Bifunctional antibodies and uses thereof

Abstract

The invention provides a bifunctional antibody or antigen binding fragment, the Fab end of the bifunctional antibody or antigen binding fragment can target B cells, and the Fc end of the bifunctional antibody or antigen binding fragment enhances the binding affinity to FcRn protein through amino acid mutation. The antibody can achieve the effect of treating autoimmune diseases through different action mechanisms, and the treatment effect of the antibody is superior to that of existing single-target-point targeting drugs.

Description

[0001] Reference to the Sequence Listing

[0002] This application contains a sequence listing in computer-readable form, which is incorporated herein by reference. Technical Field

[0003] The present disclosure relates to the field of antibodies, and more particularly to a bifunctional antibody that targets surface antigens of immune cells such as B cells (e.g., CD20) respectively and is capable of binding FcRn simultaneously. Background Art

[0004] Autoimmune diseases (sometimes abbreviated as autoimmune diseases hereinafter) refer to diseases in which the body's immune response to self-antigens leads to damage to its own tissues. A variety of autoimmune diseases often involve the production of pathogenic IgG antibodies that attack self-antigens. For example: myasthenia gravis, pemphigus, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, warm autoimmune hemolytic anemia, paroxysmal nocturnal hemoglobinuria, Graves' ophthalmopathy, autoimmune hemolytic anemia, thrombocytopenia, idiopathic thrombocytopenic purpura, chronic inflammatory demyelinating polyneuropathy, primary Sjögren's syndrome, hemolytic disease of the newborn, Guillain-Barré syndrome, membranous glomerulonephritis, lupus nephritis, postural tachycardia syndrome, demyelinating diseases, neuromyelitis optica, anti-myelin oligodendrocyte glycoprotein immunoglobulin G antibody-related diseases, etc.

[0005] For autoimmune diseases caused by endogenous pathogenic IgG antibodies, traditional treatment methods such as immunosuppressants, plasma exchange, intravenous immunoglobulin infusion, surgical intervention, etc. have disadvantages such as poor efficacy, strong side effects, complex processes, and high prices.

[0006] B cells play an important role in the occurrence and development of autoimmune diseases: First, B cells secrete a large amount of autoantibodies, which can directly bind to receptors to activate or inhibit receptor functions, or bind to self-antigens to form immune complexes and activate the complement system or effector cells to attack healthy tissues; Second, B cells can act as antigen-presenting cells to activate pathogenic T cells; Third, B cells also cause or exacerbate inflammatory responses by secreting various cytokines; Finally, B cells can promote and maintain the formation of ectopic germinal centers in target organs, continuously releasing high-affinity autoantibodies.

[0007] There are already several B cell-targeted drugs that have achieved certain effects in autoimmune diseases. For example, Rituximab (RTX) is an antibody targeting the B cell surface antigen CD20. It can specifically eliminate CD20-expressing B cells through pathways such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP). It has been approved by the US FDA for the treatment of rheumatoid arthritis that is unresponsive to one or more tumor necrosis factor (TNF) antagonists. In addition, there are many successful cases of RTX in the treatment of myasthenia gravis and systemic lupus erythematosus.

[0008] The neonatal fragment receptor (FcRn) is an MHC class I (major histocompatibility complex class I) molecule, a heterodimer formed by a non-covalent binding of an α chain and a β2-microglobulin. It can be detected in various tissue cells throughout the life cycle, such as endothelial cells, epithelial cells, stem cells, keratinocytes, and various blood cells. Its main function is to maintain the levels of IgG and serum albumin in the serum and regulate the distribution of IgG in tissues. FcRn mediates the recycling mechanism to achieve IgG transport in a pH-dependent manner, that is, under acidic conditions (pH 6.0 - 6.5), FcRn binds to the Fc fragment of IgG, and dissociates under neutral and weakly alkaline conditions (pH 7.0 - 7.5).

[0009] In recent years, studies have shown that by blocking FcRn, inhibiting the binding of endogenous IgG to FcRn under both physiological neutral and acidic pH conditions can accelerate the degradation of IgG and pathogenic autoantibodies, thereby achieving the effect of alleviating or treating autoimmune diseases. Currently, the antibody drug Efgartigimod has been approved for marketing. Its essence is the Fc fragment of human IgG1 to increase the affinity for FcRn, thereby inhibiting the binding of endogenous pathogenic IgG antibodies to FcRn. Rozanolixizumab is another marketed drug targeting FcRn and is a humanized monoclonal antibody. These two drugs have shown good therapeutic effects on autoimmune diseases such as myasthenia gravis in clinical trials.

[0010] However, the above-mentioned drugs still have the following deficiencies in clinical practice: First, they can only target the clearance of IgG subtype antibodies and cannot produce therapeutic effects on autoimmune diseases caused by autoantibodies of other subtypes; Second, in some cases, a "rebound effect" will occur, that is, in order to make up for the sudden drop of IgG antibodies in the body, the body produces more autoantibodies instead.

[0011] In addition, there is no reported drug that can both target the surface antigen of B cells and specifically bind to FcRn to block the function of FcRn at the same time. Currently, drugs for treating autoimmune diseases all target a single target or a single mechanism of action, so there is still great room for improvement in the treatment effect. For example, Rituximab (RTX) is only an antibody that can target the B cell surface antigen CD20, but cannot bind to FcRn at the same time. Efgartigimod and Rozanolixizumab can only bind to FcRn, but cannot target B cells. Summary of the Invention

[0012] In view of the above problems, the present disclosure provides antibodies, their preparation methods, compositions, etc. The benefits provided by the present disclosure are widely applicable to the fields of antibody therapy and diagnosis, and can be used in combination with antibodies that can react with various targets. The present invention provides an antibody that can specifically bind to the surface antigen of B cells (for example, specifically bind to CD19, CD20), preferably a chimeric monoclonal antibody or a humanized monoclonal antibody, and preferably can also bind to FcRn at the same time, thereby inhibiting / blocking the binding of pathogenic antibodies to FcRn.

[0013] On the one hand, the present disclosure provides an isolated antibody or its antigen-binding fragment, which is a bifunctional antibody. Among them, the Fab end of the antibody or its antigen-binding fragment specifically binds to the surface antigen of B cells (for example, specifically binds to CD19, CD20), and the Fc end of the antibody or its antigen-binding fragment can specifically bind to the FcRn protein.

[0014] In the above antibody, the Fab end contains a heavy chain variable region (HCDR) and a light chain variable region (LCDR).

[0015] The heavy chain variable region contains:

[0016] (i) HCDR1, which contains a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with SEQ ID NO: 39 or is composed of SEQ ID NO: 39;

[0017] (ii) HCDR2, which comprises a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with SEQ ID NO: 40 or SEQ ID NO: 42 or consists of SEQ ID NO: 40 or SEQ ID NO: 42; and

[0018] (iii) HCDR3, which comprises a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with SEQ ID NO: 41 or consists of SEQ ID NO: 41,

[0019] The light chain variable region comprises:

[0020] (i) LCDR1, which comprises a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with SEQ ID NO: 36 or consists of SEQ ID NO: 36;

[0021] (ii) LCDR2, which comprises a sequence having at least 60%, at least 65%, at least 75%, at least 95%, or 100% sequence identity with SEQ ID NO: 37 (ATS) or consists of SEQ ID NO: 37 (ATS); and

[0022] (iii) LCDR3, which comprises a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with SEQ ID NO: 38 or consists of SEQ ID NO: 40,

[0023] Wherein the Fc terminus comprises a heavy chain constant region, and the heavy chain constant region comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 27, and has one, two, three, four or five substitutions selected from the group consisting of: M252Y, W, F, P; S254T, C, A, L; T256E, Q, N; H433K, R; N434F, Y, W, P, L (EU numbering).

[0024] In the above antibody, preferably the substitutions are selected from the group consisting of: M252Y, W; S254T; T256E; H433K; N434F, Y, W (EU numbering).

[0025] In some embodiments, the antibody or fragment has one or more of the following properties:

[0026] (a) Bind to CD20 expressed on human or monkey B cells with an EC50 of 10.77 nM or less;

[0027] (b) Bind to human FcRn with a KD value not higher than 1.71E-08 under mildly acidic conditions (e.g., pH 5.5 - 6.5, such as pH 6);

[0028] (c) Bind to human FcRn with a KD value not higher than 2.20E-06 under neutral conditions (e.g., pH 6.5 - 7.5, such as pH 7, pH 7.4);

[0029] (d) Specifically bind to FcγR receptors, such as FcγRIIIA (CD16aA), FcγRIIA (CD32A), and FcγRI (CD64).

[0030] In some embodiments of the present invention, the antibody or fragment can induce killing and / or inhibit cells expressing human CD20 (e.g., tumor cells, such as tumor cells expressing CD20) through antibody-dependent cell-mediated cytotoxicity (ADCC); and / or, induce killing of cells expressing human CD20 (e.g., tumor cells, such as tumor cells expressing CD20) through complement-dependent cytotoxicity (CDC).

[0031] In some embodiments, the isolated antibody or its antigen-binding fragment comprises a κ-type or λ-type light chain constant region.

[0032] On the one hand, the present disclosure provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the antibody or fragment as described above.

[0033] On the one hand, the present disclosure provides a vector comprising the nucleic acid molecule as described above.

[0034] On the one hand, the present disclosure provides a host cell comprising the nucleic acid molecule as described above or the vector as described above.

[0035] On the one hand, the present disclosure provides a multispecific molecule comprising the antibody or antigen-binding fragment as described above.

[0036] In some embodiments, the multispecific molecule specifically binds to CD20 and additionally specifically binds to one or more other targets, such as FcRn.

[0037] In some embodiments, the multispecific molecule further comprises at least one molecule (e.g., a second antibody) having a second binding specificity for a second target.

[0038] In some embodiments, the multispecific molecule is a bispecific antibody.

[0039] On the other hand, the present disclosure provides a pharmaceutical composition or a kit, which comprises the antibody or fragment as described above, the nucleic acid molecule as described above, the vector as described above, the host cell as described above, the conjugate as described above, the antibody-drug conjugate as described above, the multispecific molecule as described above; and a pharmaceutically acceptable carrier.

[0040] On the other hand, the present disclosure provides a method for preparing the antibody or fragment as described above, which comprises the following steps:

[0041] (i) expressing the antibody or fragment as described above in the host cell as described above; and optionally (ii) isolating the antibody or its antigen-binding fragment from the host cell.

[0042] In yet another aspect, the present disclosure provides a method for diagnosing, detecting or monitoring a B cell-related disease, comprising administering an effective dose of the antibody or its antigen-binding fragment, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit to a subject in need thereof.

[0043] In yet another aspect, the present disclosure provides the antibody or its antigen-binding fragment, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit for use in a method for diagnosing, detecting or monitoring a B cell-related disease in a subject.

[0044] In yet another aspect, the present disclosure provides the use of the antibody or its antigen-binding fragment, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit in the preparation of a reagent for diagnosing, detecting or monitoring a B cell-related disease.

[0045] In yet another aspect, the present disclosure provides a method for treating a B cell-related disease or determining its prognosis in a subject, comprising administering an effective dose of the antibody or its antigen-binding fragment, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit to a subject in need thereof.

[0046] In some embodiments, the B cell-related disease is an autoimmune disease selected from myasthenia gravis, pemphigus, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, warm autoimmune hemolytic anemia, paroxysmal nocturnal hemoglobinuria, Graves' ophthalmopathy, autoimmune hemolytic anemia, thrombocytopenia, idiopathic thrombocytopenic purpura, chronic inflammatory demyelinating polyneuropathy, primary Sjögren's syndrome, hemolytic disease of the newborn, Guillain-Barré syndrome, membranous glomerulonephritis, lupus nephritis, postural orthostatic tachycardia syndrome, demyelinating diseases, neuromyelitis optica, and myelin oligodendrocyte glycoprotein immunoglobulin G antibody-related diseases.

[0047] In some embodiments, the B cell-related disease is cancer selected from B cell lymphomas, including NHL, pre-B cell lymphoblastic leukemia / lymphoma, and mature B cell tumors, such as B cell chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), including low-grade, intermediate-grade, and high-grade FL, cutaneous follicular center lymphoma, marginal zone B cell lymphoma (MALT type, nodal, and splenic), hairy cell leukemia, diffuse large B cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenström macroglobulinemia, and anaplastic large cell lymphoma (ALCL).

[0048] In some embodiments, the subject is a mammal, preferably a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The drawings are provided to further understand the present disclosure and form a part of the specification. Together with the embodiments of the present disclosure, they are used to explain the present disclosure and do not constitute a limitation to the present disclosure.

[0050] Figures 1A - 1D Shows the binding activity of the antibody of the present invention to the CD20 protein level.

[0051] Figures 2A - 2B Shows the binding curve of the bifunctional antibody targeting CD20 and FcRn to CD20 on the surface of Raji cells.

[0052] Figures 3A - 3B Shows the binding curve of the bifunctional antibody targeting CD20 and FcRn to CD20 on the surface of Raji cells.

[0053] Figures 4A - 4C Shows the binding curve of the bifunctional antibody targeting CD20 and FcRn to MDCK-hFcRn under the condition of pH 6.0.

[0054] Figures 5A - 5B Shows the binding curve of a bifunctional antibody targeting CD20 and FcRn to MDCK-hFcRn under the condition of pH 6.0.

[0055] Figures 6A - 6C Shows the binding curve of the bifunctional antibody to MDCK-hFcRn under the condition of pH 7.4.

[0056] Figure 7A Shows the binding curve of the bifunctional antibody to MDCK-hFcRn under the condition of pH 7.4.

[0057] Figure 7B Shows the binding curve of the bifunctional antibody to MDCK-hFcRn under the condition of pH 7.4.

[0058] Figure 7C Shows the binding curve of the bifunctional antibody to MDCK-hFcRn under the condition of pH 7.4.

[0059] Figure 8A Shows the ADCC activity of the bifunctional antibody.

[0060] Figure 8B Shows the ADCC activity of the bifunctional antibody.

[0061] Figure 8C Shows the ADCC activity of the bifunctional antibody.

[0062] Figure 8D Shows the ADCC activity of the bifunctional antibody.

[0063] Figure 8E Shows the ADCC activity of the bifunctional antibody.

