A nano antibody against BCMA and its preparation method and application

Abstract

The present invention discloses a nano antibody against BCMA and its preparation method and application, and relates to the field of biomedical technology. The present invention uses CRISPR / Cas9 technology to knock out B2M and TRAC genes in T cells to prepare universal CAR-T cells; the nano antibody targeting BCMA and the natural ligand SIRPα extracellular N-terminal V region of CD47, the transmembrane region of CD28, the intracellular signal region of 4-1BB and the intracellular signal region of CD3ζ are connected in series to form a dual-targeted chimeric antigen receptor for the treatment of multiple myeloma, so as to solve the high recurrence problem faced by CAR-T cell therapy in the treatment of multiple myeloma. The universal CAR-T cells prepared by the present invention have strong killing activity against multiple myeloma cells, effectively improve the treatment effect, and provide a key material for the subsequent development of adoptive immunotherapy with high efficiency and lasting anti-tumor activity.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an anti-BCMA nanobody and a preparation method and application thereof. Background Art

[0002] Multiple myeloma (MM) is a blood tumor disease characterized by abnormal proliferation of plasma cells (PCs) and the production of a large amount of pathological immunoglobulins in the body. Its tumor cells originate from plasma cells in the bone marrow. Among blood tumors, the incidence of MM is second only to lymphoma, accounting for about 10% of blood system malignancies, with an incidence of about 2 to 3 per 100,000 people. In the field of immunotherapy, studies have shown that CD38, CD47, CD138, SLAMF7, GPRC5D, FcRH5 and BCMA are highly expressed on the surface of malignant plasma cells, and their monoclonal antibodies or antibody-drug conjugates show good anti-tumor effects. Nevertheless, due to the high clonal heterogeneity of MM and its complex interaction with the bone marrow environment, MM patients will still develop drug resistance after standardized treatment, which ultimately leads to relapse in most patients. Therefore, in order to improve the problems of reduced efficacy during treatment, it is necessary to develop safer, more effective and diverse new drugs.

[0003] BCMA (B-cell maturation antigen; CD269; TNFRSF17) is a member of the tumor necrosis factor receptor superfamily. In normal human tissues, BCMA mRNA and protein are almost exclusively found on plasma cells, and are selectively overexpressed during the malignant transformation of plasma cells, promoting tumor cell growth, survival, and drug resistance. The consistent upregulation and uniqueness of BCMA on the surface of MM cells, whether in cell lines or patient samples, make BCMA an attractive target for MM drug discovery and development. In addition, studies have found that BCMA expression levels are similar at different stages of multiple myeloma (from untreated to relapse), suggesting that BCMA may be an effective therapeutic target throughout the disease process.

[0004] CD47 is a transmembrane glycoprotein widely expressed on a variety of normal tissue cells. In addition, it is also overexpressed on a variety of tumor cells (osteosarcoma, colorectal cancer, ovarian cancer, breast cancer, bladder cancer, head and neck cancer, etc.), and is an important tumor antigen. CD47 belongs to the immunoglobulin superfamily and binds to a variety of proteins, including integrins, thrombospondin-1, and signal regulatory protein α (SIRPα). Similar to the checkpoints PD-1 / PD-L1 axis and MHC-I / LILRB1 axis involved in regulating the phagocytic clearance escape of tumor cells, the CD47-SIRPα axis also has the function of escaping phagocytic clearance as an immune checkpoint. CD47 binds to the N-terminus of SIRPα on immune cells, sends a "don't eat me" signal, inhibits the phagocytosis of macrophages, and protects healthy cells from being destroyed by the immune system. In the natural immunity of tumors, tumor cells also use this mechanism: by overexpressing CD47 molecules on the surface of tumor cells, macrophages treat them as "normal cells" and thus escape macrophage-mediated phagocytosis. Studies have found that blocking the CD47-SIRPα interaction can promote the destruction of tumor cells by phagocytes (including macrophages and neutrophils), thereby enhancing the anti-tumor effect. Recent studies have found that compared with non-malignant plasma cells, multiple myeloma (MM) cells escape the clearance of phagocytes by widely upregulating CD47 and using the above mechanism. After blocking the interaction between CD47 and SIRPα with anti-CD47 monoclonal antibodies, it was found that the phagocytic ability of phagocytes was enhanced and the growth of MM cells was inhibited. Therefore, the high expression of CD47 on MM makes it an important target for the development of drugs to treat MM.

[0005] Camelids have a special IgG molecule that naturally lacks the light chain (Light chain) and the first constant region of the heavy chain (CH1), which is called heavy chain antibody (HcAbs, Heavy chain-only Antibodies), and its variable region is called VHH (Variable domains of Camelidae heavy chain-only antibodies). The VHH structure is rugby-shaped, with a diameter of about 2.5nm and a height of about 4.0nm. Due to its small structure and molecular weight (15kDa), it is also called nanobody (Nb, Nanobody). Nb is considered to be the smallest antibody molecule found so far that has the function of binding to a complete antigen. Compared with other small molecule genetically engineered antibodies, nanobodies have received great attention in various fields due to their good stability, solubility, affinity, specificity, flexibility in coupling with different protein molecules, and easy modification. At present, the application of nanobodies is mainly concentrated in disease treatment, disease and medical diagnosis, scientific research, etc. The humanized nanoantibody ALX-0081 (Caplacizumab), as the first nanoantibody drug, was officially launched in 2018 for the treatment of acquired thrombocytopenic purpura.

