RU2817143C2 - Anti-cd79b antibody, antigen-binding fragment thereof and pharmaceutical use thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to an anti-human CD79B antibody or an antigen-binding fragment thereof, a method for production thereof, as well as a composition and a conjugate containing same. Also disclosed is a polynucleotide coding an anti-human CD79B antibody, or an antigen-binding fragment thereof, as well as a vector and a host cell containing it.

EFFECT: invention is effective for treating cancer or tumour characterized by CD79B expression.

29 cl, 9 dwg, 4 tbl, 5 ex

Description

This application claims the benefit of the Chinese patent application “Anti-CD79B Antibody, Its Antigen Binding Fragment and Its Pharmaceutical Use” (application number 201910083330.4), filed on January 28, 2019, which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to an anti-human CD79B antibody and an antigen binding fragment thereof, a chimeric antibody and a humanized antibody containing the CDR region(s) of an anti-CD79B antibody, a pharmaceutical composition containing an anti-human CD79B antibody or an antigen binding fragment thereof, and its use as an anti-cancer drug, in particular as a drug for the treatment of lymphoma (DLBCL) (diffuse B-cell large lymphoma).

BACKGROUND ART

Malignancy (cancer) is the second leading cause of death in the world, ranking just behind heart disease. Lymphoma is a malignant tumor that arises from the lymphoid hematopoietic system and is the most common hematological tumor in the world. The incidence of lymphoma in China has been increasing in recent years, and the current incidence rate is approximately 6.68 cases per 100,000 people.

Lymphoma is divided into two types: non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma (HL). Non-Hodgkin's lymphoma is a general term for a group of diseases with abnormal proliferation of lymphocytes with strong heterogeneity. Its incidence is significantly higher than that of Hodgkin's lymphoma, accounting for more than 80% of lymphomas. Among them, diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma in adults, accounting for approximately 32.5% of all non-Hodgkin lymphomas; in the Asian population this proportion is even higher - close to 40%. It is more common in older patients with a median age of onset of 60-64 years. There are slightly more male patients than female patients.

The current standard first-line regimen for diffuse large B-cell lymphoma (DLBCL) is rituximab combined with chemotherapy (R-CHOP). Before the marketing of rituximab, the standard first-line treatment regimen against DLBCL was the anthracycline-based regimen CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone). The R-CHOP treatment regimen significantly improved long-term survival of patients with DLBCL. The results of the clinical trial show that, compared with the traditional CHOP regimen, the R-CHOP regimen can significantly prolong the median overall survival time of patients with DLBCL by 4.9 years, the median disease-free survival time by more than 6.6 years, and 5 The -year disease-free survival rate increased from 30% to 54%. However, there are still 10% to 15% of treatment-resistant patients who do not respond, and 20% to 30% of patients who relapse. And not all patients with DLBCL are suitable for the R-CHOP regimen, such as elderly patients over 80 years of age whose physical condition does not allow standard R-CHOP treatment; Another example is that of more aggressive types of lymphoma and recurrent lymphoma, and the R-CHOP regimen may not be appropriate. Therefore, the development of a new generation of immunotherapies with fewer side effects for the treatment of DLBCL is urgently needed.

According to the classification of lymphocytes by origin, diffuse large B-cell lymphoma (DLBCL) belongs to B-cell lymphoma. The B cell receptor (BCR) complex is the most important molecule on the surface of B cells. The BCR complex consists of membrane immunoglobulin (mIg), which recognizes and binds antigen, and Igα (CD79a) and Igβ (CD79B) heterodimers, which transmit antigen stimulation signals. Igα and Igβ are 47 kDa and 37 kDa glycoproteins, respectively, and they belong to the immunoglobulin superfamily. The genes encoding Igα and Igβ are called mb-1 and B29, respectively. Both Igα and Igβ have an Ig-like domain at the amino terminus of the extracellular region. Both Igα and Igβ can be used as protein tyrosine kinase substrates and are involved in BCR signal transduction. BCR is widely expressed on B-cell lymphomas and on normal B cells. Given the clinical success and robust safety of the CD20-targeted rituximab, the development of BCR-targeted therapeutics should also have good curation and safety.

In response to the unmet medical needs associated with CD79B, many international pharmaceutical companies, including Roche Pharmaceuticals, are actively developing anti-CD79B antibodies and related products. Related patents include US 9085630, WO 2009012256, WO 2009012268, WO 2009099728, WO 2014011519, WO 2014011521, WO 2016090210, WO 2016205176, WO 20 16040856, WO 2016021621, WO 2017009474, WO 2014177615, etc.

Based on the expression of CD79B, it is useful to generate therapeutic antibodies against the CD79B antigen. There is still an unmet need in the field to develop effective antibodies against human CD79B to treat hematopoietic tumors or delay progression.

SUMMARY OF THE INVENTION

The present disclosure provides an anti-CD79B antibody or an antigen-binding fragment thereof, a nucleic acid encoding the same, a vector, a host cell, an antibody-drug conjugate and a pharmaceutical composition thereof, and a method of using them for treating or delaying the development of cancer, especially hematopoietic tumors.

In a first aspect, the present disclosure provides an anti-human CD79B antibody or an antigen binding fragment thereof, which comprises an antibody heavy chain variable region and an antibody light chain variable region, wherein:

the variable region of the antibody heavy chain contains at least one complementarity determining region (HCDR), as shown in sequences selected from the following: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13 , SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25; and/or

the antibody light chain variable region contains at least one complementarity determining region (LCDR) as shown in sequences selected from the following: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 16 and SEQ ID NO: 26.

In some embodiments, an anti-human CD79B antibody or antigen binding fragment thereof is provided, wherein: the heavy chain variable region comprises:

(I) HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively; or

(II) HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively; or

(III) HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25, respectively;

and/or the light chain variable region contains:

(I) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; or

(II) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 16, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; or

(III) LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 26, SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

In some specific embodiments, an anti-human CD79B antibody or an antigen-binding fragment thereof is provided that contains any one selected from the following (l)-(lll):

(I) a heavy chain variable region containing HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively; And

a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively;

(II) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively; And

a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 16, SEQ ID NO: 11 and SEQ ID NO: 12, respectively;

(III) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25, respectively; And

a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 26, SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

In some specific embodiments, an anti-human CD79B antibody or antigen binding fragment thereof is provided, wherein:

the heavy chain variable region contains:

(I) a sequence as shown in SEQ ID NO: 3, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 3; or

(II) a sequence as shown in SEQ ID NO: 5, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 5; or

(III) a sequence as shown in SEQ ID NO: 17, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 17;

and/or the light chain variable region contains:

(I) a sequence as shown in SEQ ID NO: 4, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 4; or

(II) a sequence as shown in SEQ ID NO: 6, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 6; or

(III) a sequence as shown in SEQ ID NO: 18, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 18.

In some specific embodiments, the heavy chain variable region of an anti-human CD79B antibody or antigen binding fragment thereof is as shown in SEQ ID NO: 3, and the light chain variable region is as shown in SEQ ID NO: 4.

In some other specific embodiments, the heavy chain variable region of the anti-human CD79B antibody or antigen binding fragment is as shown in SEQ ID NO: 5, and the light chain variable region is as shown in SEQ ID NO: 6.

In some other specific embodiments, the heavy chain variable region of an anti-human CD79B antibody or antigen binding fragment is as shown in SEQ ID NO: 17, and the light chain variable region is as shown in SEQ ID NO: 18.

In some specific embodiments, an anti-human CD79B antibody or antigen binding fragment thereof is provided, wherein:

heavy chain contains:

(I) a sequence as shown in SEQ ID NO: 19, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 19; or

(II) a sequence as shown in SEQ ID NO: 21, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 21; or

and/or the light chain contains:

(I) a sequence as shown in SEQ ID NO: 20, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 20; or

(II) a sequence as shown in SEQ ID NO: 22, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 22;

In some specific embodiments, the heavy chain of the anti-human CD79B antibody or antigen binding fragment is as shown in SEQ ID NO: 19, and the light chain is as shown in SEQ ID NO: 20.

In some other specific embodiments, the heavy chain of the anti-human CD79B antibody or antigen binding fragment is as shown in SEQ ID NO: 21, and the light chain is as shown in SEQ ID NO: 22.

In some embodiments, an anti-human CD79B antibody or antigen-binding fragment thereof is provided, as described above, which is a murine antibody or fragment thereof.

In some specific embodiments, the light chain variable region of a mouse anti-CD79B antibody or antigen binding fragment thereof comprises a light chain FR region and/or a light chain constant region of a murine κ, λ, or variant thereof.

In some specific embodiments, a mouse anti-CD79B antibody or antigen binding fragment thereof comprises a heavy chain FR region and/or a heavy chain constant region of a mouse IgG1, IgG2, IgG3, IgG4, or variant thereof.