[0064] Figure 8F Shows the ADCC activity of the bifunctional antibody.

[0065] Figure 9 Shows the CDC activity of the bifunctional antibody.

[0066] Figure 10 Shows the ADCP activity of the bifunctional antibody.

[0067] Figure 11 Shows the average plasma concentration-time curve.

[0068] Figure 12 Shows the average plasma concentration-time curve.

[0069] Figure 13 Shows the average plasma concentration-time curve. Detailed implementation manners

[0070] To enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are merely illustrative embodiments of a part of the present invention, rather than all embodiments. Therefore, the present invention is not limited to the specific illustrative embodiments. In addition, any chapter headings used herein are not to be construed as limiting the subject matter described.

[0071] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention will have the meanings commonly understood by one of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms shall include plural forms and plural terms shall include singular forms. More specifically, as used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" include plural referents. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "comprising" and other forms (such as "including" and "containing") is not restrictive. In addition, the ranges provided in the specification and the appended claims include the endpoints and all values between the endpoints.

[0072] Definitions

[0073] To better understand the present invention, the definitions and explanations of relevant terms are provided as follows.

[0074] The term "antibody" or "Ab" generally refers to a Y-shaped tetrameric protein comprising two heavy chains (H) and two light chains (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. The light chains of an antibody can be divided into κ or λ light chains. The heavy chains can be divided into μ, δ, γ, α, or ε, which define the isotype of the antibody as IgM, IgD, IgG, IgA, or IgE, respectively. In both the light and heavy chains, the variable region is linked to the constant region by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further divided into hypervariable regions (termed complementarity-determining regions, abbreviated as CDRs) separated by relatively conserved regions (termed framework regions, abbreviated as FRs). Each VH and VL consists of three CDRs and four FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the N-terminus to the C-terminus. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen-binding site / portion, respectively. The distribution of amino acids in the various regions or domains follows the numbering definitions in common systems such as Kabat, IMGT, or Chothia. In the specific embodiments of the present disclosure, the numbering definition in the IMGT system is used for the determination of CDR sequences.

[0075] Antibodies in the present disclosure also include antigen-binding portions (used interchangeably with the term "antigen-binding fragment"). An antigen-binding portion refers to a polypeptide containing a fragment of a complete antibody that retains the ability to specifically bind an antigen with the same antigen specificity as the full-length or complete antibody, and / or competes with the full-length antibody for binding to the same antigen. Under some conditions, antigen-binding portions include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, bispecific antibodies, and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide. The antigen-binding portion of an antibody can be obtained from a given antibody by conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods) and can be screened for specificity in the same manner as the complete antibody.

[0076] The term "bifunctional antibody" in the present disclosure refers to an antibody (preferably a monoclonal antibody) in which the Fab segment specifically binds to one target and the Fc segment specifically binds to FcRn. For example, an example of a preferred antibody in the present disclosure is a bifunctional antibody capable of specifically binding CD20 and FcRn.

[0077] The term "isotype" refers to the class of antibodies encoded by the constant region genes of the heavy chain (e.g., IgM or IgG1).

[0078] The term "monoclonal antibody" or "mAb" refers to an antibody molecule / preparation composed of a single molecule. Monoclonal antibodies exhibit a single binding specificity and affinity for a specific epitope. The antibodies of the present invention can be derived from different species, including but not limited to mice, rats, rabbits, guinea pigs, and humans.

[0079] The term "epitope" refers to the antigenic determinant in a molecule, which is the part of the molecule recognized by the immune system (e.g., recognized by an antibody), such as a discontinuous three-dimensional site on an antigen recognized by the immune system. In the present invention, the indicated epitope is, for example, the CD20 protein.

[0080] As used herein, the term "chimeric antibody" refers to an antibody in which the variable region sequence is from one species and the constant region sequence is from another species, such as an antibody in which the variable region sequence is derived from a murine antibody and the constant region sequence is derived from a human antibody.

[0081] The term "humanized antibody" is intended to refer to an antibody in which the CDR sequences / antigen-binding portions or sites derived from the germline of another mammalian species, such as a mouse, have been grafted onto a human framework sequence. In addition, additional framework region modifications can be made within the human framework sequence.

[0082] The term "CDR grafting" refers to the transplantation of the CDR regions of a non-human antibody onto a human antibody framework region, which is a commonly used method for humanizing non-human antibodies. Generally, the human antibody framework region with the highest homology to the non-human antibody framework region is selected as the recipient for CDR grafting. This operation can be carried out using CDR grafting methods well known in the art.

[0083] The term "KD value" is the equilibrium dissociation constant between an antibody and its antigen, i.e., the ratio of koff / kon or kd / ka (measured by SPR / BLI technology). Therefore, the lower the KD value, the higher the affinity of the antibody. Thus, the "KD value" can be used to measure the binding affinity between an antibody and its antigen.

[0084] The terms "CD20" and "CD20 antigen" are used interchangeably herein and include any variant, isotype, and species homolog of human CD20 that is naturally expressed by cells or expressed on cells transfected with the CD20 gene. In some embodiments, the binding of the antibodies of the present disclosure to the CD20 antigen mediates the killing of cells expressing CD20 (e.g., tumor cells) by inactivating CD20. The killing of cells expressing CD20 can occur through one or more of the following mechanisms: induction of cell death / apoptosis, ADCC, and CDC.

[0085] The term "anti-CD20 antibody" or "CD20 antibody" refers to an antibody that can bind to the CD20 antigen or to cells expressing CD20 as defined herein. According to the binding characteristics and biological activities of anti-CD20 antibodies to the CD20 antigen, two types of anti-CD20 antibodies (type I and type II anti-CD20 antibodies) can be distinguished in accordance with Cragg, M.S. et al., Blood 103 (2004) 2738-2743; and Cragg, M.S. et al., Blood 101 (2003) 1045-1052.

[0086] The term "FcRn protein" refers to a receptor molecule located on the cell membrane surface, and its protein structure is similar to that of MHC-I molecules. FcRn is widely expressed in epithelial cells, endothelial cells, and various immune cells (such as monocytes and macrophages) in many tissues in the body. FcRn can bind to the Fc portion of IgG, prevent the IgG molecule from being lysed by lysosomes, and can play a role in increasing the in vivo half-life of IgG. Vascular endothelial cells are the main sites where FcRn protects IgG from being catabolized. When vascular endothelial cells endocytose a large amount of proteins and other substances in the blood, FcRn can effectively intercept IgG by binding to the Fc portion of IgG and return it to the blood circulation, thereby prolonging the in vivo half-life of IgG. The binding of FcRn to IgG is pH-dependent. For example, they only bind under slightly acidic conditions (such as pH 6.0-6.5) and dissociate under neutral conditions (such as pH 7.0-7.5). Therefore, in the acidified lysosome, IgG can effectively bind to FcRn and be transported to the cell membrane surface following FcRn. Since the pH value of extracellular blood is slightly alkaline (pH 7.3-7.5), IgG dissociates from FcRn and is "recycled" into the blood circulation.

[0087] The term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets. In certain embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific for an antigen) means that the antibody binds to the antigen with a KD value of less than about 10 -5 M, for example less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less.

[0088] The term "concentration-time curve graph" is sometimes abbreviated as C-T graph or concentration-time graph in this disclosure, and refers to a blood drug concentration-time curve graph, which is a curve showing the change of blood drug concentration over time with time as the abscissa and blood drug concentration as the ordinate, and can reflect the dynamic change law of drugs in the body to a certain extent. Usually, a concentration-time curve graph needs to be drawn in a bioequivalency (BE) test or a pharmacokinetic test.

[0089] The term "isolated" refers to a state obtained by artificial means from its natural state. If a certain "isolated" substance or component exists in nature, it may be due to a change in its natural environment, or the separation of the substance from its natural environment, or both. For example, an unisolated polynucleotide or polypeptide naturally exists in a living organism, and the highly purified same polynucleotide or polypeptide separated from this natural state is called an isolated polynucleotide or polypeptide. The term "isolated" does not exclude a mixture of artificial or synthetic substances, nor other impure substances that do not affect the activity of the separated substance. For example, an isolated antibody can be substantially free of other cellular materials and / or chemical substances.

[0090] The term "vector" refers to a nucleic acid mediator into which a polynucleotide can be inserted. When a vector allows the expression of a protein encoded by the polynucleotide inserted therein, the vector is called an expression vector. The vector can cause the genetic material elements carried therein to be expressed in a host cell by transformation, transduction or transfection. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phages, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); phages such as λ phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, polyomaviruses (such as SV40). A vector can contain multiple elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, a vector can contain an origin of replication. For a vector expressing an antibody, a vector type in which the heavy and light chains of the antibody are present in different vectors or a vector type in which the heavy and light chains are present in the same vector can be used.

[0091] The term "host cell" refers to a cell system that can be engineered to produce a target protein, protein fragment, or peptide. Host cells include, but are not limited to, cultured cells such as mammalian cultured cells derived from rodents (rat, mouse, guinea pig, or hamster), such as CHO, BHK, NSO, SP2 / 0, YB2 / 0; or human tissue or hybridoma cells, yeast cells, and insect cells, as well as cells contained within transgenic animals or cultured tissues. The term encompasses not only the particular cells being tested, but also the progeny of such cells. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may differ from the parental cells, but are still included within the scope of the term "host cell".

[0092] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules (or protein molecules) or two or more nucleic acid molecules determined by alignment and comparison of the sequences. "Percent identity" refers to the percentage of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest molecule being compared. For these calculations, gaps in the alignment (if any) are preferably addressed by specific mathematical models or computer programs (i.e., "algorithms"). Methods that can be used to calculate the identity of nucleic acids or polypeptides for alignment include those described in Computational Molecular Biology, (Lesk, A.M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D.W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A.M., and Griffin, H.G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al, 1988, SIAM J. Applied Math. 48:1073.

[0093] The term "immunogenicity" refers to the ability to stimulate the formation of specific antibodies or sensitized lymphocytes in an organism. It not only refers to the property of an antigen to stimulate the activation, proliferation, and differentiation of specific immune cells to ultimately produce immune effector substances such as antibodies and sensitized lymphocytes, but also refers to the formation of a specific immune response of antibodies or sensitized T lymphocytes in the immune system of an organism after antigen stimulation. Immunogenicity is the most important property of an antigen. Whether an antigen can successfully induce the generation of an immune response in a host depends on three factors: the nature of the antigen, the reactivity of the host, and the means of immunization.

[0094] The term "transfection" refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include, but are not limited to, lipid transfection and chemical and physical methods such as electroporation. Many transfection techniques are known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al, 1981, Gene 13:197.

[0095] The terms "hybridoma" and "hybridoma cell line" can be used interchangeably. When referring to the terms "hybridoma" and "hybridoma cell line", they also include subclones and progeny cells of the hybridoma.

[0096] The term "immune effector function" includes any function mediated by components of the immune system that results in inhibition of tumor growth and / or inhibition of tumorigenesis, and also includes inhibition of tumor dissemination and metastasis. Preferably, the immune effector function results in killing of tumor cells. Preferably, the immune effector function in the present invention is an antibody-mediated effector function. Such functions include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), induction of apoptosis in cells bearing tumor-associated antigens (e.g., by binding of an antibody to a surface antigen) and / or inhibition of proliferation of cells bearing tumor-associated antigens, preferably ADCC and / or CDC. Antibodies can also act simply by binding to tumor-associated antigens on the surface of tumor cells. For example, an antibody can block the function of a tumor-associated antigen or induce apoptosis simply by binding to the tumor-associated antigen on the surface of a tumor cell.

[0097] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to the killing of target cells by NK cells, macrophages, neutrophils, etc. that express IgG Fc receptors, through binding to the Fc segment of IgG antibodies that have bound to the surface of the target cells. The antibody "arms" the cytotoxic cells and is absolutely required for this killing. The main cell mediating ADCC, the NK cell, only expresses FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. To evaluate the ADCC activity of a molecule of interest, an in vitro ADCC assay can be performed, such as those described in U.S. Patent Nos. US5,500,362 or US5,821,337. Effector cells that can be used in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Optionally or additionally, the ADCC activity of a molecule of interest can be evaluated in vivo, such as in an animal model disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998).

[0098] The term "complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (appropriate subclass) that has bound its cognate antigen. To evaluate complement activation, a CDC assay can be performed, such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996).

[0099] The term "subject" includes any mammal, such as a human or non-human animal, preferably a human.

[0100] The term "cancer" refers to any tumor or malignant cell growth, proliferation, or metastasis-mediated solid and non-solid tumors such as leukemia that cause a medical condition. For example, cancers associated with B cells (e.g., related to CD20 expression) include, but are not limited to: B cell lymphomas, including NHL, pre-B cell lymphoblastic leukemia / lymphoma, and mature B cell tumors, such as B cell chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), including low-grade, intermediate-grade, and high-grade FL, cutaneous follicular center lymphoma, marginal zone B cell lymphoma (MALT type, nodal and splenic types), hairy cell leukemia, diffuse large B cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenström's macroglobulinemia, and anaplastic large cell lymphoma (ALCL).

[0101] As used herein, the terms "treatment" and "therapy" in the context of treating a medical condition generally relate to the treatment and therapy of a human or animal, in which some desired therapeutic effect is achieved, e.g., inhibiting the progression of the medical condition, including a decrease in the rate of progression, a halt in the rate of progression, regression of the medical condition, improvement of the medical condition, and cure of the medical condition. Treatment also includes treatment as a prophylactic measure (i.e., prophylaxis). For cancer, "treatment" may refer to inhibiting or slowing the growth, proliferation, or metastasis of a tumor or malignant cells or some combination thereof. For a tumor, "treatment" includes removing all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of the tumor, or some combination thereof.

[0102] The term "therapeutically effective amount" relates to the amount of an active compound or a material, composition, or dosage form comprising the active compound that is effective, when administered according to a desired treatment regimen, to produce certain desired therapeutic effects commensurate with a reasonable benefit / risk ratio. For example, when used in conjunction with treating or detecting a related disease or disorder, an "effective amount" or "effective dose" refers to the amount or concentration of an antibody or an antigen-binding fragment thereof that is effective to treat or detect the disease or disorder.

[0103] The term "pharmaceutically acceptable" means that a carrier, diluent, excipient, and / or its salt is chemically and / or physically compatible with the other ingredients in the formulation and is physiologically compatible with the recipient.

