Myeloid cell engager antibody targeting CD20 and CD89 and use thereof

Abstract

The present invention relates to a myeloid cell engager antibody targeting CD20 and CD89. The present invention also provides a method for preparing the myeloid cell engager antibody and a method for treating or preventing diseases.

Description

Myeloid cell engager antibodies targeting cd20 and cd89 and uses thereof TECHNICAL FIELD

[0001] The present invention relates to Myeloid Cell Engager antibodies (e.g., bispecific antibodies, multispecific antibodies) targeting CD20 and CD89. The present invention also provides methods of making the Myeloid Cell Engager antibodies, and methods of using the same for treating or preventing diseases. BACKGROUND

[0002] CD89, also known as Immunoglobulin A (IgA) Fc Receptor (FcαRI), is a receptor protein that is expressed in a restricted manner on myeloid cells, including neutrophils, eosinophils, monocytes, and macrophages. Binding and activation of CD89 triggers phagocytosis, trogocytosis, antigen presentation functions of myeloid cells, and initiates production and release of superoxide and various cytokines and immune mediators by myeloid cells (Bakema and Egmond, Mucosal Immunol 2011, 4:612-624). Myeloid Cell Engager (or CD89 Myeloid Cell Engager) antibodies (e.g., bispecific antibodies, multispecific antibodies) that co-target CD89 and a specific antigen on the surface of a target cell can be used to recruit and activate CD89 + Myeloid cells specifically attack target cells (Sewnath et al., Expert Opin Biol Ther 2022, 22:983-995).

[0003] CD20 is a multi-pass transmembrane protein with a molecular weight of about 35 kDa that is specifically expressed on pre-B lymphocytes and mature B lymphocytes, and is involved in regulating the cycle initiation and proliferation of B lymphocytes (Golay et al., J Immunol 1985, 135:3795-3801; Tedder et al., Eur J Immunol 1986, 16:881-887). CD20 is widely expressed in most B-cell malignancies, such as non-Hodgkin lymphoma (NHL) and B-chronic lymphocytic leukemia (B-CLL) (Anderson et al., Blood 1984, 63:1424-1433); in addition, it is also expressed in B cells involved in autoimmune diseases and inflammatory diseases, so CD20 is a target for treating B-cell malignancies and certain autoimmune diseases.

[0004] Some CD20-based targeted therapeutics have been successfully used to treat B-cell malignancies, for example, anti-CD20 monoclonal antibodies (e.g., Rituximab, Ocrelizumab, Ofatumumab, Ublituximab, Obinutuzumab), anti-CD20 / CD3 T cell engager antibodies (e.g., Glofitamab, Epcoritamab, Mosunetuzumab). However, anti-CD20 / CD3 T cell engager antibodies are often accompanied by cytokine release syndrome (CRS) caused by T cell overactivation during treatment, which leads to serious side effects (Liu et al., J Hematol Oncol 2023, 16:90).

[0005] In view of this, the present application develops a novel myeloid cell engager antibody targeting CD20 and CD89, which can not only effectively activate one or more myeloid effector cells (e.g., neutrophils and macrophages) other than T cells, selectively kill target cells (e.g., B cells) expressing CD20, but also does not trigger cytokine storm, and is easier to obtain high-purity target heterodimeric drug products in the production process. SUMMARY

[0006] The present application provides an anti-CD89 monospecific antibody and a myeloid cell engager antibody (e.g., multispecific antibody, bispecific antibody) constructed based thereon. In one aspect, the anti-CD89 monospecific antibody of the present application can specifically recognize and bind to the extracellular Ig-like domain 2 of CD89, and does not affect the binding of CD89 to IgA; on the other hand, the present application constructs a myeloid cell engager multispecific antibody, especially a bispecific antibody, based on the anti-CD89 monospecific antibody. The bispecific antibody is a non-symmetrical heterodimeric molecule containing a constant region, which can simultaneously bind and cross-link myeloid cells expressing CD89 and pathogenic / target cells or cancer cells expressing a specific antigen / protein, 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 cytokines upon activation, thus the risk of the myeloid cell engager antibody directly triggering a cytokine storm is very low. In yet another aspect, the present application introduces a series of amino acid mutations in the sequence of the antibody to optimize its CMC (Chemistry, Manufacturing, and Controls) properties, including improving the stability of the molecule, facilitating the formation of heterodimers during expression, and facilitating the separation of the target 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-CD89 antibody or antigen binding fragment thereof, which is capable of specifically recognizing and binding to CD89 extracellular Ig-like domain 2, and the binding site of which to CD89 is different from the binding site of CD89 to IgA.

[0008] In some embodiments, the anti-CD89 antibody comprises a fully human or humanized anti-CD89 antibody or a derivative / optimized antibody thereof, (e.g., the Mab 14.1 antibody described in WO2002064634 and an optimized antibody thereof). In one embodiment, the fully human or humanized anti-CD89 antibody or a derivative / optimized antibody thereof or antigen binding fragment thereof does not inhibit the binding of IgA to CD89 or does not compete with IgA for binding to CD89.

[0009] 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 (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 stability, preferably a scFv molecule with a disulfide bond at the VH-VL interface. The disulfide bond at the VH-VL interface is formed by introducing at least one cysteine mutation in the VH domain and the VL domain of the anti-CD89 scFv molecule, respectively, so that at least one disulfide bond is formed at the interface of 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.

[0010] In one aspect, the present application provides an isolated anti-CD20 antibody or antigen binding fragment thereof, which is capable of specifically recognizing and binding to CD20.

[0011] In one aspect, the present application provides a CD89 myeloid cell engager comprising a first antigen binding domain and a second antigen binding domain. In some embodiments, the myeloid cell engager is a bispecific antibody. Wherein the first antigen binding domain specifically binds to a particular membrane protein on a target cell, including a cell associated with a proliferative disease (e.g., cancer), an autoimmune disease, an inflammatory disease, and / or a fibrotic disease, including any membrane protein or receptor selectively expressed on the target cell other than CD89, e.g., CD20 expressed on the surface of a B cell, a membrane protein or antigen specifically expressed on a cell infected by a pathogen; and the second antigen binding domain specifically binds to CD89 on the surface of a myeloid cell, preferably a VH-VL interface disulfide-containing scFv domain. The bispecific antibody is capable of directing the activation of a CD89-expressing myeloid cell and is capable of effectively mediating the activated myeloid cell to direct killing / elimination of the target cell.

[0012] Further, the CD89 myeloid cell engager 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 comprising native and mutant protein forms of human IgG (including human IgG1, IgG2, IgG3, IgG4) heavy chain constant region, 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 the separation of the desired heterodimer from homodimer byproducts by purification processes, and / or mutations that reduce or enhance Fc Effector Function of the heavy chain constant region. 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 an amino acid sequence as set forth in SEQ ID NO: 59).

[0013] An exemplary CD89 myeloid cell engager of the present application is an anti-CD20 / CD89 bispecific antibody comprising a first antigen binding domain and a second antigen binding domain, the first and second antigen binding domains are capable of specifically binding to CD20 on the surface of a target cell (e.g., a B cell) and CD89 on the surface of a myeloid cell, respectively. The bispecific antibody is a structurally stable heterodimer, capable of cross-linking a CD89-expressing myeloid cell and a CD20-expressing target cell (e.g., a B cell), and is capable of effectively mediating the activated CD89 +The myeloid cells target kill the target cells, achieving the goal of clearing the target cells, and the CD89-expressing myeloid cells do not release a large amount of cytokines after activation, thus there is no or substantially no risk of triggering a cytokine storm.

[0014] In some embodiments, the first antigen binding domain of the anti-CD20 / CD89 bispecific antibody is selected from an anti-CD20 antibody or antigen binding fragment thereof that is capable of specifically binding CD20; the second antigen binding domain of the bispecific antibody is selected from an anti-CD89 antibody or antigen binding fragment thereof that is capable of specifically binding the extracellular domain of CD89, preferably an antibody that specifically binds CD89 extracellular Ig-like domain 2. In some embodiments, the bispecific antibody mediates the directed killing of target cells by myeloid cells by specifically binding CD89 on the surface of myeloid cells and CD20 on the surface of target cells (e.g., B cells). In one embodiment, the bispecific antibody induces the killing of target cells (e.g., B cells) expressing CD20 by myeloid cells (e.g., neutrophils and macrophages) by specifically binding CD89 on the surface of myeloid cells and CD20 on the surface of target cells and activating the myeloid cells.

[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, and the anti-CD20 / CD89 bispecific antibodies, 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 make the anti-CD89 antibodies or antigen binding fragments thereof, and the anti-CD20 / CD89 bispecific antibodies 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 a pharmaceutical composition comprising the anti-CD20 / CD89 bispecific antibodies described herein, and a pharmaceutically acceptable carrier.

[0017] In another aspect, the present application relates to a kit comprising the anti-CD20 / CD89 bispecific antibodies described herein, or the pharmaceutical composition of the present application, and optionally at least one additional therapeutic agent.

[0018] In another aspect, the present application relates to a method of treating and / or preventing a CD20-associated disorder, or use, comprising administering to a subject in need thereof a therapeutically effective amount of the anti-CD20 / CD89 bispecific antibody, the pharmaceutical composition, or the kit of the present application. Alternatively, the present application relates to the use of the anti-CD20 / CD89 bispecific antibody, the pharmaceutical composition, or the kit in the manufacture of a medicament for treating and / or preventing a CD20-associated disorder. Alternatively, the present application relates to the anti-CD20 / CD89 bispecific antibody, the pharmaceutical composition, or the kit for use in treating and / or preventing a CD20-associated disorder.

[0019] In some embodiments, the CD20-associated disorder comprises B-cell lymphoma (e.g., NHL, including Diffuse Large B-cell Lymphoma [DLBCL], Follicular Lymphoma [FL], Marginal Zone Lymphoma, Small Lymphocytic Lymphoma, Mantle Cell Lymphoma [MCL], and Burkitt's Lymphoma) and B-cell leukemia (e.g., CLL, ALL, HCL), and autoimmune diseases caused by B-cell auto-reactivity (e.g., systemic lupus erythematosus [SLE], cutaneous lupus, discoid lupus, lupus nephritis, scleroderma, dermatomyositis [DM], polymyositis [PM], psoriasis, psoriatic arthritis, rheumatoid arthritis, multiple sclerosis, systemic sclerosis, vasculitis, thrombocytopenia, myasthenia gravis, type I diabetes).

[0020] In another aspect, the present application relates to a method of depleting target cells expressing CD20 (e.g., pathogenic B-cells) in a subject in need thereof, comprising administering to the subject in need thereof a therapeutically effective amount of the anti-CD20 / CD89 bispecific antibody, the pharmaceutical composition, or the kit of the present application.

[0021] The present application is further illustrated by the following drawings and specific embodiments, other features and advantages of which will be apparent from the drawings and detailed description, and it is to be understood that changes in the details can be made by those skilled in the art without departing from the scope of the present application which is defined by the appended claims. All references cited in the present application, including publications, patents, and patent applications, are incorporated herein by reference in their entirety. BRIEF DESCRIPTION OF DRAWINGS

[0022] 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, with control antibody being parent single-chain antibody Mab 14.1-HL1405.

[0023] Figure 2. Schematic diagram of the structure of anti-CD20 / CD89 bispecific antibody. The configuration of the exemplary bispecific antibody 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 the VH-VL interface, the IgG domain or Fab-Fc domain is the sequence of anti-CD20 antibody, 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, and the antibody heavy chain constant region can include "knob-into-hole" mutations, "pi mutations" and L234F / L235E / P331S mutations (EU Numbering, referred to as "TM mutations").

[0024] Figure 3. Detection of the activity of anti-CD20 / CD89 bispecific antibody Cy09-GFT in co-binding CD20 and CD89 by ForteBio, wherein the control antibodies include Obinutuzumab and anti-CD89 antibody Mab14.1-HL1405.

[0025] Figure 4. Detection of whether the anti-CD20 / CD89 bispecific antibody Cy09-GFT has the activity of blocking the binding of CD89 and human IgA by ForteBio.

[0026] Figure 5. Detection of the binding activity of anti-CD20 / CD89 bispecific antibody Cy09-GFT to CD20-expressing Raji cells by flow cytometry, wherein the control antibody is Obinutuzumab.

[0027] Figure 6. Detection of the binding activity of anti-CD20 / CD89 bispecific antibody Cy09-GFT to CD89-expressing Jurkat cells by flow cytometry.

[0028] Figure 7. Detection of the biological activity of anti-CD20 / CD89 bispecific antibody Cy09-GFT by reporter gene method, wherein the target cell (T) is CD20-expressing Raji cell, and the effector cell (E) is Jurkat cell co-expressing CD89 and firefly luciferase reporter gene regulated by NFAT response element, and T+E in the figure represents the co-incubation group of target cells and effector cells, and E represents the control group containing only effector cells.

[0029] Figure 8. Changes in the proportion of B cells in the PBMC live cell population after the primary leukocytes derived from the peripheral blood of 13 healthy people were treated with the anti-CD20 / CD89 bispecific antibody Cy09-GFT, wherein the control antibody was Glofitamab. The lower right histogram shows the minimum residual amount of primary B cells from each healthy person after treatment with Cy09-GFT or Glofitamab.

[0030] Figure 9. Detection of the concentration of each cytokine in the culture supernatant after the primary leukocytes derived from the peripheral blood of 13 healthy people were treated at the highest drug concentration for about 20 hours to analyze whether the anti-CD20 / CD89 bispecific antibody Cy09-GFT would induce a large amount of cytokine release from myelocytes, wherein the negative control was blank culture medium and the positive control was Glofitamab. The content of each cytokine in the culture supernatant was expressed as the fold of its corresponding negative control (baseline value).

[0031] Figure 10. Detection of the ADCC activity of the anti-CD20 / CD89 bispecific antibody Cy09-GFT using a reporter gene method, wherein the positive control antibody was the anti-CD20 antibody Obinutuzumab with a wild-type Fc sequence.

[0032] Figure 11. Flow cytometry detection of changes in the proportion of CD19 + B cells in the peripheral blood of cynomolgus monkeys after a single intravenous injection of the anti-CD20 / CD89 bispecific antibody Cy09-GFT. The arrow in the figure indicates the administration time point. DETAILED DESCRIPTION

[0033] DEFINITIONS

[0034] 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. For the purposes of the present application, the following terms are defined below.

[0035] As used herein, the terms "CD20," "CD20 receptor," "CD20 antigen," and "CD20 protein" are used interchangeably and refer to the protein also known as B-lymphocyte surface differentiation antigen Bp35, B-lymphocyte surface antigen Bl, BM5 and LF5, Leukocyte surface antigen Leu-16. Unless otherwise indicated, as used herein "CD20" refers to any native form of human CD20, which can be the full-length CD20 amino acid sequence as set forth in UniProt Accession No. P11836, as naturally expressed by a cell (including a B cell), or as expressed by a cell transfected with a CD20 gene or cDNA. The term includes naturally occurring allelic variants and splice variants of CD20, isoforms, homologs, and species homologs. CD20 can be isolated from a human body, or can be produced recombinantly or synthetically.

[0036] As used herein, the terms "CD89," "CD89 protein," "CD89 receptor," "FcaRI," or "IgA receptor" are used interchangeably. CD89 is a glycosylated, transmembrane receptor that comprises two extracellular domains, a transmembrane domain, and an intracellular domain, which is constitutively expressed on neutrophils, eosinophils, macrophages, and most monocytes, and expression levels are influenced by inflammatory microenvironments. For example, neutrophils have significantly increased expression of CD89 after stimulation with G-CSF or GM-CSF (Weisbart et al., Nature 1988, 332:647). CD89 extracellular domain 1 (D1) binds IgAl and IgA2 with moderate affinity (Kd~5xlO6M) and extracellular domain 2 (D2) binds IgAl and IgA2 with high affinity (Kd~5xlO7M). D ~5xlO6M -7 CD89-mediated effector cell functions include ADCC and phagocytosis, as well as the release of limited inflammatory mediators and cytokines (Shen, Immunol Res 1992, 11:273; Morton et al., Crit Rev Immunol 1996, 16:423).

[0037] As used herein, a "CD20-expressing cell" or "CD20-expressing target cell" can be a naturally occurring cell or cell line (e.g., a B cell), or a recombinantly produced cell line by introduction of a nucleic acid encoding CD20 into a host cell.

[0038] As used herein, a "CD89-expressing cell" can be a naturally occurring cell or cell line (e.g., a neutrophil, eosinophil, macrophage, monocyte), or a recombinantly produced cell line by introduction of a nucleic acid encoding CD89 into a host cell.

[0039] In the present context, "bispecific" or "bi-specific" is intended to include any antibody or antigen-binding fragment capable of specifically binding two different antigens or epitopes, comprising two separate antigen-binding domains and each having a unique antigen-binding specificity. For example, where one antigen-binding domain binds to a first antigen or epitope, the other antigen-binding domain binds to a second antigen different from the first antigen, or a second different epitope of the first antigen. The first and second are used only to facilitate differentiation between binding to different antigens or epitopes, and the use of these terms is not intended to impart a particular order of binding to different antigens or epitopes unless explicitly so stated.

[0040] 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 antigenic epitope of a single antigen. In some embodiments, examples of the monospecific antibody include the anti-CD20 antibodies and anti-CD89 antibodies of the present application.

[0041] 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 with the target antigen. In the present application, "antigen-binding domain" also refers to a specific region on the antibody or antigen-binding fragment or derivative thereof that interacts with a specific epitope on CD20 or CD89, by binding, steric hindrance, stabilizing / destabilizing, spatial distribution, etc., to reach a dynamic equilibrium between the two.

[0042] 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 the amino terminus to the carboxy terminus. The term "J region" refers to the subsequence of the variable region that encodes the C-terminal portion including 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 human germline sequences, or can be naturally occurring or artificially modified.

[0043] 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-CD20 antibodies or antigen-binding fragments thereof, the anti-CD89 antibodies or antigen-binding fragments thereof, or the anti-CD20 / CD89 bispecific antibodies of the present application, and Table 1 shows the location numbering of the antibody CDR amino acid sequences determined using the 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.

[0044] 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).

[0045] 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 unambiguously apply this "Kabat numbering" system to variable region sequences of any antibody without the need for any experimental data other than the sequence itself. Unless otherwise indicated, the numbering of particular amino acid residues in the variable region of the antigen binding domain of an anti-CD20 antibody, an anti-CD89 antibody, or an anti-CD20 / CD89 bispecific antibody of the application is according to the Kabat numbering system.

