WO2021147941A1 - 一种bcma结合蛋白及其制备方法和应用 - Google Patents

一种bcma结合蛋白及其制备方法和应用 Download PDF

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WO2021147941A1
WO2021147941A1 PCT/CN2021/073026 CN2021073026W WO2021147941A1 WO 2021147941 A1 WO2021147941 A1 WO 2021147941A1 CN 2021073026 W CN2021073026 W CN 2021073026W WO 2021147941 A1 WO2021147941 A1 WO 2021147941A1
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binding protein
amino acid
seq
heavy chain
light chain
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French (fr)
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王正
何云
戎一平
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和铂医药(苏州)有限公司
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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
    • C07K16/2878—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 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • 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

Definitions

  • the invention belongs to the field of biological macromolecules, and specifically relates to a BCMA binding protein and a preparation method and application thereof.
  • MM Multiple myeloma
  • Plasma cells derived from bone marrow accompanied by abnormal secretion of immunoglobulins or free light chains. It ranks second among the most common hematological malignancies and accounts for approximately all hematological tumors. Of 10%. In the United States, there are more than 30,000 newly diagnosed patients and more than 12,000 deaths every year. Although there are new targeted therapies such as protease inhibitors (Bortezomb), it is generally believed that it is incurable, so more efforts are needed in drug development.
  • protease inhibitors Bose inhibitors
  • BCMA B cell maturation antigen
  • TNFRSF17 or CD269 B cell maturation antigen
  • the anti-BCMA antibodies in the prior art have defects such as weak internalization and weak binding to 293T-huBCMA cells. Therefore, there is an urgent need for an anti-BCMA antibody that has sufficient affinity for medicine and excellent internalization.
  • the technical problem to be solved by the present invention is to overcome the defects of weak internalization of the existing anti-BCMA antibody and weak binding with 293T-huBCMA cells in the prior art, and provide a BCMA binding protein and a preparation method and application thereof.
  • the present invention provides a BCMA binding protein, which includes a heavy chain variable region (VH) and a light chain variable region (VL).
  • the light chain variable region includes LCDR1, LCDR2 and LCDR3,
  • the amino acid sequence of LCDR1 is shown in SEQ ID NO: 1 in the sequence listing or a variant thereof
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO: 2 in the sequence listing or a variant thereof.
  • the amino acid sequence of LCDR3 is shown in SEQ ID NO: 3 in the sequence list or a variant thereof
  • the heavy chain variable region includes HCDR1, HCDR2 and HCDR3, and the amino acid sequence of HCDR1 is as shown in SEQ ID in the sequence list.
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 5 in the sequence list or a variant thereof
  • the amino acid sequence of the HCDR3 is shown as SEQ ID NO: in the sequence list 6 shown or its variants.
  • the variants are amino acid sequences obtained after 3, 2 or 1 mutations are made on the basis of the amino acid sequences shown in SEQ ID NO: 1 to 6, respectively.
  • amino acid mutations Similar to "have 3, 2 or 1 amino acid mutations" in “amino acid mutations” means that compared with the original amino acid sequence, the variant sequence has amino acid mutations, including amino acid insertions based on the original amino acid sequence, Missing or replacement.
  • the CDR mutations can include 3, 2 or 1 amino acid mutations, and the same or different numbers of amino acid residues can optionally be selected for mutations between these CDRs.
  • CDR1 can be mutated. There are no amino acid mutations for CDR2 and CDR3.
  • the mutations described in the present invention may include mutations currently known to those skilled in the art, such as some mutations that may be made to the antibody during the production or application of the antibody, such as the transcription of the CDR regions that may exist.
  • Potential post-translational modifications (PTMs) sites are mutated, including antibody aggregation, asparaginedeamidation, sites (NG, NS, and/or NH, etc.), aspartic acid isomerization ( DG, DP) sensitive sites, N-glycosylation (N- ⁇ P ⁇ S/T) sensitive sites and oxidation sensitive sites and other related mutations.
  • PTMs post-translational modifications
  • the BCMA binding protein described in the present invention also includes a framework region of an anti-BCMA antibody, and the framework region includes a heavy chain framework region (HFWR) and/or a light chain framework region (LFWR);
  • HFWR heavy chain framework region
  • LFWR light chain framework region
  • the heavy chain framework region is preferably a human heavy chain framework region, and the human heavy chain framework regions HFWR1 to 4 preferably respectively contain the amino acid sequences shown in SEQ ID NO: 13 to 16 in the sequence list, or respectively It is a variant of the amino acid sequence shown in SEQ ID NO: 13-16;
  • the light chain framework region is preferably a human light chain framework region, and the human light chain framework regions LFWR1 to 4 preferably respectively contain the amino acid sequences shown in SEQ ID NO: 17 to 20 in the sequence list, or respectively It is a variant of the amino acid sequence shown in SEQ ID NO: 17-20.
  • the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 11 in the sequence listing or a variant thereof, and/or, the light chain variable region Including the amino acid sequence shown in SEQ ID NO: 12 in the sequence listing or its variants.
  • the variant is based on the deletion, substitution or addition of one or more amino acid residues based on the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12, and the mutant amino acid sequence is consistent with the VH And/or the amino acid sequence of VL has at least 85% sequence identity, and maintains or improves the binding of the BCMA binding protein to BCMA; the at least 85% sequence identity is preferably at least 90% sequence identity, more preferably At least 95% sequence identity, most preferably at least 99% sequence identity.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 11 in the sequence listing, and the amino acid sequence of the light chain variable region is shown in the sequence listing SEQ ID NO: 12 is shown.
  • the amino acid sequences of the CDRs listed above are all shown in accordance with the Kabat definition rules (the claims of the present invention are also shown in accordance with the Kabat definition rules).
  • the CDR of an antibody can be defined in many ways in the art, such as the Kabat definition rule based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, Fifth Edition, U.S. National Institute of Health, Bethesda, Maryland (1991)) and Chothia definition rules based on the location of structural loop regions (see JMol Biol 273:927-48, 1997).
  • the Combined definition rule including Kabat definition and Chothia definition can also be used to determine the amino acid residues in the variable domain sequence.
