosal administration routes), for example, intranasally, orally, vaginally, rectally, sublingually or topically. Depending on the route of administration, the active ingredient can be coated in a material to protect it from the action of acids and other natural conditions that can cause it to lose its activity.

[0334] The pharmaceutical compositions can be sterile aqueous solutions or dispersions. They also can be formulated in microemulsions, liposomes, or other ordered structures suitable for high drug concentration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will be determined by the subject and the particular mode of administration, and will generally be the amount of the composition that produces therapeutic effects. Generally, compositions containing from about 0.01% to about 99% of active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30%, are used for pharmaceutical purposes.

[0335] Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single dose can be administered, several divided doses can be administered, or the dose can be proportionally reduced or increased as indicated by the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit contains a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Additionally, the anti-CD89 / ILT7 bispecific antibodies of the present application, or immunoconjugates, chimeric antigen receptors, engineered T cell receptors, or oncolytic viruses comprising the anti-ILT7 antibodies or antigen binding fragments thereof described herein can also be administered as sustained release formulations, which reduce the frequency of administration.

[0336] A "therapeutically effective amount" of an anti-CD89 / ILT7 bispecific antibody of the present application, or immunoconjugates, chimeric antigen receptors, engineered T cell receptors, or oncolytic viruses comprising the anti-ILT7 antibodies or antigen binding fragments thereof, preferably results in a decrease in the severity of the disease symptoms, an increase in the frequency and duration of disease symptom non-progression, or a prevention of physical damage or disability due to the disease affliction. For example, a "therapeutically effective amount" of a subject preferably is capable of inhibiting the progression of the disease by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80%, relative to an untreated subject. A therapeutically effective amount of a therapeutic antibody (including monospecific and bispecific antibodies) can ameliorate at least one symptom of a subject's condition, which subject is typically a human or other mammal. A "therapeutically effective amount" can also vary according to factors such as the dosage form, method of administration, age, body weight, gender, or pathological condition of the patient, diet, time of administration, administration interval, route of administration, excretion rate, and response sensitivity.

[0337] Pharmaceutical compositions can be selected to provide controlled release of the active ingredient, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. See, e.g., Robinson, ed., Sustained and Controlled Release Drug Delivery Systems, 1978 Marcel Dekker.

[0338] The therapeutic pharmaceutical composition can be delivered by a medical device selected from the group consisting of: (1) needle-free hypodermic injection devices (e.g., US 5,399,163; US 5,383,851; US 5,312,335; US 5,064,413; US 4,941,880; US 4,790,824; and US 4,596,556); (2) micro-infusion pumps (US 4,487,603); (3) transdermal devices (US 4,486,194); (4) infusion apparatus (US 4,447,233 and US 4,447,224); and (5) osmotic devices (US 4,439,196 and US 4,475,196); the disclosures of which are incorporated herein by reference.

[0339] In certain embodiments, the anti-CD89 / ILT7 bispecific antibodies described herein, or immunoconjugates, chimeric antigen receptors, engineered T cell receptors, or oncolytic viruses comprising the anti-ILT7 antibodies or antigen binding fragments thereof described herein, can be formulated to ensure biodistribution in vivo. For example, to ensure that the therapeutic antibodies described herein are able to cross the blood-brain barrier, they can be formulated in a liposome, which can additionally comprise a targeting moiety to enhance their selective delivery to particular cells or organs. See, e.g., US 4,522,811; US 5,374,548; US 5,416,016; and US 5,399,331; Ranade, J Clin Pharmacol 1989, 29: 685-694; Umezawa et al., Biochem Biophys Res Commun 1988, 153: 1038-1044; Bloeman et al., FEBS Lett 1995, 357: 140-144; Owais et al., Antimicrob Agents Chemother 1995, 39: 180-184; Briscoe et al., Am J Physiol 1995, 268: L374-380; Schreier et al., J Biol Chem 1994, 269: 9090-9098; Keinanen and Laukkanen, FEBS Lett 1994, 346: 123-126; and Killion and Fidler, Immunomethods 1994, 4: 273-279.

[0340] 8. A kit

[0341] In one aspect, the present application provides a kit comprising an anti-CD89 / ILT7 bispecific antibody described herein, or an immunoconjugate, chimeric antigen receptor, engineered T cell receptor, or oncolytic virus comprising the anti-ILT7 antibody or antigen binding fragment thereof described herein, or a pharmaceutical composition described herein, and optionally at least one additional therapeutic agent. The additional therapeutic agent includes, but is not limited to, a therapeutic agent for a pDC-associated disorder, a tumor therapeutic agent, and an anti-infective therapeutic agent.

[0342] 9. Methods and uses for treating a pDC-associated disorder

[0343] In one aspect, the present application relates to a method of treating a pDC-associated disorder, comprising administering to a subject in need thereof an effective amount of an anti-CD89 / ILT7 bispecific antibody described herein, or an immunoconjugate, chimeric antigen receptor, engineered T cell receptor, or oncolytic virus comprising the anti-ILT7 antibody or antigen binding fragment thereof described herein, or a pharmaceutical composition or kit described herein. Alternatively, the present application relates to the use of the above-mentioned bispecific antibody, immunoconjugate, chimeric antigen receptor, engineered T cell receptor, or oncolytic virus, pharmaceutical composition or kit for the manufacture of a medicament for treating a pDC-associated disorder. Alternatively, the present application relates to the above-mentioned bispecific antibody, immunoconjugate, chimeric antigen receptor, engineered T cell receptor, or oncolytic virus, pharmaceutical composition or kit for use in treating a pDC-associated disorder.

[0344] The pDC-associated disorder includes, but is not limited to, an autoimmune disease, a cancer, and a disorder associated with pDC tissue accumulation. The subject can be a human, a non-human primate, or other mammal such as a dog, etc.

[0345] The autoimmune disease refers to any disorder associated with the body's own immune response, including but not limited to lupus (e.g., systemic lupus erythematosus, cutaneous lupus, discoid lupus, lupus nephritis), multiple sclerosis, scleroderma, dermatomyositis, polymyositis, psoriasis, psoriatic arthritis, rheumatoid arthritis, myasthenia gravis, Goodpasture's syndrome, Sjogren's syndrome, systemic sclerosis, inflammatory bowel disease (the main types are Crohn's Disease and ulcerative colitis), irritable bowel syndrome, and type I diabetes.

[0346] The cancer includes solid tumors and hematological tumors.

[0347] In some embodiments, administration of an effective amount of the anti-CD89 / ILT7 bispecific antibody or a pharmaceutical composition or kit thereof to a subject in need thereof is capable of reducing or depleting pathogenic pDCs. In some specific embodiments, administration of the bispecific antibody is capable of significantly reducing the number of pDCs in a subject or a biological sample taken from a subject (e.g., blood and / or other tissues such as skin cells, skin biopsy samples, etc. of a healthy subject or a subject having SLE) as compared to prior to administration of the bispecific antibody. In some embodiments, the bispecific antibody is capable of reducing the Cutaneous Lupus Erythematosus Disease Activity and Severity Index (CLASI) in a subject in a damaged tissue or lesion site.

[0348] In some embodiments, administration of the anti-CD89 / ILT7 bispecific antibody or a pharmaceutical composition or kit thereof to a subject in need thereof is capable of inhibiting the production of interferons, inflammatory cytokines and / or chemokines by pDCs. The interferons include IFN-I (e.g., IFN-a), IFN-III, the inflammatory cytokines include IL-6, IL-10, TNF-a, and the chemokines include MIP-1a / CCL3, MIP-1b / CCL4, CCL5 / RANTES, IP-10 / CXCL10 and MCP-1. In some specific embodiments, administration of the bispecific antibody is capable of significantly reducing the level of IFN-a in a blood sample of a subject as compared to prior to administration of the bispecific antibody.

[0349] In some embodiments, administration of the anti-CD89 / ILT7 bispecific antibody or a pharmaceutical composition or kit thereof to a subject in need thereof is capable of activating CD89-expressing myeloid cells and mediating their killing of pDCs without triggering massive cytokine release.

[0350] 10. Detection uses

[0351] The present application also relates to the use of an anti-ILT7 antibody or antigen binding fragment thereof, or an anti-CD89 / ILT7 bispecific antibody for detecting and / or assaying (or quantifying) ILT7 or ILT7-expressing cells (e.g., pDCs) in a sample (e.g., a biological sample such as serum, tissue, biopsy sample), and for a method of screening patients with pDC-related disorders that are responsive to treatment with the anti-CD89 / ILT7 bispecific antibody described herein.

[0352] In some embodiments, the anti-ILT7 antibody or antigen-binding fragment thereof, or anti-CD89 / ILT7 bispecific antibody can be used to diagnose abnormal proliferation of pDCs to facilitate determination of a treatment regimen. For example, the antibody can be conjugated to a detectable label or reporter molecule and the labeled antibody can be contacted with a sample obtained from a patient to diagnose the level of pDCs. The detectable label or reporter molecule can be a radioisotope, such as 3 H、 14 C、 32 P、 33 P、 35 S、 123 I、 125 I、 131 I、 111 In or 188 Rh; a fluorescent material, such as umbelliferone, fluorescein, rhodamine, fluorescein isothiocyanate, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; a chemiluminescent material, such as luminol; a bioluminescent material, such as luciferase, luciferin, or aequorin; or an enzyme, such as alkaline phosphatase, b-galactosidase, acetylcholinesterase, horseradish peroxidase, or luciferase.

[0353] In some embodiments, a method that can be used to detect or determine the level of pDCs in a sample includes: (a) contacting a sample or a control sample with the anti-ILT7 antibody or antigen-binding fragment thereof, or anti-ILT7 / CD89 bispecific antibody, and (b) detecting the number of pDCs in the sample that bind to the antibody or antigen-binding fragment thereof, or bispecific antibody. Wherein the control sample includes a positive control, which can be a sample known to be able to evaluate a disease or condition, and a negative control, which can be a sample of a healthy subject. A higher number of pDCs in the sample compared to the control with a statistical difference indicates the presence of abnormal proliferation of pDCs. Specific exemplary assays that can be used to detect or determine pDCs in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunopet (e.g., 89 Zr, 64 Cu, etc.) and fluorescence-activated cell sorting (FACS).

