WO2021121250A1 - Bcma结合抗体及其用途 - Google Patents

Bcma结合抗体及其用途 Download PDF

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WO2021121250A1
WO2021121250A1 PCT/CN2020/136725 CN2020136725W WO2021121250A1 WO 2021121250 A1 WO2021121250 A1 WO 2021121250A1 CN 2020136725 W CN2020136725 W CN 2020136725W WO 2021121250 A1 WO2021121250 A1 WO 2021121250A1
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antibody
cells
bcma
seq
cdr
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PCT/CN2020/136725
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English (en)
French (fr)
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叶立军
冯婷
张嘉美
王先进
王保垒
彭亮
路力生
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深圳市菲鹏生物治疗股份有限公司
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Priority to BR112022011964A priority Critical patent/BR112022011964A2/pt
Priority to US17/785,419 priority patent/US20230039487A1/en
Publication of WO2021121250A1 publication Critical patent/WO2021121250A1/zh

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    • 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
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Definitions

  • the present invention relates to the field of biomedicine, in particular to an antibody capable of specifically binding to BCMA, a chimeric antigen receptor and immune cells containing the antibody.
  • MM Multiple myeloma
  • the current treatment options for the disease are mainly symptomatic treatment, chemotherapy, radiotherapy, and stem cell transplantation, but the nearly 100% recurrence rate makes the treatment of the disease extremely difficult.
  • B-cell maturation antigen (BCMA, B-cell maturation antigen) is a member of the TNF superfamily receptor (TNFRSF17, type III transmembrane protein, full length 185 amino acids, extracellular segment 54 amino acids). It is specifically expressed on the surface of plasma cells and multiple myeloma cells; and it is not expressed in memory B cells, hematopoietic stem cells and other normal tissue cells.
  • TNF superfamily receptor TNFRSF17, type III transmembrane protein, full length 185 amino acids, extracellular segment 54 amino acids.
  • BCMA is highly expressed on the surface of plasma cells and myeloma cells, so BCMA is an ideal target for the treatment of multiple myeloma.
  • the current clinical results show that immune cell therapy for patients with multiple myeloma is significantly better than chemotherapy and radiotherapy; in view of this, there is an urgent need in the art for a functional antibody targeting BCMA and its derived immune cell therapy products.
  • the present invention relates to an antibody that can specifically bind to BCMA and is selected from a) and/or b):
  • the present invention also relates to a chimeric antigen receptor comprising the antibody.
  • the present invention also relates to nucleic acids, vectors and host cells for expressing and producing the antibody and chimeric antigen receptor.
  • the present invention also relates to immune cells, which comprise the chimeric antigen receptor as described above.
  • the present invention also relates to a pharmaceutical composition, which includes the antibody as described above or the immune cell as described above, and one or more of pharmaceutically acceptable excipients, diluents or carriers.
  • the BCMA antibody provided by the present invention can specifically bind to the extracellular segment of BCMA, and has excellent affinity and specificity (it basically does not bind to other antigens on the cell membrane surface); and the antibody is functional Antibody, which has the activity of blocking the binding of BCMA to its ligand APRIL.
  • the immune cells constructed based on the antibody that can express the chimeric antigen receptor have a very good specific killing function for BCMA-positive tumor cells.
  • Figure 1 shows the detection of the affinity of BCMA antibodies (5E2, 5F4) for BCMA by ELISA in an embodiment of the present invention
  • Figure 2 shows the detection of the affinity of BCMA antibodies (5E2, 5F4) for BCMA by Fortebio in an embodiment of the present invention
  • FIG. 3 shows the detection of the affinity of BCMA antibodies (5E2, 5F4) for BCMA by means of FACs in an embodiment of the present invention
  • Figure 4 shows the results of a competitive binding experiment between BCMA antibody (5E2, 5F4) and BCMA ligand APRIL in an embodiment of the present invention
  • Figure 5 is a specific detection result of BCMA antibody (5E2, 5F4) in an embodiment of the present invention.
  • Figure 6 is a schematic diagram of the structure of the C11D5.3 CAR plasmid used in an embodiment of the present invention.
  • Figure 7 is a schematic diagram of the structure of the 5E2CAR plasmid used in an embodiment of the present invention.
  • Figure 8 is an experimental result of flow cytometric detection of CAR positive rate of CART cells in an embodiment of the present invention.
  • Figure 9 shows the detection result of target cell apoptosis after CART and target cells are co-cultured for 8 hours in an embodiment of the present invention
  • Figure 10 shows the results of IL-2 detection after 8 hours of co-cultivation of CART cells and target cells in an embodiment of the present invention
  • Figure 11 shows the results of IFN ⁇ detection after 8 hours of co-culture of CART cells and target cells in an embodiment of the present invention
  • Figure 12 is the result of ELISA detecting the affinity of the humanized BCMA antibody against the BCMA antigen in an embodiment of the present invention
  • Figure 13 is the result of Fortebio detecting the affinity of the humanized BCMA antibody against the BCMA antigen in an embodiment of the present invention
  • Figure 14 shows the binding result of FACs detection antibody and tumor cell line in an embodiment of the present invention
  • Figure 15 is a result of competitive binding of humanized BCMA antibody and BCMA ligand APRIL in an embodiment of the present invention
  • Figure 16 is the result of a flow cytometer in an embodiment of the present invention to detect that the humanized BCMA antibody can specifically bind to cells expressing positive BCMA;
  • Figure 17 is a schematic diagram of the structure of PCDHF-42 in an embodiment of the present invention.
  • Figure 18 is a schematic diagram of the structure of PCDHF-73 in an embodiment of the present invention.
  • Figure 19 is a schematic diagram of the structure of PCDHF-74 in an embodiment of the present invention.
  • Figure 20 shows the CAR positive rate CAR+ of CART cells in an embodiment of the present invention
  • Figure 21 shows the detection result of target cell apoptosis after CART and target cells are co-cultured for 6 hours in an embodiment of the present invention
  • Figure 22 shows the IL-2 detection results after CART cells and target cells are co-cultured for 6 hours in an embodiment of the present invention.
  • Figure 23 shows the results of IFN ⁇ detection after CART cells and target cells are co-cultured for 6 hours in an embodiment of the present invention.
  • the present invention relates to an antibody that can specifically bind to BCMA and is selected from a) and/or b):
  • antibody generally refers to all proteins/protein fragments containing CDR regions, especially full-length antibodies or antibody functional fragments.
  • full-length antibody includes polyclonal antibodies and monoclonal antibodies.
  • antibody functional fragment is a substance that contains part or all of the CDR of an antibody, which lacks at least some of the amino acids present in the full-length chain but still has specificity. Binding to antigen. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope.
  • the fragment is a fragment that has the function of blocking the binding of BCMA to its ligand APRIL.
  • fragments can block or reduce the activity of BCMA.
  • such fragments will comprise a single heavy chain and a single light chain, or parts thereof.
  • the fragments can be produced by recombinant nucleic acid technology, or can be produced by enzymatic or chemical cleavage of antigen binding molecules (including intact antibodies).
  • Variants of antibodies are also within the scope of the present invention, for example, each has at least 70% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, 95% of the amino acid sequence of the sequence described herein. -97%, 97%-99%, or higher than 99% variable light chain and/or variable heavy chain.
  • the variant of the antibody includes at least the above 6 CDRs; in some cases, the variant of the antibody includes at least one heavy chain and one light chain, while in other cases, the variant form contains two identical light chains. Chain and two identical heavy chains (or sub-portions thereof). In some cases, the variant retains the ability to block the binding of BCMA to its ligand APRIL.
  • those skilled in the art will be able to use well-known techniques to determine suitable variants of the antigen binding molecules as set forth herein. In certain embodiments, those skilled in the art can identify suitable regions of the molecule that can be altered by targeting regions that are believed to be unimportant for activity without destroying activity.
  • the antibody provided by the present invention can specifically bind to the extracellular segment of BCMA, and has excellent specificity (it basically does not bind to other antigens on the cell membrane surface).
  • an important advantage of the antibody is that it has the activity of blocking the binding of BCMA to its ligand APRIL, so it can preferably be used as an antibody drug.
  • Antibodies can be used to analyze the amount of BCMA present in a sample and/or subject.
  • the diagnostic antibody is not an antibody used to block the binding function of BCMA to its ligand APRIL.
  • the antibodies disclosed herein can be used or provided in analysis kits and/or analysis kits for detecting BCMA in tissues or cells of mammals, especially humans, for screening/diagnosing diseases or disorders related to changes in BCMA levels Or method.
  • the kit may comprise an antigen binding molecule that binds to BCMA, along with means for indicating the binding of the antigen binding molecule to BCMA (if present) and optionally the level of BCMA protein.