[0006] Chimeric Antigen Receptor-T cells (CAR-T) are a type of genetically modified T cells that can specifically recognize specific antigens independently of MHC molecules, and can continuously activate, proliferate, and specifically kill tumor cells. CAR is composed of a CD8 signal peptide, an antigen recognition region (single-chain antibody or nanoantibody or ligand or Fab segment), a transmembrane region, and a series of signal transduction regions of T cells in series. The lentiviral vector or retroviral vector is used to co-transfect mammalian cells with packaging plasmids and auxiliary plasmids to produce viral particles containing CAR. The particles infect primary T cells again to enable them to stably express CAR, and finally form CAR-T cells. The cells are then re-infused into the patient's body. The modified T cells have the function of specifically recognizing and killing tumor cells expressing related antigens, thereby achieving the purpose of clearing tumor cells.

[0007] However, due to the high cost and long preparation cycle of personalized CAR-T cells, as well as the fact that T cells from some patients are difficult to obtain and difficult to expand in vitro, CAR-T is greatly limited in clinical application.

[0008] Based on this, the application of universal CAR-T technology (allogeneic CAR-T cells) will largely solve the above problems. Existing gene editing technology is used to knock out TCR and other genes that mediate inter-individual immunogenicity in T cells from healthy donors, thereby reducing graft-versus-host disease (GVHD) and host-versus-graft reaction (HVGR), thereby realizing the industrialization of CAR-T cell therapy and reducing costs. On May 12, 2022, Caribou Biosciences announced that its CRISPR gene-editing-based spot CAR-T therapy CB-010 was effective in a phase 1 clinical trial for the treatment of relapsed and refractory B-cell non-Hodgkin's lymphoma. Published data showed that among the five evaluable patients, the overall remission rate (ORR) reached 100% and the complete remission rate (CR) reached 80%. So far, among the four patients with complete remission, this is still the case three months after treatment, with the longest record of complete remission being six months.

[0009] At present, the FDA has approved Abecma (ide-cel) and Carvykti, which are used for adult patients with RRMM who have previously received 4 lines of treatment or multiple lines of treatment (including 3 types of drugs: immunomodulators, proteasome inhibitors, and anti-CD38 antibodies). In addition, there are currently about 46 CAR-T drugs targeting BCMA in the clinical or preclinical stage worldwide, indicating that BCMA has broad prospects in MM drug discovery and development. However, BCMA-CART faces the problem of high recurrence in the clinical treatment of MM patients, and the possible cause of recurrence is the downregulation or loss of the BCMA target. In addition, analysis of clinical cases showed that the expression rate of CD47 in malignant plasma cells of MM patients was about 100%.

[0010] Therefore, developing a bispecific universal chimeric antigen receptor that simultaneously targets BCMA and CD47 can not only effectively avoid the problem of target escape but also significantly improve the therapeutic effect, which will lay a good foundation for clinical trials and clinical treatments. However, there are currently no related reports. Summary of the invention

[0011] The purpose of the present invention is to solve the above problems and provide a universal chimeric antigen receptor for the treatment of multiple myeloma and its application.

[0012] In order to achieve the above-mentioned purpose, the technical scheme of the present invention is as follows: a universal chimeric antigen receptor for the treatment of multiple myeloma, wherein the universal chimeric antigen receptor is composed of a nanobody targeting BCMA, an extracellular N-terminal V region of the natural ligand SIRPα targeting CD47, a CD28 transmembrane region, a 4-1BB intracellular signal region and a CD3ζ intracellular signal region in series.

[0013] The present invention also provides an anti-BCMA nanobody (Nb), comprising any of the following:

[0014] (1) A nanobody against BCMA protein, named BCMA-Nb381, the amino acid sequence of which is shown in SEQ ID No. 1;

[0015] (2) a nanobody against BCMA protein, named BCMA-Nb388, the amino acid sequence of which is shown in SEQ ID No. 2;

[0016] (3) a nanobody against BCMA protein, named BCMA-Nb394, the amino acid sequence of which is shown in SEQ ID No. 3;

[0017] Furthermore, a method for preparing an anti-BCMA nanobody (Nb) comprises the following steps:

[0018] (1) The BCMA extracellular domain recombinant protein was emulsified with Freund's adjuvant and then immunized with Bactrian camels for five consecutive immunizations, with an interval of 2 weeks between each immunization.

[0019] (2) 7 days after the last immunization, peripheral blood was collected to isolate lymphocytes and construct a phage display library against BCMA protein;

[0020] (3) Using phage display technology, after three rounds of screening, the sequencing results were compared and analyzed to compare the differences in the CDR regions of the nanoantibodies, and ultimately three nanoantibodies that specifically bind to BCMA were obtained.

[0021] The present invention also provides a humanized anti-BCMA nanobody (huNb), which is a humanized anti-BCMA protein nanobody modified according to BCMA-Nb381, Nb388 and Nb394, named BCMA-hu381, BCMA-hu388 and BCMA-hu394, and the amino acid sequences are shown in SEQ ID No. 4, 5 and 6.

[0022] The present invention also provides the N-terminal V region of SIRPα, a natural ligand of CD47 molecule, named SIRPαV, and the amino acid sequence is shown in SEQ ID No.7.

[0023] The present invention also provides an anti-human CD3 molecule scFv antibody OKT3, the amino acid sequence of which is shown in SEQ ID No.8.