[0046] Antibodies exist as intact immunoglobulins or as a number of fragments produced by digestion with various peptidases. Although various antibody fragments are defined in terms of the digestion of intact antibodies, one of skill will appreciate that such fragments can be synthesized de novo either chemically or by utilizing recombinant DNA methodologies. In the present context, the term "antigen binding fragment" of an antibody (or simply "antibody portion" or "antibody fragment") refers to one or more portions of an antibody that contains one or more CDRs or any other antigen binding site that is capable of binding to an antigen (e.g., CD20 or the extracellular domain of CD20, CD89 or the extracellular domain of CD89), but which does not have the structure of an intact antibody. An antigen binding fragment can have the same antigenic specificity as the intact antibody. In certain embodiments, an antigen binding fragment can contain one or more CDRs from a particular antibody grafted to a framework region from one or more different antibodies. Antigen binding fragments include, but are not limited to: (1) "Fab" fragments, a monovalent fragment consisting of the VH, VL, CL, and CHI domains; (2) "F(ab')2" fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (3) "Fv" fragments 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) "Fd" fragments consisting of the VH and CHI domains; (5) "single chain Fv antibody" ("scFv"), "single chain antibody" or "scFv molecule" refers to an engineered antibody in which the light chain variable domain is directly linked to the heavy chain variable domain either by a short linker or by a linker peptide (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 (Zapata et al., Protein Eng 1995, 8:1057-1062; US 5641870) that is linked to a complementary light chain polypeptide to form a pair of antigen binding regions; (6) "dAb" fragments (Ward et al., Nature 1989, 341 :544-546; WO 90 / 05144A1) comprise a single variable domain, e.g., VH.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).

[0047] 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-CD20 antibody, an anti-CD89 antibody, or an anti-CD20 / CD89 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 to comprise from its amino-terminus to its carboxy-terminus the following portions: Cysteine residue at position 226 or Proline residue at position 230, according to the EU numbering system / convention, as found in Kabat et al., Sequences of Proteins of Immunological Interest, 1991, 5th Ed., NIH Publication 91-3242.

[0048] The term "anti-CD20 antibody" or "an antibody that specifically binds CD20" refers to any form of antibody or fragment thereof that specifically binds CD20 and encompasses both polyclonal and monoclonal antibodies, as well as biologically functional antibody fragments.

[0049] 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. The anti-CD89 antibody, the anti-CD89 antibody portion in the anti-CD20 / CD89 bispecific antibody of the application 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.

[0050] In the context of the present application, 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 biologies (e.g., a biological sample such as blood, serum, plasma, or a tissue sample), that is, the binding is selective for the target molecule and is not significantly displaced by other molecules, e.g., by non-target molecules. For example, an antibody that specifically binds to a target molecule (which can be an antigen) binds to the target molecule with greater affinity, greater avidity, more readily, and / or with longer duration than it binds to other non-target molecules. Antibodies that react with a particular protein can be identified using a variety of techniques. For example, ELISA immunoassays, flow cytometric fluorescence-activated cell sorting (FACS), cellular fluorescence assays, and Surface Plasmon Resonance (SPR) techniques can be used to determine whether an antibody of the application or antigen-binding fragment thereof binds to a target antigen / protein. Typically, specific or selective binding of an antibody or binding agent to an antigen will produce a signal at least twice the background signal, and more typically at least 10-100 times the background signal, under the assay conditions, and will not substantially bind to other antigens / proteins present in the sample. In certain embodiments, an antibody specifically binds to a target antigen / protein with an equilibrium dissociation constant (K D ) value of <1 μΜ, <100 nM, <10 nM, <1 nM, or <0.1 nM.

[0051] In the context of the present application, the term "monoclonal antibody" 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 a single binding 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, and can be made using recombinant DNA methods (see US4816567), and can be isolated from phage antibody libraries, 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.

[0052] In the present context, the term "chimeric antibody" refers to an antibody containing 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).

[0053] In the present context, the term "humanized antibody or humanized antigen binding fragment" refers to an antibody or antigen binding fragment comprising 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 parts 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 of the antibody by substituting (e.g. by back mutation) amino acid residues in the human immunoglobulin FR regions with the corresponding amino acid residues in the non-human species antibody. In some embodiments, the FR regions derived from a human can comprise the same amino acid sequence as in the human antibody from which it is derived, or it can comprise some amino acid changes, e.g. no more than 3, 2, or 1 amino acid changes. In some embodiments, the amino acid changes can be present only in the heavy chain FR regions, only in the light chain FR regions, or in both chains.

[0054] In the context of the present disclosure, the term "corresponding human germline sequence" refers to an antibody variable region amino acid sequence or subsequence that has the highest amino acid sequence identity to a reference human germline immunoglobulin variable region amino acid sequence, as compared 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.

[0055] The anti-CD20 antibodies, anti-CD89 antibodies, anti-CD20 / CD89 bispecific antibodies of the present disclosure 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., CD20 and / or CD89).

[0056] In the context of the present disclosure, the term "antigen" refers to a molecule that is capable of being bound by a binding agent, e.g., an antibody or antigen-binding fragment thereof. In some embodiments, an antigen is capable of being used to immunize an animal and produce antibodies in the animal that are capable of binding to the antigen, which can have one or more epitopes that interact with the antibodies.

[0057] The term "epitope" or "antigen binding epitope" or "binding epitope" as used herein refers to a protein determinant, an antigenic portion of a molecule that is recognized by and specifically binds to an antibody. Epitopes are typically groups of surface groupings of molecules such as amino acids or sugar side chains, and are typically formed by secondary and tertiary structural characteristics of a molecule, as well as by primary charge characteristics. The portion of the epitope recognized by an antibody or antigen-binding fragment thereof is called the paratope.

[0058] In the context of the present disclosure, the term "CD89 myeloid engager", "myeloid engager antibody", or "myeloid engager" refers to a polypeptide comprising an anti-CD89 antibody or antigen-binding fragment thereof and at least one antibody or antigen-binding domain that targets a target cell surface specific antigen, including a multispecific antibody or bispecific antibody.

[0059] The term "affinity" or "binding affinity" refers to the intrinsic binding capacity of an interaction between molecules, 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 indicated, "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 generally be expressed by 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 those used in the present application.

[0060] The term "fusion" or "fused" as used herein in reference 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 naturally. The fused amino acid sequence can be produced recombinantly by two genes encoding the polynucleotide sequences, and can be expressed by methods of introducing the construct containing the recombinant polynucleotide into a host cell.

[0061] 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, one, two, three, four, five, and / or six CDRs of an anti-CD89 antibody, or an anti-CD20 / CD89 bispecific antibody of the present application have amino acid mutations 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 increasing the stability (e.g., reducing or eliminating the risk of aspartate isomerization and / or asparagine deamidation). In some embodiments, one, two, three, and / or four FRs of an anti-CD89 antibody, or an anti-CD20 / CD89 bispecific antibody of the present application have amino acid mutations to improve and optimize the performance of the antibody or antigen binding fragment, 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 increasing the stability (e.g., reducing or eliminating the risk of aspartate isomerization and / or asparagine deamidation). In some embodiments, CDRs and FRs of an anti-CD89 antibody, or an anti-CD20 / CD89 bispecific antibody of the present application are subjected to one or more amino acid mutations to increase the degree of humanization of the antibody or antigen binding fragment, enhance the binding affinity or binding activity to the target molecule (e.g., CD89), 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.

[0062] 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); and (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 or anti-CD20 / CD89 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.

[0063] 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 identical amino acid residue as in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as identical residues. The conservative substitutions of the amino acid residues can or can not be considered as identical residues. Alignment of sequences for the determination of percent sequence identity can be performed by tools publicly available 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 the sequence alignment.

[0064] 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 more likely to occur in an amino acid sequence comprising an Asn-Gly, Asn-Ser or Asn-Thr site, and asparagine is more susceptible 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 the antibody that are susceptible to deamidation (Sydow et al., PLoS ONE 2014, 9: e100736), and replacing these with amino acid residues less susceptible or not susceptible to deamidation.

[0065] 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 activity.

[0066] 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). The protein is substantially purified in an isolated preparation. 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" refers to an antibody that is substantially free of other antibodies of different antigenic specificities (e.g., an isolated antibody that specifically binds CD20 is substantially free of antibodies that specifically bind antigens other than CD20, or an isolated antibody that specifically binds CD89 is substantially free of antibodies that specifically bind antigens other than CD89). 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, or an anti-CD20 / CD89 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.

[0067] 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.

[0068] As used herein, the term "polypeptide" refers to polymers of amino acids and equivalents thereof, and not to a particular length of the product. Thus, "peptides" and "proteins" are encompassed within the definition of polypeptide. Also included within the definition of polypeptide are "antibodies" as defined herein.

[0069] As used herein, the term "asymmetric" means that the bispecific antibody of the present application cannot be divided into two identical halves. Specifically, certain natural antibodies (e.g., IgG) comprise two identical heavy chains and two identical light chains, wherein one heavy chain and one light chain make up one half that is mutually symmetrical with 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 composed of three different polypeptide chains, wherein two heavy chains are not identical and two light chains are identical.

[0070] The terms "heterodimer" or "heterodimeric bispecific antibody" herein refer to an asymmetric bispecific antibody composed 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 through 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, the exemplary format of the bispecific antibody of the present application is 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 halves of the bispecific antibody, and the use of these terms is not intended to impart a specific order of the two different halves of the bispecific antibody, unless explicitly so stated. Likewise, the terms "first", "second", "third", etc. herein are also introduced only for the purpose of distinguishing different moieties, and are not intended to designate the order of the relevant moieties.

[0071] Unless the context indicates otherwise, a "derivative" is a polypeptide or fragment thereof having one or more non-conservative or conservative amino acid mutations 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 natural and non-naturally occurring) known in the art.

[0072] In the present context, the term "expression vector" or "vector" refers to a vehicle into which a polynucleotide or nucleic acid encoding a certain protein can be operatively inserted and the protein is obtained expression. 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.

[0073] 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 passages. The progeny can not be identical to the parent cell in nucleic acid content, but can contain mutations. Mutant progeny that have the same function or biological activity as the originally transformed cell are included in the present invention.

[0074] In the present context, the terms "subject", "patient" or "individual" can be 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, like 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 composition involved in the use or method of the present invention are for (prophylactically and / or therapeutically) treating a non-human animal.

[0075] In the present context, "Treating", "Treatment" or "To treat" a certain disease or condition refers to alleviating, Alleviating or To alleviate a certain disease or condition, reducing the speed of onset or development of a certain disease or condition, reducing the risk of developing a certain disease or condition, or delaying the development of symptoms associated with a certain disease or condition, reducing or terminating symptoms associated with a certain disease or condition, producing complete or partial reversal of a certain disease or condition, curing a certain disease or condition, or a combination thereof.

[0076] The terms "therapeutically effective amount", "effective dose" or "effective amount" refer to the amount of agent required to achieve a desired prophylactic or ameliorative effect on symptoms associated with a disease or condition and / or to reduce the severity of a disease or condition. A therapeutically effective amount of a formulation, antibody or antigen-binding fragment thereof, bispecific antibody, or pharmaceutical composition of the application can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antigen-binding fragment thereof, bispecific antibody, or pharmaceutical composition to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the formulation, antibody or antigen-binding fragment thereof, bispecific antibody, or pharmaceutical composition are outweighed by the therapeutically beneficial effects. The term "effective amount" refers to the amount of active ingredient or pharmaceutical agent which will elicit the pharmacological and / or therapeutic effect in a subject, including but not limited to ameliorating, alleviating or reducing a disease, condition or symptom associated therewith, delaying or halting disease progression.

[0077] The terms "pharmaceutically acceptable" or "pharmaceutically acceptable" refer to those carriers, solvents, diluents, excipients, and / or salts that are, in general, chemically and / or physically compatible with the other ingredients of a formulation and are physiologically compatible with the subject.

[0078] The term "about" when used in association with a numerical value means a range of numerical values that has 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.

[0079] The term "and / or" should be understood to mean either one of the items or a combination of the items.

[0080] As used herein, the terms "comprising" or "including" or "containing" or "having" or "involving" are interchangeable with respect to the elements, integers or steps contained therein. As used herein, the terms "comprising" or "including" or "containing" or "having" or "involving" should be understood to encompass the elements, integers or steps as set forth in the specification, unless otherwise specified.

[0081] As used herein, the term "optional" means that the object modified by the term exists or does not exist, for example "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.

[0082] As used herein, the terms "some embodiments," "one embodiment," "an embodiment," or "the embodiment" or combinations thereof refer to the described features, structures, or characteristics in at least one embodiment of the application. Therefore, it is contemplated that those features, structures, or characteristics that are described in the above context can be combined in any suitable manner in one or more embodiments. Furthermore, the above described features, structures, or characteristics can be applied to one or more embodiments in any suitable manner.

[0083] Unless otherwise defined, all terms (including 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. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure, and will not be interpreted in an overly literal or overly formal sense unless expressly so defined herein.

[0084] All patents, patent applications, and other publications are herein expressly incorporated by reference for the purpose of describing and disclosing, by reference to the descriptions and disclosures of such publications, the certain information contained in such publications. Such incorporation by reference is solely in connection with the description and disclosure provided herein and is not to be construed as an admission that such publications are prior art.

[0085] Various aspects of the application will be described in further detail in the following sections.

[0086] 1. Anti-CD20 antibodies and antigen-binding fragments thereof

[0087] In one aspect, the present application provides an isolated anti-CD20 antibody or antigen-binding fragment thereof, which is capable of specifically binding to a human CD20 protein or a CD20-expressing cell; the CD20-expressing cell includes human primary B cells (including normal B cells and cancerous B cells) and a B lymphoma cell line expressing CD20, such as Raji cells.

[0088] The anti-CD20 antibody can also optionally include F(ab')2, Fab, Fab', Fv, scFv, scFv-Fc, single domain antibody (sdAb), or have an IgG type. The anti-CD20 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, and fragments thereof, as long as the antibody is capable of specifically recognizing and binding to CD20.

[0089] In some embodiments, the anti-CD20 antibody is selected from the group consisting of humanized B-Ly1 antibody. The humanized B-Ly1 antibody refers to the antibody obtained by chimerizing and humanizing the mouse anti-CD20 monoclonal antibody B-Ly1 (see Poppema et al., Biotest Bulletin 1987, 3: 131-139) with human IgG1 heavy chain constant region disclosed in WO2005044859, which has the binding specificity of the mouse B-Ly1 antibody.

[0090] In another aspect, the anti-CD20 antibody or antigen-binding fragment thereof of the present application comprises a heavy chain variable region (VH) and a light chain variable region (VL). The heavy chain variable region comprises a HCDR1 amino acid sequence as set forth in SEQ ID NO: 1, a HCDR2 amino acid sequence as set forth in SEQ ID NO: 2, and a HCDR3 amino acid sequence as set forth in SEQ ID NO: 3; and the light chain variable region comprises a LCDR1 amino acid sequence as set forth in SEQ ID NO: 4, a LCDR2 amino acid sequence as set forth in SEQ ID NO: 5, and a LCDR3 amino acid sequence as set forth in SEQ ID NO: 6.

[0091] In some embodiments, the anti-CD20 antibody comprises a VH amino acid sequence as set forth in SEQ ID NO: 7 and a VL amino acid sequence as set forth in SEQ ID NO: 8.

[0092] In some embodiments, the anti-CD20 antibody further comprises a heavy chain constant region and / or a light chain constant region, wherein the heavy chain constant region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 56-58, and the light chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 59.

[0093] 2. Anti-CD89 antibodies and antigen-binding fragments thereof

[0094] 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 human CD89 extracellular Ig-like domain 2, and does not block 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.

[0095] The anti-CD89 antibodies 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 class. The anti-CD89 antibodies of the present application can be murine, chimeric, humanized, fully human, monoclonal, polyclonal, monospecific, bispecific, multispecific, and antibody fragments, as long as the antibodies specifically recognize and bind to the extracellular Ig-like domain 2 of CD89. In some embodiments, the anti-CD89 antibodies are selected from fully human anti-CD89 antibodies or optimized antibodies or antibody fragments thereof.

[0096] In some embodiments, the anti-CD89 antibodies or antigen-binding fragments thereof comprise an anti-CD89 scFv molecule, or an anti-CD89 scFv-Fc molecule fused to an IgG Fc fragment. The scFv molecule comprises a heavy chain variable region (VH) and a 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 or can not contain a disulfide bond; the scFv molecule can be constructed in the order of VH-linker-VL, or VL-linker-VH.

[0097] Generally, scFv molecules with no 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), and the present application enhances the stability of the scFv molecule, e.g., reduces the occurrence of aggregation, by introducing a disulfide bond between the VH and VL domains.

[0098] "Introduced" or "introducing" as used in the present application refers to a mutation, including substitution, insertion, or addition, of an original or native amino acid residue in a parent polypeptide (e.g., an anti-CD89 scFv molecule of the present application). For example, an amino acid residue 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 at the same position that is different from the original / native amino acid residue in the parent polypeptide.

[0099] 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 the FR4 of the VH and the FR2 of the VL, or the CDR3 of the VH and the CDR3 of the VL, or the CDR2 of the VH and the CDR3 of the VL, or the CDR3 of the VH and the CDR1 of the VL, or 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 link the CDR2 of the VH and the CDR3 of the VL, or to 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 mutation 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, for example, by mass spectrometry.

[0100] To mutate the amino acid residues at specific positions in the VH domain and the VL domain of the anti-CD89 scFv molecule to cysteine, the coding sequences of the VH domain and the VL domain can be manipulated, for example, by replacing the codon encoding the original / natural amino acid with the codon encoding cysteine.

[0101] In one embodiment, the VH domain and the VL domain of the anti-CD89 scFv molecule of the present application are each introduced with a cysteine residue by way of amino acid mutation, preferably 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 containing a cysteine residue can be inserted or added at the position of the non-cysteine residue or at a position adjacent to the position of the non-cysteine residue. The position of substitution or insertion or addition can be determined 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 containing a cysteine residue, a disulfide bond can be formed at the interface of the VH domain and the VL domain of the anti-CD89 scFv molecule of the present application. To this end, a number of factors need to be considered, such as 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 molecule.

[0102] The distance and angle between two amino acid residues to be mutated can be determined by a person skilled in the art using 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 the target protein (e.g., the VH domain and the VL domain of an anti-CD89 antibody or an 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 can be used to determine the distance and angle between the amino acid residues based on the protein crystal structure data. Further, the amino acid residues in the target protein that are important for stabilizing the VH-VL interface need to be identified, which can be located anywhere in the VH-VL interface. In some embodiments, the site of introducing a first cysteine residue in the VH domain and the site of introducing a second cysteine residue in the VL domain of the anti-CD89 scFv molecule can be correctly paired, so that a stable disulfide bond can be formed 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 correctly paired sites are located at the edge of the VH-VL interface, and the paired amino acid residues are close enough, e.g., the spatial distance between the paired amino acid residues is less than 10 angstrom, preferably about 5 angstrom. Once the paired sites of introducing cysteine residues or the paired amino acid residues to be mutated are determined, a person skilled in the art can mutate the codon of the amino acid to be mutated to a cysteine codon by methods known in the art (e.g., PCR mutagenesis, site-directed mutagenesis or cassette mutagenesis).

