WO2020156439A1 - 抗cd79b抗体、其抗原结合片段及其医药用途 - Google Patents

抗cd79b抗体、其抗原结合片段及其医药用途 Download PDF

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WO2020156439A1
WO2020156439A1 PCT/CN2020/073803 CN2020073803W WO2020156439A1 WO 2020156439 A1 WO2020156439 A1 WO 2020156439A1 CN 2020073803 W CN2020073803 W CN 2020073803W WO 2020156439 A1 WO2020156439 A1 WO 2020156439A1
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seq
antibody
sequence
antigen
binding fragment
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PCT/CN2020/073803
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English (en)
French (fr)
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杨翠青
唐任宏
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上海拓界生物医药科技有限公司
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Priority to JP2021543401A priority Critical patent/JP2022518062A/ja
Priority to MX2021008760A priority patent/MX2021008760A/es
Priority to US17/310,204 priority patent/US20220162304A1/en
Priority to KR1020217026421A priority patent/KR20210121102A/ko
Priority to AU2020213565A priority patent/AU2020213565A1/en
Priority to BR112021014420-0A priority patent/BR112021014420A2/pt
Priority to CN202080008873.0A priority patent/CN113286823B/zh
Priority to CA3127556A priority patent/CA3127556A1/en
Priority to EP20749292.7A priority patent/EP3919516A4/en
Publication of WO2020156439A1 publication Critical patent/WO2020156439A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • This application relates to an anti-human CD79B antibody and antigen-binding fragment, a chimeric antibody, a humanized antibody containing the CDR region of the anti-CD79B antibody, and a pharmaceutical composition containing a human anti-CD79B antibody or an antigen-binding fragment thereof, and As an anticancer drug, especially for the treatment of lymphoma (DLBCL) drugs.
  • DLBCL lymphoma
  • Malignant tumor is the second leading cause of death in the world, after heart disease.
  • Lymphoma is a malignant tumor originating from the lymphoid hematopoietic system and is the most common hematological tumor in the world.
  • the incidence of lymphoma in China has been on the rise in recent years, and the current incidence is about 6.68 cases per 100,000 people.
  • Non-Hodgkin's lymphoma is a general term for a group of abnormal lymphocyte proliferation diseases with strong heterogeneity. Its incidence is much higher than Hodgkin's lymphoma, accounting for more than 80% of lymphoma.
  • DLBCL diffuse large B-cell lymphoma
  • DLBCL diffuse large B-cell lymphoma
  • the first-line standard regimen for diffuse large B-cell lymphoma is rituximab combined with chemotherapy (R-CHOP).
  • R-CHOP chemotherapy
  • the anthracycline-based CHOP cyclophosphamide, doxorubicin, vincristine, prednisone
  • the R-CHOP regimen has significantly improved the long-term survival rate of DLBCL patients.
  • the clinical trial results show that: Compared with the traditional CHOP regimen, the R-CHOP regimen can significantly extend the median overall survival time of patients with DLBCL by 4.9 years.
  • the disease-free survival time was more than 6.6 years, and the 5-year disease-free survival rate increased from 30% to 54%. However, 10% to 15% of refractory patients still do not respond, and 20% to 30% of patients have relapses. And not all DLBCL patients are suitable for R-CHOP regimen, such as elderly patients over 80 years old, their physical fitness does not allow standard R-CHOP treatment; another example, for more aggressive types of lymphoma and recurrent lymphoma , The R-CHOP program may be invalid. Therefore, the development of a new generation of immunotherapies with fewer side effects is extremely necessary for the treatment of DLBCL.
  • B-cell lymphoma belongs to B-cell lymphoma.
  • the B cell antigen receptor (BCR) complex is the most important molecule on the surface of B cells.
  • the BCR complex is composed of membrane immunoglobulin (mIg) that recognizes and binds antigen and Ig ⁇ (CD79a) and Ig ⁇ (CD79B) heterodimers that transmit antigen-stimulating signals.
  • Ig ⁇ and Ig ⁇ are 47kDa and 37kDa glycoproteins, which belong to the immunoglobulin superfamily.
  • the genes encoding Ig ⁇ and Ig ⁇ are called mb-1 and B29, respectively.
  • Both Ig ⁇ and Ig ⁇ have an Ig-like domain at the amino terminus of the extracellular region. Both Ig ⁇ and Ig ⁇ can be used as substrates of protein tyrosine kinases and participate in BCR signal transduction. BCR is widely expressed on B-cell lymphoma and normal B cells. In view of the clinical success and reliable safety of rituximab targeting CD20, the development of therapeutic methods targeting BCR should also have good efficacy and safety.
  • CD79B Based on the expression of CD79B, it is beneficial to generate therapeutic antibodies against the CD79B antigen. There is still an unmet need in the art for the development of effective anti-human CD79B antibodies for the treatment of hematopoietic tumors or to delay the progress.
  • the present disclosure provides an anti-CD79B antibody or antigen-binding fragment thereof, which encodes a nucleic acid, a vector, a host cell, an antibody-drug conjugate, a pharmaceutical composition, and a method for treating or delaying cancer (especially hematopoietic tumors) .
  • the present disclosure provides an anti-human CD79B antibody or antigen-binding fragment thereof, which comprises an antibody heavy chain variable region and an antibody light chain variable region, wherein:
  • An antibody heavy chain variable region which includes at least one determinant complementarity region (HCDR) selected from the following sequences: 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, SEQ ID NO: 25; and/or
  • HCDR determinant complementarity region
  • An antibody light chain variable region which includes at least one determinant complementary region (LCDR) selected from the following sequence: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 16 , SEQ ID NO: 26.
  • LCDR determinant complementary region
  • an anti-human CD79B antibody or antigen-binding fragment thereof is provided, wherein:
  • variable region of the antibody heavy chain including:
  • variable region of the antibody light chain comprising:
  • an anti-human CD79B antibody or antigen-binding fragment thereof comprises any one selected from the following (I) to (II):
  • the heavy chain variable region which includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 respectively;
  • the light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively;
  • the heavy chain variable region which includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively;
  • the light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 16, SEQ ID NO: 11, and SEQ ID NO: 12, respectively;
  • the light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 26, SEQ ID NO: 11, and SEQ ID NO: 12, respectively.
  • an anti-human CD79B antibody or antigen-binding fragment thereof is provided, wherein:
  • variable region of the heavy chain including:
  • SEQ ID NO: 3 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
  • And/or light chain variable region including:
  • SEQ ID NO: 4 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
  • the heavy chain variable region of the anti-human CD79B antibody or antigen-binding fragment is shown in the sequence SEQ ID NO: 3, and the light chain variable region is shown in the sequence SEQ ID NO: 4.
  • the heavy chain variable region of the anti-human CD79B antibody or antigen-binding fragment is shown in the sequence SEQ ID NO: 5
  • the light chain variable region is shown in the sequence SEQ ID NO: 6.
  • the heavy chain variable region of the anti-human CD79B antibody or antigen-binding fragment is shown in the sequence SEQ ID NO: 17, and the light chain variable region is shown in the sequence SEQ ID NO: 18.
  • the anti-human CD79B antibody or antigen-binding fragment thereof wherein:
  • Heavy chain including:
  • SEQ ID NO: 19 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
  • And/or light chain including:
  • SEQ ID NO: 20 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
  • the heavy chain of the anti-human CD79B antibody or antigen-binding fragment is shown in the sequence SEQ ID NO: 19, and the light chain is shown in the sequence SEQ ID NO: 20.
  • the heavy chain of the anti-human CD79B antibody or antigen-binding fragment is shown in the sequence SEQ ID NO: 21, and the light chain is shown in the sequence SEQ ID NO: 22.
  • the anti-human CD79B antibody or antigen-binding fragment thereof as described above is provided, which is a murine antibody or fragment thereof.
  • the light chain variable region of the murine anti-CD79B antibody or antigen-binding fragment thereof comprises the light chain FR region and/or light chain constant region of the murine ⁇ , ⁇ chain or a variant thereof.
  • the murine anti-CD79B antibody or antigen-binding fragment thereof comprises a heavy chain FR region and/or a heavy chain constant region of murine IgG1, IgG2, IgG3, IgG4 or variants thereof.
  • the anti-human CD79B antibody or antigen-binding fragment thereof as described above is provided, which is a chimeric antibody or a fragment thereof.
  • the chimeric anti-CD79B antibody or antigen-binding fragment thereof comprises a light chain FR region and/or light chain constant region of a human ⁇ , ⁇ chain or a variant thereof, and/or human IgG1, IgG2 , IgG3 or IgG4 or its variant heavy chain FR region and/or heavy chain constant region.
  • an anti-human CD79B antibody or antigen-binding fragment thereof as described above, which is a humanized antibody or a fragment thereof, a human antibody or a fragment thereof.
  • an anti-human CD79B humanized antibody or antigen-binding fragment thereof as described above, wherein the light chain sequence is shown in SEQ ID NO: 20 or its variant sequence; the variant sequence is The light chain contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes, or has at least 80%, 85%, 90%, 95%, 96%, and SEQ ID NO: 20. 97%, 98%, or 99% identity.
  • the heavy chain sequence is shown in SEQ ID NO: 19 or its variant sequence; the variant sequence contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes in the heavy chain, or It is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19.
  • a humanized antibody or antigen-binding fragment thereof against human CD79B as described above, wherein the light chain sequence is shown in SEQ ID NO: 22 or its variant sequence, or is combined with SEQ ID NO: 22 has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity; the variant sequence includes 1, 2, 3, 4, 5, 6 in the light chain , 7, 8, 9, or 10 amino acid changes.
  • the heavy chain sequence is shown in SEQ ID NO: 21 or its variant sequence; the variant sequence contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes in the heavy chain, or It has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:21.
  • an anti-CD79B human antibody or fragment thereof as described above which further comprises a constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof.
  • a human IgG1 or IgG2 constant region is included.
  • an anti-human CD79B humanized antibody or fragment thereof as described above which further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably comprising human IgG1 , IgG2 or IgG4 heavy chain FR region, more preferably comprising human IgG1 or IgG2 heavy chain FR region.
  • an anti-human CD79B antibody or antigen-binding fragment thereof as described above wherein the antigen-binding fragment is selected from Fab, Fv, sFv, F(ab') 2 , linear antibody, single chain antibody, scFv, sdAb, sdFv, Nanobody, peptide antibody (peptibody), domain antibody and multispecific antibody (bispecific antibody, diabody (diabody), triabody (triabody) and tetrabody), Series two-scFv, series three-scFv).
  • an isolated monoclonal antibody or antigen-binding fragment thereof is provided, which can compete with the aforementioned monoclonal antibody or antigen-binding fragment for binding to human CD79B or its epitope.
  • amino acid residues of the VH/VL CDR of the anti-human CD79B antibody in this disclosure are determined and annotated by the Chothia numbering system.
  • the present disclosure provides a polynucleotide encoding the anti-human CD79B antibody or antigen-binding fragment thereof as described above.
  • the polynucleotide may be DNA or RNA.
  • the present disclosure provides an expression vector containing the polynucleotide as described above.
  • the expression vector can be a eukaryotic expression vector, a prokaryotic expression vector, or a viral vector.
  • the present disclosure provides a host cell transformed with the expression vector as described above, which may be a eukaryotic cell or a prokaryotic cell.
  • the host cell is a bacterium, yeast, or mammalian cell. In some specific embodiments, the host cell is Escherichia coli, Pichia pastoris, Chinese Hamster Ovary (CHO) cells or Human Embryonic Kidney (HEK) 293 cells.