  • the combined definition rule is to combine the Kabat definition and the Chothia definition. Based on this, a larger scope is taken, as shown in Table 1-1.
  • the terms "CDR" and "complementarity determining region" of a given antibody or its region (e.g., variable region) should be understood to encompass the above-mentioned already described by the present invention. Complementary decision area defined by any one of the known schemes.
  • the BCMA binding protein described in the present invention preferably further includes a heavy chain constant region and/or a light chain constant region; preferably, the heavy chain constant region is selected from hIgG1, hIgG2, hIgG3, hIgG4 or mutations thereof,
  • the light chain constant region is selected from a kappa chain or a lambda chain or a mutation thereof; wherein the kappa chain or lambda chain is preferably a human antibody light chain kappa chain or lambda chain.
  • the heavy chain constant region is hIgG1
  • the light chain constant region is the light chain ⁇ chain of a human antibody.
  • the BCMA binding protein described in the present invention can be a full-length antibody, Fab, Fab', F(ab') 2 , Fv, scFv, bispecific antibody, multispecific antibody, single domain antibody or single domain antibody, or Monoclonal antibodies or polyclonal antibodies prepared from the above antibodies.
  • the monoclonal antibody can be developed by a variety of approaches and technologies, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc.
  • the mainstream is to prepare monoclonal antibodies from wild-type or transgenic mice through hybridoma technology.
  • the BCMA binding protein is a full-length antibody
  • the full-length antibody includes a heavy chain and a light chain
  • the heavy chain includes the amino acid sequence shown in SEQ ID NO: 9
  • the light chain includes the amino acid sequence shown in SEQ ID NO: 10.
  • the present invention also provides an isolated nucleic acid, which encodes the BCMA binding protein as described above.
  • the present invention also provides an expression vector containing the isolated nucleic acid as described above.
  • the present invention also provides a host cell comprising the expression vector as described above; preferably, the host cell is a prokaryotic cell or a eukaryotic cell.
  • the preparation method of the host cell may be a conventional preparation method in the art, for example, it is prepared by transforming the above-mentioned expression vector into a host cell.
  • the host cell is a variety of conventional host cells in the art, as long as it can make the expression vector stably replicate itself and the nucleic acid carried by it can be effectively expressed.
  • the host cell is E. coli TG1 or BL21 cell (expressing single-chain antibody or Fab antibody), or CHO-K1 cell (expressing full-length IgG antibody).
  • the transformation method is a conventional transformation method in the field, preferably a chemical transformation method, a heat shock method or an electrotransformation method.
  • the present invention also provides a method for preparing a BCMA binding protein, which comprises culturing the host cell as described above, and obtaining the BCMA binding protein from the culture.
  • the present invention also provides an immunoconjugate comprising a cytotoxic agent and the BCMA binding protein as described above.
  • the present invention also provides a pharmaceutical composition, which comprises the above-mentioned BCMA binding protein or the above-mentioned immunoconjugate.
  • the present invention also provides the use of the above-mentioned BCMA binding protein, the above-mentioned immunoconjugate and the above-mentioned pharmaceutical composition in the preparation of a medicine for the treatment and/or prevention of cancer; preferably, the cancer is bone marrow Tumor, colon cancer, lung cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, breast cancer, cervical cancer or ovarian cancer.
  • the present invention also provides a kit combination, which includes a kit A and a kit B; the kit A contains the BCMA binding protein of the invention, the host cell, and The immunoconjugate and the pharmaceutical composition; the kit B contains other targeted or the same targeted antibodies, bispecific antibodies, genetically modified cells or pharmaceutical compositions.
  • the use of the medicine box A and the medicine box B is in no particular order, or the medicine box A is used first and then the medicine box B, or the medicine box B is used first and then the medicine box A is used.
  • the BCMA binding protein, the immunoconjugate and the pharmaceutical composition or the kit combination of the present invention can be administered to patients for the treatment of related tumors.
  • compositions and methods are intended to mean that the composition and method include the described elements but do not exclude other elements, but based on the understanding of the context, it also includes the case of "consisting of”.
  • antibody in the present invention includes immunoglobulin (Ig), which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
  • Ig immunoglobulin
  • the amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different.
  • 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 a kappa chain or a lambda 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 antibody light chain variable region of the present invention may further comprise a light chain constant region, and the light chain constant region comprises human-derived kappa, lambda chains or variants thereof.
  • the antibody heavy chain variable region of the present invention may further comprise a heavy chain constant region, and the heavy chain constant region comprises human IgG1, 2, 3, 4 or variants thereof.
  • Each light chain variable region (VL) and heavy chain variable region (VH) is composed of 3 complementarity determining regions (CDR) and 4 framework regions (FWR).
  • CDR complementarity determining regions
  • FWR framework regions
  • the sequence from the amino terminus to the carboxy terminus is as follows: FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR4.
  • the 3 CDRs of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDRs of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • mutation includes substitutions, additions, and/or deletions of amino acids or nucleotides, and "amino acid substitutions” are substitutions in which an amino acid residue is replaced with another amino acid residue and an amino acid residue having a similar side chain.
  • vector or "expression vector” as used herein is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the inside of a cell.
  • vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides related to ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term “vector” includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, and the like.
  • transfection refers to the introduction of exogenous nucleic acid into eukaryotic cells. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, Liposome fusion, lipofection, protoplast fusion, retroviral infection and biolistics.
  • the preparation method of the immunoconjugate can be conventional in the art, preferably the preparation method described in Doronina, 2006, Bioconjugate Chem. 17, 114-124.
  • the preparation method produces antibody-drug conjugates with a minimum low conjugation fraction (LCF) of less than 10%.
  • the immunoconjugate can exist in any physical form known in the art, and is preferably a clear solution.
  • cancer As used in the present invention, the terms “cancer”, “cancer” and “tumor” are intended to include all types of cancerous growths or tumorigenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, regardless of histopathological type Or what is the aggressiveness stage. Examples include, but are not limited to, solid tumors, hematological cancers, soft tissue tumors, and metastatic lesions.
  • the reagents and raw materials used in the present invention are all commercially available.
  • a fully human antibody with good specific affinity to hBCMA is obtained by immunoscreening, which binds better than the control antibody on tumor cell lines.
