Anti-CD79b antibody, preparation method therefor and use thereof

Abstract

The present invention provides an anti-CD79b antibody, a preparation method therefor and a use thereof. Disclosed are a group of antibodies having a unique antigen binding moiety, capable of specifically binding to CD79b and effectively inhibiting tumor cells expressing CD79b. The antibody of the present invention can be used for inhibiting diseases related to CD79b expression or affected by CD79b functions, for example, tumors.

Description

Anti-CD79b antibody, preparation method and application thereof

[0001] This application claims priority to patent application filed on June 28, 2023, application number CN 202310771066.X; the entire contents of which are incorporated herein. Technical Field

[0002] The present invention belongs to the fields of immunology and pharmacy, and more specifically, relates to an anti-CD79b antibody, a preparation method thereof, and an application thereof. Background Art

[0003] CD79b is a component of the B cell receptor, forming a heterodimer with CD79a to mediate the signaling generated by antigen binding to the BCR. CD79b contains an extracellular immunoglobulin domain, a transmembrane domain, and an intracellular signaling domain. Both CD79b and CD79a can induce BCR endocytosis, but CD79b is dominant.

[0004] Currently, the immunotherapy drugs targeting CD79b available in the world include: Polatuzumab vedotin, which was approved by the FDA in 2019 and is the first CD79b-targeted antibody-drug conjugate (ADC), is used to treat relapsed or refractory diffuse large B-cell lymphoma; another ADC drug targeting CD79b, iladatuzumab vedotin, is used to treat non-Hodgkin's lymphoma and is currently undergoing Phase I trials.

[0005] Lymphomas are malignancies originating from the lymphopoietic system and are the most common hematological malignancies worldwide. Non-Hodgkin's lymphoma (NHL) is highly prevalent, accounting for over 80% of lymphomas. CD79b is expressed in over 90% of NHL cases. Among patients who relapse after chemotherapy, 87% of follicular lymphomas and 77% of diffuse large B-cell lymphomas express CD79b. Furthermore, CD79b is expressed only in normal pre-B cells and mature B cells, not in hematopoietic stem cells. Unlike other highly expressed B cell surface antigens, CD79b is rapidly internalized and delivered to lysosomes after antibody binding. These unique functions make CD79b a promising target for the targeted delivery of cytotoxic agents. Therefore, the development of therapeutics targeting CD79b is of particular importance in non-Hodgkin's lymphomas, such as diffuse large B-cell lymphoma and follicular lymphoma.

[0006] Based on the expression of CD79b and the unmet medical needs in the market, it is of great significance to develop antibody-targeted drugs targeting CD79b.

[0007] Summary of the Invention

[0008] The purpose of the present invention is to provide an anti-CD79b antibody, a preparation method thereof and an application thereof.

[0009] In a first aspect of the present invention, an anti-CD79b antibody or an antigen-binding fragment thereof is provided, wherein the anti-CD79b antibody has a light chain variable region and a heavy chain variable region, wherein the amino acid sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO: 7, the amino acid sequence of CDR2 is shown in SEQ ID NO: 10 or SEQ ID NO: 8, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 15, SEQ ID NO: 9 or SEQ ID NO: 22; the amino acid sequence of the light chain variable region CDR1 is shown in SEQ ID NO: 19, SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 23, the amino acid sequence of CDR2 is "LV", and the amino acid sequence of CDR3 is shown in SEQ ID NO: 5, SEQ ID NO: 20, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 24 or SEQ ID NO: 6.

[0010] In one or more preferred embodiments, the antibody or antigen-binding fragment thereof is selected from:

[0011] The amino acid sequences of the heavy chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 15, respectively; the amino acid sequences of the light chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 19, "LV," and SEQ ID NO: 5, respectively (e.g., A8-3m26, A8-3m18, or A8-3);

[0012] The amino acid sequences of the heavy chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 15, respectively; the amino acid sequences of the light chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 19, "LV," and SEQ ID NO: 20, respectively (e.g., A8);

[0013] The amino acid sequences of the heavy chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 15, respectively; the amino acid sequences of the light chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 11, "LV," and SEQ ID NO: 14, respectively (e.g., A1);

[0014] The amino acid sequences of the heavy chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 9, respectively; the amino acid sequences of the light chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 4, "LV," and SEQ ID NO: 18, respectively (e.g., A5);

[0015] The amino acid sequences of the heavy chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 22, respectively; the amino acid sequences of the light chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 23, "LV," and SEQ ID NO: 24, respectively (e.g., A18);

[0016] The amino acid sequences of the heavy chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 7, SEQ ID NO: 10, and SEQ ID NO: 9, respectively; the amino acid sequences of the light chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 4, "LV," and SEQ ID NO: 6, respectively (e.g., hmC3-3, mC3, or 101C5E9);

[0017] The amino acid sequences of the heavy chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively; the amino acid sequences of the light chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 4, "LV" and SEQ ID NO: 6, respectively (such as mC1 or 99H10D7).

[0018] In one or more preferred embodiments, the antibody or antigen-binding fragment thereof includes Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv.

[0019] In one or more preferred embodiments, the antibody includes: a humanized antibody, a chimeric antibody or a murine antibody.

[0020] In one or more preferred embodiments, the antibody is a humanized antibody having a humanized or fully human heavy chain FR region and a light chain FR region, and preferably having a humanized or fully human constant region.

[0021] In one or more preferred embodiments, the FR regions of the antibody (including heavy chain FR regions and light chain FR regions) are highly homologous, identical or similar to the FR regions of human antibody germline genes.

[0022] In one or more preferred embodiments, the antibody is a chimeric antibody having humanized or fully human constant regions, including a heavy chain constant region and a light chain constant region.

[0023] In one or more preferred embodiments, the chimeric antibody or humanized antibody has a human antibody IgG1 heavy chain constant region and a human kappa light chain constant region.

[0024] In one or more preferred embodiments, the antibody is a humanized antibody, including a heavy chain FR region and a light chain FR region.

[0025] In one or more preferred embodiments, the heavy chain FR region includes heavy chain FR1, FR2, FR3, and FR4, including FR1 with an amino acid sequence as shown at positions 1-25 in SEQ ID NO: 28, SEQ ID NO: 26, or SEQ ID NO: 13, FR2 as shown at positions 34-50 in SEQ ID NO: 13, FR3 as shown at positions 59-96 in SEQ ID NO: 13, and FR4 as shown at positions 103-113 in SEQ ID NO: 13.

[0026] In one or more preferred embodiments, the light chain FR region includes light chain FR1, FR2, FR3, and FR4, including FR1 with an amino acid sequence as shown at positions 1-26 in SEQ ID NO:12, FR2 as shown at positions 38-54 in SEQ ID NO:12, SEQ ID NO:27, or SEQ ID NO:25, FR3 as shown at positions 57-93 in SEQ ID NO:12, and FR4 as shown at positions 103-113 in SEQ ID NO:12.

[0027] In one or more preferred embodiments, the antibody is a humanized antibody, the heavy chain variable region of which has the amino acid sequence shown in SEQ ID NO: 28, and the light chain variable region of which has the amino acid sequence shown in SEQ ID NO: 27.

[0028] In one or more preferred embodiments, the antibody is a humanized antibody, the heavy chain variable region of which has the amino acid sequence shown in SEQ ID NO: 26, and the light chain variable region of which has the amino acid sequence shown in SEQ ID NO: 25

[0029] In one or more preferred embodiments, the antibody is a humanized antibody, the heavy chain variable region of which has the amino acid sequence shown in SEQ ID NO: 13, and the light chain variable region of which has the amino acid sequence shown in SEQ ID NO: 12.

[0030] In one or more preferred embodiments, the antibody further comprises a heavy chain variable region having an amino acid sequence that is more than 80% (e.g., 85%, 90%, 93%, 95%, 97% or more) identical to the sequence shown in SEQ ID NO: 28, SEQ ID NO: 26, or SEQ ID NO: 13.

[0031] In one or more preferred embodiments, the antibody further comprises a light chain variable region having an amino acid sequence that is more than 80% (e.g., 85%, 90%, 93%, 95%, 97% or more) identical to the sequence shown in SEQ ID NO: 27, SEQ ID NO: 25, or SEQ ID NO: 12.

[0032] In another aspect of the present invention, an isolated polynucleotide or a construct containing the polynucleotide is provided, wherein the polynucleotide encodes any one of the above-mentioned anti-CD79b antibodies or antigen-binding fragments thereof.

[0033] In one or more preferred embodiments, the construct is an expression vector.

[0034] In another aspect of the present invention, an expression system for an antibody or an antigen-binding fragment thereof is provided, wherein the expression system contains the construct or the exogenous polynucleotide integrated into the genome.

[0035] In one or more preferred embodiments, the expression system is a cell (expression system).

[0036] In one or more preferred embodiments, the cell (host cell) is a mammalian cell.

[0037] In one or more preferred embodiments, the mammalian cells include (but are not limited to): Chinese hamster ovary (CHO) cells (such as ExpiCHO), Vero cells, HEK-293 cells, NS0 cells, SP2 / 0 cells, BHK cells, PER-C6 cells; preferably, they are CHO cells.