  • the present disclosure provides an antibody drug conjugate.
  • the antibody-drug conjugate according to the present disclosure comprises or consists of the following: antibody, linker, drug.
  • the antibody drug conjugate according to the present disclosure is an antibody covalently coupled to the drug via a linker.
  • the antibody drug conjugate contains a cytotoxic agent.
  • the cytotoxic agent is selected from the group consisting of toxins, chemotherapeutics, antibiotics, radioisotopes, and nucleolytic enzymes.
  • the present disclosure provides a method for preparing an anti-human CD79B antibody or antigen-binding fragment thereof, comprising: expressing the antibody or antigen-binding fragment thereof in a host cell as described above, and isolating it from the host cell The antibody or antigen-binding fragment thereof.
  • the present disclosure provides a composition, such as a pharmaceutical composition, which contains a therapeutically effective amount of the anti-human CD79B antibody or antigen-binding fragment thereof as described above and a pharmaceutically acceptable excipient, dilution or carrier.
  • the unit dose of the pharmaceutical composition may contain 0.01 to 99% by weight of the anti-human CD79B antibody or antigen-binding fragment thereof or the amount of the anti-CD79B antibody or antigen-binding fragment thereof in the unit dose of the pharmaceutical composition It is 0.1 mg to 2000 mg, and in some embodiments, 1 mg to 1000 mg.
  • the present disclosure further provides the use of any one or a combination selected from the following in the preparation of a medicine: the anti-human CD79B antibody or antigen-binding fragment thereof according to the present disclosure, the pharmaceutical composition according to the present disclosure, Antibody drug conjugate; wherein the drug is used in the drug or pharmaceutical composition to treat a proliferative disease or delay the progression of a proliferative disease; the proliferative disease may be cancer or tumor.
  • the cancer or tumor is lymphoma or leukemia.
  • the lymphoma is selected from: diffuse large B-cell lymphoma, non-Hodgkin's lymphoma, small lymphocytic lymphoma, and mantle cell lymphoma.
  • the non-Hodgkin's lymphoma is selected from: aggressive NHL, recurrent aggressive NHL, recurrent painless NHL, refractory NHL, refractory painless NHL.
  • the leukemia is selected from: chronic lymphocytic leukemia, hairy cell leukemia, and acute lymphocytic leukemia.
  • the present disclosure provides a method for treating or preventing a proliferative disease or delaying the progression of a proliferative disease, the method comprising administering to a subject an effective amount of the anti-human CD79B antibody according to the present disclosure or its antigen binding
  • the fragment, or the pharmaceutical composition according to the present disclosure, or the antibody-drug conjugate according to the present disclosure wherein the proliferative disease may be cancer or tumor.
  • the cancer or tumor is lymphoma or leukemia.
  • the lymphoma is selected from: diffuse large B-cell lymphoma, non-Hodgkin's lymphoma, small lymphocytic lymphoma, and mantle cell lymphoma.
  • the non-Hodgkin's lymphoma is selected from: aggressive NHL, recurrent aggressive NHL, recurrent painless NHL, refractory NHL, refractory painless NHL.
  • the leukemia is selected from: chronic lymphocytic leukemia, hairy cell leukemia, and acute lymphocytic leukemia.
  • Figure 1 ELISA test results of serum titer of Balb/c mice immunized with human CD79B ECD-hFc protein.
  • Figure 2 FACS test results of serum titer of Balb/c mice immunized with human CD79B ECD-hFc protein.
  • Figure 3 ELISA test results of serum titer of SJL mice immunized with human CD79B ECD-hFc protein.
  • Figure 4 FACS test results of serum titer of SJL mice immunized with human CD79B ECD-hFc protein.
  • Figure 5 ELISA test results of serum titer of SJL mice immunized with human CD79B ECD-his protein.
  • Figure 6 FACS test results of serum titer of SJL mice immunized with human CD79B ECD-his protein.
  • Figure 7 ELISA test results of anti-human CD79B mouse monoclonal antibody, in which hIgG1 is the negative control antibody and SN8 is the positive control antibody.
  • Figure 8 FACS detection result of anti-human CD79B mouse monoclonal antibody, in which hIgG1 is the negative control antibody and SN8 is the positive control antibody.
  • Figure 9 FACS test results of cross-reactivity of anti-human CD79B mouse monoclonal antibody, in which mIgG is the negative control antibody; anti-cyno HR008 is anti-monkey CD79B mouse monoclonal antibody, its antibody sequence is derived from the anti-monkey in patent WO2009012268A1 CD79B mouse monoclonal antibody (clone number 10D10) was used as a positive control antibody.
  • CD79B refers to any CD79B of any vertebrate origin, including mammals, such as primates (e.g., humans, cynos) and rodents (e.g., mice and rats).
  • the term “CD79B” encompasses "full length", unprocessed CD79B and any form of CD79B processed from cells.
  • the term also encompasses 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 prepared by recombinant or synthetic methods.
  • antibody in the present disclosure refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
  • the amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE.
  • the corresponding heavy chains are ⁇ chain, ⁇ chain, and ⁇ chain. , ⁇ chain and ⁇ chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • the light chain is divided into ⁇ chain or ⁇ chain by the difference of the constant region.
  • Each of the five types of Ig can have a kappa chain or a lambda chain.
  • the sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly and is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region (C region).
  • the variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conservative sequences.
  • Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR).
  • Each light chain variable region (VL) and heavy chain variable region (VH) consists of 3 CDR regions and 4 FR regions.
  • the sequence from the amino terminal to the carboxy terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3;
  • the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2 and HCDR3.
  • the number and position of the CDR amino acid residues in the VL region and VH region of the antibody or antigen-binding fragment comply with the known Chothia (ABM) numbering rules.
  • human antibody or “recombinant human antibody” includes human antibodies prepared, expressed, created or isolated by recombinant methods, and the related techniques and methods are well known in the art, such as:
  • Antibodies prepared, expressed, created or isolated by methods such as splicing human immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant human antibodies contain variable and constant regions, which utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations that occur during antibody maturation.
  • murine antibody in the present disclosure is a monoclonal antibody to human CD79B or its epitope prepared according to the knowledge and skills in the art. During preparation, the test subject is injected with CD79B antigen (or its epitope), and then hybridomas expressing antibodies with desired sequences or functional properties are isolated.
  • the murine CD79B antibody or antigen-binding fragment thereof may further comprise the light chain constant region of murine ⁇ , ⁇ chain or variants thereof, or further comprise murine IgG1, IgG2 , IgG3 or IgG4 or its variant heavy chain constant region.
  • human antibody includes antibodies having variable and constant regions of human germline immunoglobulin sequences.
  • the human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • the term “human antibody” does not include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto human framework sequences (ie, "humanized antibodies”) .
  • humanized antibody also known as CDR-grafted antibody, refers to an antibody produced by grafting CDR sequences of non-human species into the framework of a human antibody variable region. It can overcome the strong immune response induced by the chimeric antibody carrying a large number of non-human species protein components. In order to avoid the decrease of immunogenicity and the decrease of activity at the same time, the variable region of the human antibody can be subjected to minimal reverse mutation to maintain activity.
  • chimeric antibody is an antibody formed by fusing the variable region of an antibody of the first species with the constant region of an antibody of the second species, which can reduce the immune response induced by the antibody of the first species.
  • To establish a chimeric antibody it is necessary to select a hybridoma that secretes a specific monoclonal antibody of the first species, then clone the variable region gene from the hybridoma cell of the first species (such as mouse), and then clone the second species (such as human )
  • the constant region gene of the antibody, the variable region gene of the first species and the constant region gene of the second species are connected to form a chimeric gene and then inserted into the vector, and finally the chimeric antibody molecule is expressed in the eukaryotic industrial system or the prokaryotic industrial system.
  • the constant region of the second species (for example, human) antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising the human IgG2 or IgG4 heavy chain constant region, or after using amino acid mutations IgG1 without ADCC (antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) toxicity.
  • Antigen-binding fragment refers to any fragment that retains the antigen-binding activity of an intact antibody. Specifically, there can be mentioned but not limited to Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments sFv fragments that bind to human CD79B.
  • the Fv fragment contains the variable region of the heavy chain of the antibody and the variable region of the light chain, but does not have the constant region, and has the smallest antibody fragment with all the antigen binding sites.
  • Fv antibodies also include 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 connect the variable regions of two antibodies into a polypeptide chain, which is called a single chain antibody or single chain Fv (sFv).
  • binding to CD79B refers to the ability to interact with CD79B or its epitope.
  • the CD79B or its epitope may be of human origin.
  • antigen-binding site in the present disclosure refers to linear or discontinuous on the antigen, a linear site or a three-dimensional site recognized by the antibody or antigen-binding fragment of the present disclosure.
  • epitope refers to a site on an antigen that specifically binds to an immunoglobulin or antibody.
  • Epitopes can be formed by adjacent amino acids or non-adjacent amino acids that are juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are usually maintained after exposure to a denaturing solvent, while epitopes formed by tertiary folding are usually lost after treatment with the denaturing solvent.
  • Epitopes usually include at least 3-15 amino acids in a unique spatial conformation. Methods to determine what epitope is bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation detection analysis. Methods for determining the spatial conformation of an epitope include the techniques in the art and the techniques described herein, such as X-ray crystal analysis and two-dimensional nuclear magnetic resonance.
  • Specific binding and “selective binding” refer to the binding of an antibody to an epitope on a predetermined antigen.
  • an antibody when measured by surface plasmon resonance (SPR) technology in the instrument, the antibody is approximately lower than 10 -7 M or even smaller
  • the equilibrium dissociation constant (K D ) binds to a predetermined antigen or its epitope, and its binding affinity to the predetermined antigen or its epitope (affinity) is that of its affinity with the predetermined antigen (or its epitope) or closely related antigens
  • affinity of foreign non-specific antigens such as BSA, etc.
  • Cross-reactivity refers to the ability of the antibodies of the present disclosure to bind to CD79B from different species.
  • an antibody of the present disclosure that binds human CD79B can also bind to CD79B of another species.
  • Cross-reactivity is measured by detecting specific reactivity with purified antigens in binding assays such as SPR and ELISA, or binding or functional interaction with cells that physiologically express CD79B. Methods of determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance analysis, or flow cytometry.
  • Inhibition or blocking are used interchangeably and encompasses both partial and complete inhibition/blocking. Inhibition/blocking of CD79B preferably reduces or alters the normal level or type of activity that occurs when CD79B binding occurs without inhibition or blocking. Inhibition and blocking are also intended to include any measurable reduction in binding affinity of CD79B when contacted with anti-CD79B antibody compared to CD79B not contacted with anti-CD79B antibody.
  • Inhibiting growth (e.g., referring to cells) is intended to include any measurable decrease in cell growth.
  • the methods for producing and purifying antibodies or antigen-binding fragments are well known and can be found in the prior art, such as Cold Spring Harbor's Antibody Experiment Technical Guide (chapters 5-8 and 15).
  • human CD79B or its fragments can be used to immunize mice, and the obtained antibody can be renatured, purified, and amino acid sequencing can be performed by conventional methods.
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the antibody or antigen-binding fragment of the invention is genetically engineered to add one or more human FR regions to the non-human CDR region.
  • the human FR germline sequence can be obtained from the website of ImmunoGeneTics (IMGT) http://imgt.cines.fr, or from the Journal of Immunoglobulin, 2001ISBN012441351.
  • the engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods.
  • the 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.
  • mammalian expression systems can lead to 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 expanded in the serum-free medium of the bioreactor to produce antibodies.
  • the antibody-secreted culture medium can be purified and collected by conventional techniques.