[0103] 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., Journal of Biological Chemistry 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.

[0104] 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.

[0105] 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 VH and VL of disulfide bond-containing scFv molecules with increased thermal stability include the pairing of cysteine residues at positions VH44-VL100, VH44-VL101, VH60-VL95, VH100-VL91, VH101-VL46, VH103-VL43, and VH105-VL43, preferably VH44-VL100.

[0106] In some embodiments, the VH-VL interface of the disulfide bond-containing scFv exhibits the ability to maintain maximum monomer content under a range of test conditions.

[0107] In another aspect, the anti-CD89 antibody or antigen-binding fragment thereof of the present application comprises a VH and a VL. In some embodiments, the antibody of the present application is derived from a CD89 antibody comprising a VH amino acid sequence as set forth in SEQ ID NO: 26 and a VL amino acid sequence as set forth in SEQ ID NO: 40 (i.e., parent antibody Mab 14.1, see WO2002064634, which is incorporated by reference herein), and is capable of specifically recognizing and binding to CD89 extracellular Ig-like domain 2 without blocking IgA binding to CD89.

[0108] In some embodiments, the anti-CD89 antibody of the present application is derived from an antibody comprising a VH amino acid sequence as set forth in SEQ ID NO: 26:

[0109] In some embodiments, the anti-CD89 antibody of the present application is derived from an antibody comprising a VL amino acid sequence as set forth in SEQ ID NO: 40:

[0110] In some embodiments, the anti-CD89 antibody of the present application comprises one or more CDRs of a VH amino acid sequence as set forth in SEQ ID NO: 26 or an amino acid sequence as set forth in SEQ ID NOs: 9, 10, and 16, or a variant thereof, including a humanized antibody or any other variant described herein. In some embodiments, the anti-CD89 antibody comprises a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 9, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 10, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 16.

[0111] In some embodiments, the anti-CD89 antibodies of the application further comprise one or more CDRs in the VL amino acid sequence set forth as SEQ ID NO: 40 or the amino acid sequences set forth as SEQ ID NOs: 19, 20, and 22, or variants thereof, including humanized antibodies or any other variants described herein. In some embodiments, the anti-CD89 antibodies comprise a LCDR1 having the amino acid sequence set forth as SEQ ID NO: 19, a LCDR2 having the amino acid sequence set forth as SEQ ID NO: 20, and a LCDR3 having the amino acid sequence set forth as SEQ ID NO: 22.

[0112] In some specific embodiments, the anti-CD89 antibodies comprise a HCDR1 having the amino acid sequence set forth as SEQ ID NO: 9, a HCDR2 having the amino acid sequence set forth as SEQ ID NO: 10, and a HCDR3 having the amino acid sequence set forth as SEQ ID NO: 16, and a LCDR1 having the amino acid sequence set forth as SEQ ID NO: 19, a LCDR2 having the amino acid sequence set forth as SEQ ID NO: 20, and a LCDR3 having the amino acid sequence set forth as SEQ ID NO: 22.

[0113] In another aspect, the VH and / or VL of the anti-CD89 antibodies of the application can be used as starting material for engineering to make antibodies that are more suitable for administration to humans, such as optimized antibodies of the parent antibody Mab 14.1 described herein.

[0114] In some embodiments, the anti-CD89 optimized antibodies of the 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 set forth as SEQ ID NO: 26) and / or VL (amino acid sequence set forth as SEQ ID NO: 40) 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.

[0115] In some 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 (SEQ ID NO: 26) of Mab 14.1 are mutated at one or more amino acids, 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 amino acid sequence as set forth in SEQ ID NO: 26), and / or mutating the valine residue at position 31 of HFR3 (corresponding to position 93 of the VH amino acid sequence as set forth in SEQ ID NO: 26).

[0116] In one embodiment, the anti-CD89 optimized antibodies of the present application have one or more amino acid mutations in the heavy chain FR regions (HFR) of the VH amino acid sequence of the parent antibody Mab 14.1 (SEQ ID NO: 26), including D46E and / or V93A, which are capable of significantly increasing the degree of humanization of the parent antibody Mab 14.1.

[0117] In some embodiments, the anti-CD89 optimized antibodies of the present 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: 26) and / or VL (amino acid sequence set forth in SEQ ID NO: 40) of Mab 14.1, such as an amino acid mutation at position 59 (F59) of the VH amino acid sequence set forth in SEQ ID NO: 26, and / or at position 56 (G56) of the VL amino acid sequence set forth in SEQ ID NO: 40; for example, the amino acid mutation in the VH amino acid sequence is selected from F59Y, and / or the amino acid mutation in the VL amino acid sequence is selected from G56S. Further, one or more amino acid mutations can be introduced in HCDR2 and / or LCDR2, for example, the mutation can be made to the phenylalanine residue at position 11 of HCDR2 (corresponding to position 59 of the VH amino acid sequence set forth in SEQ ID NO: 26), and / or to the glycine residue at position 7 of LCDR2 (corresponding to position 56 of the VL amino acid sequence set forth in SEQ ID NO: 40). The mutation of the phenylalanine residue at position 11 of HCDR2 (corresponding to position 59 of the VH amino acid sequence set forth in SEQ ID NO: 26) includes F59Y; the mutation of the glycine residue at position 7 of LCDR2 (corresponding to position 56 of the VL amino acid sequence set forth in SEQ ID NO: 40) includes G56A, G56S, or G56T, preferably G56S.

[0118] In some embodiments, the anti-CD89 optimized antibodies of the present 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 residues of the heavy chain variable region CDRs or light chain variable region CDRs of the parent antibody Mab 14.1 are mutated, which mutation can be an amino acid substitution, addition, or deletion, preferably an amino acid substitution.

[0119] 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 set forth as SEQ ID NO: 26) 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 comprise 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 in the aspartate residue at position 4 (corresponding to position 52a of the VH amino acid sequence set forth as SEQ ID NO: 26), the aspartate residue at position 5 (corresponding to position 53 of the VH amino acid sequence set forth as SEQ ID NO: 26), the glycine residue at position 6 (corresponding to position 54 of the VH amino acid sequence set forth as SEQ ID NO: 26), the aspartate residue at position 13 (corresponding to position 61 of the VH amino acid sequence set forth as SEQ ID NO: 26), and / or the serine residue at position 14 (corresponding to position 62 of the VH amino acid sequence set forth as SEQ ID NO: 26) of HCDR2 to reduce or eliminate the risk of aspartate isomerization of the Mab 14.1 amino acid sequence. Mutations in the aspartate residue at position 4 (corresponding to position 52a of the VH amino acid sequence set forth as SEQ ID NO: 26) of HCDR2 include D52aG, D52aA, D52aY, or D52aT; mutations in the aspartate residue at position 5 (corresponding to position 53 of the VH amino acid sequence set forth as SEQ ID NO: 26) include D53H, D53I, D53T, D53Y, D53S, or D53G; mutations in the glycine residue at position 6 (corresponding to position 54 of the VH amino acid sequence set forth as SEQ ID NO: 26) include G54D, G54A, G54S, or G54T; mutations in the aspartate residue at position 13 (corresponding to position 61 of the VH amino acid sequence set forth as SEQ ID NO: 26) include D61E, D61A, or D61Q; and mutations in the serine residue at position 14 (corresponding to position 62 of the VH amino acid sequence set forth as SEQ ID NO: 26) include S62K, S62N, S62P, or S62R.

[0120] 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: 26) 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 amino acid sequence set forth as SEQ ID NO: 26) and / or the serine residue at position 14 (corresponding to position 62 of the VH amino acid sequence set forth as SEQ ID NO: 26) 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.

[0121] In one specific embodiment, one or more amino acid mutations are made to the CDR regions in the VL of Mab 14.1 (SEQ ID NO: 40) 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 amino acid sequence set forth as SEQ ID NO: 40) and / or the serine residue at position 5 (corresponding to position 93 of the VL amino acid sequence set forth as SEQ ID NO: 40) of LCDR3 can be mutated. Mutations to the asparagine residue at position 4 (corresponding to position 92 of the VL amino acid sequence set forth as SEQ ID NO: 40) 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 amino acid sequence set forth as SEQ ID NO: 40) include S93D, S93H, S93N, S93Q, S93R, or S93T.

[0122] In some embodiments, multiple amino acid mutations can also be made to the CDR regions (including heavy chain variable region CDRs and / or 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 and monomer purity, and to enhance stability, etc.

[0123] In some embodiments, one or two CDR regions and one or two FR regions of the VH of Mab 14.1 (the amino acid sequence of which is set forth in SEQ ID NO: 26) are subjected to a plurality of amino acid mutations at positions including: the aspartic acid residue at position 5 of HCDR2 (corresponding to position 53 of the VH amino acid sequence set forth in SEQ ID NO: 26), the phenylalanine residue at position 11 (corresponding to position 59 of the VH amino acid sequence set forth in SEQ ID NO: 26), the serine residue at position 14 (corresponding to position 62 of the VH amino acid sequence set forth in SEQ ID NO: 26), and the aspartic acid residue at position 11 of HFR2 (corresponding to position 46 of the VH amino acid sequence set forth in SEQ ID NO: 26), and / or the valine residue at position 31 of HFR3 (corresponding to position 93 of the VH amino acid sequence set forth in SEQ ID NO: 26); 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.

[0124] In some embodiments, two CDR regions of the VL of Mab 14.1 (the amino acid sequence of which is set forth in SEQ ID NO: 40) are subjected to a plurality of amino acid mutations at positions including: the glycine residue at position 7 of LCDR2 (corresponding to position 56 of the VL amino acid sequence set forth in SEQ ID NO: 40), and / or the asparagine residue at position 4 of LCDR3 (corresponding to position 92 of the VL amino acid sequence set forth in SEQ ID NO: 40); 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.

[0125] 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.

[0126] In another aspect, the amino acid sequence numbering of the heavy chain CDR regions, the light chain CDR regions, and the heavy chain variable region and the 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 2, wherein the heavy chain variable region CDRs and the light chain variable region CDRs are defined by the Kabat numbering system.

[0127] Table 2. Amino acid sequence ID numbers of CDR regions, and heavy chain variable region and light chain variable region of anti-CD89 antibodies

[0128] In some embodiments, the present application provides an isolated anti-CD89 monoclonal antibody or antigen-binding fragment thereof, which comprises (1) a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 9; (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; i.e., as set forth in SEQ ID NO: 50), 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: 16; (4) a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 19; (5) a LCDR2 having an amino acid sequence of GASSLEX7 (X7= G, A, S or T; i.e., as set forth in SEQ ID NO: 51), 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; i.e., as set forth in SEQ ID NO: 52), 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.

[0129] In some specific embodiments, the anti-CD89 antibody or antigen-binding fragment thereof described above comprises a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 9, a HCDR2 having an amino acid sequence as set forth in any one of SEQ ID NOs: 10-14, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 16, and a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 19, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 20 or 21, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 22 or 23.

[0130] In one specific embodiment, the anti-CD89 antibody or antigen-binding fragment thereof of the present application comprises:

[0131] (1) a HCDR1 having an amino acid sequence of SEQ ID NO: 9, a HCDR2 having an amino acid sequence of SEQ ID NO: 10, 11, 12, or 13, a HCDR3 having an amino acid sequence of SEQ ID NO: 16; and a LCDR1 having an amino acid sequence of SEQ ID NO: 19, a LCDR2 having an amino acid sequence of SEQ ID NO: 20, a LCDR3 having an amino acid sequence of SEQ ID NO: 22, 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

[0132] (2) a HCDR1 having an amino acid sequence of SEQ ID NO: 9, a HCDR2 having an amino acid sequence of SEQ ID NO: 10, a HCDR3 having an amino acid sequence of SEQ ID NO: 16; and a LCDR1 having an amino acid sequence of SEQ ID NO: 19, a LCDR2 having an amino acid sequence of SEQ ID NO: 21, a LCDR3 having an amino acid sequence of SEQ ID NO: 22, 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

[0133] (3) a HCDR1 having an amino acid sequence of SEQ ID NO: 9, a HCDR2 having an amino acid sequence of SEQ ID NO: 10, a HCDR3 having an amino acid sequence of SEQ ID NO: 16; and a LCDR1 having an amino acid sequence of SEQ ID NO: 19, a LCDR2 having an amino acid sequence of SEQ ID NO: 20, a LCDR3 having an amino acid sequence of SEQ ID NO: 23, 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

[0134] (4) a HCDR1 having an amino acid sequence of SEQ ID NO: 9, a HCDR2 having an amino acid sequence of SEQ ID NO: 14, a HCDR3 having an amino acid sequence of SEQ ID NO: 16; and a LCDR1 having an amino acid sequence of SEQ ID NO: 19, a LCDR2 having an amino acid sequence of SEQ ID NO: 21, a LCDR3 having an amino acid sequence of SEQ ID NO: 23, 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.

[0135] Preferably, the anti-CD89 antibody or antigen-binding fragment thereof comprises: a HCDR1 having an amino acid sequence of SEQ ID NO: 9, a HCDR2 having an amino acid sequence of SEQ ID NO: 14, a HCDR3 having an amino acid sequence of SEQ ID NO: 16; and a LCDR1 having an amino acid sequence of SEQ ID NO: 19, a LCDR2 having an amino acid sequence of SEQ ID NO: 21, a LCDR3 having an amino acid sequence of SEQ ID NO: 23, 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.

[0136] 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 any one of SEQ ID NOS: 26-33, 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%, at least 99%, or 100%) identical thereto, and (2) a VL comprising an amino acid sequence of any one of SEQ ID NOS: 40-43, 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%, at least 99%, or 100%) identical thereto. Preferably, the differences in amino acids between the amino acid sequences of SEQ ID NOS: 26, 27, 28, 29, 30, 31, 32, or 33 and amino acid sequences that are 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 sequences of SEQ ID NOS: 40, 41, 42, or 43 and amino acid sequences that are at least 80% identical thereto are primarily or entirely in the FR regions of the light chain of the anti-CD89 antibody.

[0137] In some embodiments, the anti-CD89 antibodies or antigen-binding fragments thereof of the present application comprise a heavy chain variable region, VH, and a light chain variable region, VL, each comprising: a VH amino acid sequence as set forth in SEQ ID NO: 26 and a VL amino acid sequence as set forth in SEQ ID NO: 40, 41, or 42, 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, respectively, to the VH and VL amino acid sequences; or

[0138] a VH amino acid sequence as set forth in SEQ ID NO: 27, 28, 29, 30, or 31 and a VL amino acid sequence as set forth in SEQ ID NO: 40, 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, respectively, to the VH and VL amino acid sequences; or

[0139] a VH amino acid sequence as set forth in SEQ ID NO: 32 or 33 and a VL amino acid sequence as set forth in SEQ ID NO: 43, 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, respectively, to the VH and VL amino acid sequences.

[0140] In one embodiment, the anti-CD89 antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO: 32 or 33, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 43, 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, respectively, to the amino acid sequences of the VH and VL. Preferably, the antibody or antigen-binding fragment thereof comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO: 33, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 43, 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, respectively, to the amino acid sequences of the VH and VL. Preferably, the differences in amino acids between the amino acid sequence as set forth in SEQ ID NO: 32 or 33 and an amino acid sequence that is at least 85% identical thereto, and between the amino acid sequence as set forth in SEQ ID NO: 43 and an amino acid sequence that is at least 85% identical thereto, are primarily or entirely in the FR regions of the heavy and light chains, respectively, of the anti-CD89 antibody.

[0141] In another aspect, the anti-CD89 antibody or antigen-binding fragment thereof of the present application comprises a scFv molecule having a disulfide bond at the VH-VL interface, the heavy chain variable region VH and the light chain variable region 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.

[0142] In some embodiments, the VH-VL interface of an anti-CD89 scFv molecule of the application comprises two or more introduced cysteines 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: 9; (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; i.e., SEQ ID NO: 50); (3) a HCDR3 having an amino acid sequence of SEQ ID NO: 16; (4) a LCDR1 having an amino acid sequence of SEQ ID NO: 19; (5) a LCDR2 having an amino acid sequence of GASSLEX7 (X7= G, A, S, or T; i.e., SEQ ID NO: 51); (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; i.e., SEQ ID NO: 52), 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.

[0143] 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 does not comprise a disulfide bond in the VH-VL interface, the pairing of said first and second cysteine residues being selected from one or more of the following combinations of paired amino acid mutations:

[0144] (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 polypeptide anti-CD89 scFv molecule to introduce a second cysteine residue; and / or

[0145] (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; i.e. as shown in SEQ ID NO: 50) 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 QQFX8X9YPFT (X8= N, A, G, H, L, S, T or Y; X9= S, D, H, N, Q, R or T; i.e. as shown in SEQ ID NO: 52) of the VL of said parent anti-CD89 scFv molecule to introduce a second cysteine residue; and / or

[0146] (3) an amino acid mutation S6C at position 6 of HCDR3, i.e. at the amino acid sequence as shown in SEQ ID NO: 16, 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; i.e. SEQ ID NO: 52) of the VL of said parent anti-CD89 scFv molecule to introduce a second cysteine residue; and / or

[0147] (4) performing an amino acid mutation D9C at position 101 of the VH of the parent anti-CD89 scFv molecule, i.e., at position 9 of the HCDR3 of the amino acid sequence as shown in SEQ ID NO: 16, to introduce a first cysteine ​​residue; and performing 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

[0148] (5) performing an amino acid mutation W103C or Q105C at position 103 or 105 of the VH of the parent anti-CD89 scFv molecule to introduce the first cysteine ​​residue; and performing an amino acid mutation A43C at position 43 of the VL of the parent anti-CD89 scFv molecule to introduce the second cysteine ​​residue.

[0149] In other embodiments, the anti-CD89 scFv molecule containing a disulfide bond at the VH-VL interface of the present invention comprises HCDR1, HCDR2, HCDR3 of the heavy chain variable region and LCDR1, LCDR2, LCDR3 of the light chain variable region, wherein the HCDR2, HCDR3 and / or LCDR3 comprise one or more cysteine ​​mutations. The heavy chain variable region and the light chain variable region comprise: (1) HCDR1 having the amino acid sequence shown in SEQ ID NO: 9; (2) having the 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; i.e., HCDR2 shown in SEQ ID NO: 53), preferably, X1 = D, X2 = H, X3 = G, X4 = Y, X5 = D, X6 = K; (3) having an amino acid sequence of EGYSGX 11 WFX 12 Y(X 11 =S or C, X 12 =D or C; i.e., as shown in SEQ ID NO: 54); (4) a LCDR1 having an amino acid sequence as shown in SEQ ID NO: 19; (5) a LCDR2 having an amino acid sequence of GASSLEX7 (X7 = G, A, S or T; i.e., as shown in SEQ ID NO: 51), 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; i.e., as set forth in SEQ ID NO: 55), 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 the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0150] 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: 9, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 14 or 15, a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 16, 17, or 18, a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 19, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 21, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 23, 24, or 25.