[0104] The term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active agent and is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19 th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH regulators, surfactants, adjuvants, and ionic strength enhancers. For example, pH regulators include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0105] The term "adjuvant" refers to a non-specific immune enhancer that can enhance the immune response against an antigen in an organism or alter the type of immune response when delivered to the organism together with or pre-delivered to the organism with an antigen. There are various adjuvants, including, but not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., Freund's complete adjuvant and Freund's incomplete adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, etc. Freund's adjuvant is the most commonly used adjuvant in current animal experiments. Aluminum hydroxide adjuvant is more commonly used in clinical trials.

[0106] The term "concentration-time curve graph" is sometimes abbreviated as the C-T graph in this disclosure, which refers to the blood drug concentration-time curve graph. It is a curve showing the change of blood drug concentration over time with time as the abscissa and blood drug concentration as the ordinate, and can reflect the dynamic change law of drugs in the body to a certain extent. Usually, a concentration-time curve graph needs to be drawn in bioequivalency (BE) tests or early pharmacokinetic tests.

[0107] Anti - CD20 antibody

[0108] In some aspects, the invention includes isolated antibodies or antigen-binding fragments thereof.

[0109] In the context of this application, "antibody" can include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized and primatized antibodies, CDR-grafted antibodies, human antibodies, recombinantly produced antibodies, intracellular antibodies, bifunctional antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies, including their mutant proteins and variants, improved antibodies; and their derivatives (including Fc fusion proteins and other modifications), and any other immunoreactive molecules, as long as they exhibit preferential association or binding with the CD20 protein. In addition, unless the context otherwise provides, the term also includes all classes of antibodies (i.e., IgA, IgD, IgE, IgG, and IgM) and all subclasses (i.e., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). In a preferred embodiment, the antibody is a monoclonal antibody. In a more preferred embodiment, the antibody is a chimeric monoclonal antibody or a humanized monoclonal antibody or an improved chimeric monoclonal antibody.

[0110] In some embodiments, the antibody comprises a heavy chain variable region (HCDR) and a light chain variable region (LCDR), wherein

[0111] The heavy chain variable region comprises:

[0112] (i) HCDR1, which comprises a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with SEQ ID NO: 39 or consisting of SEQ ID NO: 39;

[0113] (ii) HCDR2, which comprises a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with SEQ ID NO: 40 or SEQ ID NO: 42 or consisting of SEQ ID NO: 40 or SEQ ID NO: 42; and

[0114] (iii) An HCDR3, which comprises a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with SEQ ID NO: 41 or consists of SEQ ID NO: 41,

[0115] The light chain variable region comprises:

[0116] (i) An LCDR1, which comprises a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with SEQ ID NO: 36 or consists of SEQ ID NO: 36;

[0117] (ii) An LCDR2, which comprises a sequence having at least 60%, at least 65%, at least 75%, at least 95%, or 100% sequence identity with SEQ ID NO: 37 (ATS) or consists of SEQ ID NO: 37 (ATS); and

[0118] (iii) An LCDR3, which comprises a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with SEQ ID NO: 38 or consists of SEQ ID NO: 38,

[0119] wherein the antibody or its antigen-binding fragment further comprises a heavy chain constant region, which comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 27, and has one, two, three, four or five substitutions selected from the group consisting of: M252Y, W, F, P; S254T, C, A, L; T256E, Q, N; H433K, R; N434F, Y, W, P, L (where the above amino acid numbering is according to EU numbering).

[0120] The EU numbering described in the present disclosure refers to the numbering described in Edelman, G.M. et al. Proc Natl Acad Sci USA. 1969; 63(1): 78-85.

[0121] The variable regions and CDRs in the antibody sequence can be identified according to the general rules (as described above) developed in the art, such as the IMGT numbering system or by aligning the sequence with a database of known variable regions.

[0122] In some embodiments, the substitutions in the heavy chain constant region are selected from the group consisting of: M252Y, W; S254T; T256E; H433K; N434F, Y, W (EU numbering).

[0123] In some embodiments, the heavy chain constant region of the antibody comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 27, has a substitution of H433K (EU numbering) relative to SEQ ID NO: 27, and further has at least one, two, three, four substitutions selected from the group consisting of: M252Y,W; S254T; T256E; N434F,Y,W (EU numbering).

[0124] In some embodiments, the heavy chain constant region of the antibody comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 27, has a substitution of S254T (EU numbering) relative to SEQ ID NO: 27, and further has one, two, three, four substitutions selected from the group consisting of: M252Y,W; T256E; H433K; N434F,Y,W (EU numbering).

[0125] In some embodiments, the heavy chain constant region of the antibody comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 27, has a substitution of N434F,Y,W (EU numbering) relative to SEQ ID NO: 27, and further has one, two, three, four substitutions selected from the group consisting of: M252Y,W; S254T; T256E; H433K (EU numbering).

[0126] In some embodiments, the heavy chain constant region of the antibody comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 27, has a substitution of T256E (EU numbering) relative to SEQ ID NO: 27, and further has one, two, three, four substitutions selected from the group consisting of: M252Y,W; S254T; H433K; N434F,Y,W (EU numbering).

[0127] In some embodiments, the heavy chain constant region of the antibody comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 27, and having the substitution M252Y, W (EU numbering) relative to SEQ ID NO: 27, and further having one, two, three, or four substitutions selected from the group consisting of: S254T; T256E; H433K; N434F, Y, W (EU numbering).

[0128] In some embodiments, the heavy chain constant region of the antibody comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 27, and having the substitutions:

[0129] (a) M252Y + H433K + N434F;

[0130] (b) M252W + H433K + N434F;

[0131] (c) M252Y + H433K + N434Y;

[0132] (d) M252W + H433K + N434Y;

[0133] (e) M252Y + S254T + T256E + H433K + N434Y;

[0134] (f) M252Y + S254T + T256E + H433K + N434W;

[0135] All using EU numbering.

[0136] In some embodiments, the heavy chain constant region of the antibody comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NOs: 29 - 35 or consists of one of SEQ ID NOs: 29 - 35.

[0137] In some embodiments, the heavy chain variable region of the antibody comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NOs: 19, 22, 23, 24, 25 or consists of one of SEQ ID NOs: 19, 22, 23, 24, 25.

[0138] In some embodiments, the light chain variable region of the antibody comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NOs: 18, 20, 21 or consists of one of SEQ ID NOs: 18, 20, 21.

[0139] In some embodiments, the light chain constant region of the antibody comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 26 or consists of SEQ ID NO: 26.

[0140] In some embodiments, the antibody comprises:

[0141] (a) a light chain that comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NOs: 1, 4, 5 or consists of one of SEQ ID NOs: 1, 4, 5;

[0142] (b) a heavy chain that comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NOs: 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or consists of one of SEQ ID NOs: 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17.

[0143] An antibody or fragment that comprises:

[0144] A light chain that comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NO: 1, 4, 5 or consists of one of SEQ ID NO: 1, 4, 5;

[0145] A heavy chain that comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NO: 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or consists of one of SEQ ID NO: 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17.

[0146] In some embodiments, the antibody or fragment comprises:

[0147] (a) A light chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1 or consisting of SEQ ID NO: 1; and a heavy chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NO: 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or consisting of one of SEQ ID NO: 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17;

[0148] (b) A light chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 4 or consisting of SEQ ID NO: 4; and a heavy chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to one of SEQ ID NO: 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or consisting of one of SEQ ID NO: 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17;

[0149] (c) a light chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 5 or a light chain consisting of SEQ ID NO: 4; and a heavy chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with one of SEQ ID NOs: 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or a heavy chain consisting of one of SEQ ID NOs: 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17.

[0150] In some embodiments, the isolated antibody or antigen-binding fragment thereof may comprise conservative substitutions, modifications or replacements (e.g., conservative replacements), deletions or additions of amino acids in the variable regions of the heavy and / or light chains (e.g., replacement, deletion or addition of 1, 2, 3, 4 or 5 amino acids; or conservative replacement of up to 20, up to 15, up to 10, or up to 5 amino acids), or have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% sequence identity with the antibody or antigen fragment from which it is derived. Amino acid deletion variants that contain deletions at the N-terminus and / or C-terminus of the protein are also referred to as N-terminal and / or C-terminal truncation variants. It is understood in the art that certain modifications of conservative sequences that do not eliminate antigen binding can be made. See, for example, Brummell et al. (1993) Biochem 32:1180-8; de Wildt et al. (1997) Prot. Eng. 10:835-41; Komissarov et al. (1997) J. Biol. Chem. 272:26864-26870; Hall et al. (1992) J. Immunol. 149:1605-12; Kelley and O’Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al. (1998) Int. Immunol. 10:341-6 and Beers et al. (2000) Clin. Can. Res. 6:2835-43.

[0151] As described above, the term "conservative substitution" as used herein refers to an amino acid substitution that does not adversely affect or alter the basic properties of a protein / polypeptide containing the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced with another amino acid residue having a similar side chain, such as a residue that is physically or functionally similar (e.g., having a similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, and histidine), amino acids having acidic side chains (e.g., aspartic acid and glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids having β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, the corresponding amino acid residue is preferably replaced with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997), which are incorporated herein by reference).

[0152] According to the present invention, if an antibody is mentioned as comprising a specific antibody heavy chain and / or a specific antibody light chain (e.g., a chain comprising a specific CDR sequence), the two heavy chains and / or the two light chains of the antibody are preferably each constituted by the specific antibody heavy chain and / or the specific antibody light chain, respectively.

[0153] In some embodiments, the antibody or fragment has one or more of the following properties:

[0154] (a) binds to human or monkey CD20 with an EC50 of 10.77 nM or less; preferably lower than the EC50 of Rituximab;

[0155] (b) binds to human FcRn with a KD value of no higher than 1.71E-08 under mildly acidic conditions (e.g., pH 5.5-6.5, such as pH 6);

[0156] (c) binds to human FcRn with a KD value of no higher than 2.20E-06 under neutral conditions (such as pH 6.5 - 7.5, such as pH 7, pH 7.4);;

[0157] (d) specifically binds to FcγR receptors, such as FcγRIIIA (CD16aA), FcγRIIA (CD32A), and FcγRI (CD64).

[0158] Regardless of how the antibody is produced, methods for testing the ability of an antibody to bind to an antigen (such as CD20, FcRn) are known in the art and include any antibody - antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, SPR, and competitive inhibition assays (see, for example, Janeway et al., below and U.S. Patent Application Publication No. 2002 / 0197266 and the above section on competitive assays).

[0159] According to the present invention, in a standard assay (such as the assays described in the present invention), if an antibody has a significant affinity for a predetermined target (such as CD20 protein or cells expressing CD20), the antibody is capable of binding to the predetermined target. To test the binding of a monoclonal antibody to live cells expressing CD20, flow cytometry can be used. Preferably, in a fluorescence - activated cell sorting (FACS) analysis, the binding of the antibody to the target expressed on the cell surface is assayed. If the antibody can detectably bind to the target (CD20 protein or cells expressing CD20), the antibody is capable of binding to the target and has an "affinity". Preferably, if the antibody of the present invention is present at a concentration of 10 μg / mL or lower, 5 μg / mL or lower, 3 μg / mL or lower, 2 μg / mL or lower, 1 μg / mL or lower, 0.5 μg / mL or lower, the antibody can detectably bind to the target.

[0160] In some embodiments, the binding properties of the antibody or fragment can be: binding to human or monkey CD20 or CD19 with a KD of 1×10 -7 M or lower; binding to human or monkey CD20 or CD19 with a KD of 5×10 -8 M or lower; binding to human or monkey CD20 or CD19 with a KD of 1×10 -8 M or lower; binding to human or monkey CD20 or CD19 with a KD of 5×10 -9 M or lower; binding to human or monkey CD20 or CD19 with a KD of 1×10 -9 M or lower; or binding to human or monkey CD20 or CD19 with a KD of 1×10 -9 M or lower.

[0161] The CD20 specificity described in the present invention refers to the ability to bind to one or more CD20 epitopes, especially CD20 epitopes in their native conformation, particularly human CD20 specificity.

[0162] The CD19 specificity described in the present invention refers to the ability to bind to one or more CD19 epitopes, especially CD19 epitopes in their native conformation, particularly human CD19 specificity.

[0163] In some embodiments of the present invention, the antibody or fragment can induce killing and / or inhibit cells expressing human CD20 (such as tumor cells, such as tumor cells expressing CD20) through antibody-dependent cell-mediated cytotoxicity (ADCC); and / or induce killing of cells expressing human CD20 (such as tumor cells, such as tumor cells expressing CD20) through complement-dependent cytotoxicity (CDC).

[0164] In some embodiments of the present invention, the antibody or fragment can induce killing and / or inhibit cells expressing human CD19 (such as tumor cells, such as tumor cells expressing CD19) through antibody-dependent cell-mediated cytotoxicity (ADCC); and / or induce killing of cells expressing human CD19 (such as tumor cells, such as tumor cells expressing CD19) through complement-dependent cytotoxicity (CDC).

[0165] In some embodiments, the antibody is a monoclonal antibody.

[0166] In some embodiments, the antibody is a bispecific antibody.

[0167] In some embodiments, the antibody is a chimeric antibody or a humanized antibody or a modified antibody such as a modified chimeric antibody.

[0168] In some embodiments, the isolated antibody or its antigen-binding fragment comprises a constant region of IgG. The constant region of IgG is preferably selected from the IgG1, IgG2, IgG3, or IgG4 constant regions. The constant region of IgG is more preferably selected from the constant region of IgG1.

[0169] In the art, various means for modifying without changing the desired properties of the antibody are diverse, such as the method of recombining the light and heavy chains of the antibody and performing amino acid substitutions as used in the present disclosure. For example, the sequences in the present invention, including chimeric antibody sequences or humanized antibody sequences, can be subjected to conservative amino acid substitutions.