In some embodiments, an anti-human CD79B antibody or antigen-binding fragment thereof is provided, as described above, which is a chimeric antibody or fragment thereof. In some specific embodiments, the chimeric anti-CD79B antibody or antigen binding fragment thereof comprises a light chain FR region and/or a human κ, λ or variant light chain constant region and/or a heavy chain FR region and/or a human IgG1 heavy chain constant region, IgG2, IgG3, IgG4 or variant thereof.

In some embodiments, an anti-human CD79B antibody or antigen-binding fragment thereof is provided, as described above, which is a humanized antibody, a human antibody, or a fragment thereof.

In some embodiments, a humanized anti-human CD79B antibody or antigen binding fragment thereof is provided, as described above, wherein the light chain sequence is shown in SEQ ID NO: 20 or a sequence variant thereof; this sequence variant contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions in the light chain, or is at least 80%, 85%, 90%, 95 %, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 20. The heavy chain sequence is shown in SEQ ID NO: 19 or a sequence variant thereof; this sequence variant contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions in the heavy chain, or is at least 80%, 85%, 90%, 95 %, 96%, 97%, 98% or 99% identity to SEQ ID NO: 19.

In some embodiments, a humanized anti-human CD79B antibody or antigen binding fragment thereof is provided, as described above, wherein the light chain sequence is shown in SEQ ID NO: 22 or a sequence variant thereof; or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 22; this sequence variant contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions in the light chain. The heavy chain sequence is shown in SEQ ID NO: 21 or a sequence variant thereof; this sequence variant contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions in the heavy chain or is at least 80%, 85%, 90%, 95% -th, 96%, 97%, 98% or 99% identity with SEQ ID NO: 21.

In some embodiments, the human anti-CD79B antibody or fragment thereof, as described above, further comprises a human IgG1, IgG2, IgG3 or IgG4 constant region, or a variant thereof. In some specific embodiments, the human anti-CD79B antibody or fragment thereof comprises a human IgG1 or IgG2 constant region.

In some embodiments, the humanized anti-human CD79B antibody or fragment thereof, as described above, further comprises a heavy chain constant region of a human IgG1, IgG2, IgG3 or IgG4, or a variant thereof, preferably comprising a heavy chain FR region of a human IgG1, IgG2 or IgG4, more preferably contains the FR region of the heavy chain of human IgG1 or IgG2.

In some embodiments, an anti-human CD79B antibody or antigen binding fragment thereof is provided, as described above, wherein the antigen binding fragment is selected from the group consisting of Fab, Fv, sFv, F(ab') ₂ , linear antibody, single chain antibody, scFv, sdAb, sdFv, nanobody, peptibody, domain antibody and multispecific antibody (bispecific antibody, diabody, tribody and tetrabody, tandem di-scFv and tandem tri-scFv).

In some embodiments, an isolated monoclonal antibody or antigen binding fragment thereof is provided that can compete with the aforementioned monoclonal antibody or antigen binding fragment thereof for binding to human CD79B or an epitope thereof.

The amino acid residues of the CDR VH/VL of the anti-human CD79B antibody in the present disclosure are defined and annotated by the Chothia numbering system.

In a second aspect, the present disclosure provides a polynucleotide encoding an anti-human CD79B antibody or an antigen binding fragment thereof, as described above, which may be DNA or RNA.

In a third aspect, the present disclosure provides an expression vector comprising a polynucleotide as described above, which may be a eukaryotic expression vector, a prokaryotic expression vector, or a viral vector.

In a fourth aspect, the present disclosure provides a host cell transformed with an expression vector as described above, which may be a eukaryotic cell or a prokaryotic cell.

In some embodiments, the host cell is a bacterial cell, a yeast cell, or a mammalian cell. In some specific embodiments, the host cell is Escherichia coli, Pichia pastoris, a Chinese hamster ovary (CHO) cell, or a human embryonic kidney (HEK) 293 cell.

In a fifth aspect, the present disclosure provides an antibody-drug conjugate.

An antibody-drug conjugate according to the present disclosure contains or consists of the following: an antibody, a linker, and a drug.

In a specific embodiment, an antibody-drug conjugate according to the present disclosure is an antibody covalently linked to the drug through a linker.

In some embodiments, the antibody-drug conjugate contains a cytotoxic agent. In certain specific embodiments, the cytotoxic agent is selected from the group consisting of a toxin, a chemotherapeutic agent, an antibiotic, a radioactive isotope, and a nucleolytic enzyme.

In a sixth aspect, the present disclosure provides a method for producing an antibody against human CD79B or an antigen binding fragment thereof, comprising: expressing the antibody or antigen binding fragment thereof in a host cell as described above, and isolating the antibody or antigen binding fragment thereof from the host cell.

In a seventh aspect, the present disclosure provides a composition, for example a pharmaceutical composition, which contains a therapeutically effective amount of the above-mentioned anti-human CD79B antibody or antigen binding fragment thereof and a pharmaceutically acceptable excipient, diluent or carrier.

In some specific embodiments, a unit dose of the pharmaceutical composition contains from 0.01% to 99% by weight of an anti-human CD79B antibody or an antigen-binding fragment thereof, or the amount of an anti-CD79B antibody or an antigen-binding fragment thereof in a unit dose of the pharmaceutical composition is from 0.1 mg to 2000 mg, and, in some specific embodiments, from 1 mg to 1000 mg.

An eighth aspect according to the present disclosure further provides the use in the preparation of a drug of any one or combination selected from the following: an anti-human CD79B antibody or an antigen binding fragment thereof of the present disclosure, a pharmaceutical composition of the present disclosure, an antibody-drug conjugate of the present disclosure; wherein the drug or pharmaceutical composition is used to treat a proliferative disease or delay the progression of a proliferative disease; said proliferative disease may be a cancer or a tumor. In some embodiments, the cancer or tumor is a lymphoma or leukemia. In a specific embodiment, the lymphoma is selected from the group consisting of the following: diffuse large B-cell lymphoma, non-Hodgkin lymphoma, small cell lymphocytic lymphoma, and mantle cell lymphoma. In a particular embodiment, the non-Hodgkin's lymphoma is selected from the group consisting of the following: aggressive NHL, recurrent aggressive NHL, recurrent painless NHL, difficult-to-treat NHL, and difficult-to-treat painless NHL. In a specific embodiment, the leukemia is selected from the group consisting of the following: chronic lymphocytic leukemia, hairy cell leukemia, and acute lymphocytic leukemia.

In a ninth aspect, the present disclosure further provides a method of treating or preventing a proliferative disease or delaying the progression of a proliferative disease, which comprises administering to a subject a therapeutically effective amount or a disease-delaying effective amount of an anti-human CD79B antibody or an antigen binding fragment thereof of the present disclosure, or a pharmaceutical composition of the present disclosure. , or an antibody-drug conjugate of the present disclosure; wherein the proliferative disease may be a cancer or tumor. In some embodiments, the cancer or tumor is lymphoma or leukemia. In a specific embodiment, the lymphoma is selected from the group consisting of the following: diffuse large B-cell lymphoma, non-Hodgkin lymphoma, small cell lymphocytic lymphoma, and mantle cell lymphoma. In a particular embodiment, the non-Hodgkin's lymphoma is selected from the group consisting of the following: aggressive NHL, recurrent aggressive NHL, recurrent painless NHL, difficult-to-treat NHL, and difficult-to-treat painless NHL. In a specific embodiment, the leukemia is selected from the group consisting of the following: chronic lymphocytic leukemia, hairy cell leukemia, and acute lymphocytic leukemia.

DESCRIPTION OF GRAPHIC MATERIALS

Fig. 1: ELISA (enzyme-linked immunosorbent assay) results of serum titer of Balb/c mice immunized with human CD79B-hFc ECD (extracellular domain) protein.

Fig. 2: FACS (fluorescence assisted cell sorting) detection of serum titer of Balb/c mice immunized with human CD79B-hFc ECD protein.

Fig. 3: ELISA results of serum titer of SJL mice immunized with human CD79B-hFc ECD protein.

Fig. 4: FACS results of serum titer detection of SJL mice immunized with human CD79B-hFc ECD protein.

Fig. 5: ELISA results of serum titer of SJL mice immunized with human CD79B-his ECD protein.

Fig. 6: FACS results of serum titer detection of SJL mice immunized with human CD79B-his ECD protein.

Fig. 7: ELISA results of mouse monoclonal antibodies against human CD79B, where hIgG1 is a negative control antibody and SN8 is a positive control antibody.

Fig. 8: FACS detection results of mouse monoclonal antibodies against human CD79B, where hIgG1 is a negative control antibody and SN8 is a positive control antibody.