[0354] The present application is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and all references, patents and patent documents recited herein are expressly incorporated herein by reference. The following examples are illustrative only and are not intended to limit the scope of the application. Unless otherwise indicated, the reagents, materials, instruments and procedures, as set forth in the following examples, are routine and known in the art.

[0355] Example

[0356] Example 1. Optimization of anti-CD89 antibody Mab 14.1

[0357] 1.1 Construction of Mab 14.1 single chain antibody

[0358] Anti-CD89 humanized antibody Mab 14.1 was described in PCT Publication No. WO2002064634. The construction of the expression vector of Mab 14.1 scFv-Fc fusion protein in this example was as follows: a linker sequence (as shown in SEQ ID NO: 167) was introduced at the C-terminus of the heavy chain variable region sequence of Mab 14.1 (as shown in SEQ ID NO: 90) to connect with the N-terminus of the light chain variable region sequence (as shown in SEQ ID NO: 104), and a Mab 14.1 scFv sequence was designed, and another linker sequence (as shown in SEQ ID NO: 168) was introduced at the C-terminus of the scFv sequence for connection with the wild-type human IgG1 Fc fragment (as shown in the amino acid sequence of SEQ ID NO: 163 from position 99 to position 330). The DNA fragment encoding the above sequence was obtained by chemical synthesis, and then the DNA fragment was inserted into the pcDNA3.1-human IgG plasmid by homologous recombination, and the expression vector of Mab 14.1 scFv-Fc fusion protein was obtained by screening and cloning.

[0359] 1.2 Single point / multiple point mutation optimization of Mab 14.1 single chain antibody

[0360] Using online software abYsis (http: / / abysis.org / abysis / index.html), the amino acid residues with low degree of humanization, potential aspartic acid isomerization and / or asparagine deamidation sites in the light and heavy chain variable region sequences of Mab 14.1 were analyzed, and a total of 39 single point or multiple point mutations were designed to improve the degree of humanization of the antibody and / or reduce the risk of aspartic acid isomerization and / or asparagine deamidation of the antibody molecule (as shown in Table 5). The 39 designed single point / multiple point mutations were introduced into the expression vector of Mab 14.1 scFv-Fc fusion protein by PCR method, and then antibody protein was obtained by transient expression, and AKTA Pure system was used for Protein A affinity chromatography purification to obtain antibody protein.

[0361] The transient expression of the 39 single-chain antibody mutants and their parent single-chain antibody Mab14.1 was detected by a molecular interaction instrument (ForteBio). The specific method is as follows: after transient expression by ExpiCHO-S cells, the culture supernatant was harvested and added to a 96-well black wall plate at 200 μL / well. The sample to be detected and Protein A probe were placed in the ForteBio instrument, and the binding rate of the antibody molecules in the culture supernatant and the Protein A probe was detected by a preset quantitative program. After each cycle was completed, the probe was immersed in 10 mM glycine buffer (pH 1.5) for regeneration, and then the next cycle of detection was started. After all the samples were detected, the analysis software was used for calculation according to the standard curve, and the transient expression amount of each mutant and the parent antibody was obtained.

[0362] The binding kinetics of the 39 single-chain antibody mutants and human CD89 recombinant protein were detected by ForteBio, and the control antibody was the parent single-chain antibody Mab14.1. The specific method is as follows: the antibody to be detected was diluted to 5 μg / mL with PBS solution and added to a 96-well black wall plate at 200 μL / well; human CD89 recombinant protein was diluted to 10 μg / mL with PBS solution and added to the same 96-well black wall plate at 200 μL / well. The 96-well black wall plate containing the sample to be detected and the Protein A detection probe were placed in the ForteBio instrument, and the program was set. First, the Protein A probe was combined with the sample to be detected to a signal value of 1.5 nm, and then the binding and dissociation detection was carried out, and the time was 300 s. After each cycle was completed, the probe was immersed in 10 mM glycine buffer (pH 1.5) for regeneration, and then the next cycle of detection was started. After all the samples were detected, the analysis software was used to fit and calculate the binding constant (k a ) and dissociation constant (k dis ) between the antibody and the antigen, and the affinity constant (K D ) value was calculated.

[0363] The mutation sites, transient expression amount, and the results of the binding kinetics detection with CD89 of each mutant are shown in Table 5. Compared with the parent single-chain antibody Mab14.1, most of the 39 single / multiple point mutations retained the antigen binding affinity of the parent antibody, and improved the humanization degree of the antibody and / or significantly reduced the risk of aspartate isomerization and / or asparagine deamidation of the antibody molecule, preferably 7 of the mutations, including 5 single point mutations in the heavy chain of the antibody: Mab14.1-Hu-01, Mab14.1-Hu-02, Mab14.1-Hu-03, Mab14.1-Lia-11 and Mab14.1-Lia-23, and 2 single point mutations in the light chain of the antibody: Mab14.1-Hu-05 and Mab14.1-Lia-33.

[0364] Table 5. Analysis results of single / multiple point mutations of Mab14.1, transient expression amount, and binding kinetics with CD89

[0365] L is the light chain; H is the heavy chain.

[0366] 1.3 Combination mutation optimization of Mab14.1 single-chain antibody

[0367] The above-mentioned preferred 7 single point mutations were combined and introduced into the variable region sequence of the Mab14.1 single-chain antibody to construct antibody molecules containing different combination mutations. The transient expression and purification of the antibody samples were carried out according to the method described in item 1.2 of this embodiment, and the transient expression amount and the binding kinetics with CD89 of each mutant and the control parent antibody Mab14.1 were detected by ForteBio.

[0368] The combination mutation sites, transient expression amount, purity after Protein A affinity purification, and binding kinetics with CD89 of each mutant antibody molecule are shown in Table 6. The transient expression amount and antigen binding affinity of each combination mutant are comparable to those of the parent antibody Mab14.1, but the purity of the combination mutants Mab14.1-HL1404 and Mab14.1-HL1405 after Protein A affinity purification can reach more than 90%, even 100%, which is significantly better than the parent antibody and other mutants.

[0369] Table 6. Analysis results of combination mutations of Mab14.1, transient expression amount, purity, and binding kinetics with CD89

[0370] L is the light chain; H is the heavy chain; Full R 2 The similarity of the fitted curve and the measured curve is represented.

[0371] 1.4 Introduction of disulfide bonds at the VH-VL interface of anti-CD89 single-chain antibody molecules and their effects on molecular stability and biological activity

[0372] To further enhance the structural stability of the anti-CD89 scFv molecule, the distances between amino acid residues at the VH-VL interface of the single-chain antibody Mab14.1-HL1405 were calculated using MOE software. A subset of these spatial distances were then selected. The amino acid residue pairs within the specified range were mutated to cysteine ​​residues to form disulfide bonds, thereby stabilizing the single-chain antibody molecule structure. The specific method is as follows: Twelve cysteine ​​mutations (as shown in Table 7) were introduced into the single-chain antibody Mab14.1-HL1405 expression vector using PCR. Antibody samples were then prepared according to the method described in section 1.2 of this embodiment. After purification by Protein A affinity chromatography, high proportions of polymers were found in the mutants Mab14.1-HL1405-Cy02 and Mab14.1-HL1405-Cy07. Other cysteine ​​mutants yielded high-purity scFv monomer molecules.

[0373] Table 7. Cysteine ​​combination mutation sites in Mab14.1-HL1405

[0374] Following the method described in section 1.2 of this embodiment, the binding kinetics of each cysteine ​​mutant with CD89 were detected using ForteBio. The results are shown in Table 8. The mutant Mab14.1-HL1405-Cy08 did not bind to CD89. The binding affinity of the mutants Mab14.1-HL1405-Cy01, Mab14.1-HL1405-Cy03, Mab14.1-HL1405-Cy09, and Mab14.1-HL1405-Cy10 was not significantly different from that of the maternal antibody Mab14.1-HL1405. The binding affinity of the other mutants was reduced to varying degrees.

[0375] Further, the cell binding activity of the above mutants was detected by flow cytometry using Jurkat cells expressing CD89, according to the following method: Jurkat cells expressing CD89 were collected and resuspended with FACS buffer (PBS containing 1% BSA), and then added to a 96-well U-shaped plate at 50 μL / well. Each mutant to be tested was diluted by 10 times in gradient in FACS buffer at an initial concentration of 100 μg / mL, and then added to the above-mentioned well plate at 50 μL / well, mixed and incubated on ice for 1 hour. The supernatant was centrifuged and the cells were washed 3 times with FACS buffer. AF488-labeled goat anti-human IgG (H+L) antibody (Jackson Immuno) was diluted 1:1000 (volume ratio) in FACS buffer, added to the above-mentioned well plate at 100 μL / well, mixed and incubated on ice for about 40 minutes. The supernatant was centrifuged and the cells were washed 3 times with FACS buffer. The cells were resuspended with 100 μL / well FACS buffer and detected by flow cytometry (NovoCyte 3005, Agilent). The detection results are shown in Figure 1 and Table 8, and the cell binding activity of each mutant showed varying degrees of change compared with the parent antibody Mab14.1-HL1405, wherein the cell binding activity of Mab14.1-HL1405-Cy01, Mab14.1-HL1405-Cy03, Mab14.1-HL1405-Cy09 and Mab14.1-HL1405-Cy10 was basically equivalent to that of the parent antibody, which was consistent with the above antigen binding affinity detection results.

[0376] Table 8. Binding affinity of Mab14.1-HL1405 cysteine mutants to CD89 and Jurkat cell binding activity N / A: not applicable; Full R 2 represents the similarity of the fitted curve to the measured curve.

[0377] The thermal stability of each cysteine mutant was detected by DSF. The method was as follows: each mutant was diluted to 1 mg / mL with PBS solution and loaded into a capillary. The instrument was set to detect the thermal stability of each sample at a temperature range of 25-95°C at a rate of 1°C per minute, and then data analysis was performed according to the measured curve using analysis software. The results are shown in Table 9, and compared with the parent antibody Mab14.1-HL1405, the T m and T agg values of mutant Mab14.1-HL1405-Cy09 were significantly improved.

[0378] Table 9. Thermal stability analysis results of Mab14.1-HL1405 cysteine mutants T onset: the initial temperature of protein denaturation; T m : the temperature of protein denaturation; T agg : the initial temperature of protein aggregation.