  • the antibody has an internalization effect and is better than the control, so it can be further developed as an ADC candidate.
  • the antibody According to the ELISA test, the antibody has partial blocking function, and may obtain additional curative effect when used as mAbs or CAR-T treatment.
  • Figure 1 shows the test results of the binding strength of the recombinant antibody to the cell line expressing BCMA; a. The binding of the recombinant antibody to the 293T-huBCMA cell line b. The binding of the recombinant antibody to the 293T-cynoBCMA cell line, c. The recombinant antibody and NCI- Combination of H929 cell line.
  • Figure 2 shows the test of BCMA recombinant antibody blocking the binding of BAFF and BCMA.
  • Figure 3 shows the determination of the internalization of recombinant antibodies into the BCMA cell line. a. Recombinant antibody is internalized into 293T-huBCMA cell line, b. Recombinant antibody is internalized into NCI-H929 cell line.
  • the positive control also referred to as the control antibody
  • the GSK clinical phase II ADC drug GSK2857916 that is, the CA8J6M0 humanized antibody in the US patent application US9273141B2.
  • the amino acid sequence of each functional region of the control antibody is shown in the following table (according to the Kabat numbering rule):
  • Fully human anti-BCMA antibodies were identified from hybridomas produced by H2L2 mice (and Platinum Medicine, EP2379727B1) immunized with BCMA-ECD-Fc protein.
  • the first injection of 50 ⁇ g of the above fusion protein was immunized with CFA as an immune adjuvant, and then 25 ⁇ g of protein and Ribi adjuvant (Sigma-Aldrich; Sigma Adjuvant System; Catalog) on the 15, 29, 43, 57, 71, and 86 days Number S6322) strengthened 7 times. Blood was collected on days 50, 78 and 107.
  • mice were selected for hybridoma fusion. Three days before the fusion, that is, the 132th day, the final boost was performed with 25 ⁇ g protein and Ribi adjuvant.
  • Hybridomas are produced and cloned by conventional methods, namely, by electrofusion method, the mouse spleen and lymph nodes are extracted, crushed to extract single cells, split red and washed and mixed with sp2/0 cells. The cell suspension is placed in an electrofusion tank and electroporated for fusion, and then replaced with 20% FBS HT medium after standing, and then replaced with HAT medium for culture.
  • the collected spleen B cells and the mouse myeloma cell line Sp2/0 were mixed at a ratio of 2:1 (cell number ratio), and the mixed cells were fused with an electric fusion instrument (BTX ECM2001), and the fused cells Spread it on a 96-well cell culture plate, culture it in a carbon dioxide incubator at 37°C for 10 days, and conduct a preliminary screening of hybridomas. After overnight recovery, the fused cells were seeded in a 96-well plate by limiting dilution and screened with hypoxanthine-aminopterin-thymidine. The presence of anti-BCMA antibodies in the culture supernatant of hybridomas was detected by ELISA test and flow cytometry.
  • a 96-well plate (Corning 9018) was added with newly prepared hBCMA ECD-Fc protein or hFc at 1 ⁇ g/ml in PBS, coated overnight at 4°C, then discarded and washed with PBST 3 times. The plate was blocked with 5% milk for 2 hours at room temperature and washed 3 times with PBST. 100 ⁇ l/well of hybridoma supernatant was added and incubated at room temperature for 1 hour, and then washed with PBST 3 times. Add 100 ⁇ l/well of the secondary antibody and incubate at room temperature for 1 hour, followed by washing. Add 100 ⁇ l/well of TMB to the plate and incubate for 15min at room temperature, then stop and read.
  • v TypLE (CAT#12605010, Gibco) was used to digest the adherent cells at 37°C for 3 minutes, and then the digestion was terminated with a complete medium containing 10% FBS. Wash the cells with FACS buffer (CAT#14190250, Gibco), count them, and then dilute to a density of 3 ⁇ 5 ⁇ 10 6 /ml. The cells were added to a 96-well plate (Corning 3894) at 100 ⁇ l/well. After blocking for 3 to 4 minutes, add 100 ⁇ l/well of hybridoma supernatant and incubate at 4°C for 1 hour. After washing, add secondary antibody and incubate at 4°C for one hour. The cells were then washed and analyzed by FACS.
  • FACS buffer CAT#14190250, Gibco
  • the blocking test was performed by the limiting dilution method and the ELISA method.
  • ELISA enzyme-linked immunosorbent assay
  • RT-PCR generates cDNA, and then the heavy and light chains are amplified by PCR (RT-PCR from Thermo Fisher The first chain synthesis system, Cat#: 11904018, please refer to the product manual for specific operations.
  • the PCR uses NEB's high-fidelity DNA polymerase, Cat#: M0530L, please refer to the product manual for specific operations). Then the PCR product was constructed on the T vector and sequenced (Beijing Kinco Biotechnology Co., Ltd. Shanghai Branch), and the antibody subtype was determined at the same time.
  • amino acid sequences of LCDR1 to 3 in the light chain variable region are shown in SEQ ID NOs: 1 to 3 in the sequence list; the amino acid sequences of HCDR1 to 3 in the heavy chain variable region are shown in SEQ ID NO: Shown in 4 ⁇ 6.
  • the specific amino acid sequence is shown in the table below:
  • VH antibody heavy chain variable domain sequence
  • the antibody light chain variable domain sequence (VL) is genetically synthesized and cloned into a mammalian cell expression plasmid vector encoding the human antibody Ig ⁇ light chain constant domain sequence to encode the full-length light chain of the antibody.
  • VL antibody light chain variable domain sequence
  • the heavy chain sequence of the humanized antibody of the present invention is:
  • the light chain sequence of the humanized antibody of the present invention is:
  • amino acid sequence of the heavy chain variable region of the humanized antibody of the present invention is:
  • amino acid sequence of the light chain variable region of the humanized antibody of the present invention is:
  • the plasmid encoding the antibody heavy chain (Genscript US) and the plasmid encoding the antibody light chain (Genscript US) are simultaneously transfected into mammalian host cells (such as human embryonic kidney cells HEK293), using conventional recombinant protein expression and purification techniques to obtain Purified recombinant antibody with correct pairing and assembly of light and heavy chains.