[0151] In one specific embodiment, the anti-CD89 scFv molecule with a disulfide bond in the VH-VL interface of the present application comprises:

[0152] (1) a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 9, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 14, a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 16 or 18, a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 19, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 21, a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 23, 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;

[0153] (2) HCDR1 having an amino acid sequence of SEQ ID NO: 9, HCDR2 having an amino acid sequence of SEQ ID NO: 15, HCDR3 having an amino acid sequence of SEQ ID NO: 16, and LCDR1 having an amino acid sequence of SEQ ID NO: 19, LCDR2 having an amino acid sequence of SEQ ID NO: 21, LCDR3 having an amino acid sequence of SEQ ID NO: 24, 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

[0154] (3) HCDR1 having an amino acid sequence of SEQ ID NO: 9, HCDR2 having an amino acid sequence of SEQ ID NO: 14, HCDR3 having an amino acid sequence of SEQ ID NO: 17, and LCDR1 having an amino acid sequence of SEQ ID NO: 19, LCDR2 having an amino acid sequence of SEQ ID NO: 21, LCDR3 having an amino acid sequence of 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 to the amino acid sequence of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0155] In some embodiments, the VH-VL interface containing disulfide bond- containing anti-CD89 scFv molecules of the present application further comprise a heavy chain variable region having the HCDR1, HCDR2, and HCDR3 and a light chain variable region having the LCDR1, LCDR2, and LCDR3.

[0156] In some embodiments, the VH-VL interface disulfide-containing anti-CD89 scFv of the application comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO: 34, 35, 36, 37, 38, or 39, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 44, 45, 46, 47, 48, or 49, 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%, at least 99%, or 100%) identical to the amino acid sequence of the recited VH and VL, respectively. In some preferred embodiments, the VH-VL interface disulfide-containing anti-CD89 scFv comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO: 34, 35, or 38, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 44, 45, or 48, or an amino acid sequence that is at least 80% identical to the amino acid sequence of the recited VH and VL, respectively.

[0157] In one embodiment, the VH-VL interface disulfide-containing anti-CD89 scFv molecule of the application comprises a heavy chain variable region and a light chain variable region, each comprising:

[0158] (1) a VH comprising an amino acid sequence as set forth in SEQ ID NO: 34 and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 44 or 48, 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 recited VH and VL, respectively; or

[0159] (2) a VH comprising an amino acid sequence as set forth in SEQ ID NO: 35 or 38 and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 45, 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 recited VH and VL, respectively; or

[0160] (3) a VH comprising an amino acid sequence as set forth in SEQ ID NO: 36 and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 46, 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; or

[0161] (4) a VH comprising an amino acid sequence as set forth in SEQ ID NO: 37 and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 47, 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; or

[0162] (5) a VH comprising an amino acid sequence as set forth in SEQ ID NO: 39 and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 49, 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.

[0163] In some embodiments, the VH-VL interface disulfide-containing anti-CD89 scFv molecules of the present application comprise a linker that fuses or operatively links the VH and VL of the VH-VL interface disulfide-containing anti-CD89 scFv molecules, the linker having low immunogenicity, preferably a flexible peptide as the linker, for example a linker comprising glycine, or glycine and serine.

[0164] 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 one or more linking molecules, or can include a linking molecule and at least one spacer molecule that is intended to separate the linking molecule and the compound by a specific distance.

[0165] The term "operatively 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 as described herein, or the linkage of a scFv to an Fc, or the linkage of a scFv to a Fab or IgG domain.

[0166] 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 (i.e., (G4S) n ), wherein n is an integer from 1 to 3, preferably, n is 3. In some embodiments, the linker comprises the amino acid sequence as set forth in SEQ ID NO: 60.

[0167] 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 at the VH-VL interface 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.

[0168] The anti-CD89 antibody or antigen-binding fragment thereof of the present application has a scFv form, specifically, the anti-CD89 antibody or antigen-binding fragment thereof is an anti-CD89 scFv molecule with a disulfide bond at the VH-VL interface, which significantly improves the stability of the antibody molecule while maintaining the antigen-binding specificity and binding activity of the parent anti-CD89 scFv molecule.

[0169] 3. CD89 myeloid engager antibody

[0170] In one aspect, the present application provides a CD89 myeloid engager comprising a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain specifically binds to a membrane protein or receptor on a target cell that does not express CD89, and the second antigen binding domain specifically binds to CD89 on the surface of a myeloid cell. When the first and second antigen binding domains bind to the target cell and CD89 + myeloid cell, respectively, the myeloid cell can be activated and directed to kill the target cell. In some embodiments, the myeloid engager is a bispecific antibody.

[0171] Exemplarily, the first antigen binding domain can specifically bind to a surface membrane protein or receptor of a target cell associated with a proliferative disease (e.g., cancer), an autoimmune disease, an inflammatory disease, or a fibrotic disorder, or a surface membrane protein of a target cell infected by a pathogen. For example, CD20 on the surface of a diseased B cell, a tumor-associated antigen (TAA) on the surface of a cancer cell, a surface receptor for an eosinophil, or a surface receptor for a Th2 cell. The target cell associated with a fibrotic disorder can be a cell that mediates atherosclerosis, chronic obstructive pulmonary disease (COPD), cirrhosis of the liver, scleroderma, renal transplant fibrosis, Kidney Allograft Nephropathy, or pulmonary fibrosis (including idiopathic pulmonary fibrosis and / or idiopathic pulmonary hypertension), and the target cell surface membrane protein can be a fibroblast surface receptor. The surface membrane protein of the target cell infected by the pathogen can be a membrane protein selectively expressed on the infected cell, for example, a viral envelope protein expressed on the surface of a cell infected with HIV; the target cell can also be a cell infected by a microorganism, and the target cell surface membrane protein can be a specific antigen of a microorganism.

[0172] The formats of the bispecific antibodies include, but are not limited to, bispecific formats based on BiTE or Diabody, (scFv)2-Fc, scFv-Fab, scFv-IgG or IgG-scFv fusion proteins, DVD-Ig, Quadroma, Knobs-into-holes, Common Light Chain, CrossMab, CrossFab, SEEDbody, Leucine Zipper, Duobody, IgG1 / IgG2, Dual-acting Fab (DAF)-IgG and Mab 2 Bispecific formats (see, eg, Klein et al., mAbs 2012, 4:653-663 and references cited therein) can also be symmetric or asymmetric bispecific formats.

[0173] Further, the CD89 myeloid engager antibody described in the present application is an asymmetric heterodimeric molecule containing a constant region, which comprises two different subunits (A and B), each subunit comprising a constant region, the constant region comprising a heavy chain constant region and a light chain constant region. Among them, the heavy chain constant region includes natural or mutant protein forms of the human IgG heavy chain constant region Fc region, and also includes polypeptide truncated forms containing a hinge region that promotes dimer formation. In certain embodiments, the Fc region comprises antibody CH2 and CH3 domains. The fusion protein containing the Fc portion can be purified by Protein A or Protein G affinity chromatography column, and can prolong 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: 56. 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 shown in SEQ ID NO: 59).

[0174] Because the asymmetric heterodimeric 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 heterodimeric antibody can be engineered, including but not limited to "knobs-into-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), "knobs-into-holes" and disulfide bond combination mutations (Merchant et al., Nature Biotechnology 1998, 16:677-681) and "isoelectric point (pI) mutations", so that the antibody is prone to form a higher proportion of heterodimers, and its byproducts (such as homodimers) are prone to be effectively separated by downstream purification processes, ensuring the uniformity and purity of the final product.

[0175] The "knob-and-hole" mutation is 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 a heterodimeric antibody to create 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 create a hole, thereby positioning the knob within the hole to facilitate the pairing of the heterodimeric protein / heterodimeric antibody heavy chains to form a heterodimer. The amino acid residue with a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W), and the amino acid residue with a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0176] The "pi mutation" is an amino acid mutation introduced into the constant region of one or both heavy chains of a heterodimeric antibody that changes the charge properties of 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 with 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 performed by, for example, substituting a positively or negatively charged amino acid residue with an oppositely charged amino acid residue (e.g., aspartate or glutamate is substituted for lysine or arginine), or substituting a neutral amino acid residue with a charged amino acid residue (e.g., glycine is substituted for glutamate). After the pi mutation, separation of the heterodimer (A-B) from the homodimers (A-A and B-B) by ion exchange chromatography can be achieved.

[0177] An exemplary CD89 myeloid engager of the present application is a bispecific antibody that is constructed based on an anti-CD20 antibody or antigen-binding fragment thereof and an anti-CD89 antibody or antigen-binding fragment thereof of the present application, which comprises a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain specifically binds to CD20 and the second antigen-binding domain specifically binds to CD89 extracellular Ig-like domain 2.

[0178] The anti-CD20 / CD89 bispecific antibody adopts a structure of asymmetric heterodimer 2+1 scFv-IgG or scFv-Fab-Fc:Fab-Fc, which comprises subunits A and B, wherein subunit A is a bivalent scFv-Fab-Fc form and subunit B is a monovalent Fab-Fc form; the scFv part specifically binds to CD89 extracellular Ig-like domain 2, and the VHand VLdomains of the scFv can or can not contain a disulfide bond; the Fab-Fc part specifically binds to CD20. The anti-CD20 / CD89 bispecific antibody can specifically bind to CD20, retaining the antigen binding activity of its corresponding anti-CD20 antibody or antigen binding fragment thereof. At the same time, the anti-CD20 / CD89 bispecific antibody also retains the antigen binding affinity or binding activity of its corresponding anti-CD89 antibody or antigen binding fragment thereof (K D <5 x 10 -8 M, <1 x 10 - 9 M, or <5 x 10 -10 M). In addition, the bispecific antibody also has the characteristics of high stability (e.g., thermal stability).

[0179] The anti-CD20 / CD89 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 form (i.e., one bispecific antibody molecule binds to one CD89 molecule), and binds to CD20 on the surface of target cells (e.g., B cells) in a bivalent form (i.e., one bispecific antibody molecule can bind to one or two CD20 molecules), and thus the anti-CD20 / CD89 bispecific antibody can cross-link CD89-expressing myeloid cells with CD20-expressing target cells (e.g., B cells), thereby activating the CD89-expressing myeloid cells and mediating their directed killing of the CD20-expressing target cells. 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 killing of target cells by the myeloid cells in the presence of CD20-expressing target cells (e.g., B cells), including neutrophils, eosinophils, macrophages, and monocytes. Thus, the anti-CD20 / CD89 bispecific antibody of the present application effectively avoids the non-directed activation and killing of target cells in the absence of target cells. In some embodiments, the biological activity of the anti-CD20 / CD89 bispecific antibody is detected by a reporter gene method, and the results show that the activation of CD89 by the bispecific antibody is dependent on CD20-expressing target cells, i.e., the effector cells expressing CD89 cannot be activated by the bispecific antibody in the absence of target cells. In some embodiments, the biological activity of the anti-CD20 / CD89 bispecific antibody is detected by using white blood cells isolated from the peripheral blood of healthy humans, and the results show that the bispecific antibody can effectively activate and mediate the killing / elimination of B cells by CD89-expressing human peripheral blood myeloid cells. In some embodiments, after the anti-CD20 / CD89 bispecific antibody is injected into cynomolgus monkeys, the bispecific antibody can mediate the efficient elimination of CD20-expressing B cells by CD89-expressing myeloid cells in the peripheral blood of the cynomolgus monkeys.

[0180] Since the anti-CD20 / CD89 bispecific antibody does not activate T cells, it does not trigger massive cytokine release, and thus the risk of directly triggering a cytokine storm is extremely low. Thus, the anti-CD20 / CD89 bispecific antibody of the present application has high safety for clinical application.

[0181] The heavy chain constant region of the anti-CD20 / CD89 bispecific antibody of the present application is subjected to "knob-and-hole" mutation and / or "pi mutation" to improve the homogeneity and purity of the heterodimer bispecific antibody product.

[0182] Since "knob and hole" mutations are introduced into the heavy chain CH3 region of the bispecific antibody, the homodimers formed are primarily "hole-hole" dimers. To prevent the formation of bivalent anti-CD89 homodimeric antibodies (e.g., anti-CD89 "hole-hole" homodimers), the present invention introduces a "knob" mutation into the Fc region of subunit A (scFv-Fab-Fc) containing the anti-CD89 scFv, and a "hole" mutation into the Fc region of subunit B (Fab-Fc). Furthermore, in order to be able to effectively separate the heterodimer from the "cavity-cavity" homodimer by ion exchange chromatography, a "pI mutation" can be introduced into one or both heavy chain constant regions of the bispecific antibody to significantly increase the pI difference between the two subunits (A and B), thereby increasing the pI difference between the heterodimer (AB) and the homodimer ("cavity-cavity" or BB). Preferably, different "pI mutations" are introduced simultaneously into the two heavy chain constant regions of the bispecific antibody to achieve the purpose of effectively separating the homodimer by ion exchange chromatography. In addition, introducing a "pI mutation" into the heavy chain constant region of the heterodimeric antibody is also beneficial for quality control of the antibody. For example, high-performance liquid ion exchange chromatography can be used to effectively monitor the residual homodimer in the heterodimeric final product. The specific site selected for introducing the "pI mutation" depends on many factors, including the position in the heterodimeric antibody domain, its functional role, and potential immunogenicity. The "pi mutation" has no significant effect on the biological properties of the heterodimeric antibody. In some embodiments, the introduction of the "pi mutation" into the heavy chain constant region of the anti-CD20 / CD89 bispecific antibody results in a significant difference in the pI between the heterodimer and homodimer, enabling effective separation of the two by ion exchange chromatography. As a result, the monomer (i.e., heterodimer) obtained after separation and purification of the bispecific antibody has extremely high purity (nearly 100%) and extremely low homodimer content (undetectable).

[0183] By introducing the L234F / L235E / P331S mutations (EU Numbering) into the Fc region, the anti-CD20 / CD89 bispecific antibody has significantly reduced Fc receptor binding affinity. Consequently, the Fc effector functions (including ADCC and CDC activities) of the bispecific antibody are substantially eliminated, and other immune cell types (e.g., T cells, NK cells) are not activated. Consequently, the risk of toxic side effects caused by the antibody's Fc effector functions is greatly reduced.

[0184] The first and second antigen binding domains of the anti-CD20 / CD89 bispecific antibodies of the present application can be derived from the exemplary anti-CD20 antibodies or antigen binding fragments thereof and anti-CD89 antibodies or antigen binding fragments thereof of the present application, but can also be derived from other anti-CD20 antibodies or antigen binding fragments thereof and / or other anti-CD89 antibodies or antigen binding fragments thereof known in the art.

[0185] The exemplary anti-CD20 / CD89 bispecific antibodies of the present application can be constructed based on anti-CD20 antibodies or antigen binding fragments thereof known in the art and anti-CD89 antibodies or antigen binding fragments thereof specifically binding to the extracellular Ig-like domain 2 of CD89 as shown in Table 2. The exemplary anti-CD20 / CD89 bispecific antibodies of the present application comprise 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-CD20 humanized B-Lyl antibody or antigen binding fragment thereof disclosed in WO 2005 044 859 and the second antigen binding domain comprises any of at least one CDR region and / or any of the variable regions of the anti-CD89 antibodies or antigen binding fragments thereof shown in Table 2.

[0186] In some embodiments, the first or second antigen binding domain of the anti-CD20 / CD89 bispecific antibody comprises the heavy chain variable region CDRs and / or the light chain variable region CDRs of the anti-CD20 humanized B-Lyl antibody or antigen binding fragment thereof disclosed in WO2005044859, the heavy chain variable region CDRs of the first antigen binding domain comprising the amino acid sequence of any one, two, or three of the heavy chain variable region CDRs of the anti-CD20 humanized B-Lyl antibody or antigen binding fragment thereof disclosed in WO2005044859, 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 HCDR1, HCDR2, and HCDR3 amino acid sequences, respectively; the light chain variable region CDRs of the first antigen binding domain comprising the amino acid sequence of any one, two, or three of the light chain variable region CDRs of the anti-CD20 humanized B-Lyl antibody or antigen binding fragment thereof disclosed in WO2005044859, 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 LCDR1, LCDR2, and LCDR3 amino acid sequences, respectively; the heavy chain variable region CDRs of the second antigen binding domain comprising the amino acid sequence of any one, two, or three of the heavy chain variable region CDRs of the anti-CD89 antibody or antigen binding fragment 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 HCDR1, HCDR2, and HCDR3 amino acid sequences, respectively, preferably the heavy chain variable region CDRs of the second antigen binding domain comprise the amino acid sequence of any one, two, or three of the heavy chain variable region CDRs of the anti-CD89 scFv molecule with a disulfide bond-containing VH-VL interface 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 HCDR1, HCDR2, and HCDR3 amino acid sequences, 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-CD89 antibodies or antigen binding fragments listed in Table 2, 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 LCDR1, LCDR2, and LCDR3 amino acid sequences, respectively, preferably 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-CD89 scFv molecules listed in Table 2 that have a disulfide bond at the VH-VL interface, 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 LCDR1, LCDR2, and LCDR3 amino acid sequences, respectively.

[0187] In some embodiments, the first antigen binding domain of the anti-CD20 / CD89 bispecific antibody comprises a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 1, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 2, a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 3, and a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 4, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 5, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 6, 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 the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

[0188] In some embodiments, the second antigen binding domain of the anti-CD20 / CD89 bispecific antibody comprises the heavy chain variable region CDRs and the light chain variable region CDRs of the anti-CD89 scFv molecule with disulfide bond containing VH-VL interface listed in Table 2. Specifically, the second antigen binding domain of the bispecific antibody comprises a HCDR1 having an amino acid sequence of SEQ ID NO: 9, a HCDR2 having an amino acid sequence of SEQ ID NO: 14, a HCDR3 having an amino acid sequence of SEQ ID NO: 16, and a LCDR1 having an amino acid sequence of SEQ ID NO: 19, a LCDR2 having an amino acid sequence of SEQ ID NO: 21 and a LCDR3 having an amino acid sequence of SEQ ID NO: 23, 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.