[0024] The present invention also provides a chimeric antigen receptor CAR-hu388 targeting BCMA, and the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID No.9.

[0025] The present invention also provides a chimeric antigen receptor CAR-SIRPαV targeting CD47, and the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID No.10.

[0026] The present invention also provides a chimeric antigen receptor CAR-h31 targeting BCMA and CD47, and the amino acid sequence of the chimeric antigen receptor CAR-h31 is shown in SEQ ID No.11.

[0027] The present invention also provides a chimeric antigen receptor CAR-h32 targeting BCMA and CD47, and the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID No.12.

[0028] Furthermore, the coding sequence of the chimeric antigen receptor targeting BCMA or CD47 comprises the coding sequence of the CD8α signal peptide, the coding sequence of the anti-BCMA nanobody or / and the CD47 natural ligand SIRPαV extracellular N-terminal V region, the coding sequence of the CD8 hinge region, the coding sequence of the CD28 transmembrane region, the coding sequence of the 4-1BB intracellular signal region and the coding sequence of the CD3ζ intracellular region, which are sequentially connected.

[0029] Furthermore, the coding sequence of the CD8α signal peptide is SEQ ID No.13; the coding sequence of the CD8 hinge region is SEQ ID No.14; the coding sequence of the CD28 transmembrane region is SEQ ID No.15; the coding sequence of the 4-1BB intracellular signal region is SEQ ID No.16; the coding sequence of the CD3ζ intracellular region is SEQ ID No.17; the coding sequence of the anti-BCMA humanized nanoantibody is SEQ ID No.5; the coding sequence of the CD47 natural ligand SIRPαV is SEQ IDNo.7; the coding sequence of the flexible Linker (G4S) 3 coding region is SEQ ID No.18; and the coding sequence of the rigid Linker (EAAAK) 3 coding region is SEQ ID No.19.

[0030] The present invention also provides a recombinant protein and a drug combination thereof, comprising any one of an anti-BCMA nanobody (Nb) and an anti-BCMA humanized nanobody (huNb) for use in the diagnosis of related diseases or in combination with an active agent, wherein the active agent is an immune checkpoint-related preparation and / or a bi(multi)specific antibody and / or an antibody-drug conjugate and / or a radionuclide and / or a kinase inhibitor.

[0031] Furthermore, the expression vector of the chimeric antigen receptor is selected from a lentiviral vector.

[0032] The present invention also provides a recombinant lentivirus, which is obtained by co-transfecting the above-mentioned lentiviral vector with a packaging plasmid and an envelope plasmid into mammalian cells.

[0033] Furthermore, the mammalian cell is any one of HEK293 cells, HEK293T cells or HEK293F cells, or a combination of at least two of them.

[0034] The present invention also provides an sgRNA for knocking out the TRAC gene in T cells, which is designed based on the α chain constant region of the TCR gene; the sequences of the two sgRNAs of the TRAC gene are SEQ ID No. 20-21, respectively.

[0035] The present invention also provides an sgRNA for knocking out the B2M gene in T cells, which is designed based on the functional domain of the B2M gene; the sequences of the two sgRNAs of the B2M gene are SEQ ID No.22-23, respectively.

[0036] The present invention also provides the use of the nanoantibody sequence, the N-terminal V region (SIRPαV) sequence of the natural ligand SIRPα extracellular region of CD47 molecules, the recombinant fusion protein, the chimeric antigen receptor, the host cell, etc. in the preparation of activated T cells, immune cells or in tumor treatment drugs.

[0037] The drug is used to treat tumors, including blood tumors and solid tumors. The blood tumors include at least one of multiple myeloma, acute myeloid leukemia, B-cell lymphoma, and non-Hodgkin's lymphoma; the solid tumors include at least one of osteosarcoma, colorectal cancer, ovarian cancer, breast cancer, bladder cancer, and head and neck cancer. Preferably, the tumor is multiple myeloma.

[0038] The following is the above-mentioned sequence involved in the present invention:

[0039] SEQ ID No.1

[0040] QVQLQESGGGSVQAGGSLRLSCTASGYSDSNYCMAWFRQAPGKARQGVAFINGDGVITYTDSVKGRFTISKDNAQKTLDLQMNSLKPEDTAMYYCAALTAGCVRYAAWGQGTQVTVSS

[0041] SEQ ID No.2

[0042] QVQLQESGGGSVQAGGSLRLSCAASGYTYSNYCMAWFRQAPGKLRQGVAFIDSDGTTTYADSVKGRFTISRDNAKNTLSLQMNTLKPEDTAMYYCAALRSDCSGGYRGVGQGTQVTVSS

[0043] SEQ ID No.3

[0044] QVQLQESGGGSVQGGGSLRLSCTASGFNIDDSDMGWFRQAPGDACKLVSHINSDGATYNADSVKGRFTISRDSAKNTVYLKMNSLKPEDTAVYYCHPVYNLGLIDPPACSGQGTQVTVSS

[0045] SEQ ID No.4

[0046] EVQLVESGGGSVQAGGSLRLSCTASGYSDSNYCMAWFRQAPGKGRQGVAFINGDGVITYTDSVKGRFTISKDNAQKTLDLQMNSLRAEDTAMYYCAALTAGCVRYAAWGQGTLVTVSS