[0170] Antibodies interact with target antigens mainly through amino acid residues located in six heavy-chain and light-chain complementarity-determining regions (CDRs). For this reason, the amino acid sequences of CDRs are more diverse among antibodies than other sequences. Since CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular naturally occurring antibody by constructing an expression vector that contains the CDR sequences from the particular naturally occurring antibody, which are transplanted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321:522-525; and Queen, C. et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:10029-10033). Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences. These germlines are sequences that differ from mature antibody gene sequences in that they do not contain fully assembled variable genes, which are formed by V(D)J joining during B-cell maturation. Germline gene sequences will also have sequences at individual places that are different from those of a high-affinity secondary repertoire antibody throughout the variable region.

[0171] Mouse antibodies are highly immunogenic in humans, leading to reduced therapeutic efficacy when repeatedly administered, with the major immunogenicity mediated by the heavy-chain constant region. If individual antibodies are chimerized or humanized, the immunogenicity of mouse antibodies in humans can be reduced or completely avoided.

[0172] A chimeric antibody refers to an antibody in which different parts are from different animal species, e.g., an antibody having the variable regions from a mouse antibody and the constant regions of human immunoglobulins. The variable regions of the mouse heavy and light chains are joined to the constant regions of the human heavy and light chains to obtain a chimeric antibody (e.g., as described by Kraus et al., in Methods in Molecular Biology series, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8). In a preferred embodiment, a chimeric antibody is produced by joining the human κ light-chain constant region to the mouse light-chain variable region. In another preferred embodiment, a chimeric antibody can be produced by joining the human λ light-chain constant region to the mouse light-chain variable region.

[0173] A humanized antibody refers to an antibody in which the CDR sequences / antigen-binding portions or sites derived from the germline of another mammalian species such as a mouse are transplanted onto a human framework sequence.

[0174] To reduce the immunogenicity of the antibody in humans, a humanized anti-CD20 antibody is produced using the sequence of the CD20 antibody of the present disclosure. The CDR regions of the murine anti-CD20 antibody are combined with the human framework regions (e.g., human immunoglobulins) to form the humanized anti-CD20 antibody of the present disclosure. The humanized antibody is expected to retain the function of binding to human CD20 and the function of binding to cynomolgus CD20.

[0175] Preparation or production of antibodies

[0176] The antibodies of the present invention can be produced by a variety of techniques, including conventional monoclonal antibody methods, such as the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256:495 (1975). Although hybridoma technology is preferred, other techniques for producing monoclonal antibodies can in principle be employed, such as viral or oncogene transformation of B lymphocytes or phage display techniques using antibody gene libraries, somatic cell hybridization methods, and also, for example, by genetic engineering recombinant techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibody of the present invention are obtained by chemical synthesis or PCR amplification, the resulting DNA molecules are inserted into an expression vector, then transfected into host cells, and then the transfected host cells are cultured under specific conditions to express the antibody of the present invention.

[0177] Other preferred animal systems for the preparation of hybridomas secreting monoclonal antibodies are the rat and rabbit systems (e.g., described in Spieker-Polett et al., Proc. Natl. Acad. Sci. U.S.A. 92:9348 (1995), see also Rossi et al., Am. J. Clin. Pathol. 124:295 (2005)). Hybridoma production in mice is a very well-established method. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the prior art. Fusion partners (e.g., murine myeloma cells) and fusion methods are also known.

[0178] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals, or some combination thereof. For example, monoclonal antibodies can be produced using hybridoma and well-recognized biochemical and genetic engineering techniques in the art, as described in detail in An, Zhigiang (ed.) Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley and Sons, 1st ed. 2009; Shire et al. (eds.) Current Trends in Monoclonal Antibody Development and Manufacturing, Springer Science+Business Media LLC, 1st ed. 2010; Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. 1988; Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981), each of which is hereby incorporated by reference in its entirety.

[0179] It should be understood that the selected binding sequences can be further modified, for example, to increase the affinity for the target, humanize the target binding sequences, improve their production in cell culture, reduce their immunogenicity in vivo, generate multispecific antibodies, etc., and antibodies containing the modified target binding sequences are also antibodies of the present invention.

[0180] In some embodiments, methods for producing the antibodies or fragments described in the present disclosure include the following steps:

[0181] (i) expressing the antibody or fragment in a host cell; and optionally

[0182] (ii) isolating the antibody or its antigen-binding fragment from the host cell.

[0183] In a preferred embodiment, anti-CD20 monoclonal antibodies are prepared by using hybridomas.

[0184] To obtain hybridomas that produce the antibodies of the present invention, such as the human monoclonal antibodies of the present invention, splenocytes and / or lymph node cells from immunized mice can be isolated and fused to a suitable immortalized cell line, such as a murine myeloma cell line. The resulting hybridomas are screened for the production of antigen-specific antibodies. The production of hybridomas is well known in the art. See, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

[0185] The antibodies of the present invention can also be produced in host cell transfectomas using, for example, a combination of recombinant DNA techniques and gene transfection methods well known in the art (e.g., Morrison, S. (1985) Science 229:1202). In some embodiments, DNA encoding partial or full-length light and heavy chains obtained by standard molecular biology techniques is inserted into one or more expression vectors such that the genes are operably linked to transcriptional and translational regulatory sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is linked into the vector such that the transcriptional and translational control sequences within the vector perform their intended function of regulating transcription and translation of the antibody gene.

[0186] The antibody light chain gene and the antibody heavy chain gene can be inserted into the same or different expression vectors. In some embodiments, the variable regions are used to generate full-length antibody genes of any antibody isotype by inserting them into expression vectors that already encode the heavy chain constant region and the light chain constant region of the desired isotype such that the VH segment is operably linked to the CH segment within the vector and the VL segment is operably linked to the CL segment within the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0187] To express the light and heavy chains, the expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass the various techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. The antibodies of the present invention can be expressed in prokaryotic or eukaryotic host cells, such as mammalian host cells, which can assemble and secrete properly folded and immunologically active antibodies.

[0188] Mammalian host cells for expressing the recombinant antibodies of the present invention include Chinese hamster ovary cells (CHO cells) (including dhfr CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220) used in conjunction with a DHFR selection marker (e.g., as described in R.J. Kaufman and P.A. Sharp (1982) J. Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, and SP2 cells. In particular, for use with NSO myeloma, another expression system is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338,841. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell or by secreting the antibody into the culture medium in which the host cell is growing. The antibody can be recovered from the culture medium using standard protein purification methods.

[0189] In another preferred embodiment, transgenic or transchromosomal mice with a partial human immune system (as opposed to a murine system) can be used to generate human monoclonal antibodies against CD20.

[0190] Another strategy for generating monoclonal antibodies is to directly isolate the gene encoding the antibody from antibody-producing lymphocytes of a defined strategy, see, for example, Babcock et al., 1996; A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined strategy. For details of recombinant antibody engineering also see Welschof and Krau, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8 and Benny K.C. Lo Antibody Engineering ISBN 1-58829-092-1.

[0191] To prepare a chimeric antibody, the murine immunoglobulin variable region can be linked to the human immunoglobulin constant region using methods known in the art (see, for example, U.S. Patent US4,816,567 to Cabilly et al.). By operably linking the nucleic acid encoding VH to another DNA molecule encoding the heavy chain constant regions (CH1, CH2, and CH3), the isolated nucleic acid encoding the VH region can be converted into a full-length heavy chain gene. The sequences of the human heavy chain constant region genes are known in the art (see, for example, Kabat et al. (1991), Sequences Of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is more preferably an IgG1 or IgG4 constant region. By operably linking the DNA encoding VL to another DNA molecule encoding the light chain constant region CL, the isolated nucleic acid encoding the VL region can be converted into a full-length light chain gene (and Fab light chain gene). The sequences of the human light chain constant region genes are known in the art (see, for example, Kabat et al., supra), and DNA fragments containing these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region can be a κ or λ constant region, but is generally preferably a κ constant region. Once the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. As used herein, the term "operably linked" is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0192] For the preparation of humanized antibodies, methods known in the art can be used to insert murine CDR regions into human framework sequences (see U.S. Patent No. 5,225,539 to Winter; U.S. Patent Nos. 5,530,101; 5,585,089; 5,593,762 to Queen et al.; and Lo, Benny, K.C., editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals can be utilized that are capable of not producing endogenous immunoglobulins upon immunization and are capable of generating a complete human antibody repertoire. For example, it has been reported that homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice completely suppresses endogenous antibody production, and then transfer of the human germline immunoglobulin gene array into the germline mutant mice will result in the production of human antibodies in the mice upon antigen stimulation (see, for example, Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90:2551; Jakobovits et al., 1993, Nature 362:255-258; Bruggermann et al., 1993, Year in Immunology 7:33; and Duchosal et al., 1992, Nature 355:258). Non-limiting examples of such transgenic animals include HuMAb mice (Medarex, Inc.), which contain human immunoglobulin gene miniloci encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, plus targeted mutations that inactivate the endogenous μ and κ locus genes (see, for example, Lonberg et al. (1994) Nature 368(6474):856-859); or "KM miceTM" carrying a human heavy chain transgene and a human light chain transchromosome (see patent application WO02 / 43478). Other methods for humanizing antibodies also include phage display techniques (Hoogenboom et al., 1991, J. Mol. Biol. 227:381; Marks et al., J. Mol. Biol. 1991, 222:581-597; Vaughan et al., 1996, Nature Biotech 14:309).

[0193] Nucleic acid molecule encoding the antibody of the present invention

[0194] In some aspects, the invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding an isolated antibody or a fragment thereof as disclosed herein.

[0195] The nucleic acids of the present invention can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as further described below), cDNAs encoding the light and heavy chains of the antibodies prepared by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display techniques), the nucleic acids encoding such antibodies can be recovered from the gene library.

[0196] To prepare chimeric antibodies, methods known in the art can be used to link murine immunoglobulin variable regions to human immunoglobulin constant regions (see, for example, U.S. Patent No. 4,816,567 to Cabilly et al.). By operably linking the nucleic acid encoding VH to another DNA molecule encoding the heavy chain constant regions (CH1, CH2, and CH3), the isolated nucleic acid encoding the VH region can be converted into a full-length heavy chain gene, and a DNA fragment containing these regions can be obtained by standard PCR amplification. By operably linking the DNA encoding VL to another DNA molecule encoding the light chain constant region CL, the isolated nucleic acid encoding the VL region can be converted into a full-length light chain gene (and Fab light chain gene). Once the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker.

[0197] In some embodiments, the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the heavy chain variable region of an isolated antibody as disclosed herein.

[0198] In some embodiments, the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the light chain variable region of an isolated antibody as disclosed herein.

[0199] In one aspect, the present disclosure provides a vector comprising a nucleic acid molecule as described above.

[0200] In one aspect, the present disclosure provides a host cell comprising a nucleic acid molecule as described above or a vector as described above.

[0201] Conjugate

[0202] On the one hand, the present disclosure provides a conjugate comprising the antibody or fragment thereof as described above conjugated to at least one detectable label. Detectable labels include, but are not limited to: (i) providing a detectable signal; (ii) interacting with a second label to modify the detectable signal provided by the first or second label, such as FRET (Fluorescence Resonance Energy Transfer); (iii) affecting mobility (e.g., electrophoretic mobility) by charge, hydrophobicity, shape, or other physical parameters, or (iv) providing a capture moiety, such as affinity, antibody / antigen, or ion complexation.

[0203] Structures suitable as labels include, for example, fluorescent labels, luminescent labels, chromophore labels, radioisotope labels, isotope labels, preferably stable isotope labels, isobaric labels, enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), particulate labels (especially metal particulate labels, magnetic particulate labels, polymeric particulate labels), organic small molecules (e.g., biotin, ligands of receptors or binding molecules (e.g., cell adhesion proteins or lecithin), labels that can detect sequences containing nucleic acids and / or amino acid residues by using binding agents, etc.). Labels include, without limitation, barium sulfate, ioxetamic acid, iopanoic acid, calcium iopodate, sodium diatrizoate, meglumine diatrizoate, metrizamide, sodium ioglycamate, and radiodiagnostic agents (including positron emitters (e.g., fluorine-18 and carbon-11), γ emitters (e.g., iodine-123, iodine-125, technetium-99m, iodine-131, and indium-111), nuclear magnetic resonance nuclides (e.g., fluorine and gadolinium)), luminescent substances (e.g., isoluminol and acridinium esters), fluorescent substances (e.g., fluorescein and rhodamine), colored substances (e.g., latex particles and colloidal gold).

[0204] The detectable labels as described above can be detected by methods known in the art. For example, fluorescent labels can be detected using a light detector to detect the emitted light. Enzyme labels are generally detected by providing a substrate to the enzyme and detecting the reaction product generated by the action of the enzyme on the substrate. In certain embodiments, such labels are suitable for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescent immunoassay, etc.). In certain embodiments, the detectable labels as described above can be linked to the antibody or antigen-binding fragment of the present invention through linkers of different lengths to reduce potential steric hindrance.

[0205] Multispecific molecule

[0206] The antibodies or antigen-binding fragments thereof of the present invention can be used to form multispecific molecules (e.g., bispecific molecules). The antibodies or antigen-binding fragments thereof of the present invention can be part of a multispecific molecule (e.g., bispecific molecule), and the bispecific or multispecific molecule comprises a second functional module (e.g., a second antibody) or a third functional module (e.g., a third antibody) having a binding specificity different from that of the antibodies or antigen-binding fragments thereof of the present invention, so as to be capable of binding at least two different binding sites and / or target molecules. For example, the antibodies or antigen-binding fragments thereof of the present invention can be linked to a second antibody or antigen-binding fragment thereof capable of specifically binding any protein that can be used as a potential target for combination therapy. To produce the bispecific or multispecific molecule, the antibodies or antigen-binding fragments thereof of the present invention can be linked (e.g., by chemical conjugation, gene fusion, non-covalent association or other means) to one or more other binding molecules (e.g., additional antibodies, antibody fragments, peptides or binding mimetics).

[0207] Accordingly, in some aspects, the present invention provides a multispecific molecule comprising the antibodies or antigen-binding fragments thereof of the present invention.

[0208] In certain preferred embodiments, the multispecific molecule specifically binds CD20 (e.g., human CD20 or monkey CD20), and specifically binds one or more other targets.

[0209] In certain preferred embodiments, the multispecific molecule further comprises at least one molecule (e.g., a second antibody) having a second binding specificity for a second target.

[0210] In certain preferred embodiments, the multispecific molecule is a bispecific antibody.