Fig. 9: FACS detection results for cross-reactivity of mouse monoclonal antibodies against human CD79B, where mIgG is a negative control antibody; anti-cyno HR008 is a mouse monoclonal antibody against cynomolgus CD79B, the antibody sequence of which is derived from the mouse monoclonal antibody against cynomolgus CD79B (clone number 10D10) in WO 2009012268 A1, is a positive control antibody.

DETAILED DESCRIPTION OF THE INVENTION

Terms

To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere herein, all other technical and scientific terms used herein have meanings commonly understood by those of ordinary skill in the art to which this disclosure pertains.

The three-letter codes and single-letter amino acid codes used in the present disclosure are as described in J. Biol. Chem, 243, p3558 (1968).

"CD79B" refers to any CD79B derived from any vertebrate, including a mammal such as a primate (eg, human and cynomolgus monkey) and rodent (eg, mouse or rat). The term “CD79B” includes “full-length,” unprocessed CD79B, and any form of CD79B that has been processed from cells. The term also includes naturally occurring variants of CD79B, such as splice variants, allelic variants and isoforms. The CD79B polypeptides described herein can be isolated from a variety of sources, such as human tissue types or other sources, or produced by recombinant or synthetic methods.

The term “antibody” as described in the present disclosure refers to an immunoglobulin, which is a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bond(s). The amino acid composition and sequence of the immunoglobulin heavy chain constant region are different such that their antigenicity is also excellent. According to this, immunoglobulins can be classified into five types or isotypes of immunoglobulins, namely, IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain and ε chain, respectively. The same type of Ig can be divided into different subclasses according to differences in the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3 and IgG4. The light chain is divided into κ chain or λ chain by distinguishing the constant region. Each of the five Ig types can have a κ chain or a λ chain. The sequence of approximately 110 amino acids near the N-terminus of the heavy and light chains of an antibody varies widely and is called the variable region (region V); the remaining amino acid sequence near the C-terminus is relatively stable and is called the constant region (C region). The variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. The 3 hypervariable regions determine the specificity of an antibody and are also known as complementarity determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of 3 CDR regions and 4 FR regions. The sequence from amino end to carboxy end is: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The 3 light chain CDR regions are LCDR1, LCDR2 and LCDR3; The 3 heavy chain CDR regions are HCDR1, HCDR2 and HCDR3. The number and position of the amino acid residues of the CDR region VL and VH region of the antibody or antigen binding fragment corresponds to the known rules of Chothia numbering (ABM).

The term "human antibody" or "recombinant human antibody" includes human antibodies produced, expressed, created or isolated by recombinant methods, and the techniques and methods involved are well known in the art, such as:

(1) antibodies isolated from transgenic and transchromosomal animals (for example, mice) with human immunoglobulin genes or from hybridomas obtained from them;

(2) antibodies isolated from host cells (such as transfectionomes) transformed to express antibodies;

(3) antibodies isolated from a recombinant human antibody combinatorial library; And

(4) antibodies produced, expressed, created or isolated by other techniques that are used to splice human immunoglobulin gene sequences into other DNA sequences.

Such recombinant human antibodies contain variable regions and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations, such as those occurring during antibody maturation.

The term “mouse antibody” as used herein is a monoclonal antibody against human CD79B or an epitope thereof prepared according to knowledge and skill in the art. During production, a test subject is injected with CD79B antigen or an epitope thereof, and hybridomas expressing antibodies with the desired sequence or functional properties are then isolated. In a specific embodiment of the present disclosure, the mouse anti-CD79B antibody or antigen binding fragment thereof may further comprise a mouse κ, λ chain light chain constant region, or a variant thereof, or further comprise a mouse IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, or a variant thereof.

The term "human antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. Human antibodies of the present disclosure may include amino acid residues that are not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-directed mutagenesis in vitro or through somatic mutations in vivo). However, the term “human antibody” does not include antibodies in which CDR sequences derived from the germ line of another mammalian species (such as a mouse) have been grafted onto human scaffold sequences (namely, “humanized antibodies”).

The term “humanized antibody,” also known as a CDR-grafted antibody, refers to an antibody produced by grafting CDR sequences from a non-human species into the variable region framework of a human antibody. It can overcome the strong immune responses induced by the chimeric antibody because it carries a large number of protein components from non-human species. To avoid a decrease in activity caused by a decrease in immunogenicity, the variable region of a human antibody can be subjected to minimal back mutations to maintain activity.

A "chimeric antibody" is an antibody formed by fusing the variable region of an antibody of a first type with the constant region of an antibody of a second type, which can facilitate an immune response induced by the antibody of the first type. Creating a chimeric antibody requires creating a hybridoma secreting specific monoclonal antibodies of the first species, then cloning the variable region gene from cells of the hybridoma of the first species (such as a mouse), and then cloning the constant region gene of a second species of antibody (such as a human), linking the variable region gene of the first species with a second kind of constant region gene to form a chimeric gene, which is inserted into an expression vector, and finally expressing the chimeric antibody molecule in a eukaryotic industrial system or a prokaryotic industrial system. The constant region of the second type (e.g., human) antibody may be selected from the group consisting of: a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, or a variant thereof, preferably including a human IgG2 or IgG4 heavy chain constant region, or the use IgG1 without ADCC (antibody dependent cellular cytotoxicity) after amino acid mutations.

The term "antigen binding fragment" refers to any fragment that retains the antigen binding activity of an intact antibody. In particular, mention may be made of Fab fragments, Fab' fragments, F(ab') ₂ fragments and Fv fragments or sFv fragments that will bind to human CD79B. The Fv fragment contains the heavy chain variable region and the light chain variable region of the antibody, but has no constant region and has the smallest antibody fragment with all antigen binding sites. In general, an Fv antibody also contains a polypeptide linker between the VH and VL domains, and can form the structure required for antigen binding. Different linkers can also be used to link two antibody variable regions into a polypeptide chain called a single chain antibody or single chain Fv (sFv).

The term “CD79B binding” as used herein refers to the ability to interact with CD79B or an epitope thereof. CD79B or its epitope may be of human origin. The term "antigen binding site" as used herein refers to a linear site or continuous three-dimensional site on an antigen recognized by an antibody or antigen binding fragment of the present disclosure.

The term "epitope" refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed by contiguous amino acids or by non-contiguous amino acids that come close to each other through tertiary protein folding. Epitopes formed by contiguous amino acids are usually maintained after exposure to a denaturing solvent, whereas epitopes formed by tertiary folding are usually lost after exposure to a denaturing solvent. Epitopes typically include at least 3-15 amino acids in a unique spatial conformation. Methods for determining that an epitope is bound by a given antibody are well known in the art, including detection assays by immunoblotting, immunoprecipitation, etc. Methods for determining the spatial conformation of an epitope include techniques known in the art and techniques described herein, for example, X-ray crystal analysis, two-dimensional nuclear magnetic resonance, etc.

The terms "specific binding" and "selective binding" refer to the binding of an antibody to an epitope on a given antigen. In general, when recombinant human CD79B or its epitope is used as the analyte and an antibody is used as the ligand, when measured in the instrument by surface plasmon resonance (SPR) technology, the antibody binds to the target antigen or its epitope with a dissociation constant ( _KD ) below about 10 ^-7 M or even less, and its binding affinity for a given antigen or epitope thereof is at least twice the binding affinity for non-specific antigens (such as BSA (bovine serum albumin), etc.), different, than a given antigen (or its epitope) or closely related antigens.

The term “cross-reaction” refers to the ability of the antibodies of the present disclosure to bind to CD79B from different species. For example, an antibody of the present disclosure that binds to human CD79B may also bind to CD79B of another species. Cross-reactivity is measured by detecting specific reactivity with purified antigen in binding assays (eg, SPR and ELISA) or binding or functional interaction with cells that physiologically express CD79B. Methods for determining cross-reactivity include standard binding assays as described herein, for example, surface plasmon resonance or flow cytometry analysis.

The terms "inhibit" or "block" are used interchangeably and cover both partial and complete inhibition/blocking. CD79B inhibition/blocking

preferably reduces or alters the normal level or type of activity that is observed upon CD79B binding without inhibition or blocking. It is also intended that inhibition and blocking include any measurable decrease in binding affinity for CD79B when contacted with an anti-CD79B antibody compared to CD79B not contacted with an anti-CD79B antibody.

“Growth inhibition” (eg, in relation to cells) is meant to include any measurable reduction in cell growth.

Methods for producing and purifying antibodies or antigen-binding fragments are well known and can be found in the prior art, such as Antibody: A Laboratory Manual, Cold Spring Harbor (chapters 5-8 and 15). For example, human CD79B or a fragment thereof can be used to immunize mice, and the resulting antibodies can be renatured, purified, and amino acid sequencing can be performed using conventional methods. Antigen binding fragments can also be prepared by conventional methods. An antibody or antigen binding fragment of the invention is genetically modified to introduce one or more human FR regions into non-human CDR regions. Human germline FR sequences can be obtained from the ImmunoGeneTics (IMGT) website http://imgt.cines.fr or from the Immunoglobulin FactsBook, 2001ISBN012441351.