[0047] SEQ ID No.5

[0048] EVQLVESGGGSVQAGGSLRLSCAASGYTYSNYCMAWFRQAPGKGRQGVAFIDSDGTTTYADSVKGRFTISRDNAKNTLSLQMNTLRAEDTAMYYCAALRSDCSGGYRGVGQGTLVTVSS

[0049] SEQ ID No.6

[0050] EVQLVESGGGSVQGGGSLRLSCTASGFNIDDSDMGWFRQAPGDGCKLVSHINSDGATYNADSVKGRFTISRDSAKNTVYLKMNSLRAEDTAVYYCHPVYNLGLIDPPACSGQGTLVTVSS

[0051] SEQ ID No.7

[0052] EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAK

[0053] SEQ ID No.8

[0054] DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK

[0055] SEQ ID No.9

[0056] MALPVTALLLPLALLLHAARPFEEVQLVESGGGSVQAGGSLRLSCAASGYTYSNYCMAWFRQAPGKGRQGVAFIDSDGTTTYADSVKGRFTISRDNAKNTLSLQMNTLRAEDTAMYYCAALRSDCSGGYRGVGQGTLVTVSSGTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDRRPPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELASLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0057] SEQ ID No.10

[0058] MALPVTALLLPLALLLHAARPFEEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKGTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDRRPPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELASLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0059] SEQ ID No.11

[0060] MALPVTALLLPLALLLHAARPFEEVQLVESGGGSVQAGGSLRLSCAASGYTYSNYCMAWFRQAPGKGRQGVAFIDSDGTTTYADSVKGRFTISRDNAKNTLSLQMNTLRAEDTAMYYCAALRSDCSGGYRGVGQGTLVTVSSGGGGSGGGGSGGGGSEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKGTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDRRPPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELASLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0061] SEQ ID No.12

[0062] MALPVTALLLPLALLLHAARPFEEVQLVESGGGSVQAGGSLRLSCAASGYTYSNYCMAWFRQAPGKGRQGVAFIDSDGTTTYADSVKGRFTISRDNAKNTLSLQMNTLRAEDTAMYYCAALRSDCSGGYRGVGQGTLVTVSSEAAAKEAAAKEAAAKEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKGTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDRRPPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELASLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0063] SEQ ID No.13

[0064] MALPVTALLLPLALLLHAARP

[0065] SEQ ID No.14

[0066] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0067] SEQ ID No.15

[0068] PSKPFWVLVVVGGVLACYSLLVTVAFIIFWVR

[0069] SEQ ID No.16

[0070] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE

[0071] SEQ ID No.17

[0072] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0073] SEQ ID No.18

[0074] GGGGSGGGGSGGGGS

[0075] SEQ ID No.19

[0076] EAAAKEAAAKEAAAK

[0077] SEQ ID No.20

[0078] 5'-ACAAAACTGTGCTAGACATG-3'

[0079] SEQ ID No.21

[0080] 5'-CTTCAAGAGCAACAGTGCTG-3'

[0081] SEQ ID No.22

[0082] 5'-CGCGAGCACAGCTAAGGCCA-3'

[0083] SEQ ID No.23

[0084] 5'-ACTCACGCTGGATAGCCTCC-3'

[0085] Compared with the prior art, this solution has the following beneficial effects:

[0086] The present invention uses human BCMA and CD47 molecules as targets to prepare a universal chimeric antigen receptor that can simultaneously target two surface markers of multiple myeloma, which can not only effectively solve the problem of reduced efficacy caused by target immune escape, but also reduce treatment costs and shorten treatment cycles. At the same time, it can effectively improve the killing activity of T cells, effectively reduce the recurrence rate of tumors after treatment, and prolong the survival of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 is the recombinant protein BCMA-mFc purified by SDS-PAGE analysis in the embodiment of the present invention; wherein M is Maker, 1 is reduced, and 2 is non-reduced;

[0088] Figure 2is the recombinant anti-BCMA nanobody purified by SDS-PAGE analysis in the embodiment of the present invention; wherein M is Maker;

[0089] Figure 3 This is an in vitro killing activity analysis of the bispecific antibody Nbs-OKT3 against BCMA-positive cell lines in an embodiment of the present invention;

[0090] Figure 4 The reactivity of the humanized nanobody and the camel-derived nanobody with the BCMA antigen is preferably compared using indirect ELISA in the embodiments of the present invention;

[0091] Figure 5 The affinity of BCMA-hu381, BCMA-hu388 and BCMA-hu394 to BCMA protein was determined by Biacore in the examples of the present invention;

[0092] Figure 6 In the embodiment of the present invention, the expression of CD47 molecules in natural positive cell lines is detected by FACS;

[0093] Figure 7 is a schematic diagram of a molecular model of a chimeric antigen receptor in an embodiment of the present invention;

[0094] Figure 8 In the embodiment of the present invention, FACS is used to detect the expression of CAR in T cells;

[0095] Figure 9a In the embodiment of the present invention, FACS is used to detect the knockout efficiency of TRAC gene in CAR-T cells respectively;

[0096] Figure 9b In the embodiment of the present invention, FACS is used to detect the knockout efficiency of B2M gene in CAR-T cells respectively;

[0097] Fig.9c This is a sequencing analysis diagram after TRAC gene knockout in an embodiment of the present invention;

[0098] Figure 9d This is a sequencing analysis diagram after B2M gene knockout in an embodiment of the present invention;

[0099] Fig.10 In the embodiment of the present invention, RTCA is used to detect the universal CAR-T cells for BCMA + / CD47 + -Hela cell killing activity;