  • the antibody can be filtered and concentrated by conventional methods.
  • Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange.
  • the resulting product needs to be frozen immediately, such as -70°C, or lyophilized.
  • the antibodies of the present disclosure refer to monoclonal antibodies.
  • the monoclonal antibody (mAb) described in the present disclosure refers to an antibody obtained from a single cloned cell line, and the cell line is not limited to a eukaryotic, prokaryotic or phage cloned cell line.
  • Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using, for example, hybridoma technology, recombination technology, phage display technology, synthesis technology (such as CDR-grafting), or other existing technologies.
  • Antibodies can be screened competitively for binding to the same epitope using conventional techniques known to those skilled in the art. For example, competition and cross-competition studies can be conducted to obtain antibodies that compete with each other or cross-compete with antigen binding. A high-throughput method for obtaining antibodies that bind the same epitope based on their cross-competition is described in International Patent Publication WO03/48731. Therefore, conventional techniques known to those skilled in the art can be used to obtain antibodies and antigen-binding fragments thereof that compete with the antibody molecules of the present disclosure for binding to the same epitope on CD79B.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents, compositions and animals , Human, subject, cell, tissue, organ or biological fluid contact.
  • administering can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, where the fluids are in contact with cells.
  • administering “administration” and “treatment” also mean the treatment of, for example, cells by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo.
  • Treatment when applied to human, veterinary, or research subjects refers to therapeutic treatment, preventive treatment, or preventive measures, research, and diagnostic applications.
  • Treatment means to administer an internal or external therapeutic agent to a subject, such as a composition comprising any one of the antibodies or antigen-binding fragments or conjugates thereof of the present disclosure, the subject has, or is suspected of being suffering from Yes, tend to suffer from one or more diseases or their symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms.
  • the therapeutic agent is administered in the subject or population to be treated in an amount effective to alleviate one or more symptoms of the disease, whether by inducing the regression of such symptoms or inhibiting the development of such symptoms to any clinically measured extent.
  • the amount of the therapeutic agent effective to alleviate the symptoms of any particular disease can vary depending on various factors, such as the subject’s disease state, age and weight, and the amount of the drug that produces the desired therapeutic effect in the subject. ability. Any clinical testing method commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms can evaluate whether the symptoms of the disease have been alleviated.
  • the embodiments of the present disclosure may be ineffective in alleviating the symptoms of the target disease in a subject, according to any statistical test methods known in the art such as Student's t test, chi-square test, and basis Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of subjects.
  • any statistical test methods known in the art such as Student's t test, chi-square test, and basis Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of subjects.
  • Effective amount includes an amount sufficient to improve or prevent the symptoms or conditions of the medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular subject or veterinary subject may vary depending on factors such as the condition to be treated, the general health of the subject, the method of administration and dosage, and the severity of side effects.
  • the effective amount can be the maximum dose or dosing schedule that avoids significant side effects or toxic effects.
  • Identity refers to the sequence similarity between two polynucleotide sequences or between two polypeptides.
  • positions in the two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position .
  • the percent identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared ⁇ 100%. For example, in the optimal sequence alignment, if there are 6 matches or homology in 10 positions in the two sequences, then the two sequences are 60% homologous. Generally speaking, the comparison is made when two sequences are aligned to obtain the maximum percent identity.
  • “Pharmaceutical composition” means a mixture containing one or more of the antibodies or antigen-binding fragments or conjugates described herein, or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, and other groups For example, physiological/pharmaceutically acceptable carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and thus the biological activity.
  • the antibodies (including light and heavy chains) and antigens were constructed by overlap extension PCR methods known in the art, and the DNA fragments obtained by overlap extension PCR were inserted into the expression vector pEE6.4 ((Lonza) with HindIII/BstBI restriction sites. Biologics), expressed in 293F cells (Invitrogen, Cat#R790-07).
  • the obtained recombinant protein is used for immunization or screening.
  • the human CD79B gene sequence is derived from NCBI (NP_000617.1), and its extracellular region (ECD) contains 159 amino acids (Met1-Asp159).
  • Human CD79B extracellular region (ECD) and human Fc region fusion protein (human CD79B ECD-hFc) and human CD79B extracellular region (ECD) and His-tagged fusion protein (human CD79B ECD-His) were used as immunogens, respectively. Intraperitoneal injection was used to immunize Balb/c and SJL mice, and stimulate the mice to produce antibodies against the extracellular domain (ECD) of human CD79B.
  • mice serum Collection of mouse serum. Mark the serum tube number corresponding to each mouse, check the earring number of the mouse, grab the mouse with one hand, collect about 100 ⁇ l of whole blood through the submandibular vein of the mouse face, and leave the collected whole blood sample at room temperature After about 2h, centrifuge to collect the serum on the top of the centrifuge tube.
  • the serum can be stored in a refrigerator at 4°C within a week for the detection of antibody titer and other related experiments. If the serum is stored for a long time, it can be placed in a refrigerator at -80°C to avoid repeated freezing and thawing.
  • FACS serum titer determination of immunized mice DoHH2 cells or monkey peripheral blood mononuclear cell suspensions were centrifuged and the cells were resuspended in PBS containing 0.1% BSA and counted. The test serum of each group of immunized mice was added. After 60 minutes of incubation at room temperature, the cells were washed three times and anti-mouse was added. IgG Fc-FITC secondary antibody, incubate at room temperature for 30 minutes in the dark, wash the cells three times, gently resuspend the cells in PBS containing 0.1% BSA, and test on the machine.
  • the results of serum titer ELISA test are shown in Figure 1.
  • the results of FACS detection of mouse serum are shown in Figure 2. It can be seen that the antibodies produced in mouse serum can specifically recognize CD79B protein on the surface of DoHH2 cells.
  • Human CD79b ECD-hFc protein was immunized with 5 SJL mice, numbered 5496, 5497, 5498, 5499, and 5500.
  • the results of serum titer ELISA test are shown in Figure 3.
  • the results of FACS detection of mouse serum are shown in Figure 4. It can be seen that the antibodies produced in mouse serum can specifically recognize CD79B protein on the surface of DoHH2 cells.
  • Human CD79b ECD-his protein was immunized with 5 SJL mice, numbered 5726, 5727, 5728, 5729, and 5730.
  • the results of serum titer ELISA test are shown in Figure 5.
  • the results of FACS detection of mouse serum are shown in Figure 6. It can be seen that the antibodies produced in mouse serum can specifically recognize CD79B protein on the surface of DoHH2 cells.
  • mice produced specific antibodies against CD79B, and the above mice can be used for cell fusion to produce hybridoma cell lines capable of secreting specific antibodies against CD79B.
  • Cell fusion is spontaneously or artificially induced to promote the fusion of mouse lymphocytes and myeloma cells SP2/0 (ATCC, CCL-121 TM ) into hybridoma cells.
  • Hybridoma cells have the function of antibody secretion and can proliferate indefinitely.
  • the electrofusion method was used to fuse lymphocytes and myeloma cells in the immunized group for subsequent antibody screening.
  • Subcloning by limiting dilution method The cell line to be subcloned was resuspended from 24 wells and counted. Dilute the cell concentration of each cell line to 5-10 cells/mL, add the diluted cell suspension to a 15cm disposable culture dish, add 0.2mL to each well of a 96-well culture plate, and each well contains 1- 2 pcs. Place the 96-well plate with the cells in a 37°C, 5% CO 2 incubator for culture. After 7-10 days, the subcloning plate is tested and screened according to the growth of the cells, and positive clones are selected to 24 wells for further positive confirmation.
  • hIgG1 is the negative control antibody
  • SN8 is the positive control antibody
  • SN8 is the antibody used in the antibody-conjugated drug potuzumab vedotin developed by Roche Pharmaceuticals (source of sequence reference sequence: US20170362318A).
  • Pertuzumab Vidot has been approved by the FDA for marketing. It can be seen from the results that in the ELISA experiment, the binding power of the three anti-human CD79B mouse monoclonal antibodies mAb015, mAb016 and mAb017 selected by the present disclosure is similar to that of SN8.
  • three anti-human CD79B mouse monoclonal antibodies have the strongest FACS binding capacity, including mAb015, mAb016 and mAb017 (see Figure 8 for specific data).
  • mAb015, mAb016 and mAb017 see Figure 8 for specific data.
  • hIgG1 is the negative control antibody
  • SN8 is the positive control antibody. It can be seen from the results that in the FACS experiment, the binding power of the three anti-human CD79B mouse monoclonal antibodies mAb015, mAb016, and mAb017 selected by the present disclosure is better than that of SN8.
  • the FACS test results of the cross-reactivity of the anti-human CD79B mouse monoclonal antibody are shown in Figure 9.
  • mIgG is a negative control antibody
  • anti-cyno HR008 is an anti-monkey CD79B mouse monoclonal antibody whose antibody sequence is derived from the anti-monkey CD79B mouse monoclonal antibody in patent WO2009012268A1 (clone number 10D10). It can be seen from the results that all anti-human CD79B mouse monoclonal antibodies screened in this disclosure do not recognize monkey CD79B.
  • SPR detection of anti-human CD79B mouse monoclonal antibody was used to detect the affinity between anti-human CD79B antibody and its antigen human CD79B-His.
  • the antigen human CD79B-His protein was immobilized on the CM5 chip.
  • the coupling level was set at 100RU.
  • the running buffer is HBS-EP+ (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20). Flow the diluted antibody through the experimental channel and the control channel at a flow rate of 30 ⁇ l/min for 3 minutes, and dissociate for 5 minutes. Then the regeneration buffer (10 mM glycine buffer. pH 1.5) was run at a flow rate of 30 ⁇ l/min for 30 seconds. The data is analyzed with Biacore 8K evaluation software.
  • the high-affinity hybridoma monoclonal cell line obtained in Example 2 was subjected to variable region amino acid sequence determination, and then recombinantly expressed human and mouse chimeric antibody (cAb), and further antibody identification was performed.
  • the heavy and light chain variable regions of the antibody gene were amplified by reverse transcription PCR, connected to the vector and sequenced to obtain the light and heavy chain sequence of the monoclonal antibody.
  • an RNA purification kit (Qiagen company, article number 74134, see this instruction for the steps) was used to extract total cellular RNA from the active single cell line in Example 2.
  • amino acid residues of the VH/VL CDR of the anti-human CD79B antibody in this disclosure are determined and annotated by the Chothia numbering system.
  • the optimal humanized anti-CD79B monoclonal antibody is selected as the preferred molecule by back mutation screening.
  • This method starts from the published mouse Fab crystal structure model database (such as PDB database) to find the crystal structure similar to the obtained mouse candidate molecule homology, and picks high resolution (such as ) Fab crystal structure to establish a mouse Fab model.
  • the mouse antibody light and heavy chain sequence is aligned with the sequence in the model, and the sequence consistent with the mouse antibody sequence in the model is retained to obtain the structure model of the mouse antibody; the inconsistent amino acids are the possible back mutation sites.
  • DOHH-2 cells are cultured according to the conventional suspension cell method.
  • the complete medium is composed of: 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).
  • the cells were collected by low-temperature centrifugation at 4°C, 1000 rpm, 5 minutes. Resuspend the cells in 10-15ml of FACS buffer chilled on ice.
  • the components of FACS buffer are: phosphate buffered saline (PBS), pH 7.4, plus 2% fetal bovine serum (FBS).
  • PBS phosphate buffered saline
  • FBS fetal bovine serum
  • the FACS buffer was placed on ice to pre-cool. After the cell count was centrifuged, 300,000 cells/well were added to a 96-well plate, the supernatant was discarded by centrifugation, and Fc blocking solution was added at 12.5 ⁇ g/ml (BD, Cat No.: 564220), 100 ⁇ l/well. Block for 10 minutes at room temperature.