[0038] In another aspect of the present invention, a method for preparing the antibody or antigen-binding fragment thereof is provided, comprising: expressing the antibody or antigen-binding fragment thereof using the expression system under conditions suitable for expressing the antibody or antigen-binding portion thereof, thereby expressing the antibody or antigen-binding fragment thereof.

[0039] In one or more embodiments, the method further comprises purifying and isolating the antibody or antigen-binding fragment thereof.

[0040] In another aspect of the present invention, there is provided a use of the antibody or antigen-binding fragment thereof for preparing an anti-tumor drug that specifically targets and inhibits tumor cells expressing CD79b.

[0041] In one or more preferred embodiments, the anti-tumor drug includes (but is not limited to): immunoconjugates (antibodies or their antigen-binding portions-drug conjugates); bifunctional (bispecific) or multifunctional (multispecific) antibodies (fusion proteins); and chimeric antigen receptor-modified immune cells.

[0042] In one or more preferred embodiments, the tumor includes: a non-solid tumor or a solid tumor.

[0043] In one or more preferred embodiments, the tumor includes: lymphoma, B lymphocytic leukemia.

[0044] In one or more preferred embodiments, the lymphoma includes non-Hodgkin's lymphoma.

[0045] In one or more embodiments, the non-Hodgkin's lymphoma comprises diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphoma, and Burkitt's lymphoma.

[0046] In another aspect of the present invention, a fusion protein or immunoconjugate is provided, which comprises the antibody or antigen-binding fragment thereof, and a functional molecule operatively linked thereto.

[0047] In one or more preferred embodiments, the functional molecules include: tumor-suppressing molecules, molecules targeting tumor surface markers, molecules or detectable markers targeting surface markers of immune cells, cytotoxins, radioactive isotopes, fusion partners, extracellular hinge regions, transmembrane regions and intracellular signaling regions of chimeric antigen receptors, or combinations thereof.

[0048] In one or more preferred embodiments, the tumor-suppressing molecule is an anti-tumor toxin or an anti-tumor cytokine.

[0049] In one or more preferred embodiments, the molecule targeting a tumor surface marker is an antibody or a ligand that binds to a tumor surface marker.

[0050] In one or more preferred embodiments, the molecule targeting a surface marker of an immune cell is an antibody or a ligand that binds to a surface marker of an immune cell.

[0051] In one or more preferred embodiments, the fusion partner includes (but is not limited to): a protein or active domain that has the effect of extending the half-life in vivo.

[0052] In one or more preferred embodiments, the anti-tumor toxin includes (but is not limited to): maytansine, auristatin, monomethyl auristatin, maytansinoid alkaloid, dolastatin, calicheamicin, methotrexate, vindesine, taxanes such as docetaxel, paclitaxel, lalopaclitaxel, tecitaxel or octataxel, trichothecenes, CC1065, DXd, Duocarmycin, Calicheamicin, Pyrrolobenzodiazepines or SN-38.

[0053] In one or more preferred embodiments, the anti-tumor cytokines include (but are not limited to): IL-2, IL-12, IL-15, IFN-beta, and TNF-alpha.

[0054] In one or more preferred embodiments, the antibody that binds to the tumor surface marker is an antibody that recognizes antigens other than the immunoregulatory molecule B7-H3, and the other antigens include (but are not limited to): EGFR, EGFRvIII, mesothelin, HER2, EphA2, cMet, EpCAM, MUC1, MUC16, CEA, Claudin 18.2, Claudin 6, WT1, NY-ESO-1, MAGE 3, CD47, ASGPR1 or CDH16.

[0055] In one or more preferred embodiments, the antigens bound by the antibody that binds to an immune cell surface marker include (but are not limited to): CD3, CD20, FcRH5, HER2, LYPD1, LY6G6D, PMEL17, LY6E, CD19, CD33, CD22, CD79A, CD79b, EDAR, GFRA1, MRP4, RET, Steap1 or TenB2.

[0056] In one or more preferred embodiments, the protein or active domain having the effect of extending the in vivo half-life includes (but is not limited to): immunoglobulin Fc region, preferably human immunoglobulin Fc region, serum albumin (such as human HSA) or a fragment thereof.

[0057] In one or more preferred embodiments, the immunoglobulin is a combination of one or more selected from IgG, IgA1, IgA2, IgD, IgE, and IgM, and the IgG is a combination of one or more selected from IgG1, IgG2, IgG3, or IgG4 subtypes.

[0058] In one or more preferred embodiments, a connecting peptide is provided between the antibody and the functional molecule operably linked thereto; the connecting peptide is preferably selected from a flexible polypeptide chain composed of alanine and / or serine and / or glycine, and the length of the connecting peptide is preferably 3 to 30 amino acids.

[0059] In another aspect of the present invention, a pharmaceutical composition is provided, comprising the antibody or antigen-binding fragment thereof, the fusion protein or immunoconjugate.

[0060] In one or more embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0061] In another aspect of the present invention, provided is the use of the antibody or antigen-binding fragment thereof, the fusion protein or immunoconjugate, or a pharmaceutical composition containing them in the preparation of a preparation, kit or drug kit for an anti-tumor drug that specifically targets and inhibits tumor cells expressing CD79b.

[0062] In another aspect of the present invention, a kit or medicine box is provided, comprising: a container or packaging; and the antibody, the fusion protein or immunoconjugate, or a pharmaceutical composition containing the same placed in the container or packaging.

[0063] In one or more embodiments, the test kit or medicine box may further contain: immunostimulatory antibodies, chemotherapeutic agents or anti-infective drugs, etc.

[0064] In one or more embodiments, the immunostimulatory antibodies include (but are not limited to): anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody and anti-CTLA-4 antibody.

[0065] In one or more embodiments, the kit or reagent box further contains instructions for use (package insert) describing how to use / take the antibody or antigen-binding portion thereof, fusion protein, immunoconjugate, or pharmaceutical composition.

[0066] In another aspect of the present invention, a method for stimulating an immune response in a subject is provided, the method comprising administering to the subject any of the antibodies or antigen-binding portions thereof described above, thereby stimulating the subject's immune response; preferably, the stimulation of the immune response comprises specifically targeting tumor cells expressing CD79b and inhibiting tumor cells.

[0067] In one or more embodiments, the subject is a subject bearing a tumor, and administering the antibody to the subject stimulates an immune response against the tumor.

[0068] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] 1A-C show the ELISA and FACS detection results of the chimeric antibody of the present invention against human and monkey CD79b.

[0070] FIG2 shows the tumor-killing function of the chimeric antibody-drug conjugate of the present invention.

[0071] FIG3 is the FACS detection result of the humanized antibody of the present invention.

[0072] 4A-B are the FACS detection results of the affinity matured antibodies of the present invention.

[0073] FIG5 shows the Octet detection results of the affinity matured antibodies of the present invention.

[0074] 6A-B are the ELISA detection results of the candidate molecules A8-3m18 and A8-3m26 of the present invention against human and monkey CD79b.

[0075] 7A-B are the FACS detection results of the candidate molecules A8-3m18 and A8-3m26 of the present invention.

[0076] FIG8 shows the Octet detection results of the candidate molecules A8-3m18 and A8-3m26 of the present invention on human CD79b.

[0077] FIG9 shows the Octet detection results of the binding epitopes of the candidate molecule A8-3m18 and polatuzumab of the present invention.

[0078] 10A-B are the FACS detection results of the binding epitopes of the candidate molecule A8-3m18 and polatuzumab of the present invention.

[0079] 11A-D show the tumor-killing function of the candidate molecules A8-3m18 and A8-3m26 conjugated drugs of the present invention.

[0080] 12A-B show the anti-tumor effects of the MMAE-coupled antibody-drug conjugate of the present invention on Ramos tumor-bearing mice.

[0081] FIG13 shows the anti-tumor effect of the DXd-coupled antibody-drug conjugate of the present invention on Ramos tumor-bearing mice. DETAILED DESCRIPTION

[0082] After extensive research, the inventors have discovered for the first time a group of antibodies with unique antigen-binding portions (CDR regions) that specifically bind to CD79b and effectively inhibit tumor cells expressing CD79b. The antibodies of the present invention can be used to inhibit diseases associated with CD79b expression or affected by CD79b function, such as tumors.

[0083] Existing anti-CD79b antibodies have room for further improvement in terms of affinity for the antigen, maximum binding capacity for CD79b-expressing cells, and tumor inhibition. Based on this, the present inventors conducted screening and optimization, resulting in the antibody of the present invention with a unique antigen-binding portion. This antibody exhibits significant advantages in terms of maximum antigen binding capacity and affinity, as well as excellent tumor inhibition.

[0084] the term

[0085] As used herein, "antibody" is used as a general term herein, including full-length antibodies, single-chain antibodies, and all parts, domains or fragments thereof (including but not limited to antigen-binding domains or fragments). In addition, the term "sequence" used herein (for example, in terms such as "immunoglobulin sequence", "antibody sequence", "single variable domain sequence", "VHH sequence" or "protein sequence") should generally be understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the sequence, unless a more limited explanation is required herein.