[0100] Fig.11 This is the in vitro killing activity analysis of universal CAR-T cells in the embodiments of the present invention;

[0101] Fig.12 This is an analysis of the survival of the MM mouse transplantation model using universal CAR-T cells in an embodiment of the present invention. DETAILED DESCRIPTION

[0102] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme of the present invention will be further described in detail below in combination with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0103] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0104] Example 1 Construction of eukaryotic expression vector of BCMA protein and its expression and purification

[0105] 1.1 Vector construction

[0106] Using the plasmid containing the full-length BCMA gene as a template, primers were designed to amplify the BCMA extracellular domain (ECD) gene, and it was connected to the pVax-mFc vector after double digestion with restriction endonucleases PstⅠ and XbaⅠ by homologous recombination. It was transformed into DH5α competent medium, coated with kanamycin-resistant plates, and cultured in a 37℃ incubator overnight. Single clones were picked for sequencing and identification.

[0107] 1.2 Expression and purification of recombinant proteins

[0108] The recombinant plasmid pVax-BCMA(ECD)-mFc containing the BCMA extracellular domain gene was successfully constructed and transfected into HEK293T cells. After 5 days of expression, the culture supernatant was collected and purified by affinity chromatography using an NTA-Ni column to obtain a high-purity recombinant protein BCMA-mFc. The results are as follows: Figure 1 shown.

[0109] Example 2 Screening and preparation of anti-BCMA protein nanobodies

[0110] 2.1 Protein emulsification and animal immunization

[0111] 1 mg of purified BCMA-mFc recombinant protein was emulsified with an equal volume of Freund's complete adjuvant and used for the first immunization subcutaneously in the neck of the Alxa Bactrian camel. Then, 1 mg of the protein was emulsified with incomplete Freund's adjuvant and used for 4 consecutive immunizations every 2 weeks. Peripheral anticoagulant and coagulation blood were collected on the 7th day after the shock immunization.

[0112] 2.2 Construction and panning of VHH phage antibody library

[0113] 2.2.1 Isolation of peripheral blood lymphocytes

[0114] 200 mL of peripheral anticoagulated blood was collected aseptically from the camel's jugular vein and diluted with an equal volume of RPMI-1640 medium. Peripheral blood lymphocytes were then separated by centrifugation using Ficoll-Paque Plus lymphocyte separation solution (Greinerbio-one) and lymphocyte separation tubes. The obtained lymphocytes can be directly used to extract total RNA or frozen at -80°C for later use.

[0115] 2.2.2 VHH gene amplification

[0116] First, the total RNA of lymphocytes was extracted according to the instructions (catalog 74134, RNeasy Plus Mini Kit, QIAGEN), and then the cDNA was obtained by reverse transcription using a reverse transcription kit (catalog 18080051, SuperScriptⅢ First-Strand Synthesis System, Invitrogen). The VHH gene was amplified by nested PCR using the cDNA as a template. The primers for the first round of amplification were CALL001 and CALL002 (see Table 1). The band of about 700 bp was separated and recovered by agarose gel electrophoresis. Then, the recovered 700 bp product was used as a template for the second round of PCR amplification. The primers for the second round of amplification were VHH-FOR and VHH-REV (see Table 1). The band of 400 bp was separated and recovered by agarose gel electrophoresis.

[0117] 2.2.3 Construction of VHH phage display vector

[0118] The 400 bp product recovered in 2.2.2 and the phage display vector pMECS were double-digested with endonucleases PstI and NotI and recovered, and then ligated with T4 DNA ligase.

[0119] 2.2.4 Electroporation of ligation products into TG1 competent cells and harvesting of phage antibody library

[0120] The ligation product in 2.2.3 was added to the competent E. coli TG1 cells and electroporated into TG1. After electroporation, SOC medium was added immediately and cultured at 37°C, 200 rpm for 1 hour. Then, the culture was spread on LB / Amp-Glu plates. After culture at 37°C for 6 to 8 hours, the bacterial lawn was collected and 1 / 3 volume of 50% glycerol was added to prepare the phage library.

[0121] 2.2.5 Screening of anti-BCMA protein specific nanoantibodies

[0122] Using the prepared phage library as the antibody source, phage display technology was used for three rounds of screening, and a total of three anti-BCMA specific nanoantibodies were obtained, which were named BCMA-Nb381, BCMA-Nb388 and BCMA-Nb394, respectively.

[0123] 2.2.6 Preparation of anti-BCMA protein-specific nanobodies

[0124] The VHH gene was amplified using the plasmid containing the nanobody gene in 2.2.5 as a template and constructed into the eukaryotic expression vector pcDNA3.1-hFc by homologous recombination. After sequencing, the plasmid was extracted and transfected into HEK293T cells. After 5 days of expression, the supernatant was collected and purified by affinity chromatography using an NTA-Ni column to obtain a high-purity recombinant nanobody ( Figure 2 ).

[0125] Example 3 Analysis of the in vitro cytotoxicity of bispecific antibody Nbs-OKT3 against myeloma cells

[0126] The three nanobodies in this patent were respectively connected with the anti-CD3 antibody OKT3 using (G4S)3linker to a eukaryotic expression vector, transfected into HEK293T cells for expression, and purified using a Ni column. The purified bispecific antibodies of different concentrations were co-incubated with the multiple myeloma cell line MM.1S-luciferase and activated T cells at an effector-target ratio of 3:1 for 24 hours, and the killing activity was evaluated by detecting the fluorescence intensity. The results are as follows: Figure 3 As shown, the above three bispecific antibodies all have strong killing activity against natural MM cell lines.