[0379] Example 2. Screening of murine anti-human ILT7 antibodies

[0380] 2.1 Design of human ILT7 antigen and construction of engineered cell lines expressing human ILT family protein members

[0381] The human ILT7 antigen fragment was designed and prepared as follows: by alignment of the amino acid sequences of human ILT family protein members, it was found that the amino acid sequence from position 420 to 446 of the extracellular domain near the membrane end of ILT7 had the lowest homology, and therefore this peptide segment was selected for immunization of animals in order to obtain antibodies that specifically target ILT7 without cross-binding to other members of the ILT family. The polypeptide fragment was prepared by chemical synthesis, and was coupled to keyhole limpet hemocyanin (KLH) at the N-terminus in order to enhance the immune response of the polypeptide antigen in animals.

[0382] The engineered cell lines expressing human ILT family protein members were constructed as follows: 293T cells were transfected with the plasmid pCMV-FLAG into which the FcεRIγ gene had been inserted, and a monoclonal engineered cell line overexpressing FcεRIγ was obtained by FLAG-Tag screening; then pCMV-HA expression plasmids encoding each of the activating-type protein members of the human ILT family (including LIR6, ILT1, ILT7, ILT11, ILT8) were transfected into this cell line, and pCMV-HA expression plasmids encoding each of the inhibitory-type protein members of the human ILT family (including ILT2, ILT4, ILT5, ILT3 and LIR8) were transfected into wild-type 293T cells, and 293T engineered cell lines co-expressing FcεRIγ and each of the activating-type protein members of the ILT family and 293T engineered cell lines expressing each of the inhibitory-type protein members of the ILT family were obtained by HA-Tag screening.

[0383] 2.2 Screening and sequence analysis of murine anti-human ILT7 monoclonal antibodies

[0384] The ILT7 ectodomain 420-446 amino acid sequence polypeptide coupled with KLH prepared above (396-422 of the amino acid sequence shown in SEQ ID NO: 161, i.e., the amino acid sequence shown in SEQ ID NO: 162), ILT7 ectodomain full-length protein (ILT7-his, purchased from Yiqioshengzhou), and 293T engineered cell strain overexpressing ILT7 (referred to as 293T-ILT7 cells) were used to immunize 3 BALB / c mice in an alternating manner for 5 times, and then the serum was taken, the ILT7-his was used as an antigen, and the ELISA method was used to detect the serum antibody titer. The results showed that the serum antibody titers of the 3 immunized mice were all more than 1:50000. The spleen cells of the two mice with the highest serum antibody titer were extracted and electrofused with myeloma cells SP2 / 0, and then plated for hybridoma cell screening. After immunofluorescence detection, flow cytometry detection and ELISA detection of the culture supernatant of the hybridoma cells, and after one round of main cell screening and two rounds of subcloning screening, 22 hybridoma monoclonal cells were selected for antibody sequencing, and finally 9 sequence-specific anti-ILT7 murine monoclonal antibodies were obtained.

[0385] 2.3 Construction and expression of anti-ILT7 chimeric antibodies

[0386] The light and heavy chain genes of the 9 murine anti-ILT7 antibodies obtained by screening were subjected to homologous recombination with linearized expression vectors (the heavy chain variable region VH was connected with the pcDNA3.4 vector containing the human IgG1 constant region, and the light chain variable region VL was connected with the pcDNA3.4 vector containing the human CK constant region), and the correct sequence of the chimeric antibody expression plasmid was obtained by colony PCR detection and DNA sequencing analysis. The chimeric antibody expression plasmid was extracted and purified by a conventional method; the 9 groups of chimeric antibody expression plasmids containing light and heavy chain gene fragments were transiently transfected into 293F cells, and then the chimeric antibodies were expressed by serum-free culture. After collecting the culture supernatant, the binding activity of the chimeric antibodies to 293T-ILT7 cells was detected by flow cytometry, and the results showed that 7 chimeric antibodies could bind to 293T-ILT7 cells (data not shown). The 7 chimeric antibodies with cell binding activity were purified by Protein A affinity chromatography using the AKTA Pure system, and the purified chimeric antibodies were obtained.

[0387] 2.4 Detection of the binding activity of anti-ILT7 chimeric antibodies to human ILT family protein members

[0388] The binding activity of the above-mentioned 7 anti-ILT7 chimeric antibodies with cell binding activity to human ILT family members was detected by ELISA. The specific steps are as follows: each recombinant protein (including ILT1, ILT6, ILT7, ILT8, ILT11, ILT2, ILT3, ILT4 and ILT5, all purchased from Yiqioshengzhou) was diluted with PBS solution to 1 μg / mL and added to a 96-well plate at 100 μL / well, and incubated at 4°C overnight. The plate was washed with PBS once. 100 μL of blocking solution (containing 1% BSA in PBST [0.05% Tween-20 in PBS]) was added to each well, and incubated at room temperature for 1 hour. The plate was washed with PBST once. Each chimeric antibody to be tested and the control antibody (anti-ILT7 antibody VIB7734 prepared as described in PCT Publication No. WO2017156298, designated as BM in the present application) was diluted by 4-fold gradient from 2 μg / mL in blocking solution, and then added to the above-mentioned 96-well plate at 100 μL / well, and incubated at room temperature for 2 hours. The plate was washed with PBST 3 times. HRP-goat anti-human IgG (H+L) antibody (Jackson Immuno) was diluted at 1:10000 (v / v) in blocking solution, added to the above-mentioned 96-well plate at 100 μL / well, and incubated at room temperature for 1 hour. 100 μL of TMB color developing solution was added to each well, and incubated at room temperature for 3-10 minutes in the dark. After 50 μL of stop solution (2M HCl) was added to each well, the OD450 value was read by a multifunctional enzyme label instrument (Thermo, Varioskan). The detection results are shown in FIG. 2, and the 7 anti-ILT7 chimeric antibodies only bind to ILT7, but not to other members of the ILT family.

[0389] Further, the binding activity of the above-mentioned 7 anti-ILT7 chimeric antibodies to 293T engineered cell strains expressing each protein member of the human ILT family was verified by flow cytometry according to the method described in item 1.4 of Example 1. The results are shown in FIG. 3, and the chimeric antibodies MAB21-1-12-1-A1 and MAB21-1-12-1-A6 weakly bind to certain other members of the ILT family (including ILT1, ILT8, ILT11, ILT2, ILT3, ILT4 and ILT5) at high concentrations, and the remaining 5 anti-ILT7 chimeric antibodies have no cross-binding activity to other members of the ILT family.

[0390] 2.5 Detection of the binding kinetics of anti-ILT7 chimeric antibodies to human ILT7

[0391] The binding kinetics of each anti-ILT7 chimeric antibody and the control antibody BM to human ILT7 recombinant protein was detected by ForteBio according to the method described in item 1.2 of Example 1. The results are shown in Table 10, and the binding affinity constant (Kd) of each anti-ILT7 chimeric antibody to ILT7 is shown in Table 10.D ) are all less than K D .

[0392] Table 10. Binding kinetics results of chimeric antibodies to ILT7 recombinant protein Full R 2 represents the similarity of the fitted curve to the measured curve.

[0393] Example 3 Humanization and optimization of anti-ILT7 chimeric antibodies

[0394] 3.1 Humanization of anti-ILT7 chimeric antibodies

[0395] According to the analysis by MOE software, the heavy chain framework region sequences of the chimeric antibodies MAB21-1-12-1-A4, MAB21-1-12-1-B5 and MAB21-1-12-1-C1 were replaced with the framework region sequences of Germline IGHV1-2*06 and IGHV3-72*01, respectively, and the light chain framework region sequences were replaced with the framework regions of Germline IGKV1-NL1*01, IGKV1-16*01 and IGKV2-28*01, respectively, to construct humanized antibodies HuA4, HuB5 and HuC1. The specific method is as follows: the DNA fragments encoding the variable region sequences of the humanized antibodies were obtained by gene synthesis, and the DNA fragments of the heavy chain variable region and light chain variable region sequences were homologously recombined into pcDNA3.1 vectors containing human IgG1 heavy chain constant region sequences and light chain kappa constant region sequences, respectively, to construct expression plasmids of each humanized antibody, and each humanized antibody was expressed by transiently transfecting ExpiCHO-S cells and serum-free culture, and the antibodies in the culture supernatant were purified by Protein A affinity chromatography using AKTA Pure system, to obtain humanized antibodies HuA4, HuB5 and HuC1.

[0396] According to the method described in item 1.2 of Example 1, the binding kinetics of the above humanized antibodies to ILT7 recombinant protein was detected by ForteBio. The detection results are shown in Table 11, and each humanized antibody basically retains the binding activity to ILT7 recombinant protein, wherein the antigen binding affinity of humanized antibody HuA4 is the highest.

[0397] Table 11. Analysis results of binding kinetics of humanized antibodies to ILT7 recombinant protein

[0398] Full R 2 represents the similarity of the fitted curve to the measured curve.

[0399] 3.2 Single point / multiple point mutation optimization of anti-ILT7 humanized antibody HuA4

[0400] After analyzing the amino acid residues with lower degree of humanization and potential stability risk sites in the variable region sequences of the light and heavy chains of the anti-ILT7 humanized antibody HuA4 using online software abYsis (http: / / abysis.org / abysis / index.html) and MOE software, a total of 110 single / multiple point mutations were designed to improve the degree of humanization and / or improve stability (as shown in Table 12). The specific method is as follows: the designed 110 single / multiple point mutations were introduced into the expression vector of the humanized antibody HuA4 by PCR method, and then the antibody light and heavy chain expression plasmids were combined to transfect ExpiCHO-S cells to express each single / multiple point mutant antibody. According to the method described in item 1.2 of Example 1, the content of each single / multiple point mutant antibody in the culture supernatant after 3 days of transfection culture was detected using ForteBio, and the binding affinity to ILT7 recombinant protein was detected. The detection results are summarized in Table 12. Compared with the parent antibody HuA4, a total of 30 point mutations in the 110 single / multiple point mutations improved the degree of humanization of the antibody while did not significantly affect the binding affinity of the antibody to ILT7, which include 14 point mutations in the light chain of the antibody: HuA4-Hu-L04, HuA4-Hu-L10, HuA4-Hu-L12, HuA4-Hu-L19, HuA4-Hu-L20, HuA4-Hu-L24, HuA4-Hu-L32, HuA4-Hu-L33, HuA4-Hu-L39, HuA4-Hu-L40, HuA4-Hu-L41 and HuA4-Hu-L45, and 16 point mutations in the heavy chain of the antibody: HuA4-Hu-H47, HuA4-Hu-H49, HuA4-Hu-H50, HuA4-Hu-H51, HuA4-Hu-H57, HuA4-Hu-H66, HuA4-Hu-H70, HuA4-Hu-H84, HuA4-Hu-H95, HuA4-Hu-H98, HuA4-Hu-H103, HuA4-Hu-H104, HuA4-Hu-H105, HuA4-Hu-H107, HuA4-Hu-H109 and HuA4-Hu-H110.