  • mammalian host cells such as human embryonic kidney cells HEK293
  • HEK293 cells were expanded and cultured in FreeStyle TM F17 Expression Medium medium (Thermo, Cat#: A1383504). Before the start of transient transfection, adjust the cell concentration to 6-8 ⁇ 10 5 cells/ml, and incubate for 24 hours in a 37°C 8% CO 2 shaker. The cell concentration is 1.2 ⁇ 10 6 cells/ml.
  • FACS identification Recombinant antibodies were diluted to 9 different concentrations in sequence, and incubated on 293T-huBCMA, 293T-cynoBCMA and NCI-H929 cell lines for 1 hour, and then the secondary antibody was tested after 1 hour incubation. The verification results are shown in Figure 1. Shown.
  • PR000892 binds to 293T-huBCMA cells (purchased from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.; Item No.: CAT#KC-0233) and has significant binding, and the affinity is better than that of the control antibody;
  • PR000892 binds to 293T-cynoBCMA (purchased from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.; article number: CAT#KC-0979) and its affinity is equivalent to that of the control antibody; in Figure 1c, PR000892 and the tumor cell line NCI-H929 Its affinity is comparable to that of the control antibody.
  • Recombinant antibodies are serially diluted to 7 different concentrations and tested on Corning 9018 plates pre-coated with 2 ⁇ g/ml huBCMA-ECD-FC. The subsequent steps are the same as above. As shown in Figure 2, PR000892 can partially block the binding of BAFF and BCMA under in vitro conditions.

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Abstract

一种BCMA结合蛋白及其制备方法和应用。所述BCMA结合蛋白包括重链可变区和轻链可变区，所述的轻链可变区包含LCDR1、LCDR2和LCDR3；所述的重链可变区包含HCDR1、HCDR2和HCDR3。所述BCMA结合蛋白具有良好特异性亲合力，其在肿瘤细胞系上比对照抗体结合更好，且其内化效应优于对照，因此可以作为ADC候选物进一步开发。经ELISA试验，抗体都有部分阻断功能，在作为mAbs或CAR-T治疗时可能获得额外的疗效。

Description

一种BCMA结合蛋白及其制备方法和应用
本申请要求申请日为2020/1/23的中国专利申请202010076287.1的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于生物大分子领域，具体涉及一种BCMA结合蛋白及其制备方法和应用。
背景技术
多发性骨髓瘤(MM)一直以骨髓来源的浆细胞增殖失控为特征，伴有异常分泌的免疫球蛋白或游离轻链，在最常见的血液系统恶性肿瘤中居第二位，约占所有血液肿瘤的10％。在美国，每年新诊断的患者超过30000人，死亡人数超过12000人。尽管有了蛋白酶抑制剂(Bortezomb)等新的靶向疗法，但人们普遍认为它是无法治愈的，因此需要在药物开发方面做出更多的努力。
B细胞成熟抗原(BCMA)，又称TNFRSF17或CD269，是一种仅在B细胞谱系，尤其是末端分化的B细胞中表达的特异性抗原。它通常不存在于初始和记忆B细胞，并在多发性骨髓瘤细胞上高度表达。BCMA与其配体BAFF和APRIL相互作用时，可通过NF-kB和JNK通路的信号转导，诱导B细胞成熟、增殖和存活。作为一种I型跨膜蛋白，在体液免疫中起着重要的作用，是抗体衍生免疫治疗的理想靶点。
然而现有技术中的抗BCMA的抗体存在内化作用弱以及与293T-huBCMA细胞结合弱等缺陷，因此亟需一种抗BCMA的抗体其既具有足以成药的亲和力又具有优异的内化作用。
发明内容
本发明所要解决的技术问题是为克服现有技术中存在的已有抗BCMA的抗体内化作用弱以及与293T-huBCMA细胞结合弱等缺陷，提供一种BCMA结合蛋白及其制备方法和应用。
为解决以上技术问题，本发明提供一种BCMA结合蛋白，其包括重链可变区(VH)和轻链可变区(VL)，所述的轻链可变区包含LCDR1、LCDR2和LCDR3，所述的LCDR1的氨基酸序列如序列表中SEQ ID NO:1所示或为其变体，所述的LCDR2的氨基酸序列如序列表中SEQ ID NO:2所示或为其变体，所述的LCDR3的氨基酸序列如序列表中SEQ ID NO:3所示或为其变体；所述的重链可变区包含HCDR1、HCDR2和HCDR3，所述的HCDR1的氨基酸序列如序列表中SEQ ID NO:4所示或为其变体，所述的HCDR2的氨基酸序列如序列表中SEQ ID NO:5所示或为其变体，所述的HCDR3的氨基酸序列如序列表中SEQ ID NO:6所示或为其变体。其中，所述变体为分别在SEQ ID NO:1～6所示的氨基酸序列的基础上发生3、2或者1个突变后获得的氨基酸序列。