The engineered antibodies or antigen binding fragments of the present disclosure can be prepared and purified by conventional methods. For example, cDNA sequences encoding the heavy chain (SEQ ID NO: 20) and light chain (SEQ ID NO: 21) can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more recommended prior art, mammalian expression systems can result in glycosylation of antibodies, especially at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are propagated in a serum-free bioreactor medium to produce antibodies. The culture medium into which antibodies are secreted can be purified and selected using traditional techniques. Antibodies can be filtered and concentrated using traditional methods. Soluble mixtures and polymers can also be removed by traditional methods such as molecular sieves and ion exchange. The resulting product must be immediately frozen, for example at -70°C, or lyophilized.

The antibody of the present disclosure refers to a monoclonal antibody. A monoclonal antibody (mAb) described in the present disclosure refers to an antibody derived from a cell line of a single clone, wherein said cell line is not limited to a cell line of a eukaryotic, prokaryotic, or phage cell lineage. Monoclonal antibodies or antigen binding fragments can be produced by recombination using, for example, hybridoma techniques, genetic engineering technology, phage display technology, synthesis technology (such as CDR grafting) or other existing technologies.

To screen antibodies for competitive binding to the same epitope, conventional techniques known to those skilled in the art can be used. For example, competition and cross-competition studies can be performed to produce antibodies that compete or cross-compete with each other for binding to an antigen. A high-throughput method for producing antibodies that bind to the same epitope based on their cross-competition is described in international patent publication WO 03/48731. Therefore, conventional techniques known to those skilled in the art can be used to produce antibodies and antigen binding fragments thereof that compete with antibody molecules of the present disclosure for binding to the same epitope on CD79B.

The terms "transfer", "administration" and "treatment", when applied to animals, humans, experimental subjects, cells, tissues, organs or body fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent or composition with animals, humans , subjects, cells, tissues, organs or biological fluids. The terms "transmission", "administration" and "processing" can refer, for example, to treatment, pharmacokinetics, diagnostics, research and experimental methods. Treatment of cells involves contact of reagents with cells and contact of reagents with liquid, which, in turn, is in contact with cells. The terms "transfer", "administration" and "treatment" also refer to the treatment, for example, of cells with reagents, diagnostics, binding compositions or other cells in vitro and ex vivo. The term "treatment", when applied to human, veterinary or research subjects, refers to therapeutic treatment, prophylactic treatment or prophylactic measures, research and diagnostic applications.

The term “treatment” refers to the internal or external administration of a therapeutic agent, such as a composition containing any one of the antibodies or antigen-binding fragments thereof of the present disclosure, to a subject who already has or is suspected of having, or is susceptible to, one or more than one disease or a symptom thereof, and the therapeutic agent is known to have a therapeutic effect on said symptoms. In general, a therapeutic agent is given in an effective amount to relieve one or more symptoms of a disease in a treated subject or population, either to induce regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable extent. The amount of a therapeutic agent that is effective to relieve any particular disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the subject's disease state, age and body weight, and the ability of the drug to produce the desired therapeutic effect in the subject. Whether the symptoms of a disease have been alleviated can be assessed by any of the clinical testing methods commonly used by doctors or other health care professionals to assess the severity or progression of symptoms. Although embodiments of the present disclosure (e.g., treatments or products) may be ineffective in alleviating the target disease symptom in a particular subject, when determined according to any statistical test methods known in the art, such as Student's t test, chi test -squared test, Mann and Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Tepstra test, and Wilcoxon test, they should reduce the target disease symptom in a statistically significant number of subjects.

"Effective amount" includes a sufficient amount to reduce or prevent symptoms or conditions of a medical condition. An effective amount also refers to a sufficient amount to provide or facilitate diagnosis. The effective amount for a particular subject or veterinary subject may vary depending on factors such as the condition being treated, the general health of the subject, the mode, route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage schedule that avoids significant side effects or toxic effects.

"Identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When positions in two sequences are occupied by the same monomeric subunit of a base or amino acid, for example, if each position of two DNA molecules is occupied by an adenine, then the molecules are homologous at that position. The percentage identity between two sequences is a function of the number of matching or homologous positions that share the two sequences, divided by the number of all positions to be compared, x 100%. For example, in the case of optimal sequence alignment, if there are 6 matches or homology at 10 positions in two sequences, then the two sequences are 60% homologous. Generally speaking, a comparison is made by aligning two sequences to obtain the highest percentage of identity.

The expressions “cell,” “cell line,” and “cell culture,” as used herein, may be used interchangeably, and all such names include their progeny. Therefore, the terms “transformant” and “transformed cell” include primary experimental cells and cultures derived from them, regardless of the number of passages. It should also be understood that due to intentional or unintentional mutations, all offspring may not be exactly the same in terms of DNA content. The term includes screened mutant progeny with the same function or biological activity as the original transformed cells. When the term refers to different indications, it would be obvious from the context.

The term “possible” or “perhaps” means that the described event or conditions that follow the term “possible” may (but do not necessarily) occur, and the description includes cases where the event or conditions do or do not occur. For example, the phrase "possibly containing 1 to 3 antibody heavy chain variable regions" means that antibody heavy chain variable regions of specific sequences may (but are not necessarily) present.

The term "pharmaceutical composition" means a mixture containing one or more antibodies or antigen binding fragments or conjugates described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof and other chemical component(s), as well as other components such as physiologically/pharmaceutically acceptable carrier(s) and excipient(s). The purpose of a pharmaceutical composition is to promote administration to the body, thereby facilitating the absorption of the active ingredient and thereby exerting its biological activity.

EXAMPLES

The following examples are included to further describe this disclosure, but these examples do not limit the scope of the invention.

Experimental methods that do not specify specific conditions in the Examples or Test Examples of the present disclosure generally follow traditional conditions or conditions recommended by the raw material or product manufacturer. See Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor; Current Protocols Molecular Biology, Ausubel et al., Greene Publishing Associates, Wiley Interscience, NY. Non-source specific reagents are traditional reagents purchased from the market.

Example 1 Cloning and Expression of Protein Antigens Antibodies (containing light and heavy chains) and antigens were constructed by PCR (polymerase chain reaction) methods with overlapping primers known in the art, and DNA fragments obtained by PCR with overlapping primers were inserted into the expression vector pEE6.4 (Lonza Biologies) through the use of two Hindll/BstBI enzyme cleavage sites, both antibodies and antigens were expressed in 293F cells (Invitrogen, cat. no. R790-07). The resulting recombinant protein was used for immunization or screening. The sequence of the human CD79B gene is obtained from NCBI (National Center for Biotechnology Information) (NP_000617.1), and its extracellular region (ECD) contains 159 amino acids (Met1-Asp159).

> Amino acid sequence of the fusion protein of the extracellular domain (ECD) of human CD79B and the human FC region (ECD of human CD79B-hFc):

> Amino acid sequence of the extracellular domain (ECD) fusion protein of human CD79B and His tag (ECD of human CD79B-His):

Example 2: Production of mouse monoclonal antibodies

1. Mouse immunization and serum titer detection A fusion protein of the extracellular domain (ECD) of human CD79B and the human FC region (ECD of human CD79B-hFc), and a fusion protein of the extracellular domain (ECD) of human CD79B and His tag (ECD of human CD79B-His) were used as immunogens to immunize Balb/c and SJL mice by intraperitoneal injection, respectively, to stimulate mice to produce antibodies against the extracellular domain (ECD) of human CD79B in vivo.

The experimental stages were as follows:

1) Immunization by intraperitoneal injection. The amount of antigen required for a given immunization was calculated according to the immunization procedure. The protein antigen was diluted with PBS to the appropriate antigen concentration as required, and then the antigen was emulsified. The emulsified mixture of antigen and adjuvant was transferred into a 2.0 ml sterile syringe, paying attention to air bubbles exposed to the atmosphere. The tail of the mouse was grasped with the right hand, and the skin of the mouse's head and neck was easily grasped with the thumb and index finger of the left hand. With the abdominal cavity directed upward, the injection site on the right side of the mouse's abdomen was wiped with a cotton ball containing 75% alcohol. The antigen preparation was loaded into the syringe in advance, with the point of the needle pointing upward and the mouse's head pointing downward. The tip of the needle was inserted horizontally into the skin, the syringe was inserted into the abdominal cavity of the mouse at a 45-degree angle with the abdominal cavity, and the antigen-adjuvant mixture was slowly injected. After immunization was completed, observation was carried out for at least 2 hours.