[0401] Table 12. Single / multiple point mutations of HuA4, transient expression amount and binding kinetics analysis results with ILT7 L is light chain; H is heavy chain; N / A represents not applicable; Full R 2 represents the similarity of the fitted curve and the measured curve.

[0402] 3.3 Combination mutation optimization of anti-ILT7 humanized antibody HuA4

[0403] Different combinations of mutations were introduced into the humanized antibody HuA4, i.e., combinations of the above-mentioned point mutations on the light chain were introduced into the light chain of HuA4, and combinations of the above-mentioned point mutations on the heavy chain were introduced into the heavy chain of HuA4, to construct light chain mutation combination expression plasmids and heavy chain mutation combination expression plasmids, respectively. As shown in Table 13, the constructed light and heavy chain expression plasmids were paired and then transfected into ExpiCHO-S cells to prepare antibody samples. According to the method described in item 1.2 of Example 1, the antibody content in the culture supernatant after transfection for 3 days was detected using ForteBio, and the binding affinity to ILT7 recombinant protein was detected. The results are shown in Table 13. Antibodies HuA4c-L1H4 and HuA4c-L1H11 significantly improved the degree of humanization, and their transfection expression levels and antigen binding affinities were comparable to those of the parent antibody HuA4.

[0404] Table 13. Combination mutations, transfection expression levels, and binding kinetics analysis results of anti-ILT7 humanized antibody HuA4

[0405] 3.4 Binding activity of optimized anti-ILT7 humanized antibody HuA4 to ILT family protein members

[0406] According to the methods described in item 1.4 of Example 1 and item 2.4 of Example 2, the binding activity between humanized antibodies HuA4c-L1H4 and HuA4c-L1H11 and ILT family protein members was detected by flow cytometry and ELISA, respectively. As shown in Figure 4A (flow cytometry detection) and Figure 4B (ELISA detection), HuA4c-L1H4 and HuA4c-L1H11 did not bind to other ILT family protein members except ILT7, indicating that the optimized antibodies maintained the binding specificity of the parent antibody.

[0407] 3.5 Binding activity of anti-ILT7 humanized antibody to human primary pDC cells

[0408] The binding activity of humanized antibodies HuA4c-L1H4 and HuA4c-L1H11 to human primary pDC cells was detected by flow cytometry, and the specific method was as follows: after the human peripheral blood mononuclear cells (PBMC) (purchased from Miaoshun Biotechnology) were fully resuspended with FACS buffer, 100 μL / well was added to a 96-well U-shaped plate, Fc receptor blocking reagent (Biolegend) was added for incubation for 10 minutes, and then biotin-labeled HuA4c-L1H4, HuA4c-L1H11 and control antibody BM were added at a final concentration of 10 μg / mL, and incubated on ice for 1 hour. The supernatant was centrifuged and the cells were washed twice with FACS buffer. AF488-labeled streptavidin (Jackson Immuno) was diluted 1:1000 (volume ratio) in FACS buffer, then 100 μL / well was added to the resuspended cells in the above-mentioned well plate, and 5 μL / well of APC-anti-human CD123 antibody (Biolegend) and PE-anti-human BDCA2 antibody (Biolegend) were added, and incubated on ice for about 40 minutes. The supernatant was centrifuged and the cells were washed twice with FACS buffer. Resuspend the cells in each well with 100 μL of FACS buffer, and add 3 μL / well of 7-AAD (Biolegend), incubate at room temperature for 3 minutes, and then place in a flow cytometer for detection. The signal of the pDC cell population in the AF488 channel was analyzed after the pDC cell population was circled, and the binding activity of HuA4c-L1H4, HuA4c-L1H11 and control antibody BM to human primary pDC cells was analyzed. The detection results are shown in Figure 5, and the pDC cells incubated with HuA4c-L1H4, HuA4c-L1H11 and BM have significantly enhanced fluorescence signals in the AF488 channel, indicating that they can all specifically bind to human primary pDC cells. + BDCA2 + After the pDC cell population was circled, the signal of the population in the AF488 channel was analyzed after incubation with blank control, HuA4c-L1H4 or HuA4c-L1H11 and control antibody BM, to determine the binding activity of HuA4c-L1H4, HuA4c-L1H11 and control antibody BM to human primary pDC cells. The detection results are shown in Figure 5, and the pDC cells incubated with HuA4c-L1H4, HuA4c-L1H11 and BM have significantly enhanced fluorescence signals in the AF488 channel, indicating that they can all specifically bind to human primary pDC cells.

[0409] Example 4 Construction of Anti-CD89 / ILT7 Bispecific Antibody and Identification of Its Functional Activity

[0410] 4.1 Construction of Anti-CD89 / ILT7 Bispecific Antibody

[0411] In this example, the monospecific antibody capable of specifically recognizing the extracellular Ig-like domain 2 of CD89 and the monospecific antibody specifically recognizing the amino acid residues 420-446 of the extracellular domain of ILT7 (HuA4c-L1H4 or HuA4c-L1H11) were selected to construct exemplary anti-CD89 / ILT7 bispecific antibodies. As shown in Figure 6, the configuration of the bispecific antibodies is 2+1 scFv-IgG or scFv-Fab-Fc: Fab-Fc asymmetric structure, wherein the subunit A is in the form of scFv-Fab-Fc, the subunit B is in the form of Fab-Fc, the scFv domain is selected from the anti-CD89 scFv with disulfide bond at the VH-VL interface shown in Table 7, the IgG domain or Fab-Fc domain is the anti-ILT7 antibody (HuA4c-L1H4 or HuA4c-L1H11), and the light chain variable region of the scFv domain is connected to the heavy chain variable region of the IgG domain or Fab-Fc domain via a peptide linker (G4S)2. The asymmetric structure adopted by the anti-CD89 / ILT7 bispecific antibodies ensures that they can only bind to myeloid cells expressing CD89 in a monovalent manner, but at the same time can bind to target cells (e.g., pDC) expressing ILT7 in a monovalent or bivalent manner, thus avoiding the possibility of non-targeted activation of myeloid cells and the resulting toxic side effects in the absence of target cells. In addition, the myeloid cells (e.g., neutrophils and macrophages) expressing CD89 do not release a large amount of cytokines after activation, so the risk of directly triggering a cytokine storm is extremely low.

[0412] In this example, the following different types of engineering modifications were also made to the heavy chain constant region of the bispecific antibodies: (1) the formation of heterodimers was promoted by introducing "protuberance and cavity" mutations in the heavy chain Fc region of the IgG domain, and since the bispecific antibodies only contain one light chain, there is no problem of light chain mispairing; (2) in order to be able to effectively separate the "cavity-cavity" homodimers that may be formed during expression by ion exchange chromatography, different types of "pI mutations" were introduced into the two heavy chain constant regions of the bispecific antibodies, for example, a mutation that increases the pI value was introduced into the heavy chain constant region containing the "protuberance" mutation, and a mutation that decreases the pI value was introduced into the heavy chain constant region containing the "cavity" mutation, in order to expand the pI value difference between the heterodimer bispecific antibodies and the "cavity-cavity" homodimers; (3) in order to avoid the possibility that the Fc effector function of the antibody may kill non-target cells of the bispecific antibodies, the L234F / L235E / P331S (EU Numbering) mutation (referred to as "TM mutation" for short) was introduced into the Fc region of the bispecific antibodies to reduce its binding to FcyRs and Clq, thereby eliminating the Fc effector function.

[0413] The two heavy chain constant regions of the anti-CD89 / ILT7 bispecific antibody introduce different types of engineering modifications and the amino acid mutations involved are shown in Table 14. After introducing different types of "pI mutations" in the two heavy chain constant regions, the difference in the theoretical pI values of the heterodimeric bispecific antibody and the "cavity-cavity" homodimer is at least 0.8 pH units.

[0414] Table 14. Amino acid mutations introduced in the heavy chain constant regions of the anti-CD89 / ILT7 bispecific antibody

[0415] According to the above design, first, the TM mutation was introduced in the heavy chain expression vector of HuA4c-L1H4 or HuA4c-L1H11 constructed in Example 3 using the PCR method, and then the "knob" and "cavity" mutations were introduced in the same way, respectively, and the "pI mutations" corresponding to each heavy chain were introduced, and finally the anti-CD89 scFv sequence shown in Table 7 containing a disulfide bond at the VH-VL interface was connected to the N-terminus of the "knob" heavy chain of HuA4c-L1H4 or HuA4c-L1H11 using homologous recombination, completing the construction of a series of bispecific antibody heavy chain expression plasmids. After combining the constructed heavy chain expression vector with the light chain expression vector of HuA4c-L1H4 or HuA4c-L1H11 constructed in Example 3, ExpiCHO-S cells were co-transfected and cultured in serum-free medium to express bispecific antibodies (a total of 14 bispecific antibodies, respectively named: Cy01-L1H11 ~ Cy12-L1H11, and Cy01-L1H4 and Cy09-L1H4). After the cell viability was less than 70%, the culture supernatant was collected, and the antibody was purified by Protein A affinity chromatography using the AKTA Pure system. The purified antibody obtained was subjected to SEC-HPLC analysis, and the results showed that, except for Cy02-L1H11, Cy03-L1H11 and Cy10-L1H11, the remaining bispecific antibodies all showed a high monomer proportion, and Cy01-L1H4 was close to 100% (data not shown).