[0189] In some embodiments, the first or second antigen binding domain of the anti-CD20 / CD89 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-CD20 humanized B-Lyl antibody or antigen binding fragment thereof disclosed in WO2005044859, 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 light chain variable region of the first antigen binding domain comprises any of the VL amino acid sequences of the anti-CD20 humanized B-Lyl antibody or antigen binding fragment thereof disclosed in WO2005044859, 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 heavy chain variable region of the second antigen domain comprises any of the VH amino acid sequences of the anti-CD89 antibody or antigen binding fragment thereof listed in Table 2, 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 light chain variable region of the second antigen binding domain comprises any of the VL amino acid sequences of the anti-CD89 antibody or antigen binding fragment thereof listed in Table 2, 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.

[0190] In some specific embodiments, the first antigen domain of the anti-CD20 / CD89 bispecific antibody comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO: 7, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 8, 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 to the amino acid sequences of the VH and VL, respectively.

[0191] In some embodiments, the second antigen binding domain of the anti-CD20 / CD89 bispecific antibody comprises a VH comprising an amino acid sequence as set forth in SEQ ID NO: 34, and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 48, 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.

[0192] In another aspect, the exemplary anti-CD20 / CD89 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 Fab-Fc of the bispecific antibody as the first antigen binding domain and the scFv as the second antigen binding domain, whereby the subunit A comprises the second antigen binding domain, a linker, and the first antigen binding domain, and the subunit B comprises the first antigen binding domain; the scFv domain is fused to or operably linked to the Fab-Fc domain via a linker, which not only has low immunogenicity, but also ensures the stability of the bispecific antibody, preferably a flexible peptide as the linker.

[0193] In some embodiments, the first antigen binding domain of the anti-CD20 / CD89 bispecific antibody specifically binds to CD20 and is a Fab-Fc domain, which 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-CD20 humanized B-Lyl antibody or antigen binding fragment thereof disclosed in WO2005044859; the second antigen binding domain of the anti-CD20 / CD89 bispecific antibody specifically binds to CD89 extracellular Ig-like domain 2 and is a scFv domain, which is a VH-VL interface disulfide-containing scFv molecule, 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-CD89 antibody or antigen binding fragment thereof shown in Table 2, wherein the anti-CD89 antibody or antigen binding fragment thereof is a VH-VL interface disulfide-containing scFv molecule; the C-terminus of the VL domain in the scFv domain is fused to or operably linked to the N-terminus of the VH domain of the Fab-Fc via a linker to construct a trivalent bispecific antibody.

[0194] In some embodiments, the scFv domain is linked to the Fab-Fc domain via a linker, the amino acid sequence of which is (G4S) n , n is an integer greater than 0, such as 1-3, preferably the amino acid sequence of the linker is (G4S)2.

[0195] In one embodiment, the anti-CD20 / CD89 bispecific antibody comprises a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain comprises the VH and VL of the anti-CD20 humanized B-Lyl antibody or antigen binding fragment thereof disclosed in WO2005044859, and the second antigen binding domain comprises the VH and VL of the anti-CD89 antibody or antigen binding fragment thereof shown in Table 2, which is a scFv molecule with disulfide bond in the VH-VL interface. The first antigen binding domain is a Fab-Fc domain, and the second antigen binding domain is a scFv domain. The VL domain in the scFv domain is C-terminally linked 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].

[0196] In some embodiments, the anti-CD20 / CD89 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 which, the heavy chain constant region comprises an engineered human IgG heavy chain constant region, which is engineered to be able to self-assemble to form correct heavy chain / heavy chain pairing, the engineering comprises “knobs-into-holes” mutation and / or “pl mutation”, and can further comprise amino acid mutation to the Fc region of the heavy chain to alter its effector function. 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: 59)

[0197] In some specific embodiments, the heavy chain CH3 region of the anti-CD20 / CD89 bispecific antibody introduces “knobs-into-holes” mutation, thereby facilitating the formation of heterodimer. Specifically, “knobs” mutation is introduced to the heavy chain CH3 region of subunit A (scFv-Fab-Fc), which comprises T366W; “holes” mutation is introduced to the heavy chain CH3 region of subunit B (Fab-Fc), which comprises T366S, L368A and Y407V.

[0198] In some specific embodiments, the heavy chain Fc region of the anti-CD20 / CD89 bispecific antibody further introduces "pl mutations" to facilitate the removal of homodimers by downstream purification processes. In order to minimize the immunogenicity risk induced by "pl mutations", the selection of the pl mutation sites in the heavy chain constant region of the anti-CD20 / CD89 bispecific antibody of the present application is based on the isotype differences among human IgG subtypes (i.e. IgGl, IgG2, IgG3 and IgG4). Specifically, the mutations can be selected by aligning the amino acid sequences of human IgGl, IgG2, IgG3 and IgG4 constant regions, mutating the neutral or basic amino acid residues in the human IgGl constant region to acidic or neutral amino acid residues at the corresponding sites in other human IgG subtypes to decrease the pl value; and / or mutating the neutral or acidic amino acid residues in the human IgGl constant region to basic or neutral amino acid residues to increase the pl value. For example, one or more of the mutations shown in Table 3 can be selected to change the pl value of the heavy chain. In some embodiments, the two heavy chain constant regions of the anti-CD20 / CD89 bispecific antibody of the present application have different types of "pl mutations", e.g. a mutation that decreases the pl value is introduced in one heavy chain constant region and a mutation that increases the pl value is introduced in the other heavy chain constant region, thereby significantly increasing the pl value difference between the heterodimer and homodimer of the bispecific antibody of the present application, facilitating purification separation. The pl value can be obtained by theoretical calculation or experimental determination. In one embodiment, a mutation that increases the pl value is further introduced in the heavy chain constant region of the bispecific antibody based on the "knobs-into-holes" mutation, which includes the amino acid mutations Q196K and / or N276K; and a mutation that decreases the pl value is further introduced in the other heavy chain constant region of the bispecific antibody based on the "holes-into-knobs" mutation, which includes one or more of the amino acid mutations G137E, G138S, N203D, K274Q and Q419E. Thus, the theoretical pl value of the heterodimer (A-B) of the heavy chain constant region containing "knobs-into-holes" mutation and "pl mutation" differs from the theoretical pl value of the formed homodimer (B-B, i.e. "holes-holes") by at least 0.5 pH units, preferably the difference is more than 0.8 pH units.

[0199] Table 3. Amino acid residues of the antibody heavy chain hlgGl constant region pl mutation sites

[0200] One function of the Fc region of an antibody is to generate "effector functions" when the antibody binds its target molecule with the immune system, 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 (FcRs) 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). The Fc region mediates CDC through binding to proteins of the complement system, such as Clq (Ward et al., Ther Immunol 1995, 2: 77-94).

[0201] In some specific embodiments, the anti-CD20 / CD89 bispecific antibodies of the 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: N297A or N297Q (IgGl) S267E / L328F (IgGl) L234A / L235A (IgGl) L234F / L235E / P331S (IgGl) C220S / C226S / C229S / P238S (IgGl) C226S / C229S / E233P / L234V / L235A (IgGl) V234A / G237A (IgG2) H268Q / V309L / A330S / A331S (IgG2) L235A / G237A / E318A (IgG4)

[0202] A preferred engineered Fc region is a human IgGl Fc with L234F / L235E / P331S (EU numbering system) amino acid substitutions, which can reduce binding of the Fc region 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.

[0203] In some embodiments, both heavy chain constant regions of the anti-CD20 / CD89 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, FcyRIIb, FcyRIIIa [158F], and FcyRIIIa [158V]). In some embodiments, the introduction of L234F / L235E / P331S mutations in both heavy chain constant regions of the anti-CD20 / CD89 bispecific antibody results in substantially no binding of the bispecific antibody to FcyRI, FcyRIIa (131H), FcyRIIb, FcyRIIIa (158F), and / or FcyRIIIa (158V). In some embodiments, the introduction of L234F / L235E / P331S mutations in both heavy chain constant regions of the anti-CD20 / CD89 bispecific antibody results in substantially no binding of the bispecific antibody to Clq. In some embodiments, the introduction of L234F / L235E / P331S mutations in both heavy chain constant regions of the anti-CD20 / CD89 bispecific antibody does not affect the binding affinity of the bispecific antibody to FcRn. In some embodiments, the introduction of L234F / L235E / P331S mutations in both heavy chain constant regions of the anti-CD20 / CD89 bispecific antibody significantly reduces the ADCC activity of the bispecific antibody. In one embodiment, the ADCC activity of the anti-CD20 / CD89 bispecific antibody is significantly reduced or abrogated compared to an antibody that does not comprise the amino acid mutations in the Fc region.

[0204] In another aspect, the anti-CD20 / CD89 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-CD20 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-CD20 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-CD20 antibody.

[0205] In some embodiments, the anti-CD89 scFv molecule can or can not contain a disulfide bond between the VH domain and the VL domain of the anti-CD89 scFv molecule, preferably contains a disulfide bond. In some embodiments, the anti-CD89 scFv molecule comprises a HCDR1 having an amino acid sequence of SEQ ID NO: 9, a HCDR2 having an amino acid sequence of SEQ ID NO: 14, and a HCDR3 having an amino acid sequence of SEQ ID NO: 16, and a LCDR1 having an amino acid sequence of SEQ ID NO: 19, a LCDR2 having an amino acid sequence of SEQ ID NO: 21, and a LCDR3 having an amino acid sequence of SEQ ID NO: 23, 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.

[0206] In some embodiments, the anti-CD89 scFv molecule comprises a VH comprising an amino acid sequence of SEQ ID NO: 34, 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: 48, 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.

[0207] In some embodiments, the anti-CD89 scFv molecule further comprises a linker for linking the VH domain and the VL domain thereof. In some embodiments, the linker comprises an amino acid sequence of (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: 60.

[0208] In some embodiments, the heavy chain variable region of the anti-CD20 antibody comprises a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 1, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 2, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 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 the HCDR1, HCDR2, and HCDR3, respectively.

[0209] In some embodiments, the heavy chain variable region of the anti-CD20 antibody comprises an amino acid sequence as set forth in SEQ ID NO: 7.

[0210] In some embodiments, the first and second heavy chain constant regions 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 pi mutation, TM mutation, and "bump" mutation. In some embodiments, the first heavy chain constant region has an amino acid sequence as set forth in SEQ ID NO: 58.

[0211] In some embodiments, the first polypeptide chain further comprises a linker for linking the anti-CD89 scFv molecule and the anti-CD20 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: 61. n In some embodiments, the first polypeptide chain further comprises a linker for linking the anti-CD89 scFv molecule and the anti-CD20 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: 61.

[0212] 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 pi mutation, TM mutation, and "pocket" mutation. In some embodiments, the second heavy chain constant region has an amino acid sequence as set forth in SEQ ID NO: 57.

[0213] In some embodiments, the light chain variable region of the anti-CD20 antibody comprises a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 4, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 5, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 6, 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.

[0214] In some embodiments, the light chain variable region of the anti-CD20 antibody comprises an amino acid sequence as set forth in SEQ ID NO: 8.

[0215] 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 as set forth in SEQ ID NO: 59).

[0216] In another aspect, the anti-CD20 / CD89 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 is a second antigen binding domain that specifically binds to extracellular Ig-like domain 2 of CD89, preferably an anti-CD89 scFv sequence with a disulfide bond-containing VH-VL interface, VH1 represents a heavy chain variable region of the second 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 second antigen binding domain, L2 represents a linker (such as (G4S) n , wherein n is an integer from 1 to 3, VH2 represents a heavy chain variable region of the first antigen binding domain that specifically binds to CD20, CH a represents a heavy chain constant region of subunit A; the second polypeptide chain comprises VH2-CH b , wherein CH b represents a heavy chain constant region of subunit B; and the third polypeptide chain comprises VL2-CL, wherein VL2 represents a light chain variable region of the first antigen binding domain that specifically binds to CD20, and CL represents an IgG light chain constant region.

[0217] In some embodiments, the VH1 comprises a HCDR1 having an amino acid sequence of SEQ ID NO: 9, a HCDR2 having an amino acid sequence of SEQ ID NO: 14, and a HCDR3 having an amino acid sequence of SEQ ID NO: 16, 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, and HCDR3, respectively; L1 comprises a linker amino acid sequence of SEQ ID NO: 60; VL1 comprises a LCDR1 having an amino acid sequence of SEQ ID NO: 19, a LCDR2 having an amino acid sequence of SEQ ID NO: 21, and a LCDR3 having an amino acid sequence of SEQ ID NO: 23, 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 LCDR1, LCDR2, and LCDR3, respectively; L2 comprises a linker amino acid sequence of SEQ ID NO: 61; VH2 comprises a HCDR1 having an amino acid sequence of SEQ ID NO: 1, a HCDR2 having an amino acid sequence of SEQ ID NO: 2, and a HCDR3 having an amino acid sequence of SEQ ID NO: 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 sequences of the HCDR1, HCDR2, and HCDR3, respectively; CH a comprises an engineered heavy chain constant region, including a human IgGl heavy chain constant region with a "knobs-into-holes" mutation, a mutation to increase the pi, and a mutation to decrease Fc effector function, the amino acid sequence of which is set forth in SEQ ID NO: 58.

[0218] In some specific embodiments, the VH1 comprises an amino acid sequence as set forth in SEQ ID NO: 34, 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 VL1 comprises an amino acid sequence as set forth in SEQ ID NO: 48, 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 the VH2 comprises an amino acid sequence as set forth in SEQ ID NO: 7, 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.

[0219] 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 pi, and a mutation to reduce Fc effector function, the amino acid sequence of which is set forth in SEQ ID NO: 57.

[0220] In some embodiments, the VL2 comprises a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 4, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 5, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 6, 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 LCDR1, LCDR2, and 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 as set forth in SEQ ID NO: 59).

[0221] In some specific embodiments, the VL2 comprises an amino acid sequence as set forth in SEQ ID NO: 8, 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.

[0222] In some specific embodiments, the anti-CD20 / CD89 bispecific antibody of the application comprises:

[0223] (1) one 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 as set forth in SEQ ID NO: 9, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 14, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 16, L1 comprising a linker amino acid sequence as set forth in SEQ ID NO: 60, the VL1 comprising a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 19, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 21, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 23, L2 comprising a linker amino acid sequence as set forth in SEQ ID NO: 61, the VH2 comprising a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 1, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 2, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 3, the CH a comprising an amino acid sequence as set forth in SEQ ID NO: 58;

[0224] (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: 1, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 2, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 3, the CH b comprising an amino acid sequence as set forth in SEQ ID NO: 57;

[0225] (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: 4, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 5, and a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 6, the CL comprising an amino acid sequence as set forth in SEQ ID NO: 59.

[0226] In some embodiments, the anti-CD20 / CD89 bispecific antibody of the present application comprises:

[0227] (1) one first polypeptide chain comprising: VH1 comprising an amino acid sequence as set forth in SEQ ID NO: 34, LI comprising an amino acid sequence as set forth in SEQ ID NO: 60, VL1 comprising an amino acid sequence as set forth in SEQ ID NO: 48, L2 comprising an amino acid sequence as set forth in SEQ ID NO: 61, VH2 comprising an amino acid sequence as set forth in SEQ ID NO: 7, CH a ;

[0228] (2) one second polypeptide chain comprising: VH2 comprising an amino acid sequence as set forth in SEQ ID NO: 7, CH b ; and

[0229] (3) two third polypeptide chains comprising: VL2 comprising an amino acid sequence as set forth in SEQ ID NO: 8, CL comprising an amino acid sequence as set forth in SEQ ID NO: 59.

[0230] In some embodiments, the first polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 62, the second polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 63, and the third polypeptide chain comprises an amino acid sequence as set forth in SEQ ID NO: 64.

[0231] 4. Polynucleotides, vectors, and host cells

[0232] In one aspect, the present application provides a nucleic acid encoding an anti-CD89 antibody or antigen-binding fragment thereof, and an anti-CD20 / CD89 bispecific antibody. The present application also includes polynucleotide variants encoding the amino acid sequences described herein.

[0233] One skilled in the art can determine nucleic acid sequences encoding the antibodies of the application based on the genetic code. Polymerase chain reaction (PCR) can be used to isolate and amplify DNA sequences encoding the anti-CD89 antibodies of the application or antigen-binding fragments thereof, or anti-CD20 / CD89 bispecific antibodies. Oligonucleotides can additionally contain recognition sites for restriction endonucleases to facilitate insertion of the amplified DNA fragments into expression vectors. 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.

[0234] Nucleic acid molecules of the application include single- and double-stranded DNA and RNA, and corresponding complementary sequences. Nucleic acid molecules of the application also include isolated nucleic acid molecules, preferably DNA or RNA that has been purified and whose component nucleotide sequences are identifiable, manipulatable, and recoverable 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.

[0235] The anti-CD89 antibodies or antigen-binding fragments thereof, or anti-CD20 / CD89 bispecific antibodies of the application can be prepared by mutagenizing specific nucleotides in the DNA encoding the anti-CD89 antibodies or antigen-binding fragments thereof, or anti-CD20 / CD89 bispecific antibodies using PCR mutagenesis or other techniques known to those of ordinary skill in the art to produce DNA encoding variants, followed by expression / amplification of the recombinant DNA in cell culture as outlined herein. In addition, the anti-CD89 antibodies or antigen-binding fragments thereof, or anti-CD20 / CD89 bispecific antibodies can also be prepared by in vitro synthesis using established techniques.

[0236] As is known to those skilled in the art, due to the degeneracy of the genetic code, the anti-CD89 antibodies or antigen-binding fragments thereof, or anti-CD20 / CD89 bispecific antibodies of the present application are 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, one skilled in the art can make many different nucleic acids, depending on the particular amino acid sequence identified, by simply modifying the coding sequence(s) of each in a manner that does not change the amino acid sequence of the anti-CD89 antibodies or antigen-binding fragments thereof, or anti-CD20 / CD89 bispecific antibodies of the present application.

[0237] In another aspect, the present application also provides an expression vector comprising a nucleic acid encoding the anti-CD89 antibodies or antigen-binding fragments thereof, or anti-CD20 / CD89 bispecific antibodies herein.

[0238] The nucleic acids encoding the anti-CD89 antibodies or antigen-binding fragments thereof, or anti-CD20 / CD89 bispecific antibodies of the present application can be constructed in suitable vectors for introduction into host cells 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 with the promoter.

[0239] As used herein, the term "operably linked" refers to 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, thereby placing the latter under the influence of the former.

[0240] 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 (e.g., 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 (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papova viruses (e.g., SV40). The vectors can contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vectors can contain a replication origin. The vectors can also include components that aid in their entry into cells, including but not limited to viral particles, liposomes, or protein coats.

[0241] In another aspect, the present application also provides a host cell comprising a nucleic acid or an expression vector encoding the anti-CD89 antibodies or antigen-binding fragments thereof, or anti-CD20 / CD89 bispecific antibodies described herein.