[0211] Pharmaceutical composition

[0212] In some aspects, the present invention relates to pharmaceutical compositions, and the present disclosure provides a pharmaceutical composition or kit comprising the antibodies or fragments as described above, the nucleic acid molecules as described above, the vectors as described above, the host cells as described above, the conjugates as described above, the antibody-drug conjugates as described above, the multispecific molecules as described above; and a pharmaceutically acceptable carrier.

[0213] The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical composition of the present invention may also be administered in combination with, for example, another immunostimulant, anticancer agent, antiviral agent, or vaccine, such that the anti-CD20 antibody enhances the immune response to the vaccine. Pharmaceutically acceptable carriers may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, combinations of various components known in the art, or more.

[0214] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickening agents, coloring agents, emulsifying agents, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoethanol, mercaptoacetic acid, mercaptosorbitol, butyl methyl anisole, butylated hydroxytoluene, and / or propyl gallate. As disclosed in the present invention, in a solvent of an antibody or its antigen-binding fragment in a composition containing one or more antioxidants such as methionine that discloses the composition of the present invention, the antibody or its antigen-binding fragment containing a reducing antibody or its antigen-binding fragment can be oxidized. Oxidation-reduction can prevent or reduce the decrease in binding affinity, thereby enhancing antibody stability and extending the shelf life. Therefore, in some embodiments, the present invention provides a composition comprising one or more antibodies or their antigen-binding fragments and one or more antioxidants such as methionine. The present invention further provides various methods in which an antibody or its antigen-binding fragment is mixed with one or more antioxidants such as methionine, so that the antibody or its antigen-binding fragment can be prevented from being oxidized to extend its shelf life and / or increase its activity.

[0215] For further illustration, pharmaceutically acceptable carriers can include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isosmotic dextrose injection, sterile water injection, or dextrose and lactated Ringer injection, non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isosmotic agents such as sodium chloride or dextrose, buffering agents such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose or polyvinylpyrrolidone, emulsifying agents such as polysorbate 80 (TWEEN-80), sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. The antimicrobial agents used as carriers can be added to pharmaceutical compositions in multi-dose containers containing phenols or cresols, mercury preparations, benzyl alcohol, chlorobutanol, methylparaben and propylparaben, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients can include, for example, water, saline, dextrose, glycerol or ethanol. Suitable non-toxic auxiliary substances can include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrin.

[0216] Administration, formulations and dosages

[0217] The pharmaceutical compositions of the present invention can be administered in vivo to a subject in need thereof by various routes, including but not limited to oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal and intrathecal, or by implantation or inhalation. The compositions of the present invention can be formulated into preparations in solid, semi-solid, liquid or gaseous forms; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants and aerosols. Suitable formulations and administration routes can be selected according to the intended application and treatment regimen.

[0218] Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets (including coated tablets), elixirs, suspensions, syrups or inhalants and their controlled release forms.

[0219] Formulations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). These liquids may additionally contain other pharmaceutically acceptable ingredients, such as antioxidants, buffers, preservatives, stabilizers, bacteriostatic agents, suspending agents, thickening agents, and solutes that render the formulation isotonic with the blood (or other relevant body fluid) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, etc. Examples of isotonic carriers suitable for such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Similarly, the specific dosing regimen (including dose, timing, and repetition) will depend on the particular individual and the individual's medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance rate, etc.).

[0220] The requirements for an effective pharmaceutical carrier for injectable formulations / compositions are well known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, edited by Banker and Chalmers, pages 238 - 250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th edition, pages 622 - 630 (1986)).

[0221] The frequency of administration can be determined and adjusted during the course of treatment and is based on reducing the number of proliferating or tumorigenic cells, maintaining such reduction of tumor cells, reducing the proliferation of tumor cells, or delaying the development of metastasis. In some embodiments, the dose administered can be adjusted or reduced to control potential side effects and / or toxicity. Alternatively, sustained continuous release formulations of the therapeutic compositions of the invention may be suitable.

[0222] Those skilled in the art will understand that the appropriate dose may vary from patient to patient. Determining the optimal dose generally involves balancing the level of therapeutic benefit against any risks or harmful side effects. The dose level selected will depend on a variety of factors, including but not limited to the activity of the particular compound, the route of administration, the time of administration, the rate of clearance of the compound, the duration of treatment, other drugs, compounds, and / or materials used in combination, the severity of the condition, and the species, sex, age, weight, medical condition, general health, and previous medical history of the patient. However, the dose is generally selected to achieve a local concentration at the site of action that achieves the desired effect without causing substantial harmful or adverse side effects.

[0223] Generally, the antibodies or antigen-binding fragments thereof of the present invention can be administered in a variety of ranges. These include from about 4 μg / kg body weight to about 100 mg / kg body weight per dose; from about 20 μg / kg body weight to about 50 mg / kg body weight per dose; from about 50 μg / kg body weight to about 10 mg / kg body weight per dose. Other ranges include from about 100 μg / kg body weight to about 20 mg / kg body weight and from about 0.5 mg / kg body weight to about 15 mg / kg body weight. In certain embodiments, the dose is at least about 100 μg / kg body weight, at least about 250 μg / kg body weight, at least about 750 μg / kg body weight, at least about 3 mg / kg body weight, at least about 5 mg / kg body weight, at least about 10 mg / kg body weight.

[0224] In certain preferred embodiments, the treatment process involving the antibodies or antigen-binding fragments thereof of the present invention will comprise multiple doses of the selected pharmaceutical product administered over a period of weeks or months. More specifically, the antibodies or antigen-binding fragments thereof of the present invention can be administered daily, every two days, every four days, weekly, every ten days, every two weeks, every three weeks, monthly, every six weeks, every two months, every ten weeks or every three months. In this regard, it is understood that the dose or the interval can be altered based on patient response and clinical practice.

[0225] A compatible formulation for parenteral administration (e.g., intravenous injection) will comprise an antibody or antigen-binding fragment thereof as disclosed herein at a concentration of about 5 μg / mL to about 100 mg / mL. In certain selected embodiments, the concentration of the antibody or antigen-binding fragment thereof will include 10 μg / mL, 20 μg / mL, 50 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, 200 μg / μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL, 800 μg / mL, 900 μg / mL or 1 mg / mL. In other preferred embodiments, the antibody-drug conjugate will include 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL, 12 mg / mL, 14 mg ml, 16 mg / mL, 18 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL or 100 mg / mL.

[0226] The antibodies of the invention can be co-administered with one or more other therapeutic agents (e.g., cytotoxic agents, radiotoxic agents, anti-tumor agents, anti-angiogenic agents, or immunosuppressive agents). The anti-CD20 antibodies of the invention are administered to reduce the induction of an immune response against the antibodies of the invention. The antibody can be linked to the therapeutic agent (as an immune complex) or can be administered separately from the therapeutic agent.

[0227] In the context of administering therapy, as used herein, the terms "combination" or "co-administer" refer to the use of more than one therapy or therapeutic agent. The use of the term "combination" does not limit the order in which the therapies or therapeutic agents are administered to a subject. A therapy or therapeutic agent can be administered before, at the same time as, or after a second therapy or therapeutic agent is administered to a patient. Preferably, the therapies or therapeutic agents are administered to a subject in a particular order, amount, and / or at a particular time interval such that the therapies or therapeutic agents can act together. In a specific embodiment, the therapies or therapeutic agents are administered to a subject in a particular order, amount, and / or at a particular time interval such that they provide an increased benefit compared to if they were administered in some other manner (notably, independently of each other). Preferably, the increased benefit is a synergistic effect.

[0228] Medical use

[0229] The antibodies, antibody compositions, and methods of the invention have numerous in vitro and in vivo uses, including, for example, the detection of CD20 or the enhancement of an immune response. For example, these molecules can be administered in vitro or ex vivo to cultured cells, or, for example, in vivo to a human subject.

[0230] Preferred subjects include mammals, such as humans / patients. Mammals in the context of the invention are humans, non-human primates, domesticated animals such as dogs, cats, sheep, cows, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., and captive animals, such as zoo animals.

[0231] Treatment of B - cell related diseases

[0232] In some aspects, the invention provides methods of treating a disorder in a mammal, which comprise administering to a subject in need thereof (e.g., a human) a therapeutically effective amount of an antibody or an antigen-binding fragment thereof disclosed herein.

[0233] In some aspects, the antibodies of the present disclosure have one or more of the following activities: (i) killing cells expressing CD20 and / or CD19; (ii) inhibiting the proliferation of cells expressing CD20 and / or CD19; (iii) inhibiting the colony formation of cells expressing CD20 and / or CD19; (iv) mediating tumor remission (e.g., volume reduction), preferably complete remission (e.g., complete disappearance); (v) preventing the formation or re-formation of tumors, and (vi) inhibiting the metastasis of cells expressing CD20 and / or CD19. Accordingly, the antibodies can be used for one or more of the foregoing (especially when administered to a patient).

[0234] As described herein, the antibodies of the present disclosure have one or more activities of therapeutically applying killing cells and / or inhibiting cells. In particular, killing cells, inhibiting cell proliferation, and / or inhibiting cell colony formation can be used for treating or preventing cancer (including cancer metastasis). Inhibiting cell proliferation, colony formation, and / or metastasis can be applied, especially for treating or preventing cancer metastasis and metastatic spread of cancer cells.

[0235] In some aspects, the antibodies of the present disclosure mediate the killing of cells (e.g., cells expressing human CD20, such as tumor cells, or tumor cells expressing CD20) by inducing lysis, apoptosis, homotypic adhesion, and / or phagocytosis mediated by antibody-dependent cell-mediated cytotoxicity (ADCC).

[0236] In some aspects, the antibodies of the present disclosure mediate the killing of cells (e.g., cells expressing human CD20 and / or CD19, such as tumor cells, or tumor cells expressing CD20 and / or CD19) by inducing lysis, apoptosis, homotypic adhesion, and / or phagocytosis mediated by complement-dependent cytotoxicity (CDC).

[0237] In some embodiments, the antibodies exert the activities described herein (e.g., killing cells and / or inhibiting one or more cell activities (e.g., cell proliferation and / or colony formation) without inducing lysis mediated by complement-dependent cytotoxicity (CDC), lysis mediated by antibody-dependent cell cytotoxicity (ADCC), apoptosis, homotypic adhesion, and / or phagocytosis.

[0238] The antibodies of the present invention can interact with components of the immune system, preferably through ADCC or CDC interactions. Moreover, the antibodies of the present invention can also act by simply binding to CD20 and / or CD19 on the cell surface (thus, for example, blocking cell proliferation).

[0239] In some aspects, in the presence of effector cells, ADCC-mediated cell lysis occurs. In some specific embodiments, the effector cells are selected from monocytes, mononuclear cells, NK cells, and neutrophils (PMNs).

[0240] In some specific embodiments, phagocytosis is carried out by macrophages.

[0241] In some aspects, the present disclosure provides a method for treating a B cell-related disease or determining its prognosis in a subject, comprising administering to the subject in need an effective dose of the antibody or its antigen-binding fragment, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit.

[0242] In some aspects, the present disclosure provides the antibody or its antigen-binding fragment, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit for use in a method for treating a B cell-related disease or determining its prognosis in a subject.

[0243] In some aspects, the present disclosure provides the use of the antibody or its antigen-binding fragment, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit in the preparation of a reagent (or drug) for treating a B cell-related (e.g., CD20 expression-related) disease or determining its prognosis. In one embodiment, the B cell-related (e.g., CD20 expression-related) diseases include tumor diseases, such as cancer.

[0244] In some embodiments, the B cell-related (e.g., CD20 expression-related) disease is cancer, which is preferably selected from the group consisting of: B cell lymphoma, including NHL, pre-B cell lymphoblastic leukemia / lymphoma, and mature B cell tumors, such as B cell chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), including low-grade, intermediate-grade, and high-grade FL, cutaneous follicular center lymphoma, marginal zone B cell lymphoma (MALT type, nodal, and splenic), hairy cell leukemia, diffuse large B cell lymphoma, Burkitt lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenström macroglobulinemia, and anaplastic large cell lymphoma (ALCL).

[0245] The antibody or its antigen-binding fragment can be used alone as a single therapy or can be used in combination with chemotherapy or radiotherapy.

[0246] An antibody or antigen-binding fragment thereof can be used in combination with an anti-cancer agent, cytotoxic agent, or chemotherapeutic agent.

[0247] The term "anti-cancer agent" or "anti-proliferative agent" means any agent that can be used to treat a cell proliferative disorder such as cancer and includes, but is not limited to, cytotoxic agents, cytostatic agents, anti-angiogenic agents, radiotherapy and radiotherapeutic agents, targeted anti-cancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy, anti-metastatic agents, and immunotherapeutic agents. It should be understood that in the selected embodiments as described above, such anti-cancer agents can comprise conjugates and can be bound to the disclosed site-specific antibodies prior to administration. More specifically, in certain embodiments, a selected anti-cancer agent is linked to an unpaired cysteine of an engineered antibody to provide an engineered conjugate as described herein. Thus, such engineered conjugates are expressly contemplated within the scope of the present invention. In other embodiments, the disclosed anti-cancer agents will be administered in combination with a site-specific conjugate comprising different therapeutic agents as described above.