  • frame region or "FR” region means regions other than those regions defined as CDRs that exclude antibody variable domains.
  • Each antibody variable domain framework can be further subdivided into adjacent regions (FR1, FR2, FR3, and FR4) separated by CDRs.
  • variable regions VL/VH of the heavy chain and light chain can be obtained by arranging and connecting the following numbered CDRs and FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • isolated in connection with a polypeptide or nucleic acid means that the polypeptide or nucleic acid is not in its natural medium or in its natural form. Therefore, the term “isolated” includes a polypeptide or nucleic acid taken from its original environment, for example, if it is naturally occurring, from the natural environment.
  • the antibody a includes the heavy chain framework regions FR-H1, FR- as shown in SEQ ID NO: 7-10, SEQ ID NO: 41-44, or SEQ ID NO: 45-48. H2, FR-H3, and FR-H4; and/or; sequence as shown in SEQ ID NO: 11-14, SEQ ID NO: 49-52, SEQ ID NO: 53-56 or SEQ ID NO: 57-60.
  • the antibody b comprises the heavy chain framework regions FR-H1, FR-H2, FR-H3 and FR-H4 shown in SEQ ID NO: 25-28 in sequence; and/or; sequence in sequence The light chain framework regions FR-L1, FR-L2, FR-L3, and FR-L4 shown in SEQ ID NOs: 29 to 32.
  • the antibody is one of F(ab')2, Fab, Fv, scFv, and bispecific antibodies. In a further embodiment, the antibody is a single chain variable fragment (scFv).
  • the antibody has a constant region, and the constant region sequence is selected from the sequence of any one constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.
  • the species source of the constant region is independently selected from cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, Chicken, duck, goose, turkey, cockfighting or human.
  • the present invention also relates to a chimeric antigen receptor, which comprises an antibody as described above.
  • a preferred orientation of the chimeric antigen receptor according to the present invention includes the type of antibody contained in sc-Fv.
  • the chimeric antigen receptor further comprises one or more elements selected from the group consisting of a leader peptide, a linker sequence, a transmembrane domain, a costimulatory domain, and a signaling domain.
  • the leader peptide is selected from the CD8 leader chimeric receptor signal peptide.
  • the leader peptide is selected from the linker sequence selected from the hinge region of CD8.
  • the transmembrane domain is selected from the transmembrane region of CD8.
  • the costimulatory domain is the signal transmission area of CD28, CD28T, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1 ), inducible T cell costimulatory factor (ICOS), lymphocyte function-related antigen-1 (LFA-1, CDl-la/CD18), CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig ⁇ (CD79a), DAP-10, Fc ⁇ receptor, MHC class 1 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM Protein), activated NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NK
  • the signaling domain is selected from any one of PKC ⁇ , Fc ⁇ RI ⁇ , ZAP70, CD3 ⁇ , or any combination thereof.
  • the functional fragment includes the CD8 leader chimeric receptor signal peptide, the hinge region of CD8, the transmembrane region of CD8, CD137, and CD3 ⁇ .
  • the invention also relates to an isolated nucleic acid that encodes an antibody as described above, or a chimeric antigen receptor as described above.
  • the present invention also relates to a vector, which contains the isolated nucleic acid as described above.
  • vector refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillary viruses.
  • Polyoma vacuole virus such as SV40.
  • the vector of the present invention contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or multiple Adenylation signal and poly U sequence etc.).
  • regulatory elements commonly used in genetic engineering such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or multiple Adenylation signal and poly U sequence etc.).
  • the vector of the present invention may also contain fragments such as genes used in screening (for example, antibiotic resistance genes), nucleic acids used to produce fluorescent proteins, and the like.
  • the fluorescent protein can be selected from green fluorescent protein, blue fluorescent protein, yellow fluorescent protein, orange fluorescent protein or red fluorescent protein.
  • the vector when the vector contains the nucleic acid encoding the chimeric antigen receptor as described above, the vector is preferably a retroviral vector, more preferably a lentiviral vector.
  • nucleotide sequence of the lentiviral vector is shown in SEQ ID NO: 18.
  • the present invention also relates to a host cell, which contains a vector as described above.
  • the term "host cell” refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or subtilis, fungal cells such as yeast cells or Aspergillus, such as S2 fruit fly cells or Sf9 Such as insect cells, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • the host cell is preferably a eukaryotic cell, more preferably a mammalian cell.
  • the present invention also relates to immune cells, which comprise the chimeric antigen receptor as described above;
  • the immune cells are T cells, tumor infiltrating lymphocytes (TIL cells), NK cells, dendritic cells, or NK-T cells.
  • the immune cells are autologous T cells or allogeneic T cells.
  • the immune cells are obtained from or prepared from peripheral blood.
  • the immune cells are obtained or prepared from peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • the immune cells are obtained from or prepared from bone marrow.
  • the immune cells are obtained from or prepared from cord blood.
  • the immune cell is a human cell.
  • the present invention also relates to a pharmaceutical composition, which includes the antibody as described above or the immune cell as described above, and one or more of pharmaceutically acceptable excipients, diluents or carriers.
  • pharmaceutically acceptable excipient, diluent or carrier refers to an excipient, diluent or carrier that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, which is well known in the art , Including but not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers.
  • pH adjusting agents include, but are not limited to, phosphate buffer
  • surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80
  • ionic strength enhancers include, but are not limited to, sodium chloride.
  • the pharmaceutical composition can be used for BCMA related diseases, especially multiple myeloma. Therefore, in particular, the present invention also relates to a method for treating multiple myeloma in a subject in need thereof, the method comprising:
  • an effective amount refers to an amount sufficient to obtain or at least partially obtain the desired effect.
  • an effective amount is usually an amount sufficient to prevent, prevent, or delay the occurrence of the disease. It is completely within the abilities of those skilled in the art to determine such an effective amount.
  • the effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the subject’s own immune system, the subject’s general conditions such as age, weight, and sex, the way the drug is administered, and other treatments that are administered at the same time and many more.
  • the method of administration may be injection administration or the like.
  • vocabulary such as “subject”, “subject”, “patient”, etc. can be used universally as needed.
  • the subject may be a mammal, preferably a human.
  • hBCMA-mFc mouse antibody Fc fragment
  • ACRO Mouse antibody Fc fragment
  • BCA-H5253 mouse antibody Fc fragment
  • Antibody affinity ELISA test Coat hBCMA ECD hFc, ACRO, BC7-H5254 in a 96-well enzyme-linked coated plate at a concentration of 2 ⁇ g/ml, 100 ⁇ l/well, and dilute 3 times (the former concentration is the latter concentration) 3 times that of ELISA, diluted with 1% BSA) the antibody binds to the antigen, and the EC 50 value of each antibody is detected (the specific operation steps are general ELISA operation steps).
  • R0317 is a C11D5.3BCMA mouse monoclonal antibody. Its specific sequence and information can be found publicly. The antibody to be tested was synthesized by Shenzhen Feipeng Therapeutic Co., Ltd. The results shown in Figure 1, results show that antibodies 5E2 and 5F4 with the BCMA having EC 50 R0317 at the same level.
  • Antibody affinity Fortebio detection using ProG biosensor (Pall ForteBIO, 18-1502), first Loading Buffer (PBS+0.02% Tween20), then add h BCMA ECD hFc (R0341) 5 ⁇ g/mL, and then add 3 kinds of antibodies, respectively Detect the KD, Kon and Kdis of 3 kinds of antibodies.
  • the specific operation steps are the conventional operation of the Fortebio instrument (forteBio, Serial NO: FB-40476).
  • the test results are shown in Figure 2, indicating that 5E2 5F4 has a strong affinity with the BCMA antigen and is at the same level as R0317.
  • FACs detects the binding of antibodies to tumor cell lines:
  • RPMI8226 cells CRM-CCL-155 TM
  • H929 cells CRL-9068 (TM ) is human multiple myeloma bone marrow B lymphocytes.
  • the specific detection method is as follows: harvest the cells, wash once with PBS, and then resuspend the cells in PBS according to 2E+5cells/200 ⁇ l. Three kinds of antibodies were diluted in gradients (the initial antibody concentration was 10 ⁇ g/ml, 1% BSA was diluted 3 times, a total of 9 gradients) and incubated with the cells at 4°C for 30 min.
  • HBCMA-mFc was coated on an ELISA plate at 4 ⁇ g/ml, and coated overnight at 4°C. After washing with PBS once, blocking with 1% BSA for 1 h, and then washing with PBS once. Dilute antibodies 5E2 and 5F4 stepwise, starting at a concentration of 200 ⁇ g/ml, and using 1% BSA for a 3-fold dilution, a total of 7 gradients, with h APRIL hFc 4 ⁇ g/ml (this concentration is used when APRIL binds to hBCMA-mFc).