[0242] The host cell can be a eukaryotic cell, for example, a mammalian host cell including, but not limited to, the SV40-transformed monkey kidney CV1 line (COS-7, ATCC, CRL-1651), the human embryonic kidney line 293 or 293 cells suspended in medium, Graham et al., J Gen Virol 1977, 36:59-74, baby hamster kidney cells (BHK-21, ATCC, CCL-10), Chinese hamster ovary cells (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.

[0243] 5. Preparation method

[0244] The present application provides a method for preparing the anti-CD89 antibody or antigen-binding fragment thereof of the present application, or the anti-CD20 / CD89 bispecific antibody using the host cell.

[0245] The method includes transfecting a host cell with a nucleic acid or expression vector encoding the anti-CD89 antibody or antigen-binding fragment thereof of the present application, or the anti-CD20 / CD89 bispecific antibody, and then culturing the host cell in a medium for a period of time to express the anti-CD89 antibody or antigen-binding fragment thereof of the present application, or the anti-CD20 / CD89 bispecific antibody. Without limitation, commercially available media can be used as the medium for culturing the host cell.

[0246] Preferably, the expressed anti-CD89 antibody or antigen-binding fragment thereof, or anti-CD20 / CD89 bispecific antibody can be secreted into the culture medium of the host cell. 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; other purification techniques can also be used, such as, for example, anion or cation exchange chromatography, hydrophobic interaction chromatography, and hydroxyapatite chromatography.

[0247] 6. Pharmaceutical composition

[0248] The present application provides a pharmaceutical composition comprising an anti-CD20 / CD89 bispecific antibody described herein, and a pharmaceutically acceptable carrier.

[0249] The pharmaceutical composition 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. For example, the following references set forth the selection and use of suitable carriers: Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Edition, 2003 (Lippincott Williams & Wilkins), the disclosure of which is incorporated herein by reference.

[0250] Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration. As used herein, "parenteral administration" refers to modes of administration other than enteral and topical administration, including, but not limited to, injection, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and sternal injection and infusion. 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 inactivate it.

[0251] The pharmaceutical compositions can be in the form of a sterile aqueous solution or dispersion. They can also be in the form of a microemulsion, liposome, or other ordered structure suitable for high drug concentration. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the subject and the particular mode of administration. In general, a composition 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%, in combination with a pharmaceutically acceptable carrier is desirable.

[0252] 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 suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Additionally, the anti-CD20 / CD89 bispecific antibodies of the present application can also be administered as a sustained release formulation, allowing for less frequent dosing.

[0253] A "therapeutically effective amount" of an anti-CD20 / CD89 bispecific antibody of the present application preferably results in a decrease in the severity of a disease symptom, an increase in the frequency and duration of disease symptom stability, or a prevention of physical damage or disability due to a disease affliction. For example, a "therapeutically effective amount" of a subject preferably inhibits disease progression 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 antibodies and bispecific antibodies) improves at least one symptom of a subject's condition, which subject is typically a human or other mammal. A "therapeutically effective amount" can also be determined differently by a clinician depending on various factors, including but not limited to method of formulation, 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.

[0254] Pharmaceutical compositions can be formulated to provide controlled release of the therapeutic antibody. For example, a sustained release formulation can be used. Suitable examples of sustained release formulations include a polymer matrix, a polymer coating and a lipid matrix. See, e.g., Langer, Science 249: 1527-1533 (1990); and Langer and

[0255] Therapeutic pharmaceutical compositions 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.

[0256] In certain embodiments, the anti-CD20 / CD89 bispecific antibodies described herein can be formulated to ensure bio-distribution in vivo. For example, to ensure that the therapeutic antibodies described herein cross the blood-brain barrier, they can be formulated in a liposome, which can additionally contain a targeting moiety to enhance selective delivery to a particular cell or organ. 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.

[0257] 7. A kit

[0258] In one aspect, the present application provides a kit comprising the anti-CD20 / CD89 bispecific antibody of the present application, or the pharmaceutical composition of the present application, and optionally at least one additional therapeutic agent, including but not limited to, a tumor therapeutic agent and an immunosuppressive agent for CD20-associated disorders.

[0259] In some embodiments, the tumor therapeutic agent and the immunosuppressive agent include but are not limited to: a chemotherapeutic agent, a hormone receptor modulator, a kinase inhibitor, a B-cell surface receptor antibody, an activator of tumor cell apoptosis, an anti-angiogenic drug, an aromatase inhibitor, a cytokine inhibitor.

[0260] 8. Methods and uses for treating CD20-associated disorders

[0261] In one aspect, the present application relates to a method for treating a CD20-associated disorder, comprising administering to a subject in need thereof a therapeutically effective amount of the anti-CD20 / CD89 bispecific antibody of the present application, or the pharmaceutical composition or the kit of the present application. Alternatively, use of the anti-CD20 / CD89 bispecific antibody of the present application, or the pharmaceutical composition or the kit thereof, in the manufacture of a medicament for treating a CD20-associated disorder. Alternatively, the bispecific antibody, the pharmaceutical composition or the kit of the present application for use in treating a CD20-associated disorder.

[0262] The CD20-associated disorder includes B-cell malignancies and autoimmune diseases caused by B-cell auto-reactivity. The subject can be a human, a non-human primate or other mammals such as dogs, etc.

[0263] The B-cell malignancies include B-cell lymphomas and B-cell leukemias, exemplary B-cell lymphomas are NHL, including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma, small lymphocytic lymphoma, mantle cell lymphoma (MCL) and Burkitt lymphoma; the B-cell leukemias include CLL, ALL and HCL.

[0264] The autoimmune diseases caused by B-cell auto-reactivity include but are not limited to systemic lupus erythematosus (SLE), cutaneous lupus, discoid lupus, lupus nephritis, scleroderma, dermatomyositis (DM), polymyositis (PM), psoriasis, psoriatic arthritis, rheumatoid arthritis, multiple sclerosis, systemic sclerosis, vasculitis, thrombocytopenia, myasthenia gravis, type I diabetes.

[0265] In some embodiments, administering a therapeutically effective amount of an anti-CD20 / CD89 bispecific antibody or a pharmaceutical composition or kit thereof to a subject having a CD20-associated disorder is capable of reducing or depleting pathogenic B cells. In some specific embodiments, administration of the bispecific antibody is capable of significantly reducing the number of B cells in a subject or a biological sample taken from a subject (e.g., blood of a healthy subject or a subject having a CD20-associated disorder) as compared to prior to administration of the bispecific antibody.

[0266] 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 applications cited throughout this application are expressly incorporated by reference herein. The reagents, materials, apparatus and procedures involved in the following examples are conventional in the art unless specifically indicated otherwise.

[0267] Example

[0268] Example 1. Optimization of anti-CD89 antibody Mab 14.1

[0269] 1.1 Construction of Mab 14.1 single chain antibody

[0270] Anti-CD89 humanized antibody Mab 14.1 is described in PCT Publication No. WO2002064634. The construction of Mab 14.1 scFv-Fc fusion protein expression vector in this example is as follows: a linker sequence (as shown in SEQ ID NO: 60) is introduced at the C-terminus of the heavy chain variable region of Mab 14.1 (as shown in SEQ ID NO: 26) to connect with the N-terminus of the light chain variable region (as shown in SEQ ID NO: 40), and a Mab 14.1 scFv sequence is designed, and another linker sequence (as shown in SEQ ID NO: 61) is introduced at the C-terminus of the scFv sequence for connection with the wild-type human IgGl Fc fragment (amino acid sequence as shown in SEQ ID NO: 56 from position 99 to position 330). The DNA fragment encoding the above sequence is obtained by chemical synthesis, and then the DNA fragment is inserted into the pcDNA3.1-human IgGl plasmid by homologous recombination, and a clone is screened to obtain the expression vector of Mab 14.1 scFv-Fc fusion protein.

[0271] 1.2 Single point / multiple point mutation optimization of Mab 14.1 single chain antibody

[0272] Using the online software abYsis (http: / / abysis.org / abysis / index.html), we analyzed amino acid residues with low humanization levels and potential aspartate isomerization and / or asparagine deamidation sites in the light and heavy chain variable region sequences of Mab14.1. A total of 39 single-point or multi-point mutations were designed to improve the degree of humanization of the antibody and / or reduce the risk of aspartate isomerization and / or asparagine deamidation (as shown in Table 4). These 39 single-point or multi-point mutations were introduced into the expression vector of the Mab14.1 scFv-Fc fusion protein by PCR. The antibody protein was then transiently expressed and purified by Protein A affinity chromatography using the AKTA Pure system.

[0273] The transient expression levels of the 39 single-chain antibody mutants and their parental single-chain antibody, Mab14.1, were measured using a molecular interaction analyzer (ForteBio). The specific method is as follows: After transient expression using ExpiCHO-S cells, the culture supernatant was harvested and added to a 96-well black-walled plate at 200 μL / well. The test samples and Protein A probe were placed in the ForteBio analyzer, and the binding rate of each antibody molecule in each culture supernatant to the Protein A probe was measured using a preset quantitative program. After each cycle, the probe was immersed in 10 mM glycine buffer (pH 1.5) to regenerate the probe before the next cycle of detection was started. After all samples were tested, the transient expression levels of each mutant and the parental antibody were calculated using analysis software based on the standard curve.

[0274] ForteBio was used to detect the binding kinetics of the above 39 single-chain antibody mutants with human CD89 recombinant protein, and the control antibody was the parent single-chain antibody Mab14.1. The specific method is as follows: the antibody to be tested was diluted to 5μg / mL with PBS solution and added to a 96-well black-walled plate at 200μL / well; the human CD89 recombinant protein was diluted to 10μg / mL with PBS solution and added to the same 96-well black-walled plate at 200μL / well. The 96-well black-walled plate containing the antibody to be tested and the Protein A detection probe were placed in the ForteBio instrument, and the program was set to first allow the Protein A probe to bind to the antibody to be tested until the signal value reached 1.5nm, and then the binding and dissociation detection with the antigen were performed, both for 300s. After each cycle was completed, the probe was immersed in 10mM glycine buffer (pH 1.5) to regenerate the probe, and then the next cycle of detection was started. After all samples were tested, the binding constant (k) between the antibody and the antigen was calculated using the analysis software. a ) and dissociation constant (k dis ) was fitted and the affinity constant (KD ) numerical values.

[0275] The mutation sites, transient expression amounts, and the results of the binding kinetics detection with CD89 of each mutant are shown in Table 4. Most of the 39 single / multiple point mutations retain the antigen binding affinity of the parent antibody Mab14.1, and improve the humanization degree of the antibody and / or significantly reduce the aspartate isomerization and / or asparagine deamidation risk of the antibody, preferably 7 mutations thereof, 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.

[0276] Table 4. Results of single / multiple point mutations of Mab14.1, transient expression amounts, and binding kinetics analysis with CD89

[0277] L is the light chain; H is the heavy chain.

[0278] 1.3 Combination mutation optimization of Mab14.1 single chain antibody

[0279] The above-mentioned preferred 7 single point mutations are 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 are carried out according to the method described in item 1.2 of this embodiment, and the transient expression amounts and the binding kinetics with CD89 of each mutant and the control parent antibody Mab14.1 are detected by ForteBio.

[0280] The combination mutation sites, transient expression amounts, purities after Protein A affinity purification, and the results of the binding kinetics analysis with CD89 of each mutant molecule are shown in Table 5. The transient expression amounts and antigen binding affinities of each combination mutant are comparable to those of the parent antibody Mab14.1, but the purities of the combination mutants Mab14.1-HL1404 and Mab14.1-HL1405 after Protein A affinity purification can reach more than 90%, even 100%, which are significantly better than those of the parent antibody and other mutants.

[0281] Table 5. Results of combination mutations of Mab14.1, transient expression amounts, purities, and binding kinetics analysis with CD89 L is the light chain; H is the heavy chain; Full R 2 represents the similarity of the fitted curve to the measured curve.

[0282] 1.4 Disulfide bond introduction at the VH-VL interface of anti-CD89 scFv molecules and its effect on the stability and biological activity of the molecules

[0283] To further enhance the structural stability of anti-CD89 scFv molecules, the distances between each pair of amino acid residues in the VH-VL interface of single-chain antibody Mab14.1-HL1405 were calculated using MOE software, and pairs of amino acid residues with spatial distances within 5 A were selected and mutated to cysteine in order to form disulfide bonds and achieve the purpose of stabilizing the structure of the single-chain antibody molecule. The specific method is as follows: 12 pairs of cysteine mutations (as shown in Table 6) were introduced into the expression vector of single-chain antibody Mab14.1-HL1405 by PCR, and then the preparation of antibody samples was performed according to the method described in item 1.2 of this example. After purification by Protein A affinity chromatography, it was found that mutants Mab14.1-HL1405-Cy02 and Mab14.1-HL1405-Cy07 had a high proportion of multimers, and the other cysteine mutants could obtain high-purity scFv monomer molecules.

[0284] Table 6. Mab14.1-HL1405 cysteine combination mutation sites

[0285] According to the method described in item 1.2 of this example, the binding kinetics of each cysteine mutant to CD89 was detected using ForteBio, and the results are shown in Table 7. Mutant Mab14.1-HL1405-Cy08 did not bind to CD89, and the binding affinities of mutants Mab14.1-HL1405-Cy01, Mab14.1-HL1405-Cy03, Mab14.1-HL1405-Cy09, and Mab14.1-HL1405-Cy10 were not significantly different from those of the parent antibody Mab14.1-HL1405, and the binding affinities of the other mutants were reduced to varying degrees.

[0286] ​Further, the cell binding activity of the above mutants was detected by flow cytometry using Jurkat cells expressing CD89. The specific method is as follows: collect Jurkat cells expressing CD89 and resuspend with FACS buffer (PBS containing 1% BSA), then add 50 μL per well to a 96-well U-shaped plate. Each mutant to be tested was diluted by 10 times gradient in FACS buffer at an initial concentration of 100 μg / mL, then added to the above well plate at 50 μL per well, mixed and incubated on ice for 1 hour. Centrifuge to remove the supernatant, and wash the cells with FACS buffer for 3 times. AF488 labeled goat anti-human IgG (H+L) antibody (Jackson Immuno) was diluted in FACS buffer at 1:1000 (volume ratio), added to the above well plate at 100 μL per well, mixed and incubated on ice for about 40 minutes. Centrifuge to remove the supernatant, and wash the cells with FACS buffer for 3 times. Resuspend the cells with 100 μL per well of FACS buffer, and detect using a flow cytometer (NovoCyte 3005, Agilent). The detection results are shown in Figure 1 and Table 7, and the cell binding activity of each mutant shows varying degrees of change compared to 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 is basically equivalent to that of the parent antibody, which is consistent with the above antigen binding affinity detection results.

[0287] Table 7. Binding affinity of Mab14.1-HL1405 cysteine mutants to CD89 and binding activity to Jurkat cells N / A: not applicable; Full R 2 represents the similarity of the fitted curve to the measured curve.

[0288] The thermal stability of each mutant was detected by DSF. The specific method is as follows: each mutant was diluted to 1 mg / mL with PBS and loaded into a capillary, and the instrument was set to detect the thermal stability of each sample at a temperature range of 25-95°C at a temperature increase 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 8, 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.

[0289] Table 8. Thermal stability analysis results of Mab14.1-HL1405 cysteine mutants Tonset : onset temperature of protein denaturation; T m : protein denaturation temperature; T agg : onset temperature of protein aggregation.

[0290] Example 2 Construction of anti-CD20 / CD89 bispecific antibodies and identification of their functional activities

[0291] 2.1 Construction of anti-CD20 / CD89 bispecific antibodies

[0292] The exemplary anti-CD20 / CD89 bispecific antibodies of the present application are constructed from the humanized B-Lyl antibody disclosed in WO2005044859 capable of specifically recognizing CD20 (wherein the VH amino acid sequence is shown as SEQ ID NO: 7, the VL amino acid sequence is shown as SEQ ID NO: 8, and the CH and CL amino acid sequences are shown as SEQ ID NO: 56 and SEQ ID NO: 59, respectively) and a monospecific antibody capable of specifically recognizing the extracellular Ig-like domain 2 of human CD89. As shown in Figure 2, the configuration of the anti-CD20 / CD89 bispecific antibodies is 2+1 scFv-IgG or scFv-Fab-Fc: Fab-Fc asymmetric structure, wherein subunit A represents the scFv-Fab-Fc format and subunit B represents the Fab-Fc format, the scFv domain is selected from the anti-CD89 single chain antibodies with disulfide bond-containing VH-VL interface shown in Table 6 (e.g., Mab 14.1-HL1405-Cy09), and the IgG domain or Fab-Fc domain is the anti-CD20 humanized B-Lyl antibody, 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 via a peptide linker (G4S)2. The asymmetric structure adopted by the anti-CD20 / CD89 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., B cells) expressing CD20 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, 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.

[0293] The present embodiment also performs the following different types of engineering modifications on the heavy chain constant region of the bispecific antibody: (1) to promote the formation of heterodimer by introducing "knob and hole" mutations in the heavy chain Fc region of the IgG domain, since the bispecific antibody only contains one light chain, there is no problem of light chain mispairing; (2) to be able to effectively separate the "hole-hole" homodimer that may be formed during the expression process by ion exchange chromatography, by introducing different types of "pI mutations" in the two heavy chain constant regions of the bispecific antibody, for example, introducing mutations that increase the pI value in the heavy chain constant region containing the "knob" mutation, and introducing mutations that decrease the pI value in the heavy chain constant region containing the "hole" mutation, to expand the pI value difference between the heterodimer bispecific antibody and the "hole-hole" homodimer; (3) to avoid the possibility that the Fc effector function of the antibody may cause killing of non-target cells by the bispecific antibody, by introducing L234F / L235E / P331S (EU Numbering) mutations (defined as "TM mutations") in the Fc region of the bispecific antibody to reduce its binding to FcyRs and Clq, thereby eliminating the Fc effector function.

[0294] The different types of engineering modifications introduced into the two heavy chain constant regions of the anti-CD20 / CD89 bispecific antibody and the amino acid mutations involved are shown in Table 9. After introducing different types of "pI mutations" in the two heavy chain constant regions, the difference in the theoretical pI values between the heterodimer bispecific antibody and the "hole-hole" homodimer is about 1.3 pH units.

[0295] Table 9. Amino acid mutations introduced into the heavy chain constant region of the anti-CD20 / CD89 bispecific antibody

[0296] The anti-CD20 humanized B-Ly1 antibody variable region encoding sequence was obtained using a gene synthesis method, and the heavy chain variable region and light chain variable region gene sequences were homologously recombined into pcDNA3.1 vectors containing human IgG1 heavy chain constant region sequences and light chain kappa constant region sequences (as shown in SEQ ID NO: 59), respectively, to construct the expression vector of the antibody.