[0127] Example 4 Humanization transformation and activity detection of preferred nanobodies

[0128] 4.1 Humanization, expression and purification of nanobodies

[0129] Referring to the sequence characteristics of the fully human antibody framework region, the amino acids in certain specific sites of the camel-derived nanoantibody framework region (FR region) were replaced while keeping the amino acid sequence of the CDR region unchanged, the corresponding humanized nanoantibody was designed and fully synthesized, and the vector was constructed, expressed and purified by referring to the method of 2.2.6 in Example 2.

[0130] 4.2 Biacore detection of affinity of humanized nanobodies

[0131] The recombinant humanized nanoantibodies hu381, hu388 and hu394 were respectively tested for binding affinity with the antigen BCMA-mFc coated on the CM5 chip using the Biacore 8k instrument. The results are as follows Figure 5 As shown, the affinity of humanized nanobody to BCMA is between 7.46×10 -11 M~1.04×10 -10 M.

[0132] Example 5 Identification of CD47 Expression in Multiple Myeloma Cell Lines

[0133] APC anti-human CD47 antibody (catalog 323123, Biolegend) and SIRPαV-hFc recombinant protein were co-incubated with BCMA naturally positive cell lines RPMI-8226 and MM.1S, respectively. 647-Goat@human IgG (catalog 109-603-003, Jackson) antibody was used for detection. The results were as follows Figure 6 As shown, CD47 molecules were strongly positively expressed in the above cell lines.

[0134] Example 6 Preparation of Chimeric Antigen Receptor Lentivirus

[0135] 6.1 Design of chimeric antigen receptors

[0136] In this example, chimeric antigen receptors CAR-hu388, CAR-SIRPαV, CAR-h31, and CAR-h32 against BCMA and / or CD47 are constructed, wherein the antigen binding domains of CAR-h31 and CAR-h32 are h31 and h32, respectively. The molecular model diagram of the chimeric antigen receptor is shown in FIG. Figure 7 shown.

[0137] 6.2 Construction of lentiviral vector expressing chimeric antigen receptor

[0138] Using the plasmid containing hu388 and / or SIRPαV sequences as templates, homologous recombination primers were designed to amplify the antigen binding domains respectively. The PCR products and the lentiviral vector pSLCAR-CD19-BBz digested with restriction endonucleases BamHⅠ and Kpn I were recovered according to the instructions of the gel recovery kit, and then connected using homologous recombination enzymes. The construction was confirmed to be successful after transformation and sequencing verification.

[0139] 6.3 Lentiviral packaging

[0140] The specific steps are as follows:

[0141] 6.3.1 The lentiviral plasmid containing the chimeric antigen receptor, the packaging auxiliary plasmid psPAX2 and the envelope plasmid pMD2.G were mixed at a mass ratio of 6:3:1, and then mixed with the transfection reagent at a mass volume ratio of 3:1, added to a certain volume of serum-free Opti-MEM medium, and allowed to stand at room temperature for 20 minutes after mixing. The above mixture was evenly added to the culture dish with HEK293T cells, and after mixing, it was placed in an incubator at 37°C and 5% CO2 for 6 to 8 hours, and then the fresh medium was replaced and the culture was continued for 48 hours, and the culture supernatant was collected. At the same time, the fresh medium was replaced and the culture was continued until 72 hours after transfection, and the supernatant was collected.

[0142] 6.3.2 After centrifugation at 4°C, 2000 rpm for 10 min, filter the collected culture supernatant with a 0.45 μm filter membrane and centrifuge it in an ultracentrifuge at 4°C, 35,000 rpm for 2.5 h. Discard the supernatant and resuspend the precipitate in an appropriate volume of RPMI-1640 culture medium. The virus solution can be used directly for subsequent infection of T cells or aliquoted and frozen at -80°C for later use.

[0143] Example 7 Preparation of CAR-T cells

[0144] The specific steps are as follows:

[0145] 7.1 Isolation of primary T lymphocytes

[0146] Collect an appropriate amount of human peripheral blood in an anticoagulant tube, mix by inversion, dilute the peripheral blood with an equal volume of RPMI-1640 medium, and then use Ficoll-Paque Plus lymphocyte separation solution and lymphocyte separation tube to separate peripheral blood lymphocytes by centrifugation, lyse red blood cells, and then use PanT Cell Isolation Kit (catalog 130-096-535, Miltenyi) to sort T cells.

[0147] 7.2 T cell culture

[0148] The sorted T cells were adjusted to 5×10 6 / mL, human T Cell Activation / Expansion Kit (catalog 130-091-441, Miltenyi) was used to activate T cells, and after mixing, they were cultured in an incubator at 37°C and 5% CO2 for 48 hours. After stimulation, the beads were removed by centrifugation at 300g for 10 minutes, the supernatant was discarded, and the T cells were resuspended in fresh T cell culture medium.

[0149] 7.3 CAR Lentivirus Infection

[0150] The CAR-containing lentiviral concentrate (MOI = 10) prepared in Example 6 6.3.2 was mixed with T cells, 10 μg / mL polybrene, 10 ng / mL IL-7 and 10 ng / mL IL-15 were added, and the mixture was centrifuged at 32°C and 2000 rpm for 2 hours. After centrifugation, the cell plate was taken out and placed in an incubator at 37°C and 5% CO2 for culture.