[0086] As used herein, "monoclonal antibody" or "mAb" refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope.

[0087] As used herein, a "chimeric antibody" is an antibody that is derived from one species (such as mouse) but has its antibody constant region replaced by that of another species (such as human). The "chimeric antibody" includes a "humanized antibody."

[0088] As used herein, "humanized antibody" refers to a molecule having an antigen binding site substantially derived from a non-human species immunoglobulin, wherein the rest of the immunoglobulin structure of the molecule is based on the structure and / or sequence of a human immunoglobulin. The antigen binding site may comprise a complete variable domain fused to a constant domain, or may comprise only complementarity determining regions (CDRs) transplanted into appropriate framework regions in the variable domain. The antigen binding site may be wild type or modified by one or more amino acid substitutions, for example, to be more similar to a human immunoglobulin. Certain forms of humanized antibodies retain all CDR sequences. Other forms have one or more CDRs that have been altered relative to the original antibody.

[0089] As used herein, a "detectable label" refers to a marker that can be attached to an antibody and used to determine the presence and amount of a specific target in a subject to be detected. The "detectable label" may include, but is not limited to, enzymes, fluorescent markers, radionuclides, quantum dots, colloidal gold, and the like. More specifically, it may be selected from, for example, horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, β-D-galactosidase, urease, catalase, or glucoamylase.

[0090] "Sequence identity" between two polypeptide sequences indicates the percentage of identical amino acids between the sequences. "Sequence similarity" indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions. Methods for evaluating the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, the BLAST program from the NCBI database can be used to determine identity.

[0091] An "effective amount" of an agent is that amount necessary to effect a physiological change in the cell or tissue to which it is administered.

[0092] A "therapeutically effective amount" of an agent, such as a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or preventive result. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents the adverse effects of a disease.

[0093] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the individual or subject is a human.

[0094] The term "pharmaceutical composition" refers to a preparation that is in such form that the biological activity of the active ingredient contained therein is effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0095] "Pharmaceutically acceptable carrier" refers to a component other than the active ingredient in a pharmaceutical composition that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0096] The term "treatment / prevention" (and grammatical variations thereof) refers to an attempt to alter the natural course of a disease in a treated individual and can be a clinical intervention performed for prevention or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or alleviating the disease state, and eliminating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the development of the disease or slow the progression of the condition.

[0097] In the present invention, the tumor is a tumor expressing CD79b.

[0098] Antibody

[0099] The present invention provides an anti-CD79b antibody having the aforementioned CDR region.

[0100] The antigen-binding properties of an antibody's heavy and light chains are typically determined by three complementarity-determining regions (CDRs) located in each of the heavy and light chain variable regions. These CDRs are arranged in a sequence with the framework regions (FRs), which do not directly participate in the binding reaction. These CDRs form a looped structure, spatially close to each other through the β-sheets formed by the FRs, forming the antibody's antigen-binding site. The CDR regions represent sequences of proteins of immunological interest, and the antibodies of the present invention include sequence modifications targeting both the CDR and framework regions.

[0101] In more specific embodiments, the present invention also specifies the heavy chain variable region and light chain variable region. The present invention also includes antibodies whose heavy chain variable region and / or light chain variable region have a sequence identity of 85% or more (e.g., 88%, 90%, 93%, 95%, 97% or 99% or more) with the specified sequence, and which have the same functions as the antibodies described in the embodiments of the present invention. Preferably, the antibodies of the present invention are humanized antibodies.

[0102] The anti-CD79b antibody provided by the present invention can efficiently and specifically bind to human CD79b, can efficiently block the effects of CD79b, and can efficiently carry another substance coupled thereto to target cells.

[0103] The present invention also includes an anti-CD79b antibody fusion protein comprising a first domain of an antibody as described herein and a second domain for extending in vivo half-life and / or binding to effector cells. The fusion protein can be a binding molecule that specifically binds to cells expressing CD79b.

[0104] In the second domain, the fragment for extending the half-life in vivo may include serum albumin or a fragment thereof, polyethylene glycol, a domain that binds serum albumin (such as an antibody against serum albumin), a polyethylene glycol-liposome complex, etc. In the second domain, the fragment that has a binding effect on effector cells may include an immunoglobulin Fc region, etc., preferably selected from the human immunoglobulin Fc region. The human immunoglobulin Fc region includes mutations for changing the effector function mediated by Fc, and the effector function includes a combination of one or more of CDC activity, ADCC activity, and ADCP activity. The immunoglobulin may be selected from a combination of one or more of IgG, IgA1, IgA2, IgD, IgE, IgM, etc., and the IgG may be specifically selected from a combination of one or more of IgG1, IgG2, IgG3 or IgG4 subtypes, etc. The immunoglobulin Fc region contained in the antibody fusion protein can cause the fusion protein to form a dimer, while extending the in vivo half-life of the fusion protein and increasing Fc-mediated related activities. In one embodiment of the present invention, the immunoglobulin Fc region can be the Fc region of human IgG1, more specifically, it can be a wild-type IgG1 Fc sequence, and the sequence can be introduced with mutations for altering Fc-mediated effector functions, for example, a) mutations that alter Fc-mediated CDC activity; b) mutations that alter Fc-mediated ADCC activity; or c) mutations that alter Fc-mediated ADCP activity.

[0105] In the anti-CD79b antibody fusion protein provided by the present invention, a connecting peptide may be provided between the first domain and the second domain. The connecting peptide may be a flexible polypeptide chain composed of alanine (A) and / or serine (S) and / or glycine (G). The length of the connecting peptide may be 3 to 30 amino acids, preferably 3 to 9, 9 to 12, 12 to 16, or 16 to 20 amino acids. In another specific embodiment of the present invention, the length of the connecting peptide may be 8 or 15.

[0106] The present invention also provides an isolated polynucleotide encoding the antibody of the present invention or the fusion protein. The polynucleotide may be RNA, DNA, or cDNA. Methods for providing the isolated polynucleotide should be known to those skilled in the art. For example, the isolated polynucleotide may be prepared by automated DNA synthesis and / or recombinant DNA technology, or may be isolated from a suitable natural source.

[0107] Constructs and antibody expression systems

[0108] The present invention also provides a construct comprising the isolated polynucleotide of the present invention. Methods for constructing the construct should be known to those skilled in the art. For example, the construct can be constructed by in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, or the like. More specifically, the construct can be constructed by inserting the isolated polynucleotide into the multiple cloning site of an expression vector. The expression vector herein generally refers to various commercially available expression vectors well known in the art, such as bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors. The vector may also include one or more regulatory sequences operably linked to the polynucleotide sequence, and the regulatory sequences may include a suitable promoter sequence. The promoter sequence is typically operably linked to the coding sequence for the amino acid sequence to be expressed. The promoter may be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides homologous or heterologous to the host cell. The regulatory sequences may also include a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is linked to the 3' end of the nucleotide sequence encoding the polypeptide, and any terminator that is functional in the host cell of choice can be used in the present invention.

[0109] Generally speaking, suitable vectors can contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers. For example, these promoters can include, but are not limited to, the lac or trp promoters of Escherichia coli; the lambda phage PL promoter; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the methanol oxidase promoter of Pichia pastoris, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or viruses. Marker genes can be used to provide phenotypic traits for selection of transformed host cells, and can include, for example, dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E. coli. When the polynucleotide is expressed, the expression vector can also include an enhancer sequence. Insertion of an enhancer sequence in the vector enhances transcription. Enhancers are cis-acting DNA elements, typically about 10 to 300 base pairs, that act on the promoter to enhance gene transcription.

[0110] The present invention also provides an antibody expression system, comprising a construct of the present invention or an exogenous polynucleotide of the present invention integrated into its genome. Any cell suitable for expression by an expression vector can be used as a host cell. For example, the host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell, specifically including but not limited to Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells, such as yeast, filamentous fungi, plant cells; insect cells of Drosophila S2 or Sf9; or animal cells such as CHO, COS, HEK293 cells, or Bowes melanoma cells. Methods for constructing the expression system should be known to those skilled in the art, and for example, can include but are not limited to microinjection, gene guns, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation, or a combination of one or more of the following.

[0111] Immunoconjugates

[0112] The present invention also provides an immunoconjugate comprising the antibody (including antigen-binding portion / fragment thereof) or the fusion protein described herein. The immunoconjugate generally further comprises a functional molecule linked (including but not limited to covalently linked, coupled, attached, or adsorbed) to the antibody or fusion protein. The functional molecule may include but is not limited to a detectable marker, a cytotoxin, a radioisotope, a biologically active protein, a molecule targeting a tumor surface marker, a molecule that inhibits tumors, a molecule targeting a surface marker of an immune cell, or a combination thereof.

[0113] The method for preparing the immunoconjugate should be known to those skilled in the art. For example, the antibody and / or fusion protein can be linked to the functional molecule directly or through a spacer of appropriate length. The connection method can be chemical cross-linking or genetic engineering fusion expression to obtain the immunoconjugate.