2) Selection of mouse serum. Serum tube numbers were labeled to match each mouse and the number was checked on the mouse's ear. Mice were grasped with one hand, and approximately 100 μl of whole blood was collected through the submandibular vein. The collected whole blood sample was allowed to stand at room temperature for approximately 2 hours; then the serum in the upper part of the centrifuge tube was collected by centrifugation. This serum could be stored in a refrigerator at 4°C for up to one week for antibody titer detection and other related experiments. The serum could be refrigerated at -80°C for long-term storage to avoid repeated freezing and thawing.

3) Detection of serum titer of immunized mice by ELISA. Before starting this experiment, a 96-well plate was labeled accordingly and coated with antigen at a concentration of 1 μg/ml, 50 μl per well overnight in a refrigerator at 4°C. The next day, the plate with antigen, coated a day earlier, was removed and washed with using a plate washer (wash solution: 1×PBST (phosphate buffered saline with Tween)). After washing, this plate was blocked with a blocking solution of 1% BSA (bovine serum albumin) prepared in 1×PBST at 37°C for 1 hour. After washing this plate with a wash solution of 1×PBST 3 times, different dilutions of serum were added to be assay and incubated in an incubator at 37°C for 1 hour. After washing the plate with 1×PBST wash solution, 100 μl of a 1:5000 diluted secondary goat anti-mouse antibody was added 3 times and incubated in an incubator at 37°C for 0 .5 hours. After washing this plate, the chromogenic solution A and B TMB (tetramethylbenzidine) was mixed in a 1:1 ratio and then color development occurred. The development reaction was completed using 1 N. hydrochloric acid after 15 minutes. Fluorescence values at 450 nm were detected on a Spectra Max M5 multifunctional plate reader.

4) Detection of serum titer of immunized mice by FACS. Suspensions of DoHH2 cells or monkey peripheral blood mononuclear cells were centrifuged, and the cells were resuspended in PBS containing 0.1% BSA and counted. The serum to be analyzed from each group of immunized mice was added. After 60 min of incubation at room temperature, the cells were washed three times and then a secondary anti-mouse IgG Fc antibody conjugated to FITC (fluorescein isothiocyanate) was added. After 30 min of incubation at room temperature in the dark, cells were washed three times and gently resuspended in PBS containing 0.1% BSA for detection on the instrument.

The ELISA and FACS serum titer results for each group of mice are shown in FIG. 1-Fig. 7.

Five Balb/c mice were immunized with human CD79b-hFc ECD protein numbered 5491, 5492, 5493, 5494, and 5495. The serum titer results by ELISA are shown in FIG. 1. These results showed that the serum titer in immunized mice reached more than 1:100K. The results of serum titer detection by FACS are shown in FIG. 2. It can be seen that the antibodies produced in mouse serum could specifically recognize the CD79B protein on the surface of DoHH2 cells.

Five SJL mice were immunized with human CD79b-hFc ECD protein numbered 5496, 5497, 5498, 5499 and 5500. The serum titer results by ELISA are shown in FIG. 3. These results showed that the serum titer in immunized mice reached more than 1:100K. The results of serum titer detection by FACS are shown in FIG. 4. It can be seen that the antibodies produced in mouse serum could specifically recognize the CD79B protein on the surface of DoHH2 cells.

Five SJL mice were immunized with human CD79b-his ECD protein numbered 5726, 5727, 5728, 5729 and 5730. The serum titer results by ELISA are shown in FIG. 5. These results showed that the serum titer in immunized mice reached more than 1:10K. The results of mouse serum titer detection by FACS are shown in FIG. 6. It can be seen that the antibodies produced in mouse serum could specifically recognize the CD79B protein on the surface of DoHH2 cells.

From the above results, it may be known that the immunized mice produced specific antibodies against CD79B, and the above-mentioned mice could be used for cell fusion to obtain hybridoma cell lines capable of secreting specific antibodies against CD79B.

2. Preparation of hybridoma and antibody screening

Cell fusion is induced spontaneously or artificially to stimulate the fusion of murine lymphocytes and SP2/0 myeloma cells (ATCC, CCL-121™) into hybridoma cells that have antibody secreting function and can proliferate indefinitely. Lymphocytes from the immunized group of mice and myeloma cells were fused using the electrofusion method, and the hybridoma cells were used for subsequent antibody screening.

1) Electrofusion experiment. One week before confluence, SP2/0 cells were propagated in 10% DMEM (Dulbecco's modified Eagle's medium). The sacrificed mice had their spleen and lymph nodes removed in a biosafety cabinet, washed and ground in Petri dishes, and lymphocytes collected. SP2/0 and lymphocytes were mixed in a given proportion, fusion was carried out using an electrofusion device, and the program was set. After confluence, the cells were seeded into a 96-well plate and cultured in an incubator at 37°C, 5% CO ₂ . The state of the cells was observed every day, and the cell fusion rate was calculated 5 days after fusion. The fused hybridoma cells were screened 9-14 days after fusion, and the cells in the positive well were selected for expansion in a 24-well plate.

2) Subcloning by limited breeding method. Cell lines to be subcloned were resuspended from 24-well culture wells and counted. Each cell line was diluted to a cell concentration of 5–10 cells/ml. The diluted cell suspension was added to 15 cm disposable culture dishes, and 0.2 ml was added to each well of a 96-well culture plate, with each well containing 1-2 cells. A 96-well plate inoculated with cells was placed in a 37°C, 5% CO ₂ incubator for culture. After 7-10 days, the subcloning plate was identified and screened according to the growth status of the cells, and positive clones were selected and transferred into 24 wells for further confirmation of the positive ones.

3) ELISA screening. Before the start of the experiment, a 96-well plate was labeled accordingly and coated with antigen at a concentration of 1 μg/ml, 50 μl per well overnight in a refrigerator at 4°C. The next day, the plate with antigen, coated a day earlier, was removed and washed using plate washer (wash solution: 1×PBST). After washing, the plate was blocked with 1% BSA blocking solution prepared in 1×PBST at 37°C for 1 hour. After washing the plate with 1×PBST wash solution 3 times, 50 μl of supernatant of the cells to be analyzed was added and incubated in an incubator at 37°C for 1 hour. After washing this plate with a 1×PBST wash solution, 100 μl of a 1:5000 diluted secondary goat anti-mouse antibody was added 3 times and incubated in an incubator at 37°C for 0.5 hours. After washing this plate, the chromogenic solution A and B of TMB were mixed in a 1:1 ratio, and then color development occurred. The development reaction was completed using 1 N. hydrochloric acid after 15 minutes. Fluorescence values at 450 nm were detected on a Spectra Max M5 multifunctional plate reader.

4) FACS screening. DoHH2 cell suspensions were centrifuged, and these cells were resuspended in PBS containing 0.1% BSA and counted. The cell supernatant to be analyzed was added. After 60 min of incubation at room temperature, the cells were washed three times and then a FITC-conjugated anti-mouse IgG Fc secondary antibody was added. After 30 min of incubation at room temperature in the dark, cells were washed three times and gently resuspended in PBS containing 0.1% BSA for detection on the instrument.

5) Identification of positive hybridoma clones. After fusing and screening subclones of murine spleen cells, the present inventors obtained a range of specific antibodies against the human CD79B antigen. Among them, 17 hybridomas with the best binding ability in ELISA and FACS were used for antibody production and purification. The ELISA results for the culture supernatant of anti-human CD79B positive hybridoma cell clones are shown in Table 1. The FACS results for the FACS detection of the culture supernatant of anti-human CD79B positive hybridoma cell clones are shown in Table 2. mIgG was used as a negative control in both the ELISA and FACS assays .

3. Production, purification and identification of mouse monoclonal antibodies

1) Production and purification of mouse monoclonal antibodies. The hybridoma cells used for antibody production were observed under a microscope. These cells were selected when grown to no more than 70% and in good cell condition and counted using a Countstar IC1000 cell counter. The cell concentration was adjusted to 1×10 ⁵ - 5×10 ⁵ cells/ml using well-prepared medium, and the cells were transferred to roller bottles. Roller bottles containing the transferred cells were loaded onto a roller bottle incubator for incubation at 37°C for 10-15 days. Cell growth status was monitored every day. This culture was removed for purification after the medium became orange and clear. Antibodies were purified by passing the cell supernatant through protein A columns and purification was carried out according to conventional methods.