Abstract

本公开涉及抗CD79B抗体、其抗原结合片段及其医药用途。进一步地，本公开涉及包含所述抗CD79B抗体CDR区的嵌合抗体、人源化抗体，以及包含人抗CD79B抗体或其抗原结合片段的药物组合物，以及其作为抗癌药物的用途。尤其地，本公开涉及一种人源化的抗CD79B抗体，在制备治疗淋巴瘤(如DLBCL)的药物中的用途。

Description

抗CD79B抗体、其抗原结合片段及其医药用途
本申请要求2019年1月28日提交的中国专利申请《抗CD79B抗体、其抗原结合片段及其医药用途》(申请号201910083330.4)的优先权，通过引用并入此处。
技术领域
本申请涉及一种抗人CD79B抗体及抗原结合片段，包含所述抗CD79B抗体CDR区的嵌合抗体、人源化抗体，以及包含人抗CD79B抗体或其抗原结合片段的药物组合物，以及其作为抗癌药物，特别是治疗淋巴瘤(DLBCL)药物的用途。
背景技术
恶性肿瘤(癌症)是全球第二位死因，仅排在心脏病之后。淋巴瘤是起源于淋巴造血***的恶性肿瘤，是全世界最常见的血液肿瘤。近年来中国淋巴瘤发病率呈上升趋势，目前发病率约为每10万人6.68例。
淋巴瘤分为非霍奇金淋巴瘤(non-Hodgkin’s lymphoma，NHL)和霍奇金淋巴瘤(Hodgkin’s lymphoma，HL)两类。非霍奇金淋巴瘤是一组具有较强异质性的淋巴细胞异常增殖性疾病的总称，其发病率远高于霍奇金淋巴瘤，约占淋巴瘤的80％以上。其中弥漫大B细胞淋巴瘤(DLBCL)是成人最常见的淋巴瘤类型，约占所有非霍奇金淋巴瘤的32.5％；在亚洲人群中，这一比例更高，接近40％。好发于老年患者，中位发病年龄为60-64岁，男性患者略多于女性患者。
目前，弥漫大B细胞淋巴瘤(DLBCL)的一线标准方案为利妥昔单抗联合化疗方案(R-CHOP)。在利妥昔单抗上市之前，以蒽环类为基础的CHOP(环磷酰胺、多柔比星、长春新碱、***)方案是DLBCL的一线标准治疗方案。R-CHOP治疗方案使得DLBCL患者的长期存活率得到明显改善，临床试验结果显示：相比于传统的CHOP方案，R-CHOP方案治疗DLBCL能显著延长患者的中位总生存时间达4.9年，中位无病生存时间超过6.6年，5年无疾病进展生存率从30％提高至54％。但仍有10％至15％的难治患者没有反应，并有20％至30％的患者出现复发。而且不是所有DLBCL患者都适合R-CHOP方案，如80岁以上的高龄患者，他们的体能不允许进行标准的R-CHOP治疗；又如，对于侵袭性更高的淋巴瘤类型以及复发性淋巴瘤，R-CHOP方案可能无效。因此，开发新一代副作用更小的免疫疗法对于DLBCL的治疗是极其必要的。
根据淋巴细胞的起源分类，弥漫大B细胞淋巴瘤(DLBCL)属于B细胞淋巴瘤。B细胞抗原受体(B cell receptor，BCR)复合物是B细胞表面最主要的分子。BCR复合物由识别和结合抗原的膜免疫球蛋白(mIg)和传递抗原刺激信号的Igα(CD79a)和Igβ(CD79B)异源二聚体组成。Igα和Igβ分别为47kDa和37kDa糖蛋白，属于免疫球蛋白超家族成员，编码Igα和Igβ的基因分别称为mb-1和B29。 Igα和Igβ胞膜外区氨基端处均有一个Ig样结构域。Igα和Igβ均可作为蛋白酪氨酸激酶的底物，参与BCR信号转导。BCR广泛表达于B细胞淋巴瘤以及正常B细胞上。鉴于靶向CD20的利妥昔单抗在临床上所取得的成功以及可靠的安全性，开发靶向BCR的治疗方法也应该具有良好的疗效和安全性。
针对仍未被满足的与CD79B相关的医疗需求，目前已有包括罗氏制药的多家国际制药公司在积极研发针对CD79B抗体和相关产品。相关专利如US9085630、WO2009012256、WO2009012268、WO2009099728、WO2014011519、WO2014011521、WO2016090210、WO2016205176、WO2016040856、WO2016021621、WO2017009474、WO2014177615等。
基于CD79B的表达，生成针对CD79B抗原的治疗性抗体是有益的。本领域对于开发有效的抗人CD79B抗体，用于造血***肿瘤治疗或延缓进展，仍有未满足的需求。
发明内容
本公开提供一种抗CD79B抗体或其抗原结合片段，其编码核酸、载体、宿主细胞、抗体药物偶联物、药物组合物，及其用于治疗或延缓癌症(特别是造血***肿瘤)的方法。
第一方面，本公开提供一种抗人CD79B抗体或其抗原结合片段，其包含抗体重链可变区和抗体轻链可变区，其中：
抗体重链可变区，其包含至少1个选自如以下序列所示的决定簇互补区(HCDR)：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、SEQ ID NO：25；和/或
抗体轻链可变区，其包含至少1个选自如以下序列所示的决定簇互补区(LCDR)：SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：16、SEQ ID NO：26。
一些实施方案中，提供一种抗人CD79B抗体或其抗原结合片段，其中：
抗体重链可变区，包含：
(I)分别如SEQ ID NO：7、SEQ ID NO：8和SEQ ID NO：9所示的HCDR1、HCDR2和HCDR3；或
(II)分别如SEQ ID NO：13、SEQ ID NO：14和SEQ ID NO：15所示的HCDR1、HCDR2和HCDR3；或
(III)分别如SEQ ID NO：23、SEQ ID NO：24和SEQ ID NO：25所示的HCDR1、HCDR2和HCDR3；
和/或抗体轻链可变区，包含：
(I)分别如SEQ ID NO：10、SEQ ID NO：11和SEQ ID NO：12所示的LCDR1、 LCDR2和LCDR3；或
(II)分别如SEQ ID NO：16、SEQ ID NO：11和SEQ ID NO：12所示的LCDR1、LCDR2和LCDR3；或
(III)分别如SEQ ID NO：26、SEQ ID NO：11和SEQ ID NO：12所示的LCDR1、LCDR2和LCDR3。
一些具体实施方案中，提供抗人CD79B抗体或其抗原结合片段，其包含选自以下(I)至(II)中的任一项：
(I)重链可变区，其包含分别如SEQ ID NO：7、SEQ ID NO：8和SEQ ID NO：9所示的HCDR1、HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：10、SEQ ID NO：11和SEQ ID NO：12所示的LCDR1、LCDR2和LCDR3；
(II)重链可变区，其包含分别如SEQ ID NO：13、SEQ ID NO：14和SEQ ID NO：15所示的HCDR1、HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：16、SEQ ID NO：11和SEQ ID NO：12所示的LCDR1、LCDR2和LCDR3；
(III)重链可变区，其包含分别如SEQ ID NO：23、SEQ ID NO：24和SEQ ID NO：25所示的HCDR1、HCDR2和HCDR3；和
轻链可变区，其包含分别如SEQ ID NO：26、SEQ ID NO：11和SEQ ID NO：12所示的LCDR1、LCDR2和LCDR3。
一些具体的实施方案中，提供抗人CD79B抗体或其抗原结合片段，其中：
重链可变区，包含：
(I)如SEQ ID NO：3所示的序列或与SEQ ID NO：3具有至少80％、85％、90％、95％、96％、97％、98％或99％同一性的序列；或
(II)如SEQ ID NO：5所示的序列或与SEQ ID NO：5具有至少80％、85％、90％、95％、96％、97％、98％或99％同一性的序列；或
(III)如SEQ ID NO：17所示的序列或与SEQ ID NO：17具有至少80％、85％、90％、95％、96％、97％、98％或99％同一性的序列；
和/或轻链可变区，包含：
(I)如SEQ ID NO：4所示的序列或与SEQ ID NO：4具有至少80％、85％、90％、95％、96％、97％、98％或99％同一性的序列；或
(II)如SEQ ID NO：6所示的序列或与SEQ ID NO：6具有至少80％、85％、90％、95％、96％、97％、98％或99％同一性的序列；或
(III)如SEQ ID NO：18所示的序列或与SEQ ID NO：18具有至少80％、85％、90％、95％、96％、97％、98％或99％同一性的序列。
一些具体的实施方案中，所述抗人CD79B抗体或抗原结合片段的重链可变区如序列SEQ ID NO：3所示，轻链可变区如序列SEQ ID NO：4所示。
在另一些具体的实施方案中，所述抗人CD79B抗体或抗原结合片段的重链可变区如序列SEQ ID NO：5所示，轻链可变区如序列SEQ ID NO：6所示。
在另一些具体的实施方案中，所述抗人CD79B抗体或抗原结合片段的重链可变区如序列SEQ ID NO：17所示，轻链可变区如序列SEQ ID NO：18所示。
在一些具体的实施方案中，抗人CD79B抗体或其抗原结合片段，其中：
重链，包含：
(I)如SEQ ID NO：19所示的序列或与SEQ ID NO：19具有至少80％、85％、90％、95％、96％、97％、98％或99％同一性的序列；或
(II)如SEQ ID NO：21所示的序列或与SEQ ID NO：21具有至少80％、85％、90％、95％、96％、97％、98％或99％同一性的序列；
和/或轻链，包含：
(I)如SEQ ID NO：20所示的序列或与SEQ ID NO：20具有至少80％、85％、90％、95％、96％、97％、98％或99％同一性的序列；或
(II)如SEQ ID NO：22所示的序列或与SEQ ID NO：22具有至少80％、85％、90％、95％、96％、97％、98％或99％同一性的序列；
一些具体的实施方案中，所述抗人CD79B抗体或抗原结合片段的重链如序列SEQ ID NO：19所示，轻链如序列SEQ ID NO：20所示。
在另一些具体的实施方案中，所述抗人CD79B抗体或抗原结合片段的重链如序列SEQ ID NO：21所示，轻链如序列SEQ ID NO：22所示。
一些实施方案中，提供如上所述的抗人CD79B抗体或其抗原结合片段，其为鼠源抗体或其片段。
一些具体实施方案中，所述鼠源抗CD79B抗体或其抗原结合片段的轻链可变区包含鼠源κ、λ链或其变体的轻链FR区和/或轻链恒定区。
一些具体实施方案中，所述鼠源抗CD79B抗体或其抗原结合片段包含鼠源IgG1、IgG2、IgG3、IgG4或其变体的重链FR区和/或重链恒定区。
一些实施方案中，提供如上所述的抗人CD79B抗体或其抗原结合片段，其为嵌合抗体或其片段。一些具体实施方案中，所述嵌合抗CD79B抗体或其抗原结合片段包含人源κ、λ链或其变体的轻链FR区和/或轻链恒定区，和/或人源IgG1、IgG2、IgG3或IgG4或其变体的重链FR区和/或重链恒定区。
一些实施方案中，提供一种如上所述的抗人CD79B抗体或其抗原结合片段，其为人源化抗体或其片段、人抗体或其片段。
一些实施方案中，提供一种如上所述的抗人CD79B人源化抗体或其抗原结合片段，其中轻链序列为SEQ ID NO：20或其变体序列所示；所述的变体序列在轻链包含1、2、3、4、5、6、7、8、9或10个氨基酸变化，或与SEQ ID NO：20具有至少80％、85％、90％、95％、96％、97％、98％或99％同一性。重链序列为SEQ ID NO：19或其变体序列所示；所述变体序列在重链包含1、2、3、4、5、 6、7、8、9或10个氨基酸变化，或与SEQ ID NO：19具有至少80％、85％、90％、95％、96％、97％、98％或99％同一性。