[0248] In some embodiments, the B cell-related disease is an autoimmune disease, preferably an autoimmune disease selected from the group consisting of: allogeneic islet graft rejection, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, Alzheimer's disease, antineutrophil cytoplasmic autoantibody (ANCA), autoimmune adrenal disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune myocarditis, autoimmune neutropenia, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, autoimmune urticaria, Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman's syndrome, celiac spruce-dermatitis, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, dermatomyositis, dilated cardiomyopathy, discoid lupus, acquired epidermolysis bullosa, essential mixed cryoglobulinemia, factor VIII deficiency, fibromyalgia-fibromyositis, glomerulonephritis, Grave's disease, Guillain-Barre, Goodpasture's syndrome, graft-versus-host disease (GVHD), Hashimoto's thyroiditis, hemophilia A, idiopathic membranous neuropathy, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA neuropathy, IgM polyneuropathy, immune-mediated thrombocytopenia, juvenile arthritis, Kawasaki's disease, lichen planus, lichen sclerosus, lupus erthematosis, Meniere's disease, mixed connective tissue disease, mucous membrane pemphigoid, multiple sclerosis, type 1 diabetes, multifocal motor neuropathy (MMN), myasthenia gravis, paraneoplastic bullous pemphigoid, pemphigoid gestationis, pemphigus vulgaris, pemphigus foliaceus, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, relapsing polychondritis, Reynauld's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjorgen's syndrome, solid organ transplant rejection, stiff-person syndrome, systemic lupus erythematosus, Takayasu arteritis, toxic epidermal necrolysis (TEN), Stevens Johnson syndrome (SJS), temporal arteritis / giant cell arteritis, thrombotic thrombocytopenic purpura, ulcerative colitis, uveitis, dermatitis herpetiformis vasculitis, antineutrophil cytoplasmic antibody-associated vasculitis, vitiligo, and Wegner's granulomatosis.,

[0249] Diagnosis

[0250] The present invention provides in vitro and in vivo methods for detecting, diagnosing, or monitoring a proliferative disorder and methods for screening cells from a patient to identify tumor cells including oncogenic cells. Such methods include identifying an individual with cancer for treatment or monitoring the progression of cancer, including contacting a patient or a sample obtained from the patient (in vivo or in vitro) with an antibody described herein and detecting the presence, absence, or level of binding of the antibody bound to or free of a target molecule in the sample. In some embodiments, the antibody will comprise a detectable label or a reporter molecule as described herein.

[0251] In some aspects, the present disclosure provides a method for diagnosing, detecting or monitoring a B cell-related disease, comprising administering an effective dose of the antibody or its antigen-binding fragment, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit to a subject in need thereof.

[0252] In some aspects, the present disclosure provides the antibody or its antigen-binding fragment, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit for use in a method for diagnosing, detecting or monitoring a B cell-related disease in a subject.

[0253] In yet another aspect, the present disclosure provides the use of the antibody or its antigen-binding fragment, the nucleic acid molecule, the vector, the host cell, the conjugate, the antibody-drug conjugate, the multispecific molecule, or the pharmaceutical composition or kit in the preparation of a reagent (or drug) for diagnosing, detecting or monitoring a CD20-related disease.

[0254] Samples can be analyzed by a variety of assays, such as radioimmunoassay, enzyme immunoassay (e.g., ELISA), competitive binding assay, fluorescence immunoassay, immunoblot assay, Western blot analysis, and flow cytometry assay. Compatible in vivo diagnostic or diagnostic assays can include imaging or monitoring techniques known in the art, such as magnetic resonance imaging, computed tomography (e.g., CAT scan), positron emission tomography (e.g., PET scan), radiography, ultrasound, etc., known to those skilled in the art.

[0255] The method for in vitro detecting or monitoring CD20 expression or the level of CD20-expressing cells described in the present invention can also be used for non-diagnostic purposes.

[0256] Preferred subjects include mammals, such as humans / patients in need.

[0257] Samples from the subject are blood, excreta (urine or feces), oral or nasal secretions, or bronchoalveolar lavage fluid, tissue fluid, sweat, or extracts thereof from the subject.

[0258] Drug packaging and kits

[0259] Also provided are pharmaceutical packages and kits that include one or more containers of one or more doses of an antibody or an antigen-binding fragment thereof. In certain embodiments, unit doses are provided, where a unit dose contains a predetermined amount of a composition that includes, for example, an antibody or an antigen-binding fragment thereof, with or without one or more other reagents. For other embodiments, such unit doses are supplied in single-use prefilled syringes for injection. In other embodiments, the composition contained in the unit dose can include saline, sucrose, or the like; buffers such as phosphate, etc.; and / or be formulated within a stable and effective pH range. Alternatively, in certain embodiments, the conjugate composition can be provided as a lyophilized powder, which can be reconstituted after addition of a suitable liquid (e.g., sterile water or saline solution). In certain preferred embodiments, the composition includes one or more substances that inhibit protein aggregation, including but not limited to sucrose and arginine. Any label on or associated with the container indicates that the encapsulated conjugate composition is for treating a selected oncological disease condition.

[0260] Such kits generally contain a pharmaceutically acceptable formulation of the engineered conjugate in a suitable container and optionally contain one or more anticancer agents or other agents in the same or different containers. The kit can also contain other pharmaceutically acceptable formulations for diagnostic or combination therapy.

[0261] More specifically, the kit can have a single container containing an antibody or an antigen-binding fragment thereof of the present disclosure, with or without additional components, or they can have separate containers for each desired reagent. In cases where combination therapeutic agents for conjugation are provided, a single solution can be premixed in molar equivalents or with one component in excess of the other. Alternatively, the conjugate of the kit and any optional anticancer agent can be stored separately in different containers prior to administration to a patient. The kit can also include a second / third container device for holding a sterile pharmaceutically acceptable buffer or other diluent such as bacteriostatic water for injection (BWFI), phosphate-buffered saline (PBS), Ringer's solution, and glucose solution.

[0262] When the components of the kit are provided as one or more liquid solutions, the liquid solutions are preferably aqueous solutions, particularly preferably sterile aqueous solutions or saline solutions. However, the components of the kit can be provided as dry powders. When the reagent or component is provided as a dry powder, the powder can be reconstituted by addition of a suitable solvent. It is contemplated that the solvent can also be provided in another container.

[0263] The invention is further described in the following paragraphs.

[0264] Paragraph 1. An isolated antibody or antigen-binding fragment thereof, which is a bispecific antibody, wherein the Fab end of the antibody or antigen-binding fragment specifically binds to a B cell surface antigen (e.g., specifically binds to CD19, CD20, BCMA, CD38), and the Fc end of the antibody or antigen-binding fragment specifically binds to the FcRn protein.

[0265] Paragraph 2. The antibody or fragment as described in Paragraph 1, wherein the Fab end specifically binds to the B cell surface antigen with an EC50 of less than 1 μM (e.g., less than 100 nM, 10 nM, 1 nM, 100 pM, 10 pM); and / or preferably, under neutral conditions (e.g., pH 6.5 - 7.5), the binding affinity KD of the Fc end to FcRn is less than 10 -6 M, such as less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or a KD value less than that binds to FcRn; or preferably, under acidic conditions (e.g., pH less than 6.5), the binding affinity KD of the Fc end to FcRn is less than 1.0x10 -7 M, such as less than about 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or a KD value less than that binds to FcRn.

[0266] Paragraph 3. The antibody or fragment as described in Claim 1 or 2, wherein the B cell surface antigen is CD20, CD19, BCMA, CD38, preferably CD20, CD19.

[0267] Paragraph 4. The antibody or fragment as described in any one of the preceding paragraphs, wherein the Fab end comprises a heavy chain variable region (HCDR) and a light chain variable region (LCDR),

[0268] The heavy chain variable region comprises:

[0269] (i) HCDR1, which comprises a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with SEQ ID NO: 39 or consists of SEQ ID NO: 39;

[0270] (ii) HCDR2, which comprises a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with SEQ ID NO: 40 or SEQ ID NO: 42 or consists of SEQ ID NO: 40 or SEQ ID NO: 42; and

[0271] (iii) An HCDR3, which comprises a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with SEQ ID NO: 41 or consists of SEQ ID NO: 41,

[0272] The light chain variable region comprises:

[0273] (i) An LCDR1, which comprises a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with SEQ ID NO: 36 or consists of SEQ ID NO: 36;

[0274] (ii) An LCDR2, which comprises a sequence having at least 60%, at least 65%, at least 75%, at least 95%, or 100% sequence identity with SEQ ID NO: 37(ATS) or consists of SEQ ID NO: 37(ATS); and

[0275] (iii) An LCDR3, which comprises a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with SEQ ID NO: 38 or consists of SEQ ID NO: 38,

[0276] wherein the Fc region comprises a heavy chain constant region, which comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 27, and has one, two, three, four or five substitutions selected from the group consisting of: M252Y, W, F, P; S254T, C, A, L; T256E, Q, N; H433K, R; N434F, Y, W, P, L, wherein the amino acid position numbering is according to EU numbering.

[0277] Paragraph 5. The antibody or fragment as described in any one of the preceding paragraphs, wherein the substitution is selected from the group consisting of: M252Y, W; S254T; T256E; H433K; N434F, Y, W, wherein the amino acid position numbering is according to EU numbering.

[0278] Paragraph 6. The antibody or fragment as described in any one of paragraphs 1-3, wherein the heavy chain constant region comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 27, has the substitution H433K relative to SEQ ID NO: 27, and further has at least one, two, three, four substitutions selected from the group consisting of: M252Y,W; S254T; T256E; N434F,Y,W, wherein the amino acid position numbering is according to EU numbering.

[0279] Paragraph 7. The antibody or fragment as described in any one of the preceding paragraphs, wherein the heavy chain constant region comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 27, has the substitution S254T (EU numbering) relative to SEQ ID NO: 27, and further has one, two, three, four substitutions selected from the group consisting of: M252Y,W; T256E; H433K; N434F,Y,W, wherein the amino acid position numbering is according to EU numbering.

[0280] Paragraph 8. The antibody or fragment as described in any one of the preceding paragraphs, wherein the heavy chain constant region comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 27, has the substitution N434F,Y,W relative to SEQ ID NO: 27, and further has one, two, three, four substitutions selected from the group consisting of: M252Y,W; S254T; T256E; H433K, wherein the amino acid position numbering is according to EU numbering.

[0281] Paragraph 9. The antibody or fragment as described in any one of the preceding paragraphs, wherein the heavy chain constant region comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 27, has the substitution T256E relative to SEQ ID NO: 27, and further has one, two, three, four substitutions selected from the group consisting of: M252Y,W; S254T; H433K; N434F,Y,W, wherein the amino acid position numbering is according to EU numbering.

[0282] Paragraph 10. The antibody or fragment as described in any one of the foregoing paragraphs, wherein the heavy chain constant region comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 27, and has substitutions M252Y,W relative to SEQ ID NO: 27, and further has one, two, three, or four substitutions selected from the group consisting of: S254T; T256E; H433K; N434F,Y,W, wherein the amino acid position numbering is according to EU numbering.

[0283] Paragraph 11. The antibody or fragment as described in any one of the foregoing paragraphs, wherein the heavy chain constant region comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 27, and has the following substitutions relative to SEQ ID NO: 27:

[0284] M252Y + H433K + N434F;

[0285] M252W + H433K + N434F;

[0286] M252Y + H433K + N434Y;

[0287] M252W + H433K + N434Y;

[0288] M252Y + S254T + T256E + H433K + N434Y;

[0289] M252Y + S254T + T256E + H433K + N434W;

[0290] wherein the amino acid position numbering is according to EU numbering.

[0291] Paragraph 12. The antibody or fragment as described in any one of the foregoing paragraphs, wherein the heavy chain constant region comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with one of SEQ ID NOs: 29 - 35 or consists of one of SEQ ID NOs: 29 - 35.

[0292] Paragraph 13. The antibody or fragment as described in any one of the preceding paragraphs, wherein the heavy chain variable region comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with one of SEQ ID NO: 19, 22, 23, 24, 25 or consists of one of SEQ ID NO: 19, 22, 23, 24, 25.

[0293] Paragraph 14. The antibody or fragment as described in any one of the preceding paragraphs, wherein the light chain variable region comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with one of SEQ ID NO: 18, 20, 21 or consists of one of SEQ ID NO: 18, 20, 21.

[0294] Paragraph 15. The antibody or fragment as described in any one of the preceding paragraphs, further comprising a light chain constant region, which comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 26 or consists of SEQ ID NO: 26.

[0295] Paragraph 16. The antibody or fragment as described in any one of the preceding paragraphs, which comprises:

[0296] A light chain, which comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with one of SEQ ID NO: 1, 4, 5 or consists of one of SEQ ID NO: 1, 4, 5;

[0297] A heavy chain, which comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with one of SEQ ID NO: 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or consists of one of SEQ ID NO: 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17.

[0298] Paragraph 17. The antibody or fragment as described in Paragraph 16, which comprises:

[0299] (a) A light chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 1 or a light chain consisting of SEQ ID NO: 1; and a heavy chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with one of SEQ ID NO: 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or a heavy chain consisting of one of SEQ ID NO: 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17;

[0300] (b) A light chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 4 or a light chain consisting of SEQ ID NO: 4; and a heavy chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with one of SEQ ID NO: 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or a heavy chain consisting of one of SEQ ID NO: 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17;

[0301] (c) A light chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 5 or a light chain consisting of SEQ ID NO: 4; and a heavy chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with one of SEQ ID NO: 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or a heavy chain consisting of one of SEQ ID NO: 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17.

[0302] Paragraph 18. The antibody or fragment as described in any of the foregoing paragraphs, which has one or more of the following properties:

[0303] (a) Binds to human or monkey CD20 with an EC50 of 1 μM or less; preferably lower than the EC50 of Rituximab;

[0304] (b) Under neutral conditions (e.g., pH 6.5 - 7.5), the binding affinity KD of the Fc end to FcRn is less than 10 -6M, such as less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 An antibody or fragment thereof that binds to FcRn with a KD value of M or less;

[0305] (c) Under acidic conditions (e.g., pH less than 6.5), the binding affinity KD of the Fc terminus to FcRn is less than 1.0 x 10 -7 M, such as less than about 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 An antibody or fragment thereof that binds to FcRn with a KD value of M or less.

[0306] (d) Specifically binds to FcγR receptors, such as FcγRIIIA (CD16aA), FcγRIIA (CD32A), and FcγRI (CD64).

[0307] Paragraph 19. An antibody or fragment thereof as described in any of the preceding paragraphs, wherein the antibody is a monoclonal antibody.

[0308] Paragraph 20. An antibody or fragment thereof as described in any of the preceding paragraphs, wherein the antibody is a chimeric antibody or a humanized antibody.

[0309] Paragraph 21. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding an antibody or fragment thereof as described in any of paragraphs 1 - 20.

[0310] Paragraph 22. A vector comprising the nucleic acid molecule as described in paragraph 21.

[0311] Paragraph 23. A host cell comprising the nucleic acid molecule as described in paragraph 22 or the vector as described in paragraph 22.

[0312] Paragraph 24. A conjugate comprising an antibody or fragment thereof as described in any of paragraphs 1 - 20 conjugated to at least one detectable label.