2) ELISA detection of mouse monoclonal antibodies against human CD79B. Before starting the experiment, a 96-well plate was labeled accordingly and coated with antigen at a concentration of 1 μg/ml, 50 μl per well overnight in the refrigerator at 4°C. The next day, the antigen plate coated the previous day was removed and washed once using a plate washer (wash solution: 1×PBST). After washing, the plate was blocked with a blocking solution of 1% BSA prepared in 1×PBST at 37°C for 1 hour. After washing the plate with a wash solution of 1×PBST 3 times, 50 μl of antibody diluted to 100 nM (1 :10), and incubated in an incubator at 37°C for 1 hour. After washing this plate with a 1×PBST wash solution, 100 μl of a 1:5000 diluted secondary goat anti-mouse antibody was added 3 times and incubated in an incubator at 37°C in for 0.5 hours. After washing this plate, the chromogenic solution A and B TMB were mixed in a 1:1 ratio and then color development occurred. The development reaction was completed using 1 N. hydrochloric acid after 15 minutes. Fluorescence values at 450 nm were detected on a Spectra Max M5 multifunctional plate reader. Among them, three mouse monoclonal antibodies against human CD79B had the strongest binding ability in ELISA, including mAb015, mAb016 and mAb017 (see Fig. 7 for specific data). Among them, hIgG1 was a negative control antibody, and SN8 was a positive control antibody. SN8 is an antibody used in the antibody-conjugated drug polatuzumab vedotin developed by Roche Pharmaceuticals (sequence reference: US 20170362318 A). Polatuzumab vedotin has now been approved by the US Food and Drug Administration for marketing. From the results, it can be seen that in the ELISA experiment, the binding ability of three mouse anti-human CD79B monoclonal antibodies: mAb015, mAb016 and mAb017, preferentially selected according to the present disclosure, was similar to the binding ability of SN8.

3) FACS detection of mouse monoclonal antibodies against human CD79B. After centrifugation of the DoHH2 cell suspension, the cells were resuspended in PBS containing 0.1% BSA and counted. 100 μl of antibody diluted to 100 nM (1:10) was added and incubated for 1 h at room temperature. After washing these cells three times, a secondary anti-mouse IgG Fc antibody conjugated to FITC was added. After 30 min of incubation at room temperature in the dark, cells were washed three times and gently resuspended in PBS containing 0.1% BSA for detection on the instrument. Among them, three mouse monoclonal antibodies against human CD79B had the highest binding activity in FACS, including mAb015, mAb016 and mAb017 (see Fig. 8 for specific data). Among them, hIgG1 was a negative control antibody, and SN8 was a positive control antibody. From these results, it can be known that in the FACS experiment, the binding ability of three mouse monoclonal antibodies against human CD79B: mAb015, mAb016 and mAb017, preferably selected according to the present disclosure, was better than SN8.

4) FACS detection of cross-activity of mouse monoclonal antibodies against human CD79B. 293F-cynoCD79B cells were obtained by transient transfection method. After centrifugation of the cell suspension, cells were resuspended in PBS containing 0.1% BSA and counted. 100 μl of antibody was added at concentrations of 10 μg/ml and 1 μg/ml, respectively. These cells were incubated for 1 hour at room temperature. After washing the cells three times, a secondary anti-mouse IgG Fc antibody conjugated to FITC was added. After 30 min of incubation at room temperature in the dark, these cells were washed three times and gently resuspended in PBS containing 0.1% BSA for detection on the instrument. FACS detection results showing cross-activity of mouse monoclonal antibodies against human CD79B are shown in FIG. 9, where mIgG1 is a negative control antibody, anti-cyno HR008 is a mouse monoclonal antibody against cynomolgus CD79B, the antibody sequence of which is derived from the mouse monoclonal antibody against cynomolgus CD79B (clone number 10D10) in WO 2009012268 A1. It can be seen from these results that all mouse monoclonal antibodies against human CD79B screened in this disclosure did not recognize cynomolgus CD79B.

5) Detection of SPR (surface plasmon resonance) of mouse monoclonal antibodies against human CD79B. The affinity between the anti-human CD79B antibody and its antigen, human CD79B-His, was detected by surface plasmon resonance (SPR). The human CD79B-His protein antigen was immobilized on a CM5 chip. The binding level was set to 100 RU (response units). The running buffer was HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20). The diluted antibody flowed through the experimental channel and the control channel at a flow rate of 30 μl/min for 3 minutes, and dissociation was carried out for 5 minutes. Then the regeneration buffer (10 mM glycine buffer, pH 1.5) was run at a flow rate of 30 μl/min for 30 s. Data were analyzed using Biacore 8K evaluation software.

Example 3: Determination of the amino acid sequence of the variable region of a mouse monoclonal antibody

The high affinity hybridoma monoclonal cell lines obtained in Example 2 were subjected to variable region amino acid sequencing. Human and mouse chimeric antibodies (cAb) were then recombinantly expressed and further antibody identification was then performed. The heavy and light chain variable regions of the antibody gene were amplified by reverse transcription-PCR (polymerase chain reaction), linked to a vector, and sequenced to obtain the monoclonal antibody light and heavy chain sequences. First, total cellular RNA from single cell lines with good activity in Example 2 was extracted using an RNA purification kit (Qiagen, item number 74134, see specification for steps). Single-stranded cDNA was then prepared by using a cDNA synthesis kit (Invitrogen, product number 18080-051), that is, an oligo-dT reverse transcription cDNA primer. It was used as a template to synthesize antibody light and heavy chain variable region sequences by a PCR method, and the PCR product was cloned into the pMD-18T TA vector and then submitted for sequencing. The resulting antibody light and heavy chain sequences were respectively cloned into an expression vector (see Example 1 for the method), the recombinant monoclonal antibody was expressed, confirmed for activity (see Example 2 for the method), and then humanization work was carried out.

The amino acid residues of the CDR VH/VL of the anti-human CD79B antibody in the present disclosure are defined and annotated by the Chothia numbering system.

> Variable region of the heavy chain of the mouse hybridoma monoclonal antibody mAb015:

> Mouse hybridoma monoclonal antibody light chain variable region mAb015:

> Variable region of the heavy chain of the mouse hybridoma antibody mAb017:

> Variable region of the light chain of the mouse hybridoma monoclonal antibody mAb017:

> Variable region of the heavy chain of the mouse hybridoma monoclonal antibody mAb016:

≥ Mouse hybridoma monoclonal antibody light chain variable region mAb016:

The mouse CDR sequences according to Chothia numbering rules are shown in Table 3:

Example 4 Humanization of Anti-Human CD79B Antibodies After alignment, homology of the light and heavy chain sequences of the murine anti-CD79B monoclonal antibodies obtained in Example 3 was established with respect to the humanized antibody model of the antibody database. According to this model, optimal humanized monoclonal antibodies against CD79B were selected as the preferred molecules through back mutation screening. This method began by searching a published mouse Fab crystal structure model database (such as the PDB database) for crystal structures similar or homologous to the structure of the resulting mouse Fab molecules, and high resolution Fab crystal structures (such as less than 2.5 A ) were selected to establish the Fab mouse model. The light and heavy chain sequences of the mouse antibody were compared with those in this model. Sequences consistent with those of the mouse antibody in the model were retained to obtain a structural model of the mouse antibody; discordant amino acids were potential sites of reverse mutation. The mouse antibody structural model was run using the Swiss-pdb viewer program for energy optimization (minimization). Amino acids at different positions in this model (except CDR) were reverse mutated, and the resulting (humanized) mutant antibodies were compared with the pre-humanization antibodies for activity. Humanized antibodies retained good activity. The CDR regions were then optimized, including avoidance of glycosylation, deamidation, and oxidation sites. The antibodies described above were cloned, expressed and purified through gene cloning and recombinant expression methods. After detection through SPR, etc. the humanized antibodies hAb015 and hAb017, which retained the best activity, were finally selected. See Table 4 for specific data. The humanized antibodies hAb015 and hAb017 retained similar affinities and associated functions as the murine monoclonal antibodies.

The sequences of the humanized antibodies hAb015 and hAb017 are shown below.

> Heavy chain sequence of the humanized antibody hAb015:

> Light chain sequence of the humanized antibody hAb015:

> Heavy chain sequence of the humanized antibody hAb017:

> Light chain sequence of the humanized antibody hAb017:

Example 5 Endocytosis of anti-CD79B antibodies

In order to analyze whether the anti-CD79B antibodies of the present disclosure could be endocytosed into cells along with human CD79B after binding to human CD79B, an endocytosis experiment was performed with DOHH-2 cells (DSMZ, ACC 47) with high expression of human CD79B protein to evaluate the ability of these antibodies to endocytose.

DOHH-2 cells were cultured according to a suitable conventional method for suspension cells. The composition of the complete medium was as follows: RPMI 1640 medium (GIBCO, cat. no.: 11835-030) plus 10% (v/v) fetal bovine serum (FBS) (GIBCO, cat. no.: 10099-141) and penicillin/ streptomycin (GIBCO, cat. no: 15070-063).