一些实施方案中，提供一种如上所述的抗人CD79B的人源化抗体或其抗原结合片段，其中轻链序列为SEQ ID NO：22或其变体序列所示，或与SEQ ID NO：22具有至少80％、85％、90％、95％、96％、97％、98％或99％同一性；所述的变体序列在轻链包含1、2、3、4、5、6、7、8、9或10个氨基酸变化。重链序列为SEQ ID NO：21或其变体序列所示；所述变体序列在重链包含1、2、3、4、5、6、7、8、9或10个氨基酸变化，或与SEQ ID NO：21具有至少80％、85％、90％、95％、96％、97％、98％或99％同一性。
一些实施方案中，一种如上所述的抗CD79B的人抗体或其片段，其进一步包含人IgG1、IgG2、IgG3或IgG4或其变体的恒定区。一些具体实施方案中，包含人IgG1或IgG2恒定区。
一些实施方案中，一种如上所述的抗人CD79B的人源化抗体或其片段，其进一步包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG1、IgG2或IgG4重链FR区，更优选包含人源IgG1或IgG2重链FR区。
一些实施方案中，一种如上所述的抗人CD79B抗体或其抗原结合片段，其中所述的抗原结合片段选自Fab、Fv、sFv、F(ab’) 2、线性抗体、单链抗体、scFv、sdAb、sdFv、纳米抗体、肽抗体(peptibody)、结构域抗体和多特异性抗体(双特异性抗体、双链抗体(diabody)、三链抗体(triabody)和四链抗体(tetrabody)、串联二-scFv、串联三-scFv)。
一些实施方案中，提供一种分离的单克隆抗体或其抗原结合片段，能与上述的单克隆抗体或其抗原结合片段竞争结合人CD79B或其表位。
本公开中抗人CD79B抗体的VH/VL CDR的氨基酸残基由Chothia编号***确定并注释。
第二方面，本公开提供一种编码如上所述的抗人CD79B抗体或其抗原结合片段的多核苷酸，多核苷酸可以是DNA或RNA。
第三方面，本公开提供一种含有如上所述的多核苷酸的表达载体，表达载体可以是真核表达载体、原核表达载体、病毒载体。
第四方面，本公开提供一种用如上所述的表达载体转化的宿主细胞，其可以是真核细胞、原核细胞。
一些实施方案中，所述宿主细胞为细菌、酵母菌、哺乳动物细胞。一些具体实施方案中，所述宿主细胞为大肠杆菌、毕赤酵母、中国仓鼠卵巢(CHO)细胞或人胚肾(HEK)293细胞。
第五方面，本公开提供一种抗体药物偶联物。
根据本公开的抗体药物偶联物包含或由以下组成：抗体、接头、药物。
在具体的实施方案中，根据本公开的抗体药物偶联物是通过接头共价偶联至 药物的抗体。
一些实施方案中，所述抗体药物偶联物含有细胞毒剂。一些具体实施方案中，所述细胞毒剂选自毒素、化疗剂、抗生素、放射性同位素和溶核酶。
第六方面，本公开提供一种用于制备抗人CD79B抗体或其抗原结合片段的方法包括：在如前所述的宿主细胞中表达该抗体或其抗原结合片段，并自该宿主细胞中分离该抗体或其抗原结合片段。
第七方面，本公开提供一种组合物，例如药物组合物，其含有治疗有效量的如上所述的抗人CD79B抗体或其抗原结合片段和可药用的赋形剂、稀释或载体。
在一些具体实施方式中，所述药物组合物单位剂量中可含有0.01至99重量％的抗人CD79B抗体或其抗原结合片段或药物组合物单位剂量中含抗CD79B抗体或其抗原结合片段的量为0.1mg至2000mg，在一些具体实施方式中为1mg至1000mg。
第八方面，本公开进一步提供选自以下的任一项或组合在制备药物中的用途：根据本公开的抗人CD79B抗体或其抗原结合片段、根据本公开的药物组合物、根据本公开的抗体药物偶联物；其中，所述药物用于所述药物或药物组合物用于治疗增殖性疾病或延缓增殖性疾病进展；所述增殖性疾病可以是癌症或肿瘤。在一些实施方案中，所述癌症或肿瘤是淋巴瘤或白血病。在具体的实施方案中，所述淋巴瘤选自：弥漫大B细胞淋巴瘤、非何杰金氏淋巴瘤、小淋巴细胞性淋巴瘤、套细胞淋巴瘤。在具体的实施方案中，所述非何杰金氏淋巴瘤选自：攻击性NHL、复发性攻击性NHL、复发性无痛性NHL、顽固性NHL、顽固性无痛性NHL。在具体的实施方案中，所述白血病选自：慢性淋巴细胞性白血病、毛细胞白血病、急性淋巴细胞性白血病。
第九方面，本公开提供一种治疗或预防增殖性疾病或延缓增殖性疾病进展的方法，该方法包括给予受试者治疗或延缓疾病有效量的根据本公开的抗人CD79B抗体或其抗原结合片段、或根据本公开的药物组合物、或根据本公开的抗体-药物偶联物；其中，所述增殖性疾病可以是癌症或肿瘤。在一些实施方案中，所述癌症或肿瘤是淋巴瘤或白血病。在具体的实施方案中，所述淋巴瘤选自：弥漫大B细胞淋巴瘤、非何杰金氏淋巴瘤、小淋巴细胞性淋巴瘤、套细胞淋巴瘤。在具体的实施方案中，所述非何杰金氏淋巴瘤选自：攻击性NHL、复发性攻击性NHL、复发性无痛性NHL、顽固性NHL、顽固性无痛性NHL。在具体的实施方案中，所述白血病选自：慢性淋巴细胞性白血病、毛细胞白血病、急性淋巴细胞性白血病。
附图说明
图1：用人CD79B ECD-hFc蛋白免疫Balb/c小鼠的血清效价ELISA检测结果。
图2：用人CD79B ECD-hFc蛋白免疫Balb/c小鼠的血清效价FACS检测结果。
图3：用人CD79B ECD-hFc蛋白免疫SJL小鼠的血清效价ELISA检测结果。
图4：用人CD79B ECD-hFc蛋白免疫SJL小鼠的血清效价FACS检测结果。
图5：用人CD79B ECD-his蛋白免疫SJL小鼠的血清效价ELISA检测结果。
图6：用人CD79B ECD-his蛋白免疫SJL小鼠的血清效价FACS检测结果。
图7：抗人CD79B小鼠单克隆抗体ELISA的检测结果，其中hIgG1为阴性对照抗体，SN8为阳性对照抗体。
图8：抗人CD79B小鼠单克隆抗体FACS的检测结果，其中hIgG1为阴性对照抗体，SN8为阳性对照抗体。
图9：抗人CD79B小鼠单克隆抗体交叉反应性的FACS检测结果，其中mIgG为阴性对照抗体；抗cyno HR008为抗猴CD79B小鼠单克隆抗体，其抗体序列来源于专利WO2009012268A1中的抗猴CD79B小鼠单克隆抗体(克隆号10D10)，作为阳性对照抗体。
具体实施方式
发明详述
术语
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除显而易见在本文件中的它处另有明确定义，否则本文使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
本公开所用氨基酸三字母代码和单字母代码如J.Biol.Chem，243，p3558(1968)中所述。
“CD79B”指任何脊椎动物来源(包括哺乳动物，诸如灵长类(例如人、猕猴(cyno))和啮齿类(例如小鼠和大鼠))的任何CD79B。术语“CD79B”涵盖“全长”、未加工的CD79B以及自细胞中加工得到的任何形式的CD79B。该术语还涵盖天然存在的CD79B变体，例如剪接变体、等位变体和同种型(isoform)。本文所述CD79B多肽可以从多种来源分离，诸如人组织类型或其它来源，或者通过重组或合成方法制备。
本公开所述的术语“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区 (HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(VL)和重链可变区(VH)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1，LCDR2，和LCDR3；重链的3个CDR区指HCDR1，HCDR2和HCDR3。所述的抗体或抗原结合片段的VL区和VH区的CDR氨基酸残基在数量和位置符合已知的Chothia(ABM)编号规则。
术语“人抗体”或“重组人抗体”包括通过重组方法制备、表达、创建或分离的人抗体，所涉及的技术和方法在本领域中是熟知的，诸如：
(1)从人免疫球蛋白基因的转基因、转染色体动物(例如小鼠)或由其制备的杂交瘤中分离的抗体；
(2)从经转化以表达抗体的宿主细胞如转染瘤中分离的抗体；
(3)从重组组合人抗体文库中分离的抗体；以及
(4)通过将人免疫球蛋白基因序列剪接到其他DNA序列等方法制备、表达、创建或分离的抗体。
此类重组人抗体包含可变区和恒定区，这些区域利用特定的由种系基因编码的人种系免疫球蛋白序列，但也包括随后诸如在抗体成熟过程中发生的重排和突变。
术语“鼠源抗体”在本公开中为根据本领域知识和技能制备的对人CD79B或其表位的单克隆抗体。制备时用CD79B抗原(或其表位)注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本公开一个具体的实施方案中，所述的鼠源CD79B抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区。
术语“人抗体”包括具有人种系免疫球蛋白序列的可变和恒定区的抗体。本公开的人抗体可包括不由人种系免疫球蛋白序列编码的氨基酸残基(如通过体外随机或位点特异性诱变或通过体内体细胞突变所引入的突变)。然而，术语“人抗体”不包括这样的抗体，即其中已将衍生自另一种哺乳动物物种(诸如小鼠)种系的CDR序列移植到人骨架序列上(即“人源化抗体”)。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将非人物种的CDR序列移植到人的抗体可变区框架中产生的抗体。可以克服嵌合抗体由于携带大量非人物种蛋白成分，从而诱导的强烈的免疫应答反应。为避免在免疫原性下降的同时引起活性的下降，可对所述的人抗体可变区可进行最少反向突变，以保持活性。
术语“嵌合抗体(chimeric antibody)”，是将第一物种抗体的可变区与第二物种抗体的恒定区融合而成的抗体，可以减轻第一物种抗体诱发的免疫应答反应。 建立嵌合抗体，要选建立分泌第一物种特异性单抗的杂交瘤，然后从第一物种(例如小鼠)杂交瘤细胞中克隆可变区基因，再根据需要克隆第二物种(如人)抗体的恒定区基因，将第一物种可变区基因与第二物种恒定区基因连接成嵌合基因后***载体中，最后在真核工业***或原核工业***中表达嵌合抗体分子。第二物种(例如人)抗体的恒定区可选自人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG2或IgG4重链恒定区，或者使用氨基酸突变后无ADCC(antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用)毒性的IgG1。
“抗原结合片段”，指保留了完整抗体的抗原结合活性的任何片段。具体而言，可以提及但不限于Fab片段，Fab’片段，F(ab’)2片段，以及与人CD79B结合的Fv片段sFv片段。Fv片段含有抗体重链可变区和轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般地，Fv抗体还包含在VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。也可以用不同的连接物将两个抗体可变区连接成一条多肽链，称为单链抗体(single chain antibody)或单链Fv(sFv)。
本公开的术语“与CD79B结合”，指能与CD79B或其表位相互作用。所述CD79B或其表位可以是人源的。本公开的术语“抗原结合位点”指抗原上线性的或不连续的，由本公开抗体或抗原结合片段识别的线性位点或三维空间位点。
术语“表位”是指抗原上与免疫球蛋白或抗体特异性结合的位点。表位可以由相邻的氨基酸、或通过蛋白质的三级折叠而并列的不相邻的氨基酸形成。由相邻的氨基酸形成的表位通常在暴露于变性溶剂后保持，而通过三级折叠形成的表位通常在变性溶剂处理后丧失。表位通常以独特的空间构象包括至少3-15个氨基酸。确定什么表位由给定的抗体结合的方法在本领域中是熟知的，包括免疫印迹和免疫沉淀检测分析等。确定表位的空间构象的方法包括本领域中的技术和本文所述的技术，例如X射线晶体分析法和二维核磁共振等。