[0114] For therapeutic purposes, it may be appropriate to combine with therapeutic effector groups such as radioactive groups, i.e., groups composed of radioisotopes or radionuclides (e.g. 3 H. 14 C. 15 N. 33 P. 35 S. 90 Y. 99 Tc, 111 ln、 123 1. 125 1. 131 1. 201 TI, 213Bi), a toxin or a cytotoxic group such as a cytostatic group or a group consisting of or including the same.

[0115] The immunoconjugate may comprise an antibody or fusion protein of the present invention and a detectable label. Detectable labels include, but are not limited to, fluorescent labels, chromogenic labels, and protein tags; such as enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals, and non-radioactive paramagnetic metal ions. More than one label may also be included. The label used to label the antibody for detection and / or analysis and / or diagnostic purposes depends on the specific detection / analysis / diagnostic technique and / or method used, such as immunohistochemical staining of (tissue) samples, flow cytometry, etc. Suitable labels for detection / analysis / diagnostic techniques and / or methods known in the art are well known to those skilled in the art.

[0116] The antibody of the present invention or fusion protein can be coupled with a labeling group (labeled polypeptide), which can then be used, for example, for diagnostic purposes. Suitable labeling groups can be selected from radioisotopes (such as those mentioned above) or groups containing radioisotopes, radionuclides, fluorescent groups (such as fluorescent proteins such as GFP, RFP, etc., dyes, rhodamine, fluorescein and derivatives thereof such as FITC, cyanine dyes), enzyme groups (such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase), chemiluminescent groups, biotin groups, metal particles (such as gold particles), magnetic particles (such as having a core containing magnetite (Fe3O4) and / or maghemite (Fe2O3)), predetermined polypeptide groups, etc.

[0117] The immunoconjugate may comprise an antibody or fusion protein of the present invention and a molecule that targets a surface marker of an immune cell. The molecule that targets a surface marker of an immune cell can recognize immune cells and carry the antibody of the present invention to the immune cells. The antibody of the present invention can simultaneously target the immune cells to tumor cells, thereby utilizing the killing effect of the antibody of the present invention while also inducing the immune cells to specifically kill the tumor.

[0118] The immunoconjugate may comprise an antibody or fusion protein of the present invention and at least one molecule that targets a tumor surface marker or a tumor-suppressing molecule. The tumor-suppressing molecule may be an anti-tumor cytokine or an anti-tumor toxin. For example, the cytokine may include, but is not limited to, IL-2, IL-12, IL-15, IFN-beta, TNF-alpha, etc. The molecule that targets a tumor surface marker may, for example, act synergistically with the antibody of the present invention to more precisely target tumor cells.

[0119] The immunoconjugate can also be a chimeric antigen receptor (CAR), which can be expressed in immune cells. The “immune cells” and “immune effector cells” can be used interchangeably, including: T lymphocytes, NK cells or NKT cells, etc., preferably, NK cells and T lymphocytes. The chimeric antigen receptor generally comprises an extracellular binding region, a transmembrane region and an intracellular signaling region connected in sequence, wherein the extracellular binding region comprises the antibody or fusion protein of the present invention. The design of the transmembrane region and the intracellular signaling region based on CAR technology is well known in the art: for example, the transmembrane region adopts the transmembrane region of molecules such as CD8 and CD28, and the intracellular signaling region adopts the intracellular signaling region of the immunoreceptor tyrosine activation motif (ITAM) CD3ζ chain or FcεRIγ tyrosine activation continuation and co-stimulatory signal molecules CD28, CD27, CD137, CD134, MyD88, CD40, etc. More specifically, for T lymphocytes, the first generation of CAR T lymphocytes contains only ITAM in the intracellular signaling region, in which the various parts of the chimeric antigen receptor are connected in the following form: scFv-TM-ITAM, which can stimulate anti-tumor cytotoxic effects; the second generation of CAR T lymphocytes adds the intracellular signaling region of CD28 or CD137 (also known as 4-1BB), in which the various parts of the chimeric antigen receptor are connected in the following form: scFv-TM-CD28-ITAM or scFv-TM- / CD137-ITAM; the B7 / CD28 or 4-1BBL / CD137 co-stimulation effect in the intracellular signaling region causes the continuous proliferation of T lymphocytes, and can increase the level of cytokines such as IL-2 and IFN-γ secreted by T lymphocytes, while improving the survival cycle and anti-tumor effect of CAR T in the body; the third generation of CAR T lymphocytes, in which the various parts of the chimeric antigen receptor are connected in the following form: scFv-TM-CD28-CD137-ITAM or scFv-TM-CD28-CD134-ITAM, further improving the survival cycle of CAR T in the body and its anti-tumor effect.

[0120] According to the above, the chimeric antigen receptor prepared using the antibody or fusion protein of the present invention can be a chimeric antigen receptor comprising the following sequentially connected extracellular binding regions, transmembrane regions and intracellular signaling regions: the antibody or fusion protein of the present invention, CD8 and CD3ζ; the antibody or fusion protein of the present invention, CD8, CD137 and CD3ζ; the antibody or fusion protein of the present invention, the transmembrane region (CD28a) of the CD28 molecule, the intracellular signaling region (CD28b) of the CD28 molecule and CD3ζ; or the antibody or fusion protein of the present invention, the transmembrane region of the CD28 molecule, the intracellular signaling region of the CD28 molecule, CD137 and CD3ζ.

[0121] The chimeric antigen receptor is expressed on the surface of immune effector cells, and while utilizing the killing effect of the antibody of the present invention, the immune effector cells can also have a highly specific cytotoxic effect on tumor cells expressing CD79b.

[0122] As a preferred embodiment of the present invention, the antibodies of the present invention can be linked to tumor-suppressing molecules. Preferably, the tumor-suppressing molecules are anti-tumor toxins, including toxins that act on tubulin, such as monomethyl auristatin-related compounds and their derivatives, maytansinoid-related compounds and their derivatives; toxins that act on DNA, such as duocarmycin, calicheamicin, pyrrolobenzodiazepines (PBDs), SN-38, Dxd and other related compounds and their derivatives; and related compounds and their derivatives that act on other intracellular functions, such as metabolism, transcription, translation, and signal transduction. The present invention also includes analogs, isomers, and precursors of these small molecule toxins.

[0123] Pharmaceutical compositions and kits

[0124] The present invention also provides a pharmaceutical composition comprising the anti-CD79b antibody of the present invention, or the fusion protein of the anti-CD79b antibody of the present invention, or the immunoconjugate of the present invention.

[0125] The pharmaceutical composition may also include various pharmaceutically acceptable carriers in the art. Pharmaceutically acceptable carriers are non-toxic to the recipient at the dosage and concentration used, and may include, but are not limited to: buffers such as acetate, Tris, phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parahydroxybenzoates such as methyl parahydroxybenzoate or propyl parahydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); proteins such as blood albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; tonicity modifiers such as trehalose and sodium chloride; sugars such as sucrose, mannitol, trehalose or sorbitol; surfactants such as polysorbate; salt-forming counterions such as sodium; metal complexes (such as Zn-protein complexes); and / or nonionic surfactants such as Or polyethylene glycol (PEG). Pharmaceutical preparations for in vivo administration are generally sterile. Methods for achieving sterility of pharmaceutical preparations should be known to those skilled in the art. For example, it can be achieved by filtration through a sterile filtration membrane. Those skilled in the art can also select a suitable pharmaceutically acceptable carrier according to the desired dosage form of the pharmaceutical composition to prepare it into different dosage forms. For example, the pharmaceutical composition of the present invention can be in various dosage forms including but not limited to tablets, injections, lyophilized agents, etc.

[0126] In the pharmaceutical composition, the content of the fusion protein and the immunoconjugate is generally an effective amount, and the content of the active ingredient corresponding to the effective amount can be determined according to the subject to be treated and the specific administration method. For example, based on the total mass of the pharmaceutical composition, the content of the fusion protein and the immunoconjugate can range from about 0.01 to 99%, 0.1 to 70%, 1 to 30%, 0.01 to 0.05%, 0.05 to 0.1%, 0.1 to 0.3%, 0.3 to 0.5%, 0.5 to 1%, 1 to 3%, 3 to 5%, 5 to 10%, 10 to 20%, 20 to 30%, 30 to 50%, 50 to 70%, or 70 to 99%.

[0127] The fusion proteins, immunoconjugates, and pharmaceutical compositions of the present invention can be administered as a single active ingredient or in combination therapy, i.e., in combination with other agents. For example, the combination therapy may include the fusion protein, immunoconjugate, or pharmaceutical composition in combination with at least one other anti-tumor drug. As another example, the combination therapy may include the fusion protein, immunoconjugate, or pharmaceutical composition in combination with antibodies targeting other tumor-specific antigens.

[0128] The present invention also provides a detection kit comprising the antibody, fusion protein, or immunoconjugate described herein. The kit may further include, as needed, a container, controls (negative or positive controls), a buffer, adjuvants, etc., which can be selected by those skilled in the art based on specific circumstances. The kit may also include instructions for use to facilitate operation by those skilled in the art.

[0129] The present invention further provides a method for detecting CD79b protein using the antibody, including but not limited to qualitative detection, quantitative detection and localization detection. Specifically, the detection method includes but is not limited to immunofluorescence assay, immunohistochemistry and radioimmunoassay.