In this experiment, cells were selected by low-temperature centrifugation at 4°C, 1000 rpm for 5 min. These cells were resuspended in 10-15 ml of FACS buffer, pre-chilled on ice. The composition of the FACS buffer was as follows: phosphate-buffered saline (PBS), pH 7.4, plus 2% fetal bovine serum (FBS). The FACS buffer was pre-cooled on ice throughout the experiment. After centrifugation and counting, cells were added to a 96-well plate at a rate of 300,000 cells/well. After centrifugation and discarding of the supernatant, 12.5 μg/ml Fc blocking solution (BD, cat. no.: 564220) was added in a volume of 100 μl/well. These cells were blocked at room temperature for 10 min. 20 μg/ml of anti-CD79B antibodies to be assayed were then added to the appropriate wells and incubated at 4°C in the dark for 1 hour. These cells were washed twice with pre-chilled PBS buffer to remove unbound antibodies. Complete cell medium (RPMI 1640 medium with 10% fetal calf serum) was added and the cells were incubated at 37°C and 5% CO ₂ for 0 h, 1 h, 2 h or 4 h. After centrifugation and discarding of the supernatant, 100 μl was added /well 2% PFA buffer (paraformaldehyde). These cells were resuspended and allowed to stand for 10 minutes. These cells were then washed with FACS buffer 3 times, then 100 μl of secondary antibody solution (fluorescently labeled goat anti-human secondary antibody: dilution 1:250 with a concentration of 2 μg/ml, Biolegend, cat. no. 409304) was added and incubated at 4°C in the dark for 0.5 h. Pre-cooled PBS buffer was added and centrifuged at 4°C, discarding the supernatant, repeating three times. Cells were resuspended in FACS buffer at a volume of 200 μl/well and detected by flow cytometry (BD FACS Calibur).

The results showed that none of the three antibodies: SN8, hAb015 and hAb017 could be endocytosed by DOHH-2 cells when incubated at 4°C. Meanwhile, when incubated at 37°C, most antibodies were endocytosed by DOHH-2 cells after 1 hour, and antibody endocytosis reached a maximum after 4 hours. All 3 antibodies were endocytosed relatively well.

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LIST OF SEQUENCES

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<120> ANTI-CD79B ANTIBODY, ITS ANTIGENE-BINDING FRAGMENT AND THEIR

PHARMACEUTICAL APPLICATION

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405 410 415

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

420 425 430

Lys

<210> 2

<211> 207

<212> PRT

<213> Artificial sequence

<220>

<221> PEPTIDE

<222> (0)..(207)

<223> Human CD79B ECD-His

<400> 2

Ala Arg Ser Glu Asp Arg Tyr Arg Asn Pro Lys Gly Ser Ala Cys Ser

1 5 10 15

Arg Ile Trp Gln Ser Pro Arg Phe Ile Ala Arg Lys Arg Gly Phe Thr

20 25 30

Val Lys Met His Cys Tyr Met Asn Ser Ala Ser Gly Asn Val Ser Trp

35 40 45

Leu Trp Lys Gln Glu Met Asp Glu Asn Pro Gln Gln Leu Lys Leu Glu

50 55 60

Lys Gly Arg Met Glu Glu Ser Gln Asn Glu Ser Leu Ala Thr Leu Thr

65 70 75 80

Ile Gln Gly Ile Arg Phe Glu Asp Asn Gly Ile Tyr Phe Cys Gln Gln

85 90 95

Lys Cys Asn Asn Thr Ser Glu Val Tyr Gln Gly Cys Gly Thr Glu Leu

100 105 110

Arg Val Met Gly Phe Ser Thr Leu Ala Gln Leu Lys Gln Arg Asn Thr

115 120 125

Leu Lys Asp Gly Ile Ile Met Ile Gln Thr Leu Leu Ile Ile Leu Phe

130 135 140

Ile Ile Val Pro Ile Phe Leu Leu Leu Asp Lys Asp Asp Ser Lys Ala

145 150 155 160

Gly Met Glu Glu Asp His Thr Tyr Glu Gly Leu Asp Ile Asp Gln Thr

165 170 175

Ala Thr Tyr Glu Asp Ile Val Thr Leu Arg Thr Gly Glu Val Lys Trp

180 185 190

Ser Val Gly Glu His Pro Gly Gln Glu His His His His His

195 200 205

<210> 3

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<222> (0)..(117)

<223> Monoclonal antibody heavy chain variable region

mouse hybridoma mAb015

<400> 3

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Ser Ser Phe Thr Ser Tyr

20 25 30

Gly Ile Asn Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Phe Pro Arg Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Glu Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Lys Gly Asp Leu Gly Asp Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser

115

<210> 4

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<222> (0)..(112)

<223> Monoclonal antibody light chain variable region

mouse hybridoma mAb015

<400> 4

Asp Phe Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Arg Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asp Gly Asn Thr Tyr Phe Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 5

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<222> (0)..(119)

<223> Monoclonal antibody heavy chain variable region

mouse hybridoma mAb017

<400> 5

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Gly Ile Asn Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Tyr Pro Arg Ser Gly Asn Ile Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Ser Asp Tyr Asp Gly Asp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 6

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<222> (0)..(112)

<223> Monoclonal antibody light chain variable region

mouse hybridoma mAb017

<400> 6

Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His His

20 25 30

Asp Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Gln Leu Glu Ile Lys

100 105 110

<210> 7

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (0)..(7)

<223> mAb015 heavy chain CDR1

<400> 7

Gly Ser Ser Phe Thr Ser Tyr

15

<210> 8

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (0)..(6)

<223> mAb015 heavy chain CDR2

<400> 8

Phe Pro Arg Ser Gly Asn

15

<210> 9

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (0)..(8)

<223> mAb015 heavy chain CDR3

<400> 9

Gly Asp Leu Gly Asp Phe Asp Tyr

15

<210> 10

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (0)..(16)

<223> mAb015 light chain CDR1

<400> 10

Arg Ser Ser Gln Ser Ile Val His Ser Asp Gly Asn Thr Tyr Phe Glu

1 5 10 15

<210> 11

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (0)..(7)

<223> Light chain CDR2 mAb015 mAb016 mAb017

<400> 11

Lys Val Ser Asn Arg Phe Ser

15

<210> 12

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (0)..(9)

<223> Light chain CDR3 mAb015 mAb016 mAb017

<400> 12

Phe Gln Gly Ser His Val Pro Trp Thr

15

<210> 13

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (0)..(7)

<223> mAb017 heavy chain CDR1

<400> 13

Gly Tyr Thr Phe Thr Thr Tyr

15

<210> 14

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (0)..(6)

<223> mAb017 heavy chain CDR2

<400> 14

Tyr Pro Arg Ser Gly Asn

15

<210> 15

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (0)..(10)

<223> mAb017 heavy chain CDR3

<400> 15

Gly Ser Asp Tyr Asp Gly Asp Phe Ala Tyr

1 5 10

<210> 16

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (0)..(16)

<223> mAb017 light chain CDR1

<400> 16

Arg Ser Ser Gln Ser Ile Val His His Asp Gly Asn Thr Tyr Leu Glu

1 5 10 15

<210> 17

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<222> (0)..(117)

<223> mAb016 heavy chain variable region

<400> 17

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asn Tyr

20 25 30

Gly Ile Ile Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asp Ile Phe Pro Gly Ser Gly Asn Thr Tyr Tyr Asn Glu Asn Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ser Arg Gly Glu Leu Gly Asp Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser

115

<210> 18

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<222> (0)..(112)

<223> mAb016 light chain variable region

<400> 18

Val Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val His Ser

20 25 30

Asp Gly Thr Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 19

<211> 447

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<222> (0)..(447)

<223> Humanized antibody heavy chain sequence

hAb015

<400> 19

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Ser Ser Phe Ser Ser Tyr

20 25 30

Gly Ile Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Phe Pro Arg Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Glu Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Asp Leu Gly Asp Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 20

<211> 219

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<222> (0)..(219)

<223> Humanized antibody light chain sequence

hAb015

<400> 20

Asp Phe Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asp Gly Asn Thr Tyr Phe Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 21

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<222> (0)..(449)

<223> Humanized antibody heavy chain sequence

hAb017

<400> 21

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Gly Ile Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Tyr Pro Arg Ser Gly Asn Ile Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ser Asp Tyr Asp Gly Asp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 22

<211> 219

<212> PRT

<213> Artificial sequence

<220>

<221> CIRCUIT

<222> (0)..(219)

<223> Humanized antibody light chain sequence

hAb017

<400> 22

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His His

20 25 30

Asp Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 23

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (0)..(7)

<223> mAb016 heavy chain CDR1

<400> 23

Gly Tyr Ile Phe Thr Asn Tyr

15

<210> 24

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (0)..(6)

<223> mAb016 heavy chain CDR2

<400> 24

Phe Pro Gly Ser Gly Asn

15

<210> 25

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (0)..(8)

<223> mAb016 heavy chain CDR3

<400> 25

Gly Glu Leu Gly Asp Phe Asp Tyr

15

<210> 26

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (0)..(16)