  • K562 cells (ATCC, article number CCL-243) were infected with the prepared lentivirus to construct K562-BCMA cells, and the cells were single-clonal selection and identification, and a stable cell line K562-BCMA stably expressing BCMA was obtained.
  • the 5E2 and 5F4 antibodies were incubated with K562, H929, RPMI8226 and K562-BCMA cells at 4°C for 30 min, and then washed twice with PBS, and then the secondary antibody PE goat anti-mouse IgG1 (Biolegend, B288920) was added and incubated at 4°C for 30 min. , And then washed twice with PBS. Then use the Beckman Coulter flow cytometer for detection. The test results are shown in Figure 5 below. 5E2 and 5F4 can specifically bind to cells with positive BCMA expression.
  • RNA extraction kit: TOYOBO LIFE SCIENCE, catalog number 836700, extraction steps follow the instructions.
  • Perform reverse transcription on the extracted RNA to obtain cDNA Reverse Transcription Kit: TOYOBO LIFE Science, Catalog No. 11141ES10), and perform PCR amplification on the obtained cDNA (using specific primers for the mouse IgG1 sequence) to obtain the antibody sequence and It was sequenced.
  • VH and VL were sequenced. See the VH and VL sequences labeled 5E2 and 5F4 in Example 1. Since the affinity, specificity and functional properties of the two antibodies are similar, we will take 5E2 as an example for CART function verification.
  • the scFv sequence of 5E2 antibody (obtained by connecting the C-terminus of VH with the N-terminus of VL through a connecting peptide, the nucleotide sequence of the connecting peptide is shown in SEQ ID NO: 35) and the scFv sequence of C11D5.3 (SEQ ID NO :17)
  • the CAR plasmids were constructed on the lentiviral vector (PCDHF) respectively.
  • the schematic diagrams of the structure of the C11D5.3CAR plasmid and the 5E2CAR plasmid are shown in Figure 6 and Figure 7.
  • Packaging lentivirus the packaging system and packaging steps are as follows:
  • DMEM medium containing 10% FBS, (DMEM Gibco, 11995040-1L; FBS Gibco , 10091-148), under 5% CO 2, 37 °C CO 2 Cultivate in an incubator for 48 hours;
  • "*1, *2, *3" are the lentivirus packaging plasmid PMD2.G, pMDLg/pRRE, and pRSV-Rev sequence respectively, which can be obtained through public channels (website http://www.miaolingbio.com/).
  • c Collect the cell supernatant 48h after packaging, and detect the lentivirus titer after ultracentrifugation at 25000rpm.
  • Target cells K562, K562-BCMA, H929 and RPMI8226 each 2 ⁇ 10E+06 cells, first use CytoCalceinTM Violet 550 to stain the target cells, 1 ⁇ 10E+05 cells/100 ⁇ l/well.
  • the effector cells (CAR+CART, T cell as control) and the target cells were added to a 96-well plate at a ratio of 0.25:1, 1:1, 5:1, and 10:1, and the final volume was 200 ⁇ l. After culturing for 8 hours, the cells were mixed and centrifuged.
  • the supernatant was used Human IL-2 ELISA detection kit (invitrogen, REF 88-7025-88) Human IFN gamma ELISA kit (invitrogen, REF 88-7316-88) to detect the concentration of IL-2 and IFN- ⁇ in each well, precipitation Part of it was resuspended in 100 ⁇ l Annexin V Binding Buffer (Biolegend, B274722), centrifuged at 300g for 5 min, and then 2.0 ⁇ l APC-Annexin V (Biolegend, Cat 640920) and 1.2 ⁇ l PI dye (Biolegend, Cat 421301) were added, and incubated for 15 minutes in the dark.
  • Human IL-2 ELISA detection kit invitrogen, REF 88-7025-88
  • Human IFN gamma ELISA kit invitrogen, REF 88-7316-88
  • the murine 5E2 antibody was humanized to obtain 4 antibody sequences, including 2 heavy chain variable regions (SEQ ID NO: 36-SEQ ID NO: 37) and 3 light chain variable regions (EQ ID NO: 38-SEQ ID NO: 40).
  • Antibody affinity ELISA test Coat hu (human) BCMA ECD His, ACRO, BCA-H522y in 96-well enzyme-linked coated plate at a concentration of 2 ⁇ g/mL, 100 ⁇ L/well, and use 4 humanized antibodies with 1 %BSA was prepared to an initial concentration of 20 ⁇ g/mL, and a 3-fold dilution of 1%BSA (the concentration of the former gradient antibody was 3 times that of the latter gradient), the antibody bound to the antigen, and the EC 50 values of the four antibodies were detected (specifically The operation steps are general ELISA operation steps). Among them, mVH-mVL is a murine BCMA antibody, which is a murine antibody before humanization. The results are shown in Figure 12 below. The results show that the four humanized BCMA antibodies and the murine BCMA antibody mVH-mVL have the same level of EC 50 , And huVH1-VL2 has the highest affinity.
  • FACs detects the binding of antibodies to tumor cell lines:
  • K562 cells ( CCL-243TM) are human chronic myeloid leukemia cells
  • CHO cells CRL-12023 TM
  • K562 cells and CHO cells were respectively infected with lentivirus containing the full-length human BCMA sequence. After infection, single clones were selected to obtain K562-BCMA cell lines and CHO-BCMA cells stably expressing BCMA
  • the specific detection method is as follows: harvest the cells, wash once with PBS, and then resuspend the cells in PBS at 2E+5cells/200 ⁇ L.
  • the 4 humanized BCMA antibodies were diluted 3-fold with 1% BSA, the concentration of the former gradient was 3 times that of the latter, (the initial concentration of the antibody was 30 ⁇ g/ml, 11 gradients in total), respectively, and The cells were incubated at 4°C for 30 minutes. After that, it was incubated with APC anti-human IgG Fc Antibody (Biolegend, 409306) at 4°C for 30 minutes, washed twice with 1 ⁇ PBS, and detected by a Beckman Coulter (model: CytoFLEX) flow cytometer.
  • the three humanized antibodies bind to K562-BCMA cells and CHO-BCMA cells in a concentration gradient-dependent manner and the EC 50 is at the same level as the murine BCMA antibody.
  • the EC50 of huVH1VL2 and K562-BCMA is slightly weaker.
  • the EC 50 of huVH1-VL2 and CHO-BCMA affinity is at the same level as the other three humanized antibodies.
  • the four humanized antibodies and murine BCMA antibodies bind to CHO-BCMA in terms of abundance, and the order from high to low is m VH-m VL, hu VH1-VL1, hu VH2-VL1, hu VH1-VL2, hu VH1-VL3.
  • HBCMA-mFc was coated on an ELISA plate at 4 ⁇ g/ml, and coated overnight at 4°C. After washing with 1 ⁇ PBS once, blocking with 1% BSA (Sangon Biotech, A500023-0100) for 1 h, and then washing with 1 ⁇ PBS once. Dilute the 4 humanized antibodies in a 3-fold gradient. The concentration of the former gradient is 3 times that of the latter. The initial concentration is 100 ⁇ g/ml, a total of 11 gradients.
  • the diluent is h APRIL His 0.2 ⁇ g/ml, (This concentration is between the EC 50 and saturation of APRIL and hBCMA-mFc binding), h APRIL His was dissolved in 1% BSA, incubated at 37°C for 30 min, washed 5 times with 1 ⁇ PBS, and added 1:1500 diluted HRP-labeled antibody His antibody (Anti-his tag HRP, Biolegend, 652504), incubated at 37°C for 30 min, washed 5 times, TMB developed the color, and read on the multi-functional microplate reader after termination. The results are shown in Figure 15 below.
  • the IC50 of the four humanized BCMA antibodies to block APRIL is equivalent to that of the murine BCMA antibody m VH-m VL, and the four humanized BCMA antibodies have obvious blocking effects on APRIL and hBCMA-mFc. Concentration gradient dependence.
  • the packaging system and packaging steps are as follows:
  • DMEM medium DMEM Gibco, 11995040-1L; FBS Gibco, 10091-148 containing 10% FBS, 5% CO2, CO2 incubator at 37°C Cultivate for 24h;
  • c Collect the cell supernatant 48h after packaging, and detect the lentivirus titer after ultracentrifugation at 25000rpm.
  • Ficoll Lymphatic Separation Solution separates PBMC cells from blood (50 mL of donated blood from volunteers), and separates T cells by magnetic bead positive selection coupled with CD3/CD28 antibody.