[0130] A method for detecting the presence of CD79b protein in a sample may include: contacting the sample with an antibody of the present invention; observing whether an antibody complex is formed; the formation of an antibody complex indicates the presence of CD79b protein in the sample. The sample may be a cell and / or tissue sample; the sample may be fixed or dissolved in a medium; and the level of CD79b protein in the fixed or dissolved sample is detected. In some embodiments, the subject of detection may be a cell-containing sample present in a cell preservation solution. In other embodiments, the antibody is further conjugated to a fluorescent dye, chemical substance, peptide, enzyme, isotope, label, etc. that can be used for detection or can be detected by other reagents.

[0131] use

[0132] The present invention also provides use of the antibody, fusion protein, immunoconjugate or pharmaceutical composition of the present invention in the preparation of a pharmaceutical composition for diagnosing, treating or preventing a disease associated with cells expressing (or overexpressing) CD79b.

[0133] In some embodiments, the tumor comprises: a non-solid tumor or a solid tumor; provided that the tumor expresses CD79b.

[0134] In some more specific embodiments, the tumor includes (but is not limited to) in situ or metastatic: hematological tumors such as lymphoma, leukemia, multiple myeloma; digestive system tumors such as esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, bile duct and gallbladder cancer; respiratory system tumors such as lung cancer, pleural tumors; nervous system tumors such as glioma, neuroblastoma, meningioma; head and neck tumors such as oral cancer, tongue cancer, laryngeal cancer, nasopharyngeal cancer; gynecological and reproductive system tumors such as breast cancer, ovarian cancer, cervical cancer, vulvar cancer, testicular cancer, prostate cancer, penile cancer; urinary system tumors such as kidney cancer, bladder cancer, skin and other systems such as skin cancer, melanoma, osteosarcoma, liposarcoma, thyroid cancer; provided that the tumor expresses CD79b.

[0135] In a specific embodiment, the tumor is a B-cell lymphoma. In the B-cell lymphoma, more than 85% (more preferably more than 90%) of the tumor cells express CD79b, so that the antibodies of the present invention can be targeted and enriched in such tumor cells and exert a killing effect.

[0136] In this field, the antibody polatuzumab has been used. The research results of the present inventors show that some antibodies of the present invention have a higher maximum binding capacity for tumor cells expressing CD79b (such as Daudi and Ramos). After coupling with small molecules, more drugs bind to Daudi cells, which are tumor cells with low CD79b expression, resulting in a better cell killing effect.

[0137] After being coupled to MMAE (such as A8-3m18-MMAE), the antibody of the present invention exhibits a better killing effect on tumor cells (such as Daudi) with relatively low CD79b expression, and may have better efficacy in vivo.

[0138] The antibody-conjugated DXd of the present invention (such as forming a low-dose A8-3m26-DXd) exhibits significant tumor inhibitory effects in tumor-bearing animals (such as Ramos tumor-bearing mice). It is foreseeable that the use of this conjugated preparation to treat patients who have developed resistance to polatuzumab-MMAE has great potential.

[0139] The "therapeutically effective amount" of the fusion protein, immunoconjugate, or pharmaceutical composition provided by the present invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or prevents damage or disability caused by the pain of the disease. For example, for the treatment of CD79b-related tumors (including lymphomas, etc.), a "therapeutically effective amount" preferably inhibits cell growth or tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, and more preferably at least about 80%, relative to an untreated subject. The ability to inhibit tumor growth can be evaluated in an animal model system that predicts efficacy against human tumors. Alternatively, it can be evaluated by examining the ability to inhibit cell growth, which inhibition can be determined in vitro by tests known to those skilled in the art. A therapeutically effective amount of the fusion protein, immunoconjugate, or pharmaceutical composition is generally capable of reducing tumor size or otherwise alleviating symptoms in the subject. Those skilled in the art can select an appropriate therapeutically effective amount based on actual conditions, for example, the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration selected. The prescription of treatment (e.g., determination of dosage, etc.) can be determined by a physician, typically taking into account factors including, but not limited to, the disease being treated, the individual patient's condition, the delivery site, the method of administration, and other factors. A prophylactically effective amount refers to an amount effective to achieve the desired prophylactic effect at the necessary dosage and time. Typically, but not necessarily, a prophylactic dose is administered to a subject before the onset of the disease or in the early stages of the disease, so a "prophylactically effective amount" is generally lower than a "therapeutically effective amount."

[0140] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Experimental procedures in the following examples, where specific conditions are not specified, were generally performed under conventional conditions, such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, or according to the conditions recommended by the manufacturer.

[0141] Example 1: Screening for high-affinity antibodies

[0142] Human CD79b fusion protein with a mouse Fc tag was used as an immunogen to immunize 10 SJL mice by intraperitoneal injection to stimulate the production of anti-human CD79b antibodies in the mice.

[0143] The two mice with the highest serum titer were selected, and the spleen lymphocytes of the mice were fused with myeloma cells SP2 / 0 using the electrofusion method to obtain hybridoma cells that can secrete specific anti-human CD79b antibodies and can proliferate indefinitely.

[0144] Hybridoma subclones were screened using ELISA and FACS. The ELISA assay was performed as follows: human CD79b-His protein was first adsorbed onto an ELISA plate, hybridoma cell culture supernatant was added, incubated at room temperature for 1 hour, and the plate was washed. HRP-conjugated rabbit anti-mouse full-length secondary antibody (Thermo Fisher) was then added, incubated at room temperature for 1 hour, and the plate was washed. Finally, TMB was used for color development, and the absorbance at 450 nm was recorded using a microplate reader. The FACS assay was performed as follows: Daudi cells (expressing CD79b antigen) were harvested by centrifugation, resuspended in PBS, counted, added to a 96-well plate, centrifuged, and the supernatant removed. Hybridoma cell culture supernatant was then added, incubated at room temperature for 30 minutes, and the cells were washed twice. PE-conjugated goat anti-mouse full-length secondary antibody (Jackson) was added, incubated in the dark for 30 minutes, and the cells were washed once. The cells were resuspended in PBS and analyzed by flow cytometry.

[0145] After repeated experiments and comparisons, two high-affinity antibodies were finally screened and obtained, which were produced by subclones 99H10D7 and 101C5E9, respectively. The results of ELISA and FACS tests are shown in Table 1.

[0146] Table 1

[0147] The results show that the monoclonal antibodies produced by subclones 99H10D7 and 101C5E9 can not only bind to human CD79b antigen at the protein level, but also bind to Daudi cells expressing CD79b antigen at the cellular level.

[0148] Example 2: Sequence analysis of antibodies

[0149] mRNA was extracted from the two hybridoma monoclonal cells obtained in Example 1 (Omega) and reverse-transcribed into cDNA (TAKARA). The heavy and light chain variable regions of the antibody genes were amplified from the reverse-transcribed cDNA using PCR, ligated into the pMDTM18-T vector (TAKARA), and transformed into competent DH5α cells. The heavy and light chain variable regions of the monoclonal antibodies were sequenced. Tables 2 and 3 respectively show the amino acid sequences of the variable regions of the mouse monoclonal antibody anti-human CD79b and the CDRs classified according to IMGT.

[0150] Table 2

[0151] Table 3

[0152] Sequence analysis revealed that the amino acid sequence of the light chain variable region of 99H10D7 was identical to that of 101C5E9; the amino acid sequence of the heavy chain variable region was different, and the amino acid sequence of the HCDR2 region was different.

[0153] Example 3. Preparation of chimeric antibodies

[0154] By using PrimeSTAR@Max DNA Polymerase (TAKARA), the antibody heavy chain and light chain variable region nucleic acid genes obtained in Example 2, the nucleic acid genes encoding the human antibody IgG1 heavy chain constant region (GenBank accession number of its amino acid sequence: AXN93653.1) and the human kappa light chain constant region (GenBank accession number of its amino acid sequence: AWK57456.1) were PCR cloned and combined into full-length genes of the heavy chain and light chain.

[0155] The target gene was isolated by agarose gel electrophoresis and purified using a gel extraction kit (Omega). Finally, the target gene was ligated into the expression vector using the ClonExpress Ultra One Step Cloning Kit (Vazyme). After sequencing verification of the expression vector, the chimeric antibody was transfected into ExpiCHO cells for expression and purified using Protein A.

[0156] The present inventors named the chimeric antibodies mC1 (corresponding to 99H10D7) and mC3 (corresponding to 101C5E9), respectively.

[0157] Example 4. Activity determination of chimeric antibodies mC1 and mC3

[0158] First, the binding ability of chimeric antibodies mC1 and mC3 to the extracellular domain of human and monkey CD79b proteins was tested using ELISA. Human CD79b-ECD-His and monkey CD79b-ECD-His (Sino Bio) proteins were adsorbed onto ELISA plates, respectively. 5% skim milk powder was added and incubated at room temperature for 1 hour, followed by washing. Serially diluted antibodies were added, incubated at room temperature for 1 hour, and then washed. HRP-labeled goat anti-human Fc secondary antibody (Jackson) was then added, incubated at room temperature for 1 hour, and then washed. Finally, TMB was used for color development, and absorbance at 450 nm was recorded using a microplate reader. As shown in Figure 1A, chimeric antibodies mC1 and mC3 bound to human CD79b with similar affinities. As shown in Figure 1B, mC3 bound to monkey CD79b with higher affinity than mC1.