[0416] 4.2 Binding affinity of anti-CD89 / ILT7 bispecific antibody to ILT7 and CD89

[0417] The binding kinetics of the bispecific antibodies and control antibodies (including the bispecific antibody HL1405-L1H11 constructed based on the single-chain antibody Mab14.1-HL1405 and HuA4c-L1H11 which do not contain disulfide bonds in the VH-VL interface) to ILT7 and CD89 recombinant proteins were determined by the method described in item 1.2 of Example 1. The results, as shown in Table 15, indicated that the binding affinities of each bispecific antibody to ILT7 were comparable to those of the control antibodies; the bispecific antibodies Cy07-L1H11 and Cy08-L1H11 lost the binding activity to CD89, and the other bispecific antibodies retained the binding affinities to CD89 to varying degrees.

[0418] Table 15. Results of the binding kinetics analysis of the anti-CD89 / ILT7 bispecific antibodies to ILT7

[0419] N / A means not applicable; Full R 2 The similarity of the fitted curve to the measured curve is represented.

[0420] 4.3 Biological activity of the anti-CD89 / ILT7 bispecific antibodies

[0421] The biological activity of the anti-CD89 / ILT7 bispecific antibodies was detected by the reporter gene method. The specific method is as follows: target cells (including 293T-ILT7 cells and wild-type 293T cells) were collected respectively, resuspended with DMEM medium containing 1% FBS, and then 80 μL / well of the two target cells were plated into a 96-well white wall plate and cultured at 37°C overnight. The effector cells (Jurkat cells co-expressing CD89 and firefly luciferase reporter gene regulated by NFAT response element) were collected, resuspended with DMEM medium containing 1% FBS, and then 20 μL / well of the effector cells were plated into the above-mentioned 96-well white wall plate to make the cell count ratio of target cells to effector cells 1:20; each bispecific antibody and control antibody was diluted by 5 times gradient from 6 μg / mL, and then 20 μL / well was added to the above-mentioned 96-well white wall plate and cultured at 37°C. After 6 hours of culture, the plate was taken out, Bio-Lite luciferase substrate (Nanjing Novizhan) was added, and the chemiluminescence value was read by a multifunctional enzyme label instrument (Varioskan, Thermo). The results are shown in Figure 7. The bispecific antibodies did not cause the activation of effector cells when wild-type 293T cells were used as target cells, while when 293T-ILT7 cells were used as target cells, except for Cy07-L1H11 and Cy08-L1H11, the rest of the bispecific antibodies could activate the CD89-mediated signal pathway in effector cells to different degrees, indicating that the anti-CD89 / ILT7 bispecific antibodies had target cell-dependent CD89 agonistic activity (i.e. would not non-directionally activate effector cells when target cells were absent), and the activation activity was related to the binding affinity of CD89.

[0422] 4.4 Molecular stability of anti-CD89 / ILT7 bispecific antibodies

[0423] In order to investigate the molecular stability of the anti-CD89 / ILT7 bispecific antibodies, each bispecific antibody was repeatedly frozen and thawed 5 times, or incubated at 37°C for 7 days, and then the SEC-HPLC method was used to detect the changes of the multimers of each bispecific antibody after treatment. The results are shown in Table 16. After the above treatment, the multimer ratio of the control bispecific antibody HL1405-L1H11 in the sample increased by about 4% compared with before treatment, while the bispecific antibodies Cy01-L1H11, Cy06-L1H11, Cy09-L1H11, Cy12-L1H11, Cy01-L1H4 and Cy09-L1H4 did not change significantly, indicating that the disulfide bond introduced in the VH-VL interface of the anti-CD89 single chain antibody could enhance the molecular stability of the bispecific antibodies.

[0424] Table 16. Test results of molecular stability of anti-CD89 / ILT7 bispecific antibodies

[0425] 4.5 Anti-CD89 / ILT7 bispecific antibodies co-binding activity to ILT7 and CD89

[0426] To determine whether the bispecific antibodies Cy01-L1H4 and Cy09-L1H4 can bind to both ILT7 and CD89 recombinant proteins at the same time, the following method was used: first, biotin-labeled CD89 recombinant protein was captured by SA probe, after equilibration with PBST, the bispecific antibodies and control monospecific antibodies (including anti-ILT7 humanized antibody HuA4c-L1H4 and anti-CD89 antibody Mab14.1-HL1405) were added for binding and dissociation detection, and finally ILT7 recombinant protein was added for binding and dissociation detection. The results are shown in Figure 8, and the bispecific antibodies Cy01-L1H4 and Cy09-L1H4 can bind to both ILT7 and CD89 at the same time.

[0427] 4.6 Anti-CD89 / ILT7 bispecific antibodies binding activity to ILT family protein members

[0428] According to the method described in item 1.4 of Example 1 and item 2.4 of Example 2, the binding activity of bispecific antibodies Cy01-L1H4 and Cy09-L1H4 to ILT family protein members was detected by flow cytometry (Figure 9A) and ELISA (Figure 9B). The results are shown in Figure 9, and the bispecific antibodies Cy01-L1H4 and Cy09-L1H4 do not bind to other ILT family protein members except ILT7.

[0429] 4.7 Anti-CD89 / ILT7 bispecific antibodies mediated killing activity of human primary leukocytes to ILT7 expressing target cells

[0430] To detect the specific killing activity of human primary leukocytes mediated by anti-CD89 / ILT7 bispecific antibodies to ILT7 expressing target cells, 293T-ILT7-luc and wild type 293T cells (transfected cells were named 293T-ILT7-luc and 293T-luc, respectively) transfected and expressing firefly luciferase were used as target cells. The specific method was as follows: 293T-ILT7-luc and 293T-luc cells were collected and resuspended with 10% FBS-containing RPMI-1640 medium, and then 50 μL / well was added to a 96-well U-shaped plate (as "target cells"). At the same time, according to the method described in the instruction provided by the manufacturer Stemcell, HetaSep TMLeukocytes were isolated from peripheral whole blood of healthy human, and then added into the above 96-well plate at 50 μL / well (as "effector cells") to make the ratio of effector cells to target cells in each well at 40:1. Cy01-L1H4 and Cy09-L1H4 were diluted to 12 μg / mL with RPMI-1640 medium containing 10% FBS and then added into the above 96-well plate at 20 μL / well, and the plate was incubated at 37°C overnight. Bio-Lite Luciferase Reagent (Promega) was added to detect the survival rate of target cells. The results, as shown in Figure 10, indicated that both bispecific antibodies Cy01-L1H4 and Cy09-L1H4 could induce human primary leukocytes to kill target cells expressing ILT7, and the activity was basically the same, while no killing effect was observed on cells not expressing ILT7.

[0431] Further, the killing activity of myeloid cells on pDC mediated by anti-CD89 / ILT7 bispecific antibody Cy09-L1H4 was evaluated indirectly by detecting the expression / secretion amount of IFNα in the supernatant of leukocyte culture, using leukocytes derived from peripheral whole blood of healthy human and SLE patients. The specific method was as follows: according to the above method, HetaSep TM Leukocytes were isolated from peripheral whole blood of healthy human or SLE patients, and then added into 96-well U-shaped plate at 180 μL / well, and 20 μL / well of anti-CD89 / ILT7 bispecific antibody Cy09-L1H4 diluted 10 times with RPMI-1640 medium containing 10% FBS (the initial final concentration was 0.2 μg / mL) was added. After the plate was incubated at 37°C overnight, 50 μL of CpG oligodeoxynucleotide ODN-2216 (purchased from MCE) with a concentration of 5 μM was added to each well, and then the plate was incubated at 37°C for about 20 hours. After the incubation, the supernatant was collected by centrifugation at 1000g for 10 minutes, and the concentration of IFNα in the supernatant of leukocyte culture was detected by using human IFNα quantitative ELISA kit (DuoSet). In this embodiment, peripheral whole blood of 16 healthy humans and 9 SLE patients were tested, and Figure 11 shows the representative experimental results (shown as response curve) selected from one healthy human and one SLE patient, and the activity (shown as EC50) of the bispecific antibody in inhibiting the secretion of IFNα by leukocytes derived from 16 healthy humans and 9 SLE patients is also summarized in Figure 11. The results indicated that anti-CD89 / ILT7 bispecific antibody Cy09-L1H4 could effectively induce myeloid cells in peripheral blood of healthy human or SLE patients to kill pDC, thereby significantly inhibiting the secretion of IFNα, and there was no significant difference in the activity between healthy human and SLE patient (t-test, p ＞ 0.05).

[0432] 4.8 Activity of Anti-CD89 / ILT7 Bispecific Antibody in Inducing Cytokine Release from Peripheral Blood Cells of Healthy Human and SLE Patients

[0433] While the activity of anti-CD89 / ILT7 bispecific antibody in mediating myeloid cell killing pDCs was detected using leukocytes derived from peripheral blood of healthy human and SLE patients, the bispecific antibody whether induced cytokine release was also determined. The method was as follows: peripheral whole blood derived from healthy human or SLE patients was added to 96-well U-plate at 180 μL / well, then 20 μL / well of bispecific antibody Cy09-L1H4 diluted 10-fold gradient in RPMI-1640 serum-free medium (the initial final concentration was 2 μg / mL) was added, or blank medium (i.e. RPMI-1640 serum-free medium) was added as negative control, or human CD3 / CD28 T cell activator (Human CD3 / CD28 T Cell Activator, Stemcell) was added as positive control. After the plate was incubated at 37°C for about 20 hours, the plasma supernatant was collected by centrifugation at 1000g for 10 minutes, and the cytokine concentrations (including TNFα, IL-6, IFNγ, IL-10, IL-1β, and IL-5) in the collected plasma supernatant samples were quantitatively detected using Luminex Human Discovery Assay (6-Plex) kit (R&D System). As shown in Figure 12, compared with the negative control, CD3 / CD28 T cell activator could significantly stimulate the release of a large amount of cytokines from peripheral blood cells of healthy human or SLE patients, while no significant increase in cytokine concentration was detected in samples under the action of different concentrations of Cy09-L1H4, indicating that Cy09-L1H4 mediated myeloid cells did not produce or secrete a large amount of cytokines when killing pDCs.

[0434] 4.9 Verification of Related Properties of Anti-CD89 / ILT7 Bispecific Antibody after Engineering of Heavy Chain Constant Region

[0435] 4.9.1 Fc Effector Function of Anti-CD89 / ILT7 Bispecific Antibody

[0436] To reduce the potential safety risk due to Fc-mediated effector functions, three point mutations: L234F / L235E / P331S (i.e. "TM mutations") were introduc...