[0151] 7.4 CAR-T cell culture

[0152] The density of prepared CAR-T cells was maintained at 1×10 6 / mL, half of the medium was changed every 2 to 3 days and cultured continuously for subsequent experiments.

[0153] 7.5 Detection of CAR expression in CAR-T cells

[0154] Take 5×10 4 CAR-T cells were co-incubated with antigen BCMA-mFc and / or CD47-hFc recombinant protein, centrifuged and washed 3 times at 1200 rpm, and then 647 (catalog 115-605-003, Jackson) and 647-Goat@human IgG was tested for CAR expression by FACS. The results are as follows: Figure 8 As shown, the expression rates of CAR in CAR-hu388, CAR-SIRPα, CAR-h31, and CAR-h32 were 93.43%, 93.80%, 97.51%, and 97.63%, respectively.

[0155] Example 8 Preparation of universal CAR-T cells

[0156] 8.1 sgRNA design

[0157] First, human TRAC (NG_001332.3) and B2M (NM_004048.4) gene sequences were downloaded from the NCBI database, and sgRNAs were designed for the TRAC constant region and B2M functional domain using CRISPR design software (http: / / chopchop.cbu.uib.no / ).

[0158] 8.2 CRISPR / Cas9-mediated gene knockout

[0159] The above gene sequences and their complementary chains were synthesized separately. In order to form a ribonucleoprotein complex (RNP), the recombinant Cas9 protein (ThermoFisher) and the synthesized sgRNA were mixed at a ratio of 10 μg:5 μg and then added to 10 μL 4D-Nucleofector TM Solution was incubated at 37°C for 10-15 minutes, and the prepared RNPs were mixed with 100 μL 4D-Nucleofector TM Solution resuspended 1.0×10 7 The CAR-T cells were mixed and added to the Lonza electroporator and electroporated using program E0-115. After electroporation, the cells were placed in an incubator at 37°C and 5% CO2 for further culture.

[0160] 8.3 Knockout efficiency detection

[0161] 3 to 4 days after electroporation, 1.0 × 10 5 The knockout efficiency of TRAC and B2M genes was detected by FACS in T cells, and the antibodies used were FITC anti-CD3 (clone UCTH1, BioLegend) and PE anti-β2m (clone 2M2, BioLegend). 647anti-HLA-DR,DP,DQ (clone Tü39, BioLegend), the results are as follows Figure 9a , b shows that the knockout efficiencies of TRAC and B2M genes were 46.58% and 48.85%, respectively.

[0162] 8.4 Identification of TRAC and B2M genes in CAR-T cells after knockout

[0163] Take 1.0×10 4 The whole genome of the knocked-out CAR-T cells in Example 8.2 was extracted using a total DNA extraction kit, and the TRAC and B2M genes were amplified using primers TRAC-F, TRAC-R and B2M-F, B2M-R (see Table 1), respectively. The amplified products (TRAC: 500 bp; B2M: 592 bp) were recovered from the gel and sequenced. The sequencing results are shown in Fig.9c As shown in d, the genes TRAC and B2M were successfully knocked out in CAR-T cells, indicating that universal CAR-T cells were successfully prepared.

[0164] 8.5 Purification of CAR-T cells after knockout

[0165] The present invention utilizes magnetic bead sorting to separate TRAC - / B2M-CAR-T cells were purified. Specifically: (1) 1.0×10 5 CAR-T cells were incubated with biotin anti-CD3 antibody (catalog 317319, BioLegend) at room temperature for 30 minutes, washed three times with PBS after the incubation, and incubated with Anti-biotin MicroBeads (catalog 130-090-485, Miltenyi) according to the instructions. After the incubation, the cells were washed three times and placed on a magnetic rack to collect the unadsorbed cells; (2) The unadsorbed cells in (1) were incubated with biotin anti-β2m antibody (catalog 316308, BioLegend) at room temperature for 30 minutes, washed three times with PBS after the incubation, and incubated with Anti-biotin MicroBeads. After the incubation, the cells were washed three times and placed on a magnetic rack to collect the unadsorbed cells; (3) The unadsorbed cells in (2) were incubated with biotin anti-HLA-DR antibody (catalog 307614, BioLegend) at room temperature for 30 minutes, washed 3 times with PBS after the incubation, incubated with Anti-biotin MicroBeads, washed 3 times after the incubation, and placed on a magnetic rack to collect the unadsorbed cells. Finally, 1×10 6 Resuspend the CAR-T cells at 100 μg / mL and add 10 ng / mL IL-7 and 10 ng / mL IL-15 stimulating factors, and continue to culture at 37°C and 5% CO2 for subsequent experiments.

[0166] Example 9 In vitro detection of the killing activity of universal CAR-T cells against tumor cells

[0167] 9.1 In vitro detection of CAR-T cells for BCMA + / CD47 + -Hela cell killing activity

[0168] BCMA + / CD47 +-Hela cells were slowly added to a 96-well plate dedicated to the non-labeled killing detector (RTCA) at a density of 5000 cells per well and 100 μL / well (duplicate wells), and cultured in the instrument until the Cell Index value was between 1.0 and 2.0. CAR-T cells and control cells were added at an E:T ratio of 3:1, and the instrument was started to continue detection. The results are shown in Figure 9. The killing activity of bispecific universal CAR-T (CAR-h32, CAR-h31) is stronger than that of monospecific CAR-T (CAR-hu388, CAR-SIRPαV).