[0159] Subsequently, FACS technology was used to detect the binding ability of chimeric antibodies mC1 and mC3 to human CD79b at the cellular level. Tumor cells expressing human CD79b were centrifuged in Daudi, resuspended in PBS, and counted. The cells were added to a 96-well plate and centrifuged to remove the supernatant. Serially diluted antibodies were added, incubated at 4°C for 30 minutes, and then the cells were washed twice. A secondary antibody with a PE-labeled goat anti-human IgG Fc tag (Jackson) was added. After incubation at 4°C in the dark for 30 minutes, the cells were washed once, resuspended in PBS, and detected using a flow cytometer. The results are shown in Figure 1C. Both chimeric antibodies mC1 and mC3 can bind to the CD79b antigen on the cell surface with similar affinities.

[0160] Example 5. Preparation of chimeric antibody-drug conjugates and testing of their killing effects

[0161] Chimeric antibodies mC1 and mC3 were conjugated with the small molecule drug monomethyl auristatin (MMAE) to prepare antibody-drug conjugates (ADCs) to test their cytotoxicity against Daudi tumor cells expressing human CD79b. The chimeric antibodies were first incubated with TCEP (Sigma) to break the interchain disulfide bonds, followed by conjugation with mc-vc-PAB-MMAE (medchemexpress). Finally, the conjugates were purified using a desalting column to remove excess small molecule drug. The resulting ADCs were designated mC1-MMAE and mC3-MMAE, respectively. IgG-MMAE was used as a negative control. Hydrophobic interaction chromatography analysis revealed a ratio of 1:3.7 for mC1 and 1:4.9 for mC3, while the negative control used a ratio of 1:4.0 for IgG and MMAE.

[0162] Daudi tumor cells expressing human CD79b were seeded at 5,000 cells per well in a 96-well plate. Antibody-drug conjugates mC1-MMAE and mC3-MMAE, as well as a negative control IgG-MMAE, were serially diluted and added to the 96-well plate containing Daudi cells. The cells were cultured at 37°C for 5 days. Cell viability was measured using CellTiter-Glo (Promega), thereby assessing the cell-killing effect of the antibody-drug conjugate.

[0163] The results are shown in Figure 2. Both mC1-MMAE and mC3-MMAE had significant killing effects on Daudi cells expressing human CD79b, and the killing effect of mC3-MMAE was stronger than that of mC1-MMAE.

[0164] Example 6: Humanization of chimeric antibodies

[0165] The chimeric antibody mC3 was humanized. The variable regions of the light and heavy chain sequences of the mC3 antibody were analyzed using the IMGT database. The CDR1, CDR2, and CDR3 regions, which are crucial for antigen binding, are shown in Table 3. During humanization, the CDR regions were retained, while the murine FR regions were replaced with those that closely resembled the FR regions of the human antibody germline gene. The resulting humanized antibody, named hmC3-3, has variable regions for both its light and heavy chains shown in Table 4.

[0166] Table 4

[0167] The variable region amino acid sequence of the hmC3-3 antibody was converted to a nucleotide sequence and synthesized after codon optimization. The sequence was then ligated with the constant region genes of the human IgG1 heavy and light chains and constructed into expression vectors. Once sequencing results were confirmed, the plasmids were extracted and transfected into ExpiCHO cells for expression. The humanized antibody hmC3-3 was obtained by protein A purification.

[0168] Example 7: Analysis of affinity and binding capacity of humanized antibodies

[0169] The affinity of the humanized antibody hmC3-3 for cells expressing human CD79b was determined by FACS compared to the chimeric antibody mC3. The method was the same as in Example 4, except that polatuzumab was used as a positive control antibody, the sequence of which was derived from patent US8088378.

[0170] The results are shown in Figure 3. Compared to the chimeric antibody mC3, the humanized antibody hmC3-3 exhibited a 2.6-fold lower affinity for binding to CD79b at the cellular level. Although the EC50 values ​​of mC3 and hmC3-3 were lower than those of the control antibody polatuzumab, the maximum binding capacity of both mC3 and hmC3-3 on cells was greater than that of polatuzumab.

[0171] Example 8. Affinity optimization of humanized antibody hmC3-3

[0172] To improve the affinity of the humanized antibody hmC3-3, affinity maturation of hmC3-3 was performed using phage display technology, and the antibody was modified through two rounds of screening. First, point mutations were introduced into the CDR1 and CDR3 of the hmC3-3 light chain and the CDR3 of the heavy chain. A phage display library was then constructed, and anti-CD79b Fab antibodies were expressed on phage. High-affinity anti-CD79b Fab antibodies were selected using magnetic beads. A total of six molecules with improved affinity were screened: A1, A3, A5, A8, A9, and A18. The amino acid sequences of their light chain CDR1, CDR3, and heavy chain CDR3 are shown in Table 5.

[0173] The heavy and light chain variable region genes of these six Fab antibodies were further linked to the genes for the human IgG1 heavy chain constant region and the human kappa light chain constant region, respectively, to construct expression plasmids. These were expressed in ExpiCHO cells and purified using Protein A to obtain the corresponding antibodies. FACS was used to examine whether the affinity for human CD79b-expressing Ramos cells was enhanced, using the same method as in Example 4. The results, as shown in Figure 4A, show that antibodies A1, A5, A8, and A18 all showed improved affinity for human CD79b compared to hmC3-3. Based on the EC50, A8 showed the strongest binding affinity for human CD79b, approximately 7-fold higher than that of hmC3-3. The maximum binding capacity of hmC3-3 to CD79b on the cell membrane was higher than that of A8.

[0174] Based on the A8 molecule, a second round of screening and modification was carried out to obtain the A8-3 molecule with improved affinity. The amino acid sequences of its light chain CDR1, CDR3 and heavy chain CDR3 are shown in Table 5.

[0175] Table 5

[0176] The heavy chain and light chain variable region genes of the Fab antibody A8-3 were connected to the genes of the human antibody IgG1 heavy chain constant region and the human kappa light chain constant region, respectively, to construct an expression plasmid, which was expressed in ExpiCHO cells and purified with Protein A to obtain the A8-3 full-length antibody.

[0177] FACS was used to detect the enhanced affinity for Ramos cells expressing human CD79b, using the same method as in Example 4. As shown in Figure 4B, A8-3 bound to CD79b with an affinity similar to that of the chimeric antibody mC3, and the maximum binding capacity of A8-3 on cells was 56.3% higher than that of polatuzumab.

[0178] Example 9: Affinity testing of optimized antibodies

[0179] Octet was used to detect the affinity of antibodies A8 and A8-3 to human CD79b-ECD-His protein. The results are shown in Figure 5 and Table 6.

[0180] Table 6

[0181] Judging from the KD value, the affinity of the humanized antibody hmC3-3 to the human CD79b-ECD-His protein is about 6 times lower than that of the chimeric antibody mC3. However, after modification, the affinity of A8-3 to the human CD79b-ECD-His protein is very close to that of the chimeric antibody mC3, and is also better than polatuzumab.

[0182] A higher binding constant Ka value indicates that the antibody binds to the antigen faster. A8 can bind to the antigen faster than mC3. A8-3 is similar to the chimeric antibody mC3 and faster than hmC3-3.

[0183] Judging from the dissociation constant Kd value, mC3 and A8-3 are the best. After binding to the antigen, they are not easy to dissociate from the antigen, and the dissociation constant is about 6 times higher than that of polatuzumab.

[0184] Example 10. Thermal stability analysis of optimized antibodies

[0185] The thermal stability of A8-3 and mC3 was tested using dynamic light scattering (DLS). The DLS (Wyatt) instrument detects the light scattering of the antibody when heated from 25°C to 85°C, thereby obtaining the polymerization temperature T of the antibody. agg The results are shown in Table 7.

[0186] Table 7

[0187] According to Table 7, compared with mC3, the polymerization temperature T agg The value decreased by 6.3℃.

[0188] Example 11: Improvement of Thermal Stability of Optimized Antibodies

[0189] In order to improve the thermal stability of A8-3, the different amino acids between mC3 and A8-3 were further analyzed to find the factors that affect the polymerization temperature T agg Through analysis, mutation screening was performed on the variable region of A8-3 to obtain antibodies A8-3m18 and A8-3m26, whose amino acid sequences of the light and heavy chain variable regions are shown in Table 8. The antibodies were expressed using ExpiCHO and purified using Protein A to obtain A8-3m18 and A8-3m26 antibodies.

[0190] Table 8

[0191] Example 12: Dynamic Light Scattering Analysis of Thermal Stability

[0192] The thermal stability of A8-3m18 and A8-3m26 molecules was determined using dynamic light scattering (DLS), as described in Example 10. The results are shown in Table 9.

[0193] Table 9

[0194] Compared with A8-3, the polymerization temperature Tagg values ​​of A8-3m18 and A8-3m26 increased to 65.48℃ and 65.29℃, respectively.