  • Target cells K562, K562-BCMA, RPMI8226 each 2 ⁇ 10E+06 cells, first use CytoCalcein TM Violet 550 to stain the target cells, 1 ⁇ 10E+05 cells/100ul/well. Add effector cells (CAR+CART, T cells as control) and the above target cells in a 96-well plate at a ratio of 0.25:1, 1:1, 5:1, and 10:1, and mix well. The final volume is 200ul. After culturing for 6 hours, the cells were mixed and centrifuged.
  • the supernatant was used Human IL-2 ELISA detection kit (invitrogen, REF 88-7025-88) Human IFN gamma ELISA kit (invitrogen, REF 88-7316-88) to detect the concentration of IL-2 and IFN- ⁇ in each well, precipitation Part of it was resuspended in 100ul Annexin V Binding Buffer (Biolegend, B274722), centrifuged at 300g for 5min, then 3ul APC-Annexin V (Biolegend, Cat 640920) and 1.5ul PI dye (Biolegend, Cat 421301) were added, and incubated for 15min in the dark, and added Resuspend in 100ul Annexin V Binding Buffer, and then use the Beckman Coulter flow cytometer to detect the apoptotic ratio of each target cell (the result is shown in Figure 21), and use ELISA to detect the concentration of IL-2 and IFN- ⁇ in the supernatant of each well (the result is shown in Figure
  • K562 are BCMA-negative cells
  • K562-BCMA and RPMI8226 are BCMA-positive cells.
  • the results show that PCDHF-73CART, PCDHF-74CART and PCDHF-42CART have strong specific killing effect on BCMA-positive target cells, and the killing ability of the three CART cells is basically the same, and it has almost no killing effect on BCMA-negative cells;
  • PCDHF -42CART, PCDHF-73CART and PCDHF-74CART kill BCMA positive target cells when IL-2 and IFN- ⁇ secretion are at the same level.
  • the comprehensive specific killing and factor secretion test results indicate that humanized BCMA CART PCDHF-73CART and PCDHF-74CART and mouse BCMA CART PCDHF-42CART have the same killing effect on BCMA positive target cells.

Abstract

提供了一种抗体，其能够特异性结合BCMA。所提供的BCMA抗体能够特异性地与BCMA的胞外段结合，并具有优秀的亲和力与特异性；且该抗体为功能性抗体，其具有阻断BCMA与其配体APRIL结合的活性。基于该抗体构建的免疫细胞对于BCMA阳性的肿瘤细胞有非常好的特异性杀伤功能。

Description

BCMA结合抗体及其用途 技术领域
本发明涉及生物医疗领域，具体而言，涉及一种能够特异性结合BCMA的抗体，包含所述抗体的嵌合抗原受体及免疫细胞。
背景技术
多发性骨髓瘤(MM)是常见的恶性血液病，占所有癌症死亡例的2％，据Global data 2019年统计，2017年全球发病人数为353890人，预计2027年将达到555243例，其主要病状是骨髓中的浆细胞无限增殖，进而导致骨坏死。目前对于该病的治疗方案主要为对症治疗、化疗、放疗和干细胞移植，但将近100％的复发率让对该病的治疗变得极为艰难。
B细胞成熟抗原(BCMA，B-cell maturation antigen)是TNF超家族受体成员(TNFRSF17，III型跨膜蛋白，全长185个氨基酸，胞外段54个氨基酸)。特异性高表达于浆细胞和多发性骨髓瘤细胞表面；并且记忆B细胞，造血干细胞和其他正常组织细胞中均不表达。其功能与同家族受体TACI、BAFFR以及配体APRIL/BAFF一起调节B细胞的活化，分化以及转化成浆细胞和延长浆细胞寿命；B细胞分化成浆细胞过程中，细胞表面BCMA表达上调，缺乏BCMA的小鼠有正常数量的B细胞健康且外观正常，但浆细胞的生存周期缩短。
目前对于多发性骨髓瘤患者的治疗，效果差，成本高，周期长；BCMA因其特异性高表达于浆细胞和骨髓瘤细胞的表面，所以BCMA是治疗多发性骨髓瘤非常理想的靶点，现有临床结果显示对于多发性骨髓瘤患者的免疫细胞治疗明显优于化疗和放疗；鉴于此，本领域急需一种靶向BCMA的功能抗体，以及其衍生的免疫细胞治疗产品。
发明内容
本发明涉及一种抗体，其能够特异性结合BCMA，且选自a)和/或b)：
a)包含氨基酸序列依次如SEQ ID NO:1～3所示的重链互补决定区CDR-VH1、CDR-VH2、CDR-VH3，以及氨基酸序列依次如SEQ ID NO:4～6所示的轻链互补决定区CDR-VL1、CDR-VL2、CDR-VL3；
b)包含氨基酸序列依次如SEQ ID NO:19～21所示的重链互补决定区CDR-VH1、CDR-VH2、CDR-VH3，以及氨基酸序列依次如SEQ ID NO:22～24所示的轻链互补决定区CDR-VL1、CDR-VL2、CDR-VL3。
本发明还涉及包含所述抗体的嵌合抗原受体。
本发明还涉及用于表达生产所述抗体和嵌合抗原受体的核酸、载体及宿主细胞。
本发明还涉及免疫细胞，其包含如上所述的嵌合抗原受体。
本发明还涉及药物组合物，其包括如上所述的抗体或如上所述的免疫细胞，以及药学上可接受的赋形剂、稀释剂或载体中的一种或多种。
与现有技术相比，本发明的有益效果为：
(1)本发明所提供的BCMA抗体能够特异性地与BCMA的胞外段结合，并具有优秀的亲和力与特异性(其基本不与细胞膜表面的其他抗原发生结合)；且该抗体为功能性抗体，其具有阻断BCMA与其配体APRIL结合的活性。
(2)基于该抗体构建的能够表达嵌合抗原受体的免疫细胞对于BCMA阳性的肿瘤细胞有非常好的特异性杀伤功能。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可 以根据这些附图获得其他的附图。
图1为本发明一个实施例中通过ELISA方式检测BCMA抗体(5E2、5F4)针对BCMA的亲和力；
图2为本发明一个实施例中通过Fortebio方式检测BCMA抗体(5E2、5F4)针对BCMA的亲和力；
图3为本发明一个实施例中通过FACs方式检测BCMA抗体(5E2、5F4)针对BCMA的亲和力；
图4为本发明一个实施例中BCMA抗体(5E2、5F4)与BCMA配体APRIL竞争性结合实验结果；
图5为本发明一个实施例中BCMA抗体(5E2、5F4)的特异性检测结果；
图6为本发明一个实施例中所采用的C11D5.3CAR质粒的结构示意图；
图7为本发明一个实施例中所采用的5E2CAR质粒的结构示意图；
图8为本发明一个实施例中流式检测CART细胞CAR阳性率的实验结果；
图9为本发明一个实施例中CART与靶细胞共培养8h后靶细胞凋亡检测结果；
图10为本发明一个实施例中CART细胞与靶细胞共培养8h后IL-2检测结果；
图11为本发明一个实施例中CART细胞与靶细胞共培养8h后IFNγ检测结果；
图12为本发明一个实施例中ELISA检测BCMA人源化抗体体针对BCMA抗原的亲和力结果；
图13为本发明一个实施例中Fortebio检测BCMA人源化抗体体针对BCMA抗原的亲和力结果；