[0169] 9.2 In vitro detection of the killing activity of universal CAR-T cells against RPMI-8226 and MM.1S cells

[0170] Universal CAR-T cells, control T cells, and blank cells were mixed with 2×10 4 Tumor cells RPMI-8226-luciferase and MM.1S-luciferase were mixed at an E:T ratio of 4:1, 2:1, 1:1, and 1:2 and added to a 96-well plate. Each group was set up with duplicate wells and placed in a 37°C, 5% CO2 incubator for 24 hours. After co-culture, 5 μL of luciferase substrate was added to each well, pipetted and mixed, and immediately placed in a multifunctional microplate reader to measure RLU (relative light unit), and the fluorescence intensity was used to quantify the killing efficiency. The killing efficiency calculation formula is: [1-(experimental well reading / control well reading)]×100%. The results are shown in Fig.11 As shown, they have similar killing activity in the two tumor cells, and the killing activity of bispecific universal CAR-T (CAR-h32, CAR-h31) is stronger than that of monospecific CAR-T (CAR-hu388, CAR-SIRPαV).

[0171] 9.3 In vivo detection of anti-tumor activity of CAR-T cells in MM mouse transplantation model

[0172] MM.1S-luciferase and RPMI-8226-luciferase cells were cultured at 2×10 6 10 cells were inoculated into 6- to 10-week-old NCG mice through the tail vein. Ten days after inoculation, tumor growth was detected using a live imaging device and the mice were randomly divided into five groups, with five mice in each group. On the 13th day after tumor cell inoculation, universal CAR-T and mock-T cells were inoculated at a rate of 4×10 per mouse. 6The cells were inoculated into the corresponding groups through the tail vein, and the control group was inoculated with the same volume of PBS. The survival status of each mouse in the experimental group and the control group was observed and statistically analyzed for 100 days. The Kaplan-Meier method was used to draw the mouse survival curve and the log-rank (Mantel-Cox) test was used to statistically compare the differences in the survival period of mice in each group. The results are shown in Fig.12 As shown, the survival of the bispecific universal CAR-h32 group was significantly longer than that of the monospecific CAR-T (CAR-hu388, CAR-SIRPαV), indicating that bispecific CAR-T cells effectively avoided problems such as reduced efficacy and tumor recurrence caused by target escape.

[0173] Table 1 Primer sequences required in the present invention

[0174]

[0175]

[0176] The above specific embodiments are only explanations of the present invention, and they are not limitations of the present invention. After reading this specification, those skilled in the art can make modifications to the embodiments without creative contribution as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. An anti-BCMA nanobody, characterized in that The nanobody is named BCMA-Nb388, and the amino acid sequence is shown in SEQ ID No.

2.

2. A humanized nanobody against BCMA, characterized in that: The humanized anti-BCMA Nanobody modified by the Nanobody according to claim 1 is named BCMA-hu388, and the amino acid sequence is shown in SEQ ID No.

5.

3. An isolated nucleic acid, characterized in that It encodes an anti-BCMA nanobody as described in claim 1 or a humanized anti-BCMA nanobody as described in claim 2.

4. A recombinant vector, characterized in that: It contains the isolated nucleic acid as claimed in claim 3.

5. A host cell, characterized in that It contains the recombinant vector as claimed in claim 4.

6. A method for preparing an anti-BCMA nanobody, characterized in that: It includes: Cultivate the host cell as described in claim 5 to obtain the anti-BCMA nanobody.

7. An anti-BCMA chimeric antigen receptor CAR, characterized in that: The chimeric antigen receptor CAR comprises a signal peptide, an antigen recognition region capable of recognizing BCMA antigen, a hinge region, a transmembrane region and a signal transduction domain, and the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID No.9; The antigen recognition region of the chimeric antigen receptor CAR comprises the humanized anti-BCMA nanobody as described in claim 2; The signal peptide is a CD8α signal peptide; The hinge region includes CD8; The transmembrane region includes the CD28 transmembrane domain; The signal transduction domain includes CD3ζ; The signal transduction domain also includes the 4-1BB intracellular region.

8. A bispecific chimeric antigen receptor CAR-h31 or CAR-h32 against BCMA and CD47, characterized in that: The chimeric antigen receptor CAR comprises an antigen recognition region, a hinge region, a transmembrane region and a signal transduction domain that can recognize BCMA and CD47 antigens, and the amino acid sequence of the chimeric antigen receptor is shown in any one of SEQ ID No.11 or SEQ ID No.12; The antigen recognition region of the chimeric antigen receptor CAR includes the humanized anti-BCMA nanobody as described in claim 2 and the CD47 natural ligand SIRPα extracellular V region (SIRPαV).

9. A CAR-T cell, characterized in that: It comprises the chimeric antigen receptor CAR according to claim 7 or the chimeric antigen receptor according to claim 8; The CAR-T cells are selected from universal CAR-T cells or autologous CAR-T cells.

10. Use of the anti-BCMA nanobody according to claim 1, the humanized anti-BCMA nanobody according to claim 2, the isolated nucleic acid according to claim 3, the recombinant vector according to claim 4, the host cell according to claim 5, the chimeric antigen receptor CAR according to claim 7, the bispecific chimeric antigen receptor according to claim 8, or the CAR-T cell according to claim 9 in the preparation of a drug for treating tumors; The tumor is multiple myeloma.

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