[0313] Paragraph 25. An antibody - drug conjugate comprising an antibody, which comprises one or more drug moieties, wherein the drug moiety is covalently linked directly or via a linker to an antibody or fragment thereof as described in any of paragraphs 1 - 20.

[0314] Paragraph 26. A multispecific molecule comprising the antibody or antigen-binding portion described in any one of paragraphs 1-20; preferably, the multispecific molecule specifically binds to CD20 and additionally specifically binds to one or more other targets (such as CD19, BCMA, CD38); more preferably, the multispecific molecule further comprises at least one molecule having a second binding specificity for a second target.

[0315] Paragraph 27. A pharmaceutical composition or kit comprising the antibody or fragment described in any one of paragraphs 1-20, or the nucleic acid molecule described in paragraph 21, or the vector described in paragraph 22, or the host cell described in paragraph 23, or the conjugate described in paragraph 24, or the antibody-drug conjugate described in paragraph 25, or the multispecific molecule described in paragraph 26; and a pharmaceutically acceptable carrier.

[0316] Paragraph 28. A method for preparing the antibody or fragment described in any one of paragraphs 1-20, comprising the steps of:

[0317] (i) expressing the antibody or fragment described in any one of paragraphs 1-20 in the host cell described in paragraph 23; and optionally

[0318] (ii) isolating the antibody or its antigen-binding fragment from the host cell.

[0319] Paragraph 29. Use of the antibody or fragment described in any one of paragraphs 1-20, or the nucleic acid molecule described in paragraph 21, or the vector described in paragraph 22, or the host cell described in paragraph 23, or the conjugate described in paragraph 24, or the antibody-drug conjugate described in paragraph 25, or the multispecific molecule described in paragraph 26, or the pharmaceutical composition or kit described in paragraph 27 in the preparation of a kit for diagnosing, detecting or monitoring B cell-related diseases.

[0320] Paragraph 30. Use of the antibody or fragment described in any one of paragraphs 1-20, or the nucleic acid molecule described in paragraph 21, or the vector described in paragraph 22, or the host cell described in paragraph 23, or the conjugate described in paragraph 24, or the antibody-drug conjugate described in paragraph 25, or the multispecific molecule described in paragraph 26, or the pharmaceutical composition or kit described in paragraph 27 in the preparation of a drug for treating B cell-related diseases or determining their prognosis.

[0321] Use according to paragraph 29 or 30, wherein the B cell-related disease is cancer, and the cancer is selected from the group consisting of: B cell lymphoma, including NHL, pre-B cell lymphoblastic leukemia / lymphoma, and mature B cell tumors, such as B cell chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), including low-grade, intermediate-grade, and high-grade FL, cutaneous follicular center lymphoma, marginal zone B cell lymphoma (MALT type, nodal and splenic types), hairy cell leukemia, diffuse large B cell lymphoma, Burkitt lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenström macroglobulinemia, and anaplastic large cell lymphoma (ALCL).

[0322] Use according to paragraph 29 or 30, wherein the B cell-related disease is an autoimmune disease, and the autoimmune disease is selected from the following group: allogeneic islet graft rejection, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, Alzheimer's disease, antineutrophil cytoplasmic autoantibody (ANCA), autoimmune adrenal disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune myocarditis, autoimmune neutropenia, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, autoimmune urticaria, Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman's syndrome, celiac spruce-dermatitis, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, dermatomyositis, dilated cardiomyopathy, discoid lupus, acquired epidermolysis bullosa, essential mixed cryoglobulinemia, factor VIII deficiency, fibromyalgia-fibromyositis, glomerulonephritis, Grave's disease, Guillain-Barre, Goodpasture's syndrome, graft-versus-host disease (GVHD), Hashimoto's thyroiditis, hemophilia A, idiopathic membranous neuropathy, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura,ITP), IgA neuropathy, IgM polyneuropathy, immune-mediated thrombocytopenia, juvenile arthritis, Kawasaki's disease, lichen planus, lichen sclerosus, lupus erthematosis, Meniere's disease, mixed connective tissue disease, mucous membrane pemphigoid, multiple sclerosis, type 1 diabetes, multifocal motor neuropathy (MMN), myasthenia gravis, paraneoplastic bullous pemphigoid, pemphigoid gestationis, pemphigus vulgaris, pemphigus foliaceus, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, relapsing polychondritis, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, solid organ transplant rejection, stiff-person syndrome, systemic lupus erythematosus, Takayasu arteritis, toxic epidermal necrolysis (TEN), Stevens Johnson syndrome (SJS), temporal arteritis / giant cell arteritis, thrombotic thrombocytopenic purpura, ulcerative colitis, uveitis, dermatitis herpetiformis vasculitis, anti-neutrophil cytoplasmic antibody-associated vasculitis, vitiligo and Wegner's granulomatosis.,

[0323] Examples

[0324] The present invention generally described herein will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention. In addition, the experimental methods in the following examples are all conventional methods unless otherwise specified. The raw materials, reagent materials, etc. used in the following examples are all commercially available products unless otherwise specified.

[0325] Tables 1, 2, and 3 below provide an overview of some of the sequences contained in the antibodies of the examples. The exemplary antibodies of the present disclosure are anti-CD20 monoclonal antibodies.

[0326] Table 1 Variable region amino acid sequence of rituximab after heavy chain humanization

[0327]

[0328]

[0329] Table 2 Full-length amino acid sequence after humanization of the light chain of rituximab

[0330]

[0331] Table 3 Amino acid sequence after mutation of the constant region of human IgG1 antibody

[0332]

[0333]

[0334] In the embodiments of the present disclosure, the light chain constant regions of all antibodies are the same, and their sequences are shown in SEQ ID NO: 26. In the following embodiments, the antibody names are named in the order of "heavy chain variable region - heavy chain constant region - light chain". For example, the antibody named "zH2P2-11-L3" means that the heavy chain variable region sequence of this antibody is the sequence shown as "zH2P2 (SEQ ID NO: 24)" in Table 1, the heavy chain constant region sequence of this antibody is the sequence shown as "11 (SEQ ID NO: 30)" in Table 3, and the light chain of this antibody is the sequence shown as "L3 (SEQ ID NO: 4)" in Table 2.

[0335] Example 1 Construction, expression and purification of bispecific antibody

[0336] Humanize the heavy and light chains of rituximab: Select the human germline gene IGHV1-69*02 / IGHJ2*01 with the highest homology for heavy chain CDR transplantation; select human IGKV1-16*01 / IGKJ4*01 for light chain CDR transplantation. Then, through amino acid mutation of key residues and PTM (Post-translational modification) removal of mutations, a total of 4 different heavy chain variable regions (see Table 1) and 2 different light chain humanized sequences (see Table 2) are designed.

[0337] Amino acid mutations were designed for the Fc region of human IgG1 antibody to obtain 7 different Fc mutant sequences shown in Table 3. Among them, the antibody named RTX - YTEKF (its light chain sequence is SEQ ID NO:1 and heavy chain sequence is SEQ ID NO:3) is based on the rituximab sequence, with five amino acid mutations of M252Y, S254T, T256E, H433K, and N434F added to the Fc segment, which is the mutant sequence of efgartigimod. The heavy and light chain gene fragments of the antibody of the present invention and the reference antibody were cloned into the pcDNA3.1 vector, transfected into CHO cells for expression, and the cells were collected after 7 days of culture. The antibody was obtained by affinity purification using a Protein A column.

[0338] Example 2 Binding Activity of Bifunctional Antibody to CD20 Protein Level

[0339] The above - purified candidate antibodies were subjected to ELISA to detect the protein - level binding activity. Human CD20 antigen protein was dispensed into 96 - well enzyme - linked immunosorbent assay (ELISA) plates at a concentration of 1 μg / mL, 100 μl / well, and incubated overnight at 4°C. Then, after washing 3 times with PBST, 100 μl of 3% BSA blocking solution was added and incubated at 37°C for 1 hour. After washing 3 times with PBST, 100 μl of the antibody sample diluted in a gradient (starting concentration of 666 ng / ml, 3 - fold serial dilution, a total of 9 concentration points) was added and incubated at 37°C for 1 hour. After washing 3 times with PBST, 100 μl of Anti - Human IgG HRP (diluted 1:10000) was added and incubated at 37°C for 0.5 hour. After washing 4 times with PBST, 100 μl of TMB chromogenic solution was added and developed at 37°C for 8 min, and 100 μl / well of ELISA stop solution was added to terminate the reaction. Then, the absorbance was measured with an enzyme - linked immunosorbent assay reader at a wavelength of 450 nm.

[0340] The results are as Figures 1A - 1D shown. The binding activities of the bifunctional antibody and rituximab to CD20 protein are basically at the same level.

[0341] Example 3 Binding of Bifunctional Antibody to Endogenous CD20 Expressed on the Membrane Surface of Raji Cells

[0342] Flow cytometry was used to detect the binding of the CD20 and FcRn bifunctional antibody to endogenous CD20 expressed on the tumor cell membrane. Specific operation: 1×10 5A number of Raji cells (from Procell) were incubated with anti-CD20 antibodies (500 nM or 250 nM, 5-fold serial dilution) in the dark at 4 °C for 1 hour. After washing, goat anti-human IgG Fc-specific-FITC diluted at a ratio of 1:300 (1:300, sigma-Aldrich, F9512-1ML) was added at 4 °C and incubated in the dark for 0.5 hour. After washing, the cells were resuspended and the fluorescence intensity of the cells was detected using a FACS instrument (BD Lyric). The data was processed using GraphPad Prism software.

[0343] The results are as Figures 2A - 2B and Figures 3A - 3B shown: The EC50 values of most of the antibodies (3 - 7 nM) were significantly better than that of the RTX monoclonal antibody (7.762 nM).

[0344] Example 4 Binding of the bifunctional antibody to hFcRn (human FcRn) on the surface of transfected cell lines at pH 6.0

[0345] The activity of the CD20 and FcRn bifunctional antibody binding to hFcRn on the membrane of transfected MDCK-hFcRn cells at pH 6.0 was detected using flow cytometry. Specific operation: 1x10 5 MDCK-FcRn cells were incubated with the CD20 and FcRn bifunctional antibody constructed in Example 1 (100 nM, 5-fold serial dilution) in the dark at 4 °C for 1 hour. After washing, goat anti-human IgG Fab-specific-FITC diluted at a ratio of 1:300 (sigma-Aldrich, F5512-1ML) was added at 4 °C and incubated in the dark for 0.5 hour. After washing, the cells were resuspended and the fluorescence intensity (median value) of the cells was detected using a FACS instrument (BD Lyric). The data was processed using Graphpad Prism software.

[0346] The results are as Figures 4A - 4C and Figures 5A - 5B shown: The antibodies with heavy chain Fc numbered 10 or 11 were comparable to RTX-YTEKF, and the other antibodies decreased slightly. Except for the slightly lower upper plateau of the binding curves of zH2P2-10-L3, zH2P2-23-L3, and zH2P2-25-L3, there was no significant difference between the other antibodies and the YTEKF antibody.

[0347] Example 5 Binding of the bifunctional antibody to hFcRn on the surface of transfected cell lines at pH 7.4

[0348] The activity of the Fc end of the antibody binding to hFcRn on the membrane of transfected MDCK-hFcRn cells at pH 7.4 was detected using flow cytometry. Specific operation: 1x10 5One MDCK-FcRn cell was incubated with a CD20 and FcRn bifunctional antibody (starting concentration 500 nM, 5-fold serial dilution or 556 nM, 3-fold serial dilution) at 4 °C in the dark for 1 hour. After washing, sheep anti-human IgG Fab specific-FITC (sigma-Aldrich, F5512-1ML) diluted at a ratio of 1:300 was added at 4 °C and incubated in the dark for 0.5 hour. After washing, the cells were resuspended and the fluorescence intensity (median) of the cells was detected using a FACS instrument (BD Lyric). The data was processed using Graphpad Prism software.

[0349] The results showed ( Figures 6A - 6C and Figures 7A - 7C ): The binding ability of antibodies zH2P2-10-L3 and zH2P2-10-L4 to MDCK-hFcRn was better than that of the reference antibody RTX-YTEKF at pH 7.4.

[0350] Example 6 Affinity of bifunctional antibodies for FcRn

[0351] Using a Biacore 8K biomolecular interaction analyzer, the affinities between different candidate antibodies and FcRn receptors (FCGRT&B2M, Acro) of different species (human, cynomolgus monkey, mouse) were compared. The candidate antibodies were coupled to the CM5 chip (Cytiva) via amino groups, and the FcRn receptor proteins diluted with different pH (pH 6.0, pH 7.4) HBS-EP+ buffer were passed over the CM5 chip. The affinity results were fitted using Biacore 8K Evaluation software, and the affinity data are shown in Tables 4 - 6 below.

[0352] The affinities of antibodies zH2P2-10-L3 and zH2P2-10-L4 for human FcRn were higher than that of the reference antibody RTX-YTEKF under acidic and neutral conditions; the affinities of the remaining candidate antibodies were comparable to that of the reference antibody. For mouse FcRn, the affinities of the bifunctional antibodies were comparable to that of RTX-YTEKF under acidic and neutral pH conditions. The affinities of the bifunctional antibodies for cynomolgus monkey FcRn protein were similar to those for human FcRn, and the affinities of antibodies zH2P2-10-L3 and zH2P2-10-L4 for human FcRn were slightly higher than that of RTX-YTEKF under acidic and neutral conditions.

[0353] Table 4 Results of SPR analysis of the affinity of candidate antibodies for human FcRn receptor protein

[0354]

[0355] Table 5 Results of SPR analysis of the affinity of candidate antibodies for mouse FcRn receptor protein

[0356]

[0357]

[0358] Table 6 Affinity results of candidate antibodies for cynomolgus FcRn receptor protein in SPR analysis

[0359]

[0360] Example 7 Affinity of bifunctional antibody for FcγR

[0361] Using a Biacore 8K biomolecular interaction analyzer, the affinities between different candidate antibodies and FcγR receptors of different species (human, cynomolgus monkey, mouse) were compared. The candidate antibodies were conjugated to the CM5 chip (Cytiva) via amino groups, and the Fcγ receptor proteins diluted in a gradient with HBS-EP+ Buffer were passed over the CM5 chip. The affinity results were fitted using the Biacore 8K Evaluation software, and the affinity data are shown in Tables 7 - 9 below. The affinities of antibodies zH2P2-10-L3 and zH2P2-11-L4 for FcγR proteins such as human / cynomolgus monkey / mouse CD16a, human / cynomolgus monkey CD32a, and mouse CD16-2 were slightly lower than those of RTX, which might lead to a decrease in the ADCC activity of B cell killing. However, the affinity for CD64 protein was basically consistent with that of RTX.