图14为本发明一个实施例中FACs检测抗体与肿瘤细胞系的结合结果；
图15为本发明一个实施例中人源化BCMA抗体与BCMA配体APRIL竞争性结合结果；
图16为本发明一个实施例中流式细胞仪进行检测人源化BCMA抗体能够特异性地结合BCMA表达阳性的细胞的结果；
图17为本发明一个实施例中PCDHF-42结构示意图；
图18为本发明一个实施例中PCDHF-73结构示意图；
图19为本发明一个实施例中PCDHF-74结构示意图；
图20为本发明一个实施例中CART细胞CAR阳性率CAR+；
图21为本发明一个实施例中CART与靶细胞共培养6h后靶细胞凋亡检测结果；
图22为本发明一个实施例中CART细胞与靶细胞共培养6h后IL-2检测结果；以及
图23为本发明一个实施例中CART细胞与靶细胞共培养6h后IFNγ检测结果。
具体实施方式
现将详细地提供本发明实施方式的参考，其一个或多个实例描述于下文。提供每一实例作为解释而非限制本发明。实际上，对本领域技术人员而言，显而易见的是，可以对本发明进行多种修改和变化而不背离本发明的范围或精神。例如，作为一个实施方式的部分而说明或描述的特征可以用于另一实施方式中，来产生更进一步的实施方式。
因此，旨在本发明覆盖落入所附权利要求的范围及其等同范围中的此类修改和变化。本发明的其它对象、特征和方面公开于以下详细描述中或从中是显而易见的。本领域普通技术人员应理解本讨论仅是示例性实施方式的描述，而非意在限制本发明更广阔的方面。
本发明涉及一种抗体，其能够特异性结合BCMA，且选自a)和/或b)：
a)包含氨基酸序列依次如SEQ ID NO:1～3所示的重链互补决定区CDR-VH1、CDR-VH2、CDR-VH3，以及氨基酸序列依次如SEQ ID NO:4～6所示的轻链互补决定区CDR-VL1、CDR-VL2、CDR-VL3；
b)包含氨基酸序列依次如SEQ ID NO:19～21所示的重链互补决定区CDR-VH1、CDR-VH2、CDR-VH3，以及氨基酸序列依次如SEQ ID NO:22～24所示的轻链互补决定区 CDR-VL1、CDR-VL2、CDR-VL3。。
在本发明中，“抗体”泛指包含CDR区的一切蛋白/蛋白片段，特别是全长抗体或抗体功能片段。“全长抗体”此用语包括多克隆抗体及单克隆抗体，术语“抗体功能片段”是包含抗体CDR的一部分或全部的物质，其缺乏至少一些存在于全长链中的氨基酸但仍能够特异性结合至抗原。此类片段具生物活性，因为其结合至靶抗原，且可与其他抗原结合分子(包括完整抗体)竞争结合至给定表位。在一些实施方式中，片段是具有阻断BCMA与其配体APRIL结合功能的片段。在一些实施方案中，片段可阻断或降低BCMA的活性。在一个方面中，此类片段将包含单个重链和单个轻链，或其部分。所述片段可通过重组核酸技术产生，或可通过抗原结合分子(包括完整抗体)的酶裂解或化学裂解产生。
抗体的变体也在本发明范围内，例如各自与本文所述的序列的氨基酸序列具有至少70％～80％、80％～85％、85％～90％、90％～95％、95％～97％、97％～99％或高于99％同一性的可变轻链和/或可变重链。在一些情况下，抗体的变体至少包括上述6个CDR；在一些情况下，抗体的变体至少包括一个重链和一个轻链，而在其他情况下，变体形式含有两个相同的轻链和两个相同的重链(或其子部分)。在一些情况下，变体保留阻断BCMA与其配体APRIL结合的能力。所属领域技术人员将能够使用熟知的技术确定如本文所阐明的抗原结合分子的合适变体。在某些实施方案中，所属领域技术人员可鉴别分子的可通过靶据信对于活性而言不重要的区来改变而不破坏活性的合适区域。
本发明所提供的抗体能够特异性地与BCMA的胞外段结合，并具有优秀的特异性(其基本不与细胞膜表面的其他抗原发生结合)。特别地，该抗体的一个重要优点在于其具有阻断BCMA与其配体APRIL结合的活性，因而优选可作为抗体药物使用。
在另外一些实施方案中，鉴于本发明已经验证了抗体与BCMA的亲和力，因而本领域技术人员也可以在不付出创造性的前提下判断其也可用作诊断或验证工具。抗体可用于分析样品和/或受试者中存在的BCMA的量。在一些实施方案中，诊断性抗体不是用于阻断BCMA与其配体APRIL结合功能的抗体。在一些实施方案中，本文所公开的抗体可用于或提供于用于检测哺乳动物特别是人组织或细胞中的BCMA以筛检/诊断与BCMA水平变化相关的疾病或病症的分析试剂盒和/或方法中。试剂盒可包含结合BCMA的抗原结合分子，连同用于指示抗原结合分子与BCMA的结合(若存在)及任选地BCMA蛋白水平的构件。
当在本文中使用时，“骨架区”或“FR”区意味着抗体可变结构域的排除被定义为CDR的那些区域之外的区域。每个抗体可变结构域构架可以被进一步细分成被CDR分隔开的毗邻区域(FR1、FR2、FR3和FR4)。
通常情况下，重链和轻链的可变区VL/VH可由以下编号的CDR与FR按如下组合排列连接获得：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
当在本文中使用时，与多肽或核酸相关联的术语“分离的”是指多肽或核酸不是处于其天然介质中或天然形式下。因此，术语“分离的”包括从其原始环境，例如如果它是天然存在的，从天然环境取出的多肽或核酸。
在一些实施方式中，所述抗体a包含序列如SEQ ID NO:7～10、SEQ ID NO:41～44、或SEQ ID NO:45～48所示的重链骨架区FR-H1、FR-H2、FR-H3及FR-H4；和/或；序列依次如SEQ ID NO:11～14、SEQ ID NO:49～52、SEQ ID NO:53～56或SEQ ID NO:57～60所示的轻链骨架区FR-L1、FR-L2、FR-L3及FR-L4。
在一些实施方式中，所述抗体b包含序列依次如SEQ ID NO:25～28所示的重链骨架区FR-H1、FR-H2、FR-H3及FR-H4；和/或；序列依次如SEQ ID NO:29～32所示的轻链骨架区FR-L1、FR-L2、FR-L3及FR-L4。
在一些实施方式中，所述抗体为F(ab’)2、Fab、Fv、scFv以及双特异抗体中的一种。在进一步实施方案中，所述抗体是单链可变片段(scFv)。
在一些实施方式中，所述抗体具有恒定区，恒定区序列选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE、IgD任何其中之一恒定区的序列。
在一些实施方式中，所述恒定区的种属来源独立地选自牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人。
本发明还涉及嵌合抗原受体，其包含如上所述的抗体。
应当理解，根据本发明的嵌合抗原受体的一个优选取向包含的抗体类型为sc-Fv。
在一些实施方式中，所述嵌合抗原受体还包含一个或多个选自由以下组成的组的元件：前导肽、接头序列、跨膜结构域、共刺激结构域和信号传导结构域。
在一些实施方式中，所述前导肽选自CD8leader嵌合受体信号肽。
在一些实施方式中，所述前导肽选自接头序列选自CD8的hinge区。
在一些实施方式中，所述跨膜结构域选自CD8的跨膜区。
在一些实施方式中，共刺激域是以下项的信号传送区：CD28、CD28T、OX-40、4-1BB/CD137、CD2、CD7、CD27、CD30、CD40、程序性死亡-1(PD-1)、可诱导型T细胞共刺激因子(ICOS)、淋巴细胞功能相关抗原-1(LFA-1、CDl-la/CD18)、CD3γ、CD3δ、CD3ε、CD247、CD276(B7-H3)、LIGHT、(TNFSF14)、NKG2C、Igα(CD79a)、DAP-10、Fcγ受体、MHC 1类分子、TNF受体蛋白、免疫球蛋白蛋白质、细胞因子受体、整联蛋白、信号淋巴细胞活化分子(SLAM蛋白)、活化NK细胞受体、BTLA、Toll配体受体、ICAM-1、B7-H3、CDS、ICAM-1、GITR、BAFFR、LIGHT、HVEM(LIGHTR)、KIRDS2、SLAMF7、NKp80(KLRF1)、NKp44、NKp30、NKp46、CD19、CD4、CD8α、CD8β、IL-2Rβ、IL-2Rγ、IL-7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CDl ld、ITGAE、CD103、ITGAL、CDl la、LFA-1、ITGAM、CDl lb、ITGAX、CDl lc、ITGBl、CD29、ITGB2、CD18、LFA-1、ITGB7、NKG2D、TNFR2、TRANCE/RANKL、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRT AM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、CD69、SLAMF6(NTB-A、Lyl08)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、CD19a、与CD83特异性结合的配体或其任何组合。
在一些实施方式中，所述信号传导结构域选自PKCθ、FcεRIγ、ZAP70、CD3ζ中的任一种，或其任意组合。
在一些实施方式中，所述功能片段包括CD8leader嵌合受体信号肽、CD8的hinge区、CD8的跨膜区、CD137以及CD3ζ。