在类似“具有3、2或1个氨基酸突变”中“氨基酸突变”是指相较于原氨基酸序列而言，变体的序列存在氨基酸的突变，包括在原氨基酸序列的基础上发生氨基酸的插入、缺失或替换。示例性的解释是对CDR的突变可以包含3个、2个或1个氨基酸的突变，这些CDR之间可以任选地选择相同或不同数目的氨基酸残基进行突变，例如可以是对CDR1进行1个氨基酸的突变，对CDR2和CDR3不进行氨基酸突变。
本发明中所述的突变可以包括目前如本领域技术人员公知的突变，例如在抗体的生产或者应用过程中，可能会对抗体进行的一些突变，例如对可能存在的，特别是CDR区的转录后修饰(Potential post-translational modifications，PTMs)的位点进行突变，包括抗体的聚集、脱酰胺基敏感(asparagine deamidation，位点(NG、NS和/或NH等)、天冬氨酸异构(DG、DP)敏感位点、N糖基化(N-{P}S/T)敏感位点及氧化敏感位点等相关突变。
较佳地，本发明中所述的BCMA结合蛋白还包括抗BCMA抗体的框架区，所述框架区包括重链框架区(HFWR)和/或轻链框架区(LFWR)；
所述的重链框架区优选人源重链框架区，所述的人源重链框架区HFWR1～4优选分别包含分别如序列表中SEQ ID NO:13～16所示的氨基酸序列，或者分别为SEQ ID NO:13～16所示的氨基酸序列的变体；
所述的轻链框架区优选人源轻链框架区，所述的人源轻链框架区LFWR1～4优选分别包含分别如序列表中SEQ ID NO:17～20所示的氨基酸序列，或者分别为SEQ ID NO:17～20所示的氨基酸序列的变体。
在本发明一较佳实施例中，所述的重链可变区包括如序列表中SEQ ID NO:11所示的氨基酸序列或其变体，和/或，所述的轻链可变区包括如序列表中SEQ ID NO:12所示的氨基酸序列或其变体。所述的变体为基于如SEQ ID NO:11或者SEQ ID NO:12所示的氨基酸序列发生一个或者多个氨基酸残基的缺失、取代或添加，且所述突变的氨基酸序列与所述VH和/或VL的氨基酸序列具有至少85％序列同一性，并保持或改善了所述BCMA结合蛋白与BCMA的结合；所述至少85％序列同一性优选为至少90％序列同一性，更优选为至少95％序列同一性，最优选为至少99％序列同一性。
在本发明一更佳的实施例中，所述的重链可变区的氨基酸序列如序列表中SEQ ID NO:11所示，且所述的轻链可变区的氨基酸序列如序列表中SEQ ID NO:12所示。
在本申请中，上述所列CDR的氨基酸序列均是按照Kabat定义规则所示出的(本发明的权利要求中也是按照Kabat定义规则所示出的序列)。但是，本领域人员公知，在本领域中可以通过多种方法来定义抗体的CDR，例如基于序列可变性的Kabat定义规则(参见，Kabat等人，免疫学的蛋白质序列，第五版，美国国立卫生研究院，贝塞斯达，马里兰州(1991))和基于结构环区域位置的Chothia定义规则(参见JMol Biol 273:927-48,1997)。在本申请中，还可以使用包含了Kabat定义和Chothia定义的Combined定义 规则确定可变结构域序列中的氨基酸残基。其中Combined定义规则即是将Kabat定义和Chothia定义的范围相结合，基于此取了一个更大的范围，详见表1-1。本领域技术人员应当理解的是，除非另有规定，否则术语给定抗体或其区(例如可变区)的“CDR”及“互补决定区”应了解为涵盖如通过本发明描述的上述已知方案中的任何一种界定的互补决定区。虽然本发明的权利要求中请求保护的范围是基于Kabat定义规则所示出的序列，但是根据其他CDR的定义规则所对应的氨基酸序列也应当落在本发明的保护范围中。
本发明中所述的BCMA结合蛋白，较佳地还包括重链恒定区和/或轻链恒定区；较佳地，所述重链恒定区选自hIgG1、hIgG2、hIgG3或hIgG4或其突变，所述轻链恒定区选自κ链或者λ链或其突变；其中所述κ链或者λ链优选人源抗体轻链κ链或者λ链。
在本发明一较佳实施例中，所述的重链恒定区为hIgG1，且所述轻链恒定区为人源抗体的轻链κ链。
本发明中所述的BCMA结合蛋白可为全长抗体、Fab、Fab’、F(ab’) 2、Fv、scFv、双特异性抗体、多特异性抗体、单域抗体或单区抗体，或由上述抗体制得的单克隆抗体或多克隆抗体。所述单克隆抗体可以由多种途径和技术进行研制，包括杂交瘤技术、噬菌体展示技术、单淋巴细胞基因克隆技术等，主流是通过杂交瘤技术从野生型或转基因小鼠制备单克隆抗体。
在本发明一具体实施例中，所述的BCMA结合蛋白为全长抗体，所述全长抗体包括重链和轻链，所述重链包括如SEQ ID NO:9所示的氨基酸序列，所述轻链包括如SEQ ID NO:10所示的氨基酸序列。
本发明还提供一种分离的核酸，其编码如上所述的BCMA结合蛋白。
本发明还提供一种包含如上所述的分离的核酸的表达载体。
本发明还提供一种宿主细胞，其包含如上所述的表达载体；优选地，所述宿主细胞为原核细胞或真核细胞。所述宿主细胞的制备方法可为本领域常规的制备方法，例如为：将上述表达载体转化至宿主细胞中制得。所述的宿 主细胞为本领域常规的各种宿主细胞，只要能满足使上述表达载体稳定地自行复制，且所携带所述的核酸可被有效表达即可。优选地，所述宿主细胞为E.coli TG1或BL21细胞(表达单链抗体或Fab抗体)，或者CHO-K1细胞(表达全长IgG抗体)。其中所述转化方法为本领域常规转化方法，较佳地为化学转化法，热激法或电转法。
本发明还提供一种BCMA结合蛋白的制备方法，其包含培养如上所述的宿主细胞，从培养物中获得所述BCMA结合蛋白。
本发明还提供一种免疫偶联物，其包含细胞毒性剂，以及如上所述的BCMA结合蛋白。
本发明还提供一种药物组合物，其包含如上所述的BCMA结合蛋白或如上所述的免疫偶联物。
本发明还提供如上所述的BCMA结合蛋白、如上所述的免疫偶联物和如上所述的药物组合物在制备治疗和/或预防癌症的药物中的应用；优选地，所述癌症为骨髓瘤、结肠癌、肺癌、前列腺癌、肝癌、肾癌、胰腺癌、乳腺癌、宫颈癌或卵巢癌。