[0297] According to the above design, first, the TM mutation was introduced into the constructed heavy chain expression vector of the anti-CD20 humanized B-Ly1 antibody by PCR method, then the "knobs" and "holes" mutations were introduced in the same way, respectively, and the "pl mutation" corresponding to each heavy chain (wherein the amino acid sequence of the heavy chain constant region containing the "knobs" mutation is shown in SEQ ID NO: 58, and the amino acid sequence of the heavy chain constant region containing the "holes" mutation is shown in SEQ ID NO: 57), and finally the sequence of the anti-CD89 scFv (for example, Mab14.1-HL1405-Cy09) containing disulfide bond in the VH-VL interface shown in Table 6 was connected to the N-terminus of the "knobs" heavy chain of the anti-CD20 humanized B-Ly1 antibody by homologous recombination, to complete the construction of the bispecific antibody heavy chain expression vector. After combining the constructed bispecific antibody heavy chain expression vector with the above-constructed light chain expression vector of the anti-CD20 humanized B-Ly1 antibody, the ExpiCHO-S cells were co-transfected and cultured in serum-free medium to express the bispecific antibody (named Cy09-GFT). After the cell viability was less than 70%, the culture supernatant was collected, and the antibody was purified by Protein A affinity chromatography using AKTA Pure system. The purified antibody obtained was subjected to SEC-HPLC analysis, and the results showed that the bispecific antibody exhibited a high monomer proportion, about 80%.

[0298] 2.2 Binding affinity of anti-CD20 / CD89 bispecific antibody to CD20

[0299] According to the method described in item 1.2 of Example 1, the binding kinetics of the bispecific antibody Cy09-GFT to human CD20 and CD89 recombinant proteins, respectively, were detected by ForteBio. The results are shown in Table 10, and the bispecific antibody retains the antigen binding affinity of its corresponding monospecific antibody.

[0300] Table 10. Results of binding kinetics analysis of anti-CD20 / CD89 bispecific antibody to CD20 and CD89, respectively Full R 2 Indicates the similarity of the fitted curve to the measured curve.

[0301] 2.3 Co-binding activity of anti-CD20 / CD89 bispecific antibody to CD20 and CD89

[0302] To detect whether the bispecific antibody Cy09-GFT can bind to both human CD20 and CD89 recombinant proteins simultaneously, the following method was used: first, biotin-labeled human CD89 recombinant protein was captured by SA probe, and after equilibration with PBST (0.05% Tween-20 in PBS), the bispecific antibody and control antibodies (including anti-CD20 antibody Obinutuzumab and anti-CD89 antibody Mab14.1-HL1405) were added for binding detection, and finally human CD20 recombinant protein was added for binding detection. The results are shown in Figure 3, and the bispecific antibody Cy09-GFT can bind to both CD20 and CD89.

[0303] 2.4 Anti-CD20 / CD89 bispecific antibody does not block the binding of CD89 to IgA

[0304] To detect whether the bispecific antibody Cy09-GFT can block the binding of CD89 to IgA, the following method was used: first, biotin-labeled human CD89 recombinant protein was captured by SA probe fixed on the detector, and after equilibration with PBST, one group of human serum-derived IgA (Sigma) was directly loaded to verify the binding of IgA to CD89, and the other group was first loaded with bispecific antibody Cy09-GFT and allowed to bind to CD89 to saturation, and then IgA was loaded to detect whether Cy09-GFT can interfere with the binding of CD89 to IgA. The results are shown in Figure 4, and the binding of IgA to CD89 recombinant protein is the same in the presence or absence of bispecific antibody Cy09-GFT (labeled as ① and ② in the figure), indicating that the bispecific antibody and its anti-CD89 antibody part do not block the binding of IgA to CD89.

[0305] 2.5 Binding activity of anti-CD20 / CD89 bispecific antibody to Raji cells expressing CD20 and Jurkat cells expressing CD89, respectively

[0306] The cell binding activity of the bispecific antibody Cy09-GFT was detected by flow cytometry using Raji cell line expressing CD20 and Jurkat cell line expressing CD89, respectively, and the control antibodies included Obinutuzumab. The specific method was as follows: the cells were collected and resuspended with FACS buffer (PBS containing 1% BSA), and then added to 96-well U-shaped plates at 50 μL / well. The test antibody with an initial concentration of 600 nM or 100 μg / mL was diluted with FACS buffer by 5-fold gradient, and then added to the above-mentioned well plates 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 or AF647 labeled goat anti-human IgG (H+L) antibody (Jackson Immuno) was diluted in FACS buffer at a volume ratio of 1:1000, added to the above-mentioned well plates at 100 μL / well, mixed and incubated on ice for 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. The detection results are shown in FIG. 5 and FIG. 6, and the bispecific antibody Cy09-GFT has specific binding activity to cells, which is consistent with the above-mentioned antigen binding affinity detection results.

[0307] 2.6 Biological activity of anti-CD20 / CD89 bispecific antibody

[0308] Biological activity of anti-CD20 / CD89 bispecific antibody was detected by reporter gene method. The specific method is as follows: target cells (i.e. Raji cells expressing CD20) were collected, resuspended with DMEM medium containing 1% FBS, and then plated into 96-well white wall plates at 80 μL / well; 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 added into the 96-well white wall plates containing target cells at 20 μL / well, so that the cell count ratio of target cells to effector cells was 1:20, and a culture well containing only effector cells was set as a control group. The bispecific antibody Cy09-GFT with an initial concentration of 10 μg / mL was diluted by 5 times gradient, and then added into the 96-well plates at 20 μL / well, and incubated at 37°C for 6 hours. Then, the 96-well plates were taken out, and Bio-Lite luciferase substrate (Nanjing Novizan) was added, and the chemiluminescence value was read by using a multifunctional enzyme label instrument (Varioskan, Thermo). As shown in FIG. 7, the bispecific antibody cannot activate the CD89-mediated signal pathway in the presence of only effector cells, but can activate the CD89-mediated signal pathway in the presence of both target cells and effector cells, indicating that the anti-CD20 / CD89 bispecific antibody has target cell-dependent CD89 agonistic activity, i.e. the anti-CD20 / CD89 bispecific antibody cannot non-directionally activate myeloid effector cells expressing CD89 in the absence of target cells.

[0309] 2.7 Activity of anti-CD20 / CD89 bispecific antibody in mediating killing of human primary B cells by human primary leukocytes and activity of inducing cytokine release

[0310] According to the method described in the instruction provided by the manufacturer Stemcell, HetaSep TMLeukocytes were isolated from peripheral blood of 13 healthy people, and resuspended in RPMI-1640 medium containing 10% FBS, and then added into 96-well U-shaped plates at 180 μL / well. Anti-CD20 / CD89 bispecific antibody Cy09-GFT and control antibody (anti-CD20 / CD3 bispecific antibody Glofitamab [manufacturer: Roche]) were diluted in RPMI-1640 medium containing 10% FBS to 20 μg / mL, and then diluted by 10 times gradient, and then added into the above 96-well U-shaped plates at 20 μL / well, and a blank medium group (i.e. only the same volume of RPMI-1640 medium containing 10% FBS was added) was set as a negative control. After the 96-well plates were incubated at 37°C for about 20 hours, the culture supernatant in the highest concentration (2 μg / mL) treatment wells and the negative control wells of Cy09-GFT and Glofitamab was collected, centrifuged at 1000g at 4°C for 10 minutes, and then the upper supernatant was aspirated and stored at -80°C for subsequent cytokine release detection. The cells in each treatment well and control well of the 96-well plate were stained with FITC or PE labeled anti-human CD19 antibody (Biolegend) and 7-AAD (Biolegend), and detected and analyzed by flow cytometry. The cells in each well were first circled out by forward scatter light (FSC-H) and side scatter light (SSC-H), and then the viable cells in the cell population were circled out by 7-AAD, and finally the ratio of B cells in the PBMC viable cell population was analyzed by CD19 + B cells in the PBMC viable cell population. The results, as shown in FIG. 8, show that Cy09-GFT can induce CD89 expressing myelocytes to kill CD20 expressing B cells, and the killing effect is superior to the killing effect of T effector cells mediated by Glofitamab (the killing effect refers to the minimum residual ratio of B cells in PBMC).

[0311] After the above culture supernatant stored at -80°C was thawed, the concentrations of each cytokine (including TNFα, IL-6, IFNγ, IL-10, IL-1β, and IL-5) contained therein were quantitatively detected by using Luminex Human Discovery Assay (6-Plex) kit (R&D System). The results, as shown in FIG. 9, show that compared with the negative control, Glofitamab can significantly stimulate the release of a large amount of cytokines from healthy human primary leukocytes, while no significant increase in the concentration of cytokines is detected in the supernatant treated by Cy09-GFT, indicating that the myelocytes mediated by Cy09-GFT do not produce or release a large amount of cytokines when killing target cells.

[0312] 2.8 Fc effector function of anti-CD20 / CD89 bispecific antibody

[0313] To reduce the potential safety risk due to Fc-mediated effector functions, the present embodiment introduces three point mutations in the Fc region of the anti-CD20 / CD89 bispecific antibody: L234F / L235E / P331S (i.e. "TM mutations") to reduce the binding activity of the antibody to Fc receptors and Clq. It is confirmed by Fortebio assay that the bispecific antibody Cy09-GFT containing TM mutations has no significant binding activity to FcyRI, FcyRIIa (131H), FcyRIIb, FcyRIIIa (158V), FcyRIIIa (158F) and Clq, while the binding affinity to FcRn has no significant difference compared with the IgGl isotype control antibody (data not shown).

[0314] In addition, the present embodiment also uses Jurkat cells expressing FcyRIIIa-158V receptor and NFAT response element regulated luciferase reporter gene (Promega) as effector cells, Raji cells expressing CD20 as target cells, and anti-CD20 antibody Obinutuzumab with wild-type Fc sequence as positive control to further verify whether the bispecific antibody Cy09-GFT containing "TM mutations" has ADCC activity. The specific method is as follows: centrifugal collection of Raji cells, resuspension with ADCC detection buffer (RPMI-1640 containing 0.5% FBS), and then 1x10 4 Cells / well were added to a 96-well plate. The test antibody was diluted with ADCC detection buffer gradient, and 20 μL / well was added to the above 96-well plate. The effector cells were centrifugally collected and resuspended with ADCC detection buffer, and then 1x10 5 The above 96-well plate was placed in a 37°C / 5% CO2 incubator for 6 hours. After the 96-well plate was equilibrated at room temperature, the detection reagent Bio-Lite Luciferase Reagent (Novagen) was added, and the chemiluminescence value was read by a multifunctional enzyme label instrument. The detection results are shown in Figure 10. Unlike the positive control antibody Obinutuzumab, Cy09-GFT did not induce any luciferase expression even at high concentrations, indicating that the CD20 / CD89 bispecific antibody with "TM mutations" introduced in the Fc region has no ADCC activity.

[0315] 2.9 Anti-CD20 / CD89 bispecific antibody in cynomolgus monkey in vivo efficacy evaluation

[0316] The anti-CD20 / CD89 bispecific antibody Cy09-GFT was analyzed by flow cytometry for its binding to CD19 + B cells and CD89+ The results showed that Cy09-GFT and CD19 + The binding activity of B cells was almost the same in cynomolgus monkeys and humans (EC 50 0.396μg / mL and 0.390μg / mL respectively); at the same time, it + There was no significant difference in the binding activity of myeloid cells between cynomolgus monkeys and humans (EC 50 Therefore, cynomolgus monkeys are a pharmacologically / toxicologically relevant species for the anti-CD20 / CD89 bispecific antibody.

[0317] Two cynomolgus macaques, one male and one female, were given a single intravenous injection of the bispecific antibody Cy09-GFT at a dose of 10 mg / kg. Peripheral blood was collected 120h and 72h before administration, and 24h, 72h, and 168h after administration for flow cytometry analysis to observe changes in the number of B cells. The flow cytometry assay was performed as follows: 5 μL of Human TruStain FcX (Biolegend) was added to the flow tubes, followed by 100 μL of cynomolgus macaque whole blood containing an anticoagulant and incubated at room temperature for 10 min. 10 μL of FITC anti-human CD19 (Beckman, clone: ​​J3-119) was then added to each tube, gently flicked to mix, and incubated on ice for 60 min. 1× RBC buffer (Biolegend) was added to each tube, gently shaken, and incubated at room temperature in the dark for 10 min. Cells were collected by centrifugation and washed once with pre-cooled FACS buffer (PBS buffer containing 1% BSA). Cells in each tube were resuspended in FACS buffer and analyzed using a flow cytometer. The experimental data were expressed as CD19. + The percentage of B cells in PBMCs is presented.

[0318] The results are shown in Figure 11. Compared with 120h and 72h before administration, the CD19 in the peripheral blood of cynomolgus monkeys 24h after administration was significantly higher than that in the control group. + B cells were almost completely eliminated, and there was no sign of recovery within 168 hours after administration. + Myeloid cells express CD20 + During the experiment, two cynomolgus monkeys, one male and one female, showed no abnormal clinical manifestations within 168 hours of dosing, and no significant changes in body weight were observed (data not shown).

Claims

1. 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: 9; (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: 50), 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: 16; (4) a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 19; (5) a LCDR2 having an amino acid sequence of GASSLEX7 (X7 = G, A, S or T; as set forth in SEQ ID NO: 51), 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: 52), 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 the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

2. The anti-CD89 antibody or antigen-binding fragment thereof of claim 1, comprising: (1) a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 9, (2) a HCDR2 having an amino acid sequence as set forth in any one of SEQ ID NOs: 10-14, and (3) a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 16; and (4) a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 19, (5) a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 20 or 21, and (6) a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 22 or 23; Further, the anti-CD89 antibody or antigen-binding fragment thereof comprises: (1) a HCDR1 having an amino acid sequence of SEQ ID NO: 9, a HCDR2 having an amino acid sequence of SEQ ID NO: 14, a HCDR3 having an amino acid sequence of SEQ ID NO: 16; and a LCDR1 having an amino acid sequence of SEQ ID NO: 19, a LCDR2 having an amino acid sequence of SEQ ID NO: 21, a LCDR3 having an amino acid sequence of SEQ ID NO: 23, 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 (2) a HCDR1 having an amino acid sequence of SEQ ID NO: 9, a HCDR2 having an amino acid sequence of SEQ ID NO: 10, 11, 12, or 13, a HCDR3 having an amino acid sequence of SEQ ID NO: 16; and a LCDR1 having an amino acid sequence of SEQ ID NO: 19, a LCDR2 having an amino acid sequence of SEQ ID NO: 20, a LCDR3 having an amino acid sequence of SEQ ID NO: 22, 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 (3) a HCDR1 having an amino acid sequence of SEQ ID NO: 9, a HCDR2 having an amino acid sequence of SEQ ID NO: 10, a HCDR3 having an amino acid sequence of SEQ ID NO: 16; and a LCDR1 having an amino acid sequence of SEQ ID NO: 19, a LCDR2 having an amino acid sequence of SEQ ID NO: 21, a LCDR3 having an amino acid sequence of SEQ ID NO: 22, 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 (4) HCDR1 having an amino acid sequence of SEQ ID NO: 9, HCDR2 having an amino acid sequence of SEQ ID NO: 10, HCDR3 having an amino acid sequence of SEQ ID NO: 16; and LCDR1 having an amino acid sequence of SEQ ID NO: 19, LCDR2 having an amino acid sequence of SEQ ID NO: 20, LCDR3 having an amino acid sequence of SEQ ID NO: 23, 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.

3. The anti-CD89 antibody or antigen-binding fragment thereof of claim 1 or 2, comprising: HCDR1 having an amino acid sequence of SEQ ID NO: 9, HCDR2 having an amino acid sequence of SEQ ID NO: 14, HCDR3 having an amino acid sequence of SEQ ID NO: 16; and LCDR1 having an amino acid sequence of SEQ ID NO: 19, LCDR2 having an amino acid sequence of SEQ ID NO: 21, LCDR3 having an amino acid sequence of SEQ ID NO: 23, 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.

4. The anti-CD89 antibody or antigen-binding fragment thereof of any one of claims 1-3, comprising a VH having an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 26-33, and a VL having an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 40-43; Further, the anti-CD89 antibody or antigen-binding fragment thereof comprises: (1) a VH having an amino acid sequence of SEQ ID NO: 26 and a VL having an amino acid sequence of SEQ ID NO: 40, 41, or 42, 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 VH and VL, respectively; or (2) a VH having an amino acid sequence of SEQ ID NO: 27 and a VL having an amino acid sequence of SEQ ID NO: 40, 41, or 42, 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 VH and VL, respectively; or (3) a VH having an amino acid sequence of SEQ ID NO: 28 and a VL having an amino acid sequence of SEQ ID NO: 40, 41, or 42, 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 VH and VL, respectively; or (4) a VH having an amino acid sequence of SEQ ID NO: 29 and a VL having an amino acid sequence of SEQ ID NO: 40, 41, or 42, 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 VH and VL, respectively; or (5) a VH having an amino acid sequence of SEQ ID NO: 30 and a VL having an amino acid sequence of SEQ ID NO: 40, 41, or 42, 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 VH and VL, respectively; or (6) a VH having an amino acid sequence of SEQ ID NO: 31 and a VL having an amino acid sequence of SEQ ID NO: 40, 41, or 42, 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 VH and VL, respectively; or (7) a VH having an amino acid sequence of SEQ ID NO: 32 and a VL having an amino acid sequence of SEQ ID NO: 40, 41, or 42, 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 VH and VL, respectively; or (8) a VH having an amino acid sequence of SEQ ID NO: 33 and a VL having an amino acid sequence of SEQ ID NO: 40, 41, or 42, 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 VH and VL, respectively. (2) a VH comprising an amino acid sequence as set forth in SEQ ID NO: 27, 28, 29, 30, or 31 and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 40, 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, respectively, to the amino acid sequences of the VH and VL; (3) a VH comprising an amino acid sequence as set forth in SEQ ID NO: 32 or 33 and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 43, 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, respectively, to the amino acid sequences of the VH and VL; Preferably, the anti-CD89 antibody or antigen-binding fragment thereof comprises: a VH comprising an amino acid sequence as set forth in SEQ ID NO: 32 or 33 and a VL comprising an amino acid sequence as set forth in SEQ ID NO: 43, 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, respectively, to the amino acid sequences of the VH and VL.