本发明还涉及分离的核酸，其编码如上所述的抗体，或如上所述的嵌合抗原受体。
本发明还涉及载体，其包含如上所述的分离的核酸。
术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。在一些实施方式中，本发明所述载体中包含基因工程中常用的调控元件，例如增强子、启动子、内部核糖体进入位点(IRES)和其他表达控制元件(例如转录终止信号，或者多腺苷酸化信号和多聚U序列等)。
在一些实施方式中，本发明所述载体中还可以包含筛选所用的基因(例如抗生素抗性基因)、用于生成荧光蛋白的核酸等片段。荧光蛋白可以选择绿色荧光蛋白、蓝色荧光蛋白、黄色荧光蛋白、橙色荧光蛋白或红色荧光蛋白。
特别地，当所述载体中包含编码如上所述的嵌合抗原受体的核酸时，所述载体优选为逆转录病毒载体，更优选为慢病毒载体。
在一个具体的实施方式中，所述慢病毒载体的核苷酸序列为SEQ ID NO:18所示。
本发明还涉及宿主细胞，其包含如上所述的载体。
术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。宿主细胞优选为真核细胞，更优选为哺乳动物细胞。
本发明还涉及免疫细胞，其包含如上所述的嵌合抗原受体；
在一些实施方式中，所述免疫细胞为T细胞、肿瘤浸润淋巴细胞(TIL细胞)、NK细胞、树突状细胞或NK-T细胞。
在一些实施方式中，所述免疫细胞为自体T细胞或同种异体T细胞。
在一些实施方案中，所述免疫细胞是获自外周血或由其制备。
在一些实施方案中，所述免疫细胞是获自外周血单核细胞(PBMC)或由其制备。
在一些实施方案中，所述免疫细胞是获自骨髓或由其制备。
在一些实施方案中，所述免疫细胞是获自脐带血或由其制备。
在一些实施方案中，所述免疫细胞是人细胞。
本发明还涉及药物组合物，其包括如上所述的抗体或如上所述的免疫细胞，以及药学上可接受的赋形剂、稀释剂或载体中的一种或多种。
术语“药学上可接受的赋形剂、稀释剂或载体”是指在药理学和/或生理学上与受试者和活性成分相容的赋形剂、稀释剂或载体，其是本领域公知的，包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。例如，pH调节剂包括但不限于磷酸盐缓冲液；表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80；离子强度增强剂包括但不限于氯化钠。
所述药用组合物可用于BCMA相关疾病，特别是多发性骨髓瘤。因而特别地，本发明还是涉及一种用于治疗有此需要的主体中的多发性骨髓瘤的方法，所述方法包括：
a)提供所述药用组合物；以及
b)向所述主体的施用治疗有效量的所述药用组合物。
术语“有效量”是指足以获得或至少部分获得期望的效果的量。期望的效果例如，预防或治疗多发性骨髓瘤，有效量通常是足以预防，阻止，或延迟疾病的发生的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、主体自己的免疫系统的总体状态、主体的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
在一些实施方式中，施用的方法可以采用注射给予等。
在本发明中“主体”、“受试者”、“患者”等词汇可根据需要通用。主体可以为哺乳动物，优选为人。
下面将结合实施例对本发明的实施方案进行详细描述。
实施例1 靶向BCMA抗体的获得
将人BCMA胞外段与小鼠抗体Fc片段(Fragment crystallizable)的缀合物(以下简称hBCMA-mFc，ACRO，BCA-H5253)腹腔注射BALB/c小鼠(广东省医学实验动物中心)，每周一次，每次100μg/200μl/只，免疫3周后每周取小鼠尾血并检测血清中BCMA抗体的表达；选择血清中BCMA抗体表达量高的小鼠取脾细胞与瘤细胞(SP20，ATCC HB-12546)融合形成融合子，融合子培养10～14天后选择培养上清中表达BCMA抗体的融合子进行单克隆；选择表达BCMA抗体的单克隆杂交瘤细胞株进行扩大培养，培养7～10天后收集细胞培养液纯化获得BCMA抗体，对所得的大量抗体经过筛选后，获得两个候选抗体(5E2和5F4)，5E2测序重链可变区氨基酸序列VH为SEQ ID NO:15所示，轻链可变区氨基酸序列VL为序列SEQ ID NO:16所示；5F4测序重链可变区氨基酸序列VH为SEQ ID NO:33所示，轻链可变区氨基酸序列VL为序列SEQ ID NO:34所示。
实施例2、BCMA抗体的筛选
1)BCMA抗体(5E2,5F4)亲和力检测：
通过ELISA、Fortebio和FACs三种方式检测不同抗体针对BCMA的亲和力，具体方法如下：
抗体亲和力ELISA检测：将hBCMA ECD hFc，ACRO，BC7-H5254包被在96孔酶联包被板中，浓度为2μg/ml，100μl/孔，将3倍梯度稀释(前一个浓度是后一个浓度的3倍，用1％BSA稀释)的抗体与抗原结合，检测各抗体的EC 50值(具体操作步骤为一般ELISA操作步骤)。R0317为C11D5.3BCMA鼠单抗，其具体序列和信息可以公开查找到，待测抗体由深圳市菲鹏治疗股份有限公司合成。结果如图1所示，结果显示BCMA抗体5E2和5F4与R0317具有处于同一水平的EC 50。
抗体亲和力Fortebio检测，利用带ProG biosensor(Pall ForteBIO,18-1502)，先Loading Buffer(PBS+0.02％Tween20),再加入h BCMA ECD hFc(R0341)5μg/mL,然后分别加入3种抗体，分别检测3种抗体的KD，Kon和Kdis。具体操作步骤为使用Fortebio仪器(forteBio，Serial NO:FB-40476)的常规操作。检测结果如图2所示，说明5E2 5F4与BCMA抗原的亲和力较强并且和R0317处于同一水平。
FACs检测抗体与肿瘤细胞系的结合：
RPMI8226细胞(
Figure PCTCN2020136725-appb-000001
CRM-CCL-155 TM)为人多发性骨髓瘤外周血B淋巴细胞，H929细胞(
Figure PCTCN2020136725-appb-000002
CRL-9068 TM)为人多发性骨髓瘤骨髓B淋巴细胞。具体检测方法如下：收获细胞，PBS洗涤1次，然后用PBS按照2E+5cells/200μl将细胞重悬。将3种抗体梯度稀释(抗体起始浓度为10μg/ml，1％BSA 3倍稀释，共9个梯度)后分别与细胞4℃孵育30min。其后与PE标记的抗小鼠IgG第二抗体(Biolegend，B288920)孵育，洗涤2次，Beckman Coulter(型号：CytoFLEX)流式细胞仪检测。如图3所示，5E2、5F4与RPMI8226和H929细胞有浓度梯度依赖的结合并且EC50显著低于R0317，说明5E2、5F4和RPMI8226和H929细胞的亲和力优于R0317。
2)BCMA抗体(5E2、5F4)与BCMA配体APRIL竞争性结合实验：
购买ACRO重组人APRIL Ala-Leu 250+N端连接人IgG1 Fc(货号APL-H5267)。将hBCMA-mFc以4μg/ml包被于ELISA板中，4℃包被过夜。PBS洗涤1次后，用1％BSA封闭1h，然后再PBS洗涤1次。将抗体5E2和5F4梯度稀释，起始浓度为200μg/ml，1％BSA进行3倍梯度稀释，共7个梯度，稀释液用h APRIL hFc 4μg/ml(此浓度在APRIL与hBCMA-mFc结合的EC50和饱和之间)，37℃孵育30min，洗涤5次，加入1:1500稀释的HRP标记的羊抗人Fc抗体(Goat Anti-Human IgG Antibody,Fc,HRP conjugate Sigma，AP113P)，37℃孵育30min，洗涤5次，TMB显色，终止后多功能酶标仪读数。结果如图4所示，5E2、5F4阻断APRIL的IC50与R0317相当，并且5E2、5F4对APRIL与hBCMA-mFc的阻断效果明显且呈浓度梯度依赖性。
3)BCMA抗体(5E2,5F4)特异性
对本发明的BCMA抗体进行特异性流式检测。体外合成人BCMA全长基因，并引入酶切位点，通过双酶切将合成的人BCMA全长基因插入慢病毒包装质粒载体(PCDHF)中，进行慢病毒包装(具体操作步骤为一般4质粒慢病毒包装步骤，具体可参考吉凯基因官网中关于慢病毒包装的部分)。将制备得到的慢病毒侵染K562细胞(ATCC，货号CCL-243)以构建K562-BCMA细胞，对所述细胞进行单克隆挑取和鉴定，获得稳定表达BCMA的稳定细胞株K562-BCMA。将5E2，5F4抗体分别与K562，H929，RPMI8226和K562-BCMA细胞于4℃孵育30min，之后用PBS洗涤2次，之后加入二抗PE山羊抗小鼠IgG1(Biolegend，B288920)于4℃孵育30min，之后用PBS洗涤2次。然后利用Beckman Coulter流式细胞仪进行检测。检测结果如下图5所示，5E2，5F4能够特异性地结合BCMA表达阳性的细胞。
实施例3、构建BCMA CART细胞并进行体外功能验证
1)获取5E2和5F4抗体序列：