“特异性结合”、“选择性结合”是指抗体与预定的抗原上的表位结合。通常，当使用重组人CD79B或其表位作为分析物并使用抗体作为配体，在仪器中通过表面等离子体共振(SPR)技术测定时，抗体以大约低于10 -7M或甚至更小的平衡解离常数(K D)与预定的抗原或其表位结合，并且其与预定抗原或其表位结合的亲和力(affinity)是其与预定抗原(或其表位)或紧密相关的抗原之外的非特异性抗原(如BSA等)结合的亲和力的至少两倍。
“交叉反应”是指本公开的抗体与来自不同物种的CD79B结合的能力。例如，结合人CD79B的本公开的抗体也可以结合另一物种的CD79B。交叉反应性是通过在结合测定(例如SPR和ELISA)中检测与纯化抗原的特异性反应性，或与生理表达CD79B的细胞的结合或功能性相互作用来测量。确定交叉反应性的方法包括如本文所述的标准结合测定，例如表面等离子体共振分析，或流式细胞术。
“抑制”或“阻断”可互换使用，并涵盖部分和完全抑制/阻断这两者。对CD79B的抑制/阻断优选地降低或改变无抑制或阻断的情况下发生CD79B结合时出现活性的正常水平或类型。抑制和阻断也旨在包括与抗CD79B抗体接触时，与未与抗CD79B抗体接触的CD79B相比，任何可测量的CD79B结合亲和力降低。
“抑制生长”(例如涉及细胞)旨在包括细胞生长任何可测量的降低。
生产和纯化抗体或抗原结合片段的方法在现有技术中熟知和能找到，如冷泉港的抗体实验技术指南(5-8章和15章)。如，可以用人CD79B或其片段免疫小鼠，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人FR区。人FR种系序列可以从ImMunoGeneTics(IMGT)的网站http：//imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
本公开工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链(SEQ ID NO：20)和轻链(SEQ ID NO：21)的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达***会导致抗体的糖基化，特别是在Fc区的高度保守N端。通过表达与人源抗原特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化、收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛，离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
本公开的抗体指单克隆抗体。本公开所述的单克隆抗体(mAb)，指由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的、原核的或噬菌体的克隆细胞株。单克隆抗体或抗原结合片段可以用如杂交瘤技术、重组技术、噬菌体展示技术，合成技术(如CDR-嫁接)，或其它现有技术进行重组得到。
可使用本领域技术人员已知的常规技术，就与相同表位的结合竞争性筛选抗体。例如，可进行竞争和交叉竞争研究，以获得彼此竞争或交叉竞争与抗原结合的抗体。基于它们的交叉竞争来获得结合相同表位的抗体的高通量方法描述于国际专利公开WO03/48731中。因此，可使用本领域技术人员已知的常规技术，获得与本公开的抗体分子竞争结合CD79B上的相同表位的抗体及其抗原结合片段。
“给予”、“施用”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂、组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“给予”、“施用”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”、“施用”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处 理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防性处理、或预防性措施、研究和诊断应用。
“治疗”意指给予受试者内用或外用治疗剂，诸如包含本公开的任一种抗体或其抗原结合片段或其偶联物的组合物，所述受试者已经患有、疑似患有、倾向于患有一种或多种疾病或其症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗受试者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，无论是通过诱导这类症状退化还是抑制这类症状发展到任何临床右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如受试者的疾病状态、年龄和体重，以及药物在受试者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽本公开的实施方案(例如治疗方法或制品)在缓解某个受试者中目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的受试者中应当减轻目标疾病症状。
“有效量”包含足以改善或预防医学病症的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定受试者或兽医学受试者的有效量可依据以下因素而变化：如待治疗的病症、受试者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“同一性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同一性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100％的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源。一般而言，当比对两个序列而得到最大的同一性百分率时进行比较。
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括其后代。因此，术语“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
“药物组合物”表示含有一种或多种本文所述抗体或抗原结合片段或偶联物、或 其生理学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
实施例
以下结合实施例用于进一步描述，但这些实施例并非限制的范围。
实施例或测试例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。参见Sambrook等，分子克隆，实验室手册，冷泉港实验室；当代分子生物学方法，Ausubel等著，Greene出版协会，Wiley Interscience，NY。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1.蛋白抗原的克隆表达
用本领域公知的重叠延伸PCR方法构建抗体(包含轻、重链)和抗原，将重叠延伸PCR得到的DNA片段用HindIII/BstBI这两个酶切位点***到表达载体pEE6.4((Lonza Biologics)中，在293F细胞(Invitrogen，Cat#R790-07)中表达得到。所得重组蛋白用于免疫或筛选。人CD79B基因序列来源于NCBI(NP_000617.1)，其细胞外区域(ECD)包含159个氨基酸(Met1-Asp159)。
＞人CD79B细胞外区域(ECD)和人Fc区域融合蛋白(人CD79B ECD-hFc)的氨基酸序列：
Figure PCTCN2020073803-appb-000001
＞人CD79B细胞外区域(ECD)和His标签的融合蛋白(人CD79B ECD-His)的氨基酸序列：
Figure PCTCN2020073803-appb-000002
实施例2.小鼠单克隆抗体的制备
1、小鼠免疫和血清效价检测
以人CD79B细胞外区域(ECD)和人Fc区域融合蛋白(人CD79B ECD-hFc)以及人CD79B细胞外区域(ECD)和His标签的融合蛋白(人CD79B ECD-His)分别作为免疫原，通过腹腔注射法对Balb/c和SJL小鼠进行免疫，刺激小鼠体内产生针对人CD79B细胞外区域(ECD)的抗体。
实验步骤如下：
1)腹腔注射免疫法。根据免疫程序计算出该次免疫所需的抗原量。蛋白抗原按照要求用PBS稀释抗原到相应浓度，随后进行抗原的乳化。将乳化好的抗原和佐剂混合物转移到2.0mL无菌注射器中，注意排空气泡。右手抓住小鼠尾巴，左手的大拇指和食指轻轻抓住小鼠的头颈部皮肤，腹腔向上，用75％酒精棉球擦拭小鼠右侧腹部注射部位。将事先吸好抗原药物的注射器针尖斜面向上，小鼠头部向下，平行刺入皮内后，注射器与腹腔呈45度角刺入小鼠腹腔，缓慢注入抗原和佐剂混合物，免疫完成后进行至少2h观察。
2)小鼠血清的收集。将每只小鼠对应的血清管号做好标记，检查小鼠耳钉号，单手抓取小鼠，通过小鼠面部颌下静脉采取大约100μl全血，将收集的全血样品室温静置大约2h后离心收集离心管上部血清。一周内可将血清存放于4℃冰箱，用于抗体效价等相关实验检测。若长期保存血清可置于-80℃冰箱，避免反复冻融。
3)免疫小鼠ELISA血清效价测定。实验开始前将96孔板做好相应的标记，以1μg/mL抗原浓度，每孔50μl，4℃冰箱过夜包被。次日，将前天包被好的抗原板取出，洗板机清洗一次(清洗液：1×PBST)。清洗后以1×PBST配置的1％BSA封闭液37℃封闭1小时。1×PBST清洗液洗板3次后，加入不同稀释浓度的待检血清37℃温箱，孵育1小时。1×PBST清洗液洗板3次后，加入100μl 1∶5000稀释的羊抗鼠二抗，37℃温箱孵育0.5小时洗板后，取TMB显色液A液和B液1∶1比例混合后显色。15分钟用1N盐酸终止显色反应。在Spectra Max M5多功能读板机上，检测450nm的荧光值。
4)免疫小鼠FACS血清效价测定。DoHH2细胞或猴外周血单核细胞悬液离心后以含0.1％BSA的PBS重悬细胞后计数，加入各组免疫小鼠的待测血清，室温孵育60分钟后清洗细胞三次后加入抗小鼠IgG Fc-FITC二抗，避光室温孵育30分钟后清洗细胞三次，用含0.1％BSA的PBS轻轻重悬细胞，上机检测。
各组小鼠血清效价ELISA和FACS检测结果见图1至图7。
人CD79b ECD-hFc蛋白共免疫5只Balb/c小鼠，编号为5491、5492、5493、5494、5495。血清效价ELISA检测结果见图1。结果表明，小鼠免疫血清效价达到1∶100K以上。小鼠血清FACS检测结果见图2，可见小鼠血清中产生的抗体可特异性识别DoHH2细胞表面的CD79B蛋白。
人CD79b ECD-hFc蛋白共免疫5只SJL小鼠，编号为5496、5497、5498、5499、5500。血清效价ELISA检测结果见图3。结果表明，小鼠免疫血清效价达到1∶100K 以上。小鼠血清FACS检测结果见图4，可见小鼠血清中产生的抗体可特异性识别DoHH2细胞表面的CD79B蛋白。
人CD79b ECD-his蛋白共免疫5只SJL小鼠，编号为5726、5727、5728、5729、5730。血清效价ELISA检测结果见图5。结果表明，小鼠免疫血清效价达到1∶10K以上。小鼠血清FACS检测结果见图6，可见小鼠血清中产生的抗体可特异性识别DoHH2细胞表面的CD79B蛋白。
通过以上结果可知，免疫的小鼠中产生了针对CD79B的特异性抗体，以上小鼠可用于细胞融合以产生能够分泌针对CD79B的特异性抗体的杂交瘤细胞系。
2、杂交瘤制备和抗体筛选
细胞融合是在自发或人工诱导下，促使小鼠的淋巴细胞与骨髓瘤细胞SP2/0(ATCC，CCL-121 TM)融合成杂交瘤细胞，杂交瘤细胞既具有抗体分泌功能又能无限增殖。利用电融合方法对免疫组小鼠的淋巴细胞和骨髓瘤细胞进行融合，用于后续抗体筛选。