Claims

1. An isolated anti-ILT7 antibody or its antigen-binding fragment thereof, said anti-ILT7 antibody or its antigen-binding fragment comprising at least one of the following characteristics: (1) said anti-ILT7 antibody or its antigen-binding fragment is capable of specifically binding amino acid residues 420-446 of the extracellular domain of ILT7; (2) the binding affinity (K) of said anti-ILT7 antibody or its antigen-binding fragment to ILT7 is high. D Value < 5 × 10 -8 M is preferably <1×10 -8 M, or <5×10 -9 M, more preferably <5×10 -9 M; and (3) the anti-ILT7 antibody or its antigen-binding fragment has no cross-binding activity with ILT1, ILT2, ILT3, ILT4, ILT5, ILT6, ILT8, ILT11, LIR6 or LIR8.

2. The anti-ILT7 antibody or its antigen-binding fragment as described in claim 1, comprising: (1) having an amino acid sequence of GYX 15 MI(X 15 HCDR1 of (=N or Y), preferably, X 15 =Y; (2) Has an amino acid sequence of X 16 IDPFX 17 GGX 18 X 19 YNQKX 20 KG(X 16 =N, W, or Y; X 17 =Y or F; X 18 =S, A, I, K, N, or T; X 19 =S, N, K, D, or G; X 20 =F or V) of HCDR2, preferably, X 16 =Y,X 17 =F,X 18 =T,X 19 =D,X 20 =F; (3) HCDR3 having the amino acid sequence shown in SEQ ID NO:25; (4) having the amino acid sequence RASX 21 X 22 IX 23 NYLA(X 21 =G, A, K, N, Q, R, S, or T; X 22 =N, G, I, or S; X 23 LCDR1 (=H, G, N, S, or Y), preferably, X 21 =Q,X 22 =S,X 23 =G; (5) Has an amino acid sequence of X 24 AX 25 TLX 26 X 27 (X 24 =N, D, E, G, S, or Y; X 25 =K, D, N, or Y; X 26 =A, Q, or E; X 27 LCDR2 (=E, T, or S), preferably, X 24 =N,X 25 =S or N, X 26 =E,X 27 =S; (6) Has amino acid sequence X 28 X 29 YYSTPLT(X 28 =H or Q; X 29 LCDR3 (=H or Q), preferably, X 28 =Q,X 29 =Q, or an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

3. The anti-ILT7 antibody or its antigen-binding fragment as described in claim 1 or 2, comprising: (1) HCDR1 having the amino acid sequence shown in SEQ ID NO:16, HCDR2 having the amino acid sequence shown in SEQ ID NO:24 or 23, HCDR3 having the amino acid sequence shown in SEQ ID NO:25, and LCDR1 having the amino acid sequence shown in SEQ ID NO:30, LCDR2 having the amino acid sequence shown in SEQ ID NO:36, and LCDR3 having the amino acid sequence shown in SEQ ID NO:38, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or (2) HCDR1 having the amino acid sequence shown in SEQ ID NO:15, HCDR2 having the amino acid sequence shown in SEQ ID NO:17, HCDR3 having the amino acid sequence shown in SEQ ID NO:25, and LCDR1 having the amino acid sequence shown in SEQ ID NO:26, LCDR2 having the amino acid sequence shown in SEQ ID NO:32, 33, 34, or 35, LCDR3 having the amino acid sequence shown in SEQ ID NO:37, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or (3) HCDR1 having the amino acid sequence shown in SEQ ID NO:15, HCDR2 having the amino acid sequence shown in SEQ ID NO:18, 19, 20, 21, or 22, HCDR3 having the amino acid sequence shown in SEQ ID NO:25, and LCDR1 having the amino acid sequence shown in SEQ ID NO:26, LCDR2 having the amino acid sequence shown in SEQ ID NO:31, and LCDR3 having the amino acid sequence shown in SEQ ID NO:37, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or (4) HCDR1 having the amino acid sequence shown in SEQ ID NO:15, HCDR2 having the amino acid sequence shown in SEQ ID NO:17, HCDR3 having the amino acid sequence shown in SEQ ID NO:25, and LCDR1 having the amino acid sequence shown in SEQ ID NO:26, LCDR2 having the amino acid sequence shown in SEQ ID NO:31, and LCDR3 having the amino acid sequence shown in SEQ ID NO:38, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or (5) HCDR1 having the amino acid sequence shown in SEQ ID NO:16, HCDR2 having the amino acid sequence shown in SEQ ID NO:17, HCDR3 having the amino acid sequence shown in SEQ ID NO:25, and LCDR1 having the amino acid sequence shown in SEQ ID NO:26, LCDR2 having the amino acid sequence shown in SEQ ID NO:31, and LCDR3 having the amino acid sequence shown in SEQ ID NO:37, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or (6) HCDR1 having the amino acid sequence shown in SEQ ID NO:15, HCDR2 having the amino acid sequence shown in SEQ ID NO:17, HCDR3 having the amino acid sequence shown in SEQ ID NO:25, and LCDR1 having the amino acid sequence shown in SEQ ID NO:26, 27, 28, or 29, LCDR2 having the amino acid sequence shown in SEQ ID NO:31, LCDR3 having the amino acid sequence shown in SEQ ID NO:37, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

4. The anti-ILT7 antibody or its antigen-binding fragment as described in any one of claims 1-3, comprising: HCDR1 having the amino acid sequence shown in SEQ ID NO:16, HCDR2 having the amino acid sequence shown in SEQ ID NO:23 or 24, HCDR3 having the amino acid sequence shown in SEQ ID NO:25, and LCDR1 having the amino acid sequence shown in SEQ ID NO:30, LCDR2 having the amino acid sequence shown in SEQ ID NO:36, LCDR3 having the amino acid sequence shown in SEQ ID NO:38, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; Preferably, the anti-ILT7 antibody or its antigen-binding fragment comprises: HCDR1 having the amino acid sequence shown in SEQ ID NO:16, HCDR2 having the amino acid sequence shown in SEQ ID NO:24, HCDR3 having the amino acid sequence shown in SEQ ID NO:25, and LCDR1 having the amino acid sequence shown in SEQ ID NO:30, LCDR2 having the amino acid sequence shown in SEQ ID NO:36, LCDR3 having the amino acid sequence shown in SEQ ID NO:38, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

5. The anti-ILT7 antibody or its antigen-binding fragment as described in claim 1, comprising: (1) HCDR1 having the amino acid sequence shown in SEQ ID NO:114, HCDR2 having the amino acid sequence shown in SEQ ID NO:115, HCDR3 having the amino acid sequence shown in SEQ ID NO:116, and LCDR1 having the amino acid sequence shown in SEQ ID NO:117, LCDR2 having the amino acid sequence shown in SEQ ID NO:118, LCDR3 having the amino acid sequence shown in SEQ ID NO:119, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or (2) HCDR1 having the amino acid sequence shown in SEQ ID NO:120, HCDR2 having the amino acid sequence shown in SEQ ID NO:121, HCDR3 having the amino acid sequence shown in SEQ ID NO:122, and LCDR1 having the amino acid sequence shown in SEQ ID NO:123, LCDR2 having the amino acid sequence shown in SEQ ID NO:124, and LCDR3 having the amino acid sequence shown in SEQ ID NO:125, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or (3) HCDR1 having the amino acid sequence shown in SEQ ID NO:126, HCDR2 having the amino acid sequence shown in SEQ ID NO:127, HCDR3 having the amino acid sequence shown in SEQ ID NO:128, and LCDR1 having the amino acid sequence shown in SEQ ID NO:129, LCDR2 having the amino acid sequence shown in SEQ ID NO:130, and LCDR3 having the amino acid sequence shown in SEQ ID NO:131, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or (4) HCDR1 having the amino acid sequence shown in SEQ ID NO:132, HCDR2 having the amino acid sequence shown in SEQ ID NO:133, HCDR3 having the amino acid sequence shown in SEQ ID NO:134, and LCDR1 having the amino acid sequence shown in SEQ ID NO:135, LCDR2 having the amino acid sequence shown in SEQ ID NO:136, and LCDR3 having the amino acid sequence shown in SEQ ID NO:137, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or (5) HCDR1 having the amino acid sequence shown in SEQ ID NO:138, HCDR2 having the amino acid sequence shown in SEQ ID NO:139, HCDR3 having the amino acid sequence shown in SEQ ID NO:140, and LCDR1 having the amino acid sequence shown in SEQ ID NO:141, LCDR2 having the amino acid sequence shown in SEQ ID NO:142, and LCDR3 having the amino acid sequence shown in SEQ ID NO:143, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or (6) HCDR1 having the amino acid sequence shown in SEQ ID NO:144, HCDR2 having the amino acid sequence shown in SEQ ID NO:145, HCDR3 having the amino acid sequence shown in SEQ ID NO:146, LCDR1 having the amino acid sequence shown in SEQ ID NO:147, LCDR2 having the amino acid sequence shown in SEQ ID NO:148, LCDR3 having the amino acid sequence shown in SEQ ID NO:149, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

6. The anti-ILT7 antibody or its antigen-binding fragment as described in any one of claims 1-5, comprising: (1) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with VH containing the amino acid sequence shown in SEQ ID NO:3 and VL containing the amino acid sequence shown in SEQ ID NO:4, respectively; or (2) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 99%, or 100% identity with VH containing the amino acid sequence shown in SEQ ID NO:3 and VL containing the amino acid sequence shown in SEQ ID NO:4, respectively; VL containing the amino acid sequences shown in SEQ ID NO: 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or 71 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity; or (3) VH containing the amino acid sequences shown in SEQ ID NO: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55, and VH containing the amino acid sequences shown in SEQ ID NO: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55, respectively. The VL containing the amino acid sequence shown in NO:60 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:56, 57, 58, or 59, and the VL containing the amino acid sequence shown in SEQ ID NO:72, respectively, having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:60; preferably, the anti-ILT7 antibody or its antigen-binding fragment comprises the VH containing the amino acid sequence shown in SEQ ID NO:58 or 59, and the VL containing the amino acid sequence shown in SEQ ID NO:72, respectively, having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence shown in SEQ ID NO:60; The VL containing the amino acid sequence shown in NO:72 has an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical; more preferably, the anti-ILT7 antibody or its antigen-binding fragment has an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH containing the amino acid sequence shown in SEQ ID NO:58 and the VL containing the amino acid sequence shown in SEQ ID NO:72; or, The anti-ILT7 antibody or its antigen-binding fragment comprises: (1) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with VH containing the amino acid sequence shown in SEQ ID NO:1 and VL containing the amino acid sequence shown in SEQ ID NO:2, respectively; or (2) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with VH containing the amino acid sequence shown in SEQ ID NO:5 and VL containing the amino acid sequence shown in SEQ ID NO:6, respectively; or (3) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with VH containing the amino acid sequence shown in SEQ ID NO:7 and VL containing the amino acid sequence shown in SEQ ID NO:6, respectively. The VL containing the amino acid sequence shown in SEQ ID NO:8 has an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical; or (4) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH containing the amino acid sequence shown in SEQ ID NO:9 and the VL containing the amino acid sequence shown in SEQ ID NO:10; or (5) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH containing the amino acid sequence shown in SEQ ID NO:11 and the VL containing the amino acid sequence shown in SEQ ID NO:

10. VL containing the amino acid sequence shown in NO:12 has an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical; or (6) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to VH containing the amino acid sequence shown in SEQ ID NO:13 and VL containing the amino acid sequence shown in SEQ ID NO:

14.