复苏表达5E2和5F4的杂交瘤细胞株，正常培养72h后，裂解细胞提取RNA(提取试剂盒:TOYOBO LIFE SCIENCE，货号836700，提取步骤按说明书)。对提取的RNA进行逆转录以获得cDNA(逆转录试剂盒:TOYOBO LIFE SCIENCE,货号11141ES10)，对获得的cDNA进行PCR扩增(使用针对小鼠IgG1序列的特异性引物)以获得抗体序列并对其进行测序，抗体测序VH和VL见实施例1中标记5E2和5F4的VH，VL序列。由于两个抗体的亲和力、特异性及功能特性相似，后续以5E2为例做CART功能验证。
2)慢病毒包装：
将5E2抗体的scFv序列(通过连接肽将VH的C端与VL的N端相连得到，连接肽的核苷酸序列如SEQ ID NO:35所示)以及C11D5.3的scFv序列(SEQ ID NO:17)分别构建在慢病毒载体(PCDHF)上获得CAR质粒，C11D5.3CAR质粒和5E2CAR质粒的结构示意图如图6和图7所示。利用293T细胞(
Figure PCTCN2020136725-appb-000003
CRL-3216 TM)包装慢病毒，包装体系和包装步骤如下所示：
a，接种293T细胞5E6于10cm细胞培养皿中，加入10mL含10％FBS的DMEM培养基(DMEM Gibco，11995040-1L；FBS Gibco，10091-148)，5％CO 2，37℃条件下CO 2培养箱中培养48h；
b，慢病毒包装体系
Figure PCTCN2020136725-appb-000004
其中“*1、*2、*3”分别为慢病毒包装质粒PMD2.G，pMDLg/pRRE，pRSV-Rev序列，可以通过公开途径(网址http://www.miaolingbio.com/)获得。
c，包装后48h收细胞上清，25000rpm超速离心后检测慢病毒滴度，检测方法如下：将收集的慢病毒原液梯度体积同样条件下感染293T细胞，48h后流式检测293T细胞GFP阳性率百分比，根据计算公式原液滴度(TU/mL)＝1.5×10E+05×293T细胞GFP阳性率百分比/慢病毒原液体积μl×1000来计算慢病毒原液滴度。
3)CART细胞制备
Ficoll淋巴分离液(达科为，AS1114546)从血液(菲鹏生物员工0038号志愿者献血50mL)中分离PBMC细胞，偶联CD3/CD28抗体的磁珠正选法分离获得T细胞，将慢病毒按MOI＝5:1感染T细胞以制备CART细胞，CART细胞培养7天后用二抗APC山羊抗小鼠IgG(H+L)(Jackson，115-136-146)流式检测CART细胞CAR阳性率，如图8所示。
4)CART细胞体外功能评价
分别取4种靶细胞K562，K562-BCMA，H929和RPMI8226各2×10E+06个细胞，先利用CytoCalceinTM Violet 550对靶细胞进行染色，1×10E+05细胞/100μl/孔。将效应细胞(CAR+CART，T细胞为对照)分别与上述靶细胞按照0.25:1、1:1、5:1及10:1的比率加入96孔板中 混匀，终体积200μl。培养8h后，将细胞混匀离心。上清利用Human IL-2ELISA检测试剂盒(invitrogen，REF 88-7025-88)Human IFN gamma ELISA试剂盒(invitrogen，REF 88-7316-88)检测各孔中IL-2及IFN-γ浓度，沉淀部分用100μl Annexin V Binding Buffer(Biolegend，B274722)重悬，300g离心5min，之后添加2.0μl APC-Annexin V(Biolegend，Cat 640920)和1.2μl PI染料(Biolegend，Cat 421301)，避光孵育15min，添加100μl Annexin V Binding Buffer重悬，之后利用Beckman Coulter流式细胞仪检测各靶细胞凋亡比例(结果如图9所示)，利用ELISA检测各孔上清IL-2及IFN-γ浓度(结果如图10和图11所示)。其中K562为BCMA阴性细胞，K562-BCMA，H929,RPMI8226均为BCMA阳性细胞。结果显示5E2CART和C11D5.3CART对BCMA阳性靶细胞有较强的特异性杀伤，并且2种CART细胞的杀伤阳性靶细胞能力基本一致，对BCMA阴性细胞几乎无杀伤作用。
实施例4 靶向BCMA人源化抗体的获得
将鼠源5E2抗体进行人源化后获得4个抗体序列，包括2条重链可变区(SEQ ID NO:36-SEQ ID NO:37)和3条轻链可变区(EQ ID NO:38-SEQ ID NO:40)。
实施例5 BCMA人源化抗体评价
1)人源化BCMA抗体亲和力检测
通过ELISA、Fortebio和FACs三种方式检测4个BCMA人源化抗体(hu VH1-VL1；huVH2-VL1；hu VH1-VL2；huVH1-VL3)针对BCMA抗原的亲和力，具体方法如下：
抗体亲和力ELISA检测：将hu(人)BCMA ECD His，ACRO，BCA-H522y包被在96孔酶联包被板中，浓度为2μg/mL，100μL/孔，将4个人源化抗体分别用1％BSA配制成起始浓度20μg/mL，1％BSA 3倍梯度稀释(前一梯度抗体浓度为后一梯度抗体浓度的3倍)，抗体与抗原结合，检测4个抗体的EC 50值(具体操作步骤为一般ELISA操作步骤)。其中mVH-mVL为鼠源BCMA抗体，是人源化之前的鼠源抗体，结果如下图12所示，结果显示4个人源化BCMA抗体与鼠源BCMA抗体mVH-mVL具有处于同一水平的EC 50，并且huVH1-VL2亲和力最高。
抗体亲和力Fortebio检测，利用带AMC biosensor(Pall,lot:1907292)先Loading h BCMA ECD mFc 3ug/mL,然后分别结合4种人源化抗体，分别检测4种抗体的KD，Kon和Kdis。具体操作步骤为使用Fortebio仪器(forteBio，Serial NO:FB-40476)的常规操作。检测结果如下图13所示，说明4个BCMA人源化抗体与抗原的亲和力较强并且和鼠源BCMA抗体mVH-mVL处于同一水平，huVH1-VL2亲和力最高。
FACs检测抗体与肿瘤细胞系的结合：
K562细胞(
Figure PCTCN2020136725-appb-000005
CCL-243TM)为人慢性髓系白血病细胞，CHO细胞(
Figure PCTCN2020136725-appb-000006
CRL-12023 TM)为中国仓鼠卵巢细胞，分别将含有人BCMA全长序列的慢病毒感染K562细胞和CHO细胞，感染后挑单克隆，获得稳定表达BCMA的K562-BCMA细胞株和CHO-BCMA细胞株，检测4人源化抗体分别与K562-BCMA和CHO-BCMA的亲和力(EC50)，具体检测方法如下：收获细胞，PBS洗涤1次，然后用PBS按照2E+5cells/200μL将细胞重悬。将4种BCMA人源化抗体用1％BSA 3倍梯度稀释，前一梯度抗体浓度为后一梯度抗体浓度的3倍，(抗体起始浓度为30μg/ml，共11个梯度)后分别与细胞4℃孵育30min。其后与APC anti-human IgG Fc Antibody(Biolegend，409306)4℃孵育30min，1×PBS洗涤2次，Beckman Coulter(型号：CytoFLEX)流式细胞仪检测。如下图14所示，3个人源化抗体与K562-BCMA细胞和CHO-BCMA细胞有浓度梯度依赖的结合并且EC 50与鼠源BCMA抗体处于同一水平，huVH1VL2与K562-BCMA亲和力EC50稍弱一些，但huVH1-VL2与CHO-BCMA亲和力EC 50与其它3个人源化抗体处于同一水平，4个人源化抗体和鼠源BCMA抗体与CHO-BCMA结合表达丰度上，从高到低的顺序为m VH-m VL,hu VH1-VL1，hu VH2-VL1，hu VH1-VL2，hu VH1-VL3。
2)4个人源化BCMA抗体与BCMA配体APRIL竞争性结合实验：
购买ACRO重组人APRIL Ala-Leu 250+N端His标签(Human APRIL/TNFSF13 Protein,His Tag货号APL-H5244)。将hBCMA-mFc以4μg/ml包被于ELISA板中，4℃包被过夜。1×PBS洗涤1次后，用1％BSA(Sangon Biotech,A500023-0100)封闭1h，然后再用1×PBS洗涤1次。将4个人源化抗体3倍梯度稀释，前一梯度抗体浓度为后一梯度抗体浓度的3倍，起始浓度为100μg/ml，共11个梯度，稀释液用h APRIL His 0.2μg/ml，(此浓度在APRIL与hBCMA-mFc结合的EC 50和饱和之间),h APRIL His用1％BSA溶解，37℃孵育30min，1×PBS洗涤5次，加入1:1500稀释的HRP标记的抗His抗体(Anti-his tag HRP,Biolegend，652504)，37℃孵育30min，洗涤5次，TMB显色，终止后多功能酶标仪读数。结果如下图15所示，4个人源化BCMA抗体阻断APRIL的IC50与鼠源BCMA抗体m VH-m VL相当，并且4个人源化BCMA抗体对APRIL与hBCMA-mFc的阻断效果明显且呈浓度梯度依赖性。