此外，为解决上述技术问题，本发明还提供一种药盒组合，其包括药盒A和药盒B；所述药盒A包含本发明所述的BCMA结合蛋白、所述的宿主细胞、所述的免疫偶联物和所述的药物组合物；所述药盒B包含其它靶向的或相同靶向的抗体、双特异性抗体、基因修饰的细胞或药物组合物。所述药盒A和药盒B的使用不分先后顺序，或先使用药盒A再使用药盒B，或先使用药盒B再使用药盒A。
本发明所述的BCMA结合蛋白、所述的免疫偶联物和所述的药物组合物或所述的药盒组合可施用于病人，用于治疗相关肿瘤。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。 同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
本发明所用氨基酸三字母代码和单字母代码如本领域技术人员知晓，或J.Biol.Chem,243,p3558(1968)中所述。
如本文使用的，术语“包括”或“包含”旨在表示组合物和方法包括所述的元素但不排除其他元素，但根据上下文的理解，也包括“由……组成”的情况。
本发明所述的术语“抗体”包括免疫球蛋白(Ig)，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和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。
在本发明中，本发明所述的抗体轻链可变区可进一步包含轻链恒定区，所述的轻链恒定区包含人源的κ、λ链或其变体。在本发明中，本发明所述的抗体重链可变区可进一步包含重链恒定区，所述的重链恒定区包含人源的IgG1、2、3、4或其变体。
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)。每条轻链可变区(VL)和重链可变区(VH)由3个互补决定区(CDR)和4个框架区(FWR)组成，从氨基端到羧基端依次排列的顺序为：FWR1、CDR1、FWR2、CDR2、FWR3、CDR3、FWR4。轻链的3个CDR指LCDR1、LCDR2和LCDR3；重链的3个CDR指HCDR1、HCDR2和HCDR3。
术语“突变”包括氨基酸或核苷酸的取代、添加和/或缺失，“氨基酸的取代”是其中氨基酸残基以另一种氨基酸残基置换和以具有相似侧链的氨基 酸残基置换。
本文使用的术语“载体”或“表达载体”是包含分离的核酸并可用于将分离的核酸递送至细胞内部的组合物。在本领域中已知许多载体，包括但不限于线性多核苷酸、与离子或两亲化合物相关的多核苷酸、质粒和病毒。因此，术语“载体”包括自主复制的质粒或病毒。该术语还应被解释为包括促进核酸转移到细胞中的非质粒和非病毒化合物，例如聚赖氨酸化合物、脂质体等。病毒载体的实例包括但不限于腺病毒载体、腺相关病毒载体、逆转录病毒载体等。
术语“转染”是指将外源核酸引入真核细胞。转染可以通过本领域已知的各种手段来实现，包括磷酸钙-DNA共沉淀、DEAE-葡聚糖介导的转染、聚凝胺介导的转染、电穿孔、显微注射、脂质体融合、脂质转染、原生质体融合、逆转录病毒感染和生物弹道技术(biolistics)。
所述的免疫偶联物的制备方法可为本领域常规，较佳地采用Doronina，2006，Bioconjugate Chem.17,114-124所记载的制备方法。优选地，所述的制备方法产生具有最低限度的低偶联级分(LCF)小于10％的抗体药物偶联物。所述的免疫偶联物能够以本领域所知的任何物理形态而存在，较佳地为澄清溶液。
如本发明所用，术语“癌”、“癌症”、“肿瘤”意在包括全部类型的癌性生长物或致瘤过程、转移性组织或恶性转化的细胞、组织或器官，无论组织病理学类型或侵袭力阶段是什么。例子包括但不限于实体瘤、血液学癌、软组织肿瘤和转移性病灶。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
本发明免疫筛选出1种与hBCMA具有良好特异性亲合力的全人源抗 体，其在肿瘤细胞系上比对照抗体结合更好。抗体具有内化效应且优于对照，因此可以作为ADC候选物进一步开发。经ELISA试验，抗体都有部分阻断功能，在作为mAbs或CAR-T治疗时可能获得额外的疗效。
附图说明
图1为重组抗体与表达BCMA的细胞系结合强度的测试结果图；a.重组抗体与293T-huBCMA细胞系的结合b.重组抗体与293T-cynoBCMA细胞系的结合，c.重组抗体与NCI-H929细胞系的结合。
图2为BCMA重组抗体阻断BAFF与BCMA结合的测试。
图3为重组抗体内化至BCMA细胞系的测定。a.重组抗体内化至293T-huBCMA细胞系，b.重组抗体内化至NCI-H929细胞系。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
以下实施例中所使用的阳性对照(亦称对照抗体)为GSK临床II期ADC药物GSK2857916(即美国专利申请US9273141B2中的CA8J6M0人源化抗体)。对照抗体的各功能区氨基酸序列如下表所示(依据Kabat编号规则)：
Figure PCTCN2021073026-appb-000001
Figure PCTCN2021073026-appb-000002
重链氨基酸序列：
Figure PCTCN2021073026-appb-000003
轻链氨基酸序列：
Figure PCTCN2021073026-appb-000004
实施例1抗原制备、小鼠免疫及杂交瘤制备
1.抗原制备
(ACRO cat.no:BCA-C52H7)
供应商 名称 NCBI ID Cat#
Acrobio huBCMA-ECD-Fc Q02223 BCA-H522y
Acrobio BCMA-His   BCA-C52H7
Acrobio CynoBCMA-Fc G7NPN8 BCA-C5253
Acrobio CynoBCMA-His   BCA-C5225
Acrobio BAFF-his-avitag Q9Y275 BAF-H82Q2
2.免疫