5. The anti-CD89 antibody or antigen-binding fragment thereof of any one of claims 1-4, comprising a disulfide bond-containing anti-CD89 scFv molecule whose VH and VL comprise, respectively, one or more cysteine residues introduced, wherein, one or more amino acid mutations at positions 44, 45, 60, 100, 100a, 101, 103, 105, and 106 of the VH of the scFv molecule relative to the VH of the anti-CD89 antibody or antigen-binding fragment thereof of any one of claims 1-4 to introduce a first cysteine residue, and one or more amino acid mutations at positions 34, 43, 46, 91, 95, 96, 98, 100, and 101 of the VL of the scFv molecule relative to the VL of the anti-CD89 antibody or antigen-binding fragment thereof of any one of claims 1-4 to introduce a second cysteine residue, the pairing of the first and second cysteine residues being capable of forming one or more disulfide bonds at the interface of the VH and VL of the scFv molecule, the pairing of the first and second cysteine residues being selected from the following one or more paired amino acid mutations: (1) an amino acid mutation G44C at position 44 of the VH of the anti-CD89 antibody or antigen-binding fragment thereof to introduce a first cysteine residue, and an amino acid mutation P100C or G101C at position 100 or 101 of the VL of the anti-CD89 antibody or antigen-binding fragment thereof to introduce a second cysteine residue; (2) an amino acid mutation A12C at position 60 of the VH of the anti-CD89 antibody or antigen-binding fragment thereof to introduce a first cysteine residue, and an amino acid mutation P7C at position 95 of the VL of the anti-CD89 antibody or antigen-binding fragment thereof to introduce a second cysteine residue, wherein the VH and VL have the amino acid sequences set forth in SEQ ID NOs: 50 and 52, respectively; (3) an amino acid mutation S6C at position 100 of the VH of the anti-CD89 antibody or antigen-binding fragment thereof to introduce a first cysteine residue, and an amino acid mutation F3C at position 91 of the VL of the anti-CD89 antibody or antigen-binding fragment thereof to introduce a second cysteine residue, wherein the VH and VL have the amino acid sequences set forth in SEQ ID NOs: 16 and 52, respectively; (4) an amino acid mutation D9C at position 101 of the VH of the anti-CD89 antibody or antigen-binding fragment thereof to introduce a first cysteine residue, and an amino acid mutation K46C at position 46 of the VL of the anti-CD89 antibody or antigen-binding fragment thereof to introduce a second cysteine residue, wherein the VH and VL have the amino acid sequences set forth in SEQ ID NOs: 16 and 52, respectively; (5) an amino acid mutation W103C or Q105C at position 103 or 105 of the VH of the anti-CD89 antibody or antigen-binding fragment thereof to introduce a first cysteine residue, and an amino acid mutation A43C at position 43 of the VL of the anti-CD89 antibody or antigen-binding fragment thereof to introduce a second cysteine residue, wherein the VH and VL have the amino acid sequences set forth in SEQ ID NOs: 16 and 52, respectively.

6. The anti-CD89 antibody or antigen-binding fragment thereof of claim 5, wherein the anti-CD89 scFv molecule with a disulfide bond at the VH-VL interface comprises HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, LCDR3, each of said CDRs comprising: (1) HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 9; (2) 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: 53), preferably, X1= D, X2= H, X3= G, X4= Y, X5= D, X6= K; (3) 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: 54); (4) LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 19; (5) LCDR2 having an amino acid sequence of GASSLEX7 (X7= G, A, S or T; as set forth in SEQ ID NO: 51), preferably, X7= S; (6) 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; X 14 = P or C; as set forth in SEQ ID NO: 55), preferably, X8= Y, X9= S, 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, respectively, to the amino acid sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3.

7. The anti-CD89 antibody or antigen-binding fragment thereof of claim 5 or 6, wherein the anti-CD89 scFv molecule having a disulfide bond-containing VH-VL interface comprises: (1) a HCDR1 having an amino acid sequence set forth in SEQ ID NO: 9, (2) a HCDR2 having an amino acid sequence set forth in SEQ ID NO: 14 or 15, (3) a HCDR3 having an amino acid sequence set forth in SEQ ID NO: 16, 17, or 18, and (4) a LCDR1 having an amino acid sequence set forth in SEQ ID NO: 19, (5) a LCDR2 having an amino acid sequence set forth in SEQ ID NO: 21, and (6) a LCDR3 having an amino acid sequence set forth in SEQ ID NO: 23, 24, or 25; Further, the anti-CD89 antibody or antigen-binding fragment thereof having a disulfide bond-containing VH-VL interface comprises: (1) HCDR1 having an amino acid sequence of SEQ ID NO: 9, HCDR2 having an amino acid sequence of SEQ ID NO: 14, HCDR3 having an amino acid sequence of SEQ ID NO: 16 or 18, and LCDR1 having an amino acid sequence of SEQ ID NO: 19, LCDR2 having an amino acid sequence of SEQ ID NO: 21, LCDR3 having an amino acid sequence of SEQ ID NO: 23, 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 (2) HCDR1 having an amino acid sequence of SEQ ID NO: 9, HCDR2 having an amino acid sequence of SEQ ID NO: 15, HCDR3 having an amino acid sequence of SEQ ID NO: 16, and LCDR1 having an amino acid sequence of SEQ ID NO: 19, LCDR2 having an amino acid sequence of SEQ ID NO: 21, LCDR3 having an amino acid sequence of SEQ ID NO: 24, 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 (3) HCDR1 having an amino acid sequence of SEQ ID NO: 9, HCDR2 having an amino acid sequence of SEQ ID NO: 14, HCDR3 having an amino acid sequence of SEQ ID NO: 17, and LCDR1 having an amino acid sequence of SEQ ID NO: 19, LCDR2 having an amino acid sequence of SEQ ID NO: 21, LCDR3 having an amino acid sequence of 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 to the amino acid sequence of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively.

8. The anti-CD89 antibody or antigen-binding fragment thereof of any one of claims 5-7, wherein the anti-CD89 scFv molecule with a disulfide bond in the VH-VL interface comprises a VH comprising an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 34, 35, 36, 37, 38, or 39, and a VL comprising an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 44, 45, 46, 47, 48, or 49. Further, the anti-CD89 scFv molecules with disulfide bond at the VH-VL interface comprise: (1) a VH comprising an amino acid sequence of SEQ ID NO: 34 and a VL comprising an amino acid sequence of SEQ ID NO: 48 or 44, 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; or (2) a VH comprising an amino acid sequence of SEQ ID NO: 35 or 38 and a VL comprising an amino acid sequence of SEQ ID NO: 45, 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; or (3) a VH comprising an amino acid sequence of SEQ ID NO: 36 and a VL comprising an amino acid sequence of SEQ ID NO: 46, 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; or (4) a VH comprising an amino acid sequence of SEQ ID NO: 37 and a VL comprising an amino acid sequence of SEQ ID NO: 47, 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; or (5) a VH comprising an amino acid sequence of SEQ ID NO: 39 and a VL comprising an amino acid sequence of SEQ ID NO: 49, 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.

9. The anti-CD89 antibody or antigen-binding fragment thereof of any one of claims 5-8, wherein the anti-CD89 scFv molecule with a disulfide bond-containing VH-VL interface comprises a linker comprising (GGGGS) n , wherein n is an integer from 1 to 3, preferably n is 3.

10. A CD89 myeloid engager comprising a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain specifically binds CD20, the second antigen binding domain specifically binds CD89 extracellular Ig-like domain 2, and does not block IgA binding to CD89.

11. The CD89 myeloid cell engager of claim 10, wherein the CD89 myeloid cell engager is an anti-CD20 / CD89 bispecific antibody comprising at least one of the following properties: (1) the bispecific antibody is an asymmetric trivalent molecule capable of specifically binding CD20; the bispecific antibody or antigen binding fragment thereof is further capable of specifically binding CD89 with a binding affinity (KD D ) value < 5 x 10 -8 M, < 1 x 10 -9 M, or < 5 x 10 - 10 M; (2) the bispecific antibody is capable of cross-linking a CD89-expressing myeloid cell to a CD20-expressing target cell, thereby activating the myeloid cell and mediating its directed killing of the CD20-expressing target cell; (3) the bispecific antibody is capable of avoiding non-directed activation of the myeloid cell in the absence of a CD20-expressing target cell; and the myeloid cell, upon activation, does not elicit a cytokine storm; (4) the bispecific antibody has high stability, and is easy to obtain a high homogeneity and high purity of heterodimer; (5) the bispecific antibody has significantly reduced Fc receptor binding affinity.

12. The CD89 myeloid cell engager of claim 10 or 11, wherein the first antigen binding domain comprises (1) a HCDR1 having an amino acid sequence of SEQ ID NO: 1, (2) a HCDR2 having an amino acid sequence of SEQ ID NO: 2, (3) a HCDR3 having an amino acid sequence of SEQ ID NO: 3, and (4) a LCDR1 having an amino acid sequence of SEQ ID NO: 4, (5) a LCDR2 having an amino acid sequence of SEQ ID NO: 5, and (6) a LCDR3 having an amino acid sequence of SEQ ID NO: 6, 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.

13. The CD89 myeloid accretor of any one of claims 10-12, wherein the second antigen binding domain comprises: (1) a HCDR1 having an amino acid sequence of SEQ ID NO: 9, (2) a HCDR2 having an amino acid sequence of SEQ ID NO: 14, (3) a HCDR3 having an amino acid sequence of SEQ ID NO: 16, and (4) a LCDR1 having an amino acid sequence of SEQ ID NO: 19, (5) a LCDR2 having an amino acid sequence of SEQ ID NO: 21 and (6) a LCDR3 having an amino acid sequence of SEQ ID NO: 23, 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.

14. The CD89 myeloid accretor of any one of claims 10-13, wherein the first antigen binding domain comprises: a VH comprising an amino acid sequence of SEQ ID NO: 7, and a VL comprising an amino acid sequence of SEQ ID NO: 8, 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 VH and VL, respectively.

15. The CD89 myeloid accretor of any one of claims 10-14, wherein the second antigen binding domain comprises: a VH comprising an amino acid sequence of SEQ ID NO: 34, and a VL comprising an amino acid sequence of SEQ ID NO: 48, 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 VH and VL, respectively.

16. The CD89 myeloid linker of any one of claims 10-15, comprising a 2+1 scFv-IgG or scFv-Fab-Fc:Fab-Fc structure comprising subunits A and B, wherein, Subunit A is a bivalent scFv-Fab-Fc format, the Fab-Fc domain and the scFv domain are the first and second antigen binding domains, respectively, and subunit B is a monovalent Fab-Fc format, the Fab-Fc domain is the first antigen binding domain, and the scFv domain is fused to or operably linked to the Fab-Fc domain by a linker.

17. The CD89 myeloid cell engager of any one of claims 10-16, wherein, Each subunit comprises a 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 introduction of a "knob and hole mutation” and / or an "isoelectric point (pi) mutation” into a human IgG heavy chain constant region having an amino acid sequence as set forth in SEQ ID NO:

56.

18. The CD89 myeloid linker of claim 17, wherein, The "knob” mutation in the Fc region of the subunit A comprises amino acid mutation T366W, the "hole” mutation in the Fc region of the subunit B comprises amino acid mutations T366S, L368A and Y407V; the "pi mutation” comprises introduction of a pi-increasing mutation in one of the heavy chain constant regions of the myeloid engager comprising the "knob” mutation, which comprises amino acid mutations Q196K and / or N276K, and introduction of a pi-decreasing mutation in the other heavy chain constant region of the myeloid engager comprising the "hole” mutation, which comprises one or more of amino acid mutations G137E, G138S, N203D, K274Q and Q419E.

19. The CD89 myeloid engager of claim 17 or 18, wherein the two heavy chain constant regions of the CD89 myeloid engager can further comprise a mutation that reduces Fc effector function, further wherein the heavy chain constant region comprises a human IgGl Fc with L234F / L235E / P331S (EU numbering system) amino acid substitutions.

20. The CD89 myeloid engager of any one of claims 17-19, wherein the light chain constant region is a human kappa light chain constant region.

21. The CD89 myeloid cell accesor of any one of claims 10-20, comprising one first polypeptide chain, one second polypeptide chain, and two identical third polypeptide chains, wherein, The first polypeptide chain comprises from N- to C-terminus the anti-CD89 antibody or antigen binding fragment thereof of any one of claims 5-9, which is an anti-CD89 scFv molecule, the heavy chain variable region of an anti-CD20 antibody, and a first heavy chain constant region, the second polypeptide chain comprises from N- to C-terminus the heavy chain variable region of the anti-CD20 antibody, and a second heavy chain constant region, and the third polypeptide chain comprises from N- to C-terminus the light chain variable region of the anti-CD20 antibody, and a light chain constant region.

22. The CD89 myeloid cell engager of any one of claims 10-21, comprising: one first polypeptide chain, one second polypeptide chain, and two third polypeptide chains; wherein the first polypeptide chain and the third polypeptide chain comprise subunit A of the bispecific antibody; the second polypeptide chain and the third polypeptide chain comprise subunit B of the bispecific antibody; and, (1) the first polypeptide chain comprises VH1-L1-VL1-L2-VH2-CH a , wherein VH1-L1-VL1 specifically binds CD89 extracellular Ig-like domain 2, L1, L2 represent linkers, and VH2-CH a specifically binds CD20; (2) the second polypeptide chain comprises VH2-CH b , which VH2-CH b specifically binds CD20; (3) the third polypeptide chain comprises VL2-CL, which VL2-CL specifically binds CD20.

23. The CD89 myeloid engager of claim 22, wherein the VH1 comprises a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 9, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 14, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 16, 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 amino acid sequence as set forth in SEQ ID NO: 60; the VL1 comprises a LCDR1 having an amino acid sequence of SEQ ID NO: 19, a LCDR2 having an amino acid sequence of SEQ ID NO: 21, and a LCDR3 having an amino acid sequence of SEQ ID NO: 23, 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 LCDR1, LCDR2, and LCDR3, respectively; the L2 comprises a linker amino acid sequence of SEQ ID NO: 61; the VH2 comprises a HCDR1 having an amino acid sequence of SEQ ID NO: 1, a HCDR2 having an amino acid sequence of SEQ ID NO: 2, and a HCDR3 having an amino acid sequence of SEQ ID NO: 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 sequences of the HCDR1, HCDR2, and HCDR3, respectively; The CH a comprises a human IgGl heavy chain constant region having the amino acid sequence of SEQ ID NO:

58.

24. The CD89 myeloid engager of claim 22 or 23, wherein the VH1 comprises an amino acid sequence of SEQ ID NO: 34, 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 VL1 comprises an amino acid sequence of SEQ ID NO: 48, 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 VH2 comprises an amino acid sequence of SEQ ID NO: 7, 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.

25. The CD89 myeloid adaptor of any one of claims 22-24, wherein the CH b comprises a human IgGl heavy chain constant region of the amino acid sequence set forth in SEQ ID NO:

57.

26. The CD89 myeloid engager of any one of claims 22-24, wherein the VL2 comprises a LCDR1 having an amino acid sequence of SEQ ID NO: 4, a LCDR2 having an amino acid sequence of SEQ ID NO: 5, and a LCDR3 having an amino acid sequence of SEQ ID NO: 6, 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 LCDR1, LCDR2, and LCDR3, respectively; the CL is selected from a human kappa constant region or a human lambda constant region, preferably a human kappa constant region having an amino acid sequence of SEQ ID NO:

59.

27. The CD89 myeloid engager of any one of claims 22-26, wherein the VL2 comprises an amino acid sequence of SEQ ID NO: 8, 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.

28. The CD89 myeloid engager of any one of claims 22-27, comprising: (1) one first polypeptide chain comprising: a VH1 comprising an amino acid sequence as set forth in SEQ ID NO: 34, a LI comprising an amino acid sequence as set forth in SEQ ID NO: 60, a VL1 comprising an amino acid sequence as set forth in SEQ ID NO: 48, a L2 comprising an amino acid sequence as set forth in SEQ ID NO: 61, a VH2 comprising an amino acid sequence as set forth in SEQ ID NO: 7, a CH a ; (2) a second polypeptide chain, the second polypeptide chain comprising: a VH2 comprising an amino acid sequence as set forth in SEQ ID NO: 7, a CH b ; and (3) two third polypeptide chains comprising: a VL2 comprising an amino acid sequence of SEQ ID NO: 8, a CL comprising an amino acid sequence of SEQ ID NO:

59.

29. The CD89 myeloid engager of any one of claims 22-28, wherein the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 62, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 63, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO:

64.

30. A nucleic acid encoding the anti-CD89 antibody or antigen-binding fragment thereof of any one of claims 1-9, or the CD89 myeloid engager of any one of claims 10-29.

31. An expression vector capable of expressing the nucleic acid of claim 30.

32. A host cell comprising the nucleic acid of claim 30 or the expression vector of claim 31.

33. A method of using the host cell of claim 32 to make the anti-CD89 antibody or antigen-binding fragment thereof of any one of claims 1-9, or the CD89 myeloid syndecan of any one of claims 10-29, comprising: (1) expressing the anti-CD89 antibody or antigen-binding fragment thereof, or CD89 myeloid engager in the host cell, and (2) isolating the anti-CD89 antibody or antigen-binding fragment thereof, or CD89 myeloid engager from the host cell or cell culture.

34. A pharmaceutical composition comprising the CD89 myeloid engager of any one of claims 10-29, and a pharmaceutically acceptable carrier.

35. A kit comprising an effective amount of the CD89 myeloid engager of any one of claims 10-29, or the pharmaceutical composition of claim 34, and optionally at least one additional therapeutic agent, including a tumor therapeutic agent and an immunosuppressive agent for a CD20- associated disorder.

36. The kit of claim 35, wherein the tumor therapeutic agent and the immunosuppressive agent comprise: chemotherapeutic agents, hormone receptor modulators, kinase inhibitors, B-cell surface receptor antibodies, activators of tumor cell apoptosis, anti-angiogenic agents, aromatase inhibitors, cytokine inhibitors.

37. A method of treating a CD20-associated disorder, the method comprising administering to a subject in need thereof a therapeutically effective amount of the CD89 myeloid engager of any one of claims 10-29, the pharmaceutical composition of claim 34, or the kit of claim 35 or 36.

38. The method of claim 37, wherein the CD20-associated disorder comprises B-cell malignancies and autoimmune diseases resulting from B-cell auto-reactivity; the subject is a human, non-human primate, or other mammal.

39. The method of claim 37, wherein the B-cell malignancies comprise B-cell lymphomas and B-cell leukemias; the autoimmune diseases resulting from B-cell auto-reactivity comprise systemic lupus erythematosus (SLE), cutaneous lupus, discoid lupus, lupus nephritis, scleroderma, dermatomyositis (DM), polymyositis (PM), psoriasis, psoriatic arthritis, rheumatoid arthritis, multiple sclerosis, systemic sclerosis, vasculitis, thrombocytopenia, myasthenia gravis, type I diabetes.

40. The method of any one of claims 37-39, wherein administration of the CD89 myeloid engager, the pharmaceutical composition, or the kit is capable of reducing or depleting pathogenic B-cells in the subject.

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