3)4个人源化BCMA抗体特异性
对4个人源化BCMA抗体进行特异性流式检测，将4个人源化BCMA抗体10ug/ml 100ul分别与K562，K562-BCMA，CHO，CHO-BCMA和K562-BCMA细胞于4℃孵育30min，之后用1×PBS洗涤2次，之后加入APC anti-human IgG Fc Antibody(Biolegend，409306)于4℃孵育30min，之后用1×PBS洗涤2次。然后利用Beckman Coulter流式细胞仪进行检测。检测结果如下图16所示，4个人源化BCMA抗体能够特异性地结合BCMA表达阳性的细胞。
实施例6 构建BCMA CART细胞并进行体外功能验证
1)慢病毒包装：
经过亲和力，阻断功能和特异性比较，优选将m VH-m VL,hu VH1-VL1，hu VH2-VL1抗体的scFv序列分别构建在慢病毒载体(PCDHF,含有GFP序列)上获得CAR质粒，m VH-m VL,hu VH1-VL1，hu VH2-VL1抗体的scFv序列构建得CAR质粒分别为PCDHF-42,PCDHF-73，PCDHF-74，结构示意图如图17、18、19所示。利用293T细胞(
Figure PCTCN2020136725-appb-000007
CRL-3216 TM)包装慢病毒，包装体系和包装步骤如下所示：
a接种293T细胞5E6于10cm细胞培养皿中，加入10mL含10％FBS的DMEM培养基(DMEM Gibco，11995040-1L；FBS Gibco，10091-148)，5％CO2，37℃条件下CO2培养箱中培养24h；
b慢病毒包装体系
Figure PCTCN2020136725-appb-000008
c包装后48h收细胞上清，25000rpm超速离心后检测慢病毒滴度，检测方法如下：将收集的慢病毒原液梯度体积同样条件下感染293T细胞，48h后流式检测293T细胞GFP阳性率 百分比，根据计算公式原液滴度(TU/mL)＝1.5×10E+05×293T细胞GFP阳性率百分比/慢病毒原液体积μl×1000来计算慢病毒原液滴度。
2)CART细胞制备
Ficoll淋巴分离液(达科为，AS1114546)从血液(志愿者献血50mL)中分离PBMC细胞，偶联CD3/CD28抗体的磁珠正选法分离获得T细胞，将慢病毒按MOI＝5:1感染T细胞以制备CART细胞，CART细胞培养7天后通过检测CART细胞的GFP阳性率来确定CART细胞CAR阳性率，如图20所示，
3)CART细胞体外功能评价
分别取4种靶细胞K562，K562-BCMA，RPMI8226各2×10E+06个细胞，先利用CytoCalcein TM Violet 550对靶细胞进行染色，1×10E+05细胞/100ul/孔。将效应细胞(CAR+CART，T细胞为对照)分别与上述靶细胞按照0.25:1、1:1、5:1及10:1的比率加入96孔板中混匀，终体积200ul。培养6h后，将细胞混匀离心。上清利用Human IL-2ELISA检测试剂盒(invitrogen，REF 88-7025-88)Human IFN gamma ELISA试剂盒(invitrogen，REF 88-7316-88)检测各孔中IL-2及IFN-γ浓度，沉淀部分用100ul Annexin V Binding Buffer(Biolegend，B274722)重悬，300g离心5min，之后添加3ul APC-Annexin V(Biolegend，Cat 640920)和1.5ul PI染料(Biolegend，Cat 421301)，避光孵育15min，添加100ul Annexin V Binding Buffer重悬，之后利用Beckman Coulter流式细胞仪检测各靶细胞凋亡比例(结果如图21所示)，利用ELISA检测各孔上清IL-2及IFN-γ浓度(结果如图22和图23所示)。其中K562为BCMA阴性细胞，K562-BCMA，RPMI8226均为BCMA阳性细胞。结果显示PCDHF-73CART、PCDHF-74CART和PCDHF-42CART对BCMA阳性靶细胞有较强的特异性杀伤，并且3种CART细胞的杀伤阳性靶细胞能力基本一致，对BCMA阴性细胞几乎无杀伤作用；PCDHF-42CART,PCDHF-73CART和PCDHF-74CART杀伤BCMA阳性靶细胞时IL-2和IFN-γ分泌量处于同一水平，综合特异性杀伤和因子分泌的检测结果说明，人源化BCMA CART PCDHF-73CART和PCDHF-74CART与鼠源BCMA CART PCDHF-42CART对BCMA阳性靶细胞有同等的杀伤效果。
本发明所涉及的序列如下表所示
Figure PCTCN2020136725-appb-000009
Figure PCTCN2020136725-appb-000010
Figure PCTCN2020136725-appb-000011
Figure PCTCN2020136725-appb-000012
Figure PCTCN2020136725-appb-000013
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种BCMA抗体或其抗原结合片段，其能够特异性结合BCMA，且选自a)和/或b)：
    a)包含氨基酸序列依次如SEQ ID NO:1～3所示的重链互补决定区CDR-VH1、CDR-VH2、CDR-VH3，以及氨基酸序列依次如SEQ ID NO:4～6所示的轻链互补决定区CDR-VL1、CDR-VL2、CDR-VL3；
    b)包含氨基酸序列依次如SEQ ID NO:19～21所示的重链互补决定区CDR-VH1、CDR-VH2、CDR-VH3，以及氨基酸序列依次如SEQ ID NO:22～24所示的轻链互补决定区CDR-VL1、CDR-VL2、CDR-VL3。
  2. 根据权利要求1所述的抗体或其抗原结合片段，a还包含序列如SEQ ID NO:7～10、SEQ ID NO:41～44、或SEQ ID NO:45～48所示的重链骨架区FR-H1、FR-H2、FR-H3及FR-H4；和/或；序列如SEQ ID NO:11～14、SEQ ID NO:49～52、SEQ ID NO:53～56或SEQ ID NO:57～60所示的轻链骨架区FR-L1、FR-L2、FR-L3及FR-L4；
    b还包含序列依次如SEQ ID NO:25～28所示的重链骨架区FR-H1、FR-H2、FR-H3及FR-H4；和/或；序列依次如SEQ ID NO:29～32所示的轻链骨架区FR-L1、FR-L2、FR-L3及FR-L4。
  3. 根据权利要求1或2所述的抗体或其抗原结合片段，所述抗体为F(ab’) 2、Fab、Fv、scFv以及双特异抗体中的一种。
  4. 根据权利要求1或2所述的抗体或其抗原结合片段，所述抗体具有恒定区，恒定区序列选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE、IgD任何其中之一恒定区的序列；
    可选的，所述恒定区的种属来源独立地选自牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人。
  5. 嵌合抗原受体，其包含权利要求1～3任一项所述的抗体；
    可选的，所述嵌合抗原受体还包含一个或多个选自由以下组成的组的元件：前导肽、接头序列、跨膜结构域、共刺激结构域和信号传导结构域。
  6. 分离的核酸，其编码权利要求1～4任一项所述的抗体，或权利要求5所述的嵌合抗原受体。
  7. 载体，其包含权利要求6所述的分离的核酸；
    可选的，所述载体为慢病毒载体；
    可选的，所述慢病毒载体的核苷酸序列为SEQ ID NO:18所示。
  8. 宿主细胞，其包含权利要求7所述的载体。
  9. 免疫细胞，其包含权利要求5所述的嵌合抗原受体；
    可选的，所述免疫细胞为T细胞、肿瘤浸润淋巴细胞、NK细胞、树突状细胞或NK-T细胞；
    可选的，所述免疫细胞为自体T细胞或同种异体T细胞。
  10. 药物组合物，其包括权利要求1～4任一项所述的抗体或权利要求9所述的免疫细胞，以及药学上可接受的赋形剂、稀释剂或载体中的一种或多种。
PCT/CN2020/136725 2019-12-17 2020-12-16 Bcma结合抗体及其用途 WO2021121250A1 (zh)

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CN114634568B (zh) * 2020-12-16 2024-04-09 深圳市菲鹏生物治疗股份有限公司 Bcma的抗体及其应用
CN115521381A (zh) * 2021-06-24 2022-12-27 益科思特(北京)医药科技发展有限公司 结合bcma和cd3的双特异性抗体及其制备方法与应用
CN117164714B (zh) * 2023-10-08 2024-04-23 北京奇迈永华生物科技有限公司 一种靶向bcma的抗体及其应用

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