从用BCMA-ECD-Fc蛋白免疫的H2L2小鼠(和铂医药，EP2379727B1)产生的杂交瘤中鉴定出全人源抗BCMA抗体。第一次注射50μg上述融合蛋白，以CFA为免疫佐剂进行免疫，然后在第15、29、43、57、71和86天以25μg蛋白和Ribi佐剂(Sigma-Aldrich；Sigma Adjuvant System；Catalog Number S6322)再加强7次。在第50、78和107天采血测试，小鼠血清的结合亲和力通过FACS使用表达人BCMA的HEK293T细胞或肿瘤细胞系(HEK293T-BCMA，购买自kyinno或Capan-2细胞，购自ATCC)进行检测，同时平行使用BCMA-His蛋白进行ELISA检测。根据免疫小鼠血清效价的检测结果，挑选小鼠进行杂交瘤融合，融合前3天，即第132天，以25μg蛋白和Ribi佐剂进行最后一次加强。
3.融合
杂交瘤用常规方法产生和克隆，即通过电融合法，提取小鼠脾脏和淋巴结，研碎提取单细胞，裂红并洗涤后与sp2/0细胞混合。细胞混悬液放入电融合槽电击融合，静置后换20％FBS的HT培养基，之后更换HAT培养基培养。
将收集的脾脏B细胞与小鼠骨髓瘤细胞系Sp2/0按2:1(细胞数量比)的比例混合，混合后的细胞用电融合仪(BTX ECM2001)进行细胞融合，将融合后的细胞铺在96孔细胞培养板，在二氧化碳培养箱中37℃培养10天后进行杂交瘤的初筛。过夜复原后，将融合细胞以有限稀释法接种到96孔板中，并用次黄嘌呤-氨基蝶呤-胸苷进行筛选。通过ELISA试验和流式细胞术检测杂交瘤培养上清液中抗BCMA抗体的存在。
实施例2抗体筛选及测序
1.ELISA筛选
在96孔板(Corning 9018)中加入在PBS中1μg/ml的新制备的hBCMA ECD-Fc蛋白或hFc，在4℃下涂覆过夜，然后弃去并用PBST清洗3次。在 室温下用5％的牛奶封闭平板2小时，并用PBST清洗3次。加入100μl/孔的杂交瘤上清液并在室温下孵育1小时，然后用PBST清洗3次。加入100μl/孔的第二抗体并在室温下孵育1小时，随后清洗。平板中加入100μl/孔的TMB并在室温下孵育15min，然后停止并读取。
2.FACS筛选
对于流式细胞术筛选，v用TypLE(CAT#12605010，Gibco)在37℃消化粘附细胞3分钟后，用含10％FBS的完全培养基终止消化。用FACS缓冲液(CAT#14190250，Gibco)清洗细胞并计数，然后稀释至3～5×10 6/ml密度。将细胞按100μl/孔添加到96孔板(Corning 3894)。阻断3～4分钟后，加入100μl/孔杂交瘤上清液，在4℃孵育1小时。洗涤后加入二抗，在4℃孵育一个小时。然后洗涤细胞并进行FACS分析。
3.亚克隆及筛选
用有限稀释法用ELISA法进行阻断试验。
阻断筛选：
对于阻断酶联免疫吸附试验(ELISA)，用在PBS中2μg/ml的hBCMA-ECD-FC涂覆平板过夜，洗涤、封闭和孵育与上述步骤相同。然后在平板中加入6ng/ml的生物素化的BAFF，在室温下孵育1小时。第二抗体的孵育和检测的后续步骤与以上相同。
4.测序
挑选阳性单克隆，提取总RNA。RT-PCR产生cDNA，随后重链和轻链分别用PCR扩增(RT-PCR来自赛默飞的
Figure PCTCN2021073026-appb-000005
第一链合成系统，Cat#：11904018，具体操作见产品说明书。PCR使用的是NEB的高保真DNA聚合酶，Cat#：M0530L，具体操作见产品说明书)。然后将PCR产物构建到T载体上，并进行测序(北京擎科生物科技有限公司上海分公司)，同时进行抗体亚型测定。
5.抗体序列信息
测序结果如下所示(根据Kabat编号规则)：
轻链可变区中的LCDR1～3的氨基酸序列分别如序列表中SEQ ID NO:1～3所示；重链可变区中的HCDR1～3的氨基酸序列分别如序列表中SEQ ID NO:4～6所示。具体氨基酸序列见下表：
功能区 氨基酸序列
LCDR1 RASQSISSWLA
LCDR2 KASSLES
LCDR3 QQYNSYLFT
HCDR1 SYGMY
HCDR2 AIWNDGSNNYYADSVKG
HCDR3 DRLPMASLRYFDWLGVMDA
实施例3.抗体生产、纯化和验证
1.重组抗体生产和纯化
在得到编码抗体分子的轻、重链可变结构域序列以后，可以采用常规的重组DNA技术，将轻、重链可变结构域序列和相应的人的抗体轻、重链恒定结构域序列进行融合表达，得到重组抗体分子。在本实施例中，抗体重链可变结构域序列(VH)通过基因合成并克隆到编码人IgG1抗体重链恒定结构域序列的哺乳动物细胞表达质粒载体中，以编码产生IgG1抗体的全长重链。抗体轻链可变结构域序列(VL)通过基因合成并克隆到编码人抗体Igκ轻链恒定结构域序列的哺乳动物细胞表达质粒载体中，以编码产生抗体的全长轻链。在本实施例中，由于从免疫的Harbour H2L2小鼠得到的单克隆抗体分子可变结构域的序列是人源抗体序列，因而本实施例也得到全人源的抗BCMA重组IgG1抗体。
例如，本发明人源化抗体的重链序列为：
Figure PCTCN2021073026-appb-000006
Figure PCTCN2021073026-appb-000007
本发明人源化抗体轻链序列为：
Figure PCTCN2021073026-appb-000008
本发明人源化抗体的重链可变区的氨基酸序列为：
Figure PCTCN2021073026-appb-000009
本发明人源化抗体的轻链可变区的氨基酸序列为：
Figure PCTCN2021073026-appb-000010
将编码抗体重链的质粒(Genscript US)和编码抗体轻链的质粒(Genscript US)同时转染哺乳动物宿主细胞(如人胚肾细胞HEK293)，利用常规的重组蛋白表达和纯化技术，可以得到具有轻重链正确配对组装的纯化的重组抗体。具体来说，将HEK293细胞在FreeStyle TM F17 Expression Medium培养基 (Thermo，Cat#：A1383504)中扩充培养。瞬时转染开始之前，调节细胞浓度至6～8×10 5细胞/ml，于37℃ 8％CO 2摇床中培养24小时，细胞浓度在1.2×10 6细胞/ml。准备30ml培养的细胞。将上述编码抗体重链的质粒和编码抗体轻链的质粒以2:3(质量比)的比例混合共计30μg质粒溶解于1.5ml Opti-MEM减血清培养基(Thermo，Cat#：31985088)，并用0.22μm滤膜过滤除菌。再取1.5ml Opti-MEM溶入1mg/ml PEI(Polysciences，Cat#：23966-2)120μl，静置5分钟。