7. A myeloid cell adaptor targeting ILT7 and CD89, said myeloid cell adaptor being an anti-CD89 / ILT7 bispecific antibody comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain specifically binds to the CD89 extracellular Ig-like domain 2 and does not block the binding of IgA to CD89, and the second antigen-binding domain specifically binds to amino acid residues 420-446 of the ILT7 extracellular domain, said bispecific antibody comprising at least one of the following characteristics: (1) said bispecific antibody or its antigen-binding fragment is an asymmetric trivalent molecule capable of specifically binding to the ILT7 extracellular domain, and not cross-binding with ILT1, ILT2, ILT3, ILT4, ILT5, ILT6, ILT8, ILT11, LIR6 and LIR8. (1) The bispecific antibody or its antigen-binding fragment can specifically bind to CD89; (2) The bispecific antibody can cross-link CD89-expressing myeloid cells and ILT7-expressing target cells, thereby activating the myeloid cells and mediating their targeted killing of the target cells; (3) The myeloid cell activation mediated by the bispecific antibody must be achieved through cross-linking with ILT7-expressing target cells, thus avoiding non-targeted activation of the myeloid cells in the absence of target cells; at the same time, the myeloid cells do not release a large number of cytokines after activation, thus not triggering a cytokine storm; (4) The bispecific antibody has high molecular stability, is easy to form heterodimers, and is easy to obtain high-purity heterodimers through downstream purification processes; and (5) The bispecific antibody has significantly reduced Fc receptor binding affinity.

8. The myeloid cell adaptor targeting ILT7 and CD89 as described in claim 7, wherein, The first antigen-binding domain includes: (1) HCDR1 having the amino acid sequence shown in SEQ ID NO:73, HCDR2 having the amino acid sequence shown in SEQ ID NO:78 or 79, HCDR3 having the amino acid sequence shown in SEQ ID NO:80, 81, or 82, LCDR1 having the amino acid sequence shown in SEQ ID NO:83, LCDR2 having the amino acid sequence shown in SEQ ID NO:85, and LCDR3 having the amino acid sequence shown in SEQ ID NO:87, 88, or 89, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or, (2) HCDR1 having the amino acid sequence shown in SEQ ID NO:73, HCDR2 having the amino acid sequence shown in SEQ ID NO:78, HCDR3 having the amino acid sequence shown in SEQ ID NO:80 or 81, LCDR1 having the amino acid sequence shown in SEQ ID NO:83, LCDR2 having the amino acid sequence shown in SEQ ID NO:85, and LCDR3 having the amino acid sequence shown in SEQ ID NO:87 or 89, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or, (3) HCDR1 having the amino acid sequence shown in SEQ ID NO:73, HCDR2 having the amino acid sequence shown in SEQ ID NO:79, HCDR3 having the amino acid sequence shown in SEQ ID NO:80, LCDR1 having the amino acid sequence shown in SEQ ID NO:83, LCDR2 having the amino acid sequence shown in SEQ ID NO:85, and LCDR3 having the amino acid sequence shown in SEQ ID NO:88, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; or, (4) HCDR1 having the amino acid sequence shown in SEQ ID NO:73, HCDR2 having the amino acid sequence shown in SEQ ID NO:78, HCDR3 having the amino acid sequence shown in SEQ ID NO:82, LCDR1 having the amino acid sequence shown in SEQ ID NO:83, LCDR2 having the amino acid sequence shown in SEQ ID NO:85, and LCDR3 having the amino acid sequence shown in SEQ ID NO:87, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively; Preferably, the first antigen-binding domain comprises: HCDR1 having the amino acid sequence shown in SEQ ID NO:73, HCDR2 having the amino acid sequence shown in SEQ ID NO:78, HCDR3 having the amino acid sequence shown in SEQ ID NO:80 or 81, LCDR1 having the amino acid sequence shown in SEQ ID NO:83, LCDR2 having the amino acid sequence shown in SEQ ID NO:85, and LCDR3 having the amino acid sequence shown in SEQ ID NO:87 or 89, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

9. The myeloid cell adaptor targeting ILT7 and CD89 as described in claim 7 or 8, wherein, The second antigen-binding domain comprises: HCDR1 having the amino acid sequence shown in SEQ ID NO:16, HCDR2 having the amino acid sequence shown in SEQ ID NO:24 or 23, HCDR3 having the amino acid sequence shown in SEQ ID NO:25, LCDR1 having the amino acid sequence shown in SEQ ID NO:30, LCDR2 having the amino acid sequence shown in SEQ ID NO:36, and LCDR3 having the amino acid sequence shown in SEQ ID NO:38, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively. Preferably, the second antigen-binding domain comprises: HCDR1 having the amino acid sequence shown in SEQ ID NO:16, HCDR2 having the amino acid sequence shown in SEQ ID NO:24, HCDR3 having the amino acid sequence shown in SEQ ID NO:25, LCDR1 having the amino acid sequence shown in SEQ ID NO:30, LCDR2 having the amino acid sequence shown in SEQ ID NO:36, and LCDR3 having the amino acid sequence shown in SEQ ID NO:38, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

10. The myeloid cell adaptor targeting ILT7 and CD89 as described in any one of claims 7-9, wherein, The first antigen-binding domain includes: (1) A VH containing an amino acid sequence as shown in SEQ ID NO:98, and a VL containing an amino acid sequence as shown in SEQ ID NO:108 or 112, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of the VH and VL, respectively; (2) A VH containing the amino acid sequence shown in SEQ ID NO:101, and a VL containing the amino acid sequence shown in SEQ ID NO:111, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of the VH and VL, respectively; (3) A VH containing an amino acid sequence as shown in SEQ ID NO: 99 or 102, and a VL containing an amino acid sequence as shown in SEQ ID NO: 109, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of the VH and VL, respectively; (4) A VH containing the amino acid sequence shown in SEQ ID NO:103, and a VL containing the amino acid sequence shown in SEQ ID NO:113, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of the VH and VL, respectively; (5) A VH containing the amino acid sequence shown in SEQ ID NO:100, and a VL containing the amino acid sequence shown in SEQ ID NO:110, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of the VH and VL, respectively; (6) A VH containing the amino acid sequence shown in SEQ ID NO:97, and a VL containing the amino acid sequence shown in SEQ ID NO:107, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of the VH and VL, respectively; Preferably, the first antigen-binding domain comprises: a VH containing the amino acid sequence shown in SEQ ID NO:98, and a VL containing the amino acid sequence shown in SEQ ID NO:108 or 112, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of the VH and VL, respectively; or a VH containing the amino acid sequence shown in SEQ ID NO:101, and a VL containing the amino acid sequence shown in SEQ ID NO:108 or 112. The amino acid sequence VL shown in NO:111, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of VH and VL, respectively.

11. The myeloid cell adaptor targeting ILT7 and CD89 as described in any one of claims 7-10, wherein, The second antigen-binding domain comprises: a VH containing the amino acid sequence shown in SEQ ID NO: 56, 57, 58, or 59, and a VL containing the amino acid sequence shown in SEQ ID NO: 72, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of the VH and VL, respectively; preferably, the second antigen-binding domain comprises: a VH containing the amino acid sequence shown in SEQ ID NO: 58 or 59, and a VL containing the amino acid sequence shown in SEQ ID NO:

72. The amino acid sequence VL shown in NO:72, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of VH and VL, respectively.

12. The myeloid cell adaptor targeting ILT7 and CD89 as described in any one of claims 7-11, having a 2+1scFv-IgG or scFv-Fab-Fc:Fab-Fc structure, said structure comprising subunits A and B, wherein, Subunit A is a bivalent scFv-Fab-Fc form, with the scFv domain and the Fab-Fc domain being the first and second antigen-binding domains, respectively. Subunit B is a monovalent Fab-Fc form, serving as the second antigen-binding domain. The scFv domain is fused to or operatively connected to the Fab-Fc domain via a linker.

13. The myeloid cell adaptor targeting ILT7 and CD89 as described in any one of claims 7-12, wherein each subunit comprises a constant region, the constant region comprising a heavy chain constant region and a light chain constant region, the heavy chain constant region comprising an engineered human IgG heavy chain constant region, the engineering comprising introducing "bumps and cavities" mutations and / or "isoelectric point (pI)" mutations into the human IgG heavy chain constant region having the amino acid sequence shown in SEQ ID NO:

163.

14. The myeloid cell adaptor targeting ILT7 and CD89 as described in claim 13, wherein, A "protrusion" mutation is introduced in the Fc region of subunit A, and a "cavity" mutation is introduced in the Fc region of subunit B. The "protrusion" mutation includes the amino acid mutation T366W, and the "cavity" mutation includes the amino acid mutations T366S, L368A, and Y407V. The "pI mutation" includes introducing a mutation that increases the pI value in one heavy chain constant region containing the "protrusion" mutation of the myelocyte adaptor, which includes the amino acid mutations Q196K and / or N276K, and introducing a mutation that decreases the pI value in another heavy chain constant region containing the "cavity" mutation of the myelocyte adaptor, which includes one or more of the amino acid mutations G137E, G138S, N203D, K274Q, and Q419E.