1)电融合实验。融合前一周将SP2/0细胞于10％DMEM培养基中进行扩大培养。将已处死的小鼠于生物安全柜中摘取脾脏和***于培养皿中进行冲洗研磨，收集淋巴细胞。将SP2/0和淋巴细胞按比例混合，启动电融合仪，设置程序，进行融合。融合后细胞铺于96孔板中，37℃，5％CO 2培养箱中培养，每天观察细胞状态，融合后5天统计细胞融合率。融合后9-14天对融合的杂交瘤细胞进行筛选，挑选阳性孔细胞于24孔板中扩大培养。
2)有限稀释法进行亚克隆。将需要亚克隆的细胞株自24孔培养孔中重悬起来，进行计数。稀释每株细胞株的细胞浓度至5-10个/mL，将稀释好的细胞悬液加入15cm一次性培养皿中，于96孔培养板中每孔加0.2mL，每孔含细胞为1-2个。将铺好细胞的96孔板放进37℃，5％CO 2培养箱中培养。7-10天后根据细胞的生长情况对亚克隆板进行检测筛选，并挑选阳性克隆至24孔进行进一步阳性确认。
3)ELISA筛选。实验开始前将96孔板做好相应的标记，以1μg/mL抗原浓度，每孔50μl，4℃冰箱过夜包被。次日，将前天包被好的抗原板取出，洗板机清洗一次(清洗液：1x PBST)。清洗后以1×PBST配置的1％BSA封闭液37℃封闭1小时。1×PBST清洗液洗板3次后，加入50μl待检细胞上清37℃温箱，孵育1小时。1×PBST清洗液洗板3次后，加入100μl 1∶5000稀释的羊抗鼠二抗，37℃温箱孵育0.5小时洗板后，取TMB显色液A液和B液1∶1比例混合后显色。15分钟用1N盐酸终止显色反应。在Spectra Max M5多功能读板机上，检测450nm的荧光值。
4)FACS筛选。DOHH2细胞悬液离心后以含0.1％BSA的PBS重悬细胞后计数，加入待测细胞上清，室温孵育60分钟后清洗细胞三次后加入抗小鼠IgG Fc-FITC二抗，避光室温孵育30分钟后清洗细胞三次，用含0.1％BSA的PBS轻 轻重悬细胞，上机检测。
5)杂交瘤阳性克隆鉴定。通过对小鼠脾细胞的融合和亚克隆筛选后，我们获得多个针对人CD79B抗原的特异性抗体，对其中ELISA和FACS结合力最好的17株杂交瘤进行抗体生产纯化。抗人CD79B杂交瘤阳性克隆细胞培养上清ELISA检测结果见表1。抗人CD79B杂交瘤阳性克隆细胞培养上清FACS检测结果见表2。均使用mIgG作为阴性对照。
表1.抗人CD79B杂交瘤阳性克隆的ELISA检测结果
抗体编号 克隆号 检测结果(OD450)
阴性对照 mIgG 0.05
mAb001 12A11-1G1 3.26
mAb002 19F10-1D7 3.69
mAb003 51E5G6 3.02
mAb004 67B10C1 3.41
mAb005 78A9F4 3.73
mAb006 48F11D6 3.34
mAb007 61A11F1 3.40
mAb008 63G2A2 3.56
mAb009 75F1E2 3.57
mAb010 66G3E7 3.83
mAb011 66E12H3 3.41
mAb012 73A8F3 3.45
mAb013 74C4F3 3.31
mAb014 70B8B3 3.10
mAb015 83B2G2 3.41
mAb016 83C2D4 3.46
mAb017 86F11F6 3.80
表2.抗人CD79B杂交瘤阳性克隆的FACS检测结果
Figure PCTCN2020073803-appb-000003
Figure PCTCN2020073803-appb-000004
3、小鼠单克隆抗体的生产、纯化及鉴定
1)小鼠单克隆抗体生产和纯化。显微镜下观察需要抗体生产的杂交瘤细胞，长至≥70％以上且细胞状态良好，收集细胞并用Countstar IC1000型细胞计数仪进行计数。用配制好的培养基将细胞浓度调至1×10 5至5×10 5个/mL，转移至Roller Bottle。将转好细胞的Roller Bottle送到滚瓶培养箱中37℃培养10-15天，每天观察细胞生长状况，待培养液变橙黄且透明，取出待纯化。细胞上清通过Protein A柱子进行抗体纯化，按照常规方法操作。
2)抗人CD79B小鼠单克隆抗体ELISA检测。实验开始前将96孔板做好相应的标记，以1μg/mL抗原浓度，每孔50μl，4℃冰箱过夜包被。次日，将前天包被好的抗原板取出，洗板机清洗一次(清洗液：1×PBST)。清洗后以1×PBST配置的1％BSA封闭液37℃封闭1小时。1×PBST清洗液洗板3次后，加入50μl按照100nM，1∶10稀释过的抗体，放入37℃温箱，孵育1小时。×PBST清洗液洗板3次后，加入100μl 1∶5000稀释的羊抗鼠二抗，37℃温箱孵育0.5小时。洗板后，取TMB显色液A液和B液1∶1比例混合后显色。15分钟用1N盐酸终止显色反应。在Spectra Max M5多功能读板机上，检测450nm的荧光值。其中，有三株抗人CD79B小鼠单克隆抗体的ELISA结合力最强，包括mAb015、mAb016和mAb017(具体数据见图7)。其中hIgG1为阴性对照抗体，SN8为阳性对照抗体。SN8是罗氏制药开发的抗体偶联药物波妥珠单抗维多丁(polatuzumab vedotin)中所使用的抗体(序列参考序列来源：US20170362318A)。目前波妥珠单抗维多丁已被FDA批准上市。从结果可以看出，在ELISA实验中，本公开优选出的三株抗人CD79B小鼠单克隆抗体mAb015、mAb016和mAb017的结合力与SN8类似。
3)抗人CD79B小鼠单克隆抗体FACS检测。DOHH2细胞悬液离心后以含0.1％BSA的PBS重悬细胞后计数，加入100μl按照100nM，1∶10稀释过的抗体，室温孵育1小时。清洗细胞三次后加入抗小鼠IgG Fc-FITC二抗，避光室温孵育30分钟后清洗细胞三次，用含0.1％BSA的PBS轻轻重悬细胞，上机检测。其中，有3个抗人CD79B小鼠单克隆抗体的FACS结合力最强，包括mAb015、mAb016和mAb017(具体数据见图8)。其中hIgG1为阴性对照抗体，SN8为阳性对照抗体。从结果可知，在FACS实验中，本公开优选出的三株抗人CD79B小鼠单克隆抗体mAb015、mAb016和mAb017的结合力均优于SN8。
4)抗人CD79B小鼠单克隆抗体交叉反应性FACS检测。采用瞬时转染的方 法得到293F-cynoCD79B细胞，细胞悬液离心后以含0.1％BSA的PBS重悬细胞后计数，加入100μl抗体，浓度分别为10μg/mL和1μg/mL。室温孵育1小时。清洗细胞三次后加入抗小鼠IgG Fc-FITC二抗，避光室温孵育30分钟后清洗细胞三次，用含0.1％BSA的PBS轻轻重悬细胞，上机检测。抗人CD79B小鼠单克隆抗体交叉反应性FACS检测结果见图9。其中mIgG为阴性对照抗体，抗-cyno HR008为抗猴CD79B小鼠单克隆抗体，其抗体序列来源于专利WO2009012268A1中的抗猴CD79B小鼠单克隆抗体(克隆号10D10)。由结果可知，本公开所筛选到的所有抗人CD79B小鼠单克隆抗体均不识别猴CD79B。
5)抗人CD79B小鼠单克隆抗体SPR检测。采用表面等离子共振技术(surface plasmon resonance，SPR)检测抗人CD79B抗体与其抗原人CD79B-His之间的亲和力。将抗原人CD79B-His蛋白固定化至CM5芯片。偶联水平设定在100RU。运行缓冲液为HBS-EP+(10mM HEPES、150mM NaCl、3mM EDTA、0.05％表面活性剂P20)。将稀释好的抗体在30μl/min的流速下流过实验通道和对比通道3分钟，解离5分钟。然后再生缓冲液(10mM甘氨酸缓冲液。pH1.5)在30μl/min的流速下运行30秒。数据用Biacore 8K evaluation software软件进行分析。
实施例3.小鼠单克隆抗体可变区氨基酸序列的测定
将实施例2中得到的高亲和力的杂交瘤单克隆细胞株进行可变区氨基酸序列测定，然后重组表达出人鼠嵌合抗体(chimeric antibody，cAb)，再进行进一步的抗体鉴定。用逆转录PCR扩增抗体基因的重链和轻链可变区，连接到载体测序得到单克隆抗体轻重链序列。首先采用RNA纯化试剂盒(Qiagen公司，货号74134，步骤见此说明书)提取实施例2中活性好的单细胞株的细胞总RNA。然后使用Invitrogen公司的货号为18080-051cDNA合成试剂盒制备cDNA单链，即Oligo-dT引物cDNA反转录。以此为模版，采用PCR方法合成抗体轻重链可变区序列，PCR产物克隆到TA载体pMD-18T，然后送去测序。将得到的抗体轻重链序列分别克隆到表达载体(方法见实施例1)，表达重组单克隆抗体，验证活性(方法见实施例2)后，进行人源化工作。
本公开中抗人CD79B抗体的VH/VL CDR的氨基酸残基由Chothia编号***确定并注释。
＞小鼠杂交瘤细胞单克隆抗体mAb015重链可变区：
Figure PCTCN2020073803-appb-000005
＞小鼠杂交瘤细胞单克隆抗体mAb015轻链可变区：
Figure PCTCN2020073803-appb-000006
＞小鼠杂交瘤细胞单克隆抗体mAb017的重链可变区：
Figure PCTCN2020073803-appb-000007
＞小鼠杂交瘤细胞单克隆抗体mAb017的轻链可变区：
Figure PCTCN2020073803-appb-000008
＞小鼠杂交瘤细胞单克隆抗体mAb016的重链可变区：
Figure PCTCN2020073803-appb-000009
＞小鼠杂交瘤细胞单克隆抗体mAb016的轻链可变区：
Figure PCTCN2020073803-appb-000010
依据Chothia编号规则的鼠源的CDR序列如表3所示：
表3.鼠源抗人CD79B抗体的CDR序列
Figure PCTCN2020073803-appb-000011
实施例4.抗人CD79B抗体的人源化
将实施例3得到的鼠源抗CD79B单克隆抗体轻重链序列在抗体数据库里进行同源性比较后，建立人源化抗体模型。根据模型选择回复突变筛选最优的人源化抗CD79B单克隆抗体为优选分子。该方法从已经发表的小鼠Fab晶体结构模型数据库(比如PDB数据库)中查找与所得鼠源候选分子同源性相似的晶体结构开始，挑取高分辨率(比如
Figure PCTCN2020073803-appb-000012
)的Fab晶体结构，建立小鼠Fab模型。将鼠源抗体轻重链序列和模型中的序列比对，保留和模型中和鼠源抗体序列一致的序列，得到鼠抗体的结构模型；其中不一致的氨基酸为可能的回复突变位点。用Swiss-pdb viewer软件运行鼠抗体结构模型，优化能量(最小化)。将模型中除CDR外的不 同氨基酸位点进行回复突变，将所得的突变抗体(人源化)和人源化之前抗体对比进行活性检测。保留活性好的人源化抗体。之后，对CDR区域优化，包括避免糖基化、脱酰胺化、氧化位点等。用基因克隆、重组表达的方法分别克隆、表达、纯化上述抗体，经SPR等检测，最终选出活性保持最好的人源化抗体hAb015和hAb017，具体数据见表4。人源化抗体hAb015和hAb017保持了跟小鼠单克隆抗体相似的亲和力和相关功能。
表4.人源化抗CD79B抗体的鉴定结果
Figure PCTCN2020073803-appb-000013
(注：/表示未进行检测)
人源化抗体hAb015和hAb017的序列见下。
＞hAb015人源化抗体的重链序列：
Figure PCTCN2020073803-appb-000014
＞hAb015人源化抗体的轻链序列：
Figure PCTCN2020073803-appb-000015
＞hAb017人源化抗体的重链序列：
Figure PCTCN2020073803-appb-000016
Figure PCTCN2020073803-appb-000017
＞hAb017人源化抗体的轻链序列：
Figure PCTCN2020073803-appb-000018
实施例5.抗CD79B抗体的内吞作用
为了检测本公开中的CD79B抗体结合人CD79B后，是否能够和人CD79B一起内吞到细胞内，用高表达人CD79B蛋白的DOHH-2细胞(DSMZ，ACC 47)进行细胞内吞实验以评估抗体的内吞能力。