把PEI缓慢加入质粒中，室温孵育10分钟，边摇晃培养瓶边缓慢滴入质粒PEI混合溶液，于37℃ 8％CO 2摇床中培养5天。5天后观测细胞活率。收集培养物，以3300g转速离心10分钟后取上清；然后将上清高速离心去除杂质。用PBS(pH7.4)平衡含有MabSelect TM(GE Healthcare Life Science，Cat#：71-5020-91AE)的重力柱(Bio-Rad，Cat#：7311550)，2-5倍柱体积冲洗。将上清样品过柱；用5-10倍柱体积的PBS冲洗柱子，再用pH3.5的0.1M甘氨酸洗脱目的蛋白，后用pH 8.0的Tris-HCl调节至中性，最后用超滤管(Millipore，Cat#：UFC901024)浓缩换液至PBS缓冲液，得到纯化的抗体溶液。最后用NanoDrop(Thermo Scientific TM NanoDrop TM One)测定浓度，分装、存储备用。
取上述纯化的样品适量分别上样至分析型SEC柱TSKgel G3000SWxl(HPLC仪器型号:安捷伦1260Infinity II)，检测样品的纯度，保证均一样品的纯度在95％以上。该方法流动相为1×PBS，pH7.4(生工，Cat#：E607016)，室温，流速1.0ml/min，样品浓度1mg/ml，进样体积20μl，检测波长280nm。采集后用ChemStation软件对色谱图进行积分并计算相关数据。
2.重组抗体验证
FACS鉴定：重组抗体依次稀释至9种不同浓度，并在293T-huBCMA、293T-cynoBCMA和NCI-H929细胞系上进行1小时孵育，然后第二抗体进行1小时孵育后检测，验证结果如图1所示。在图1a中，PR000892结合293T-huBCMA细胞(购自康源博创生物科技(北京)有限公司；货号为： CAT#KC-0233)有显著结合，且亲和力要优于对照抗体；图1b中，PR000892结合293T-cynoBCMA(购自康源博创生物科技(北京)有限公司；货号为：CAT#KC-0979)的亲和力与对照抗体相当；在图1c中，PR000892与肿瘤细胞系NCI-H929的亲和力与对照抗体相当。
阻断：重组抗体被连续稀释至7种不同浓度，在预先涂覆2μg/ml的huBCMA-ECD-FC的Corning 9018板上检测，后续步骤与以上相同。如图2所示，PR000892可以部分阻断BAFF与BCMA在体外条件下的结合。
内化：计数293T-huBCMA细胞或NCI-H929细胞，接种5000或10000个/孔至96孔黑壁透明底的平板(PE6005225)。重组抗体连续稀释至9种不同浓度并加入，最终浓度从100nM开始。加入a-hFc-MMAF使最终浓度为1μg/ml。平板在37℃、5％CO 2条件下孵育72小时，然后用CTG试剂盒裂解，并用Enspire检测发光。如图3a中所示，在293T-huBCMA细胞系中，PR000892的内化作用与对照抗体相当；如图3b中所示，PR000892在NCI-H929细胞系中的内化作用显著优于对照抗体。
另外：经验证，本发明抗体和对照抗体与BCMA抗原(Cat.BCA-H522y)的亲和力相当。

Claims (12)

  1. 一种BCMA结合蛋白，其特征在于，其包括重链可变区和轻链可变区，所述的轻链可变区包含LCDR1、LCDR2和LCDR3，所述的LCDR1的氨基酸序列如序列表中SEQ ID NO:1所示，所述的LCDR2的氨基酸序列如序列表中SEQ ID NO:2所示，所述的LCDR3的氨基酸序列如序列表中SEQ ID NO:3所示；
    所述的重链可变区包含HCDR1、HCDR2和HCDR3，所述的HCDR1的氨基酸序列如序列表中SEQ ID NO:4所示，所述的HCDR2的氨基酸序列如序列表中SEQ ID NO:5所示，所述的HCDR3的氨基酸序列如序列表中SEQ ID NO:6所示。
  2. 如权利要求1所述的BCMA结合蛋白，其特征在于，所述的BCMA结合蛋白还包括抗BCMA抗体的框架区，所述框架区包括重链框架区和/或轻链框架区；
    所述的重链框架区优选人源重链框架区，所述的人源重链框架区HFWR1～4包含分别如序列表中SEQ ID NO:13～16所示的氨基酸序列；
    所述的轻链框架区优选人源轻链框架区，所述的人源轻链框架区LFWR1～4包含分别如序列表中SEQ ID NO:17～20所示的氨基酸序列。
  3. 如权利要求2所述的BCMA结合蛋白，其特征在于，所述的重链可变区包括如序列表中SEQ ID NO:11所示的氨基酸序列，且所述的轻链可变区包括如序列表中SEQ ID NO:12所示的氨基酸序列。
  4. 如权利要求1～3任一项所述的BCMA结合蛋白，其特征在于，还包括重链恒定区和/或轻链恒定区；较佳地，所述重链恒定区选自hIgG1、hIgG2、hIgG3或hIgG4或其突变，所述轻链恒定区选自κ链或者λ链或其突变。
  5. 如权利要求1～4任一项所述的BCMA结合蛋白，其特征在于，其为全长抗体、Fab、Fab’、F(ab’)2、Fv、scFv、双特异性抗体、多特异性抗体、 单域抗体或单区抗体，或由上述抗体制得的单克隆抗体或多克隆抗体。
  6. 如权利要求5所述的BCMA结合蛋白，其特征在于，所述全长抗体包括重链和轻链，所述重链包括如SEQ ID NO:9所示的氨基酸序列，所述轻链包括如SEQ ID NO:10所示的氨基酸序列。
  7. 一种分离的核酸，其编码如权利要求1～6任一项所述的BCMA结合蛋白。
  8. 一种包含如权利要求7所述的分离的核酸的表达载体。
  9. 一种宿主细胞，其包含根据权利要求8所述的表达载体；优选地，所述宿主细胞为原核细胞或真核细胞。
  10. 一种BCMA结合蛋白的制备方法，其包含培养如权利要求9所述的宿主细胞，从培养物中获得所述BCMA结合蛋白。
  11. 一种药物组合物，其包含如权利要求1～6任一项所述的BCMA结合蛋白。
  12. 如权利要求1～6任一项所述的BCMA结合蛋白和如权利要求11所述的药物组合物在制备治疗和/或预防癌症的药物中的应用；优选地，所述癌症为骨髓瘤、结肠癌、肺癌、前列腺癌、肝癌、肾癌、胰腺癌、乳腺癌、宫颈癌或卵巢癌。
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