[0362] Table 7 Affinity results of candidate antibodies for human, mouse, and cynomolgus monkey CD16a receptor proteins in SPR analysis

[0363]

[0364] Table 8 Affinity results of candidate antibodies for human, mouse, and cynomolgus monkey CD32a receptor proteins in SPR analysis

[0365]

[0366] Table 9 Affinity results of candidate antibodies for human, mouse, and cynomolgus monkey CD64 receptor proteins in SPR analysis

[0367]

[0368] Example 8 ADCC activity of CD20 and FcRn bifunctional antibody against B cell killing

[0369] The antibody ADCC activity was detected using the reporter gene method. Raji cells were from Procell; Jurkat-FcγRIIIa-V158 Effector Cells were from Novoprotein. Specific operation: Raji cells (Procell) at the logarithmic growth phase were adjusted to a cell density of 5x10 5 cells / ml with 1640 + 10% FBS + 1% PS, and added to a white 96-well flat bottom plate, 25 μl / well; CD20 and FcRn bifunctional antibodies (72 nM, diluted in 5-10-fold gradients, 7 concentration points) were added, 50 μl / well; Jurkat-FcγRIIIa-V158 Effector Cells with a cell density of 3x10 6 cells / ml were added, 25 μl / well; and incubated at 37 °C and 5% CO2 for 16 hours. The luciferase chromogenic solution (Beyotime) was taken out 30 min in advance, thawed in room temperature water and equilibrated to room temperature; the 96-well plate was taken out and equilibrated at room temperature for 5-10 min, and the chromogenic solution was added, 80 μl / well; incubated at room temperature in the dark for 10 min; the luciferase activity was measured using a multifunctional microplate reader. The data was processed using GraphPad Prism software, and the final concentration of the antibody was used as the abscissa and the RLU value as the ordinate for non-linear fitting to calculate the EC50 value. Results( Figures 8A - 8F ) showed that compared with RTX, the high point platforms of the S-shaped curves of the ADCC activities of RTX-YTEKF and all bifunctional antibodies decreased, but the IC50 of the ADCC activities of most bifunctional antibodies was better than that of the reference antibodies RTX and RTX-YTEKF.

[0370] Example 9 CDC Activity of CD20 and FcRn Bifunctional Antibodies against B Cell Killing

[0371] The CDC activity of the antibody was detected using commercial plasma-derived complement (Quidel). Specific operation: Raji cells (Procell) at the logarithmic growth phase were washed once with 1640 + 1% PS and adjusted to a cell density of 5x10 5cells / ml, add to a white 96-well flat bottom plate, 50 μl / well; add the CD20 and FcRn bifunctional antibody diluted with 1640 + 1% PS + 10% complement plasma (666.67 nM, 5-fold serial dilution, 8 concentration points), 50 μl / well; set up a cell control group containing only Raji cells. Incubate at 37 °C and 5% CO2 for 4 hours. Take out the Cell Counting-Lite2.0 Luminescent Cell Viability Assay (Vazyme) 30 min in advance, thaw it in room temperature water and equilibrate to room temperature; take out the 96-well plate and equilibrate at room temperature for 5 - 10 min, add the chromogenic solution, 100 μl / well; incubate at room temperature in the dark for 5 - 10 min; measure the luciferase activity with a multifunctional microplate reader. Use the calculation formula: Cell lysis rate (Lysis%) = (1 - RLU sample / RLU cell control) * 100% to calculate the cell lysis percentage. Process the data with GraphPad Prism software, use the final concentration of the antibody as the abscissa and the cell lysis percentage as the ordinate for non-linear fitting, and calculate the IC50 value.

[0372] Results ( Figure 9 ) showed that: for the reference RTX antibody, RTX-YTEKF had comparable CDC activity. However, the CDC activities (IC50) of the antibodies ZH2P2-10-L3 and ZH2P2-11-L4 in the present disclosure were superior to RTX-YTEKF.

[0373] Example 10 ADCP activity of CD20 and FcRn bifunctional antibody against B cells

[0374] Use the reporter gene method to detect the ADCP activity of the antibody. Raji cells are from Procell; Jurkat-FcγRIIa-H131 Effector Cells are from Novoprotein. The cell surface is stably transfected with FcγRIIa (CD32a) R131H and carries the NFAT-RE-Luc reporter gene system. FcγRIIa (CD32a), FcγRI (CD64), and FcγRIIIa (CD16a) expressed on monocytes, macrophages, neutrophils, and dendritic cells can mediate ADCP, but FcγRIIa (CD32a) is the most important Fc receptor involved in ADCP, so it can be used to evaluate the ADCP activity of the antibody. Specific operation: Adjust the cell density of Raji cells growing to the logarithmic growth phase to 5◇10 5cells / ml, add to a white 96-well flat bottom plate, 25 μl / well; add the CD20 and FcRn bifunctional antibody (200 nM, 5-fold serial dilution, 8 concentration points), 50 μl / well; add Jurkat-FcγRIIa-H131 Effector Cells cells at a density of 3◇10 6 cells / ml in the logarithmic growth phase, 25 μl / well; incubate at 37 °C and 5% CO2 for 6 hours. Take out the Bright-Lite Luciferase Assay System (Vazyme) 30 min in advance, thaw it in room temperature water and equilibrate to room temperature; take out the 96-well plate and equilibrate at room temperature for 5 - 10 min, add the chromogenic solution, 100 μl / well; incubate at room temperature in the dark for 5 - 10 min; measure the luciferase activity with a multifunctional microplate reader. Process the data with GraphPad Prism software, use the final antibody concentration as the abscissa and the RLU value as the ordinate for non-linear fitting, and calculate the EC50 value.

[0375] The results showed ( Figure 10 ): The high point platform of the ADCP activity of RTX-YTEKF decreased by 30 - 40% (compared with RTX). The high point platforms of the ADCP activities of the antibodies ZH2P2-10-L3 and ZH2P2-11-L4 of the present disclosure were basically the same as that of RTX-YTEKF, and the EC50 was better than that of RTX-YTEKF (3 - 5 times).

[0376] Example 11 Pharmacokinetic (PK) study of bifunctional antibodies in hCD20 mice

[0377] Investigate the pharmacokinetic characteristics of zH2P2-10-L3 in hCD20 mice through a single intravenous administration pharmacokinetic study. The experimental animals were injected via the tail vein with 20 mg / kg of the candidate molecule zH2P2-10-L3 or zH2P2-11-L4, and the administration volume was 5 ml / kg for both. There were 9 animals in each group, and cross-blood sampling was performed, 3 animals at each time point. The blood sampling time points were before administration, 5 min, 1 h, 6 h, 24 h, 72 h, 144 h, 240 h, and 336 h after administration.

[0378] The serum concentrations of the antibodies zH2P2-10-L3 and zH2P2-11-L4 were detected by ELISA. The average concentration-time curve (C-T curve) is shown in Figure 11 . Among them, both the antibodies zH2P2-10-L3 and zH2P2-11-L4 were rapidly eliminated in hCD20 mice, with a half-life of about 2 - 2.5 h. The short half-life of the candidate molecule may be due to its too strong affinity for murine FcRn.

[0379] Example 12. PK / PD (Pharmacodynamics) Study of Bifunctional Antibody in hFcRn Mice

[0380] Based on the large difference in the affinity of zH2P2-10-L3 for murine FcRn receptor and human FcRn receptor, and the too strong affinity for murine FcRn may lead to too rapid clearance of zH2P2-10-L3, affecting its effect of accelerating IgG elimination. Therefore, hFcRn mice were first intravenously administered with a tracer human IgG antibody and then intravenously administered with zH2P2-10-L3 to investigate its pharmacokinetic situation in hFcRn mice and its effect of accelerating IgG clearance. Experimental animals were first injected with 500 mg / kg of tracer human IgG antibody via the tail vein, and 24 hours later, low and high doses of zH2P2-10-L3 (10 mg / kg, 30 mg / kg) or vehicle (PBS) or control drug (10 mg / kg efgartigimod) were injected via the tail vein, with a dosing volume of 5 ml / kg. There were 9 animals in each group, and cross-blood sampling was performed, with 3 animals at each time point. The blood sampling time points were 12 hours before dosing, 0 hour before dosing, 6 hours, 24 hours, 48 hours, 72 hours, 96 hours, 144 hours, and 192 hours after dosing.

[0381] The concentration of tracer IgG was detected by ELISA, and the results are shown in Figure 12 . Compared with the vehicle control group, low and high doses of the antibody zH2P2-10-L3 and Efgartigimod both showed significant effects of accelerating the clearance of tracer IgG. Compared with Efgartigimod, the effect of low-dose zH2P2-10-L3 was slightly weaker, and the effect of high-dose zH2P2-10-L3 was equivalent or slightly better.

[0382] The serum concentration of the antibody zH2P2-10-L3 was detected by ELISA. The average concentration-time curve (average C-T) is shown in Figure 13 , in which both low and high doses of the antibody zH2P2-10-L3 showed approximately linear elimination, and the half-life was about 35 hours.

[0383] Incorporated by reference

[0384] The entire content of each patent document and scientific document mentioned in this article is incorporated herein by reference for all purposes.

[0385] Equivalence

[0386] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the above-described embodiments are to be considered in all respects as illustrative and not restrictive of the invention described herein. Accordingly, the scope of the invention is indicated by the appended claims rather than by the foregoing description, and is intended to include all changes within the meaning and scope of equivalents of the claims.

Claims

1. A separated antibody or its antigen-binding fragment, which is a bispecific antibody, wherein, (1) The Fab end of the antibody or its antigen-binding fragment specifically binds to a B cell surface antigen, and its EC50 is less than 1 μM, such as less than 100 nM, 10 nM, 1 nM, 100 pM, 10 pM; (2) The Fc region of the antibody or its antigen-binding fragment specifically binds to human FcRn protein; preferably, under neutral conditions (e.g., pH 6.5 - 7.5), the binding affinity KD of the Fc region to FcRn is less than 10 -6 M, such as less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or a KD value less than this binds to FcRn; or preferably, under acidic conditions (e.g., pH less than 6.5), the binding affinity KD of the Fc region to FcRn is less than 1.0x10 -7 M, such as less than approximately 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or a KD value less than this binds to FcRn.

2. The antibody or fragment according to claim 1, wherein the B cell surface antigen is CD20, CD19, BCMA, CD38, preferably CD20, CD19.

3. The antibody or fragment according to claim 1 or 2, wherein the Fab end comprises a heavy chain variable region (HCDR) and a light chain variable region (LCDR), The heavy chain variable region comprises: (i) HCDR1, which comprises a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with SEQ ID NO: 39 or consists of SEQ ID NO: 39; (ii) HCDR2, which comprises a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with SEQ ID NO: 40 or SEQ ID NO: 42 or consists of SEQ ID NO: 40 or SEQ ID NO: 42; and (iii) HCDR3, which comprises a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with SEQ ID NO: 41 or consists of SEQ ID NO: 41, The light chain variable region comprises: (i) LCDR1, which comprises a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with SEQ ID NO: 36 or consists of SEQ ID NO: 36; (ii) LCDR2, which comprises a sequence having at least 60%, at least 65%, at least 75%, at least 95%, or 100% sequence identity with SEQ ID NO: 37 (ATS) or consists of SEQ ID NO: 37 (ATS); and (iii) LCDR3, which comprises a sequence having at least 80%, at least 85%, at least 95%, or 100% sequence identity with SEQ ID NO: 38 or consists of SEQ ID NO:

38.

4. The antibody or fragment according to claim 1, wherein the Fc region comprises a heavy chain constant region, and the heavy chain constant region comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 27, and has one, two, three, four or five substitutions selected from the group consisting of: M252Y, W, F, P; S254T, C, A, L; T256E, Q, N; H433K, R; N434F, Y, W, P, L, wherein the amino acid position numbering is according to the EU numbering (Edelman, G.M. et al. Proc Natl Acad Sci USA. 1969; 63(1): 78-85.).

5. The antibody or fragment according to claim 4, wherein the substitutions are selected from the group consisting of: M252Y, W; S254T; T256E; H433K; N434F, Y, W, wherein the amino acid position numbering is according to the EU numbering.

6. The antibody or fragment according to claim 4 or 5, wherein the Fc region thereof can specifically bind to FcγR (such as FcγRIIIA (CD16A), FcγRIIA (CD32A) and FcγRI (CD64)).

7. The antibody or fragment according to any one of the preceding claims, wherein the light chain variable region comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with one of SEQ ID NO: 18, 20, 21 or consists of one of SEQ ID NO: 18, 20, 21.

8. The antibody or fragment according to any one of the preceding claims, wherein the heavy chain variable region comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with one of SEQ ID NO: 19, 22-25 or consists of one of SEQ ID NO: 19-25.

9. The antibody or fragment according to any one of the preceding claims, which comprises: (a) A light chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 1 or a light chain consisting of SEQ ID NO: 1; and a heavy chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with one of SEQ ID NO: 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or a heavy chain consisting of one of SEQ ID NO: 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17; (b) A light chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 4 or a light chain consisting of SEQ ID NO: 4; and a heavy chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with one of SEQ ID NO: 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or a heavy chain consisting of one of SEQ ID NO: 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17; (c) A light chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 5 or a light chain consisting of SEQ ID NO: 4; and a heavy chain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with one of SEQ ID NO: 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or a heavy chain consisting of one of SEQ ID NO: 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17.

10. The antibody or fragment according to any one of the preceding claims, wherein the antibody is a monoclonal antibody.

11. The antibody or fragment according to any one of the preceding claims, wherein the antibody is a chimeric antibody or a humanized antibody.

12. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the antibody or fragment according to any one of claims 1-11.

13. A pharmaceutical composition or kit comprising the antibody or fragment according to any one of claims 1-11, or the nucleic acid molecule according to claim 12; and a pharmaceutically acceptable carrier.

Images