[0195] Example 13: Affinity determination for human CD79b and monkey CD79b

[0196] The affinity changes of A8-3m18, A8-3m26, mC3, and polatuzumab for human CD79b and monkey CD79b were detected using ELISA technology, using the same method as in Example 4.

[0197] As shown in Figure 6A, A8-3m18, A8-3m26, and mC3 antibodies all showed similar binding abilities to human CD79b, but polatuzumab showed stronger binding to human CD79b than A8-3m18, A8-3m26, and mC3. As shown in Figure 6B, A8-3m18, A8-3m26, and mC3 antibodies all showed binding abilities to monkey CD79b, while polatuzumab showed no binding abilities to monkey CD79b.

[0198] Example 14: FACS method for detecting antibody affinity

[0199] The affinity of A8-3m18, A8-3m26, mC3 and polatuzumab to Ramos and Daudi cells expressing human CD79b on their cell surface was detected by FACS, using the same method as in Example 4. The results are shown in Figures 7A-B.

[0200] From the EC50 values, A8-3m18, A8-3m26 and mC3 have similar binding to CD79b on tumor cells Ramos and Daudi expressing human CD79b. 50 It is about 6 times lower than that of polatuzumab, but the maximum binding amount of A8-3m18 and A8-3m26 to CD79b on cells is 1.3 to 1.9 times stronger than that of polatuzumab.

[0201] Example 15: Detection of Antibody Affinity by Octet Method

[0202] Octet was used to detect the affinity of A8-3m18, A8-3m26, and polatuzumab to human CD79b-ECD-His protein. The results are shown in FIG8 and Table 10.

[0203] Table 10

[0204] Ka values ​​indicate that polatuzumab binds to CD79b 2-5 times faster than A8-3m18 and A8-3m26. Kd values ​​indicate that A8-3m18 and A8-3m26 bind to CD79b and then dissociate more slowly than polatuzumab. A comprehensive analysis of Kd values ​​indicates that A8-3m18 and A8-3m26 have significantly greater affinity for the human CD79b-ECD-His protein than polatuzumab.

[0205] Example 16. Antibody Binding Epitope Analysis (In-Tandem Method)

[0206] The binding of A8-3m18 and polatuzumab to human CD79b epitopes was analyzed using the In-Tandem assay using an Octet instrument. First, 10 μg / mL of mouse Fc-tagged human CD79b-ECD protein was bound to the AMC sensor. The first antibody was then added until the signal reached saturation. Finally, the second antibody was added to observe whether the signal increased. If the signal increased, the first and second antibodies bound to different epitopes on human CD79b. If the signal did not change, the first and second antibodies bound to the same epitope on human CD79b.

[0207] The results obtained by this method are shown in Figure 9. When the signal of the first antibody polatuzumab binding to CD79b-ECD-mFc reaches saturation, there is no signal increase when the second antibody A8-3m18 binds. Conversely, when the signal of the first antibody A8-3m18 binding to CD79b-ECD-mFc reaches saturation, there is no signal increase when the second antibody polatuzumab binds, indicating that A8-3m18 and polatuzumab compete for binding to CD79b, that is, A8-3m18 and polatuzumab have the same binding site on human CD79b.

[0208] Example 17. Binding epitope analysis (FACS method)

[0209] FACS was used to detect the binding epitopes of human CD79b on cells by A8-3m18 and the control antibody polatuzumab. Daudi cells expressing human CD79b were centrifuged, resuspended in PBS, counted, and placed in a 96-well plate. The supernatant was removed by centrifugation. Four groups were divided into the experiment. Groups 1 and 2 received 100 nM A8-3m18 and 100 nM polatuzumab, respectively. Groups 3 and 4 received PBS and were incubated at 4°C for 30 minutes. Then, groups 1 and 3 received 3 nM biotin-polatuzumab, and groups 2 and 4 received 3 nM biotin-A8-3m18. After incubation at 4°C for 30 minutes, the cells were washed twice, and a PE-conjugated Streptavidin secondary antibody (Biolegend) was added. After incubation at 4°C for 30 minutes, the cells were washed once, resuspended in PBS, and analyzed by flow cytometry.

[0210] The results are shown in FIG10A . When 100 nM A8-3m18 antibody binds to CD79 on the cell surface, the addition of 3 nM biotin-polatuzumab fails to bind to the cells, while the control group with only 3 nM biotin-polatuzumab can bind to the cells.

[0211] As shown in FIG10B , when polatuzumab first binds to CD79 on the cell surface, it cannot bind to CD79b on the cell surface when 3 nM biotin-A8-3m18 is added.

[0212] The experimental results in Figures 10A and 10B together indicate that A8-3m18 and the control antibody polatuzumab compete for binding to the same epitope of CD79b on the cell surface.

[0213] Example 18. Preparation of a conjugate drug of an antibody and a toxin (MMAE) and its killing effect test

[0214] The killing effect of the antibody A8-3m18, A8-3m26 and polatuzumab conjugate on Daudi and Ramos tumor cells expressing human CD79b was further tested. The method for conjugating the antibody and the small molecule drug MMAE is described in Example 5.

[0215] Hydrophobic interaction chromatography analysis showed that the ratios of antibody to small molecule MMAE for the four antibody-drug conjugates A8-3m18-MMAE, mC3-MMAE, polatuzumab-MMAE, and negative control IgG-MMAE in Figure 11A were 1:4.5, 1:3.6, 1:4.3, and 1:3.5, respectively. The ratios of antibody to small molecule MMAE for the three antibody-drug conjugates A8-3m26-MMAE, polatuzumab-MMAE, and negative control IgG-MMAE in Figure 11B were 1:4.1, 1:4.7, and 1:3.9, respectively.

[0216] The killing effects of these antibody-drug conjugates on Ramos cells expressing human CD79b were tested. The results are shown in Figures 11A and 11B. mC3, A8-3m18-MMAE, A8-3m26-MMAE, and polatuzumab-MMAE all had significant killing effects, and the killing effects were similar.

[0217] As shown in Figures 11C and 11D , the killing effects on Daudi cells with relatively low expression of human CD79b showed that mC3-MMAE, A8-3m18-MMAE, and A8-3m26-MMAE were better than polatuzumab-MMAE in killing Daudi cells.

[0218] From the EC50 point of view, mC3 and A8-3m18-MMAE have a 5-fold higher killing effect on Daudi cells than polatuzumab-MMAE; A8-3m26-MMAE has a 1-fold higher killing effect on Daudi cells than polatuzumab-MMAE.

[0219] Example 19: Antitumor Effects of Antibody-Toxin Conjugate Drugs in Animals

[0220] 1. Antitumor efficacy of A8-3m26-MMAE in Ramos tumor-bearing mice.

[0221] SCID female mice were selected and inoculated with Ramos cells subcutaneously in the axilla. Thirteen days later, they were randomly divided into four groups, with five mice in each group. The average group volume was 198 mm. 3Two groups served as blank controls, one group was injected with normal saline (PBS), and the other was injected with 6 mg / kg IgG-MMAE. The two experimental groups were injected with 6 mg / kg A8-3m26-MMAE and 6 mg / kg polatuzumab-MMAE, respectively. The mice were given a single dose, and the body weight and tumor volume of each group were measured. The antibody-to-small molecule ratios of the antibody-drug conjugates IgG-MMAE, A8-3m26-MMAE, and polatuzumab-MMAE were 1:3.9, 1:4.1, and 1:4.3, respectively.

[0222] The results in Figure 12A show that compared with PBS and IgG-MMAE, A8-3m26-MMAE has a significant inhibitory effect on the tumor growth of Ramos; and all tumors have regressed at about 17 days, indicating that the tumor growth inhibitory effect of A8-3m26-MMAE is very significant.

[0223] 2. Antitumor efficacy of A8-3m18-MMAE in Ramos tumor-bearing mice

[0224] SCID female mice were selected and inoculated with Ramos cells subcutaneously in the axilla. Twelve days later, they were randomly divided into seven groups, with five mice in each group. The average group volume was 211 mm. 3 Three groups served as blank controls, one group was injected with normal saline (PBS), and the other two groups were injected with 1 mg / kg IgG-MMAE and 3 mg / kg IgG-MMAE. Four experimental groups were injected with low-dose 1 mg / kg A8-3m18-MMAE and 1 mg / kg polatuzumab-MMAE, respectively, and the high-dose group was injected with 3 mg / kg A8-3m18-MMAE and 3 mg / kg polatuzumab-MMAE. The mice were given a single dose, and the body weight and tumor volume of each group were measured. The ratios of antibody to small molecule MMAE for the conjugated drugs A8-3m18-MMAE, polatuzumab-MMAE, and IgG-MMAE were 1:4.1, 1:3.9, and 1:3.9, respectively.

[0225] The results in Figure 12B show that compared with the PBS and IgG-MMAE control groups, A8-3m18-MMAE has a significant inhibitory effect on Ramos tumor growth. Regardless of whether the dosage is 1 mg / kg or 3 mg / kg, the inhibitory effect of A8-3m18-MMAE is very good. In addition, the tumors of two mice treated with 3 mg / kg A8-3m18-MMAE were completely eliminated, indicating that A8-3m18-MMAE has a very significant inhibitory effect on Ramos tumor growth.