15. The myeloid cell adaptor targeting ILT7 and CD89 as described in claim 13 or 14, wherein, The two heavy chain constant regions of the myeloid cell adaptor may also contain mutations that reduce the function of the Fc effector. Furthermore, the heavy chain constant regions contain human IgG1 Fc with amino acid substitutions of L234F / L235E / P331S (EU numbering system).

16. The myeloid cell adaptor targeting ILT7 and CD89 as described in any one of claims 7-15, wherein, The light chain constant region is the human κ light chain constant region of the amino acid sequence shown in SEQ ID NO:

166.

17. The myeloid cell adaptor targeting ILT7 and CD89 as described in any one of claims 7-16, wherein the myeloid cell adaptor comprises one first polypeptide chain, one second polypeptide chain, and two identical third polypeptide chains, wherein, The first polypeptide chain comprises, from the N-terminus to the C-terminus, an anti-CD89 antibody or its antigen-binding fragment, a heavy chain variable region and a first heavy chain constant region of an anti-ILT7 antibody or its antigen-binding fragment as described in any one of claims 1-6, wherein the anti-CD89 antibody or its antigen-binding fragment is an anti-CD89 scFv molecule; the second polypeptide chain comprises, from the N-terminus to the C-terminus, a heavy chain variable region and a second heavy chain constant region of the anti-ILT7 antibody or its antigen-binding fragment as described in any one of claims 1-6; and the third polypeptide chain comprises, from the N-terminus to the C-terminus, a light chain variable region and a light chain constant region of the anti-ILT7 antibody or its antigen-binding fragment as described in any one of claims 1-6.

18. The myeloid cell adaptor targeting ILT7 and CD89 as described in any one of claims 7-17, wherein the myeloid cell adaptor comprises one first polypeptide chain, one second polypeptide chain, and two third polypeptide chains, wherein, The first polypeptide chain and the third polypeptide chain constitute subunit A of the myeloid cell adaptor; the second polypeptide chain and the third polypeptide chain constitute subunit B of the myeloid cell adaptor, and: (1) the first polypeptide chain contains VH1-L1-VL1-L2-VH2-CH a VH1-L1-VL1 specifically binds to CD89 extracellular Ig-like domain 2, where L1 and L2 represent linkers, and VH2-CH a (1) Specifically binds to amino acid residues 420-446 of the extracellular domain of ILT7; (2) The second polypeptide chain contains VH2-CH b The VH2-CH b (3) The third polypeptide chain contains VL2-CL, which specifically binds to amino acid residues 420-446 of the extracellular domain of ILT7.

19. The myeloid cell adaptor targeting ILT7 and CD89 as described in claim 18, wherein, The VH1 comprises HCDR1 having the amino acid sequence shown in SEQ ID NO:73, HCDR2 having the amino acid sequence shown in SEQ ID NO:78, and HCDR3 having the amino acid sequence shown in SEQ ID NO:80 or 81, or amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of the HCDR1, HCDR2, and HCDR3, respectively. L1 represents a linker containing the amino acid sequence shown in SEQ ID NO:167; The VL1 comprises LCDR1 having the amino acid sequence shown in SEQ ID NO:83, LCDR2 having the amino acid sequence shown in SEQ ID NO:85, and LCDR3 having the amino acid sequence shown in SEQ ID NO:87 or 89, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of the LCDR1, LCDR2, and LCDR3, respectively. The L2 represents a linker containing the amino acid sequence shown in SEQ ID NO:168; the VH2 contains HCDR1 having the amino acid sequence shown in SEQ ID NO:16, HCDR2 having the amino acid sequence shown in SEQ ID NO:24, and HCDR3 having the amino acid sequence shown in SEQ ID NO:25, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of the HCDR1, HCDR2, and HCDR3, respectively. The CH a It contains the constant region of the human IgG1 heavy chain, as shown in SEQ ID NO:

165.

20. The myeloid cell adaptor targeting ILT7 and CD89 as described in claim 18 or 19, wherein, VH1 comprises an amino acid sequence as shown in SEQ ID NO: 98 or 101, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it; VL1 comprises an amino acid sequence as shown in SEQ ID NO: 108, 111, or 112, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it; VH2 comprises SEQ ID NO: 98 or 101. The amino acid sequence shown in NO:58 or 59, or an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to it.

21. The myeloid cell adaptor targeting ILT7 and CD89 as described in any one of claims 18-20, wherein, The CH b Includes the constant region of the human IgG1 heavy chain, as shown in SEQ ID NO:

164.

22. The myeloid cell adaptor targeting ILT7 and CD89 as described in any one of claims 18-21, wherein, The VL2 comprises LCDR1 having the amino acid sequence shown in SEQ ID NO:30, LCDR2 having the amino acid sequence shown in SEQ ID NO:36, and LCDR3 having the amino acid sequence shown in SEQ ID NO:38, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequences of the LCDR1, LCDR2, and LCDR3, respectively. The CL is selected from the human κ constant region or the human λ constant region, preferably with an amino acid sequence like the human κ constant region shown in SEQ ID NO:

166.

23. The myeloid cell adaptor targeting ILT7 and CD89 as described in any one of claims 18-22, wherein, The VL2 comprises an amino acid sequence as shown in SEQ ID NO:72, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it.

24. The myeloid cell adaptor targeting ILT7 and CD89 as described in any one of claims 17-23, comprising: (1) A first polypeptide chain, the first polypeptide chain comprising: VH1 containing the amino acid sequence shown in SEQ ID NO: 98 or 101, L1 containing the amino acid sequence shown in SEQ ID NO: 167, VL1 containing the amino acid sequence shown in SEQ ID NO: 108, 111 or 112, L2 containing the amino acid sequence shown in SEQ ID NO: 168, VH2 containing the amino acid sequence shown in SEQ ID NO: 58 or 59, and CH containing the amino acid sequence shown in SEQ ID NO:

165. a ; (2) A second polypeptide chain, the second polypeptide chain comprising: VH2 containing the amino acid sequence shown in SEQ ID NO:58 or 59, CH containing the amino acid sequence shown in SEQ ID NO:

164. b ;as well as (3) Two third polypeptide chains, the third polypeptide chains comprising: VL2 containing the amino acid sequence shown in SEQ ID NO:72, and CL containing the amino acid sequence shown in SEQ ID NO:

166.

25. The myeloid cell adaptor targeting ILT7 and CD89 as described in any one of claims 17-24, wherein the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 169 or 172, the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 170, and the third polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 171; or, The first polypeptide chain contains an amino acid sequence as shown in SEQ ID NO:173, 175 or 176, the second polypeptide chain contains an amino acid sequence as shown in SEQ ID NO:174, and the third polypeptide chain contains an amino acid sequence as shown in SEQ ID NO:

171.

26. A nucleic acid encoding an anti-ILT7 antibody or an antigen-binding fragment thereof as described in any one of claims 1-6, or a myeloid cell adaptor targeting ILT7 and CD89 as described in any one of claims 7-25.

27. An expression vector capable of expressing the nucleic acid of claim 26.

28. A host cell comprising the nucleic acid of claim 26 or the expression vector of claim 27.

29. A method for preparing an anti-ILT7 antibody or its antigen-binding fragment as described in any one of claims 1-6, or a myeloid cell adaptor targeting ILT7 and CD89 as described in any one of claims 7-25, using the host cell of claim 28, comprising: (1) Express the anti-ILT7 antibody or its antigen-binding fragment, or myeloid cell adaptor targeting ILT7 and CD89, in the host cells, and (2) Isolate the anti-ILT7 antibody or its antigen-binding fragment, or myeloid cell adaptor targeting ILT7 and CD89, from the host cells or cell cultures.

30. An immunoconjugate, chimeric antigen receptor, engineered T-cell receptor, or oncolytic virus comprising the anti-ILT7 antibody or its antigen-binding fragment as described in any one of claims 1-6.

31. A pharmaceutical composition comprising a myeloid cell adaptor targeting ILT7 and CD89 as described in any one of claims 7-25, or an immunoconjugate as described in claim 30, a chimeric antigen receptor, an engineered T-cell receptor, or an oncolytic virus, and a pharmaceutically acceptable carrier.

32. A kit comprising an effective amount of a myeloid cell adaptor targeting ILT7 and CD89 as described in any one of claims 7-25, an immunoconjugate as described in claim 30, a chimeric antigen receptor, an engineered T-cell receptor or an oncolytic virus, or a pharmaceutical composition as described in claim 31, and optionally at least one additional therapeutic agent, said additional therapeutic agent including therapeutic agents for pDC-related conditions, tumor therapeutic agents, and anti-infective therapeutic agents.

33. A method for treating pDC-related conditions, the method comprising administering to a subject in need an effective amount of a myeloid cell adaptor targeting ILT7 and CD89 as described in any one of claims 7-25, an immunoconjugate as described in claim 30, a chimeric antigen receptor, an engineered T-cell receptor or oncolytic virus, a pharmaceutical composition as described in claim 31, or a kit as described in claim 32.

34. The method of claim 33, wherein, The pDC-related conditions include autoimmune diseases, cancer, and conditions associated with pDC tissue accumulation; the subjects may be humans, non-human primates, or other mammals.

35. The method of claim 34, wherein, The autoimmune diseases include systemic lupus erythematosus, cutaneous lupus, discoid lupus nephritis, lupus nephritis, multiple sclerosis, morphine scleroderma, dermatomyositis, polymyositis, psoriasis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, irritable bowel syndrome, and type 1 diabetes; the cancers include solid tumors and hematologic malignancies.

36. A method for detecting and / or quantifying ILT7 or ILT7-expressing cells in a sample, or a method for screening patients with pDC-related conditions who respond to treatment with a myeloid cell adaptor targeting ILT7 and CD89 as described in any one of claims 7-25, comprising incubating the sample or a biological sample isolated from the patient with an anti-ILT7 antibody or its antigen-binding fragment as described in any one of claims 1-6, or a myeloid cell adaptor targeting ILT7 and CD89 as described in any one of claims 7-25, and detecting whether the antibody binds to the sample or the biological sample.

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