<223> mAb016 light chain CDR1

<400> 26

Arg Ser Ser Gln Asn Ile Val His Ser Asp Gly Thr Thr Tyr Leu Glu

1 5 10 15

<210> 27

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (0)..(7)

<223> hAb015 heavy chain CDR1

<400> 7

Gly Ser Ser Phe Ser Ser Tyr

15

<---

Claims (57)

1. An anti-human CD79B antibody or an antigen-binding fragment thereof, which contains any one selected from the following (I)-(III): (I) a heavy chain variable region containing HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 7 or 27, SEQ ID NO: 8 and SEQ ID NO: 9, respectively; And a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; (II) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively; And a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 16, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; (III) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25, respectively; And a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 26, SEQ ID NO: 11 and SEQ ID NO: 12, respectively. 2. Antibody against human CD79B or antigen-binding fragment thereof according to claim 1, in which: the heavy chain variable region contains: a sequence as shown in SEQ ID NO: 3, or a sequence with at least 90%, 95%, 98%, 99% identity to SEQ ID NO: 3; or a sequence as shown in SEQ ID NO: 5, or a sequence with at least 90%, 95%, 98%, 99% identity to SEQ ID NO: 5; a sequence as shown in SEQ ID NO: 17, or a sequence with at least 90%, 95%, 98%, 99% identity to SEQ ID NO: 17; and/or the light chain variable region contains: a sequence as shown in SEQ ID NO: 4, or a sequence with at least 90%, 95%, 98%, 99% identity to SEQ ID NO: 4; or a sequence as shown in SEQ ID NO: 6, or a sequence with at least 90%, 95%, 98%, 99% identity to SEQ ID NO: 6; a sequence as shown in SEQ ID NO: 18, or a sequence with at least 90%, 95%, 98%, 99% identity to SEQ ID NO: 18. 3. Antibody against human CD79B or antigen-binding fragment thereof according to claim 2, in which: the heavy chain variable region of the anti-human CD79B antibody or antigen binding fragment is as shown in SEQ ID NO: 3, and the light chain variable region is as shown in SEQ ID NO: 4; or the heavy chain variable region of the anti-human CD79B antibody or antigen binding fragment is as shown in SEQ ID NO: 5, and the light chain variable region is as shown in SEQ ID NO: 6; or the heavy chain variable region of the anti-human CD79B antibody or antigen binding fragment is as shown in SEQ ID NO: 17, and the light chain variable region is as shown in SEQ ID NO: 18. 4. An anti-human CD79B antibody or an antigen binding fragment thereof according to claim 1 or 2, which is a murine antibody, a chimeric antibody, a human antibody or a humanized antibody or a fragment thereof. 5. The anti-human CD79B antibody or antigen binding fragment thereof according to claim 4, which is a humanized antibody, with a heavy chain thereof comprising: the sequence as shown in SEQ ID NO: 19, or a sequence with at least 90% strength, 95%, 98% or 99% identity with SEQ ID NO: 19; or a sequence as shown in SEQ ID NO: 21, or a sequence with at least 90%, 95%, 98% or 99% identity to SEQ ID NO: 21; and/or the light chain contains: a sequence as shown in SEQ ID NO: 20, or a sequence with at least 90%, 95%, 98% or 99% identity to SEQ ID NO: 20 ; or a sequence as shown in SEQ ID NO: 22, or a sequence with at least 90%, 95%, 98% or 99% identity to SEQ ID NO: 22. 6. The anti-human CD79B antibody or antigen binding fragment thereof according to claim 5, wherein the heavy chain of the anti-CD79B antibody or antigen binding fragment is as shown in SEQ ID NO: 19 and the light chain is as shown in SEQ ID NO: 20; or the heavy chain of the anti-human CD79B antibody or antigen binding fragment is as shown in SEQ ID NO: 21, and the light chain is as shown in SEQ ID NO: 22. 7. Antibody against human CD79B or antigen-binding fragment thereof according to claim 5, in which: the humanized antibody further comprises a human IgG1, IgG2, IgG3 or IgG4 heavy chain constant region or a variant thereof; the antigen binding fragment is selected from the group consisting of Fab, Fab'-SH, Fv, scFv and/or fragment (Fab') ₂ . 8. The anti-human CD79B antibody or antigen binding fragment thereof according to claim 7, wherein the humanized antibody further comprises a human IgG1, IgG2 or IgG4 heavy chain constant region. 9. The anti-human CD79B antibody or antigen-binding fragment thereof according to claim 8, wherein the humanized antibody further comprises a human IgG1 or IgG2 heavy chain constant region. 10. An anti-human CD79B antibody conjugate is a medicinal product for use in the treatment of a cancer or tumor characterized by the expression of CD79B, in which the antibody comprises an anti-human CD79B antibody or an antigen-binding fragment thereof according to any one of claims. 1-9. 11. The anti-human CD79B antibody-drug conjugate of claim 10, wherein said antibody-drug conjugate comprises a cytotoxic agent. 12. The anti-human CD79B antibody-drug conjugate of claim 11, wherein said cytotoxic agent is selected from the group consisting of a toxin, a chemotherapeutic agent, an antibiotic, a radioactive isotope, and a nucleolytic enzyme. 13. A polynucleotide encoding an antibody against human CD79B or an antigen-binding fragment thereof according to any one of claims. 1-9. 14. An expression vector comprising the polynucleotide of claim 13, which is a eukaryotic vector, a prokaryotic vector or a viral vector. 15. A host cell for expressing an antibody against human CD79B or an antigen-binding fragment thereof, comprising the vector of claim 14. 16. The host cell of claim 15, wherein said host cell is a bacterial cell, a yeast cell, or a mammalian cell. 17. The host cell of claim 16, wherein said host cell is Escherichia coli, Pichia pastoris, a Chinese hamster ovary cell, or a human embryonic kidney 293 cell. 18. A method for producing an antibody against human CD79B or an antigen-binding fragment thereof, comprising: expressing the anti-human CD79B antibody or an antigen-binding fragment thereof in a host cell according to claim 15; and isolating the anti-human CD79B antibody or an antigen-binding fragment thereof from the culture. 19. Pharmaceutical composition for use in the treatment of cancer or tumor characterized by the expression of CD79B, containing: any one of or any combination thereof selected from the following: an anti-CD79B antibody or an antigen-binding fragment thereof according to any one of paragraphs. 1-9, antibody-drug conjugate according to claims. 10-12, polynucleotide according to claim 13, vector according to claim 14; And a pharmaceutically acceptable excipient, diluent or carrier. 20. The use of an antibody against human CD79B or an antigen-binding fragment thereof according to any one of paragraphs. 1-9, antibody-drug conjugate according to claims. 10-12, a polynucleotide according to claim 13 or a vector according to claim 14 in the preparation of a medicinal product or pharmaceutical composition, where: the drug or pharmaceutical composition is used to treat a cancer or tumor characterized by the expression of CD79B. 21. Use according to claim 20, wherein the cancer or tumor characterized by CD79B expression is lymphoma or leukemia. 22. The use of claim 21, wherein the lymphoma is selected from the group consisting of the following: diffuse large B-cell lymphoma, non-Hodgkin lymphoma, small cell lymphocytic lymphoma and mantle cell lymphoma. 23. The use of claim 22, wherein the non-Hodgkin's lymphoma is selected from the group consisting of the following: aggressive NHL, recurrent aggressive NHL, recurrent painless NHL, refractory NHL, refractory painless NHL. 24. Use according to claim 21, wherein the leukemia is selected from the group consisting of the following: chronic lymphocytic leukemia, hairy cell leukemia and acute lymphocytic leukemia. 25. A method of treating cancer or tumor characterized by CD79B expression, comprising: administering to a subject an effective amount of an anti-human CD79B antibody or an antigen-binding fragment thereof according to any one of claims. 1-9, antibody-drug conjugate according to claims. 10-12, a polynucleotide according to claim 13, a vector according to claim 14, a pharmaceutical composition according to claim 19, or any combination thereof. 26. The method of claim 25, wherein the cancer or tumor characterized by CD79B expression is lymphoma or leukemia. 27. The method of claim 26, wherein said lymphoma is selected from the group consisting of the following: diffuse large B-cell lymphoma, non-Hodgkin lymphoma, small cell lymphocytic lymphoma, and mantle cell lymphoma. 28. The method of claim 27, wherein the non-Hodgkin's lymphoma is selected from the group consisting of the following: aggressive NHL, relapsed aggressive NHL, relapsed painless NHL, refractory NHL and refractory painless NHL. 29. The method of claim 26, wherein the leukemia is selected from the group consisting of the following: chronic lymphocytic leukemia, hairy cell leukemia, and acute lymphocytic leukemia.