[0226] Example 20. Preparation of a conjugate drug of an antibody and a small molecule drug DXd and testing of its killing effect

[0227] A8-3m26 was conjugated to the small molecule drug DXd to produce an antibody-drug conjugate. The specific steps involved first incubating A8-3m26 with TCEP (Sigma) to break the interchain disulfide bonds, then adding maleimide-GGFG-DXd (MedChemExpress) for conjugation. Finally, the conjugates were purified using a desalting column to remove excess small molecule drug. The resulting antibody-drug conjugates were named A8-3m26-DXd, and the negative control was IgG-DXd. Hydrophobic interaction chromatography analysis revealed that the antibody-to-small molecule ratio for both IgG-DXd and A8-3m26-DXd was 1:8.

[0228] The inventors investigated the anti-tumor efficacy of A8-3m26-DXd in Ramos tumor-bearing mice. SCID female mice were subcutaneously inoculated with Ramos cells in the axilla and randomly divided into four groups 13 days later, with 5 mice in each group. The average group volume was 198 mm 3 Three groups served as negative controls, one group was injected with normal saline (PBS), the other two groups were injected with 0.3 mg / kg IgG-DXd and 1.0 mg / kg IgG-DXd, respectively, and the two experimental groups were injected with 0.3 mg / kg A8-3m26-DXd and 1.0 mg / kg A8-3m26-DXd, respectively. The drug was administered once in total, and the body weight and tumor volume of each group of mice were measured.

[0229] The results in Figure 13 show that compared with PBS and IgG-DXd, administration of 1 mg / kg A8-3m26-DXd has a significant tumor inhibitory effect on Ramos tumor growth, indicating that A8-3m26-coupled small molecule DXd has a significant inhibitory effect on tumor growth.

[0230] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the scope of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be based on the appended claims. At the same time, all documents mentioned in this application are cited as references in this application, just as if each document was cited as a reference individually.

Claims

1. An anti-CD79b antibody or an antigen-binding fragment thereof, wherein the anti-CD79b antibody has a light chain variable region and a heavy chain variable region, wherein: The amino acid sequences of the heavy chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 7, SEQ ID NO: 10 and SEQ ID NO: 15, respectively; The amino acid sequences of the light chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 19, "LV", and SEQ ID NO: 5, respectively; The amino acid sequences of the heavy chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 7, SEQ ID NO: 10 and SEQ ID NO: 15, respectively; The amino acid sequences of the light chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 19, "LV", and SEQ ID NO: 20, respectively; The amino acid sequences of the heavy chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 7, SEQ ID NO: 10 and SEQ ID NO: 15, respectively; The amino acid sequences of the light chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 11, "LV", and SEQ ID NO: 14, respectively; The amino acid sequences of the heavy chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 7, SEQ ID NO: 10 and SEQ ID NO: 9, respectively; The amino acid sequences of the light chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 4, "LV", and SEQ ID NO: 18, respectively; The amino acid sequences of the heavy chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 7, SEQ ID NO: 10 and SEQ ID NO: 22, respectively; The amino acid sequences of the light chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 23, "LV", and SEQ ID NO: 24, respectively; The amino acid sequences of the heavy chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 7, SEQ ID NO: 10 and SEQ ID NO: 9, respectively; The amino acid sequences of the light chain variable region CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 4, "LV", and SEQ ID NO: 6, respectively; The amino acid sequences of the heavy chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively; The amino acid sequences of the light chain variable region CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 4, "LV" and SEQ ID NO: 6, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibodies or antigen-binding fragments thereof include Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv; or The antibodies include: humanized antibodies, chimeric antibodies or mouse antibodies.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody is a humanized antibody, including a heavy chain FR region and a light chain FR region; The heavy chain FR region includes heavy chain FR1, FR2, FR3, and FR4, including an amino acid sequence such as FR1 as shown at positions 1-25 in SEQ ID NO:28, SEQ ID NO:26, or SEQ ID NO:13, FR2 as shown at positions 34-50 in SEQ ID NO:13, FR3 as shown at positions 59-96 in SEQ ID NO:13, and FR4 as shown at positions 103-113 in SEQ ID NO:13; The light chain FR region includes light chain FR1, FR2, FR3, and FR4, including an amino acid sequence as shown in SEQ ID NO:

12. FR1 as shown at positions 1-26, FR2 as shown at positions 38-54 in SEQ ID NO: 12, SEQ ID NO: 27 or SEQ ID NO: 25, FR3 as shown at positions 57-93 in SEQ ID NO: 12, FR4 as shown at positions 103-113 in SEQ ID NO:

12.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein: The antibody is a humanized antibody selected from: An antibody having a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 28, or a heavy chain variable region with an amino acid sequence greater than 85% identical to that sequence, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 27, or a light chain variable region with an amino acid sequence greater than 85% identical to that sequence; or An antibody having a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 26, or a heavy chain variable region with an amino acid sequence of more than 85% identity to that sequence, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 25, or a light chain variable region with an amino acid sequence of more than 85% identity to that sequence; or An antibody having a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 13 or having 85% or more identity thereto, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 12 or having 85% or more identity thereto.

5. An isolated polynucleotide or a construct containing the polynucleotide, wherein the polynucleotide encodes the anti-CD79b antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. An expression system for an antibody or an antigen-binding fragment thereof, said expression system comprising the construct as claimed in claim 5 or the polynucleotide as claimed in claim 5 integrated exogenously in its genome.

7. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising: Under conditions suitable for expressing the antibody or its antigen-binding portion, the expression system of claim 6 is used for expression, thereby expressing the antibody or its antigen-binding fragment.

8. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 for preparing an anti-tumor drug that specifically targets and inhibits tumor cells expressing CD79b.

9. The use according to claim 8, characterized in that The anti-tumor drugs include: Immunoconjugates; Bifunctional or multifunctional antibodies; Chimeric antigen receptor-modified immune cells.

10. The use according to claim 8, characterized in that The tumors include: lymphoma, B lymphocytic leukemia.

11. A fusion protein or immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, and a functional molecule operably linked thereto.

12. The fusion protein or immunoconjugate according to claim 11, characterized in that The functional molecules include: tumor-suppressing molecules, molecules targeting tumor surface markers, molecules or detectable markers targeting surface markers of immune cells, cytotoxins, radioactive isotopes, fusion partners, extracellular hinge regions, transmembrane regions and intracellular signal regions of chimeric antigen receptors, or combinations thereof.

13. The fusion protein or immunoconjugate according to claim 12, characterized in that The tumor-suppressing molecule is an anti-tumor toxin or an anti-tumor cytokine; or The molecule targeting the tumor surface marker is an antibody or a ligand that binds to the tumor surface marker; or The molecule targeting the surface marker of an immune cell is an antibody or a ligand that binds to the surface marker of an immune cell; or The fusion partner includes: a protein or an active domain having the function of prolonging the half-life in vivo.

14. The fusion protein or immunoconjugate according to claim 13, characterized in that The anti-tumor toxins include: Maytansine, Aulistatin, Monomethyl Auristatin, Maytansinoid, Dolastatin, Calcheamicin, Methotrexate, Vindesine, Taxanes such as Docetaxel, Paclitaxel, Lalopaclitaxel, Tecitaxel or Otataxel, Trichothecenes, CC1065, DXd, Duocarmycin, Calicheaamicin, Pyrrolobenzodiazepines or SN-38; or The anti-tumor cytokines include: IL-2, IL-12, IL-15, IFN-beta, TNF-alpha; or The antibody binding to the tumor surface marker is an antibody that recognizes other antigens besides the immunomodulatory molecule B7-H3, and the other antigens include: EGFR, EGFRvIII, mesothelin, HER2, EphA2, cMet, EpCAM, MUC1, MUC16, CEA, Claudin 18.2, Claudin 6, WT1, NY-ESO-1, MAGE 3, CD47, ASGPR1 or CDH16; or The antigens bound by the antibody that binds to the immune cell surface marker include: CD3, CD20, FcRH5, HER2, LYPD1, LY6G6D, PMEL17, LY6E, CD19, CD33, CD22, CD79A, CD79b, EDAR, GFRA1, MRP4, RET, Steap1 or TenB2; or The protein or active domain having the function of prolonging the half-life in vivo includes: immunoglobulin Fc region, preferably human immunoglobulin Fc region, and serum albumin.

15. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or the fusion protein or immunoconjugate according to any one of claims 11 to 14.

16. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the fusion protein or immunoconjugate according to any one of claims 11 to 14, or a pharmaceutical composition containing them in the preparation of a preparation, a kit or a drug kit for an anti-tumor drug that specifically targets and inhibits tumor cells expressing CD79b.

17. A test kit or a medicine kit, comprising: Container or packaging; as well as, Placed in the container or packaging: The antibody according to any one of claims 1 to 4, the fusion protein or immunoconjugate according to any one of claims 11 to 14, or a pharmaceutical composition containing them.

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