DOHH-2细胞按照常规悬浮细胞的方法进行培养，完全培养基的成分为：RPMI 1640培养基(GIBCO，Cat No.：11835-030)，加10％(v/v)胎牛血清(FBS)(GIBCO，CatNo.：10099-141)和青霉素/链霉素(GIBCO，Cat No.：15070-063)。
实验时4℃低温离心收集细胞，1000rpm，5分钟。将细胞重悬到10-15ml在冰上预冷的FACS缓冲液中。FACS缓冲液的成分为：磷酸盐缓冲液(PBS)，pH7.4，加2％胎牛血清(FBS)。整个实验过程中，FACS缓冲液放在冰上预冷。细胞计数离心后以300,000细胞/孔加入96孔板中，离心弃去上清后加入Fc封闭液，12.5μg/ml(BD，Cat No.：564220)，100μl/孔。在室温下封闭10分钟。然后加入20μg/ml的待测CD79B抗体至对应的孔中，4℃避光孵育1小时。用预冷的PBS缓冲液洗2遍以除去未结合的抗体。加入细胞完全培养基(具有10％胎牛血清的RPMI 1640培养基)，在37℃，5％CO 2下孵育0小时、1小时、2小时、4小时。离心弃去上清后加入100μl/孔2％PFA缓冲液，重悬细胞后放置10分钟。然后用FACS缓冲液清洗3遍，然后加入100μl二抗溶液(荧光标记羊抗人二抗：1∶250稀释，浓度为2μg/ml，Biolegend，Cat#409304)，在4℃避光孵育半小时。加入预冷的PBS缓冲液，4℃离心弃上清，重复三次。细胞重悬在FACS缓冲液中，200μl/孔，使用流式细胞仪(BD FACS Calibur)检测。
结果表明，在4℃孵育时，SN8、hAb015和hAb017这3个抗体均不能被DOHH-2细胞内吞。而在37℃孵育时，1小时后大部分抗体已经被DOHH-2细胞内吞，4小时后抗体内吞达到最大值。3个抗体均具有较好的内吞作用。

Claims (13)

  1. 一种抗人CD79B抗体或其抗原结合片段，其包含选自以下(I)至(II)中的任一项：
    (I)重链可变区，其包含分别如SEQ ID NO：7、SEQ ID NO：8和SEQ ID NO：9所示的HCDR1、HCDR2和HCDR3；和
    轻链可变区，其包含分别如SEQ ID NO：10、SEQ ID NO：11和SEQ ID NO：12所示的LCDR1、LCDR2和LCDR3；
    (II)重链可变区，其包含分别如SEQ ID NO：13、SEQ ID NO：14和SEQ ID NO：15所示的HCDR1、HCDR2和HCDR3；和
    轻链可变区，其包含分别如SEQ ID NO：16、SEQ ID NO：11和SEQ ID NO：12所示的LCDR1、LCDR2和LCDR3；
    (III)重链可变区，其包含分别如SEQ ID NO：23、SEQ ID NO：24和SEQ ID NO：25所示的HCDR1、HCDR2和HCDR3；和
    轻链可变区，其包含分别如SEQ ID NO：26、SEQ ID NO：11和SEQ ID NO：12所示的LCDR1、LCDR2和LCDR3。
  2. 根据权利要求1所述的抗人CD79B抗体或抗原结合片段，其中：
    重链可变区，包含：
    如SEQ ID NO：3所示的序列或与SEQ ID NO：3具有至少90％、95％、98％、99％同一性的序列；或
    如SEQ ID NO：5所示的序列或与SEQ ID NO：5具有至少90％、95％、98％、99％同一性的序列；
    如SEQ ID NO：17所示的序列或与SEQ ID NO：17具有至少90％、95％、98％、99％同一性的序列；
    和/或轻链可变区，包含：
    如SEQ ID NO：4所示的序列或与SEQ ID NO：4具有至少90％、95％、98％、99％同一性的序列；或
    如SEQ ID NO：6所示的序列或与SEQ ID NO：6具有至少90％、95％、98％、99％同一性的序列；
    如SEQ ID NO：18所示的序列或与SEQ ID NO：18具有至少90％、95％、98％、99％同一性的序列；优选地，所述抗人CD79B抗体或抗原结合片段的重链可变区如序列SEQ ID NO：3所示，且轻链可变区如序列SEQ ID NO：4所示；或
    优选地，所述抗人CD79B抗体或抗原结合片段的重链可变区如序列SEQ ID NO：5所示，且轻链可变区如序列SEQ ID NO：6所示；或
    优选地，所述抗人CD79B抗体或抗原结合片段的重链可变区如序列SEQ ID  NO：17所示，且轻链可变区如序列SEQ ID NO：18所示。
  3. 根据权利要求1或2所述的抗人CD79B抗体或其抗原结合片段，其为鼠源抗体、嵌合抗体、人抗体、人源化抗体或其片段。
  4. 根据权利要求3所述的抗人CD79B抗体或其抗原结合片段，其是人源化抗体，其重链包含：如SEQ ID NO：19所示的序列或与SEQ ID NO：19具有至少90％、95％、98％或99％同一性的序列；或
    如SEQ ID NO：21所示的序列或与SEQ ID NO：21具有至少90％、95％、98％、或99％同一性的序列；
    和/或轻链包含：如SEQ ID NO：20所示的序列或与SEQ ID NO：20具有至少90％、95％、98％或99％同一性的序列；或
    如SEQ ID NO：22所示的序列或与SEQ ID NO：22具有至少90％、95％、98％或99％同一性的序列；
    优选地，所述抗CD79B抗体或抗原结合片段的重链如序列SEQ ID NO：19所示，且轻链如序列SEQ ID NO：20所示；或
    优选地，所述抗CD79B抗体或抗原结合片段的重链如序列SEQ ID NO：21所示，且轻链如序列SEQ ID NO：22所示。
  5. 根据权利要求3或4所述的抗人CD79B抗体或其抗原结合片段，其中：
    所述人源化抗体的重链可变区包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链框架区；
    所述抗原结合片段选自Fab、Fab’-SH、Fv、scFv和/或(Fab’)2片段；
    优选地，所述人源化抗体的重链可变区包含人源IgG1、IgG2或IgG4的重链框架区；
    更优选地，所述人源化抗体的重链可变区包含人源IgG1或IgG2的重链框架区。
  6. 一种抗体药物偶联物，其中所述抗体含有权利要求1-5任一项所限定的抗人CD79B抗体或其抗原结合片段，
    优选地，所述抗体药物偶联物含有细胞毒剂；
    更优选地，所述细胞毒剂选自毒素、化疗剂、抗生素、放射性同位素和溶核酶。
  7. 一种多核苷酸，其编码权利要求1至5任一项所述的抗人CD79B抗体或其抗原结合片段。
  8. 一种载体，其含有如权利要求7所述的多核苷酸，其为真核表达载体、原核表达载体或病毒载体。
  9. 一种宿主细胞，其包含权利要求8所述的载体，
    优选地，所述宿主细胞为细菌、酵母、哺乳动物细胞；
    更优选地，所述宿主细胞为大肠杆菌、毕赤酵母、中国仓鼠卵巢细胞或人胚肾293细胞。
  10. 一种制备抗人CD79B抗体或其抗原结合片段的方法，包括：
    在权利要求9所述的宿主细胞中表达抗人CD76B抗体或其抗原结合片段，以及从培养物中分离所述抗人CD79B抗体或其抗原结合片段。
  11. 一种药物组合物，其含有：
    选自以下的任一项或其任意组合：权利要求1至5任一项中所述的抗CD79B抗体或其抗原结合片段、权利要求6所述的抗体药物偶联物、权利要求7所述的多核苷酸、权利要求8所述的载体；以及，
    任选地，可药用的赋形剂、稀释剂或载体。
  12. 选自以下的任一项在制备药物或药物组合物中的用途：
    权利要求1至5任一项中所述的抗人CD79B抗体或其抗原结合片段、权利要求6所述的抗体-药物偶联物、权利要求7所述的多核苷酸、权利要求8所述的载体，其中：
    所述药物或药物组合物用于治疗增殖性疾病或用于延缓增殖性疾病进展，
    优选地，所述增殖性疾病是癌症或肿瘤；
    更优选地，所述癌症或肿瘤是淋巴瘤或白血病；
    所述淋巴瘤选自：弥漫大B细胞淋巴瘤、非何杰金氏淋巴瘤、小淋巴细胞性淋巴瘤、套细胞淋巴瘤；
    所述非何杰金氏淋巴瘤选自：攻击性NHL、复发性攻击性NHL、复发性无痛性NHL、顽固性NHL、顽固性无痛性NHL；
    所述白血病选自：慢性淋巴细胞性白血病、毛细胞白血病、急性淋巴细胞性白血病。
  13. 一种治疗或预防增殖性疾病或延缓增殖性疾病进展的方法，所述方法包括：
    向受试者施用治疗或延缓疾病有效量的权利要求1至5任一项中所述的抗人CD79B抗体或其抗原结合片段、权利要求6所述的抗体药物偶联物、权利要求7所述的多核苷酸、权利要求8所述的载体、权利要求11所述的药物组合物、或其 任意组合，
    优选地，所述增殖性疾病是癌症或肿瘤；
    更优选地，所述癌症或肿瘤是淋巴瘤或白血病；
    所述淋巴瘤选自：弥漫大B细胞淋巴瘤、非何杰金氏淋巴瘤、小淋巴细胞性淋巴瘤、套细胞淋巴瘤；
    所述非何杰金氏淋巴瘤选自：攻击性NHL、复发性攻击性NHL、复发性无痛性NHL、顽固性NHL、顽固性无痛性NHL；
    所述白血病选自：慢性淋巴细胞性白血病、毛细胞白血病、急性淋巴细胞性白血病。
PCT/CN2020/073803 2019-01-28 2020-01-22 抗cd79b抗体、其抗原结合片段及其医药用途 WO2020156439A1 (zh)

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JP2021543401A JP2022518062A (ja) 2019-01-28 2020-01-22 抗ｃｄ７９ｂ抗体、その抗原結合フラグメントおよびそれらの医薬用途
MX2021008760A MX2021008760A (es) 2019-01-28 2020-01-22 Anticuerpo anti-cd79b, fragmento de union a antigeno del mismo y uso farmaceutico del mismo.
US17/310,204 US20220162304A1 (en) 2019-01-28 2020-01-22 Anti-cd79b antibody, antigen-binding fragment thereof, and pharmaceutical use thereof
KR1020217026421A KR20210121102A (ko) 2019-01-28 2020-01-22 항-cd79b 항체, 이의 항원-결합 단편, 및 이의 약학적 용도
AU2020213565A AU2020213565A1 (en) 2019-01-28 2020-01-22 Anti-CD79B antibody, antigen-binding fragment thereof, and pharmaceutical use thereof
BR112021014420-0A BR112021014420A2 (pt) 2019-01-28 2020-01-22 Anticorpo anti-cd79b, fragmento de ligação ao antígeno do mesmo e uso farmacêutico do mesmo
CN202080008873.0A CN113286823B (zh) 2019-01-28 2020-01-22 抗cd79b抗体、其抗原结合片段及其医药用途
CA3127556A CA3127556A1 (en) 2019-01-28 2020-01-22 Anti-cd79b antibody, antigen-binding fragment thereof, and pharmaceutical use thereof
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